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Sample records for albicans hyphal morphogenesis

  1. Role of the RAM Network in Cell Polarity and Hyphal Morphogenesis in Candida albicans

    PubMed Central

    Song, Yunkyoung; Cheon, Seon Ah; Lee, Kyung Eun; Lee, So-Yeon; Lee, Byung-Kyu; Oh, Doo-Byung; Kang, Hyun Ah

    2008-01-01

    RAM (regulation of Ace2p transcription factor and polarized morphogenesis) is a conserved signaling network that regulates polarized morphogenesis in yeast, worms, flies, and humans. To investigate the role of the RAM network in cell polarity and hyphal morphogenesis of Candida albicans, each of the C. albicans RAM genes (CaCBK1, CaMOB2, CaKIC1, CaPAG1, CaHYM1, and CaSOG2) was deleted. All C. albicans RAM mutants exhibited hypersensitivity to cell-wall- or membrane-perturbing agents, exhibiting cell-separation defects, a multinucleate phenotype and loss of cell polarity. Yeast two-hybrid and in vivo functional analyses of CaCbk1p and its activator, CaMob2p, the key factors in the RAM network, demonstrated that the direct interaction between the SMA domain of CaCbk1p and the Mob1/phocein domain of CaMob2p was necessary for hyphal growth of C. albicans. Genome-wide transcription profiling of a Camob2 mutant suggested that the RAM network played a role in serum- and antifungal azoles–induced activation of ergosterol biosynthesis genes, especially those involved in the late steps of ergosterol biosynthesis, and might be associated, at least indirectly, with the Tup1p-Nrg1p pathway. Collectively, these results demonstrate that the RAM network is critically required for hyphal growth as well as normal vegetative growth in C. albicans. PMID:18843050

  2. cAMP-independent signal pathways stimulate hyphal morphogenesis in Candida albicans.

    PubMed

    Parrino, Salvatore M; Si, Haoyu; Naseem, Shamoon; Groudan, Kevin; Gardin, Justin; Konopka, James B

    2017-03-01

    The fungal pathogen Candida albicans can transition from budding to hyphal growth, which promotes biofilm formation and invasive growth into tissues. Stimulation of adenylyl cyclase to form cAMP induces hyphal morphogenesis. The failure of cells lacking adenylyl cyclase (cyr1Δ) to form hyphae has suggested that cAMP signaling is essential for hyphal growth. However, cyr1Δ mutants also grow slowly and have defects in morphogenesis, making it unclear whether hyphal inducers must stimulate cAMP, or if normal basal levels of cAMP are required to maintain cellular health needed for hyphal growth. Interestingly, supplementation of cyr1Δ cells with low levels of cAMP enabled them to form hyphae in response to the inducer N-acetylglucosamine (GlcNAc), suggesting that a basal level of cAMP is sufficient for stimulation. Furthermore, we isolated faster-growing cyr1Δ pseudorevertant strains that can be induced to form hyphae even though they lack cAMP. The pseudorevertant strains were not induced by CO2 , consistent with reports that CO2 directly stimulates adenylyl cyclase. Mutational analysis showed that induction of hyphae in a pseudorevertant strain was independent of RAS1, but was dependent on the EFG1 transcription factor that acts downstream of protein kinase A. Thus, cAMP-independent signals contribute to the induction of hyphal responses.

  3. Hbr1 Activates and Represses Hyphal Growth in Candida albicans and Regulates Fungal Morphogenesis under Embedded Conditions

    PubMed Central

    Pendrak, Michael L.; Roberts, David D.

    2015-01-01

    Transitions between yeast and hyphae are essential for Candida albicans pathogenesis. The genetic programs that regulate its hyphal development can be distinguished by embedded versus aerobic surface agar invasion. Hbr1, a regulator of white-opaque switching, is also a positive and negative regulator of hyphal invasion. During embedded growth at 24°C, an HBR1/hbr1 strain formed constitutively filamentous colonies throughout the matrix, resembling EFG1 null colonies, and a subset of long unbranched hyphal aggregates enclosed in a spindle-shaped capsule. Inhibition of adenylate cyclase with farnesol perturbed the filamentation of HBR1/hbr1 cells producing cytokinesis-defective hyphae whereas farnesol treated EFG1 null cells produced abundant opaque-like cells. Point mutations in the Hbr1 ATP-binding domain caused distinct filamentation phenotypes including uniform radial hyphae, hyphal sprouts, and massive yeast cell production. Conversely, aerobic surface colonies of the HBR1 heterozygote on Spider and GlcNAc media lacked filamentation that could be rescued by growth under low (5%) O2. Consistent with these morphogenesis defects, the HBR1 heterozygote exhibited attenuated virulence in a mouse candidemia model. These data define Hbr1 as an ATP-dependent positive and negative regulator of hyphal development that is sensitive to hypoxia. PMID:26039220

  4. Modulation of Phagosomal pH by Candida albicans Promotes Hyphal Morphogenesis and Requires Stp2p, a Regulator of Amino Acid Transport

    PubMed Central

    Vylkova, Slavena; Lorenz, Michael C.

    2014-01-01

    Candida albicans, the most important fungal pathogen of humans, has a unique interaction with macrophages in which phagocytosis induces a switch from the yeast to hyphal form, allowing it to escape by rupturing the immune cell. While a variety of factors induce this switch in vitro, including neutral pH, it is not clear what triggers morphogenesis within the macrophage where the acidic environment should inhibit this transition. In vitro, C. albicans grown in similar conditions in which amino acids are the primary carbon source generate large quantities of ammonia to raise the extracellular pH and induce the hyphal switch. We show here that C. albicans cells neutralize the macrophage phagosome and that neutral pH is a key inducer of germination in phagocytosed cells by using a mutant lacking STP2, a transcription factor that regulates the expression of multiple amino acid permeases, that is completely deficient in alkalinization in vitro. Phagocytosed stp2Δ mutant cells showed significant reduction in hypha formation and escaped from macrophages less readily compared to wild type cells; as a result stp2Δ mutant cells were killed at a higher rate and caused less damage to RAW264.7 macrophages. Stp2p-regulated import leads to alkalinization of the phagosome, since the majority of the wild type cells fail to co-localize with acidophilic dyes, whereas the stp2Δ mutant cells were located in acidic phagosomes. Furthermore, stp2Δ mutant cells were able to form hyphae and escape from neutral phagosomes, indicating that the survival defect in these cells was pH dependent. Finally, these defects are reflected in an attenuation of virulence in a mouse model of disseminated candidiasis. Altogether our results suggest that C. albicans utilizes amino acids to promote neutralization of the phagosomal pH, hyphal morphogenesis, and escape from macrophages. PMID:24626429

  5. Gymnemic Acids Inhibit Hyphal Growth and Virulence in Candida albicans

    PubMed Central

    Vediyappan, Govindsamy; Dumontet, Vincent; Pelissier, Franck; d’Enfert, Christophe

    2013-01-01

    Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs) as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine. PMID:24040201

  6. Gymnemic acids inhibit hyphal growth and virulence in Candida albicans.

    PubMed

    Vediyappan, Govindsamy; Dumontet, Vincent; Pelissier, Franck; d'Enfert, Christophe

    2013-01-01

    Candida albicans is an opportunistic and polymorphic fungal pathogen that causes mucosal, disseminated and invasive infections in humans. Transition from the yeast form to the hyphal form is one of the key virulence factors in C. albicans contributing to macrophage evasion, tissue invasion and biofilm formation. Nontoxic small molecules that inhibit C. albicans yeast-to-hypha conversion and hyphal growth could represent a valuable source for understanding pathogenic fungal morphogenesis, identifying drug targets and serving as templates for the development of novel antifungal agents. Here, we have identified the triterpenoid saponin family of gymnemic acids (GAs) as inhibitor of C. albicans morphogenesis. GAs were isolated and purified from Gymnema sylvestre leaves, the Ayurvedic traditional medicinal plant used to treat diabetes. Purified GAs had no effect on the growth and viability of C. albicans yeast cells but inhibited its yeast-to-hypha conversion under several hypha-inducing conditions, including the presence of serum. Moreover, GAs promoted the conversion of C. albicans hyphae into yeast cells under hypha inducing conditions. They also inhibited conidial germination and hyphal growth of Aspergillus sp. Finally, GAs inhibited the formation of invasive hyphae from C. albicans-infected Caenorhabditis elegans worms and rescued them from killing by C. albicans. Hence, GAs could be useful for various antifungal applications due to their traditional use in herbal medicine.

  7. Hyphal growth in Candida albicans does not require induction of hyphal-specific gene expression

    PubMed Central

    Naseem, Shamoon; Araya, Esteban; Konopka, James B.

    2015-01-01

    Various stimuli, including N-acetylglucosamine (GlcNAc), induce the fungal pathogen Candida albicans to switch from budding to hyphal growth. Previous studies suggested that hyphal morphogenesis is stimulated by transcriptional induction of a set of genes that includes known virulence factors. To better understand hyphal development, we examined the role of GlcNAc metabolism using a triple mutant lacking the genes required to metabolize exogenous GlcNAc (hxk1Δ nag1Δ dac1Δ). Surprisingly, at low ambient pH (∼pH 4), GlcNAc stimulated this mutant to form hyphae without obvious induction of hyphal genes. This indicates that GlcNAc can stimulate a separate signal to induce hyphae that is independent of transcriptional responses. Of interest, GlcNAc could induce the triple mutant to express hyphal genes when the medium was buffered to a higher pH (>pH 5), which normally occurs after GlcNAc catabolism. Catabolism of GlcNAc raises the ambient pH rather than acidifying it, as occurs after dextrose catabolism. This synergy between alkalinization and GlcNAc to induce hyphal genes involves the Rim101 pH-sensing pathway; GlcNAc induced rim101Δ and dfg16Δ mutants to form hyphae, but hyphal gene expression was partially defective. These results demonstrate that hyphal morphogenesis and gene expression can be regulated independently, which likely contributes to pathogenesis at different host sites. PMID:25609092

  8. Inhibitory effect of verapamil on Candida albicans hyphal development, adhesion and gastrointestinal colonization.

    PubMed

    Yu, Qilin; Ding, Xiaohui; Zhang, Bing; Xu, Ning; Jia, Chang; Mao, Jiwei; Zhang, Biao; Xing, Laijun; Li, Mingchun

    2014-06-01

    Candida albicans morphogenesis and gastrointestinal colonization are closely associated with the pathogenicity of this pathogen. This study investigated the in vitro and in vivo effect of verapamil, a calcium channel blocker, on these processes. Exposure to ≥ 10 μg mL(-1) verapamil led to a significant decrease of C. albicans hyphal cells. The ability to adhere to a polystyrene surface and buccal epithelial cells was inhibited by exposure to ≥ 20 μg mL(-1) verapamil. Detection of the Hwp1-green fluorescent protein fusion protein showed that verapamil inhibited expression and transport of Hwp1, indicating its activity against both the regulation network of morphogenesis-associated proteins and the secretory pathway in C. albicans. Moreover, treatment with verapamil at 10 mg (kg day)(-1) led to a remarkable decrease in gastrointestinal-colonizing fungal cells. This study revealed the inhibitory effect of verapamil on C. albicans hyphal development, adhesion and gastrointestinal colonization, which is relevant to decreased expression and abnormal transport of the proteins required for morphogenesis. Therefore, verapamil may be taken into account when choosing an antifungal therapy against C. albicans colonization and infection.

  9. Septin Function in Candida albicans MorphogenesisD⃞

    PubMed Central

    Warenda, Amy J.; Konopka, James B.

    2002-01-01

    The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 and CDC12 septins are essential for viability. In contrast, the C. albicans cdc10Δ and cdc11Δ mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. The cdc11Δ mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth. PMID:12181342

  10. Image analysis of hyphal morphogenesis in Saprolegniaceae (Oomycetes).

    PubMed

    Diéguez-Uribeondo, Javier; Gierz, Gerhard; Bartnicki-García, Salomon

    2004-03-01

    Because of their wide range of apical morphology, several members of saprolegniaceous fungi (Oomycetes) were chosen to examine concordance with the vesicle supply center (VSC) model of hyphal morphogenesis. Two computer routines were devised to measure diameter changes over long stretches of hyphae and to test compatibility with the theoretical hyphoid shape, y = xcot(xV/N). In all four genera examined, the apex followed closely the contour described by the hyphoid equation; divergences became evident in the subapex. The hyphae of Saprolegnia parasitica showed maximum concordance with the VSC model, i.e., their profile matched a hyphoid curve from the apex to the entire length of the mature hyphal tube. In Aphanomyces and Leptolegnia, growth in the subapical region subsided becoming less than that specified by the hyphoid equation. In Achlya bisexualis, the reverse was true, the subapical region expanded beyond that specified by the hyphoid equation. The two divergent subapical tendencies gave the hyphal tips a cylindroid or conoid appearance, respectively. Since the hyphal apex of all four species conformed to the curvature dictated by the hyphoid equation, we concluded that a basic VSC mechanism operates in all of these oomycetous fungi. Accordingly, we suggest that the shape of an oomycetous hypha is generated by a VSC-driven gradient of wall formation, which is subject to additional modification in the subapex to produce a range of hyphal tip morphologies. The mathematical basis for generating a conoid hyphal tip by elongating the VSC is described in Appendix A.

  11. Hypoxia and Temperature Regulated Morphogenesis in Candida albicans

    PubMed Central

    Kurtz, Dagmar; Juchimiuk, Mateusz; Ernst, Joachim F.

    2015-01-01

    Candida albicans is a common commensal in the human gut but in predisposed patients it can become an important human fungal pathogen. As a commensal, C. albicans adapts to low-oxygen conditions and represses its hyphal development by the transcription factor Efg1, which under normoxia activates filamentation. The repressive hypoxic but not the normoxic function of Efg1 required its unmodified N-terminus, was prevented by phosphomimetic residues at normoxic phosphorylation sites T179 and T206 and occurred only at temperatures ≤35°C. Genome-wide binding sites for native Efg1 identified 300 hypoxia-specific target genes, which overlapped partially with hypoxic binding sites for Ace2, a known positive regulator of hypoxic filamentation. Transcriptional analyses revealed that EFG1, ACE2 and their identified targets BCR1 and BRG1 encode an interconnected regulatory hub, in which Efg1/Bcr1 act as negative and Ace2/Brg1 act as positive regulators of gene expression under hypoxia. In this circuit, the hypoxic function of Ace2 was stimulated by elevated CO2 levels. The hyperfilamentous phenotype of efg1 and bcr1 mutants depended on Ace2/Brg1 regulators and required increased expression of genes encoding Cek1 MAP kinase and its downstream target Cph1. The intricate temperature-dependent regulatory mechanisms under hypoxia suggest that C. albicans restricts hyphal morphogenesis in oxygen-poor body niches, possibly to persist as a commensal in the human host. PMID:26274602

  12. Hyphal formation of Candida albicans is controlled by electron transfer system

    SciTech Connect

    Watanabe, Toshihiko . E-mail: twatanab@tohoku-pharm.ac.jp; Ogasawara, Ayako; Mikami, Takeshi; Matsumoto, Tatsuji

    2006-09-15

    Most Candida albicans cells cultured in RPMI1640 medium at 37 deg. C grow in hyphal form in aerobic conditions, but they grow in yeast form in anaerobic conditions. The hyphal growth of C. albicans was inhibited in glucose-deficient conditions. Malonic acid, an inhibitor of succinate dehydrogenase, enhanced the yeast proliferation of C. albicans, indicating that the hyphal-formation signal was derived from the glycolysis system and the signal was transmitted to the electron transfer system via the citric acid cycle. Thenoyl trifluoro acetone (TTFA), an inhibitor of the signal transmission between complex II and Co Q, significantly inhibited the hyphal growth of C. albicans. Antimycin, KCN, and oligomycin, inhibitors of complex III, IV, and V, respectively, did not inhibit the hyphal growth of C. albicans. The production of mRNAs for the hyphal formation signal was completely inhibited in anaerobic conditions. These results indicate that the electron transfer system functions upstream of the RAS1 signal pathway and activates the expression of the hyphal formation signal. Since the electron transfer system is inactivated in anaerobic conditions, C. albicans grew in yeast form in this condition.

  13. Systemic Staphylococcus aureus infection mediated by Candida albicans hyphal invasion of mucosal tissue

    PubMed Central

    Schlecht, Lisa Marie; Peters, Brian M.; Krom, Bastiaan P.; Freiberg, Jeffrey A.; Hänsch, Gertrud M.; Filler, Scott G.

    2015-01-01

    Candida albicans and Staphylococcus aureus are often co-isolated in cases of biofilm-associated infections. C. albicans can cause systemic disease through morphological switch from the rounded yeast to the invasive hyphal form. Alternatively, systemic S. aureus infections arise from seeding through breaks in host epithelial layers although many patients have no documented portal of entry. We describe a novel strategy by which S. aureus is able to invade host tissue and disseminate via adherence to the invasive hyphal elements of Candida albicans. In vitro and ex vivo findings demonstrate a specific binding of the staphylococci to the candida hyphal elements. The C. albicans cell wall adhesin Als3p binds to multiple staphylococcal adhesins. Furthermore, Als3p is required for C. albicans to transport S. aureus into the tissue and cause a disseminated infection in an oral co-colonization model. These findings suggest that C. albicans can facilitate the invasion of S. aureus across mucosal barriers, leading to systemic infection in co-colonized patients. PMID:25332378

  14. Effect of cinnamaldehyde on hyphal growth of C. albicans under various treatment conditions.

    PubMed

    Taguchi, Yuuki; Hasumi, Yayoi; Hayama, Kazumi; Arai, Ryo; Nishiyama, Yayoi; Abe, Shigeru

    2012-01-01

    This study investigated the effects of cinnamaldehyde in combatting the hyphal growth of Candida albicans under varying concentrations, treatment times, and temperatures to determine the potential benefits of applying this substance to anti-Candida foods or gargles. From the results of pretreatment with cinnamaldehyde against Candida hyphae, we found that its inhibitory activity seemed to be strengthened in parallel with prolonged pretreatment time and a rise in temperature, and that pretreatment of 2,000 μg/ml for only 1 minute significantly inhibited the hyphal growth of C. albicans. We also demonstrated by XTT assay that pretreatment with cinnamaldehyde affected the metabolic activity of Candida hyphal cells. These findings suggest that a warm drink or mouthwash containing cinnamaldehyde might be a candidate as a prophylactic or therapeutic tool against oral Candida infection.

  15. Minocycline inhibits the Candida albicans budded-to-hyphal-form transition and biofilm formation.

    PubMed

    Kurakado, Sanae; Takatori, Kazuhiko; Sugita, Takashi

    2017-03-28

    Candida albicans frequently causes bloodstream infections; the budded-to-hyphal-form transition (BHT) and biofilm formation are major contributors to virulence. In a survey of antibacterial compounds that inhibit C. albicans BHT, we found that the tetracycline derivative minocycline inhibited BHT and subsequent biofilm formation. Minocycline downregulates expression of the hypha-specific genes HWP1 and ECE1, and the adhesion factor gene ALS3 of C. albicans. In addition, minocycline decreases cell surface hydrophobicity and the extracellular β-glucan level in biofilms. Minocycline has been widely used for catheter antibiotic lock therapy in efforts to prevent bacterial infection; the compound might also be prophylactically effective against Candida infection.

  16. Boric acid destabilizes the hyphal cytoskeleton and inhibits invasive growth of Candida albicans.

    PubMed

    Pointer, Benjamin R; Boyer, Michael P; Schmidt, Martin

    2015-04-01

    Exposure of Candida albicans to sub-lethal concentrations of boric acid (BA) restricts the dimorphic fungus to its yeast morphology and prevents the formation of invasive hyphae on solid substrates. Exposure to BA causes a rapid and reversible disappearance of polarisome and Spitzenkörper in growing hyphae. In BA-treated hyphae of C. albicans, actin quickly reorganizes from cytoplasmic cables to cortical patches and cell wall growth switches from an apical to an isotropic pattern. As a result of the cytoskeletal changes, the hyphal tips broaden and directional growth of hyphae ceases in the presence of BA. An analysis of homozygous deletion strains showed that mutants with constitutive or enhanced hyphal growth (tup1, nrg1, ssn6, rbf1) are BA-sensitive, demonstrating that cellular morphology is a major determinant of BA tolerance. The screening of deletion mutants also showed that deficiencies of the main activator of hyphal gene expression, Efg1, and the Rim101-signalling cascade, leading to Efg1 activation, cause BA resistance. Taken together, the data presented show that the selective inhibitory effect on BA on C. albicans hyphae is rooted in a disruption of apical cytoskeletal elements of growing hyphae.

  17. PHR1, a pH-regulated gene of Candida albicans, is required for morphogenesis.

    PubMed Central

    Saporito-Irwin, S M; Birse, C E; Sypherd, P S; Fonzi, W A

    1995-01-01

    Candida albicans, like many fungi, exhibits morphological plasticity, a property which may be related to its biological capacity as an opportunistic pathogen of humans. Morphogenesis and alterations in cell shape require integration of many cellular functions and occur in response to environmental signals, most notably pH and temperature in the case of C. albicans. In the course of our studies of differential gene expression associated with dimorphism of C. albicans, we have isolated a gene, designated PHR1, which is regulated in response to the pH of the culture medium. PHR1 expression was repressed at pH values below 5.5 and induced at more alkaline pH. The predicted amino acid sequence of the PHR1 protein was 56% identical to that of the Saccharomyces cerevisiae Ggp1/Gas1 protein, a highly glycosylated cell surface protein attached to the membrane via glycosylphosphatidylinositol. A homozygous null mutant of PHR1 was constructed and found to exhibit a pH-conditional morphological defect. At alkaline pH, the mutant, unlike the parental type, was unable to conduct apical growth of either yeast or hyphal growth forms. This morphological aberration was not associated with defective cytoskeletal polarization or secretion. The results suggest that PHR1 defines a novel function required for apical cell growth and morphogenesis. PMID:7823929

  18. Regulation of the Cdc42/Cdc24 GTPase Module during Candida albicans Hyphal Growth

    PubMed Central

    Bassilana, Martine; Hopkins, Julie; Arkowitz, Robert A.

    2005-01-01

    The Rho G protein Cdc42 and its exchange factor Cdc24 are required for hyphal growth of the human fungal pathogen Candida albicans. Previously, we reported that strains ectopically expressing Cdc24 or Cdc42 are unable to form hyphae in response to serum. Here we investigated the role of these two proteins in hyphal growth, using quantitative real-time PCR to measure induction of hypha-specific genes together with time lapse microscopy. Expression of the hypha-specific genes examined depends on the cyclic AMP-dependent protein kinase A pathway culminating in the Efg1 and Tec1 transcription factors. We show that strains with reduced levels of CDC24 or CDC42 transcripts induce hypha-specific genes yet cannot maintain their expression in response to serum. Furthermore, in serum these mutants form elongated buds compared to the wild type and mutant budding cells, as observed by time lapse microscopy. Using Cdc24 fused to green fluorescent protein, we also show that Cdc24 is recruited to and persists at the germ tube tip during hyphal growth. Altogether these data demonstrate that the Cdc24/Cdc42 GTPase module is required for maintenance of hyphal growth. In addition, overexpression studies indicate that specific levels of Cdc24 and Cdc42 are important for invasive hyphal growth. In response to serum, CDC24 transcript levels increase transiently in a Tec1-dependent fashion, as do the G-protein RHO3 and the Rho1 GTPase activating protein BEM2 transcript levels. These results suggest that a positive feedback loop between Cdc24 and Tec1 contributes to an increase in active Cdc42 at the tip of the germ tube which is important for hypha formation. PMID:15755921

  19. PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance.

    PubMed

    Prill, Stephan K-H; Klinkert, Birgit; Timpel, Claudia; Gale, Cheryl A; Schröppel, Klaus; Ernst, Joachim F

    2005-01-01

    Protein O-mannosyltransferases (Pmt proteins) initiate O-mannosylation of secretory proteins. The PMT gene family of the human fungal pathogen Candida albicans consists of PMT1 and PMT6, as well as three additional PMT genes encoding Pmt2, Pmt4 and Pmt5 isoforms described here. Both PMT2 alleles could not be deleted and growth of conditional strains, containing PMT2 controlled by the MET3- or tetOScHOP1-promoters, was blocked in non-permissive conditions, indicating that PMT2 is essential for growth. A homozygous pmt4 mutant was viable, but synthetic lethality of pmt4 was observed in combination with pmt1 mutations. Hyphal morphogenesis of a pmt4 mutant was defective under aerobic induction conditions, yet increased in embedded or hypoxic conditions, suggesting a role of Pmt4p-mediated O-glycosylation for environment-specific morphogenetic signalling. Although a PMT5 transcript was detected, a homozygous pmt5 mutant was phenotypically silent. All other pmt mutants showed variable degrees of supersensitivity to antifungals and to cell wall-destabilizing agents. Cell wall composition was markedly affected in pmt1 and pmt4 mutants, showing a significant decrease in wall mannoproteins. In a mouse model of haematogenously disseminated infection, PMT4 was required for full virulence of C. albicans. Functional analysis of the first complete PMT gene family in a fungal pathogen indicates that Pmt isoforms have variable and specific roles for in vitro and in vivo growth, morphogenesis and antifungal resistance.

  20. Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p.

    PubMed

    Peters, Brian M; Ovchinnikova, Ekaterina S; Krom, Bastiaan P; Schlecht, Lisa Marie; Zhou, Han; Hoyer, Lois L; Busscher, Henk J; van der Mei, Henny C; Jabra-Rizk, Mary Ann; Shirtliff, Mark E

    2012-12-01

    The bacterium Staphylococcus (St.) aureus and the opportunistic fungus Candida albicans are currently among the leading nosocomial pathogens, often co-infecting critically ill patients, with high morbidity and mortality. Previous investigations have demonstrated preferential adherence of St. aureus to C. albicans hyphae during mixed biofilm growth. In this study, we aimed to characterize the mechanism behind this observed interaction. C. albicans adhesin-deficient mutant strains were screened by microscopy to identify the specific receptor on C. albicans hyphae recognized by St. aureus. Furthermore, an immunoassay was developed to validate and quantify staphylococcal binding to fungal biofilms. The findings from these experiments implicated the C. albicans adhesin agglutinin-like sequence 3 (Als3p) in playing a major role in the adherence process. This association was quantitatively established using atomic force microscopy, in which the adhesion force between single cells of the two species was significantly reduced for a C. albicans mutant strain lacking als3. Confocal microscopy further confirmed these observations, as St. aureus overlaid with a purified recombinant Als3 N-terminal domain fragment (rAls3p) exhibited robust binding. Importantly, a strain of Saccharomyces cerevisiae heterologously expressing Als3p was utilized to further confirm this adhesin as a receptor for St. aureus. Although the parental strain does not bind bacteria, expression of Als3p on the cell surface conferred upon the yeast the ability to strongly bind St. aureus. To elucidate the implications of these in vitro findings in a clinically relevant setting, an ex vivo murine model of co-infection was designed using murine tongue explants. Fluorescent microscopic images revealed extensive hyphal penetration of the epithelium typical of C. albicans mucosal infection. Interestingly, St. aureus bacterial cells were only seen within the epithelial tissue when associated with the invasive

  1. Proper Sterol Distribution Is Required for Candida albicans Hyphal Formation and Virulence

    PubMed Central

    McCourt, Paula; Liu, Hsing-Yin; Parker, Josie E.; Gallo-Ebert, Christina; Donigan, Melissa; Bata, Adam; Giordano, Caroline; Kelly, Steven L.; Nickels, Joseph T.

    2016-01-01

    Candida albicans is an opportunistic fungus responsible for the majority of systemic fungal infections. Multiple factors contribute to C. albicans pathogenicity. C. albicans strains lacking CaArv1 are avirulent. Arv1 has a conserved Arv1 homology domain (AHD) that has a zinc-binding domain containing two cysteine clusters. Here, we explored the role of the CaAHD and zinc-binding motif in CaArv1-dependent virulence. Overall, we found that the CaAHD was necessary but not sufficient for cells to be virulent, whereas the zinc-binding domain was essential, as Caarv1/Caarv1 cells expressing the full-length zinc-binding domain mutants, Caarv1C3S and Caarv1C28S, were avirulent. Phenotypically, we found a direct correlation between the avirulence of Caarv1/Caarv1, Caarrv1AHD, Caarv1C3S, and Caarv1C28S cells and defects in bud site selection, septa formation and localization, and hyphal formation and elongation. Importantly, all avirulent mutant strains lacked the ability to maintain proper sterol distribution. Overall, our results have established the importance of the AHD and zinc-binding domain in fungal invasion, and have correlated an avirulent phenotype with the inability to maintain proper sterol distribution. PMID:27587298

  2. Garcinia xanthochymus Benzophenones Promote Hyphal Apoptosis and Potentiate Activity of Fluconazole against Candida albicans Biofilms.

    PubMed

    Jackson, Desmond N; Yang, Lin; Wu, ShiBiao; Kennelly, Edward J; Lipke, Peter N

    2015-10-01

    Xanthochymol and garcinol, isoprenylated benzophenones purified from Garcinia xanthochymus fruits, showed multiple activities against Candida albicans biofilms. Both compounds effectively prevented emergence of fungal germ tubes and were also cytostatic, with MICs of 1 to 3 μM. The compounds therefore inhibited development of hyphae and subsequent biofilm maturation. Xanthochymol treatment of developing and mature biofilms induced cell death. In early biofilm development, killing had the characteristics of apoptosis, including externalization of phosphatidyl serine and DNA fragmentation, as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) fluorescence. These activities resulted in failure of biofilm maturation and hyphal death in mature biofilms. In mature biofilms, xanthochymol and garcinol caused the death of biofilm hyphae, with 50% effective concentrations (EC50s) of 30 to 50 μM. Additionally, xanthochymol-mediated killing was complementary with fluconazole against mature biofilms, reducing the fluconazole EC50 from >1,024 μg/ml to 13 μg/ml. Therefore, xanthochymol has potential as an adjuvant for antifungal treatments as well as in studies of fungal apoptosis.

  3. A Single Nucleotide Polymorphism Uncovers a Novel Function for the Transcription Factor Ace2 during Candida albicans Hyphal Development

    PubMed Central

    Orellana-Muñoz, Sara; Gutiérrez-Escribano, Pilar; Arnáiz-Pita, Yolanda; Dueñas-Santero, Encarnación; Suárez, M. Belén; Bougnoux, Marie-Elisabeth; del Rey, Francisco; Sherlock, Gavin; d’Enfert, Christophe; Correa-Bordes, Jaime; de Aldana, Carlos R. Vázquez

    2015-01-01

    Candida albicans is a major invasive fungal pathogen in humans. An important virulence factor is its ability to switch between the yeast and hyphal forms, and these filamentous forms are important in tissue penetration and invasion. A common feature for filamentous growth is the ability to inhibit cell separation after cytokinesis, although it is poorly understood how this process is regulated developmentally. In C. albicans, the formation of filaments during hyphal growth requires changes in septin ring dynamics. In this work, we studied the functional relationship between septins and the transcription factor Ace2, which controls the expression of enzymes that catalyze septum degradation. We found that alternative translation initiation produces two Ace2 isoforms. While full-length Ace2, Ace2L, influences septin dynamics in a transcription-independent manner in hyphal cells but not in yeast cells, the use of methionine-55 as the initiation codon gives rise to Ace2S, which functions as the nuclear transcription factor required for the expression of cell separation genes. Genetic evidence indicates that Ace2L influences the incorporation of the Sep7 septin to hyphal septin rings in order to avoid inappropriate activation of cell separation during filamentous growth. Interestingly, a natural single nucleotide polymorphism (SNP) present in the C. albicans WO-1 background and other C. albicans commensal and clinical isolates generates a stop codon in the ninth codon of Ace2L that mimics the phenotype of cells lacking Ace2L. Finally, we report that Ace2L and Ace2S interact with the NDR kinase Cbk1 and that impairing activity of this kinase results in a defect in septin dynamics similar to that of hyphal cells lacking Ace2L. Together, our findings identify Ace2L and the NDR kinase Cbk1 as new elements of the signaling system that modify septin ring dynamics in hyphae to allow cell-chain formation, a feature that appears to have evolved in specific C. albicans lineages

  4. Antimicrobial effect of farnesol, a Candida albicans quorum sensing molecule, on Paracoccidioides brasiliensis growth and morphogenesis

    PubMed Central

    Derengowski, Lorena S; De-Souza-Silva, Calliandra; Braz, Shélida V; Mello-De-Sousa, Thiago M; Báo, Sônia N; Kyaw, Cynthia M; Silva-Pereira, Ildinete

    2009-01-01

    Background Farnesol is a sesquiterpene alcohol produced by many organisms, and also found in several essential oils. Its role as a quorum sensing molecule and as a virulence factor of Candida albicans has been well described. Studies revealed that farnesol affect the growth of a number of bacteria and fungi, pointing to a potential role as an antimicrobial agent. Methods Growth assays of Paracoccidioides brasiliensis cells incubated in the presence of different concentrations of farnesol were performed by measuring the optical density of the cultures. The viability of fungal cells was determined by MTT assay and by counting the colony forming units, after each farnesol treatment. The effects of farnesol on P. brasiliensis dimorphism were also evaluated by optical microscopy. The ultrastructural morphology of farnesol-treated P. brasiliensis yeast cells was evaluated by transmission and scanning electron microscopy. Results In this study, the effects of farnesol on Paracoccidioides brasiliensis growth and dimorphism were described. Concentrations of this isoprenoid ranging from 25 to 300 μM strongly inhibited P. brasiliensis growth. We have estimated that the MIC of farnesol for P. brasiliensis is 25 μM, while the MLC is around 30 μM. When employing levels which don't compromise cell viability (5 to 15 μM), it was shown that farnesol also affected the morphogenesis of this fungus. We observed about 60% of inhibition in hyphal development following P. brasiliensis yeast cells treatment with 15 μM of farnesol for 48 h. At these farnesol concentrations we also observed a significant hyphal shortening. Electron microscopy experiments showed that, despite of a remaining intact cell wall, P. brasiliensis cells treated with farnesol concentrations above 25 μM exhibited a fully cytoplasmic degeneration. Conclusion Our data indicate that farnesol acts as a potent antimicrobial agent against P. brasiliensis. The fungicide activity of farnesol against this pathogen is

  5. Cdc24, the GDP-GTP exchange factor for Cdc42, is required for invasive hyphal growth of Candida albicans.

    PubMed

    Bassilana, Martine; Blyth, James; Arkowitz, Robert A

    2003-02-01

    Candida albicans, the most common human fungal pathogen, is particularly problematic for immunocompromised individuals. The reversible transition of this fungal pathogen to a filamentous form that invades host tissue is important for its virulence. Although different signaling pathways such as a mitogen-activated protein kinase and a protein kinase A cascade are critical for this morphological transition, the function of polarity establishment proteins in this process has not been determined. We examined the role of four different polarity establishment proteins in C. albicans invasive growth and virulence by using strains in which one copy of each gene was deleted and the other copy expressed behind the regulatable promoter MET3. Strikingly, mutants with ectopic expression of either the Rho G-protein Cdc42 or its exchange factor Cdc24 are unable to form invasive hyphal filaments and germ tubes in response to serum or elevated temperature and yet grow normally as a budding yeast. Furthermore, these mutants are avirulent in a mouse model for systemic infection. This function of the Cdc42 GTPase module is not simply a general feature of polarity establishment proteins. Mutants with ectopic expression of the SH3 domain containing protein Bem1 or the Ras-like G-protein Bud1 can grow in an invasive fashion and are virulent in mice, albeit with reduced efficiency. These results indicate that a specific regulation of Cdc24/Cdc42 activity is required for invasive hyphal growth and suggest that these proteins are required for pathogenicity of C. albicans.

  6. Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface

    PubMed Central

    Marín, Elvira; Parra-Giraldo, Claudia M.; Hernández-Haro, Carolina; Hernáez, María L.; Nombela, César; Monteoliva, Lucía; Gil, Concha

    2015-01-01

    Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in

  7. The actin-related protein Sac1 is required for morphogenesis and cell wall integrity in Candida albicans.

    PubMed

    Zhang, Bing; Yu, Qilin; Jia, Chang; Wang, Yuzhou; Xiao, Chenpeng; Dong, Yijie; Xu, Ning; Wang, Lei; Li, Mingchun

    2015-08-01

    Candida albicans is a common pathogenic fungus and has aroused widespread attention recently. Actin cytoskeleton, an important player in polarized growth, protein secretion and organization of cell shape, displays irreplaceable role in hyphal development and cell integrity. In this study, we demonstrated a homologue of Saccharomyces cerevisiae Sac1, in C. albicans. It is a potential PIP phosphatase with Sac domain which is related to actin organization, hyphal development, biofilm formation and cell wall integrity. Deletion of SAC1 did not lead to insitiol-auxotroph phenotype in C. albicans, but this gene rescued the growth defect of S. cerevisiae sac1Δ in the insitiol-free medium. Hyphal induction further revealed the deficiency of sac1Δ/Δ in hyphal development and biofilm formation. Fluorescence observation and real time PCR (RT-PCR) analysis suggested both actin and the hyphal cell wall protein Hwp1 were overexpressed and mislocated in this mutant. Furthermore, cell wall integrity (CWI) was largely affected by deletion of SAC1, due to the hypersensitivity to cell wall stress, changed content and distribution of chitin in the mutant. As a result, the virulence of sac1Δ/Δ was seriously attenuated. Taken together, this study provides evidence that Sac1, as a potential PIP phosphatase, is essential for actin organization, hyphal development, CWI and pathogenicity in C. albicans.

  8. Inhibitory Effect of Sophorolipid on Candida albicans Biofilm Formation and Hyphal Growth

    PubMed Central

    Haque, Farazul; Alfatah, Md.; Ganesan, K.; Bhattacharyya, Mani Shankar

    2016-01-01

    Candida albicans causes superficial and life-threatening systemic infections. These are difficult to treat often due to drug resistance, particularly because C. albicans biofilms are inherently resistant to most antifungals. Sophorolipid (SL), a glycolipid biosurfactant, has been shown to have antimicrobial and anticancer properties. In this study, we investigated the effect of SL on C. albicans biofilm formation and preformed biofilms. SL was found to inhibit C. albicans biofilm formation as well as reduce the viability of preformed biofilms. Moreover, SL, when used along with amphotericin B (AmB) or fluconazole (FLZ), was found to act synergistically against biofilm formation and preformed biofilms. Effect of SL on C. albicans biofilm formation was further visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed absence of hyphae, typical biofilm architecture and alteration in the morphology of biofilm cells. We also found that SL downregulates the expression of hypha specific genes HWP1, ALS1, ALS3, ECE1 and SAP4, which possibly explains the inhibitory effect of SL on hyphae and biofilm formation. PMID:27030404

  9. Inhibitory Effect of Sophorolipid on Candida albicans Biofilm Formation and Hyphal Growth.

    PubMed

    Haque, Farazul; Alfatah, Md; Ganesan, K; Bhattacharyya, Mani Shankar

    2016-03-31

    Candida albicans causes superficial and life-threatening systemic infections. These are difficult to treat often due to drug resistance, particularly because C. albicans biofilms are inherently resistant to most antifungals. Sophorolipid (SL), a glycolipid biosurfactant, has been shown to have antimicrobial and anticancer properties. In this study, we investigated the effect of SL on C. albicans biofilm formation and preformed biofilms. SL was found to inhibit C. albicans biofilm formation as well as reduce the viability of preformed biofilms. Moreover, SL, when used along with amphotericin B (AmB) or fluconazole (FLZ), was found to act synergistically against biofilm formation and preformed biofilms. Effect of SL on C. albicans biofilm formation was further visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed absence of hyphae, typical biofilm architecture and alteration in the morphology of biofilm cells. We also found that SL downregulates the expression of hypha specific genes HWP1, ALS1, ALS3, ECE1 and SAP4, which possibly explains the inhibitory effect of SL on hyphae and biofilm formation.

  10. Regulated proteolysis of Candida albicans Ras1 is involved in morphogenesis and quorum sensing regulation

    PubMed Central

    Piispanen, Amy; Grahl, Nora; Hollomon, Jeffrey M.; Hogan, Deborah A.

    2013-01-01

    Summary In Candida albicans, a fungal pathogen, the small G-protein Ras1 regulates many important behaviors including white-opaque switching, biofilm formation, and the induction and maintenance of hyphal growth. Like other Ras proteins, Ras1 is activated upon guanine triphosphate binding, and its activity is further modulated by post-translational lipid modifications. Here, we report that the levels of membrane-associated, full-length Ras1 were higher in hyphae than in yeast, and that yeast contained a shorter, soluble Ras1 species that resulted from cleavage. Deletion of the putative cleavage site led to more rapid induction of hyphal growth and delayed hypha-to-yeast transitions. The cleaved Ras1 species was less able to activate its effector, adenylate cyclase (Cyr1), unless tethered to the membrane by a heterologous membrane-targeting domain. Ras1 cleavage was repressed by cAMP-signaling, indicating the presence of a positive feedback loop in which Cyr1 and cAMP influence Ras1. The C. albicans quorum sensing molecule farnesol, which inhibits Cyr1 and represses filamentation, caused an increase in the fraction of Ras1 in the cleaved form, particularly in nascent yeast formed from hyphae. This newly recognized mode of Ras regulation may control C. albicans Ras1 activity in important ways. PMID:23692372

  11. Alcohols inhibit translation to regulate morphogenesis in C. albicans.

    PubMed

    Egbe, Nkechi E; Paget, Caroline M; Wang, Hui; Ashe, Mark P

    2015-04-01

    Many molecules are secreted into the growth media by microorganisms to modulate the metabolic and physiological processes of the organism. For instance, alcohols like butanol, ethanol and isoamyl alcohol are produced by the human pathogenic fungus, Candida albicans and induce morphological differentiation. Here we show that these same alcohols cause a rapid inhibition of protein synthesis. More specifically, the alcohols target translation initiation, a complex stage of the gene expression process. Using molecular techniques, we have identified the likely translational target of these alcohols in C. albicans as the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, which supports the exchange reaction where eIF2.GDP is converted to eIF2.GTP. Even minimal regulation at this step will lead to alterations in the levels of specific proteins that may allow the exigencies of the fungus to be realised. Indeed, similar to the effects of alcohols, a minimal inhibition of protein synthesis with cycloheximide also causes an induction of filamentous growth. These results suggest a molecular basis for the effect of various alcohols on morphological differentiation in C. albicans.

  12. Single-Molecule Imaging and Functional Analysis of Als Adhesins and Mannans during Candida albicans Morphogenesis

    PubMed Central

    Beaussart, Audrey; Alsteens, David; El-Kirat-Chatel, Sofiane; Lipke, Peter N.; Kucharíková, Sona; Van Dijck, Patrick; Dufrêne, Yves F.

    2012-01-01

    Cellular morphogenesis in the fungal pathogen Candida albicans is associated with changes in cell wall composition that play important roles in biofilm formation and immune responses. Yet, how fungal morphogenesis modulates the biophysical properties and interactions of the cell surface molecules is poorly understood, mainly owing to the paucity of high-resolution imaging techniques. Here, we use single-molecule atomic force microscopy to localize and analyze the key components of the surface of living C. albicans cells during morphogenesis. We show that the yeast-to-hypha transition leads to a major increase in the distribution, adhesion, unfolding and extension of Als adhesins and their associated mannans on the cell surface. We also find that morphogenesis dramatically increases cell surface hydrophobicity. These molecular changes are critical for microbe-host interactions, including adhesion, colonization, and biofilm formation. The single-molecule experiments presented here offer promising prospects for understanding how microbial pathogens use cell surface molecules to modulate biofilm and immune interactions. PMID:23145462

  13. [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies].

    PubMed

    Aydın, Cevahir; Ataoğlu, Haluk

    2015-01-01

    Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method

  14. Terpenoids of plant origin inhibit morphogenesis, adhesion, and biofilm formation by Candida albicans.

    PubMed

    Raut, Jayant S; Shinde, Ravikumar B; Chauhan, Nitin M; Karuppayil, S Mohan

    2013-01-01

    Biofilm-related infections caused by Candida albicans and associated drug resistant micro-organisms are serious problems for immunocompromised populations. Molecules which can prevent or remove biofilms are needed. Twenty-eight terpenoids of plant origin were analysed for their activity against growth, virulence attributes, and biofilms of C. albicans. Eighteen molecules exhibited minimum inhibitory concentrations of <2 mg ml(-1) for planktonic growth. Selected molecules inhibited yeast to hyphal dimorphism at low concentrations (0.031-0.5 mg ml(-1)), while adhesion to a solid surface was prevented at 0.5-2 mg ml(-1). Treatment with 14 terpenoids resulted in significant (p < 0.05) inhibition of biofilm formation, and of these, linalool, nerol, isopulegol, menthol, carvone, α-thujone, and farnesol exhibited biofilm-specific activity. Eight terpenoids were identified as inhibitors of mature biofilms. This study demonstrated the antibiofilm potential of terpenoids, which need to be further explored as therapeutic strategy against biofilm associated infections of C. albicans.

  15. Aft2, a Novel Transcription Regulator, Is Required for Iron Metabolism, Oxidative Stress, Surface Adhesion and Hyphal Development in Candida albicans

    PubMed Central

    Xu, Ning; Cheng, Xinxin; Yu, Qilin; Qian, Kefan; Ding, Xiaohui; Liu, Ruming; Zhang, Biao; Xing, Laijun; Li, Mingchun

    2013-01-01

    Morphological transition and iron metabolism are closely relevant to Candida albicans pathogenicity and virulence. In our previous study, we demonstrated that C. albicans Aft2 plays an important role in ferric reductase activity and virulence. Here, we further explored the roles of C. albicans Aft2 in numerous cellular processes. We found that C. albicans Aft2 exhibited an important role in iron metabolism through bi-directional regulation effects on iron-regulon expression. Deletion of AFT2 reduced cellular iron accumulation under iron-deficient conditions. Furthermore, both reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity were remarkably increased in the aft2Δ/Δ mutant, which were thought to be responsible for the defective responses to oxidative stress. However, we found that over-expression of C. albicans AFT2 under the regulation of the strong PGK1 promoter could not effectively rescue Saccharomyces cerevisiae aft1Δ mutant defects in some cellular processes, such as cell-wall assembly, ion homeostasis and alkaline resistance, suggesting a possibility that C. albicans Aft2 weakened its functional role of regulating some cellular metabolism during the evolutionary process. Interestingly, deletion of AFT2 in C. albicans increased cell surface hydrophobicity, cell flocculation and the ability of adhesion to polystyrene surfaces. In addition, our results also revealed that C. albicans Aft2 played a dual role in regulating hypha-specific genes under solid and liquid hyphal inducing conditions. Deletion of AFT2 caused an impaired invasive growth in solid medium, but an increased filamentous aggregation and growth in liquid conditions. Moreover, iron deficiency and environmental cues induced nuclear import of Aft2, providing additional evidence for the roles of Aft2 in transcriptional regulation. PMID:23626810

  16. The essential phosphoinositide kinase MSS-4 is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa.

    PubMed

    Mähs, Anette; Ischebeck, Till; Heilig, Yvonne; Stenzel, Irene; Hempel, Franziska; Seiler, Stephan; Heilmann, Ingo

    2012-01-01

    Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P(2) that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N

  17. Diagnosis of invasive candidiasis by enzyme-linked immunosorbent assay using the N-terminal fragment of Candida albicans hyphal wall protein 1

    PubMed Central

    Laín, Ana; Elguezabal, Natalia; Brena, Sonia; García-Ruiz, Juan Carlos; del Palacio, Amalia; Moragues, María D; Pontón, José

    2007-01-01

    Background The diagnosis of invasive candidiasis is difficult because there are no specific clinical manifestations of the disease and colonization and infection are difficult to distinguish. In the last decade, much effort has been made to develop reliable tests for rapid diagnosis of invasive candidiasis, but none of them have found widespread clinical use. Results Antibodies against a recombinant N-terminal fragment of the Candida albicans germ tube-specific antigen hyphal wall protein 1 (Hwp1) generated in Escherichia coli were detected by both immunoblotting and ELISA tests in a group of 36 hematological or Intensive Care Unit patients with invasive candidiasis and in a group of 45 control patients at high risk for the mycosis who did not have clinical or microbiological data to document invasive candidiasis. Results were compared with an immunofluorescence test to detect antibodies to C. albicans germ tubes (CAGT). The sensitivity, specificity, positive and negative predictive values of a diagnostic test based on the detection of antibodies against the N-terminal fragment of Hwp1 by immunoblotting were 27.8 %, 95.6 %, 83.3 % and 62.3 %, respectively. Detection of antibodies to the N-terminal fragment of Hwp1 by ELISA increased the sensitivity (88.9 %) and the negative predictive value (90.2 %) but slightly decreased the specificity (82.6 %) and positive predictive values (80 %). The kinetics of antibody response to the N-terminal fragment of Hwp1 by ELISA was very similar to that observed by detecting antibodies to CAGT. Conclusion An ELISA test to detect antibodies against a recombinant N-terminal fragment of the C. albicans germ tube cell wall antigen Hwp1 allows the diagnosis of invasive candidiasis with similar results to those obtained by detecting antibodies to CAGT but without the need of treating the sera to adsorb the antibodies against the cell wall surface of the blastospore. PMID:17448251

  18. Imaging morphogenesis of Candida albicans during infection in a live animal

    NASA Astrophysics Data System (ADS)

    Mitra, Soumya; Dolan, Kristy; Foster, Thomas H.; Wellington, Melanie

    2010-01-01

    Candida albicans is an opportunistic human fungal pathogen that requires an intact host immune response to prevent disease. Thus, studying host-pathogen interactions is critical to understanding and preventing this disease. We report a new model infection system in which ongoing C. albicans infections can be imaged at high spatial resolution in the ears of living mice. Intradermal inoculation into mouse ears with a C. albicans strain expressing green fluorescent protein results in systemic C. albicans infection that can be imaged in vivo using confocal microscopy. We observed filamentous growth of the organism in vivo as well as formation of microabscesses. This model system will allow us to gain significant new information about C. albicans pathogenesis through studies of host-C. albicans interactions in the native environment.

  19. Extensive Functional Redundancy in the Regulation of Candida albicans Drug Resistance and Morphogenesis by Lysine Deacetylases Hos2, Hda1, Rpd3, and Rpd31

    PubMed Central

    Li, Xinliu; Robbins, Nicole; O’Meara, Teresa R.; Cowen, Leah E.

    2016-01-01

    Summary Current treatment efforts for fungal infections are hampered by the limited availability of antifungal drugs and by the emergence of drug resistance. A powerful strategy to enhance the efficacy of antifungal drugs is to inhibit the molecular chaperone Hsp90. Hsp90 governs drug resistance, morphogenesis, and virulence in a leading fungal pathogen of humans, Candida albicans. Our previous work with Saccharomyces cerevisiae established acetylation as a novel mechanism of posttranslational control of Hsp90 function in fungi. We implicated lysine deacetylases (KDACs) as key regulators of resistance to the most widely deployed class of antifungals, the azoles, in both S. cerevisiae and C. albicans. Here, we demonstrate high levels of functional redundancy among the KDACs that are important for regulating Hsp90 function. We identify Hos2, Hda1, Rpd3, and Rpd31 as the KDACs mediating azole resistance and morphogenesis in C. albicans. Furthermore, we identify lysine 30 and 271 as critical acetylation sites on C. albicans Hsp90, and substitutions at these residues compromised Hsp90 function. Finally, we show that pharmacological inhibition of KDACs phenocopies pharmacological inhibition of Hsp90 and abrogates Hsp90-dependent azole resistance in numerous Candida species. This work illuminates new facets to the impact of KDACs on fungal drug resistance and morphogenesis, provides important insights into the divergence of the C. albicans Hsp90 regulatory network, and reveals new targets for development of antifungal drugs. PMID:27868254

  20. Signaling through Lrg1, Rho1 and Pkc1 Governs Candida albicans Morphogenesis in Response to Diverse Cues

    PubMed Central

    Leach, Michelle D.; Hogan, Deborah A.; Robbins, Nicole; Cowen, Leah E.

    2016-01-01

    The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1 or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation

  1. The pathogen Candida albicans hijacks pyroptosis for escape from macrophages.

    PubMed

    Uwamahoro, Nathalie; Verma-Gaur, Jiyoti; Shen, Hsin-Hui; Qu, Yue; Lewis, Rowena; Lu, Jingxiong; Bambery, Keith; Masters, Seth L; Vince, James E; Naderer, Thomas; Traven, Ana

    2014-03-25

    The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection. IMPORTANCE Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macrophage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent

  2. Effect of Piper betle and Brucea javanica on the Differential Expression of Hyphal Wall Protein (HWP1) in Non-Candida albicans Candida (NCAC) Species.

    PubMed

    Wan Harun, Wan Himratul Aznita; Jamil, Nur Alyaa; Jamaludin, Nor Hazwani; Nordin, Mohd-Al-Faisal

    2013-01-01

    The study aimed to identify the HWP1 gene in non-Candida albicans Candida species and the differential expression of HWP1 following treatment with Piper betle and Brucea javanica aqueous extracts. All candidal suspensions were standardized to 1 × 10(6) cells/mL. The suspension was incubated overnight at 37 °C (C. parapsilosis, 35°C). Candidal cells were treated with each respective extract at 1, 3, and 6 mg/mL for 24 h. The total RNA was extracted and reverse transcription-polymerase chain reaction was carried out with a specific primer of HWP1. HWP1 mRNAs were only detected in C. albicans, C. parapsilosis, and C. tropicalis. Exposing the cells to the aqueous extracts has affected the expression of HWP1 transcripts. C. albicans, C. parapsilosis, and C. tropicalis have demonstrated different intensity of mRNA. Compared to P. betle, B. javanica demonstrated a higher suppression on the transcript levels of HWP1 in all samples. HWP1 was not detected in C. albicans following the treatment of B. javanica at 1 mg/mL. In contrast, C. parapsilosis and C. tropicalis were shown to have HWP1 regulation. However, the expression levels were reduced upon the addition of higher concentration of B. javanica extract. P. betle and B. javanica have potential to be developed as oral health product.

  3. Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation

    PubMed Central

    Frazer, Corey; Joshi, Monika; Delorme, Caroline; Davis, Darlene; Bennett, Richard J.

    2015-01-01

    Candida albicans is a major fungal pathogen whose virulence is associated with its ability to transition from a budding yeast form to invasive hyphal filaments. The kinesin-14 family member CaKar3 is required for transition between these morphological states, as well as for mitotic progression and karyogamy. While kinesin-14 proteins are ubiquitous, CaKar3 homologs in hemiascomycete fungi are unique because they form heterodimers with noncatalytic kinesin-like proteins. Thus, CaKar3-based motors may represent a novel antifungal drug target. We have identified and examined the roles of a kinesin-like regulator of CaKar3. We show that orf19.306 (dubbed CaCIK1) encodes a protein that forms a heterodimer with CaKar3, localizes CaKar3 to spindle pole bodies, and can bind microtubules and influence CaKar3 mechanochemistry despite lacking an ATPase activity of its own. Similar to CaKar3 depletion, loss of CaCik1 results in cell cycle arrest, filamentation defects, and an inability to undergo karyogamy. Furthermore, an examination of the spindle structure in cells lacking either of these proteins shows that a large proportion have a monopolar spindle or two dissociated half-spindles, a phenotype unique to the C. albicans kinesin-14 homolog. These findings provide new insights into mitotic spindle structure and kinesin motor function in C. albicans and identify a potentially vulnerable target for antifungal drug development. PMID:26024903

  4. Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans

    PubMed Central

    Barbosa, Júnia Oliveira; Rossoni, Rodnei Dennis; Vilela, Simone Furgeri Godinho; de Alvarenga, Janaína Araújo; Velloso, Marisol dos Santos; Prata, Márcia Cristina de Azevedo; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2016-01-01

    Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model. PMID:26934196

  5. Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans.

    PubMed

    Barbosa, Júnia Oliveira; Rossoni, Rodnei Dennis; Vilela, Simone Furgeri Godinho; de Alvarenga, Janaína Araújo; Velloso, Marisol dos Santos; Prata, Márcia Cristina de Azevedo; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2016-01-01

    Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.

  6. Candida albicans and Enterococcus faecalis in the gut

    PubMed Central

    Garsin, Danielle A; Lorenz, Michael C

    2013-01-01

    The fungus Candida albicans and the gram-positive bacterium Enterococcus faecalis are both normal residents of the human gut microbiome and cause opportunistic disseminated infections in immunocompromised individuals. Using a nematode infection model, we recently showed that co-infection resulted in less pathology and less mortality than infection with either species alone and this was partly explained by an interkingdom signaling event in which a bacterial-derived product inhibits hyphal morphogenesis of C. albicans. In this addendum we discuss these findings in the contest of other described bacterial-fungal interactions and recent data suggesting a potentially synergistic relationship between these two species in the mouse gut as well. We suggest that E. faecalis and C. albicans promote a mutually beneficial association with the host, in effect choosing a commensal lifestyle over a pathogenic one. PMID:23941906

  7. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

    PubMed

    Chang, Peng; Fan, Xueyi; Chen, Jiangye

    2015-08-01

    Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains.

  8. Regulation of the Candida albicans Hypha-Inducing Transcription Factor Ume6 by the CDK1 Cyclins Cln3 and Hgc1

    PubMed Central

    Mendelsohn, Sigal; Pinsky, Mariel; Weissman, Ziva

    2017-01-01

    ABSTRACT The ability to switch between proliferation as yeast cells and development into hyphae is a hallmark of Candida albicans. The switch to hyphal morphogenesis depends on external inducing conditions, but its efficiency is augmented in stationary-phase cells. Ume6, a transcription factor that is itself transcriptionally induced under hypha-promoting conditions, is both necessary and sufficient for hyphal morphogenesis. We found that Ume6 is regulated posttranslationally by the cell cycle kinase Cdc28/Cdk1, which reduces Ume6 activity via different mechanisms using different cyclins. Together with the cyclin Hgc1, Cdk1 promotes degradation of Ume6 via the SCFCDC4 ubiquitin ligase. Since HGC1 is a key transcriptional target of Ume6, this results in a negative-feedback loop between Hgc1 and Ume6. In addition, we found that Cln3, a G1 cyclin that is essential for cell cycle progression and yeast proliferation, suppresses hyphal morphogenesis and that Cln3 suppresses Ume6 activity both in the heterologous Saccharomyces cerevisiae system and in C. albicans itself. This activity of Cln3 may provide the basis for the antagonistic relationship between yeast proliferation and hyphal development in C. albicans. IMPORTANCE The yeast to hypha (mold) morphogenetic switch of Candida albicans plays a role in its virulence and constitutes a diagnostic trait for this organism, the most prevalent systemic fungal pathogen in industrialized countries. It has long been known that hyphae are most efficiently induced from stationary cultures. Here, a molecular basis for this observation is provided. The G1 cyclin Cln3, an essential promoter of yeast proliferation, was found to suppress hyphal induction. Suppression of hyphal induction is achieved by inhibition of the activity of the central activator of hyphal morphogenesis, the transcription factor Ume6. Thus, levels of Cln3 control the switch between proliferation of C. albicans as individual yeast cells and development into

  9. The GRF10 homeobox gene regulates filamentous growth in the human fungal pathogen Candida albicans.

    PubMed

    Ghosh, Anup K; Wangsanut, Tanaporn; Fonzi, William A; Rolfes, Ronda J

    2015-12-01

    Candida albicans is the most common human fungal pathogen and can cause life-threatening infections. Filamentous growth is critical in the pathogenicity of C. albicans, as the transition from yeast to hyphal forms is linked to virulence and is also a pivotal process in fungal biofilm development. Homeodomain-containing transcription factors have been linked to developmental processes in fungi and other eukaryotes. We report here on GRF10, a homeobox transcription factor-encoding gene that plays a role in C. albicans filamentation. Deletion of the GRF10 gene, in both C. albicans SN152 and BWP17 strain backgrounds, results in mutants with strongly decreased hyphal growth. The mutants are defective in chlamydospore and biofilm formation, as well as showing dramatically attenuated virulence in a mouse infection model. Expression of the GRF10 gene is highly induced during stationary phase and filamentation. In summary, our study emphasizes a new role for the homeodomain-containing transcription factor in morphogenesis and pathogenicity of C. albicans.

  10. Hydroxyurea enhances post-fusion hyphal extension during sexual development in C. neoformans var. grubii.

    PubMed

    Zulkifli, M Naim; Kaur, Jan Naseer; Panepinto, John C

    2012-03-01

    Mating and sexual development in C. neoformans var. grubii strains of the H99 background is often less robust than that laboratory generated isogenic C. neoformans var. neoformans strains in the JEC21 background. In Candida albicans and Saccharomyces serevisiae, slowing of DNA synthesis and engagement of the replication stress response, such as that caused by treatment with hydroxyurea (HU), induces filamentation and pseudohyphal growth, respectively. In this study, we investigated the effect of HU treatment on C. neoformans var. grubii morphogenesis. Treatment with HU did not induce filamentation of yeast cells either in liquid culture or on solid YPD or V8 agar. In the presence of the opposite mating partner, we observed early emergence of hyphae in the presence of HU. Semi-quantitative analysis of fusion using marked strains demonstrated that no significant enhancement of fusion in the presence of HU. Transfer of fusion colonies from crosses performed in the absence of HU to V8 + HU revealed enhanced hyphal growth in the presence of HU. Analysis of expression of the target of HU, ribonucleotide reductase, revealed that a phylogenetically divergent catalytic subunit is replication stress responsive in C. neoformans. These results suggest that induction of replication stress promotes post-fusion hyphal growth of C. neoformans var. grubii strains in the H99 background.

  11. Essential Functional Modules for Pathogenic and Defensive Mechanisms in Candida albicans Infections

    PubMed Central

    Tsai, I-Chun; Lin, Che; Chuang, Yung-Jen

    2014-01-01

    The clinical and biological significance of the study of fungal pathogen Candida albicans (C. albicans) has markedly increased. However, the explicit pathogenic and invasive mechanisms of such host-pathogen interactions have not yet been fully elucidated. Therefore, the essential functional modules involved in C. albicans-zebrafish interactions were investigated in this study. Adopting a systems biology approach, the early-stage and late-stage protein-protein interaction (PPI) networks for both C. albicans and zebrafish were constructed. By comparing PPI networks at the early and late stages of the infection process, several critical functional modules were identified in both pathogenic and defensive mechanisms. Functional modules in C. albicans, like those involved in hyphal morphogenesis, ion and small molecule transport, protein secretion, and shifts in carbon utilization, were seen to play important roles in pathogen invasion and damage caused to host cells. Moreover, the functional modules in zebrafish, such as those involved in immune response, apoptosis mechanisms, ion transport, protein secretion, and hemostasis-related processes, were found to be significant as defensive mechanisms during C. albicans infection. The essential functional modules thus determined could provide insights into the molecular mechanisms of host-pathogen interactions during the infection process and thereby devise potential therapeutic strategies to treat C. albicans infection. PMID:24757665

  12. Perithecium morphogenesis in Sordaria macrospora.

    PubMed

    Lord, Kathryn M; Read, Nick D

    2011-04-01

    The perithecium of the self-fertile ascomycete Sordaria macrospora provides an excellent model in which to analyse fungal multicellular development. This study provides a detailed analysis of perithecium morphogenesis in the wild type and eight developmental mutants of S. macrospora, using a range of correlative microscopical techniques. Fundamentally, perithecia and other complex multicellular structures produced by fungi arise by hyphal aggregation and adhesion, and these processes are followed by specialization and septation of hyphal compartments within the aggregates. Perithecial morphogenesis can be divided into the ascogonial, protoperithecial, and perithecial stages of development. At least 13 specialized, morphologically distinct cell-types are involved in perithecium morphogenesis, and these fall into three basic classes: hyphae, conglutinate cells and spores. Conglutinate cells arise from hyphal adhesion and certain perithecial hyphae develop from conglutinate cells. Various hypha-conglutinate cell transitions play important roles during the development of the perithecial wall and neck.

  13. The Pathogen Candida albicans Hijacks Pyroptosis for Escape from Macrophages

    PubMed Central

    Uwamahoro, Nathalie; Verma-Gaur, Jiyoti; Shen, Hsin-Hui; Qu, Yue; Lewis, Rowena; Lu, Jingxiong; Bambery, Keith; Masters, Seth L.; Vince, James E.; Naderer, Thomas; Traven, Ana

    2014-01-01

    ABSTRACT The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection. PMID:24667705

  14. Identification of small molecules that disrupt vacuolar function in the pathogen Candida albicans

    PubMed Central

    Tournu, Helene; Carroll, Jennifer; Latimer, Brian; Dragoi, Ana-Maria; Dykes, Samantha; Cardelli, James; Peters, Tracy L.; Eberle, Karen E.; Palmer, Glen E.

    2017-01-01

    The fungal vacuole is a large acidified organelle that performs a variety of cellular functions. At least a sub-set of these functions are crucial for pathogenic species of fungi, such as Candida albicans, to survive within and invade mammalian tissue as mutants with severe defects in vacuolar biogenesis are avirulent. We therefore sought to identify chemical probes that disrupt the normal function and/or integrity of the fungal vacuole to provide tools for the functional analysis of this organelle as well as potential experimental therapeutics. A convenient indicator of vacuolar integrity based upon the intracellular accumulation of an endogenously produced pigment was adapted to identify Vacuole Disrupting chemical Agents (VDAs). Several chemical libraries were screened and a set of 29 compounds demonstrated to reproducibly cause loss of pigmentation, including 9 azole antifungals, a statin and 3 NSAIDs. Quantitative analysis of vacuolar morphology revealed that (excluding the azoles) a sub-set of 14 VDAs significantly alter vacuolar number, size and/or shape. Many C. albicans mutants with impaired vacuolar function are deficient in the formation of hyphal elements, a process essential for its pathogenicity. Accordingly, all 14 VDAs negatively impact C. albicans hyphal morphogenesis. Fungal selectivity was observed for approximately half of the VDA compounds identified, since they did not alter the morphology of the equivalent mammalian organelle, the lysosome. Collectively, these compounds comprise of a new collection of chemical probes that directly or indirectly perturb normal vacuolar function in C. albicans. PMID:28151949

  15. Characterization of genetic determinants that modulate Candida albicans filamentation in the presence of bacteria.

    PubMed

    Fox, Sean J; Shelton, Bryce T; Kruppa, Michael D

    2013-01-01

    In the human body, fungi and bacteria share many niches where the close contact of these organisms maintains a balance among the microbial population. However, when this microbial balance is disrupted, as with antibiotic treatment, other bacteria or fungi can grow uninhibited. C. albicans is the most common opportunistic fungal pathogen affecting humans and can uniquely control its morphogenesis between yeast, pseudohyphal, and hyphal forms. Numerous studies have shown that C. albicans interactions with bacteria can impact its ability to undergo morphogenesis; however, the genetics that govern this morphological control via these bacterial interactions are still relatively unknown. To aid in the understanding of the cross-kingdom interactions of C. albicans with bacteria and the impact on morphology we utilized a haploinsufficiency based C. albicans mutant screen to test for the ability of C. albicans to produce hyphae in the presence of three bacterial species (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). Of the 18,144 mutant strains tested, 295 mutants produced hyphae in the presence of all three bacterial species. The 295 mutants identified 132 points of insertion, which included identified/predicted genes, major repeat sequences, and a number of non-coding/unannotated transcripts. One gene, CDR4, displayed increased expression when co-cultured with S. aureus, but not E. coli or P. aeruginosa. Our data demonstrates the ability to use a large scale library screen to identify genes involved in Candida-bacterial interactions and provides the foundation for comprehending the genetic pathways relating to bacterial control of C. albicans morphogenesis.

  16. Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S-Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis

    PubMed Central

    Kraidlova, Lucie; Schrevens, Sanne; Tournu, Hélène; Van Zeebroeck, Griet; Sychrova, Hana

    2016-01-01

    ABSTRACT Amino acids are key sources of nitrogen for growth of Candida albicans. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to ScGap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae, where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of ScSam3, the only S-adenosylmethionine (SAM) transporter in S. cerevisiae, and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCE Candida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans. PMID

  17. Fungal Morphogenetic Pathways Are Required for the Hallmark Inflammatory Response during Candida albicans Vaginitis

    PubMed Central

    Palmer, Glen E.; Nash, Andrea K.; Lilly, Elizabeth A.; Fidel, Paul L.; Noverr, Mairi C.

    2014-01-01

    Vulvovaginal candidiasis, caused primarily by Candida albicans, presents significant health issues for women of childbearing age. As a polymorphic fungus, the ability of C. albicans to switch between yeast and hyphal morphologies is considered its central virulence attribute. Armed with new criteria for defining vaginitis immunopathology, the purpose of this study was to determine whether the yeast-to-hypha transition is required for the hallmark inflammatory responses previously characterized during murine vaginitis. Kinetic analyses of vaginal infection with C. albicans in C57BL/6 mice demonstrated that fungal burdens remained constant throughout the observation period, while polymorphonuclear leukocyte (PMN), S100A8, and interleukin-1β levels obtained from vaginal lavage fluid increased by day 3 onward. Lactate dehydrogenase activity was also positively correlated with increased effectors of innate immunity. Additionally, immunodepletion of neutrophils in infected mice confirmed a nonprotective role for PMNs during vaginitis. Determination of the importance of fungal morphogenesis during vaginitis was addressed with a two-pronged approach. Intravaginal inoculation of mice with C. albicans strains deleted for key transcriptional regulators (bcr1Δ/Δ, efg1Δ/Δ, cph1Δ/Δ, and efg1Δ/Δ cph1Δ/Δ) controlling the yeast-to-hypha switch revealed a crucial role for morphogenetic signaling through the Efg1 and, to a lesser extent, the Bcr1 pathways in contributing to vaginitis immunopathology. Furthermore, overexpression of transcription factors NRG1 and UME6, to maintain yeast and hyphal morphologies, respectively, confirmed the importance of morphogenesis in generating innate immune responses in vivo. These results highlight the yeast-to-hypha switch and the associated morphogenetic response as important virulence components for the immunopathogenesis of Candida vaginitis, with implications for transition from benign colonization to symptomatic infection. PMID

  18. Inhibition of Candida albicans Biofilm Formation by the Synthetic Lactoferricin Derived Peptide hLF1-11

    PubMed Central

    Morici, Paola; Fais, Roberta; Rizzato, Cosmeri

    2016-01-01

    The aim of this study was to evaluate the in vitro activity of the synthetic peptide hLF1-11 against biofilm produced by clinical isolates of Candida albicans with different fluconazole susceptibility. The antibiofilm activity of the peptide hLF1-11 was assessed in terms of reduction of biofilm cellular density, metabolic activity and sessile cell viability. The extent of morphogenesis in hLF1-11 treated and untreated biofilms was also investigated microscopically. Transcription levels of genes related to cell adhesion, hyphal development and extracellular matrix production were analysed by qRT-PCR in hLF1-11 treated and untreated biofilms. Exogenous dibutyryl-cAMP (db-cAMP) was used to rescue morphogenesis in cells exposed to the peptide. The results revealed that hLF1-11 exhibited an inhibitory effect on biofilm formation by all C. albicans isolates tested in a dose-dependent manner, regardless of their fluconazole susceptibility. Visual inspection of treated or untreated biofilm cells with an inverted microscope revealed a significant reduction in hyphal formation by hLF1-11 treated cells, as early as 3 hours of incubation. Moreover, hLF1-11 showed a reduced activity on preadherent cells. hLF1-11 induced the down-regulation of biofilm and hyphal-associated genes, which were predominantly regulated via the Ras1-cAMP-Efg1 pathway. Indeed, exogenous db-cAMP restored morphogenesis in hLF1-11 treated cells. The hLF1-11 peptide significantly inhibited biofilm formation by C. albicans mainly at early stages, interfering with biofilm cellular density and metabolic activity, and affected morphogenesis through the Ras1-cAMP-Efg1 pathway. Our findings provide the first evidence that hLF1-11 could represent a potential candidate for the prevention of biofilm formation by C. albicans. PMID:27902776

  19. A 5′ UTR-mediated Translational Efficiency Mechanism Inhibits the Candida albicans Morphological Transition

    PubMed Central

    Childers, Delma S.; Mundodi, Vasanthakrishna; Banerjee, Mohua; Kadosh, David

    2014-01-01

    SUMMARY While virulence properties of Candida albicans, the most commonly isolated human fungal pathogen, are controlled by transcriptional and post-translational mechanisms, considerably little is known about the role of post-transcriptional, and particularly translational, mechanisms. We demonstrate that UME6, a key filament-specific transcriptional regulator whose expression level is sufficient to determine C. albicans morphology and promote virulence, has one of the longest 5′ untranslated regions (UTRs) identified in fungi to date, which is predicted to form a complex and extremely stable secondary structure. The 5′ UTR inhibits the ability of UME6, when expressed at constitutive high levels, to drive complete hyphal growth, but does not cause a reduction in UME6 transcript. Deletion of the 5′ UTR increases C. albicans filamentation under a variety of conditions but does not affect UME6 transcript level or induction kinetics. We show that the 5′ UTR functions to inhibit Ume6 protein expression under several filament-inducing conditions and specifically reduces association of the UME6 transcript with polysomes. Overall, our findings suggest that translational efficiency mechanisms, known to regulate diverse biological processes in bacterial and viral pathogens as well as higher eukaryotes, have evolved to inhibit and fine-tune morphogenesis, a key virulence trait of many human fungal pathogens. PMID:24601998

  20. Robust Extracellular pH Modulation by Candida albicans during Growth in Carboxylic Acids

    PubMed Central

    Danhof, Heather A.; Vylkova, Slavena; Vesely, Elisa M.; Ford, Amy E.; Gonzalez-Garay, Manuel

    2016-01-01

    ABSTRACT The opportunistic fungal pathogen Candida albicans thrives within diverse niches in the mammalian host. Among the adaptations that underlie this fitness is an ability to utilize a wide array of nutrients, especially sources of carbon that are disfavored by many other fungi; this contributes to its ability to survive interactions with the phagocytes that serve as key barriers against disseminated infections. We have reported that C. albicans generates ammonia as a byproduct of amino acid catabolism to neutralize the acidic phagolysosome and promote hyphal morphogenesis in a manner dependent on the Stp2 transcription factor. Here, we report that this species rapidly neutralizes acidic environments when utilizing carboxylic acids like pyruvate, α-ketoglutarate (αKG), or lactate as the primary carbon source. Unlike in cells growing in amino acid-rich medium, this does not result in ammonia release, does not induce hyphal differentiation, and is genetically distinct. While transcript profiling revealed significant similarities in gene expression in cells grown on either carboxylic or amino acids, genetic screens for mutants that fail to neutralize αKG medium identified a nonoverlapping set of genes, including CWT1, encoding a transcription factor responsive to cell wall and nitrosative stresses. Strains lacking CWT1 exhibit retarded αKG-mediated neutralization in vitro, exist in a more acidic phagolysosome, and are more susceptible to macrophage killing, while double cwt1Δ stp2Δ mutants are more impaired than either single mutant. Together, our observations indicate that C. albicans has evolved multiple ways to modulate the pH of host-relevant environments to promote its fitness as a pathogen. PMID:27935835

  1. Effect of Tetrandrine against Candida albicans Biofilms

    PubMed Central

    Zhao, Lan-Xue; Li, De-Dong; Hu, Dan-Dan; Hu, Gan-Hai; Yan, Lan; Wang, Yan; Jiang, Yuan-Ying

    2013-01-01

    Candida albicans is the most common human fungal pathogen and has a high propensity to develop biofilms that are resistant to traditional antifungal agents. In this study, we investigated the effect of tetrandrine (TET) on growth, biofilm formation and yeast-to-hypha transition of C. albicans. We characterized the inhibitory effect of TET on hyphal growth and addressed its possible mechanism of action. Treatment of TET at a low concentration without affecting fungal growth inhibited hyphal growth in both liquid and solid Spider media. Real-time RT-PCR revealed that TET down-regulated the expression of hypha-specific genes ECE1, ALS3 and HWP1, and abrogated the induction of EFG1 and RAS1, regulators of hyphal growth. Addition of cAMP restored the normal phenotype of the SC5314 strain. These results indicate that TET may inhibit hyphal growth through the Ras1p-cAMP-PKA pathway. In vivo, at a range of concentrations from 4 mg/L to 32 mg/L, TET prolonged the survival of C. albicans-infected Caenorhabditis elegans significantly. This study provides useful information for the development of new strategies to reduce the incidence of C. albicans biofilm-associated infections. PMID:24260276

  2. Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis.

    PubMed

    Chen, Ying-Lien; Brand, Alexandra; Morrison, Emma L; Silao, Fitz Gerald S; Bigol, Ursela G; Malbas, Fedelino F; Nett, Jeniel E; Andes, David R; Solis, Norma V; Filler, Scott G; Averette, Anna; Heitman, Joseph

    2011-06-01

    Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.

  3. Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans

    PubMed Central

    Wang, Hong X.; Douglas, Lois M.; Veselá, Petra; Rachel, Reinhard; Malinsky, Jan; Konopka, James B.

    2016-01-01

    The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7. These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization. PMID:27009204

  4. A Cellulose Synthase-Like Protein Involved in Hyphal Tip Growth and Morphological Differentiation in Streptomyces▿

    PubMed Central

    Xu, Hongbin; Chater, Keith F.; Deng, Zixin; Tao, Meifeng

    2008-01-01

    Cellulose synthase and cellulose synthase-like proteins, responsible for synthesizing β-glucan-containing polysaccharides, play a fundamental role in cellular architectures, such as plant cell and tissue morphogenesis, bacterial biofilm formation, and fruiting-body development. However, the roles of the proteins involved in the developmental process are not well understood. Here, we report that a cellulose synthase-like protein (CslASc) in Streptomyces has a function in hyphal tip growth and morphological differentiation. The cslASc replacement mutant showed pleiotropic defects, including the severe delay of aerial-hyphal formation and altered cell wall morphology. Calcofluor white fluorescence analysis demonstrated that polysaccharide synthesis at hyphal tips was dependent on CslASc. cslASc was constitutively transcribed, and an enhanced green fluorescent protein-CslASc fusion protein was mostly located at the hyphal tips. An extract enriched in morphogenetic chaplin proteins promoted formation of aerial hyphae by the mutant. Furthermore, a two-hybrid experiment indicated that the glycosyltransferase domain of CslASc interacted with the tropomyosin-like polarity-determining DivIVA protein, suggesting that the tip-located DivIVA governed tip recruitment of the CslASc membrane protein. These results imply that the cellulose synthase-like protein couples extracellular and cytoskeletal components functioning in tip growth and cell development. PMID:18487344

  5. Modulation of Candida albicans virulence by bacterial biofilms on titanium surfaces.

    PubMed

    Cavalcanti, Yuri Wanderley; Wilson, Melanie; Lewis, Michael; Del-Bel-Cury, Altair Antoninha; da Silva, Wander José; Williams, David W

    2016-01-01

    Whilst Candida albicans occurs in peri-implant biofilms, its role in peri-implantitis remains unclear. This study therefore examined the virulence of C. albicans in mixed-species biofilms on titanium surfaces. Biofilms of C. albicans (Ca), C. albicans with streptococci (Streptococcus sanguinis, S. mutans) (Ca-Ss-Sm) and those incorporating Porphyromonas gingivalis (Ca-Pg and Ca-Ss-Sm-Pg) were developed. Expression of C. albicans genes associated with adhesion (ALS1, ALS3, HWP1) and hydrolytic enzymes (SAP2, SAP4, SAP6, PLD1) was measured and hyphal production by C. albicans quantified. Compared with Ca biofilms, significant (p<0.05) up-regulation of ALS3, HWP1, SAP2 and SAP6, and hyphal production occurred in biofilms containing streptococci (Ca-Ss-Sm). In Ca-Pg biofilms, down-regulation of HWP1 and SAP4 expression, with reduced hyphal production occurred. Ca-Ss-Sm-Pg biofilms had increased hyphal proportions and up-regulation of ALS3, SAP2 and SAP6. In conclusion, C. albicans expressed virulence factors in biofilms that could contribute to peri-implantitis, but this was dependent on associated bacterial species.

  6. The Endoplasmic Reticulum-Mitochondrion Tether ERMES Orchestrates Fungal Immune Evasion, Illuminating Inflammasome Responses to Hyphal Signals

    PubMed Central

    Tucey, Timothy M.; Verma-Gaur, Jiyoti; Nguyen, Julie; Hewitt, Victoria L.; Lo, Tricia L.; Shingu-Vazquez, Miguel; Robertson, Avril A. B.; Hill, James R.; Pettolino, Filomena A.; Beddoe, Travis; Cooper, Matthew A.; Naderer, Thomas

    2016-01-01

    ABSTRACT The pathogenic yeast Candida albicans escapes macrophages by triggering NLRP3 inflammasome-dependent host cell death (pyroptosis). Pyroptosis is inflammatory and must be tightly regulated by host and microbe, but the mechanism is incompletely defined. We characterized the C. albicans endoplasmic reticulum (ER)-mitochondrion tether ERMES and show that the ERMES mmm1 mutant is severely crippled in killing macrophages despite hyphal formation and normal phagocytosis and survival. To understand dynamic inflammasome responses to Candida with high spatiotemporal resolution, we established live-cell imaging for parallel detection of inflammasome activation and pyroptosis at the single-cell level. This showed that the inflammasome response to mmm1 mutant hyphae is delayed by 10 h, after which an exacerbated activation occurs. The NLRP3 inhibitor MCC950 inhibited inflammasome activation and pyroptosis by C. albicans, including exacerbated inflammasome activation by the mmm1 mutant. At the cell biology level, inactivation of ERMES led to a rapid collapse of mitochondrial tubular morphology, slow growth and hyphal elongation at host temperature, and reduced exposed 1,3-β-glucan in hyphal populations. Our data suggest that inflammasome activation by C. albicans requires a signal threshold dependent on hyphal elongation and cell wall remodeling, which could fine-tune the response relative to the level of danger posed by C. albicans. The phenotypes of the ERMES mutant and the lack of conservation in animals suggest that ERMES is a promising antifungal drug target. Our data further indicate that NLRP3 inhibition by MCC950 could modulate C. albicans-induced inflammation. IMPORTANCE The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1

  7. O-Mannosylation in Candida albicans Enables Development of Interkingdom Biofilm Communities

    PubMed Central

    Dutton, Lindsay C.; Nobbs, Angela H.; Jepson, Katy; Jepson, Mark A.; Vickerman, M. Margaret; Aqeel Alawfi, Sami; Munro, Carol A.; Lamont, Richard J.; Jenkinson, Howard F.

    2014-01-01

    ABSTRACT Candida albicans is a fungus that colonizes oral cavity surfaces, the gut, and the genital tract. Streptococcus gordonii is a ubiquitous oral bacterium that has been shown to form biofilm communities with C. albicans. Formation of dual-species S. gordonii-C. albicans biofilm communities involves interaction of the S. gordonii SspB protein with the Als3 protein on the hyphal filament surface of C. albicans. Mannoproteins comprise a major component of the C. albicans cell wall, and in this study we sought to determine if mannosylation in cell wall biogenesis of C. albicans was necessary for hyphal adhesin functions associated with interkingdom biofilm development. A C. albicans mnt1Δ mnt2Δ mutant, with deleted α-1,2-mannosyltransferase genes and thus defective in O-mannosylation, was abrogated in biofilm formation under various growth conditions and produced hyphal filaments that were not recognized by S. gordonii. Cell wall proteomes of hypha-forming mnt1Δ mnt2Δ mutant cells showed growth medium-dependent alterations, compared to findings for the wild type, in a range of protein components, including Als1, Als3, Rbt1, Scw1, and Sap9. Hyphal filaments formed by mnt1Δ mnt2Δ mutant cells, unlike wild-type hyphae, did not interact with C. albicans Als3 or Hwp1 partner cell wall proteins or with S. gordonii SspB partner adhesin, suggesting defective functionality of adhesins on the mnt1Δ mnt2Δ mutant. These observations imply that early stage O-mannosylation is critical for activation of hyphal adhesin functions required for biofilm formation, recognition by bacteria such as S. gordonii, and microbial community development. PMID:24736223

  8. Tpd3-Pph21 phosphatase plays a direct role in Sep7 dephosphorylation in Candida albicans.

    PubMed

    Liu, Qizheng; Han, Qi; Wang, Na; Yao, Guangyin; Zeng, Guisheng; Wang, Yanming; Huang, Zhenxing; Sang, Jianli; Wang, Yue

    2016-07-01

    Septins are a component of the cytoskeleton and play important roles in diverse cellular processes including cell cycle control, cytokinesis and polarized growth. In fungi, septin organization, dynamics and function are regulated by phosphorylation, and several kinases responsible for the phosphorylation of several septins have been identified. However, little is known about the phosphatases that dephosphorylate septins. Here, we report the characterization of Tpd3, a structural subunit of the PP2A family of phosphatases, in the pathogenic fungus Candida albicans. We found that tpd3Δ/Δ cells are defective in hyphal growth and grow as pseudohyphae under yeast growth conditions with aberrant septin organization. Western blotting detected hyperphosphorylation of the septin Sep7 in cells lacking Tpd3. Tpd3 and Sep7 colocalize at the bud neck and can coimmunoprecipitate. Furthermore, we discovered similar defects in cells lacking Pph21, a catalytic subunit of the PP2A family, and its physical association with Tpd3. Importantly, purified Tpd3-Pph21 complexes can dephosphorylate Sep7 in vitro. Together, our findings strongly support the idea that the Tpd3-Pph21 complex dephosphorylates Sep7 and regulates morphogenesis and cytokinesis. The tpd3Δ/Δ mutant is greatly reduced in virulence in mice, providing a potential antifungal target.

  9. Hyphal ontogeny in Neurospora crassa: a model organism for all seasons

    PubMed Central

    Riquelme, Meritxell; Martínez-Núñez, Leonora

    2016-01-01

    Filamentous fungi have proven to be a better-suited model system than unicellular yeasts in analyses of cellular processes such as polarized growth, exocytosis, endocytosis, and cytoskeleton-based organelle traffic. For example, the filamentous fungus Neurospora crassa develops a variety of cellular forms. Studying the molecular basis of these forms has led to a better, yet incipient, understanding of polarized growth. Polarity factors as well as Rho GTPases, septins, and a localized delivery of vesicles are the central elements described so far that participate in the shift from isotropic to polarized growth. The growth of the cell wall by apical biosynthesis and remodeling of polysaccharide components is a key process in hyphal morphogenesis. The coordinated action of motor proteins and Rab GTPases mediates the vesicular journey along the hyphae toward the apex, where the exocyst mediates vesicle fusion with the plasma membrane. Cytoplasmic microtubules and actin microfilaments serve as tracks for the transport of vesicular carriers as well as organelles in the tubular cell, contributing to polarization. In addition to exocytosis, endocytosis is required to set and maintain the apical polarity of the cell. Here, we summarize some of the most recent breakthroughs in hyphal morphogenesis and apical growth in N. crassa and the emerging questions that we believe should be addressed. PMID:27990280

  10. Hyphal ontogeny in Neurospora crassa: a model organism for all seasons.

    PubMed

    Riquelme, Meritxell; Martínez-Núñez, Leonora

    2016-01-01

    Filamentous fungi have proven to be a better-suited model system than unicellular yeasts in analyses of cellular processes such as polarized growth, exocytosis, endocytosis, and cytoskeleton-based organelle traffic. For example, the filamentous fungus Neurospora crassa develops a variety of cellular forms. Studying the molecular basis of these forms has led to a better, yet incipient, understanding of polarized growth. Polarity factors as well as Rho GTPases, septins, and a localized delivery of vesicles are the central elements described so far that participate in the shift from isotropic to polarized growth. The growth of the cell wall by apical biosynthesis and remodeling of polysaccharide components is a key process in hyphal morphogenesis. The coordinated action of motor proteins and Rab GTPases mediates the vesicular journey along the hyphae toward the apex, where the exocyst mediates vesicle fusion with the plasma membrane. Cytoplasmic microtubules and actin microfilaments serve as tracks for the transport of vesicular carriers as well as organelles in the tubular cell, contributing to polarization. In addition to exocytosis, endocytosis is required to set and maintain the apical polarity of the cell. Here, we summarize some of the most recent breakthroughs in hyphal morphogenesis and apical growth in N. crassa and the emerging questions that we believe should be addressed.

  11. Mitochondrial Activity and Cyr1 Are Key Regulators of Ras1 Activation of C. albicans Virulence Pathways

    PubMed Central

    Grahl, Nora; Demers, Elora G.; Lindsay, Allia K.; Harty, Colleen E.; Willger, Sven D.; Piispanen, Amy E.; Hogan, Deborah A.

    2015-01-01

    Candida albicans is both a major fungal pathogen and a member of the commensal human microflora. The morphological switch from yeast to hyphal growth is associated with disease and many environmental factors are known to influence the yeast-to-hyphae switch. The Ras1-Cyr1-PKA pathway is a major regulator of C. albicans morphogenesis as well as biofilm formation and white-opaque switching. Previous studies have shown that hyphal growth is strongly repressed by mitochondrial inhibitors. Here, we show that mitochondrial inhibitors strongly decreased Ras1 GTP-binding and activity in C. albicans and similar effects were observed in other Candida species. Consistent with there being a connection between respiratory activity and GTP-Ras1 binding, mutants lacking complex I or complex IV grew as yeast in hypha-inducing conditions, had lower levels of GTP-Ras1, and Ras1 GTP-binding was unaffected by respiratory inhibitors. Mitochondria-perturbing agents decreased intracellular ATP concentrations and metabolomics analyses of cells grown with different respiratory inhibitors found consistent perturbation of pyruvate metabolism and the TCA cycle, changes in redox state, increased catabolism of lipids, and decreased sterol content which suggested increased AMP kinase activity. Biochemical and genetic experiments provide strong evidence for a model in which the activation of Ras1 is controlled by ATP levels in an AMP kinase independent manner. The Ras1 GTPase activating protein, Ira2, but not the Ras1 guanine nucleotide exchange factor, Cdc25, was required for the reduction of Ras1-GTP in response to inhibitor-mediated reduction of ATP levels. Furthermore, Cyr1, a well-characterized Ras1 effector, participated in the control of Ras1-GTP binding in response to decreased mitochondrial activity suggesting a revised model for Ras1 and Cyr1 signaling in which Cyr1 and Ras1 influence each other and, together with Ira2, seem to form a master-regulatory complex necessary to integrate

  12. Laser microbeam manipulation of cell morphogenesis growing in fungal hyphae

    NASA Astrophysics Data System (ADS)

    Bracker, Charles E.; Murphy, Douglas J.; Lopez-Franco, Rosamaria

    1997-05-01

    Laser microbeam irradiation at 820 nm predictably and reproducibly altered morphogenetic patterns in fungal cells. Optical tweezers were highly effective as localized, noninvasive, and largely nondestructive probes under precise spatial and temporal control. In growing hyphae, the position of the Spitzenkorper (a multicomponent complex containing mainly secretory vesicles in the hyphal apex), is correlated with the site of maximum cell expansion during tip growth. The Spitzenkorper was not trapped by the laser, but moved away from the trap, and could be `chased' around the cell by the laser beam. Consequently, the direction of cell elongation was readily changed by moving the Spitzenkorper. When the laser was held steady at the cytoplasmic surface immediately beside the Spitzenkorper, an adventitious branch hypha was initiated on the same side of the hypha, suggesting that unilateral disturbance of vesicle traffic initiated a new lateral Spitzenkorper and hyphal branch near the original hyphal apex. If moving vesicles were trapped by the laser beam and transported to a different area of the cytoplasm near the cell surface, the cell profile bulged where the vesicles were newly concentrated. Variations in the mode of vesicle transfer caused: (1) single and multiple bulges, (2) adventitious branch hyphae, (3) increased cell diameter, and (4) changing directions of hyphal elongation. Thus, laser tweezers emerge as a powerful tool for controlling patterns of cell morphogenesis. The findings strongly support the hypothesis that sites of vesicle concentration and release to the cell surface are important determinants of cell morphogenesis in fungi. This conclusion lends support to the basic premises of a modern mathematical model of hyphal tip growth (the hyphoid/VSC model) but does not in itself provide the information needed for a comprehensive and integrated explanation of the mechanism of cell growth in fungi.

  13. Short peptides allowing preferential detection of Candida albicans hyphae.

    PubMed

    Kaba, Hani E J; Pölderl, Antonia; Bilitewski, Ursula

    2015-09-01

    Whereas the detection of pathogens via recognition of surface structures by specific antibodies and various types of antibody mimics is frequently described, the applicability of short linear peptides as sensor molecules or diagnostic tools is less well-known. We selected peptides which were previously reported to bind to recombinant S. cerevisiae cells, expressing members of the C. albicans Agglutinin-Like-Sequence (ALS) cell wall protein family. We slightly modified amino acid sequences to evaluate peptide sequence properties influencing binding to C. albicans cells. Among the selected peptides, decamer peptides with an "AP"-N-terminus were superior to shorter peptides. The new decamer peptide FBP4 stained viable C. albicans cells more efficiently in their mature hyphal form than in their yeast form. Moreover, it allowed distinction of C. albicans from other related Candida spp. and could thus be the basis for the development of a useful tool for the diagnosis of invasive candidiasis.

  14. Inhibition of Candida albicans virulence factors by novel levofloxacin derivatives.

    PubMed

    Shafreen, Raja Mohamed Beema; Raja Mohamed, Beema Shafreen; Muthamil, Subramanian; Subramanian, Muthamil; Pandian, Shunmugiah Karutha; Shunmugiah, Karutha Pandian

    2014-08-01

    Candida albicans is an important opportunistic fungal pathogen, responsible for biofilm associated infections in immunocompromised patients. The aim of the present study was to investigate the antibiofilm properties of novel levofloxacin derivatives on C. albicans biofilms. The levofloxacin derivatives at their Biofilm Inhibitory Concentrations (BIC) were able to inhibit the biofilms of C. albicans, the yeast-to-hyphal transition and were also able to disrupt their mature biofilms. Furthermore, Real-time PCR analysis showed that the expression of ergosterol biosynthesis pathway gene (ERG11) and the efflux pump-encoding genes (CDR1 and MDR1) was decreased upon treatment with the levofloxacin derivatives. The total ergosterol content quantified using UV spectrophotomer showed decrease in ergosterol in the presence of levofloxacin derivatives. Overall, levofloxacin derivatives (6a, 6c and 7d) are capable of inhibiting C. albicans virulence factors. Therefore, these compounds with potential therapeutic implications can be used as new strategy to treat biofilm-related candidal infections.

  15. Role of Arf GTPases in fungal morphogenesis and virulence

    PubMed Central

    Labbaoui, Hayet; Bogliolo, Stéphanie; Ghugtyal, Vikram; Solis, Norma V.

    2017-01-01

    Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth, which requires sustained membrane traffic and polarized growth. In many organisms, small GTPases of the Arf (ADP-ribosylation factor) family regulate membrane/protein trafficking, yet little is known about their role in fungal filamentous growth. To investigate these GTPases in C. albicans, we generated loss of function mutants in all 3 Arf proteins, Arf1-Arf3, and 2 Arf-like proteins, Arl1 and Arl3. Our results indicate that of these proteins, Arf2 is required for viability and sensitivity to antifungal drugs. Repressible ARF2 expression results in defects in filamentous growth, cell wall integrity and virulence, likely due to alteration of the Golgi. Arl1 is also required for invasive filamentous growth and, although arl1/arl1 cells can initiate hyphal growth, hyphae are substantially shorter than that of the wild-type, due to the inability of this mutant to maintain hyphal growth at a single site. We show that this defect does not result from an alteration of phospholipid distribution and is unlikely to result from the sole Golgin Imh1 mislocalization, as Imh1 is not required for invasive filamentous growth. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion in this mutant. Strikingly, the arl1/arl1 mutant is drastically reduced in virulence during oropharyngeal candidiasis. Together, our results highlight the importance of Arl1 and Arf2 as key regulators of hyphal growth and virulence in C. albicans and identify a unique function of Arl1 in secretion. PMID:28192532

  16. Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses

    PubMed Central

    Alanazi, Humidah; Semlali, Abdelhabib; Perraud, Laura; Chmielewski, Witold; Zakrzewski, Andrew

    2014-01-01

    The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC) on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P < 0.01) sensitive to oxidation but significantly (P < 0.01) resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P < 0.01) slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers. PMID:25302312

  17. Cigarette smoke-exposed Candida albicans increased chitin production and modulated human fibroblast cell responses.

    PubMed

    Alanazi, Humidah; Semlali, Abdelhabib; Perraud, Laura; Chmielewski, Witold; Zakrzewski, Andrew; Rouabhia, Mahmoud

    2014-01-01

    The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC) on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P < 0.01) sensitive to oxidation but significantly (P < 0.01) resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P < 0.01) slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers.

  18. Hyphal Growth in Human Fungal Pathogens and Its Role in Virulence

    PubMed Central

    Brand, Alexandra

    2012-01-01

    Most of the fungal species that infect humans can grow in more than one morphological form but only a subset of pathogens produce filamentous hyphae during the infection process. This subset is phylogenetically unrelated and includes the commonly carried yeasts, Candida albicans, C. dubliniensis, and Malassezia spp., and the acquired pathogens, Aspergillus fumigatus and dermatophytes such as Trichophyton rubrum and T. mentagrophytes. The primary function of hypha formation in these opportunistic pathogens is to invade the substrate they are adhered to, whether biotic or abiotic, but other functions include the directional translocation between host environments, consolidation of the colony, nutrient acquisition and the formation of 3-dimensional matrices. To support these functions, polarised hyphal growth is co-regulated with other factors that are essential for normal hypha function in vivo. PMID:22121367

  19. Interspecies Interactions between Clostridium difficile and Candida albicans

    PubMed Central

    van Leeuwen, Pim T.; van der Peet, Jasper M.; Bikker, Floris J.; Hoogenkamp, Michel A.; Oliveira Paiva, Ana M.; Kostidis, Sarantos; Mayboroda, Oleg A.

    2016-01-01

    ABSTRACT The facultative anaerobic polymorphic fungus Candida albicans and the strictly anaerobic Gram-positive bacterium Clostridium difficile are two opportunistic pathogens residing in the human gut. While a few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, the nature of the interactions between these two microbes has not been studied thus far. In the current study, both chemical and physical interactions between C. albicans and C. difficile were investigated. In the presence of C. albicans, C. difficile was able to grow under aerobic, normally toxic, conditions. This phenomenon was neither linked to adherence of bacteria to hyphae nor to biofilm formation by C. albicans. Conditioned medium of C. difficile inhibited hyphal growth of C. albicans, which is an important virulence factor of the fungus. In addition, it induced hypha-to-yeast conversion. p-Cresol, a fermentation product of tyrosine produced by C. difficile, also induced morphological effects and was identified as an active component of the conditioned medium. This study shows that in the presence of C. albicans, C. difficile can persist and grow under aerobic conditions. Furthermore, p-cresol, produced by C. difficile, is involved in inhibiting hypha formation of C. albicans, directly affecting the biofilm formation and virulence of C. albicans. This study is the first detailed characterization of the interactions between these two gut pathogens. IMPORTANCE Candida albicans and Clostridium difficile are two opportunistic pathogens that reside in the human gut. A few studies have focused on the prevalence of C. albicans in C. difficile-infected patients, but none have shown the interaction(s) that these two organisms may or may not have with each other. In this study, we used a wide range of different techniques to better understand this interaction at a macroscopic and microscopic level. We found that in the presence of C. albicans, C

  20. The Sho1 Adaptor Protein Links Oxidative Stress to Morphogenesis and Cell Wall Biosynthesis in the Fungal Pathogen Candida albicans† ‡

    PubMed Central

    Román, Elvira; Nombela, César; Pla, Jesús

    2005-01-01

    The Sho1 adaptor protein is an important element of one of the two upstream branches of the high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway in Saccharomyces cerevisiae, a signal transduction cascade involved in adaptation to stress. In the present work, we describe its role in the pathogenic yeast Candida albicans by the construction of mutants altered in this gene. We report here that sho1 mutants are sensitive to oxidative stress but that Sho1 has a minor role in the transmission of the phosphorylation signal to the Hog1 MAP kinase in response to oxidative stress, which mainly occurs through a putative Sln1-Ssk1 branch of the HOG pathway. Genetic analysis revealed that double ssk1 sho1 mutants were still able to grow on high-osmolarity media and activate Hog1 in response to this stress, indicating the existence of alternative inputs of the pathway. We also demonstrate that the Cek1 MAP kinase is constitutively active in hog1 and ssk1 mutants, a phenotypic trait that correlates with their resistance to the cell wall inhibitor Congo red, and that Sho1 is essential for the activation of the Cek1 MAP kinase under different conditions that require active cell growth and/or cell wall remodeling, such as the resumption of growth upon exit from the stationary phase. sho1 mutants are also sensitive to certain cell wall interfering compounds (Congo red, calcofluor white), presenting an altered cell wall structure (as shown by the ability to aggregate), and are defective in morphogenesis on different media, such as SLAD and Spider, that stimulate hyphal growth. These results reveal a role for the Sho1 protein in linking oxidative stress, cell wall biogenesis, and morphogenesis in this important human fungal pathogen. PMID:16287872

  1. Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis

    PubMed Central

    Tati, Swetha; Davidow, Peter; McCall, Andrew; Hwang-Wong, Elizabeth; Rojas, Isolde G.; Cormack, Brendan; Edgerton, Mira

    2016-01-01

    Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata. PMID:27029023

  2. Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis.

    PubMed

    Tati, Swetha; Davidow, Peter; McCall, Andrew; Hwang-Wong, Elizabeth; Rojas, Isolde G; Cormack, Brendan; Edgerton, Mira

    2016-03-01

    Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.

  3. Contact-induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae

    PubMed Central

    Thomson, Darren D; Wehmeier, Silvia; Byfield, FitzRoy J; Janmey, Paul A; Caballero-Lima, David; Crossley, Alison; Brand, Alexandra C

    2015-01-01

    Filamentous hyphae of the human pathogen, Candida albicans, invade mucosal layers and medical silicones. In vitro, hyphal tips reorient thigmotropically on contact with small obstacles. It is not known how surface topography is sensed but hyphae lacking the cortical marker, Rsr1/Bud1, are unresponsive. We show that, on surfaces, the morphology of hyphal tips and the position of internal polarity protein complexes are asymmetrically skewed towards the substratum and biased towards the softer of two surfaces. In nano-fabricated chambers, the Spitzenkörper (Spk) responded to touch by translocating across the apex towards the point of contact, where its stable maintenance correlated with contour-following growth. In the rsr1Δ mutant, the position of the Spk meandered and these responses were attenuated. Perpendicular collision caused lateral Spk oscillation within the tip until after establishment of a new growth axis, suggesting Spk position does not predict the direction of growth in C. albicans. Acute tip reorientation occurred only in cells where forward growth was countered by hyphal friction sufficient to generate a tip force of ∼ 8.7 μN (1.2 MPa), more than that required to penetrate host cell membranes. These findings suggest mechanisms through which the organization of hyphal tip growth in C. albicans facilitates the probing, penetration and invasion of host tissue. PMID:25262778

  4. Candida parapsilosis Protects Premature Intestinal Epithelial Cells from Invasion and Damage by Candida albicans

    PubMed Central

    Gonia, Sara; Archambault, Linda; Shevik, Margaret; Altendahl, Marie; Fellows, Emily; Bliss, Joseph M.; Wheeler, Robert T.; Gale, Cheryl A.

    2017-01-01

    Candida is a leading cause of late-onset sepsis in premature infants and is thought to invade the host via immature or damaged epithelial barriers. We previously showed that the hyphal form of Candida albicans invades and causes damage to premature intestinal epithelial cells (pIECs), whereas the non-hyphal Candida parapsilosis, also a fungal pathogen of neonates, has less invasion and damage abilities. In this study, we investigated the potential for C. parapsilosis to modulate pathogenic interactions of C. albicans with the premature intestine. While a mixed infection with two fungal pathogens may be expected to result in additive or synergistic damage to pIECs, we instead found that C. parapsilosis was able to protect pIECs from invasion and damage by C. albicans. C. albicans-induced pIEC damage was reduced to a similar extent by multiple different C. parapsilosis strains, but strains differed in their ability to inhibit C. albicans invasion of pIECs, with the inhibitory activity correlating with their adhesiveness for C. albicans and epithelial cells. C. parapsilosis cell-free culture fractions were also able to significantly reduce C. albicans adhesion and damage to pIECs. Furthermore, coadministration of C. parapsilosis cell-free fractions with C. albicans was associated with decreased infection and mortality in zebrafish. These results indicate that C. parapsilosis is able to reduce invasion, damage, and virulence functions of C. albicans. Additionally, the results with cellular and cell-free fractions of yeast cultures suggest that inhibition of pathogenic interactions between C. albicans and host cells by C. parapsilosis occurs via secreted molecules as well as by physical contact with the C. parapsilosis cell surface. We propose that non-invasive commensals can be used to inhibit virulence features of pathogens and deserve further study as a non-pharmacological strategy to protect the fragile epithelial barriers of premature infants. PMID:28382297

  5. Gold Nanoparticle-Photosensitizer Conjugate Based Photodynamic Inactivation of Biofilm Producing Cells: Potential for Treatment of C. albicans Infection in BALB/c Mice

    PubMed Central

    Sherwani, Mohd. Asif; Tufail, Saba; Khan, Aijaz Ahmed; Owais, Mohammad

    2015-01-01

    Background Photodynamic therapy (PDT) has been found to be effective in inhibiting biofilm producing organisms. We investigated the photodynamic effect of gold nanoparticle (GNP) conjugated photosensitizers against Candida albicans biofilm. We also examined the photodynamic efficacy of photosensitizer (PS) conjugated GNPs (GNP-PS) to treat skin and oral C. albicans infection in BALB/c mice. Methods The biomimetically synthesized GNPs were conjugated to photosensitizers viz. methylene blue (MB) or toluidine blue O (TB). The conjugation of PSs with GNPs was characterized by spectroscopic and microscopic techniques. The efficacy of gold nanoparticle conjugates against C. albicans biofilm was demonstrated by XTT assay and microscopic studies. The therapeutic efficacy of the combination of the GNP conjugates against cutaneous C. albicans infection was examined in mouse model by enumerating residual fungal burden and histopathological studies. Results The GNP-PS conjugate based PDT was found to effectively kill both C. albicans planktonic cells and biofilm populating hyphal forms. The mixture of GNPs conjugated to two different PSs significantly depleted the hyphal C. albicans burden against superficial skin and oral C. albicans infection in mice. Conclusion The GNP-PS conjugate combination exhibits synergism in photodynamic inactivation of C. albicans. The GNP conjugate based PDT can be employed effectively in treatment of cutaneous C. albicans infections in model animals. The antibiofilm potential of PDT therapy can also be exploited in depletion of C. albicans on medical appliances such as implants and catheters etc. PMID:26148012

  6. The quorum-sensing molecules farnesol/homoserine lactone and dodecanol operate via distinct modes of action in Candida albicans.

    PubMed

    Hall, Rebecca A; Turner, Kara J; Chaloupka, James; Cottier, Fabien; De Sordi, Luisa; Sanglard, Dominique; Levin, Lonny R; Buck, Jochen; Mühlschlegel, Fritz A

    2011-08-01

    Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C(12)-homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C(12)-homoserine lactone, may be used by other quorum-sensing molecules.

  7. Candida-streptococcal mucosal biofilms display distinct structural and virulence characteristics depending on growth conditions and hyphal morphotypes.

    PubMed

    Bertolini, M M; Xu, H; Sobue, T; Nobile, C J; Del Bel Cury, A A; Dongari-Bagtzoglou, A

    2015-08-01

    Candida albicans and streptococci of the mitis group form communities in multiple oral sites, where moisture and nutrient availability can change spatially or temporally. This study evaluated structural and virulence characteristics of Candida-streptococcal biofilms formed on moist or semidry mucosal surfaces, and tested the effects of nutrient availability and hyphal morphotype on dual-species biofilms. Three-dimensional models of the oral mucosa formed by immortalized keratinocytes on a fibroblast-embedded collagenous matrix were used. Infections were carried out using Streptococcus oralis strain 34, in combination with a C. albicans wild-type strain, or pseudohyphal-forming mutant strains. Increased moisture promoted a homogeneous surface biofilm by C. albicans. Dual biofilms had a stratified structure, with streptococci growing in close contact with the mucosa and fungi growing on the bacterial surface. Under semidry conditions, Candida formed localized foci of dense growth, which promoted focal growth of streptococci in mixed biofilms. Candida biofilm biovolume was greater under moist conditions, albeit with minimal tissue invasion, compared with semidry conditions. Supplementing the infection medium with nutrients under semidry conditions intensified growth, biofilm biovolume and tissue invasion/damage, without changing biofilm structure. Under these conditions, the pseudohyphal mutants and S. oralis formed defective superficial biofilms, with most bacteria in contact with the epithelial surface, below a pseudohyphal mass, resembling biofilms growing in a moist environment. The presence of S. oralis promoted fungal invasion and tissue damage under all conditions. We conclude that moisture, nutrient availability, hyphal morphotype and the presence of commensal bacteria influence the architecture and virulence characteristics of mucosal fungal biofilms.

  8. Ras GTPase-Activating Protein Regulation of Actin Cytoskeleton and Hyphal Polarity in Aspergillus nidulans▿ †

    PubMed Central

    Harispe, Laura; Portela, Cecilia; Scazzocchio, Claudio; Peñalva, Miguel A.; Gorfinkiel, Lisette

    2008-01-01

    Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenetic analyses strongly indicate that GapA regulates one or more “true” Ras proteins. A gapAΔ strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapAΔ strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germ tube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapAΔ disrupts the normal coupling of isotropic growth, polarity establishment, and mitosis, resulting in a highly heterogeneous cell population, including malformed germlings and a class of giant cells with no germ tubes and a multitude of nuclei. Unlike wild-type conidia, gapAΔ conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been reported in strains carrying a rasA dominant active allele, indicating that GapA downregulates RasA. gapAΔ cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapAΔ cells. As GapA-green fluorescent protein shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis. PMID:18039943

  9. Structural requirements of strigolactones for hyphal branching in AM fungi.

    PubMed

    Akiyama, Kohki; Ogasawara, Shin; Ito, Seisuke; Hayashi, Hideo

    2010-07-01

    Strigolactones are a group of terpenoid lactones that act as a host-derived signal in the rhizosphere communication of plants with arbuscular mycorrhizal (AM) fungi and root parasitic weeds as well as an endogenous plant hormone regulating shoot branching in plants. Strigolactones induce hyphal branching in AM fungi at very low concentrations, suggesting a highly sensitive perception system for strigolactones present in AM fungi. However, little is known about the structural requirements of strigolactones for hyphal branching in AM fungi. Here, we tested a series of natural and synthetically modified strigolactones as well as non-strigolactone-type germination stimulants for hyphal branching-inducing activity in germinating spores of the AM fungus Gigaspora margarita. All tested compounds with a tricyclic lactone coupled to a methylbutenolide via an enol ether bond showed activity, but differed in the active concentration and in the branching pattern of hyphae. Truncation of the A- and AB-rings in the tricyclic ABC lactone of strigolactones resulted in a drastic reduction in hyphal branching activity. Although the connection of the C-ring in the tricyclic lactone to the methylbutenolide D-ring was shown to be essential for hyphal branching, the bridge structure in the C-D part was found not necessarily to be enol ether, being replaceable with either alkoxy or imino ethers. These structural requirements in AM fungi are very similar but not identical to those observed in root parasitic weeds, especially with respect to the enol ether bridge in the C-D part.

  10. Catechin inhibits Candida albicans dimorphism by disrupting Cek1 phosphorylation and cAMP synthesis.

    PubMed

    Saito, Hideo; Tamura, Muneaki; Imai, Kenichi; Ishigami, Tomohiko; Ochiai, Kuniyasu

    2013-03-01

    Candida albicans is a fungal pathogen that undergoes dimorphism (transformation from a yeast form to a hyphal form), wherein, the yeast form is identified as a disseminating form that plays a critical role in the early stages of Candida disease progression, while the hyphal form is found to exert additional pathogenicity by adapting to various environmental conditions. Here, we elucidated the effects of catechin on C. albicans hyphal formation. Flow cytometry analysis showed catechin inhibited FCS-induced hyphal formation. Moreover, hypha-specific gene expression in MAP kinase cascade and cAMP pathway was decreased ascribable to catechin. Furthermore, through Western blotting and cAMP synthesis analysis, we found catechin obstructs Cek1 phosphorylation in MAP kinase cascade and suppresses cAMP synthesis. These results suggest that catechin possesses anti-dimorphism activity by interfering with in vitro signal transduction. Similarly, this highlights the possible application of catechin in clinical therapy for the management and prevention of candidosis.

  11. Candida albicans HWP1 gene expression and host antibody responses in colonization and disease.

    PubMed

    Naglik, Julian R; Fostira, Florentia; Ruprai, Jasmeet; Staab, Janet F; Challacombe, Stephen J; Sundstrom, Paula

    2006-10-01

    In vivo expression of the developmentally regulated Candida albicans hyphal wall protein 1 (HWP1) gene was analysed in human subjects who were culture positive for C. albicans and had oral symptoms (n=40) or were asymptomatic (n=29), or had vaginal symptoms (n=40) or were asymptomatic (n=29). HWP1 mRNA was present regardless of symptoms, implicating hyphal and possibly pseudohyphal forms in mucosal carriage as well as disease. As expected, in control subjects without oral symptoms (n=10) and without vaginal symptoms (n=10) who were culture negative in oral and vaginal samples, HWP1 mRNA was not detected. However, exposure to Hwp1 in healthy culture-negative controls, as well as in oral candidiasis and asymptomatic mucosal infections, was shown by the existence of local salivary and systemic adaptive antibody responses to Hwp1. The results are consistent with a role for Hwp1 in gastrointestinal colonization as well as in mucosal symptomatic and asymptomatic infections. Overall, Hwp1 and hyphal growth forms appear to be important factors in benign and invasive interactions of C. albicans with human hosts.

  12. Craniofacial morphogenesis workshop report.

    PubMed

    Solursh, M; Murray, J

    1994-05-01

    The following report highlights the discussions and interaction at the workshop on craniofacial morphogenesis, sponsored by The Human Frontier Science Program, held in April 1993 at the University of Iowa. A brief summary of selected sessions is included to exemplify the benefits of bringing together individuals from various disciplines and backgrounds in order to establish a unified theory of craniofacial morphogenesis. The synthesis of information and experience of a wide range of approaches made the 4-day period an invaluable experience for the participants from nine different countries.

  13. Coccidioides immitis presenting as a hyphal form in cerebrospinal fluid.

    PubMed Central

    Zepeda, M. R.; Kobayashi, G. K.; Appleman, M. D.; Navarro, A.

    1998-01-01

    This article reports a case of Coccidioides immitis that presented as a hyphal form in a 38-year-old patient. The organism was observed growing exclusively as hyphae in the cerebrospinal fluid by microscopic examination. Coccidioides immitis was the only organism cultured. The identification of C immitis was confirmed by both standard culture methods and DNA probe studies. Images Figure PMID:9685779

  14. Antimicrobial effects of liquid anesthetic isoflurane on Candida albicans

    PubMed Central

    Barodka, Viachaslau M; Acheampong, Edward; Powell, Garry; Lobach, Ludmila; Logan, David A; Parveen, Zahida; Armstead, Valerie; Mukhtar, Muhammad

    2006-01-01

    Candida albicans is a dimorphic fungus that can grow in yeast morphology or hyphal form depending on the surrounding environment. This ubiquitous fungus is present in skin and mucus membranes as a potential pathogen that under opportunistic conditions causes a series of systemic and superficial infections known as candidiasis, moniliasis or simply candidiasis. There has been a steady increase in the prevalence of candidiasis that is expressed in more virulent forms of infection. Although candidiasis is commonly manifested as mucocutaneous disease, life-threatening systemic invasion by this fungus can occur in every part of the body. The severity of candidal infections is associated with its morphological shift such that the hyphal morphology of the fungus is most invasive. Of importance, aberrant multiplication of Candida yeast is also associated with the pathogenesis of certain mucosal diseases. In this study, we assessed the anti-candidal activity of the volatile anesthetic isoflurane in liquid form in comparison with the anti-fungal agent amphotericin B in an in vitro culture system. Exposure of C. albicans to isoflurane (0.3% volume/volume and above) inhibited multiplication of yeast as well as formation of hyphae. These data suggest development of potential topical application of isoflurane for controlling a series of cutaneous and genital infections associated with this fungus. Elucidiation of the mechanism by which isoflurane effects fungal growth could offer therapeutic potential for certain systemic fungal infections. PMID:17094810

  15. Structural Requirements of Strigolactones for Hyphal Branching in AM Fungi

    PubMed Central

    Akiyama, Kohki; Ogasawara, Shin; Ito, Seisuke; Hayashi, Hideo

    2010-01-01

    Strigolactones are a group of terpenoid lactones that act as a host-derived signal in the rhizosphere communication of plants with arbuscular mycorrhizal (AM) fungi and root parasitic weeds as well as an endogenous plant hormone regulating shoot branching in plants. Strigolactones induce hyphal branching in AM fungi at very low concentrations, suggesting a highly sensitive perception system for strigolactones present in AM fungi. However, little is known about the structural requirements of strigolactones for hyphal branching in AM fungi. Here, we tested a series of natural and synthetically modified strigolactones as well as non-strigolactone-type germination stimulants for hyphal branching-inducing activity in germinating spores of the AM fungus Gigaspora margarita. All tested compounds with a tricyclic lactone coupled to a methylbutenolide via an enol ether bond showed activity, but differed in the active concentration and in the branching pattern of hyphae. Truncation of the A- and AB-rings in the tricyclic ABC lactone of strigolactones resulted in a drastic reduction in hyphal branching activity. Although the connection of the C-ring in the tricyclic lactone to the methylbutenolide D-ring was shown to be essential for hyphal branching, the bridge structure in the C–D part was found not necessarily to be enol ether, being replaceable with either alkoxy or imino ethers. These structural requirements in AM fungi are very similar but not identical to those observed in root parasitic weeds, especially with respect to the enol ether bridge in the C–D part. PMID:20418334

  16. Multicellular Models of Morphogenesis

    EPA Science Inventory

    EPA’s Virtual Embryo project (v-Embryo™), in collaboration with developers of CompuCell3D, aims to create computer models of morphogenesis that can be used to address the effects of chemical perturbation on embryo development at the cellular level. Such computational (in silico) ...

  17. Differentiation of Candida dubliniensis from Candida albicans on rosemary extract agar and oregano extract agar.

    PubMed

    de Loreto, Erico Silva; Pozzatti, Patrícia; Alves Scheid, Liliane; Santurio, Deise; Morais Santurio, Janio; Alves, Sydney Hartz

    2008-01-01

    Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans and therefore, may be misidentified in microbiological laboratories. Because molecular methods can be onerous and unfeasible in routine mycological laboratories with restricted budgets such as those in developing countries, phenotypic techniques have been encouraged in the development of differential media for the presumptive identification of these species. We examined the colony morphology and chlamydospore production of 30 C. dubliniensis isolates and 100 C. albicans isolates on two new proposed media: rosemary (Rosmarinus officinalis) extract agar (REA) and oregano (Origanum vulgare) extract agar (OEA). These substrates are traditionally used as spices and medicinal herbs. In both of these media, all C. dubliniensis isolates (100%) showed rough colonies with peripheral hyphal fringes and abundant chlamydospores after 24 to 48 hr of incubation at 25 degrees C. In contrast, under the same conditions, all isolates of C. albicans (100%) showed smooth colonies without hyphal fringes or chlamydospores. In conclusion, REA and OEA offer a simple, rapid, and inexpensive screening media for the differentiation of C. albicans and C. dubliniensis.

  18. Effects of Clitoria ternatea leaf extract on growth and morphogenesis of Aspergillus niger.

    PubMed

    Kamilla, L; Mansor, S M; Ramanathan, S; Sasidharan, S

    2009-08-01

    Clitoria ternatea is known for its antimicrobial activity but the antifungal effects of leaf extract on growth and morphogenesis of Aspergillus niger have not been observed. The extract showed a favorable antifungal activity against A. niger with a minimum inhibition concentration 0.8 mg/mL and minimum fungicidal concentration 1.6 mg/mL, respectively. The leaf extract exhibited considerable antifungal activity against filamentous fungi in a dose-dependent manner with 0.4 mg/mL IC50 value on hyphal growth of A. niger. The main changes observed under scanning electron microscopy after C. ternatea extract treatment were loss of cytoplasm in fungal hyphae and the hyphal wall and its diameter became markedly thinner, distorted, and resulted in cell wall disruption. In addition, conidiophore alterations were also observed when A. niger was treated with C. ternatea leaf extract.

  19. Fungal quorum sensing molecules: Role in fungal morphogenesis and pathogenicity.

    PubMed

    Wongsuk, Thanwa; Pumeesat, Potjaman; Luplertlop, Natthanej

    2016-05-01

    When microorganisms live together in high numbers, they need to communicate with each other. To achieve cell-cell communication, microorganisms secrete molecules called quorum-sensing molecules (QSMs) that control their biological activities and behaviors. Fungi secrete QSMs such as farnesol, tyrosol, phenylethanol, and tryptophol. The role of QSMs in fungi has been widely studied in both yeasts and filamentous fungi, for example in Candida albicans, C. dubliniensis, Aspergillus niger, A. nidulans, and Fusarium graminearum. QSMs impact fungal morphogenesis (yeast-to-hypha formation) and also play a role in the germination of macroconidia. QSMs cause fungal cells to initiate programmed cell death, or apoptosis, and play a role in fungal pathogenicity. Several types of QSMs are produced during stages of biofilm development to control cell population or morphology in biofilm communities. This review article emphasizes the role of fungal QSMs, especially in fungal morphogenesis, biofilm formation, and pathogenicity. Information about QSMs may lead to improved measures for controlling fungal infection.

  20. Interaction of Candida albicans cell wall Als3 protein with Streptococcus gordonii SspB adhesin promotes development of mixed-species communities.

    PubMed

    Silverman, Richard J; Nobbs, Angela H; Vickerman, M Margaret; Barbour, Michele E; Jenkinson, Howard F

    2010-11-01

    Candida albicans colonizes human mucosa and prosthetic surfaces associated with artificial joints, catheters, and dentures. In the oral cavity, C. albicans coexists with numerous bacterial species, and evidence suggests that bacteria may modulate fungal growth and biofilm formation. Streptococcus gordonii is found on most oral cavity surfaces and interacts with C. albicans to promote hyphal and biofilm formation. In this study, we investigated the role of the hyphal-wall protein Als3p in interactions of C. albicans with S. gordonii. Utilizing an ALS3 deletion mutant strain, it was shown that cells were not affected in initial adherence to the salivary pellicle or in hyphal formation in the planktonic phase. However, the Als3(-) mutant was unable to form biofilms on the salivary pellicle or deposited S. gordonii DL1 wild-type cells, and after initial adherence, als3Δ/als3Δ (ΔALS3) cells became detached concomitant with hyphal formation. In coaggregation assays, S. gordonii cells attached to, and accumulated around, hyphae formed by C. albicans wild-type cells. However, streptococci failed to attach to hyphae produced by the ΔALS3 mutant. Saccharomyces cerevisiae S150-2B cells expressing Als3p, but not control cells, supported binding of S. gordonii DL1. However, S. gordonii Δ(sspA sspB) cells deficient in production of the surface protein adhesins SspA and SspB showed >50% reduced levels of binding to S. cerevisiae expressing Als3p. Lactococcus lactis cells expressing SspB bound avidly to S. cerevisiae expressing Als3p, but not to S150-2B wild-type cells. These results show that recognition of C. albicans by S. gordonii involves Als3 protein-SspB protein interaction, defining a novel mechanism in fungal-bacterial communication.

  1. Neutrophil Interactions Stimulate Evasive Hyphal Branching by Aspergillus fumigatus

    PubMed Central

    Jorgensen, Julianne; Frydman, Galit H.; Jones, Caroline N.

    2017-01-01

    Invasive aspergillosis (IA), primarily caused by Aspergillus fumigatus, is an opportunistic fungal infection predominantly affecting immunocompromised and neutropenic patients that is difficult to treat and results in high mortality. Investigations of neutrophil-hypha interaction in vitro and in animal models of IA are limited by lack of temporal and spatial control over interactions. This study presents a new approach for studying neutrophil-hypha interaction at single cell resolution over time, which revealed an evasive fungal behavior triggered by interaction with neutrophils: Interacting hyphae performed de novo tip formation to generate new hyphal branches, allowing the fungi to avoid the interaction point and continue invasive growth. Induction of this mechanism was independent of neutrophil NADPH oxidase activity and neutrophil extracellular trap (NET) formation, but could be phenocopied by iron chelation and mechanical or physiological stalling of hyphal tip extension. The consequence of branch induction upon interaction outcome depends on the number and activity of neutrophils available: In the presence of sufficient neutrophils branching makes hyphae more vulnerable to destruction, while in the presence of limited neutrophils the interaction increases the number of hyphal tips, potentially making the infection more aggressive. This has direct implications for infections in neutrophil-deficient patients and opens new avenues for treatments targeting fungal branching. PMID:28076396

  2. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi.

    PubMed

    Akiyama, Kohki; Matsuzaki, Ken-ichi; Hayashi, Hideo

    2005-06-09

    Arbuscular mycorrhizal (AM) fungi form mutualistic, symbiotic associations with the roots of more than 80% of land plants. The fungi are incapable of completing their life cycle in the absence of a host root. Their spores can germinate and grow in the absence of a host, but their hyphal growth is very limited. Little is known about the molecular mechanisms that govern signalling and recognition between AM fungi and their host plants. In one of the first stages of host recognition, the hyphae of AM fungi show extensive branching in the vicinity of host roots before formation of the appressorium, the structure used to penetrate the plant root. Host roots are known to release signalling molecules that trigger hyphal branching, but these branching factors have not been isolated. Here we have isolated a branching factor from the root exudates of Lotus japonicus and used spectroscopic analysis and chemical synthesis to identify it as a strigolactone, 5-deoxy-strigol. Strigolactones are a group of sesquiterpene lactones, previously isolated as seed-germination stimulants for the parasitic weeds Striga and Orobanche. The natural strigolactones 5-deoxy-strigol, sorgolactone and strigol, and a synthetic analogue, GR24, induced extensive hyphal branching in germinating spores of the AM fungus Gigaspora margarita at very low concentrations.

  3. Interkingdom cooperation between Candida albicans, Streptococcus oralis and Actinomyces oris modulates early biofilm development on denture material.

    PubMed

    Cavalcanti, Indira M G; Nobbs, Angela H; Ricomini-Filho, Antônio Pedro; Jenkinson, Howard F; Del Bel Cury, Altair A

    2016-04-01

    Candida-associated stomatitis affects up to 60% of denture wearers, and Candida albicans remains the most commonly isolated fungal species. The oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque. The aims of this study were to determine the effects of S. oralis and A. oris on the development of C. albicans biofilms on denture material. Resin discs were coated with saliva and at early (1.5 h) or later (24 h) stages of biofilm development, cell numbers of each species were determined. Spatial distribution of microorganisms was visualized by confocal scanning laser microscopy of biofilms labelled by differential fluorescence or by fluorescence in situ hybridization. Interkingdom interactions underpinning biofilm development were also evaluated planktonically utilizing fluorescence microscopy. Synergistic interactions between all three species occurred within biofilms and planktonically. Bacterial cells coaggregated with each other and adhered singly or in coaggregates to C. albicans hyphal filaments. Streptococcus oralis appeared to enhance hyphal filament production and C. albicans biovolume was increased 2-fold. Concomitantly, cell numbers of S. oralis and A. oris were enhanced by C. albicans. Thus, cooperative physical and metabolic processes occurring between these three microbial species intensify pathogenic plaque communities on denture surfaces.

  4. Antifungal Action of Methylene Blue Involves Mitochondrial Dysfunction and Disruption of Redox and Membrane Homeostasis in C. albicans

    PubMed Central

    Ansari, Moiz A.; Fatima, Zeeshan; Hameed, Saif

    2016-01-01

    Candida albicans is known to cause infections ranging from superficial and systemic in immunocompromised person. In this study, we explored that the antifungal action of Methylene blue (MB) is mediated through mitochondrial dysfunction and disruption of redox and membrane homeostasis against C. albicans. We demonstrated that MB displayed its antifungal potential against C. albicans and two clinical isolates tested. We also showed that MB is effective against two non- albicans species as well. Notably, the antifungal effect of MB seems to be independent of the major drug efflux pumps transporter activity. We explored that MB treated Candida cells were sensitive on non-fermentable carbon source leading us to propose that MB inhibits mitochondria. This sensitive phenotype was reinforced with the fact that sensitivity of Candida cells to MB could be rescued upon the supplementation of ascorbic acid, an antioxidant. This clearly suggests that disturbances in redox status are linked with MB action. We further demonstrated that Candida cells were susceptible to membrane perturbing agent viz. SDS which was additionally confirmed by transmission electron micrographs showing disruption of membrane integrity. Moreover, the ergosterol levels were significantly decreased by 66% suggesting lipid compositional changes due to MB. Furthermore, we could demonstrate that MB inhibits the yeast to hyphal transition in C. albicans which is one of the major virulence attribute in most of the hyphal inducing conditions. Taken together, the data generated from present study clearly establishes MB as promising antifungal agent that could be efficiently employed in strategies to treat Candida infections. PMID:27006725

  5. Inhibitory effects of several saturated fatty acids and their related fatty alcohols on the growth of Candida albicans.

    PubMed

    Hayama, Kazumi; Takahashi, Miki; Yui, Satoru; Abe, Shigeru

    2015-12-01

    We examined the effect of 5 saturated fatty acids and their related alcohols on the growth of Candida albicans. The inhibitory effects of these compounds against the yeast and hyphal growth forms of C. albicans were examined using the modified NCCLS method and crystal violet staining, respectively. Among these compounds, capric acid inhibited both types of growth at the lowest concentration. The IC(80), i.e., the concentration at which the compounds reduced the growth of C. albicans by 80% in comparison with the growth of control cells, of capric acid for the hyphal growth of this fungus, which is indispensable for its mucosal invasion, was 16.7 μM. These fatty acids, including capric acid, have an unpleasant smell, which may limit their therapeutic use. To test them at reduced concentrations, the combined effect of these fatty acids and oligonol, a depolymerized polyphenol, was evaluated in vitro. These combinations showed potent synergistic inhibition of hyphal growth [fractional inhibitory concentration (FIC) index = 0.319]. Our results demonstrated that capric acid combined with oligonol could be used as an effective anti-Candida compound. It may be a candidate prophylactic or therapeutic tool against mucosal Candida infection.

  6. Human serum potentiates the expression of genes associated with antifungal drug resistance in C. albicans biofilms on central venous catheters.

    PubMed

    Samaranayake, L P; Anil, S; Hashem, M; Vellappally, S; Cheung, B P K

    2015-04-01

    Candida albicans is a major agent of fungaemias and frequently causes systemic disease through seeded, blood stream dissemination. These infections, particularly common in hospitalized patients with central venous catheters (CVCs), appear to persevere due to biofilm reservoirs of the yeast that tend to develop on the device. Although it is known that candidal biofilms are intrinsically resistant to antifungals compared with their planktonic counterparts, there is a paucity of data on the expression of antifungal drug resistance genes (DRGs) in candidal biofilms in CVC reservoirs. Furthermore, notwithstanding the fact that CVCs are constantly bathed in human serum, there are no studies on the effect of the latter on the DRG expression in candidal biofilms. Hence, we developed in vitro biofilms of three different C. albicans strains on silicone CVC discs immersed in human serum and evaluated the temporal expression of nine antifungal DRGs. In an attempt to evaluate the effect of hyphal elements on DRG expression, we incorporated a hyphal mutant (HM) and its wild-type (WT) counterpart, as well as a fresh clinical isolate in the studies. Human serum significantly up-regulated DRG transcripts in Candida biofilms on CVCs, at different stages of biofilm growth, while the WT strain over-expressed more DRGs than the HM strain. Here, we report, for the first time, that both human serum and the hyphal elements of the yeast have a profound modulatory effect on DRG expression in C. albicans biofilms on CVCs.

  7. Germ tube-specific antigens of Candida albicans cell walls

    SciTech Connect

    Sundstrom, P.R.

    1986-01-01

    Studies were performed to characterize the surface differences between blastospores and germ tubes of the pathogenic, dimorphic yeast, Candida albicans, and to identify components of yeast cells responsible for these differences. Investigation of surfaces differences of the two growth forms was facilitated by the production of rabbit antiserum prepared against Formalin-treated yeast possessing germ tubes. To prepare antiserum specific for germ tubes, this serum was adsorbed with stationary phase blastospores. Whereas the unadsorbed antiserum reacted with both blastospore and germ tube forms by immunofluorescence and Enzyme-Linked Immunosorbent Assay, the adsorbed antiserum did not react with blastospores but detected germ tube-specific antigens in hyphal forms. The differences between blastospores and germ tubes of Candida albicans, were further studied by comparing enzymatic digests of cell walls of both growth forms in radiolabeled organisms. Organisms were labeled either on the surface with /sup 125/I, or metabolically with (/sup 35/S) methionine or (/sup 3/H) mannose. Three-surface-located components (as shown by antibody adsorption and elution experiments) were precipitated from Zymolase digests. All three components were mannoproteins as shown by their ability to bind Concanavalin A, and to be labeled in protein labeling procedures, and two of these (200,000 and 155,000 molecular weight) were germ tube specific, as shown by their ability to be precipitated by germ tube-specific antiserum. Monoclonal antibodies were prepared to C. albicans, using blastospores bearing germ tubes as immunogen.

  8. Sea Urchin Morphogenesis.

    PubMed

    McClay, David R

    2016-01-01

    In the sea urchin morphogenesis follows extensive molecular specification. The specification controls the many morphogenetic events and these, in turn, precede patterning steps that establish the larval body plan. To understand how the embryo is built it was necessary to understand those series of molecular steps. Here an example of the historical sequence of those discoveries is presented as it unfolded over the last 50 years, the years during which major progress in understanding development of many animals and plants was documented by CTDB. In sea urchin development a rich series of experimental studies first established many of the phenomenological components of skeletal morphogenesis and patterning without knowledge of the molecular components. The many discoveries of transcription factors, signals, and structural proteins that contribute to the shape of the endoskeleton of the sea urchin larva then followed as molecular tools became available. A number of transcription factors and signals were discovered that were necessary for specification, morphogenesis, and patterning. Perturbation of the transcription factors and signals provided the means for assembling models of the gene regulatory networks used for specification and controlled the subsequent morphogenetic events. The earlier experimental information informed perturbation experiments that asked how patterning worked. As a consequence it was learned that ectoderm provides a series of patterning signals to the skeletogenic cells and as a consequence the skeletogenic cells secrete a highly patterned skeleton based on their ability to genotypically decode the localized reception of several signals. We still do not understand the complexity of the signals received by the skeletogenic cells, nor do we understand in detail how the genotypic information shapes the secreted skeletal biomineral, but the current knowledge at least outlines the sequence of events and provides a useful template for future

  9. Candida albicans pathogenicity mechanisms

    PubMed Central

    Mayer, François L.; Wilson, Duncan; Hube, Bernhard

    2013-01-01

    The polymorphic fungus Candida albicans is a member of the normal human microbiome. In most individuals, C. albicans resides as a lifelong, harmless commensal. Under certain circumstances, however, C. albicans can cause infections that range from superficial infections of the skin to life-threatening systemic infections. Several factors and activities have been identified which contribute to the pathogenic potential of this fungus. Among them are molecules which mediate adhesion to and invasion into host cells, the secretion of hydrolases, the yeast-to-hypha transition, contact sensing and thigmotropism, biofilm formation, phenotypic switching and a range of fitness attributes. Our understanding of when and how these mechanisms and factors contribute to infection has significantly increased during the last years. In addition, novel virulence mechanisms have recently been discovered. In this review we present an update on our current understanding of the pathogenicity mechanisms of this important human pathogen. PMID:23302789

  10. Co-occurence of filamentation defects and impaired biofilms in Candida albicans protein kinase mutants.

    PubMed

    Konstantinidou, Nina; Morrissey, John Patrick

    2015-12-01

    Pathogenicity of Candida albicans is linked with its developmental stages, notably the capacity switch from yeast-like to hyphal growth, and to form biofilms on surfaces. To better understand the cellular processes involved in C. albicans development, a collection of 63 C. albicans protein kinase mutants was screened for biofilm formation in a microtitre plate assay. Thirty-eight mutants displayed some degree of biofilm impairment, with 20 categorised as poor biofilm formers. All the poor biofilm formers were also defective in the switch from yeast to hyphae, establishing it as a primary defect. Five genes, VPS15, IME2, PKH3, PGA43 and CEX1, encode proteins not previously reported to influence hyphal development or biofilm formation. Network analysis established that individual components of some processes, most interestingly MAP kinase pathways, are not required for biofilm formation, most likely indicating functional redundancy. Mutants were also screened for their response to bacterial supernatants and it was found that Pseudomonas aeruginosa supernatants inhibited biofilm formation in all mutants, regardless of the presence of homoserine lactones (HSLs). In contrast, Candida morphology was only affected by supernatant containing HSLs. This confirms the distinct HSL-dependent inhibition of filamentation and the HSL-independent impairment of biofilm development by P. aeruginosa.

  11. Different virulence of candida albicans is attributed to the ability of escape from neutrophil extracellular traps by secretion of DNase

    PubMed Central

    Zhang, Xiaohuan; Zhao, Sainan; Sun, Luping; Li, Wenqing; Wei, Qiao; Ashman, Robert B; Hu, Yan

    2017-01-01

    Candida albicans is an important opportunistic fungus causing both disseminated and local infections. The discovery of neutrophil extracellular traps (NETs) has presented a new strategy to kill microorganisms in host’s innate immune response. Although it has been reported that NETs can trap and kill both yeast and hyphal forms of C. albicans, the mechanism by which C. albicans escape from NETs has not been fully understood. In this study, the ability of two strains of C. albicans SC5314 and 3683 to escape NETs-mediated killing was compared. It was found that SC5314 induced higher levels of reactive oxygen species (ROS) and expressions of Rac1/2 and more NETs formation by neutrophils, and also generated more deoxyribonucleases (DNase) than 3683 did. However, resistance to neutrophils killing was greater in SC5314 than that of 3683. When extracellular traps were degraded by exogenous DNase I or catalase, and neutrophil phagocytic activity blocked by cytochalasin D, the killing capacity of neutrophils co-cultured with either C. albicans SC5314 or 3683 was significantly decreased. This study indicates that C. albicans can escape from the trapping and killing of NETs by secreting DNase, which offers further insights into the basis for differences in virulence of different strains of C. albicans. PMID:28123633

  12. Interactions between Streptococcus oralis, Actinomyces oris, and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle.

    PubMed

    Cavalcanti, I M G; Del Bel Cury, A A; Jenkinson, H F; Nobbs, A H

    2017-02-01

    The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual-species biofilms, or three-species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non-fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.

  13. SR-like RNA-binding protein Slr1 affects Candida albicans filamentation and virulence.

    PubMed

    Ariyachet, Chaiyaboot; Solis, Norma V; Liu, Yaoping; Prasadarao, Nemani V; Filler, Scott G; McBride, Anne E

    2013-04-01

    Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.

  14. Isolation and characterization of yeast monomorphic mutants of Candida albicans.

    PubMed Central

    Elorza, M V; Sentandreu, R; Ruiz-Herrera, J

    1994-01-01

    A method was devised for the isolation of yeast monomorphic (LEV) mutants of Candida albicans. By this procedure, about 20 stable yeast-like mutants were isolated after mutagenesis with ethyl methane sulfonate. The growth rate of the mutants in different carbon sources, both fermentable and not, was indistinguishable from that of the parental strain, but they were unable to grow as mycelial forms after application of any of the common effective inducers, i.e., heat shock, pH alterations, proline addition, or use of GlcNAc as the carbon source. Studies performed with one selected strain demonstrated that it had severe alterations in the chemical composition of the cell wall, mainly in the levels of chitin and glucans, and in specific mannoproteins, some of them recognizable by specific polyclonal and monoclonal antibodies. It is suggested that these structural alterations hinder the construction of a normal hyphal wall. Images PMID:8157600

  15. Two CDC42 paralogs modulate C. neoformans thermotolerance and morphogenesis under host physiological conditions

    PubMed Central

    Ballou, Elizabeth R.; Nichols, Connie B.; Miglia, Kathleen J; Kozubowski, Lukasz; Alspaugh, J. Andrew

    2013-01-01

    The precise regulation of morphogenesis is a key mechanism by which cells respond to a variety of stresses, including those encountered by microbial pathogens in the host. The polarity protein Cdc42 regulates cellular morphogenesis throughout eukaryotes, and we explore the role of Cdc42 proteins in the host survival of the human fungal pathogen Cryptococcus neoformans. Uniquely, C. neoformans has two functional Cdc42 paralogs, Cdc42 and Cdc420. Here we investigate the contribution of each paralog to resistance to host stress. In contrast to non-pathogenic model organisms, C. neoformans Cdc42 proteins are not required for viability under non-stress conditions. In the presence of cell stress, strains deleted for either paralog show defects in thermotolerance and morphogenesis, likely as a result of their roles in the organization of actin and septin structures during bud growth and cytokinesis. These proteins act downstream of C. neoformans Ras1 to regulate its morphogenesis subpathway, but not its effects on mating. Cdc42, and not Cdc420, is required for virulence in a murine model of cryptococcosis. The C. neoformans Cdc42 proteins likely perform complementary functions with other Rho-like GTPases to control cell polarity, septin organization, and hyphal transitions that allow survival in the environment and in the host. PMID:20025659

  16. Examination of the pathogenic potential of Candida albicans filamentous cells in an animal model of haematogenously disseminated candidiasis.

    PubMed

    Cleary, Ian A; Reinhard, Sara M; Lazzell, Anna L; Monteagudo, Carlos; Thomas, Derek P; Lopez-Ribot, Jose L; Saville, Stephen P

    2016-03-01

    The opportunistic fungal pathogen Candida albicans is an increasingly common threat to human health. Candida albicans grows in several morphologies and mutant strains locked in yeast or filamentous forms have attenuated virulence in the murine model of disseminated candidiasis. Thus, the ability to change shape is important for virulence. The transcriptional repressors Nrg1p and Tup1p are required for normal regulation of C. albicans morphology. Strains lacking either NRG1 or TUP1 are constitutively pseudohyphal under yeast growth conditions, and display attenuated virulence in the disseminated model. To dissect the relative importance of hyphae and pseudohyphae during an infection, we used strains in which the morphological transition could be externally manipulated through controlled expression of NRG1 or TUP1. Remarkably, hyphal form inocula retain the capacity to cause disease. Whilst induction of a pseudohyphal morphology through depletion of TUP1 did result in attenuated virulence, this was not due to a defect in the ability to escape the bloodstream. Instead, we observed that pseudohyphal cells are cleared from tissues much more efficiently than either hyphal (virulent) or yeast form (avirulent) cells, indicating that different C. albicans morphologies have distinct interactions with host cells during an infection.

  17. Examination of the pathogenic potential of Candida albicans filamentous cells in an animal model of haematogenously disseminated candidiasis

    PubMed Central

    Cleary, Ian A.; Reinhard, Sara M.; Lazzell, Anna L.; Monteagudo, Carlos; Thomas, Derek P.; Lopez-Ribot, Jose L.; Saville, Stephen P.

    2016-01-01

    The opportunistic fungal pathogen Candida albicans is an increasingly common threat to human health. Candida albicans grows in several morphologies and mutant strains locked in yeast or filamentous forms have attenuated virulence in the murine model of disseminated candidiasis. Thus, the ability to change shape is important for virulence. The transcriptional repressors Nrg1p and Tup1p are required for normal regulation of C. albicans morphology. Strains lacking either NRG1 or TUP1 are constitutively pseudohyphal under yeast growth conditions, and display attenuated virulence in the disseminated model. To dissect the relative importance of hyphae and pseudohyphae during an infection, we used strains in which the morphological transition could be externally manipulated through controlled expression of NRG1 or TUP1. Remarkably, hyphal form inocula retain the capacity to cause disease. Whilst induction of a pseudohyphal morphology through depletion of TUP1 did result in attenuated virulence, this was not due to a defect in the ability to escape the bloodstream. Instead, we observed that pseudohyphal cells are cleared from tissues much more efficiently than either hyphal (virulent) or yeast form (avirulent) cells, indicating that different C. albicans morphologies have distinct interactions with host cells during an infection. PMID:26851404

  18. On Buckling Morphogenesis.

    PubMed

    Nelson, Celeste M

    2016-02-01

    Cell-generated mechanical forces drive many of the tissue movements and rearrangements that are required to transform simple populations of cells into the complex three-dimensional geometries of mature organs. However, mechanical forces do not need to arise from active cellular movements. Recent studies have illuminated the roles of passive forces that result from mechanical instabilities between epithelial tissues and their surroundings. These mechanical instabilities cause essentially one-dimensional epithelial tubes and two-dimensional epithelial sheets to buckle or wrinkle into complex topologies containing loops, folds, and undulations in organs as diverse as the brain, the intestine, and the lung. Here, I highlight examples of buckling and wrinkling morphogenesis, and suggest that this morphogenetic mechanism may be broadly responsible for sculpting organ form.

  19. On Buckling Morphogenesis

    PubMed Central

    Nelson, Celeste M.

    2016-01-01

    Cell-generated mechanical forces drive many of the tissue movements and rearrangements that are required to transform simple populations of cells into the complex three-dimensional geometries of mature organs. However, mechanical forces do not need to arise from active cellular movements. Recent studies have illuminated the roles of passive forces that result from mechanical instabilities between epithelial tissues and their surroundings. These mechanical instabilities cause essentially one-dimensional epithelial tubes and two-dimensional epithelial sheets to buckle or wrinkle into complex topologies containing loops, folds, and undulations in organs as diverse as the brain, the intestine, and the lung. Here, I highlight examples of buckling and wrinkling morphogenesis, and suggest that this morphogenetic mechanism may be broadly responsible for sculpting organ form. PMID:26632268

  20. C. albicans growth, transition, biofilm formation, and gene expression modulation by antimicrobial decapeptide KSL-W

    PubMed Central

    2013-01-01

    Background Antimicrobial peptides have been the focus of much research over the last decade because of their effectiveness and broad-spectrum activity against microbial pathogens. These peptides also participate in inflammation and the innate host defense system by modulating the immune function that promotes immune cell adhesion and migration as well as the respiratory burst, which makes them even more attractive as therapeutic agents. This has led to the synthesis of various antimicrobial peptides, including KSL-W (KKVVFWVKFK-NH2), for potential clinical use. Because this peptide displays antimicrobial activity against bacteria, we sought to determine its antifungal effect on C. albicans. Growth, hyphal form, biofilm formation, and degradation were thus examined along with EFG1, NRG1, EAP1, HWP1, and SAP 2-4-5-6 gene expression by quantitative RT-PCR. Results This study demonstrates that KSL-W markedly reduced C. albicans growth at both early and late incubation times. The significant effect of KSL-W on C. albicans growth was observed beginning at 10 μg/ml after 5 h of contact by reducing C. albicans transition and at 25 μg/ml by completely inhibiting C. albicans transition. Cultured C. albicans under biofilm-inducing conditions revealed that both KSL-W and amphotericin B significantly decreased biofilm formation at 2, 4, and 6 days of culture. KSL-W also disrupted mature C. albicans biofilms. The effect of KSL-W on C. albicans growth, transition, and biofilm formation/disruption may thus occur through gene modulation, as the expression of various genes involved in C. albicans growth, transition and biofilm formation were all downregulated when C. albicans was treated with KSL-W. The effect was greater when C. albicans was cultured under hyphae-inducing conditions. Conclusions These data provide new insight into the efficacy of KSL-W against C. albicans and its potential use as an antifungal therapy. PMID:24195531

  1. Ash1 Protein, an Asymmetrically Localized Transcriptional Regulator, Controls Filamentous Growth and Virulence of Candida albicans

    PubMed Central

    Inglis, Diane O.; Johnson, Alexander D.

    2002-01-01

    In response to a number of distinct environmental conditions, the fungal pathogen Candida albicans undergoes a morphological transition from a round, yeast form to a series of elongated, filamentous forms. This transition is believed to be critical for virulence in a mouse model of disseminated candidiasis. Here we describe the characterization of C. albicans ASH1, a gene that encodes an asymmetrically localized transcriptional regulatory protein involved in this response. We show that C. albicans ash1 mutants are defective in responding to some filament-inducing conditions. We also show that Ash1p is preferentially localized to daughter cell nuclei in the budding-yeast form of C. albicans cell growth and to the hyphal tip cells in growing filaments. Thus, Ash1p “marks” newly formed cells and presumably directs a specialized transcriptional program in these cells. Finally, we show that ASH1 is required for full virulence of C. albicans in a mouse model of disseminated candidiasis. PMID:12446785

  2. Candida albicans adhesin Als3p is dispensable for virulence in the mouse model of disseminated candidiasis

    PubMed Central

    Cleary, Ian A.; Reinhard, Sara M.; Miller, C. Lindsay; Murdoch, Craig; Thornhill, Martin H.; Lazzell, Anna L.; Monteagudo, Carlos; Thomas, Derek P.

    2011-01-01

    The presence of specific proteins, including Ece1p, Hwp1p and Als3p, distinguishes the Candida albicans hyphal cell wall from that of yeast-form cells. These proteins are thought to be important for the ability of C. albicans cells to adhere to living and non-living surfaces and for the cell-to-cell adhesion necessary for biofilm formation, and also to be pivotal in mediating C. albicans interactions with endothelial cells. Using an in vitro flow adhesion assay, we previously observed that yeast cells bind in greater numbers to human microvascular endothelial cells than do hyphal or pseudohyphal cells. This is consistent with previous observations that, in a murine model of disseminated candidiasis, cells locked in the yeast form can efficiently escape the bloodstream and invade host tissues. To more precisely explore the role of Als3p in adhesion and virulence, we deleted both copies of ALS3 in a wild-type C. albicans strain. In agreement with previous studies, our als3Δ null strain formed hyphae normally but was defective in biofilm formation. Whilst ALS3 was not expressed in our null strain, hypha-specific genes such as ECE1 and HWP1 were still induced appropriately. Both the yeast form and the hyphal form of the als3Δ strain adhered to microvascular endothelial cells to the same extent as a wild-type strain under conditions of flow, indicating that Als3p is not a significant mediator of the initial interaction between fungal cells and the endothelium. Finally, in a murine model of haematogenously disseminated candidiasis the mutant als3Δ remained as virulent as the wild-type parent strain. PMID:21436220

  3. Two components of a velvet-like complex control hyphal morphogenesis, conidiophore development, and penicillin biosynthesis in Penicillium chrysogenum.

    PubMed

    Hoff, Birgit; Kamerewerd, Jens; Sigl, Claudia; Mitterbauer, Rudolf; Zadra, Ivo; Kürnsteiner, Hubert; Kück, Ulrich

    2010-08-01

    Penicillium chrysogenum is the industrial producer of the antibiotic penicillin, whose biosynthetic regulation is barely understood. Here, we provide a functional analysis of two major homologues of the velvet complex in P. chrysogenum, which we have named P. chrysogenum velA (PcvelA) and PclaeA. Data from array analysis using a DeltaPcvelA deletion strain indicate a significant role of PcVelA on the expression of biosynthesis and developmental genes, including PclaeA. Northern hybridization and high-performance liquid chromatography quantifications of penicillin titers clearly show that both PcVelA and PcLaeA play a major role in penicillin biosynthesis in a producer strain that underwent several rounds of UV mutagenesis during a strain improvement program. Both regulators are further involved in different developmental processes. While PcvelA deletion leads to light-independent conidial formation, dichotomous branching of hyphae, and pellet formation in shaking cultures, a DeltaPclaeA strain shows a severe impairment in conidiophore formation under both light and dark conditions. Bimolecular fluorescence complementation assays provide evidence for a velvet-like complex in P. chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators in genera other than Aspergillus.

  4. Dynamics of Biofilm Formation and the Interaction between Candida albicans and Methicillin-Susceptible (MSSA) and -Resistant Staphylococcus aureus (MRSA)

    PubMed Central

    Zago, Chaiene Evelin; Silva, Sónia; Sanitá, Paula Volpato; Barbugli, Paula Aboud; Dias, Carla Maria Improta; Lordello, Virgínia Barreto; Vergani, Carlos Eduardo

    2015-01-01

    Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms. PMID:25875834

  5. Terpenoids inhibit Candida albicans growth by affecting membrane integrity and arrest of cell cycle.

    PubMed

    Zore, Gajanan B; Thakre, Archana D; Jadhav, Sitaram; Karuppayil, S Mohan

    2011-10-15

    Anti-Candida potential of six terpenoids were evaluated in this study against various isolates of Candida albicans (n=39) and non-C. albicans (n=9) that are differentially susceptible to fluconazole. All the six terpenoids tested, showed excellent activity and were equally effective against isolates of Candida sps., tested in this study. Linalool and citral were the most effective ones, inhibiting all the isolates at ≤0.064% (v/v). Five among the six terpenoids tested were fungicidal. Time dependent kill curve assay showed that MFCs of linalool and eugenol were highly toxic to C. albicans, killing 99.9% inoculum within seven min of exposure, while that of citronellal, linalyl acetate and citral required 15min, 1h and 2h, respectively. FIC index values (Linalool - 0.140, benzyl benzoate - 0.156, eugenol - 0.265, citral - 0.281 and 0.312 for linalyl acetate and citronellal) and isobologram obtained by checker board assay showed that all the six terpenoids tested exhibit excellent synergistic activity with fluconazole against a fluconazole resistant strain of C. albicans. Terpenoids tested arrested C. albicans cells at different phases of the cell cycle i.e. linalool and LA at G1, citral and citronellal at S phase and benzyl benzoate at G2-M phase and induced apoptosis. Linalool, citral, citronellal and benzyl benzoate caused more than 50% inhibition of germ tube induction at 0.008%, while eugenol and LA required 0.032 and 0.016% (v/v) concentrations, respectively. MICs of all the terpenoids for the C. albicans growth were non toxic to HeLa cells. Terpenoids tested exhibited excellent activity against C. albicans yeast and hyphal form growth at the concentrations that are non toxic to HeLa cells. Terpenoids tested in this study may find use in antifungal chemotherapy, not only as antifungal agents but also as synergistic agents along with conventional drugs like fluconazole.

  6. Bioelectromagnetics in morphogenesis.

    PubMed

    Levin, Michael

    2003-07-01

    Understanding the factors that allow biological systems to reliably self-assemble consistent, highly complex, four dimensional patterns on many scales is crucial for the biomedicine of cancer, regeneration, and birth defects. The role of chemical signaling factors in controlling embryonic morphogenesis has been a central focus in modern developmental biology. While the role of tensile forces is also beginning to be appreciated, another major aspect of physics remains largely neglected by molecular embryology: electromagnetic fields and radiations. The continued progress of molecular approaches to understanding biological form and function in the post genome era now requires the merging of genetics with functional understanding of biophysics and physiology in vivo. The literature contains much data hinting at an important role for bioelectromagnetic phenomena as a mediator of morphogenetic information in many contexts relevant to embryonic development. This review attempts to highlight briefly some of the most promising (and often underappreciated) findings that are of high relevance for understanding the biophysical factors mediating morphogenetic signals in biological systems. These data originate from contexts including embryonic development, neoplasm, and regeneration.

  7. Probiotic lactobacilli inhibit early stages of Candida albicans biofilm development by reducing their growth, cell adhesion, and filamentation.

    PubMed

    Matsubara, Victor Haruo; Wang, Yi; Bandara, H M H N; Mayer, Marcia Pinto Alves; Samaranayake, Lakshman P

    2016-07-01

    We evaluated the inhibitory effects of the probiotic Lactobacillus species on different phases of Candida albicans biofilm development. Quantification of biofilm growth and ultrastructural analyses were performed on C. albicans biofilms treated with Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus acidophilus planktonic cell suspensions as well as their supernatants. Planktonic lactobacilli induced a significant reduction (p < 0.05) in the number of biofilm cells (25.5-61.8 %) depending on the probiotic strain and the biofilm phase. L. rhamnosus supernatants had no significant effect on the mature biofilm (p > 0.05), but significantly reduced the early stages of Candida biofilm formation (p < 0.01). Microscopic analyses revealed that L. rhamnosus suspensions reduced Candida hyphal differentiation, leading to a predominance of budding growth. All lactobacilli negatively impacted C. albicans yeast-to-hyphae differentiation and biofilm formation. The inhibitory effects of the probiotic Lactobacillus on C. albicans entailed both cell-cell interactions and secretion of exometabolites that may impact on pathogenic attributes associated with C. albicans colonization on host surfaces and yeast filamentation. This study clarifies, for the first time, the mechanics of how Lactobacillus species may antagonize C. albicans host colonization. Our data elucidate the inhibitory mechanisms that define the probiotic candicidal activity of lactobacilli, thus supporting their utility as an adjunctive therapeutic mode against mucosal candidal infections.

  8. The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

    PubMed Central

    Thevissen, Karin; de Mello Tavares, Patricia; Xu, Deming; Blankenship, Jill; Vandenbosch, Davy; Idkowiak-Baldys, Jolanta; Govaert, Gilmer; Bink, Anna; Rozental, Sonia; de Groot, Piet W.J.; Davis, Talya R.; Kumamoto, Carol A.; Vargas, Gabriele; Nimrichter, Leonardo; Coenye, Tom; Mitchell, Aaron; Roemer, Terry; Hannun, Yusuf A.; Cammue, Bruno P.A.

    2012-01-01

    Summary The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2,868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2-hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast-to-hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analyzed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24-ceramides in membranes of RsAFP2-treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation. PMID:22384976

  9. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    SciTech Connect

    Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

    1987-05-01

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of /sup 51/Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of /sup 51/Cr release from radiolabeled monolayers.

  10. Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development.

    PubMed

    Bandara, H M H N; K Cheung, B P; Watt, R M; Jin, L J; Samaranayake, L P

    2013-02-01

    Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P < 0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation.

  11. Hyaluronan in limb morphogenesis.

    PubMed

    Li, Yingcui; Toole, Bryan P; Dealy, Caroline N; Kosher, Robert A

    2007-05-15

    Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.

  12. Global Role of Cyclic AMP Signaling in pH-Dependent Responses in Candida albicans

    PubMed Central

    Hollomon, Jeffrey M.; Grahl, Nora; Willger, Sven D.; Koeppen, Katja

    2016-01-01

    ABSTRACT Candida albicans behaviors are affected by pH, an important environmental variable. Filamentous growth is a pH-responsive behavior, where alkaline conditions favor hyphal growth and acid conditions favor growth as yeast. We employed filamentous growth as a tool to study the impact of pH on the hyphal growth regulator Cyr1, and we report that downregulation of cyclic AMP (cAMP) signaling by acidic pH contributes to the inhibition of hyphal growth in minimal medium with GlcNAc. Ras1 and Cyr1 are generally required for efficient hyphal growth, and the effects of low pH on Ras1 proteolysis and GTP binding are consistent with diminished cAMP output. Active alleles of ras1 do not suppress the hyphal growth defect at low pH, while dibutyryl cAMP partially rescues filamentous growth at low pH in a cyr1 mutant. These observations are consistent with Ras1-independent downregulation of Cyr1 by low pH. We also report that extracellular pH leads to rapid and prolonged decreases in intracellular pH, and these changes may contribute to reduced cAMP signaling by reducing intracellular bicarbonate pools. Transcriptomics analyses found that the loss of Cyr1 at either acidic or neutral pH leads to increases in transcripts involved in carbohydrate catabolism and protein translation and glycosylation and decreases in transcripts involved in oxidative metabolism, fluconazole transport, metal transport, and biofilm formation. Other pathways were modulated in pH-dependent ways. Our findings indicate that cAMP has a global role in pH-dependent responses, and this effect is mediated, at least in part, through Cyr1 in a Ras1-independent fashion. IMPORTANCE Candida albicans is a human commensal and the causative agent of candidiasis, a potentially invasive and life-threatening infection. C. albicans experiences wide changes in pH during both benign commensalism (a common condition) and pathogenesis, and its morphology changes in response to this stimulus. Neutral pH is considered an

  13. Global Role of Cyclic AMP Signaling in pH-Dependent Responses in Candida albicans.

    PubMed

    Hollomon, Jeffrey M; Grahl, Nora; Willger, Sven D; Koeppen, Katja; Hogan, Deborah A

    2016-01-01

    Candida albicans behaviors are affected by pH, an important environmental variable. Filamentous growth is a pH-responsive behavior, where alkaline conditions favor hyphal growth and acid conditions favor growth as yeast. We employed filamentous growth as a tool to study the impact of pH on the hyphal growth regulator Cyr1, and we report that downregulation of cyclic AMP (cAMP) signaling by acidic pH contributes to the inhibition of hyphal growth in minimal medium with GlcNAc. Ras1 and Cyr1 are generally required for efficient hyphal growth, and the effects of low pH on Ras1 proteolysis and GTP binding are consistent with diminished cAMP output. Active alleles of ras1 do not suppress the hyphal growth defect at low pH, while dibutyryl cAMP partially rescues filamentous growth at low pH in a cyr1 mutant. These observations are consistent with Ras1-independent downregulation of Cyr1 by low pH. We also report that extracellular pH leads to rapid and prolonged decreases in intracellular pH, and these changes may contribute to reduced cAMP signaling by reducing intracellular bicarbonate pools. Transcriptomics analyses found that the loss of Cyr1 at either acidic or neutral pH leads to increases in transcripts involved in carbohydrate catabolism and protein translation and glycosylation and decreases in transcripts involved in oxidative metabolism, fluconazole transport, metal transport, and biofilm formation. Other pathways were modulated in pH-dependent ways. Our findings indicate that cAMP has a global role in pH-dependent responses, and this effect is mediated, at least in part, through Cyr1 in a Ras1-independent fashion. IMPORTANCECandida albicans is a human commensal and the causative agent of candidiasis, a potentially invasive and life-threatening infection. C. albicans experiences wide changes in pH during both benign commensalism (a common condition) and pathogenesis, and its morphology changes in response to this stimulus. Neutral pH is considered an activator

  14. Transcript profiling reveals rewiring of iron assimilation gene expression in Candida albicans and C. dubliniensis.

    PubMed

    Moran, Gary P

    2012-12-01

    Hyphal growth is repressed in Candida albicans and Candida dubliniensis by the transcription factor Nrg1. Transcript profiling of a C. dubliniensis NRG1 mutant identified a common group of 28 NRG1-repressed genes in both species, including the hypha-specific genes HWP1, ECE1 and the regulator of cell elongation UME6. Unexpectedly, C. dubliniensis NRG1 was required for wild-type levels of expression of 10 genes required for iron uptake including seven ferric reductases, SIT1, FTR1 and RBT5. However, at alkaline pH and during filamentous growth in 10% serum, most of these genes were highly induced in C. dubliniensis. Conversely, RBT5, PGA10, FRE10 and FRP1 did not exhibit induction during hyphal growth when NRG1 is downregulated, indicating that in C. dubliniensis NRG1 is also required for optimal expression of these genes in alkaline environments. In iron-depleted medium at pH 4.5, reduced growth of the NRG1 mutant relative to wild type was observed; however, growth was restored to wild-type levels or greater at pH 6.5, indicating that alkaline induction of iron assimilation gene expression could rescue this phenotype. These data indicate that transcriptional control of iron assimilation and pseudohypha formation has been separated in C. albicans, perhaps promoting growth in a wider range of niches.

  15. NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

    PubMed Central

    Brothers, Kimberly M.; Gratacap, Remi L.; Barker, Sarah E.; Newman, Zachary R.; Norum, Ashley; Wheeler, Robert T.

    2013-01-01

    Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis. PMID:24098114

  16. Virulence and pathogenicity of Candida albicans is enhanced in biofilms containing oral bacteria.

    PubMed

    Cavalcanti, Yuri Wanderley; Morse, Daniel James; da Silva, Wander José; Del-Bel-Cury, Altair Antoninha; Wei, Xiaoqing; Wilson, Melanie; Milward, Paul; Lewis, Michael; Bradshaw, David; Williams, David Wynne

    2015-01-01

    This study examined the influence of bacteria on the virulence and pathogenicity of candidal biofilms. Mature biofilms (Candida albicans-only, bacteria-only, C. albicans with bacteria) were generated on acrylic and either analysed directly, or used to infect a reconstituted human oral epithelium (RHOE). Analyses included Candida hyphae enumeration and assessment of Candida virulence gene expression. Lactate dehydrogenase (LDH) activity and Candida tissue invasion following biofilm infection of the RHOE were also measured. Candida hyphae were more prevalent (p < 0.05) in acrylic biofilms also containing bacteria, with genes encoding secreted aspartyl-proteinases (SAP4/SAP6) and hyphal-wall protein (HWP1) up-regulated (p < 0.05). Candida adhesin genes (ALS3/EPA1), SAP6 and HWP1 were up-regulated in mixed-species biofilm infections of RHOE. Multi-species infections exhibited higher hyphal proportions (p < 0.05), up-regulation of IL-18, higher LDH activity and tissue invasion. As the presence of bacteria in acrylic biofilms promoted Candida virulence, consideration should be given to the bacterial component when managing denture biofilm associated candidoses.

  17. Lipid synthesis during morphogenesis of Mucor racemosus.

    PubMed Central

    Ito, E T; Cihlar, R L; Inderlied, C B

    1982-01-01

    Lipid synthesis increases coordinately with protein and RNA synthesis during morphogenesis of Mucor racemosus. The lipid synthesis inhibitor cerulenin can completely block morphogenesis under conditions in which cell growth continues. An increase in phospholipid turnover may be an important correlate to morphogenesis of Mucor spp., especially the turnover of phosphotidyl inositol and phosphatidyl ethanolamine. The increase in ornithine decarboxylase, which occurs during morphogenesis, is inhibited by the addition of cerulenin. Images PMID:7130131

  18. Candida albicans Shed Msb2 and Host Mucins Affect the Candidacidal Activity of Salivary Hst 5

    PubMed Central

    Puri, Sumant; Friedman, Justin; Saraswat, Darpan; Kumar, Rohitashw; Li, Rui; Ruszaj, Donna; Edgerton, Mira

    2015-01-01

    Salivary Histatin 5 (Hst 5) is an antimicrobial peptide that exhibits potent antifungal activity towards Candida albicans, the causative agent of oral candidiasis. However, it exhibits limited activity in vivo, largely due to inactivation by salivary components of both host and pathogen origin. Proteins secreted by C. albicans during infection such as secreted aspartyl proteases (Saps) and shed mucin Msb2 can reduce Hst 5 activity; and human salivary mucins, while suggested to protect Hst 5 from proteolytic degradation, can entrap peptides into mucin gels, thereby reducing bioavailability. We show here that Sap6 that is secreted during hyphal growth reduces Hst 5 activity, most likely a result of proteolytic degradation of Hst 5 since this effect is abrogated with heat inactivated Sap 6. We further show that just like C. albicans shedding Msb2, mammalian mucins, fetuin and porcine gut mucin (that is related to salivary mucins), also reduce Hst 5 activity. However, we identify mucin-like protein-induced changes in C. albicans cell morphology and aggregation patterns, suggesting that the effect of such proteins on Hst 5 cannot be interpreted independently of their effect on yeast cells. PMID:26529023

  19. Regulation of cellulase expression, sporulation, and morphogenesis by velvet family proteins in Trichoderma reesei.

    PubMed

    Liu, Kuimei; Dong, Yanmei; Wang, Fangzhong; Jiang, Baojie; Wang, Mingyu; Fang, Xu

    2016-01-01

    Homologs of the velvet protein family are encoded by the ve1, vel2, and vel3 genes in Trichoderma reesei. To test their regulatory functions, the velvet protein-coding genes were disrupted, generating Δve1, Δvel2, and Δvel3 strains. The phenotypic features of these strains were examined to identify their functions in morphogenesis, sporulation, and cellulase expression. The three velvet-deficient strains produced more hyphal branches, indicating that velvet family proteins participate in the morphogenesis in T. reesei. Deletion of ve1 and vel3 did not affect biomass accumulation, while deletion of vel2 led to a significantly hampered growth when cellulose was used as the sole carbon source in the medium. The deletion of either ve1 or vel2 led to the sharp decrease of sporulation as well as a global downregulation of cellulase-coding genes. In contrast, although the expression of cellulase-coding genes of the ∆vel3 strain was downregulated in the dark, their expression in light condition was unaffected. Sporulation was hampered in the ∆vel3 strain. These results suggest that Ve1 and Vel2 play major roles, whereas Vel3 plays a minor role in sporulation, morphogenesis, and cellulase expression.

  20. In Vitro and In Vivo Activities of Pterostilbene against Candida albicans Biofilms

    PubMed Central

    Li, De-Dong; Zhao, Lan-Xue; Mylonakis, Eleftherios; Hu, Gan-Hai; Zou, Yong; Huang, Tong-Kun; Yan, Lan

    2014-01-01

    Pterostilbene (PTE) is a stilbene-derived phytoalexin that originates from several natural plant sources. In this study, we evaluated the activity of PTE against Candida albicans biofilms and explored the underlying mechanisms. In 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assays, biofilm biomass measurement, confocal laser scanning microscopy, and scanning electron microscopy, we found that ≤16 μg/ml PTE had a significant effect against C. albicans biofilms in vitro, while it had no fungicidal effect on planktonic C. albicans cells, which suggested a unique antibiofilm effect of PTE. Then we found that PTE could inhibit biofilm formation and destroy the maintenance of mature biofilms. At 4 μg/ml, PTE decreased cellular surface hydrophobicity (CSH) and suppressed hyphal formation. Gene expression microarrays and real-time reverse transcription-PCR showed that exposure of C. albicans to 16 μg/ml PTE altered the expression of genes that function in morphological transition, ergosterol biosynthesis, oxidoreductase activity, and cell surface and protein unfolding processes (heat shock proteins). Filamentation-related genes, especially those regulated by the Ras/cyclic AMP (cAMP) pathway, including ECE1, ALS3, HWP1, HGC1, and RAS1 itself, were downregulated upon PTE treatment, indicating that the antibiofilm effect of PTE was related to the Ras/cAMP pathway. Then, we found that the addition of exogenous cAMP reverted the PTE-induced filamentous growth defect. Finally, with a rat central venous catheter infection model, we confirmed the in vivo activity of PTE against C. albicans biofilms. Collectively, PTE had strong activities against C. albicans biofilms both in vitro and in vivo, and these activities were associated with the Ras/cAMP pathway. PMID:24514088

  1. The Functions of Mediator in Candida albicans Support a Role in Shaping Species-Specific Gene Expression

    PubMed Central

    Jelicic, Branka; Lo, Tricia L.; Beaurepaire, Cecile; Bantun, Farkad; Quenault, Tara; Boag, Peter R.; Ramm, Georg; Callaghan, Judy; Beilharz, Traude H.; Nantel, André; Peleg, Anton Y.; Traven, Ana

    2012-01-01

    The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species. PMID:22496666

  2. Mechanotransduction: getting morphogenesis down pat.

    PubMed

    Hardin, Jeff

    2011-05-10

    Embryonic morphogenesis requires the coordination of forces across multiple tissues and their associated extracellular matrices. A new study reports a mechanical feedback loop in the Caenorhabditis elegans embryo between muscle and epidermis that may provide a model for understanding how tissues coordinate morphogenetic events in the embryo.

  3. Chemotactic collapse and mesenchymal morphogenesis

    NASA Astrophysics Data System (ADS)

    Escudero, Carlos

    2005-08-01

    We study the effect of chemotactic signaling among mesenchymal cells. We show that the particular physiology of the mesenchymal cells allows one-dimensional collapse in contrast to the case of bacteria, and that the mesenchymal morphogenesis represents thus a more complex type of pattern formation than those found in bacterial colonies. We compare our theoretical predictions with recent in vitro experiments.

  4. Candida albicans commensalism in the gastrointestinal tract.

    PubMed

    Neville, B Anne; d'Enfert, Christophe; Bougnoux, Marie-Elisabeth

    2015-11-01

    Candida albicans is a polymorphic yeast species that often forms part of the commensal gastrointestinal mycobiota of healthy humans. It is also an important opportunistic pathogen. A tripartite interaction involving C. albicans, the resident microbiota and host immunity maintains C. albicans in its commensal form. The influence of each of these factors on C. albicans carriage is considered herein, with particular focus on the mycobiota and the approaches used to study it, models of gastrointestinal colonization by C. albicans, the C. albicans genes and phenotypes that are necessary for commensalism and the host factors that influence C. albicans carriage.

  5. Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans.

    PubMed

    Ketelaar, Tijs; Meijer, Harold J G; Spiekerman, Marjolein; Weide, Rob; Govers, Francine

    2012-12-01

    The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin

  6. Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum.

    PubMed

    Corral-Ramos, Cristina; Roca, M Gabriela; Di Pietro, Antonio; Roncero, M Isabel G; Ruiz-Roldán, Carmen

    2015-01-01

    In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.

  7. Mucosal biofilms of Candida albicans.

    PubMed

    Ganguly, Shantanu; Mitchell, Aaron P

    2011-08-01

    Biofilms are microbial communities that form on surfaces and are embedded in an extracellular matrix. C. albicans forms pathogenic mucosal biofilms that are evoked by changes in host immunity or mucosal ecology. Mucosal surfaces are inhabited by many microbial species; hence these biofilms are polymicrobial. Several recent studies have applied paradigms of biofilm analysis to study mucosal C. albicans infections. These studies reveal that the Bcr1 transcription factor is a master regulator of C. albicans biofilm formation under diverse conditions, though the most relevant Bcr1 target genes can vary with the biofilm niche. An important determinant of mucosal biofilm formation is the interaction with host defenses. Finally, studies of interactions between bacterial species and C. albicans provide insight into the communication mechanisms that endow polymicrobial biofilms with unique properties.

  8. Soft X-ray tomography of phenotypic switching and the cellular response to antifungal peptoids in Candida albicans

    PubMed Central

    Uchida, Maho; McDermott, Gerry; Wetzler, Modi; Le Gros, Mark A.; Myllys, Markko; Knoechel, Christian; Barron, Annelise E.; Larabell, Carolyn A.

    2009-01-01

    The opportunistic pathogen Candida albicans can undergo phenotypic switching between a benign, unicellular phenotype and an invasive, multicellular form that causes candidiasis. Increasingly, strains of Candida are becoming resistant to antifungal drugs, making the treatment of candidiasis difficult, especially in immunocompromised or critically ill patients. Consequently, there is a pressing need to develop new drugs that circumvent fungal drug-resistance mechanisms. In this work we used soft X-ray tomography to image the subcellular changes that occur as a consequence of both phenotypic switching and of treating C. albicans with antifungal peptoids, a class of candidate therapeutics unaffected by drug resistance mechanisms. Peptoid treatment suppressed formation of the pathogenic hyphal phenotype and resulted in striking changes in cell and organelle morphology, most dramatically in the nucleus and nucleolus, and in the number, size, and location of lipidic bodies. In particular, peptoid treatment was seen to cause the inclusion of lipidic bodies into the nucleus. PMID:19880740

  9. Ras1 and Ras2 play antagonistic roles in regulating cellular cAMP level, stationary-phase entry and stress response in Candida albicans.

    PubMed

    Zhu, Yong; Fang, Hao-Ming; Wang, Yan-Ming; Zeng, Gui-Sheng; Zheng, Xin-De; Wang, Yue

    2009-11-01

    The GTPase Ras1 activates the yeast-to-hypha transition in Candida albicans by activating cAMP synthesis. Here, we have characterized Ras2. Ras2 belongs to a group of atypical Ras proteins in some fungal species that share poor identity with other Ras GTPases with many variations in conserved motifs thought to be crucial for Ras-associated activities. We find that recombinant Ras2 is enzymatically as active as Ras1. However, only RAS1 can rescue the lethality of the Saccharomyces cerevisiae ras1 ras2 mutant, suggesting functional divergence of the two genes. ras2Delta is normal in hyphal growth, but deleting RAS2 in the ras1Delta background greatly aggravates the hyphal defect, indicating that Ras2 also has a role in hyphal development. Strikingly, while RAS1 deletion causes a approximately 20-fold decrease in cellular cAMP, further deletion of RAS2 restores it to approximately 30% of the wild-type level. Consistently, while the ras1Delta mutant enters the stationary phase prematurely, the double mutant does so normally. Moreover, ras1Delta cells exhibit increased resistance to H(2)O(2) and higher sensitivity to the heavy metal Co(2+), whereas ras2Delta cells show the opposite phenotypes. Together, our data reveal a novel regulatory mechanism by which two antagonizing Ras GTPases balance each other in regulating multiple cellular processes in C. albicans.

  10. Vegetative hyphal fusion and subsequent nuclear behavior in Epichloë grass endophytes.

    PubMed

    Shoji, Jun-Ya; Charlton, Nikki D; Yi, Mihwa; Young, Carolyn A; Craven, Kelly D

    2015-01-01

    Epichloë species (including the former genus Neotyphodium) are fungal symbionts of many agronomically important forage grasses, and provide their grass hosts with protection from a wide range of biotic and abiotic stresses. Epichloë species include many interspecific hybrids with allodiploid-like genomes, which may provide the potential for combined traits or recombination to generate new traits. Though circumstantial evidence suggests that such interspecific hybrids might have arisen from nuclear fusion events following vegetative hyphal fusion between different Epichloë strains, this hypothesis has not been addressed empirically. Here, we investigated vegetative hyphal fusion and subsequent nuclear behavior in Epichloë species. A majority of Epichloë strains, especially those having a sexual stage, underwent self vegetative hyphal fusion. Vegetative fusion also occurred between two hyphae from different Epichloë strains. Though Epichloë spp. are uninucleate fungi, hyphal fusion resulted in two nuclei stably sharing the same cytoplasm, which might ultimately lead to nuclear fusion. In addition, protoplast fusion experiments gave rise to uninucleate putative hybrids, which apparently had two markers, one from each parent within the same nucleus. These results are consistent with the notion that interspecific hybrids arise from vegetative hyphal fusion. However, we also discuss additional factors, such as post-hybridization selection, that may be important to explain the recognized prevalence of hybrids in Epichloë species.

  11. Synergic effect of combination of glycyrol and fluconazole against experimental cutaneous candidiasis due to Candida albicans.

    PubMed

    Rhew, Zheong-Imm; Han, Yongmoon

    2016-10-01

    In this study, we investigated the anti-fungal activity of glycyrol, a coumarine isolated from licorice (Glycyrrhizae Radix), in a murine model of cutaneous candidiasis caused by Candida albicans. Compared to the infected sites, located on the mice's back, of the untreated control mice, the infected sites treated with glycyrol had reduced CFU (colony forming unit) values up to 60 and 85.5 % at 20 and 40 μg/mouse of glycyrol, respectively (P < 0.01). The antifungal activity of glycyrol was synergistically increased when glycyrol (10 μg/mouse) was combined with fluconazole (10 μg/mouse), demonstrating that the combination therapy is approximately 4 times more effective than fluconazole alone at 20 μg/mouse (P < 0.01). Additionally, the combination activity was 1.65 times greater than the antifungal activity of fluconazole alone at 40 μg/mouse (P < 0.05). In seeking glycyrol's antifungal mechanism, we determined that glycyrol inhibited hyphal induction and cell wall adherence of C. albicans. Thus, it is very likely that, by damaging the cell wall, glycyrol helps fluconazole invade C. albicans more readily and attack fluconazole's target in the fungus membrane. In summary, our data indicate that glycyrol may contribute to the development of a novel agent that possesses antifungal activity against cutaneous candidiasis.

  12. Expression of firefly luciferase in Candida albicans and its use in the selection of stable transformants.

    PubMed

    Doyle, Timothy C; Nawotka, Kevin A; Purchio, Anthony F; Akin, Ali R; Francis, Kevin P; Contag, Pamela R

    2006-02-01

    The infectious yeast Candida albicans is a model organism for understanding the mechanisms of fungal pathogenicity. We describe the functional expression of the firefly luciferase gene, a reporter commonly used to tag genes in many other cellular systems. Due to a non-standard codon usage by this yeast, the CUG codons were first mutated to UUG to allow functional expression. When integrated into the chromosome of C. albicans with a strong constitutive promoter, cells bioluminesce when provided with luciferin substrate in their media. When fused to the inducible promoter from the HWP1 gene, expression and bioluminescence was only detected in cultures conditioning hyphal growth. We further used the luciferase gene as a selection to isolate transformed cell lines from clinical isolates of C. albicans, using a high-density screening strategy that purifies transformed colonies by virtue of light emission. This strategy requires no drug or auxotrophic selectable marker, and we were thus able to generate stable transformants of clinical isolates that are identical to the parental strain in all aspects tested, other than their bioluminescence. The firefly luciferase gene can, therefore, be used as a sensitive reporter to analyze gene function both in laboratory and clinical isolates of this medically important yeast.

  13. A Phenotypic Profile of the Candida albicans Regulatory Network

    PubMed Central

    Homann, Oliver R.; Dea, Jeanselle; Noble, Suzanne M.; Johnson, Alexander D.

    2009-01-01

    Candida albicans is a normal resident of the gastrointestinal tract and also the most prevalent fungal pathogen of humans. It last shared a common ancestor with the model yeast Saccharomyces cerevisiae over 300 million years ago. We describe a collection of 143 genetically matched strains of C. albicans, each of which has been deleted for a specific transcriptional regulator. This collection represents a large fraction of the non-essential transcription circuitry. A phenotypic profile for each mutant was developed using a screen of 55 growth conditions. The results identify the biological roles of many individual transcriptional regulators; for many, this work represents the first description of their functions. For example, a quarter of the strains showed altered colony formation, a phenotype reflecting transitions among yeast, pseudohyphal, and hyphal cell forms. These transitions, which have been closely linked to pathogenesis, have been extensively studied, yet our work nearly doubles the number of transcriptional regulators known to influence them. As a second example, nearly a quarter of the knockout strains affected sensitivity to commonly used antifungal drugs; although a few transcriptional regulators have previously been implicated in susceptibility to these drugs, our work indicates many additional mechanisms of sensitivity and resistance. Finally, our results inform how transcriptional networks evolve. Comparison with the existing S. cerevisiae data (supplemented by additional S. cerevisiae experiments reported here) allows the first systematic analysis of phenotypic conservation by orthologous transcriptional regulators over a large evolutionary distance. We find that, despite the many specific wiring changes documented between these species, the general phenotypes of orthologous transcriptional regulator knockouts are largely conserved. These observations support the idea that many wiring changes affect the detailed architecture of the circuit, but

  14. Effects of Aeration of Sawdust Cultivation Bags on Hyphal Growth of Lentinula edodes.

    PubMed

    Lee, Hwa-Yong; Ham, Eun-Ju; Yoo, Young-Jin; Kim, Eui-Sung; Shim, Kyu-Kwang; Kim, Myung-Kon; Koo, Chang-Duck

    2012-09-01

    The effects of aeration through lid filters on the hyphal growth of Lentinula edodes (oak mushroom) in sawdust cultivation bags were investigated. The aeration treatment levels were traditional 27 mm hole cotton plugs, cotton balls and combinations of seven hole sizes × two hole positions (up and under) in the lids covering plastic bags containing 1.4 kg sawdust medium at 63% moisture that had been autoclaved for one hour and inoculated with sawdust spawn of L. edodes strain 921. Aeration treatment effects were measured based on the CO(2) concentration at the 15th wk, as well as the hyphal growth rate and degree of weight loss of bags every 14 days for 15 wk. In bags with traditional cotton plugs, the CO(2) concentration was 3.8 ± 1.3%, daily mean hyphal growth was 2.3 ± 0.6 mm and daily mean weight loss was 0.84 ± 0.26 g. In the bags with 15 mm diameter holes, the CO(2) concentration was 6.0 ± 1.6%, daily hyphal growth was 2.8 ± 0.2 mm and daily weight loss was 0.86 ± 0.4 g. The bags with 15 mm holes had a higher CO(2) concentration and lower water loss than bags with other hole sizes, but the hyphal growth was not significantly different from that of other bags. The weight loss of bags increased proportionally relative to the lid hole sizes. Taken together, these results indicate that traditional cotton plugs are economically efficient, but 15 mm hole lids are the most efficient at maintaining hyphal growth and controlling water loss while allowing CO(2) emissions.

  15. New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Candida albicans.

    PubMed

    Sadowska, Beata; Budzyńska, Aleksandra; Więckowska-Szakiel, Marzena; Paszkiewicz, Małgorzata; Stochmal, Anna; Moniuszko-Szajwaj, Barbara; Kowalczyk, Mariusz; Różalska, Barbara

    2014-08-01

    The antifungal activity of the saponin-rich fractions (SFs) from Medicago sativa (aerial parts and roots) and Saponaria officinalis (used as a well-known source of plant saponins) against Candida albicans reference and clinical strains, their yeast-to-hyphal conversion, adhesion, and biofilm formation was investigated. Direct fungicidal/fungistatic properties of the tested phytochemicals used alone, as well as their synergy with azoles (probably resulting from yeast cell wall instability) were demonstrated. Here, to the best of our knowledge, we report for the first time the ability of saponin-rich extracts of M. sativa and S. officinalis to inhibit C. albicans germ tube formation, limit hyphal growth, reduce yeast adherence and biofilm formation, and eradicate mature (24 h) Candida biofilm. Moreover, M. sativa SFs (mainly obtained from aerial parts), in the range of concentrations which were active modulators of Candida virulence factors, exhibited low cytotoxicity against the mouse fibroblast line L929. These properties seem to be very promising in the context of using plant-derived SFs as potential novel antifungal therapeutics supporting classic drugs or as ingredients of disinfectants.

  16. Molecular mechanisms of dendrite morphogenesis

    PubMed Central

    Arikkath, Jyothi

    2012-01-01

    Dendrites are key integrators of synaptic information in neurons and play vital roles in neuronal plasticity. Hence, it is necessary that dendrite arborization is precisely controlled and coordinated with synaptic activity to ensure appropriate functional neural network integrity. In the past several years, it has become increasingly clear that several cell intrinsic and extrinsic mechanisms contribute to dendritic arborization. In this review, we will discuss some of the molecular mechanisms that regulate dendrite morphogenesis, particularly in cortical and hippocampal pyramidal neurons and some of the implications of aberrant dendritic morphology for human disease. Finally, we will discuss the current challenges and future directions in the field. PMID:23293584

  17. The Transcription Factor Con7-1 Is a Master Regulator of Morphogenesis and Virulence in Fusarium oxysporum.

    PubMed

    Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; González-Reyes, José Antonio; Roncero, M Isabel G

    2015-01-01

    Previous studies have demonstrated the essential role of morphogenetic regulation in Fusarium oxysporum pathogenesis, including processes such as cell-wall biogenesis, cell division, and differentiation of infection-like structures. We identified three F. oxysporum genes encoding predicted transcription factors showing significant identities to Magnaporthe oryzae Con7p, Con7-1, plus two identical copies of Con7-2. Targeted deletion of con7-1 produced nonpathogenic mutants with altered morphogenesis, including defects in cell wall structure, polar growth, hyphal branching, and conidiation. By contrast, simultaneous inactivation of both con7-2 copies caused no detectable defects in the resulting mutants. Comparative microarray-based gene expression analysis indicated that Con7-1 modulates the expression of a large number of genes involved in different biological functions, including host-pathogen interactions, morphogenesis and development, signal perception and transduction, transcriptional regulation, and primary and secondary metabolism. Taken together, our results point to Con7-1 as general regulator of morphogenesis and virulence in F. oxysporum.

  18. Intestinal Cell Tight Junctions Limit Invasion of Candida albicans through Active Penetration and Endocytosis in the Early Stages of the Interaction of the Fungus with the Intestinal Barrier

    PubMed Central

    Bon, Fabienne; L’Ollivier, Coralie; Laue, Michael; Holland, Gudrun; Bonnin, Alain; Dalle, Frederic

    2016-01-01

    C. albicans is a commensal yeast of the mucous membranes in healthy humans that can also cause disseminated candidiasis, mainly originating from the digestive tract, in vulnerable patients. It is necessary to understand the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In this study, we investigated the kinetics of tight junction (TJ) formation in parallel with the invasion of C. albicans into the Caco-2 intestinal cell line. Using invasiveness assays on Caco-2 cells displaying pharmacologically altered TJ (i.e. differentiated epithelial cells treated with EGTA or patulin), we were able to demonstrate that TJ protect enterocytes against invasion of C. albicans. Moreover, treatment with a pharmacological inhibitor of endocytosis decreased invasion of the fungus into Caco-2 cells displaying altered TJ, suggesting that facilitating access of the yeast to the basolateral side of intestinal cells promotes endocytosis of C. albicans in its hyphal form. These data were supported by SEM observations of differentiated Caco-2 cells displaying altered TJ, which highlighted membrane protrusions engulfing C. albicans hyphae. We furthermore demonstrated that Als3, a hypha-specific C. albicans invasin, facilitates internalization of the fungus by active penetration and induced endocytosis by differentiated Caco-2 cells displaying altered TJ. However, our observations failed to demonstrate binding of Als3 to E-cadherin as the trigger mechanism of endocytosis of C. albicans into differentiated Caco-2 cells displaying altered TJ. PMID:26933885

  19. Control of Candida albicans morphology and pathogenicity by post-transcriptional mechanisms.

    PubMed

    Kadosh, David

    2016-11-01

    Candida albicans is a major human fungal pathogen responsible for both systemic and mucosal infections in a wide variety of immunocompromised individuals. Because the ability of C. albicans to undergo a reversible morphological transition from yeast to filaments is important for virulence, significant research efforts have focused on mechanisms that control this transition. While transcriptional and post-translational mechanisms have been well-studied, considerably less is known about the role of post-transcriptional mechanisms. However, in recent years several discoveries have begun to shed light on this important, but understudied, area. Here, I will review a variety of post-transcriptional mechanisms that have recently been shown to control C. albicans morphology, virulence and/or virulence-related processes, including those involving alternative transcript localization, mRNA stability and translation. I will also discuss the role that these mechanisms play in other pathogens as well as the potential they may hold to serve as targets for new antifungal strategies. Ultimately, gaining a better understanding of C. albicans post-transcriptional mechanisms will significantly improve our knowledge of how morphogenesis and virulence are controlled in fungal pathogens and open new avenues for the development of novel and more effective antifungals.

  20. Candida albicans VPS11 is required for vacuole biogenesis and germ tube formation.

    PubMed

    Palmer, Glen E; Cashmore, Annette; Sturtevant, Joy

    2003-06-01

    The Candida albicans vacuole has previously been observed to undergo rapid expansion during the emergence of a germ tube from a yeast cell, to occupy the majority of the parent yeast cell. Furthermore, the yeast-to-hypha switch has been implicated in the virulence of this organism. The class C vps (vacuolar protein sorting) mutants of Saccharomyces cerevisiae are defective in multiple protein delivery pathways to the vacuole and prevacuole compartment. In this study C. albicans homologues of the S. cerevisiae class C VPS genes have been identified. Deletion of a C. albicans VPS11 homologue resulted in a number of phenotypes that closely resemble those of the class C vps mutants of S. cerevisiae, including the absence of a vacuolar compartment. The C. albicans vps11Delta mutant also had much-reduced secreted lipase and aspartyl protease activities. Furthermore, vps11Delta strains were defective in yeast-hypha morphogenesis. Upon serum induction of filamentous growth, mutants showed delayed emergence of germ tubes, had a reduced apical extension rate compared to those of control strains, and were unable to form mature hyphae. These results suggest that Vps11p-mediated trafficking steps are necessary to support the rapid emergence and extension of the germ tube from the parent yeast cell.

  1. DNA methylation regulates phenotype-dependent transcriptional activity in Candida albicans

    PubMed Central

    Mishra, Prashant K.; Baum, Mary; Carbon, John

    2011-01-01

    DNA methylation is a common epigenetic signaling mechanism associated with silencing of repeated DNA and transcriptional regulation in eukaryotes. Here we report that DNA methylation in the human fungal pathogen Candida albicans is primarily localized within structural genes and modulates transcriptional activity. Major repeat sequences and multigene families are largely free of DNA methylation. Among the genes subject to DNA methylation are those associated with dimorphic transition between yeast and hyphal forms, switching between white and opaque cells, and iron metabolism. Transcriptionally repressed methylated loci showed increased frequency of C-to-T transitions during asexual growth, an evolutionarily stable pattern of repression associated mutation that could bring about genetic alterations under changing environmental or host conditions. Dynamic differential DNA methylation of structural genes may be one factor contributing to morphological plasticity that is cued by nutrition and host interaction. PMID:21730141

  2. Linking cellular actin status with cAMP signaling in Candida albicans.

    PubMed

    Wang, Yue; Zou, Hao; Fang, Hao-Ming; Zhu, Yong

    2010-01-01

    The fungal pathogen Candida albicans has a remarkable ability to switch growth forms. Particularly, the yeast-to-hyphae switch is closely linked with its virulence. A range of chemicals and conditions can promote hyphal growth including serum, peptidoglycan, CO2, neutral pH, and elevated temperature. All these signals act essentially through the adenylyl cyclase Cyr1 that synthesizes cAMP. Cells lacking Cyr1 are completely defective in hyphal growth. Recently, cellular actin status is found to influence cAMP synthesis. However, how Cyr1 senses and processes multiple external and internal signals to produce a contextually proper level of cAMP remains unclear. We hypothesized that Cyr1 itself possesses multiple sensors for different signals and achieves signal integration through a combined allosteric effect on the catalytic center. To test this hypothesis, we affinity-purified a Cyr1-containing complex and found that it could enhance cAMP synthesis upon treatment with serum, peptidoglycan or CO2 in vitro. The data indicate that the complex is an essentially intact sensor/effector apparatus for cAMP synthesis. The complex contains two more subunits, the cyclase-associated protein Cap1 and G-actin. We discovered that G-actin plays a regulatory role, rendering cAMP synthesis responsive to actin dynamics. These findings shed new lights on the mechanisms that regulate cAMP-mediated responses in fungi.

  3. Candida albicans: adapting to succeed.

    PubMed

    Kadosh, David; Lopez-Ribot, Jose L

    2013-11-13

    In this issue of Cell Host & Microbe, Lu et al. (2013) report on the redundancy of signaling pathways controlling Candida albicans filamentation and pathogenicity. In the process, they provide important insight into how this normal commensal of humans adapts to different host microenvironments to become a highly successful opportunistic pathogen.

  4. Encapsulation of Antifungals in Micelles Protects Candida albicans during Gall-Bladder Infection

    PubMed Central

    Hsieh, Shih-Hung; Brunke, Sascha; Brock, Matthias

    2017-01-01

    Candida albicans is a dimorphic fungus that colonizes human mucosal surfaces with the potential to cause life-threatening invasive candidiasis. Studies on systemic candidiasis in a murine infection model using in vivo real-time bioluminescence imaging revealed persistence of C. albicans in the gall bladder under antifungal therapy. Preliminary analyses showed that bile conferred resistance against a wide variety of antifungals enabling survival in this cryptic host niche. Here, bile and its components were studied for their ability to reduce antifungal efficacy in order to elucidate the underlying mechanism of protection. While unconjugated bile salts were toxic to C. albicans, taurine, or glycine conjugated bile salts were well tolerated and protective against caspofungin and amphotericin B when exceeding their critical micellar concentration. Microarray experiments indicated that upregulation of genes generally known to mediate antifungal protection is not involved in the protection process. In contrast, rhodamine 6G and crystal violet in- and efflux experiments indicated encapsulation of antifungals in micelles, thereby reducing their bioavailability. Furthermore, farnesol sensing was abolished in the presence of conjugated bile salts trapping C. albicans cells in the hyphal morphology. This suggests that bioavailability of amphiphilic and hydrophobic compounds is reduced in the presence of bile. In contrast, small and hydrophilic molecules, such as cycloheximide, flucytosine, or sodium azide kept their antifungal properties. We therefore conclude that treatment of gall bladder and bile duct infections is hampered by the ability of bile salts to encapsulate antifungals in micelles. As a consequence, treatment of gall bladder or bile duct infections should favor the use of small hydrophilic drugs that are not solubilised in micelles. PMID:28203228

  5. A Novel Small Molecule Methyltransferase is Important for Virulence in Candida albicans

    PubMed Central

    Lissina, Elena; Weiss, David; Young, Brian; Rella, Antonella; Cheung-Ong, Kahlin; Del Poeta, Maurizio; Clarke, Steven G.; Giaever, Guri; Nislow, Corey

    2014-01-01

    Candida albicans is an opportunistic pathogen capable of causing life-threatening infections in immunocompromised individuals. Despite its significant health impact, our understanding of C. albicans pathogenicity is limited, particularly at the molecular level. One of the largely understudied enzyme families in C. albicans is small molecule AdoMet-dependent methyltransferases (smMTases), which are important for maintenance of cellular homeostasis by clearing toxic chemicals, generating novel cellular intermediates and regulating intra- and interspecies interactions. Putative smMTase orf19.633 has little homology to any known protein and was previously identified based on its ability to functionally complement a baker’s yeast crg1 mutant in response to protein phosphatase inhibitor cantharidin. In this study, we demonstrated that C. albicans Crg1 (CaCrg1) is a bona fide smMTase that interacts with the toxin in vitro and in vivo. We report that CaCrg1 is important for virulence-related processes such as adhesion, hyphal elongation and membrane trafficking in response to this toxin. Using biochemical and genetic analysis we also found that CaCrg1 plays a role in complex sphingolipid pathway: it binds to exogenous short-chain ceramides in vitro, it interacts genetically with genes of glucosylceramide pathway and the deletion of CaCRG1 leads to significant changes in the abundance of phytoceramides. Finally we found that this novel lipid-related smMTase is required for virulence in the waxmoth Galleria mellonella, a model of infection. PMID:24083538

  6. Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

    PubMed

    Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

    2015-07-01

    Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

  7. Convergent Regulation of Candida albicans Aft2 and Czf1 in Invasive and Opaque Filamentation.

    PubMed

    Xu, Ning; Dong, Yi-Jie; Yu, Qi-Lin; Zhang, Bing; Zhang, Meng; Jia, Chang; Chen, Yu-Lu; Zhang, Biao; Xing, Lai-Jun; Li, Ming-Chun

    2015-09-01

    Candida albicans is the most common fungal pathogen of mucosal infections and invasive diseases in immuno-compromised humans. The abilities of yeast-hyphal growth and white-opaque switching affect C. albicans physiology and virulence. Here, we showed that C. albicans Aft2 regulator was required for embedded filamentous growth and opaque cell-type formation. Under low-temperature matrix embedded conditions, Aft2 functioned downstream of Czf1-mediated pathway and was required for invasive filamentation. Moreover, deletion of AFT2 significantly reduced opaque cell-type formation under N-acetylglucosamine (GlcNAc) inducing conditions. Ectopic expression of CZF1 slightly increased the white-opaque switching frequency in the aft2Δ/Δ mutant, but did not completely restore to wild-type levels, suggesting that Czf1 at least partially bypassed the essential requirement for Aft2 in response to opaque-inducing cues. In addition, multiple environmental cues altered AFT2 mRNA and protein levels, such as low temperature, physical environment and GlcNAc. Although the absence of Czf1 or Efg1 also increased the expression level of AFT2 gene, deletion of CZF1 remarkably reduced the stability of Aft2 protein. Furthermore, C. albicans Aft2 physically interacted with Czf1 under all tested conditions, whereas the interaction between Aft2 and Efg1 was barely detectable under embedded conditions, supporting the hypothesis that Aft2, together with Czf1, contributed to activate filamentous growth by antagonizing Efg1-mediated repression under matrix-embedded conditions.

  8. Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

    PubMed Central

    Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha

    2015-01-01

    Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis. PMID:25870228

  9. Beyond cell proliferation in avian facial morphogenesis

    PubMed Central

    Linde-Medina, Marta; Hallgrímsson, Benedikt; Marcucio, Ralph

    2016-01-01

    The upper jaw in vertebrates forms from several prominences that arise around the stomodeum or primitive mouth. These prominences undergo coordinated growth and morphogenesis to fuse and form the face. Undirected, regionalized cell proliferation is thought to be the driving force behind the morphogenesis of the facial prominences. However, recent findings suggest that directed cell behaviors in the mesenchyme (e.g., directed cell division, directed cell movement, convergent extension) might be required for successful face formation. Here we discuss the evidence for this view and how directed behaviors may interact with the basement membrane to regulate morphogenesis of the facial region. We believe that future research in these largely unexplored areas could significantly impact our understanding of facial morphogenesis. PMID:26637960

  10. Cell-intrinsic drivers of dendrite morphogenesis.

    PubMed

    Puram, Sidharth V; Bonni, Azad

    2013-12-01

    The proper formation and morphogenesis of dendrites is fundamental to the establishment of neural circuits in the brain. Following cell cycle exit and migration, neurons undergo organized stages of dendrite morphogenesis, which include dendritic arbor growth and elaboration followed by retraction and pruning. Although these developmental stages were characterized over a century ago, molecular regulators of dendrite morphogenesis have only recently been defined. In particular, studies in Drosophila and mammalian neurons have identified numerous cell-intrinsic drivers of dendrite morphogenesis that include transcriptional regulators, cytoskeletal and motor proteins, secretory and endocytic pathways, cell cycle-regulated ubiquitin ligases, and components of other signaling cascades. Here, we review cell-intrinsic drivers of dendrite patterning and discuss how the characterization of such crucial regulators advances our understanding of normal brain development and pathogenesis of diverse cognitive disorders.

  11. Candida albicans ISW2 Regulates Chlamydospore Suspensor Cell Formation and Virulence In Vivo in a Mouse Model of Disseminated Candidiasis

    PubMed Central

    Lionakis, Michail S.; Nickerson, Kenneth W.

    2016-01-01

    Formation of chlamydospores by Candida albicans was an established medical diagnostic test to confirm candidiasis before the molecular era. However, the functional role and pathological relevance of this in vitro morphological transition to pathogenesis in vivo remain unclear. We compared the physical properties of in vitro-induced chlamydospores with those of large C. albicans cells purified by density gradient centrifugation from Candida-infected mouse kidneys. The morphological and physical properties of these cells in kidneys of mice infected intravenously with wild type C. albicans confirmed that chlamydospores can form in infected kidneys. A previously reported chlamydospore-null Δisw2/Δisw2 mutant was used to investigate its role in virulence and chlamydospore induction. Virulence of the Δisw2/Δisw2 mutant strain was reduced 3.4-fold compared to wild type C. albicans or the ISW2 reconstituted strain. Altered host inflammatory reactions to the null mutant further indicate that ISW2 is a virulence factor in C. albicans. ISW2 deletion abolished chlamydospore formation within infected mouse kidneys, whereas the reconstituted strain restored chlamydospore formation in kidneys. Under chlamydospore inducing conditions in vitro, deletion of ISW2 significantly delayed chlamydospore formation, and those late induced chlamydospores lacked associated suspensor cells while attaching laterally to hyphae via novel spore-hypha septa. Our findings establish the induction of chlamydospores by C. albicans during mouse kidney colonization. Our results indicate that ISW2 is not strictly required for chlamydospores formation but is necessary for suspensor cell formation. The importance of ISW2 in chlamydospore morphogenesis and virulence may lead to additional insights into morphological differentiation and pathogenesis of C. albicans in the host microenvironment. PMID:27727302

  12. Azole resistance in Candida albicans.

    PubMed

    Smith, K J; Warnock, D W; Kennedy, C T; Johnson, E M; Hopwood, V; Van Cutsem, J; Vanden Bossche, H

    1986-04-01

    An isolate of Candida albicans from a patient with chronic mucocutaneous candidosis who relapsed during ketoconazole treatment was compared with a number of other azole-sensitive and azole-resistant isolates by tests in vitro and in three animal models of vaginal or disseminated infection. In-vitro tests indicated that the isolate was cross-resistant to all imidazole and triazole antifungals tested. In the animal models, treatment with miconazole, ketoconazole, itraconazole or fluconazole failed to influence the infection.

  13. Unisexual reproduction enhances fungal competitiveness by promoting habitat exploration via hyphal growth and sporulation.

    PubMed

    Phadke, Sujal S; Feretzaki, Marianna; Heitman, Joseph

    2013-08-01

    Unisexual reproduction is a novel homothallic sexual cycle recently discovered in both ascomycetous and basidiomycetous pathogenic fungi. It is a form of selfing that induces the yeast-to-hyphal dimorphic transition in isolates of the α mating type of the human fungal pathogen Cryptococcus neoformans. Unisexual reproduction may benefit the pathogen by facilitating sexual reproduction in the absence of the opposite a mating type and by generating infectious propagules called basidiospores. Here, we report an independent potential selective advantage of unisexual reproduction beyond genetic exchange and recombination. We competed a wild-type strain capable of undergoing unisexual reproduction with mutants defective in this developmental pathway and found that unisexual reproduction provides a considerable dispersal advantage through hyphal growth and sporulation. Our results show that unisexual reproduction may serve to facilitate access to both nutrients and potential mating partners and may provide a means to maintain the capacity for dimorphic transitions in the environment.

  14. Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A.

    PubMed

    Xie, Jinglin L; O'Meara, Teresa R; Polvi, Elizabeth J; Robbins, Nicole; Cowen, Leah E

    2017-01-01

    Protein kinases are key regulators of signal transduction pathways that participate in diverse cellular processes. In fungal pathogens, kinases regulate signaling pathways that govern drug resistance, stress adaptation, and pathogenesis. The impact of kinases on the fungal regulatory circuitry has recently garnered considerable attention in the opportunistic fungal pathogen Candida albicans, which is a leading cause of human morbidity and mortality. Complex regulatory circuitry governs the C. albicans morphogenetic transition between yeast and filamentous growth, which is a key virulence trait. Here, we report that staurosporine, a promiscuous kinase inhibitor that abrogates fungal drug resistance, also influences C. albicans morphogenesis by inducing filamentation in the absence of any other inducing cue. We further establish that staurosporine exerts its effect via the adenylyl cyclase Cyr1 and the cyclic AMP (cAMP)-dependent protein kinase A (PKA). Strikingly, filamentation induced by staurosporine does not require the known upstream regulators of Cyr1, Ras1 or Pkc1, or effectors downstream of PKA, including Efg1. We further demonstrate that Cyr1 is capable of activating PKA to enable filamentation in response to staurosporine through a mechanism that does not require degradation of the transcriptional repressor Nrg1. We establish that staurosporine-induced filamentation is accompanied by a defect in septin ring formation, implicating cell cycle kinases as potential staurosporine targets underpinning this cellular response. Thus, we establish staurosporine as a chemical probe to elucidate the architecture of cellular signaling governing fungal morphogenesis and highlight the existence of novel circuitry through which the Cyr1 and PKA govern a key virulence trait. IMPORTANCE The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream

  15. Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A

    PubMed Central

    Xie, Jinglin L.; O’Meara, Teresa R.; Polvi, Elizabeth J.; Robbins, Nicole

    2017-01-01

    ABSTRACT Protein kinases are key regulators of signal transduction pathways that participate in diverse cellular processes. In fungal pathogens, kinases regulate signaling pathways that govern drug resistance, stress adaptation, and pathogenesis. The impact of kinases on the fungal regulatory circuitry has recently garnered considerable attention in the opportunistic fungal pathogen Candida albicans, which is a leading cause of human morbidity and mortality. Complex regulatory circuitry governs the C. albicans morphogenetic transition between yeast and filamentous growth, which is a key virulence trait. Here, we report that staurosporine, a promiscuous kinase inhibitor that abrogates fungal drug resistance, also influences C. albicans morphogenesis by inducing filamentation in the absence of any other inducing cue. We further establish that staurosporine exerts its effect via the adenylyl cyclase Cyr1 and the cyclic AMP (cAMP)-dependent protein kinase A (PKA). Strikingly, filamentation induced by staurosporine does not require the known upstream regulators of Cyr1, Ras1 or Pkc1, or effectors downstream of PKA, including Efg1. We further demonstrate that Cyr1 is capable of activating PKA to enable filamentation in response to staurosporine through a mechanism that does not require degradation of the transcriptional repressor Nrg1. We establish that staurosporine-induced filamentation is accompanied by a defect in septin ring formation, implicating cell cycle kinases as potential staurosporine targets underpinning this cellular response. Thus, we establish staurosporine as a chemical probe to elucidate the architecture of cellular signaling governing fungal morphogenesis and highlight the existence of novel circuitry through which the Cyr1 and PKA govern a key virulence trait. IMPORTANCE The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream

  16. A microscopy study of hyphal growth of Penicillium rubens on gypsum under dynamic humidity conditions.

    PubMed

    van Laarhoven, Karel A; Huinink, Hendrik P; Adan, Olaf C G

    2016-05-01

    To remediate indoor fungal growth, understanding the moisture relations of common indoor fungi is crucial. Indoor moisture conditions are commonly quantified by the relative humidity (RH). RH is a major determinant of the availability of water in porous indoor surfaces that fungi grow on. The influence of steady-state RH on growth is well understood. Typically, however, the indoor RH constantly changes so that fungi have to endure frequent periods of alternating low and high RH. Knowledge of how common indoor fungi survive and are affected by the low-RH periods is limited. In particular, the specific effects of a drop in RH on the growth of the mycelium remain unclear. In this work, video microscopy was used to monitor hyphal growth of Penicillium rubens on gypsum substrates under controlled dynamic humidity conditions. The effect of a single period of low RH (RH = 50-90%) interrupting favourable conditions (RH = 97%) was tested. It was found that hyphal tips ceased to extend when exposed to any tested decrease in RH. However, new hyphal growth always emerges, seemingly from the old mycelium, suggesting that this indoor fungus does not rely only on conidia to survive the humidity patterns considered. These findings are a fundamental step in unravelling the effect of RH on indoor fungal growth.

  17. Candida albicans Mycofilms Support Staphylococcus aureus Colonization and Enhances Miconazole Resistance in Dual-Species Interactions

    PubMed Central

    Kean, Ryan; Rajendran, Ranjith; Haggarty, Jennifer; Townsend, Eleanor M.; Short, Bryn; Burgess, Karl E.; Lang, Sue; Millington, Owain; Mackay, William G.; Williams, Craig; Ramage, Gordon

    2017-01-01

    Polymicrobial inter-kingdom biofilm infections represent a clinical management conundrum. The presence of co-isolation of bacteria and fungi complicates the ability to routinely administer single antimicrobial regimens, and synergy between the microorganisms influences infection severity. We therefore investigated the nosocomial pathogens Staphylococcus aureus and Candida albicans with respect to antimicrobial intervention. We characterized the interaction using biofilm assays and evaluated the effect of miconazole treatment using in vitro and in vivo assays. Finally, we assessed the impact of biofilm extracellular matrix (ECM) on these interactions. Data indicated that the C. albicans mycofilms supported adhesion and colonization by S. aureus through close interactions with hyphal elements, significantly increasing S. aureus biofilm formation throughout biofilm maturation. Miconazole sensitivity was shown to be reduced in both mono- and dual-species biofilms compared to planktonic cells. Within a three-dimensional biofilm model sensitivity was also hindered. Galleria mellonella survival analysis showed both enhanced pathogenicity of the dual-species infection, which was concomitantly desensitized to miconazole treatment. Analysis of the ECM revealed the importance of extracellular DNA, which supported the adhesion of S. aureus and the development of the dual-species biofilm structures. Collectively, these data highlight the clinical importance of dual-species inter-kingdom biofilm infections, though also provides translational opportunities to manage them more effectively. PMID:28280487

  18. Cell wall protection by the Candida albicans class I chitin synthases

    PubMed Central

    Preechasuth, Kanya; Anderson, Jeffrey C.; Peck, Scott C.; Brown, Alistair J.P.; Gow, Neil A.R.; Lenardon, Megan D.

    2015-01-01

    Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2Δchs8Δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses. PMID:26257018

  19. Candida albicans RHO1 is required for cell viability in vitro and in vivo.

    PubMed

    Smith, Susan E; Csank, Csilla; Reyes, Guadalupe; Ghannoum, Mahmoud A; Berlin, Vivian

    2002-05-01

    In Saccharomyces cerevisiae, Rho1p plays an important role in cell wall integrity by regulating beta-1,3-glucan synthase, Pkc1p and the actin cytoskeleton. To determine the physiological role of Rho1p in the dimorphic fungus Candida albicans, the major human fungal pathogen, we constructed mutants that conditionally express Rho1p from the glucose-repressible phosphoenolpyruvate carboxykinase promoter (pPCK1). We examined the growth of these cells in a range of conditions. Depletion of Rho1p from yeast cells resulted in cell death, lysis, and aggregation. The Rho1p conditional mutant was inviable on 10% serum indicating that Rho1p was also required for hyphal viability. Furthermore, in a mouse model of systemic candidiasis, strains dependent on pPCK1-driven RHO1 expression failed to colonise the kidneys and establish disease, suggesting that the level of glucose in serum was sufficient to repress the pPCK1 and that Rho1p-depleted strains were inviable within the host. Therefore, Rho1p is essential for the viability of C. albicans in vitro and in vivo.

  20. Cellular dynamics and embryonic morphogenesis

    NASA Astrophysics Data System (ADS)

    Zallen, Jennifer

    2007-11-01

    The elongated body axis is a characteristic feature of many multicellular animals. Axis elongation occurs largely through cell rearrangements that are coordinated across a large cell population and driven by an asymmetric distribution of cytoskeletal and junctional proteins [1]. To visualize cellular dynamics during this process, we performed time-lapse confocal imaging of cell behavior in the Drosophila embryo. These studies revealed that rearranging cells display a steady increase in topological disorder that is accompanied by the formation of transient structures where 5-11 cells meet [2,3]. These multicellular rosettes form and resolve in a directional fashion to produce a local change in the aspect ratio of the cellular assembly, contributing to an overall change in tissue structure. We propose that higher-order rosette structures link local cell interactions to global tissue reorganization during morphogenesis. [1] J. Zallen and E. Wieschaus, Developmental Cell 6, 343 (2004). [2] J. Zallen and R. Zallen, J. Phys.: Condens. Matter 16, S5073 (2004). [3] J. Blankenship et al., Developmental Cell 11, 459 (2006).

  1. On the Morphogenesis of Feathers

    PubMed Central

    Yu, Mingke; Wu, Ping; Widelitz, Randall B.; Chuong, Cheng-Ming

    2015-01-01

    The most unique character of the feather is its highly ordered hierarchical branched structure1, 2. This evolutionary novelty confers flight function to birds3–5. Recent discoveries of fossils in China have prompted keen interest in the origin and evolution of feathers6–14. However, controversy arises whether the irregularly branched integumentary fibers on dinosaurs such as Sinornithosaurus are truly feathers6, 11, and whether an integumentary appendage with a major central shaft and notched edges is a non-avian feather or a proto-feather8–10. Here we take a developmental approach to analyze molecular mechanisms in feather branching morphogenesis. We have used the replication competent avian sarcoma (RCAS) retrovirus15 to efficiently deliver exogenous genes to regenerating chicken flight feather follicles. We show that the antagonistic balance between noggin and bone morphogenetic protein 4 (BMP4) plays a critical role in feather branching, with BMP4 promoting rachis formation and barb fusion, and noggin enhancing rachis and barb branching. Furthermore we show that sonic hedgehog (SHH) is essential for apoptosis of the marginal plate epithelia to become spaces between barbs. Our analyses show the molecular pathways underlying the topological transformation of feathers from cylindrical epithelia to the hierarchical branched structures, and provide first clues on the possible developmental mechanisms in the evolution of feather forms. PMID:12442169

  2. Vertex Models of Epithelial Morphogenesis

    PubMed Central

    Fletcher, Alexander G.; Osterfield, Miriam; Baker, Ruth E.; Shvartsman, Stanislav Y.

    2014-01-01

    The dynamic behavior of epithelial cell sheets plays a central role during numerous developmental processes. Genetic and imaging studies of epithelial morphogenesis in a wide range of organisms have led to increasingly detailed mechanisms of cell sheet dynamics. Computational models offer a useful means by which to investigate and test these mechanisms, and have played a key role in the study of cell-cell interactions. A variety of modeling approaches can be used to simulate the balance of forces within an epithelial sheet. Vertex models are a class of such models that consider cells as individual objects, approximated by two-dimensional polygons representing cellular interfaces, in which each vertex moves in response to forces due to growth, interfacial tension, and pressure within each cell. Vertex models are used to study cellular processes within epithelia, including cell motility, adhesion, mitosis, and delamination. This review summarizes how vertex models have been used to provide insight into developmental processes and highlights current challenges in this area, including progressing these models from two to three dimensions and developing new tools for model validation. PMID:24896108

  3. Candida species distribution, genotyping and virulence factors of Candida albicans isolated from the oral cavity of kidney transplant recipients of two geographic regions of Brazil

    PubMed Central

    2014-01-01

    Background Candida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. This investigation aimed to study the prevalence of Candida spp. and to analyze the ABC genotypes of 76 clinical isolates of C. albicans obtained from the oral cavity of kidney transplant patients from two distinct geographic regions of Brazil. Methods We typed 48 strains with ABC genotyping and Microsatelitte using primer M13 and tested three virulence factors in vitro: phospholipase activity, morphogenesis and the ability to evade from polymorphonuclear neutrophils phagocytosis. Results C. albicans was the most prevalent species (86.4%), followed by C. tropicalis (4.5%). C. albicans genotype A was the most prevalent (58 isolates; 76.4%), followed by genotype C (15 isolates; 19.7%) and genotype B (3 isolates; 3.9%). When Microsatellite technique with primer M13 was applied, 80% of the isolates from the South were placed within the same cluster. The majority of Genotype C strains were grouped together within two different clusters. Genotype C was considered more resistant to PMNs attack than genotypes A and B. Strains isolated from the South of Brazil showed also better ability to combat PMNs phagocytosis. Conclusions We found a high rate of C. albicans genotype C strains isolated from the oral cavity of this group of patients. This study characterized oral C. albicans strains isolated from kidney transplant recipients and will contribute to a better understanding of the pathogenesis of oral candidiasis. PMID:24628850

  4. The effects of triclosan on spore germination and hyphal growth of the arbuscular mycorrhizal fungus Glomus intraradices.

    PubMed

    Twanabasu, Bishnu R; Stevens, Kevin J; Venables, Barney J

    2013-06-01

    The effect of triclosan (5-chloro-2-[2,4-dichlorophenoxy]phenol; TCS), on spore germination, hyphal growth, and hyphal branching of the arbuscular mycorrhizal (AM) fungus, Glomus intraradices spores was evaluated at exposure concentrations of 0.4 and 4.0 μg/L in a static renewal exposure system. To determine if potential effects were mycotoxic or a consequence of impaired signaling between a host plant and the fungal symbiont, spores were incubated with and without the addition of a root exudate. Exposed spores were harvested at days 7, 14, and 21. AM spore germination, hyphal growth, and hyphal branching were significantly lower in both TCS concentrations compared to controls in non-root exudate treatments suggesting direct mycotoxic effects of TCS on AM development. Greater hyphal growth and hyphal branching in controls and 0.4μg/L TCS treatments with root exudate compared to non-root exudate treatments demonstrated growth stimulation by signaling chemicals present in the root exudate. This stimulatory effect was absent in the 4.0 μg/L TCS treatments indicating a direct effect on plant signaling compounds or plant signal response.

  5. Disparate effects of interferon-gamma and tumor necrosis factor-alpha on early neutrophil respiratory burst and fungicidal responses to Candida albicans hyphae in vitro.

    PubMed Central

    Diamond, R D; Lyman, C A; Wysong, D R

    1991-01-01

    We examined effects of priming with recombinant human interferon-gamma (IFN) or tumor necrosis factor-alpha (TNF) on neutrophil responses to Candida albicans hyphae. Both cytokines increased early superoxide generation after hyphal stimulation. The more pronounced effects of TNF were accompanied by an augmented surface membrane depolarization rate and were insensitive to both pertussis toxin and calcium ion chelation, but were negated by concomitant incubation with puromycin or cycloheximide during priming. IFN augmented hyphal killing despite its only minor enhancement of early respiratory burst responses, but TNF reduced neutrophil fungicidal activity to nearly 40% below those by unprimed control cells even though it enhanced early superoxide responses more dramatically. Though TNF-primed neutrophils killed hyphae at normal initial rates, IFN-primed or even unprimed cells manifested more fungicidal sustained activity. These disparate consequences of cytokine priming on hyphal destruction were paralleled by differences in late generation of potentially candidacidal oxidants, hydrogen peroxide, and hypochlorous acid. IFN added during priming failed to correct TNF-associated functional defects in neutrophil anti-Candida responses. Thus, augmentation of early respiratory burst responses to oxidant-sensitive organisms need not necessarily reflect concomitant salutary effects on microbicidal activity. Images PMID:1846880

  6. Mixed biofilms formed by C. albicans and non-albicans species: a study of microbial interactions.

    PubMed

    Santos, Jéssica Diane dos; Piva, Elisabete; Vilela, Simone Furgeri Godinho; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2016-01-01

    Most Candida infections are related to microbial biofilms often formed by the association of different species. The objective of this study was to evaluate the interactions between Candida albicans and non-albicans species in biofilms formed in vitro. The non-albicans species studied were:Candida tropicalis, Candida glabrata and Candida krusei. Single and mixed biofilms (formed by clinical isolates of C. albicans and non-albicans species) were developed from standardized suspensions of each strain (10(7) cells/mL), on flat-bottom 96-well microtiter plates for 48 hour. These biofilms were analyzed by counting colony-forming units (CFU/mL) in Candida HiChrome agar and by determining cell viability, using the XTT 2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide colorimetric assay. The results for both the CFU/mL count and the XTT colorimetric assay showed that all the species studied were capable of forming high levels of in vitro biofilm. The number of CFU/mL and the metabolic activity of C. albicans were reduced in mixed biofilms with non-albicans species, as compared with a single C. albicans biofilm. Among the species tested, C. krusei exerted the highest inhibitory action against C. albicans. In conclusion, C. albicans established antagonistic interactions with non-albicans Candida species in mixed biofilms.

  7. Impact of environmental conditions on the form and function of Candida albicans biofilms.

    PubMed

    Daniels, Karla J; Park, Yang-Nim; Srikantha, Thyagarajan; Pujol, Claude; Soll, David R

    2013-10-01

    Candida albicans, like other pathogens, can form complex biofilms on a variety of substrates. However, as the number of studies of gene regulation, architecture, and pathogenic traits of C. albicans biofilms has increased, so have differences in results. This suggests that depending upon the conditions employed, biofilms may vary widely, thus hampering attempts at a uniform description. Gene expression studies suggest that this may be the case. To explore this hypothesis further, we compared the architectures and traits of biofilms formed in RPMI 1640 and Spider media at 37°C in air. Biofilms formed by a/α cells in the two media differed to various degrees in cellular architecture, matrix deposition, penetrability by leukocytes, fluconazole susceptibility, and the facilitation of mating. Similar comparisons of a/a cells in the two media, however, were made difficult given that in air, although a/a cells form traditional biofilms in RPMI medium, they form polylayers composed primarily of yeast cells in Spider medium. These polylayers lack an upper hyphal/matrix region, are readily penetrated by leukocytes, are highly fluconazole susceptible, and do not facilitate mating. If, however, air is replaced with 20% CO2, a/a cells make a biofilm in Spider medium similar architecturally to that of a/α cells, which facilitates mating. A second, more cursory comparison is made between the disparate cellular architectures of a/a biofilms formed in air in RPMI and Lee's media. The results demonstrate that C. albicans forms very different types of biofilms depending upon the composition of the medium, level of CO2 in the atmosphere, and configuration of the MTL locus.

  8. Identification and Functional Characterization of Rca1, a Transcription Factor Involved in both Antifungal Susceptibility and Host Response in Candida albicans

    PubMed Central

    Vandeputte, Patrick; Pradervand, Sylvain; Ischer, Françoise; Coste, Alix T.; Ferrari, Sélène; Harshman, Keith

    2012-01-01

    The identification of novel transcription factors associated with antifungal response may allow the discovery of fungus-specific targets for new therapeutic strategies. A collection of 241 Candida albicans transcriptional regulator mutants was screened for altered susceptibility to fluconazole, caspofungin, amphotericin B, and 5-fluorocytosine. Thirteen of these mutants not yet identified in terms of their role in antifungal response were further investigated, and the function of one of them, a mutant of orf19.6102 (RCA1), was characterized by transcriptome analysis. Strand-specific RNA sequencing and phenotypic tests assigned Rca1 as the regulator of hyphal formation through the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway and the transcription factor Efg1, but also probably through its interaction with a transcriptional repressor, most likely Tup1. The mechanisms responsible for the high level of resistance to caspofungin and fluconazole observed resulting from RCA1 deletion were investigated. From our observations, we propose that caspofungin resistance was the consequence of the deregulation of cell wall gene expression and that fluconazole resistance was linked to the modulation of the cAMP/PKA signaling pathway activity. In conclusion, our large-scale screening of a C. albicans transcription factor mutant collection allowed the identification of new effectors of the response to antifungals. The functional characterization of Rca1 assigned this transcription factor and its downstream targets as promising candidates for the development of new therapeutic strategies, as Rca1 influences host sensing, hyphal development, and antifungal response. PMID:22581526

  9. A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

    PubMed

    Nieminen, Mikko T; Novak-Frazer, Lily; Rautemaa, Wilma; Rajendran, Ranjith; Sorsa, Timo; Ramage, Gordon; Bowyer, Paul; Rautemaa, Riina

    2014-01-01

    The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 μM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm

  10. A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

    PubMed

    Nieminen, Mikko T; Novak-Frazer, Lily; Rautemaa, Vilma; Rajendran, Ranjith; Sorsa, Timo; Ramage, Gordon; Bowyer, Paul; Rautemaa, Riina

    2014-01-01

    The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm

  11. CAP1, an Adenylate Cyclase-Associated Protein Gene, Regulates Bud-Hypha Transitions, Filamentous Growth, and Cyclic AMP Levels and Is Required for Virulence of Candida albicans

    PubMed Central

    Bahn, Yong-Sun; Sundstrom, Paula

    2001-01-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis. PMID:11325951

  12. CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans.

    PubMed

    Bahn, Y S; Sundstrom, P

    2001-05-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

  13. Regulation of dendrite morphogenesis by extrinsic cues.

    PubMed

    Valnegri, Pamela; Puram, Sidharth V; Bonni, Azad

    2015-07-01

    Dendrites play a central role in the integration and flow of information in the nervous system. The morphogenesis and maturation of dendrites is hence an essential step in the establishment of neuronal connectivity. Recent studies have uncovered crucial functions for extrinsic cues in the development of dendrites. We review the contribution of secreted polypeptide growth factors, contact-mediated proteins, and neuronal activity in distinct phases of dendrite development. We also highlight how extrinsic cues influence local and global intracellular mechanisms of dendrite morphogenesis. Finally, we discuss how these studies have advanced our understanding of neuronal connectivity and have shed light on the pathogenesis of neurodevelopmental disorders.

  14. Imaging embryonic morphogenesis in C. elegans.

    PubMed

    Hardin, Jeff

    2011-01-01

    The Caenorhabditis elegans embryo is well suited to morphogenetic analysis via modern microscopy, due to its short generation time, transparency, invariant lineage, and the ability to generate transgenic embryos expressing various fluorescent proteins. This chapter provides an overview of microscopy techniques for imaging embryonic morphogenesis, including making agar mounts, capturing four-dimensional (4D) data using Nomarski microscopy, imaging of actin in embryos, factors important for optimizing 4D fluorescence microscopy, and recent techniques that leverage fluorescence microscopy for intracellular imaging of cellular components during morphogenesis.

  15. Differential chlorate inhibition of Chaetomium globosum germination, hyphal growth, and perithecia synthesis.

    PubMed

    Biles, Charles L; Wright, Desiree; Fuego, Marianni; Guinn, Angela; Cluck, Terry; Young, Jennifer; Martin, Markie; Biles, Josiah; Poudyal, Shubhra

    2012-12-01

    Chaetomium globosum Kunze:Fr is a dermatophytic, dematiaceous fungus that is ubiquitous in soils, grows readily on cellulolytic materials, and is commonly found on water-damaged building materials. Chlorate affects nitrogen metabolism in fungi and is used to study compatibility among anamorphic fungi by inducing nit mutants. The effect of chlorate toxicity on C. globosum was investigated by amending a modified malt extract agar (MEA), oat agar, and carboxymethyl cellulose agar (CMC) with various levels of potassium chlorate (KClO(3)). C. globosum perithecia production was almost completely inhibited (90-100 %) at low levels of KClO(3) (0.1 mM) in amended MEA. Inhibition of perithecia production was also observed on oat agar and CMC at 1 and 10 mM, respectively. However, hyphal growth in MEA was only inhibited 20 % by 0.1-100 mM KClO(3) concentrations. Hyphal growth was never completely inhibited at the highest levels tested (200 mM). Higher levels of KClO(3) were needed on gypsum board to inhibit perithecia synthesis. In additional experiments, KClO(3) did not inhibit C. globosum, Fusarium oxysporum, Aspergillus niger, Penicillum expansum, and airborne fungal spore germination. The various fungal spores were not inhibited by KClO(3) at 1-100 mM levels. These results suggest that C. globosum perithecia synthesis is more sensitive to chlorate toxicity than are hyphal growth and spore germination. This research provides basic information that furthers our understanding about perithecia formation and may help in developing control methods for fungal growth on building materials.

  16. Nematode-trapping fungi and fungus-associated bacteria interactions: the role of bacterial diketopiperazines and biofilms on Arthrobotrys oligospora surface in hyphal morphogenesis.

    PubMed

    Li, Lei; Yang, Min; Luo, Jun; Qu, Qing; Chen, Ying; Liang, Lianming; Zhang, Keqin

    2016-11-01

    In soil, nematode-trapping fungi and bacteria often share microhabitats and interact with each other, but effects of fungus-associated bacteria on its trap formation are underestimated. We have ascertained the presence of Stenotrophomonas and Rhizobium genera associated with A. oligospora GJ-1. After A. oligospora GJ-1 without associated bacteria (cured Arthrobotrys) was co-cultivated with Stenotrophomonas and its supernatant extract, microscopic study of hyphae from co-cultivation indicated that bacterial biofilm formation on hyphae was related to trap formation in fungi and Stenotrophomonas supernatant extract. Four diketopiperazines (DKPs) were purified from Stenotrophomonas supernatant extract that could not induce traps in the cured Arthrobotrys. When cured Arthrobotrys was cultured with Stenotrophomonas and one of DKPs, polar attachment, bacterial biofilms on hyphae and trap formation in fungi were observed. After cured Arthrobotrys with bacterial biofilms was consecutively transferred several times on nutrient poor medium, trap formation disappeared with the disappearance of bacterial biofilms on hyphae. DKPs could facilitate chemotaxis of Stenotrophomonas towards fungal extract which was suggested to contribute to bacterial biofilms on hyphae. Furthermore, when cured Arthrobotrys was cultured with Stenotrophomonas and DKPs in soil, trap formation in fungi and bacterial biofilms on hyphae were also observed, and the fungal activity against nematode was enhanced.

  17. Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation

    PubMed Central

    Ikeh, Mélanie A. C.; Haider, Mohammed; Brown, Alistair J. P.; Morgan, Brian A.; Erwig, Lars P.; Quinn, Janet

    2017-01-01

    The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the ‘p38-related’ Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation. PMID:28135328

  18. Candida albicans strain-dependent modulation of pro-inflammatory cytokine release by in vitro oral and vaginal mucosal models.

    PubMed

    Whiley, Robert A; Cruchley, Alan T; Gore, Carelyn; Hagi-Pavli, Eleni

    2012-01-01

    Candida albicans is a commensal organism at several sites and is a versatile, opportunistic pathogen. The underlying factors of pathogen and host associated with commensalism and pathogenicity in C. albicans are complex and their importance is largely unknown. We aimed to study the responses of oral epithelial (OEM) and vaginal epithelial models (VEM) to infection by oral and vaginal C. albicans strains to obtain evidence of inter-strain differences in pathogenicity and of site-specificity. Following inoculation of models, proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 and prostaglandin E2 (PGE2) release were monitored and histological staining undertaken. Striking differences in strain behaviour and epithelial responses were observed. IL-1α, IL-1β and IL-8 release were significantly increased from the OEM in response to denture stomatitis strain NCYC 1467. Increased IL-8 release also followed infection of the OEM with both vaginal strains. Overall the VEM was relatively unresponsive to infection with either oral or vaginal strains under these conditions. Adherence and hyphal development were observed for all strains on both models although extensive, uniform tissue penetration was seen only with stomatitis strain NCYC 1467 on the OEM. Candidal strains were assayed for phospholipase (PL) and secreted aspartyl proteinase (SAP) activities where phospholipase (PL) activity was highest for strain NCYC 1467 although highest SAP activity was observed for vaginal strain NCPF 8112 in this assay. This is the first study to concurrently investigate cytokine production from oral and epithelial models using candidal strains originating from these respective mucosal sites from healthy and disease states. These data demonstrate significant differences in inflammatory responses of host epithelia to individual C. albicans strains.

  19. GDP-mannose pyrophosphorylase is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

    PubMed

    Jiang, Hechun; Ouyang, Haomiao; Zhou, Hui; Jin, Cheng

    2008-09-01

    GDP-mannose pyrophosphorylase (GMPP) catalyses the synthesis of GDP-mannose, which is the precursor for the mannose residues in glycoconjugates, using mannose 1-phosphate and GTP as substrates. Repression of GMPP in yeast leads to phenotypes including cell lysis, defective cell wall, and failure of polarized growth and cell separation. Although several GMPPs have been isolated and characterized in filamentous fungi, the physiological consequences of their actions are not clear. In this study, Afsrb1, which is a homologue of yeast SRB1/PSA1/VIG9, was identified in the Aspergillus fumigatus genome. The Afsrb1 gene was expressed in Escherichia coli, and recombinant AfSrb1 was functionally confirmed as a GMPP. By the replacement of the native Afsrb1 promoter with an inducible Aspergillus nidulans alcA promoter, the conditional inactivation mutant strain YJ-gmpp was constructed. The presence of 3 % glucose completely blocked transcription of P(alcA)-Afsrb1, and was lethal to strain YJ-gmpp. Repression of Afsrb1 expression in strain YJ-gmpp led to phenotypes including hyphal lysis, defective cell wall, impaired polarity maintenance, and branching site selection. Also, rapid germination and reduced conidiation were documented. However, in contrast to yeast, strain YJ-gmpp retained the ability to direct polarity establishment and septation. Our results showed that the Afsrb1 gene is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

  20. Function of a p24 Heterodimer in Morphogenesis and Protein Transport in Penicillium oxalicum

    PubMed Central

    Wang, Fangzhong; Liu, Kuimei; Han, Lijuan; Jiang, Baojie; Wang, Mingyu; Fang, Xu

    2015-01-01

    The lignocellulose degradation capacity of filamentous fungi has been widely studied because of their cellulase hypersecretion. The p24 proteins in eukaryotes serve important functions in this secretory pathway. However, little is known about the functions of the p24 proteins in filamentous fungi. In this study, four p24 proteins were identified in Penicillium oxalicum. Six p24 double-deletion strains were constructed, and further studies were carried out with the ΔerpΔpδ strain. The experimental results suggested that Erp and Pδ form a p24 heterodimer in vivo. This p24 heterodimer participates in important morphogenetic events, including sporulation, hyphal growth, and lateral branching. The results suggested that the p24 heterodimer mediates protein transport, particularly that of cellobiohydrolase. Analysis of the intracellular proteome revealed that the ΔerpΔpδ double mutant is under secretion stress due to attempts to remove proteins that are jammed in the endomembrane system. These results suggest that the p24 heterodimer participates in morphogenesis and protein transport. Compared with P. oxalicum Δerp, a greater number of cellular physiological pathways were impaired in ΔerpΔpδ. This finding may provide new insights into the secretory pathways of filamentous fungi. PMID:26149342

  1. The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity

    PubMed Central

    Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y.; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops. PMID:27625637

  2. Neurogenesis and Morphogenesis in the Cerebellar Cortex*

    PubMed Central

    Eccles, J. C.

    1970-01-01

    The cerebellum presents the best site in the central nervous system for defining fundamental problems concerning the origin and differentiation of neurones, and their growth and development. The many recent experimental investigations of these problems are reviewed, and hypotheses based upon them are developed in relation to neurogenesis, morphogenesis, and synaptogenesis. PMID:4915885

  3. A developmental stage of hyphal cells shows riboflavin overproduction instead of sporulation in Ashbya gossypii.

    PubMed

    Nieland, Susanne; Stahmann, K-Peter

    2013-12-01

    The hemiascomycete Ashbya gossypii develops a mycelium. Nutritional stress leads to its differentiation into sporangia. These generate spores. In parallel, the yellow pigment riboflavin is produced. Intracellularly accumulated riboflavin, made visible as a bright green fluorescence, was observed in only 60% of the hyphal cells. For the remaining 40%, it was unclear whether these cells simply export riboflavin or its biosynthesis remains down-regulated in contrast to the accumulating cells. The approach followed in this work was to convert the hyphae into protoplasts by enzymatic degradation of the cell wall. Afterwards, the protoplasts were sorted by fluorescence-activated cell sorting on the basis of riboflavin accumulation. When a reporter strain expressing lacZ under the control of the most important riboflavin biosynthesis promoter, RIB3, was used, green protoplasts were found to have more than tenfold greater reporter activity than hyaline protoplasts. This was true on the basis of total protein as well as on the basis of hexokinase specific activity, a marker for constitutive expression. These results allow the conclusion that hyphal cells of A. gossypii differ in phenotype regarding riboflavin overproduction and accumulation.

  4. Nuclear dynamics during germination, conidiation, and hyphal fusion of Fusarium oxysporum.

    PubMed

    Ruiz-Roldán, M Carmen; Köhli, Michael; Roncero, M Isabel G; Philippsen, Peter; Di Pietro, Antonio; Espeso, Eduardo A

    2010-08-01

    In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.

  5. Differences between appressoria formed by germ tubes and appressorium-like structures developed by hyphal tips in Magnaporthe oryzae.

    PubMed

    Kong, Ling-An; Li, Guo-Tian; Liu, Yun; Liu, Mei-Gang; Zhang, Shi-Jie; Yang, Jun; Zhou, Xiao-Ying; Peng, You-Liang; Xu, Jin-Rong

    2013-07-01

    Melanized appressoria are highly specialized infection structures formed by germ tubes of the rice blast fungus Magnaporthe oryzae for plant infection. M. oryzae also forms appressorium-like structures on hyphal tips. Whereas appressorium formation by conidial germ tubes has been well characterized, formation of appressorium-like structures by hyphal tips is under-investigated. In a previous study, we found that the chs7 deletion mutant failed to form appressoria on germ tubes but were normal in the development of appressorium-like structures on artificial hydrophobic surfaces. In this study, we compared the differences between the formation of appressoria by germ tubes and appressorium-like structures by hyphal tips in M. oryzae. Structurally, both appressoria and appressorium-like structures had a melanin layer that was absent in the pore region. In general, the latters were 1.4-fold larger in size but had lower turgor pressure than appressoria, which is consistent with its lower efficiency in plant penetration. Treatments with cAMP, IBMX, or a cutin monomer efficiently induced appressorium formation but not the development of appressorium-like structures. In contrast, coating surfaces with waxes stimulated the formation of both infection structures. Studies with various signaling mutants indicate that Osm1 and Mps1 are dispensable but Pmk1 is essential for both appressorium formation and development of appressorium-like structures on hyphal tips. Interestingly, the cpkA mutant was reduced in the differentiation of appressorium-like structures but not appressorium formation. We also observed that the con7 mutant generated in our lab failed to form appressorium-like structures on hyphal tips but still produced appressoria by germ tubes on hydrophobic surfaces. Con7 is a transcription factor regulating the expression of CHS7. Overall, these results indicate that the development of appressorium-like structures by hyphal tips and formation of appressoria by germ

  6. Adaptive immune responses to Candida albicans infection

    PubMed Central

    Richardson, Jonathan P; Moyes, David L

    2015-01-01

    Fungal infections are becoming increasingly prevalent in the human population and contribute to morbidity and mortality in healthy and immunocompromised individuals respectively. Candida albicans is the most commonly encountered fungal pathogen of humans, and is frequently found on the mucosal surfaces of the body. Host defense against C. albicans is dependent upon a finely tuned implementation of innate and adaptive immune responses, enabling the host to neutralise the invading fungus. Central to this protection are the adaptive Th1 and Th17 cellular responses, which are considered paramount to successful immune defense against C. albicans infections, and enable tissue homeostasis to be maintained in the presence of colonising fungi. This review will highlight the recent advances in our understanding of adaptive immunity to Candida albicans infections. PMID:25607781

  7. C. albicans Colonization of Human Mucosal Surfaces

    PubMed Central

    Southern, Peter; Horbul, Julie; Maher, Diane; Davis, Dana A.

    2008-01-01

    Background Candida albicans is a low level commensal organism in normal human populations with the continuous potential to expand and cause a spectrum of clinical conditions. Methodology/Principal Findings Using ex vivo human organ cultures and populations of primary human cells, we have developed several related experimental systems to examine early-stage interactions between C. albicans and mucosal surfaces. Experiments have been conducted both with exogenously added C. albicans and with overtly normal human mucosal surfaces supporting pre-existing infections with natural isolates of Candida. Under different culture conditions, we have demonstrated the formation of C. albicans colonies on human target cells and filament formation, equivalent to tissue invasion. Conclusions/Significance These organ culture systems provide a valuable new resource to examine the molecular and cellular basis for Candida colonization of human mucosal surfaces. PMID:18446191

  8. Candida albicans Biofilms and Human Disease

    PubMed Central

    Nobile, Clarissa J.; Johnson, Alexander D.

    2016-01-01

    In humans, microbial cells (including bacteria, archaea, and fungi) greatly outnumber host cells. Candida albicans is the most prevalent fungal species of the human microbiota; this species asymptomatically colonizes many areas of the body, particularly the gastrointestinal and genitourinary tracts of healthy individuals. Alterations in host immunity, stress, resident microbiota, and other factors can lead to C. albicans overgrowth, causing a wide range of infections, from superficial mucosal to hematogenously disseminated candidiasis. To date, most studies of C. albicans have been carried out in suspension cultures; however, the medical impact of C. albicans (like that of many other microorganisms) depends on its ability to thrive as a biofilm, a closely packed community of cells. Biofilms are notorious for forming on implanted medical devices, including catheters, pacemakers, dentures, and prosthetic joints, which provide a surface and sanctuary for biofilm growth. C. albicans biofilms are intrinsically resistant to conventional antifungal therapeutics, the host immune system, and other environmental perturbations, making biofilm-based infections a significant clinical challenge. Here, we review our current knowledge of biofilms formed by C. albicans and closely related fungal species. PMID:26488273

  9. Transcription Factors Efg1 and Bcr1 Regulate Biofilm Formation and Virulence during Candida albicans-Associated Denture Stomatitis

    PubMed Central

    Yano, Junko; Yu, Alika; Fidel, Paul L.; Noverr, Mairi C.

    2016-01-01

    Denture stomatitis (DS) is characterized by inflammation of the oral mucosa in direct contact with dentures and affects a significant number of otherwise healthy denture wearers. The disease is caused by Candida albicans, which readily colonizes and form biofilms on denture materials. While evidence for biofilms on abiotic and biotic surfaces initiating Candida infections is accumulating, a role for biofilms in DS remains unclear. Using an established model of DS in immunocompetent animals, the purpose of this study was to determine the role of biofilm formation in mucosal damage during pathogenesis using C. albicans or mutants defective in morphogenesis (efg1-/-) or biofilm formation (bcr1-/-). For in vivo analyses, rats fitted with custom dentures, consisting of fixed and removable parts, were inoculated with wild-type C. albicans, mutants or reconstituted strains and monitored weekly for fungal burden (denture and palate), body weight and tissue damage (LDH) for up to 8 weeks. C. albicans wild-type and reconstituted mutants formed biofilms on dentures and palatal tissues under in vitro, ex vivo and in vivo conditions as indicated by microscopy demonstrating robust biofilm architecture and extracellular matrix (ECM). In contrast, both efg1-/- and bcr1-/- mutants exhibited poor biofilm growth with little to no ECM. In addition, quantification of fungal burden showed reduced colonization throughout the infection period on dentures and palates of rats inoculated with efg1-/-, but not bcr1-/-, compared to controls. Finally, rats inoculated with efg1-/- and bcr1-/- mutants had minimal palatal tissue damage/weight loss while those inoculated with wild-type or reconstituted mutants showed evidence of tissue damage and exhibited stunted weight gain. These data suggest that biofilm formation is associated with tissue damage during DS and that Efg1 and Bcr1, both central regulators of virulence in C. albicans, have pivotal roles in pathogenesis of DS. PMID:27453977

  10. Proteolytic activity and cytokine up-regulation by non-albicans Candida albicans.

    PubMed

    Nawaz, Ali; Pärnänen, Pirjo; Kari, Kirsti; Meurman, Jukka H

    2015-05-01

    Mouth is an important source of infections and oral infections such as Candida infections increase the risk of mortality. Our purpose was to investigate differences in proteolytic activity of non-albicans Candida albicans (non-albicans Candida) between clinical isolates and laboratory samples. The second aim was to assess the concentration of pro- and anti-inflammatory cytokine levels IL-1β, IL-10, and TNF-α in saliva of patients with the non-albicans Candida and Candida-negative saliva samples. Clinical yeast samples from our laboratory were used for analyses. Candida strains were grown in YPG at 37 °C for 24 h in water bath with shaking. The activity of Candida proteinases of cell and cell-free fractions were analyzed by MDPF-gelatin zymography. The levels of IL-1β, IL-10, and TNF-α were measured from saliva with ELISA. The study showed differences in the proteolytic activity among the non-albicans Candida strains. C. tropicalis had higher proteolytic activity when compared to the other strains. Significant difference was found in salivary IL-1β levels between the non-albicans Candida and control strains (P < 0.002). The present findings showed differences in proteolytic activity among the non-albicans Candida strains. The increased IL-1β concentration may be one of the host response components associated with non-albicans Candida infection.

  11. Planar cell polarity in vertebrate limb morphogenesis.

    PubMed

    Gao, Bo; Yang, Yingzi

    2013-08-01

    Studies of the vertebrate limb development have contributed significantly to understanding the fundamental mechanisms underlying growth, patterning, and morphogenesis of a complex multicellular organism. In the limb, well-defined signaling centers interact to coordinate limb growth and patterning along the three axes. Recent analyses of live imaging and mathematical modeling have provided evidence that polarized cell behaviors governed by morphogen gradients play an important role in shaping the limb bud. Furthermore, the Wnt/planar cell polarity (PCP) pathway that controls uniformly polarized cell behaviors in a field of cells has emerged to be critical for directional morphogenesis in the developing limb. Directional information coded in the morphogen gradient may be interpreted by responding cells through regulating the activities of PCP components in a Wnt morphogen dose-dependent manner.

  12. Mechanics of epithelial tissue homeostasis and morphogenesis.

    PubMed

    Guillot, Charlène; Lecuit, Thomas

    2013-06-07

    Epithelia are robust tissues that support the structure of embryos and organs and serve as effective barriers against pathogens. Epithelia also chemically separate different physiological environments. These vital functions require tight association between cells through the assembly of junctions that mechanically stabilize the tissue. Remarkably, epithelia are also dynamic and can display a fluid behavior. Cells continuously die or divide, thereby allowing functional tissue homeostasis. Epithelial cells can change shape or intercalate as tissues deform during morphogenesis. We review the mechanical basis of tissue robustness and fluidity, with an emphasis on the pivotal role of junction dynamics. Tissue fluidity emerges from local active stresses acting at cell interfaces and allows the maintenance of epithelial organization during morphogenesis and tissue renewal.

  13. Peripheral nerve morphogenesis induced by scaffold micropatterning

    PubMed Central

    Memon, Danish; Boneschi, Filippo Martinelli; Madaghiele, Marta; Brambilla, Paola; Del Carro, Ubaldo; Taveggia, Carla; Riva, Nilo; Trimarco, Amelia; Lopez, Ignazio D.; Comi, Giancarlo; Pluchino, Stefano; Martino, Gianvito; Sannino, Alessandro; Quattrini, Angelo

    2014-01-01

    Several bioengineering approaches have been proposed for peripheral nervous system repair, with limited results and still open questions about the underlying molecular mechanisms. We assessed the biological processes that occur after the implantation of collagen scaffold with a peculiar porous microstructure of the wall in a rat sciatic nerve transection model compared to commercial collagen conduits and nerve crush injury using functional, histological and genome wide analyses. We demonstrated that within 60 days, our conduit had been completely substituted by a normal nerve. Gene expression analysis documented a precise sequential regulation of known genes involved in angiogenesis, Schwann cells/axons interactions and myelination, together with a selective modulation of key biological pathways for nerve morphogenesis induced by porous matrices. These data suggest that the scaffold’s microstructure profoundly influences cell behaviors and creates an instructive micro-environment to enhance nerve morphogenesis that can be exploited to improve recovery and understand the molecular differences between repair and regeneration. PMID:24559639

  14. Planar Cell Polarity in vertebrate limb morphogenesis

    PubMed Central

    Gao, Bo; Yang, Yingzi

    2013-01-01

    Studies of the vertebrate limb development have contributed significantly to understanding the fundamental mechanisms underlying growth, patterning and morphogenesis of a complex multicellular organism. In the limb, well-defined signaling centers interact to coordinate limb growth and patterning along the three axes. Recent analyses of live imaging and mathematical modeling have provided evidence that polarized cell behaviors governed by morphogen gradients play an important role in shaping the limb bud. Furthermore, the Wnt/Planar Cell Polarity (PCP) pathway that controls uniformly polarized cellular behaviors in a field of cells has emerged to be critical for directional morphogenesis in the developing limb. Directional information coded in the morphogen gradient may be interpreted by responding cells through regulating the activities of PCP components in a Wnt morphogen dose-dependent manner. PMID:23747034

  15. Feedback, Lineages and Self-Organizing Morphogenesis

    PubMed Central

    Calof, Anne L.; Lowengrub, John S.; Lander, Arthur D.

    2016-01-01

    Feedback regulation of cell lineage progression plays an important role in tissue size homeostasis, but whether such feedback also plays an important role in tissue morphogenesis has yet to be explored. Here we use mathematical modeling to show that a particular feedback architecture in which both positive and negative diffusible signals act on stem and/or progenitor cells leads to the appearance of bistable or bi-modal growth behaviors, ultrasensitivity to external growth cues, local growth-driven budding, self-sustaining elongation, and the triggering of self-organization in the form of lamellar fingers. Such behaviors arise not through regulation of cell cycle speeds, but through the control of stem or progenitor self-renewal. Even though the spatial patterns that arise in this setting are the result of interactions between diffusible factors with antagonistic effects, morphogenesis is not the consequence of Turing-type instabilities. PMID:26989903

  16. Beyond Candida albicans: Mechanisms of immunity to non-albicans Candida species.

    PubMed

    Whibley, Natasha; Gaffen, Sarah L

    2015-11-01

    The fungal genus Candida encompasses numerous species that inhabit a variety of hosts, either as commensal microbes and/or pathogens. Candida species are a major cause of fungal infections, yet to date there are no vaccines against Candida or indeed any other fungal pathogen. Our knowledge of immunity to Candida mainly comes from studies on Candida albicans, the most frequent species associated with disease. However, non-albicans Candida (NAC) species also cause disease and their prevalence is increasing. Although research into immunity to NAC species is still at an early stage, it is becoming apparent that immunity to C. albicans differs in important ways from non-albicans species, with important implications for treatment, therapy and predicted demographic susceptibility. This review will discuss the current understanding of immunity to NAC species in the context of immunity to C. albicans, and highlight as-yet unanswered questions.

  17. Two amino acid sequences direct Aspergillus nidulans protein kinase C (PkcA) localization to hyphal apices and septation sites.

    PubMed

    Jackson-Hayes, Loretta; Hill, Terry W; Loprete, Darlene M; DelBove, Claire E; Shapiro, Justin A; Henley, Jordan L; Dawodu, Omolola O

    2015-01-01

    The Aspergillus nidulans ortholog of protein kinase C (pkcA) is involved in the organism's putative cell wall integrity (CWI) pathway, and PkcA also is highly localized at growing tips and forming septa. In the present work we identify the regions within PkcA that are responsible for its localization to hyphal tips and septation sites. To this end, we used serially truncated pkcA constructs and expressed them as green fluorescent protein (GFP) chimeras and identified two regions that direct PkcA localization. The first region is a 10 amino-acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips. Proteins containing this sequence also localize to septation sites. A second region between C2 and C1B (encompassing C1A) is sufficient for localization to septation sites but not to hyphal tips. We also report that localization to hyphal tips and septation sites alone is not sufficient for truncated constructs to complement hypersensitivity to the cell wall compromising agent calcofluor white in a strain bearing a mutation in the pkcA gene. Taken together, these results suggest that localization and stress response might be independent.

  18. Morphogenesis of the C. elegans vulva

    PubMed Central

    Schindler, Adam J

    2012-01-01

    Understanding how cells move, change shape, and alter cellular behaviors to form organs, a process termed morphogenesis, is one of the great challenges of developmental biology. Formation of the C. elegans vulva is a powerful, simple, and experimentally accessible model for elucidating how morphogenetic processes produce an organ. In the first step of vulval development, three epithelial precursor cells divide and differentiate to generate 22 cells of seven different vulval subtypes. The 22 vulval cells then rearrange from a linear array into a tube, with each of the seven cell types undergoing characteristic morphogenetic behaviours that construct the vulva. Vulval morphogenesis entails many of the same cellular activities that underlie organogenesis and tissue formation across species, including invagination, lumen formation, oriented cell divisions, cell-cell adhesion, cell migration, cell fusion, extracellular matrix remodelling and cell invasion. Studies of vulval development have led to pioneering discoveries in a number of these processes and are beginning to bridge the gap between the pathways that specify cells and their connections to morphogenetic behaviors. The simplicity of the vulva and the experimental tools available in C. elegans will continue to make vulval morphogenesis a powerful paradigm to further our understanding of the largely mysterious mechanisms that build tissues and organs. PMID:23418408

  19. Laminin is required for Schwann cell morphogenesis.

    PubMed

    Yu, Wei-Ming; Chen, Zu-Lin; North, Alison J; Strickland, Sidney

    2009-04-01

    Development of the peripheral nervous system requires radial axonal sorting by Schwann cells (SCs). To accomplish sorting, SCs must both proliferate and undergo morphogenetic changes such as process extension. Signaling studies reveal pathways that control either proliferation or morphogenesis, and laminin is essential for SC proliferation. However, it is not clear whether laminin is also required for SC morphogenesis. By using a novel time-lapse live-cell-imaging technique, we demonstrated that laminins are required for SCs to form a bipolar shape as well as for process extension. These morphological deficits are accompanied by alterations in signaling pathways. Phosphorylation of Schwannomin at serine 518 and activation of Rho GTPase Cdc42 and Rac1 were all significantly decreased in SCs lacking laminins. Inhibiting Rac1 and/or Cdc42 activities in cultured SCs attenuated laminin-induced myelination, whereas forced activation of Rac1 and/or Cdc42 in vivo improved sorting and hypomyelinating phenotypes in SCs lacking laminins. These findings indicate that laminins play a pivotal role in regulating SC cytoskeletal signaling. Coupled with previous results demonstrating that laminin is critical for SC proliferation, this work identifies laminin signaling as a central regulator coordinating the processes of proliferation and morphogenesis in radial axonal sorting.

  20. Mechanobiological simulations of prenatal joint morphogenesis.

    PubMed

    Giorgi, Mario; Carriero, Alessandra; Shefelbine, Sandra J; Nowlan, Niamh C

    2014-03-21

    Joint morphogenesis is the process in which prenatal joints acquire their reciprocal and interlocking shapes. Despite the clinical importance of the process, it remains unclear how joints acquire their shapes. In this study, we simulate 3D mechanobiological joint morphogenesis for which the effects of a range of movements (or lack of movement) and different initial joint shapes are explored. We propose that static hydrostatic compression inhibits cartilage growth while dynamic hydrostatic compression promotes cartilage growth. Both pre-cavitational (no muscle contractions) and post-cavitational (with muscle contractions) phases of joint development were simulated. Our results showed that for hinge type motion (planar motion from 45° to 120°) the proximal joint surface developed a convex profile in the posterior region and the distal joint surface developed a slightly concave profile. When 3D movements from 40° to -40° in two planes were applied, simulating a rotational movement, the proximal joint surface developed a concave profile whereas the distal joint surface rudiment acquire a rounded convex profile, showing an interlocking shape typical of a ball and socket joint. The significance of this research is that it provides new and important insights into normal and abnormal joint development, and contributes to our understanding of the mechanical factors driving very early joint morphogenesis. An enhanced understanding of how prenatal joints form is critical for developing strategies for early diagnosis and preventative treatments for congenital musculoskeletal abnormalities such as developmental dysplasia of the hip.

  1. Punctuated evolution and robustness in morphogenesis

    PubMed Central

    Grigoriev, D.; Reinitz, J.; Vakulenko, S.; Weber, A.

    2014-01-01

    This paper presents an analytic approach to the pattern stability and evolution problem in morphogenesis. The approach used here is based on the ideas from the gene and neural network theory. We assume that gene networks contain a number of small groups of genes (called hubs) controlling morphogenesis process. Hub genes represent an important element of gene network architecture and their existence is empirically confirmed. We show that hubs can stabilize morphogenetic pattern and accelerate the morphogenesis. The hub activity exhibits an abrupt change depending on the mutation frequency. When the mutation frequency is small, these hubs suppress all mutations and gene product concentrations do not change, thus, the pattern is stable. When the environmental pressure increases and the population needs new genotypes, the genetic drift and other effects increase the mutation frequency. For the frequencies that are larger than a critical amount the hubs turn off; and as a result, many mutations can affect phenotype. This effect can serve as an engine for evolution. We show that this engine is very effective: the evolution acceleration is an exponential function of gene redundancy. Finally, we show that the Eldredge-Gould concept of punctuated evolution results from the network architecture, which provides fast evolution, control of evolvability, and pattern robustness. To describe analytically the effect of exponential acceleration, we use mathematical methods developed recently for hard combinatorial problems, in particular, for so-called k-SAT problem, and numerical simulations. PMID:24996115

  2. Distinct Roles of Myosins in Aspergillus fumigatus Hyphal Growth and Pathogenesis

    PubMed Central

    Renshaw, Hilary; Vargas-Muñiz, José M.; Richards, Amber D.; Asfaw, Yohannes G.; Juvvadi, Praveen R.

    2016-01-01

    Myosins are a family of actin-based motor proteins found in many organisms and are categorized into classes based on their structures. Class II and V myosins are known to be important for critical cellular processes, including cytokinesis, endocytosis, exocytosis, and organelle trafficking, in the model fungi Saccharomyces cerevisiae and Aspergillus nidulans. However, the roles of myosins in the growth and virulence of the pathogen Aspergillus fumigatus are unknown. We constructed single- and double-deletion strains of the class II and class V myosins in A. fumigatus and found that while the class II myosin (myoB) is dispensable for growth, the class V myosin (myoE) is required for proper hyphal extension; deletion of myoE resulted in hyperbranching and loss of hyphal polarity. Both myoB and myoE are necessary for proper septation, conidiation, and conidial germination, but only myoB is required for conidial viability. Infection with the ΔmyoE strain in the invertebrate Galleria mellonella model and also in a persistently immunosuppressed murine model of invasive aspergillosis resulted in hypovirulence, while analysis of bronchoalveolar lavage fluid revealed that tumor necrosis factor alpha (TNF-α) release and cellular infiltration were similar compared to those of the wild-type strain. The ΔmyoE strain showed fungal growth in the murine lung, while the ΔmyoB strain exhibited little fungal burden, most likely due to the reduced conidial viability. These results show, for the first time, the important role these cytoskeletal components play in the growth of and disease caused by a known pathogen, prompting future studies to understand their regulation and potential targeting for novel antifungal therapies. PMID:26953327

  3. ZCF32, a fungus specific Zn(II)2 Cys6 transcription factor, is a repressor of the biofilm development in the human pathogen Candida albicans

    PubMed Central

    Kakade, Pallavi; Sadhale, Parag; Sanyal, Kaustuv; Nagaraja, Valakunja

    2016-01-01

    As a human fungal pathogen, Candida albicans can cause a wide variety of disease conditions ranging from superficial to systemic infections. Many of these infections are caused by an inherent ability of the pathogen to form biofilms on medical devices resulting in high mortality. Biofilms formed by C. albicans are a complex consortium of yeast and hyphal cells embedded in an extracellular matrix and are regulated by a network of transcription factors. Here, we report the role of a novel Zn(II)2-Cys6 binuclear cluster transcription factor, ZCF32, in the regulation of biofilm formation. Global transcriptome analysis reveals that biofilm development is the most altered pathway in the zcf32 null mutant. To delineate the functional correlation between ZCF32 and biofilm development, we determined the set of genes directly regulated by Zcf32. Our data suggests that Zcf32 regulates biofilm formation by repressing the expression of adhesins, chitinases and a significant number of other GPI-anchored proteins. We establish that there is the lesser recruitment of Zcf32 on the promoters of biofilm genes in biofilm condition compared to the planktonic mode of growth. Taking together, we propose that the transcription factor ZCF32 negatively regulates biofilm development in C. albicans. PMID:27498700

  4. The FgHOG1 Pathway Regulates Hyphal Growth, Stress Responses, and Plant Infection in Fusarium graminearum

    PubMed Central

    Zhou, Xiaoying; Wang, Chenfang; Xiang, Ping; Zheng, Qian; Xu, Jin-Rong

    2012-01-01

    Fusarium head blight (FHB) caused by Fusarium graminearum is a destructive disease of wheat and barley worldwide. In a previous study of systematic characterization of protein kinase genes in F. graminearum, mutants of three putative components of the osmoregulation MAP kinase pathway were found to have distinct colony morphology and hyphal growth defects on PDA plates. Because the osmoregulation pathway is not known to regulate aerial hyphal growth and branching, in this study we further characterized the functions of the FgHog1 pathway in growth, pathogenesis, and development. The Fghog1, Fgpbs2, and Fgssk2 mutants were all reduced in growth rate, aerial hyphal growth, and hyphal branching angle. These mutants were not only hypersensitive to osmotic stress but also had increased sensitivity to oxidative, cytoplasm membrane, and cell wall stresses. The activation of FgHog1 was blocked in the Fgpbs2 and Fgssk2 mutants, indicating the sequential activation of FgSsk2-FgPbs2-FgHog1 cascade. Interestingly, the FgHog1 MAPK pathway mutants appeared to be sensitive to certain compounds present in PDA. They were female sterile but retained male fertility. We also used the metabolomics profiling approach to identify compatible solutes that were accumulated in the wild type but not in the Fghog1 deletion mutant. Overall, our results indicate that the FgSsk2-FgPbs2-FgHog1 MAPK cascade is important for regulating hyphal growth, branching, plant infection, and hyperosmotic and general stress responses in F. graminearum. PMID:23166686

  5. The effects of hydroxy fatty acids on the hyphal branching of germinated spores of AM fungi.

    PubMed

    Nagahashi, Gerald; Douds, David D

    2011-01-01

    Two hydroxy fatty acids, tentatively identified previously in carrot root exudates, were tested for their effects on hyphal growth of the arbuscular mycorrhizal (AM) fungus, Gigaspora gigantea (Nicol. and Gerd.) Gerdemann and Trappe. Best results were achieved with a long-term bioassay (7-8d) with nanomolar concentrations throughout the Petri dish in contrast to the rapid microinjection bioassay (16-24h) in which nanogram quantities were injected near growing hyphal tips. When 5nM 2-hydroxy fatty acids of various chain length were tested, the length of the hydroxyl fatty acid was significant since only 2-hydroxytetradecanoic acid (2OH-TDA) and to a slightly lesser degree, 2-hydroxydodecanoic acid (2OH-DDA) induced a hyphal growth response while 2-hydroxydecanoic acid (2OH-DA) and 2-hydroxyhexadecanoic (2OH-HDA) acid did not. The position of the hydroxyl group was critical since 5nM 3-hydroxytetradecanoic acid (3OH-TDA) had no effect on hyphal growth. The length of the non-hydroxy containing straight chain fatty acid, per se, did not appear significant since none of these fatty acids had an effect on hyphal growth. The morphological growth response promoted by 2OH-TDA consisted of multiple lateral branches, spaced fairly regularly apart, along the primary germ tubes as well as some lateral branch formation off the major secondary hyphae. This growth response was identical to that observed when germinated spores were allowed to grow towards cultured carrot roots in vitro. This response to 2OH-TDA also was observed with an unidentified Gigaspora species but no morphological response was observed with Glomus intraradices Schenck and Smith. The results indicate that 2-hydroxy fatty acids are another putative category of root exudate signals perceived by Gigaspora species, stimulating an increase in elongated lateral branches.

  6. Urinary tract infections and Candida albicans

    PubMed Central

    Behzadi, Payam; Behzadi, Elham

    2015-01-01

    Introduction Urinary tract candidiasis is known as the most frequent nosocomial fungal infection worldwide. Candida albicans is the most common cause of nosocomial fungal urinary tract infections; however, a rapid change in the distribution of Candida species is undergoing. Simultaneously, the increase of urinary tract candidiasis has led to the appearance of antifungal resistant Candida species. In this review, we have an in depth look into Candida albicans uropathogenesis and distribution of the three most frequent Candida species contributing to urinary tract candidiasis in different countries around the world. Material and methods For writing this review, Google Scholar –a scholarly search engine– (http://scholar.google.com/) and PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) were used. The most recently published original articles and reviews of literature relating to the first three Candida species causing urinary tract infections in different countries and the pathogenicity of Candida albicans were selected and studied. Results Although some studies show rapid changes in the uropathogenesis of Candida species causing urinary tract infections in some countries, Candida albicans is still the most important cause of candidal urinary tract infections. Conclusions Despite the ranking of Candida albicans as the dominant species for urinary tract candidiasis, specific changes have occurred in some countries. At this time, it is important to continue the surveillance related to Candida species causing urinary tract infections to prevent, control and treat urinary tract candidiasis in future. PMID:25914847

  7. Resveratrol lacks antifungal activity against Candida albicans.

    PubMed

    Collado-González, Mar; Guirao-Abad, José P; Sánchez-Fresneda, Ruth; Belchí-Navarro, Sarai; Argüelles, Juan-Carlos

    2012-06-01

    The putative candicidal activity of resveratrol is currently a matter of controversy. Here, the antifungal activity as well as the antioxidant response of resveratrol against Candida albicans, have been tested in a set of strains with a well-established genetic background At the doses usually employed in antifungal tests (10-40 μg/ml), resveratrol has no effect on the exponential growth of the C. albicans CAI.4 strain, a tenfold increase (400 μg/ml) was required in order to record a certain degree of cell killing, which was negligible in comparison with the strong antifungal effect caused by the addition of amphotericin B (5 μg/ml). An identical pattern was recorded in the prototrophic strains of C. albicans SC5314 and RM-100, whereas the oxidative sensitive trehalose-deficient mutant (tps1/tps1 strain) was totally refractory to the presence of resveratrol. In turn, the serum-induced yeast-to-hypha transition remained unaffected upon addition of different concentrations of resveratrol. Determination of endogenous trehalose and catalase activity, two antioxidant markers in C. albicans; revealed no significant changes in their basal contents induced by resveratrol. Collectively, our results seem to dismiss a main antifungal role as well as the therapeutic application of resveratrol against the infections caused by C. albicans.

  8. Normal morphogenesis of epithelial tissues and progression of epithelial tumors

    PubMed Central

    Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A.

    2011-01-01

    Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted. PMID:21898857

  9. Normal morphogenesis of epithelial tissues and progression of epithelial tumors.

    PubMed

    Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A

    2012-01-01

    Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted.

  10. Isolation and characterization of Candida albicans morphological mutants derepressed for the formation of filamentous hypha-type structures

    SciTech Connect

    Gil, C.; Pomes, R.; Nombela, C. )

    1990-05-01

    Several Candida albicans morphological mutants were obtained by a procedure based on a combined treatment with nitrous acid plus UV irradiation and a double-enrichment step to increase the proportion of mutants growing as long filamentous structures. Altered cell morphogenesis in these mutants correlated with an altered colonial phenotype. Two of these mutants, C. albicans NEL102 and NEL103, were selected and characterized. Mutant blastoconidia initiated budding but eventually gave rise to filamentous hypha-type formations. These filaments were long and septate, and they branched very regularly at positions near septa. Calcofluor white (which is known to bind chitin-rich areas) stained septa, branching zones, and filament tips very intensely, as observed under the fluorescence microscope. Wild-type hybrids were obtained by fusing protoplasts of strain NEL102 with B14, another morphological mutant previously described as being permanently pseudomycelial, indicating that genetic determinants responsible for the two altered phenotypes are different. The mutants characterized in this work seemed to sequentially express the morphogenic characteristics of C. albicans, from blastoconidia to hyphae, in the absence of any inducer. Further characterization of these strains could be relevant to gain understanding of the genetic control of dimorphism in this species.

  11. [Prevalence of Candida albicans and Candida non-albicans in clinical samples during 1999-2001].

    PubMed

    Mujica, M T; Finquelievich, J L; Jewtuchowicz, V; Iovannitti, C A

    2004-01-01

    The importance of epidemiological monitoring of yeasts involved in pathologic processes is unquestionable due to the increase of these infections over the last decade, the changes observed in species causing candidiasis, and empirical antifungal treatment. At the Mycology Center, 1006 isolates from a wide range of clinical samples were studied during 1999-2001. Candida albicans (40.3%) was the most isolated species, although, the Candida no albicans species with 54.9% showed the major prevalence. In blood cultures Candida parapsilosis (34.9%), C. albicans (30.2%) and C. tropicalis (25.6%) were recovered most frequently while C. glabrata represented only 2.3%. C. albicans with 60%-80% was the predominant specie in mucosal surface. We also detected Candida mediastinistis, which alert us over the importance at this location. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, being C. albicans (47.7%), the most commonly isolated, followed by C. glabrata (24.8%) and C. tropicalis (20.0%). In the candidal onychomycoses, C. parapsilosis (37.7%) outplaced C. albicans (22.0%). Fluconazole susceptibility studies of Candida species allowed us to conclude that the majority of C. albicans islolates are susceptible, and that the highest resistance averages were observed in C. glabrata (21.41%) and C. krusei (69.23%).

  12. Analysis of Candida albicans Mutants Defective in the Cdk8 Module of Mediator Reveal Links between Metabolism and Biofilm Formation

    PubMed Central

    Lindsay, Allia K.; Morales, Diana K.; Liu, Zhongle; Grahl, Nora; Zhang, Anda; Willger, Sven D.; Myers, Lawrence C.; Hogan, Deborah A.

    2014-01-01

    Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO), a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module—Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development. PMID:25275466

  13. Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms.

    PubMed

    Harrison, Joe J; Ceri, Howard; Yerly, Jerome; Rabiei, Maryam; Hu, Yaoping; Martinuzzi, Robert; Turner, Raymond J

    2007-08-01

    Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO(4)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), AsO(2)(-), and SeO(3)(2-)) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated "domed," "layer cake," "flat," and "mycelial." This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.

  14. Looking into Candida albicans infection, host response, and antifungal strategies

    PubMed Central

    Wang, Yan

    2015-01-01

    Candida albicans, a commonly encountered fungal pathogen, causes diseases varying from superficial mucosal complaints to life-threatening systemic disorders. Among the virulence traits of C. albicans, yeast-to-hypha transition is most widely acknowledged. Host innate immunity to C. albicans critically requires pattern recognition receptors (PRRs), and defence against C. albicans infection is provided by an exquisite interplay between the innate and adaptive arms of the host immune system. PMID:25590793

  15. Looking into Candida albicans infection, host response, and antifungal strategies.

    PubMed

    Wang, Yan

    2015-01-01

    Candida albicans, a commonly encountered fungal pathogen, causes diseases varying from superficial mucosal complaints to life-threatening systemic disorders. Among the virulence traits of C. albicans, yeast-to-hypha transition is most widely acknowledged. Host innate immunity to C. albicans critically requires pattern recognition receptors (PRRs), and defence against C. albicans infection is provided by an exquisite interplay between the innate and adaptive arms of the host immune system.

  16. Rapid identification of Candida albicans by using Albicans ID and fluoroplate agar plates.

    PubMed Central

    Rousselle, P; Freydiere, A M; Couillerot, P J; de Montclos, H; Gille, Y

    1994-01-01

    Two commercially available agar media, Albicans ID and Fluoroplate, that use a chromogenic or a fluorogenic substrate for the detection and identification of Candida albicans were evaluated. From 1,006 clinical samples containing 723 yeast strains, 352 C. albicans strains were detected with either of the two media. The sensitivity of each of the two media was 93.8% and the specificity was 98.6%, with five false-positive reactions for Candida tropicalis and no false-negative reactions. PMID:7883894

  17. Influence of Bacterial Presence on Biofilm Formation of Candida albicans

    PubMed Central

    Park, Su Jung; Han, Kyoung-Hee; Park, Joo Young; Choi, Sun Ju

    2014-01-01

    Purpose Candida albicans is an opportunistic pathogen that is commonly found in human microflora. Biofilm formation (BF) is known as a major virulence factor of C. albicans. The aim of this study was to examine the influence of bacterial presence on biofilm formation of C. albicans. Materials and Methods The BF of Candida was investigated when it was co-cultured with C. albicans (C. albicans 53, a yeast with a low BF ability, and C. albicans 163, a yeast with high BF ability) and bacteria. BF was assessed with XTT reduction assay. A scanning electron microscope was used to determine the structure of the biofilm, and real-time reverse transcriptase polymerase chain reaction was used to amplify and quantify hyphae-associated genes. Results Co-culturing with two different types of bacteria increased the BF value. Co-culturing with C. albicans 53 and 163 also increased the BF value compared to the value that was obtained when the C. albicans was cultured individually. However, co-culturing with bacteria decreased the BF value of C. albicans, and the BF of C. albicans 163 was markedly inhibited. The expression of adherence and morphology transition related genes were significantly inhibited by co-culturing with live bacteria. Conclusion Bacteria have a negative effect on the formation of biofilm by C. albicans. This mechanism is the result of the suppression of genes associated with the hyphae transition of C. albicans, and bacteria particles physically affected the biofilm architecture and biofilm formation. PMID:24532517

  18. Inhibition of Candida albicans by methanethiol produced by Brevibacterium linens.

    PubMed

    Lewis, B A

    1985-10-01

    Brevibacterium linens was screened for antifungal activity against Candida albicans using several antibiotic assay methods. The growth of C. albicans was inhibited only when the dual culture assay method was employed and using methionine-supplemented media. Results suggest that methanethiol which is produced by B. linens' utilization of methionine is the agent inhibitory to C. albicans' growth.

  19. Mitochondrial type 2C protein phosphatases CaPtc5p, CaPtc6p, and CaPtc7p play vital roles in cellular responses to antifungal drugs and cadmium in Candida albicans.

    PubMed

    Zhao, Yunying; Feng, Jinrong; Li, Jing; Jiang, Linghuo

    2012-12-01

    Type 2C protein phosphatases (PP2C) are monomeric enzymes that require magnesium or manganese ions for their activities. There are seven PP2C genes in Candida albicans. Here, we demonstrate that CaPTC5 encodes a mitochondrial PP2C enzyme. Expression of CaPTC5 transcripts remains constant during the serum-induced hyphal development. Deletion of CaPTC5 does not affect the in vitro filamentation but renders C. albicans cells sensitive to terbinafine and cadmium, and this sensitivity is complemented by the Saccharomyces cerevisiae ScPTC5. Deletion of CaPTC6 does not have any additive effect on, but deletion of CaPTC7 blocks, the terbinafine sensitivity owing to deletion of CaPTC5. In addition, we have shown that deletion of CaPTC6 also renders C. albicans cells sensitive to cadmium, while deletion of CaPTC7 leads to a high cadmium tolerance, and this tolerance is abolished by further deletion of CaPTC5 or CaPTC6. Furthermore, double deletion of CaPTC6 and CaPTC7 renders C. albicans cells more tolerant to azoles, but deletion of CaPTC5 and CaPTC7 slightly increases the azole sensitivity of C. albicans cells. Our results demonstrate that three mitochondrial PP2C genes CaPTC5, CaPTC6 and CaPTC7 interact differentially in the response of C. albicans cells to antifungal drugs and cadmium.

  20. Tissue invasiveness and non-acidic pH in human candidiasis correlate with “in vivo” expression by Candida albicans of the carbohydrate epitope recognised by new monoclonal antibody 1H4

    PubMed Central

    Monteagudo, C; Viudes, A; Lazzell, A; Martinez, J P; Lopez-Ribot, J L

    2004-01-01

    Background: The morphogenetic conversion between yeast and hyphal growth forms appears to be crucial in the pathogenesis of invasive candidiasis, and can be regulated by environmental signals such as extracellular pH. Aims: To characterise the epitope recognised by monoclonal antibody 1H4, and to evaluate the expression of its corresponding epitope in Candida albicans cells under different conditions of pH and temperature, and “in vivo”, in tissue samples from patients with human candidiasis. Methods: Monoclonal antibody 1H4 was generated against the 58 kDa cell wall mannoprotein of C albicans (mp58), and was further characterised by immunoblot analysis, periodate treatment of the antigenic preparations, and agglutination experiments of C albicans strains 3153A, SC5314, and 412, cultured under different environmental conditions (growth media and pH). An immunohistochemical study was performed in 24 human tissue samples from patients with mucocutaneous and systemic candidiasis. Results: 1H4 recognises a pH sensitive carbohydrate epitope on the surface of C albicans cells, and this epitope is not restricted to mp58, but is shared with other cell wall mannoproteins. Immunohistochemical findings indicated that expression of the 1H4 epitope on C albicans cells in tissue sections from human candidiasis correlates with tissue invasion and pH of the niche. 1H4 immunoreactivity was also found in candida remnants within macrophages. Conclusions: The fact that 1H4 epitope expression selectively identifies invasive forms of C albicans, in addition to candida remnants within macrophages, supports its potential value in the diagnosis and management of human candidiasis. PMID:15166263

  1. Extracellular matrix signaling in morphogenesis and repair.

    PubMed

    Clause, Kelly C; Barker, Thomas H

    2013-10-01

    The extracellular matrix (ECM) is critically important for many cellular processes including growth, differentiation, survival, and morphogenesis. Cells remodel and reshape the ECM by degrading and reassembling it, playing an active role in sculpting their surrounding environment and directing their own phenotypes. Both mechanical and biochemical molecules influence ECM dynamics in multiple ways; by releasing small bioactive signaling molecules, releasing growth factors stored within the ECM, eliciting structural changes to matrix proteins which expose cryptic sites and by degrading matrix proteins directly. The dynamic reciprocal communication between cells and the ECM plays a fundamental roll in tissue development, homeostasis, and wound healing.

  2. Review: cornification, morphogenesis and evolution of feathers.

    PubMed

    Alibardi, Lorenzo

    2017-05-01

    Feathers are corneous microramifications of variable complexity derived from the morphogenesis of barb ridges. Histological and ultrastructural analyses on developing and regenerating feathers clarify the three-dimensional organization of cells in barb ridges. Feather cells derive from folds of the embryonic epithelium of feather germs from which barb/barbule cells and supportive cells organize in a branching structure. The following degeneration of supportive cells allows the separation of barbule cells which are made of corneous beta-proteins and of lower amounts of intermediate filament (IF)(alpha) keratins, histidine-rich proteins, and corneous proteins of the epidermal differentiation complex. The specific protein association gives rise to a corneous material with specific biomechanic properties in barbules, rami, rachis, or calamus. During the evolution of different feather types, a large expansion of the genome coding for corneous feather beta-proteins occurred and formed 3-4-nm-thick filaments through a different mechanism from that of 8-10 nm IF keratins. In the chick, over 130 genes mainly localized in chromosomes 27 and 25 encode feather corneous beta-proteins of 10-12 kDa containing 97-105 amino acids. About 35 genes localized in chromosome 25 code for scale proteins (14-16 kDa made of 122-146 amino acids), claws and beak proteins (14-17 kDa proteins of 134-164 amino acids). Feather morphogenesis is periodically re-activated to produce replacement feathers, and multiple feather types can result from the interactions of epidermal and dermal tissues. The review shows schematic models explaining the translation of the morphogenesis of barb ridges present in the follicle into the three-dimensional shape of the main types of branched or un-branched feathers such as plumulaceous, pennaceous, filoplumes, and bristles. The temporal pattern of formation of barb ridges in different feather types and the molecular control from the dermal papilla through

  3. Mechanisms of thymus organogenesis and morphogenesis

    PubMed Central

    Gordon, Julie; Manley, Nancy R.

    2011-01-01

    The thymus is the primary organ responsible for generating functional T cells in vertebrates. Although T cell differentiation within the thymus has been an area of intense investigation, the study of thymus organogenesis has made slower progress. The past decade, however, has seen a renewed interest in thymus organogenesis, with the aim of understanding how the thymus develops to form a microenvironment that supports T cell maturation and regeneration. This has prompted modern revisits to classical experiments and has driven additional genetic approaches in mice. These studies are making significant progress in identifying the molecular and cellular mechanisms that control specification, early organogenesis and morphogenesis of the thymus. PMID:21862553

  4. The Botrytis cinerea PAK kinase BcCla4 mediates morphogenesis, growth and cell cycle regulating processes downstream of BcRac.

    PubMed

    Minz-Dub, Anna; Sharon, Amir

    2017-02-06

    Rac proteins are involved in a variety of cellular processes. Effector proteins that interact with active Rac convey the GTPase-generated signal to downstream developmental cascades and processes. Here we report on the analysis of the main effector and signal cascade downstream of BcRac, the Rac homolog of the grey mold fungus Botrytis cinerea. Several lines of evidence highlighted the p21-activated kinase Cla4 as an important effector of Rac in fungi. Analysis of Δbccla4 strains revealed that the BcCla4 protein was sufficient to mediate all of the examined BcRac-driven processes, including hyphal growth and morphogenesis, conidia production and pathogenicity. In addition, the Δbccla4 strains had altered nuclei content, a phenomenon that was previously observed in Δbcrac isolates, thus connecting the BcRac/BcCla4 module with cell cycle control. Further analyses revealed that BcRac/BcCla4 control mitotic entry through changes in phosphorylation status of the cyclin dependent kinase BcCdk1. The complete cascade includes the kinase BcWee1, which is downstream of BcCla4 and upstream of BcCdk1. These results provide a mechanistic insight on the connection of cell cycle, morphogenesis and pathogenicity in fungi, and position BcCla4 as the most essential effector and central regulator of all of these processes downstream of BcRac.

  5. To shape a cell: an inquiry into the causes of morphogenesis of microorganisms.

    PubMed Central

    Harold, F M

    1990-01-01

    We recognize organisms first and foremost by their forms, but how they grow and shape themselves still largely passes understanding. The objective of this article is to survey what has been learned of morphogenesis of walled eucaryotic microorganisms as a set of problems in cellular heredity, biochemistry, physiology, and organization. Despite the diversity of microbial forms and habits, some common principles can be discerned. (i) That the form of each organism represents the expression of a genetic program is almost universally taken for granted. However, reflection on the findings with morphologically aberrant mutants suggests that the metaphor of a genetic program is misleading. Cellular form is generated by a web of interacting chemical and physical processes, whose every strand is woven of multiple gene products. The relationship between genes and form is indirect and cumulative; therefore, morphogenesis must be addressed as a problem not of molecular genetics but of cellular physiology. (ii) The shape of walled cells is determined by the manner in which the wall is laid down during growth and development. Turgor pressure commonly, perhaps always, supplies the driving force for surface enlargement. Cells yield to this scalar force by localized, controlled wall synthesis; their forms represent variations on the theme of local compliance with global force. (iii) Growth and division in bacteria display most immediately the interplay of hydrostatic pressure, localized wall synthesis, and structural constraints. Koch's surface stress theory provides a comprehensive and quantitative framework for understanding bacterial shapes. (iv) In the larger and more versatile eucaryotic cells, expansion is mediated by the secretion of vesicles. Secretion and ancillary processes, such as cytoplasmic transport, are spatially organized on the micrometer scale. The diversity of vectorial physiology and of the forms it generates is illustrated by examples: apical growth of fungal

  6. Ras GTPases Modulate Morphogenesis, Sporulation and Cellulase Gene Expression in the Cellulolytic Fungus Trichoderma reesei

    PubMed Central

    Zhang, Jiwei; Zhang, Yanmei; Zhong, Yaohua; Qu, Yinbo; Wang, Tianhong

    2012-01-01

    Background The model cellulolytic fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is capable of responding to environmental cues to compete for nutrients in its natural saprophytic habitat despite its genome encodes fewer degradative enzymes. Efficient signalling pathways in perception and interpretation of environmental signals are indispensable in this process. Ras GTPases represent a kind of critical signal proteins involved in signal transduction and regulation of gene expression. In T. reesei the genome contains two Ras subfamily small GTPases TrRas1 and TrRas2 homologous to Ras1 and Ras2 from S. cerevisiae, but their functions remain unknown. Methodology/Principal Findings Here, we have investigated the roles of GTPases TrRas1 and TrRas2 during fungal morphogenesis and cellulase gene expression. We show that both TrRas1 and TrRas2 play important roles in some cellular processes such as polarized apical growth, hyphal branch formation, sporulation and cAMP level adjustment, while TrRas1 is more dominant in these processes. Strikingly, we find that TrRas2 is involved in modulation of cellulase gene expression. Deletion of TrRas2 results in considerably decreased transcription of cellulolytic genes upon growth on cellulose. Although the strain carrying a constitutively activated TrRas2G16V allele exhibits increased cellulase gene transcription, the cbh1 and cbh2 expression in this mutant still strictly depends on cellulose, indicating TrRas2 does not directly mediate the transmission of the cellulose signal. In addition, our data suggest that the effect of TrRas2 on cellulase gene is exerted through regulation of transcript abundance of cellulase transcription factors such as Xyr1, but the influence is independent of cAMP signalling pathway. Conclusions/Significance Together, these findings elucidate the functions for Ras signalling of T. reesei in cellular morphogenesis, especially in cellulase gene expression, which contribute to deciphering the

  7. Comparison of albicans vs. non-albicans candidemia in French intensive care units

    PubMed Central

    2010-01-01

    Introduction Candidemia raises numerous therapeutic issues for intensive care physicians. Epidemiological data that could guide the choice of initial therapy are still required. This analysis sought to compare the characteristics of intensive care unit (ICU) patients with candidemia due to non-albicans Candida species with those of ICU patients with candidemia due to Candida albicans. Methods A prospective, observational, multicenter, French study was conducted from October 2005 to May 2006. Patients exhibiting candidemia developed during ICU stay and exclusively due either to one or more non-albicans Candida species or to C. albicans were selected. The data collected included patient characteristics on ICU admission and at the onset of candidemia. Results Among the 136 patients analyzed, 78 (57.4%) had candidemia caused by C. albicans. These patients had earlier onset of infection (11.1 ± 14.2 days after ICU admission vs. 17.4 ± 17.7, p = 0.02), higher severity scores on ICU admission (SOFA: 10.4 ± 4.7 vs. 8.6 ± 4.6, p = 0.03; SAPS II: 57.4 ± 22.8 vs. 48.7 ± 15.5, P = 0.015), and were less often neutropenic (2.6% vs. 12%, p = 0.04) than patients with candidemia due to non-albicans Candida species. Conclusions Although patients infected with Candida albicans differed from patients infected with non-albicans Candida species for a few characteristics, no clinical factor appeared pertinent enough to guide the choice of empirical antifungal therapy in ICU. PMID:20507569

  8. Comparative proteomic analysis reveals intracellular targets for bacillomycin L to induce Rhizoctonia solani Kühn hyphal cell death.

    PubMed

    Zhang, Bao; Qin, Yuxuan; Han, Yuzhu; Dong, Chunjuan; Li, Pinglan; Shang, Qingmao

    2016-09-01

    Bacillomycin L, a natural iturinic lipopeptide produced by Bacillus amyloliquefaciens, is characterized by strong antifungal activity against a variety of agronomically important filamentous fungi including Rhizoctonia solani Kühn. To further understand its antifungal actions, proteomes were comparatively studied within R. solani hyphal cells treated with or without bacillomycin L. The results show that 39 proteins were alternatively expressed within cells in response to this lipopeptide, which are involved in stress response, carbohydrate, amino acid and nucleotide metabolism, cellular component organization, calcium homeostasis, protein degradation, RNA processing, gene transcription, and others, suggesting that, in addition to inducing cell membrane permeabilization, iturin exhibits antibiotic activities by targeting intracellular molecules. Based on these results, a model of action of bacillomycin L against R. solani hyphal cells was proposed. Our study provides new insight into the antibiotic mechanisms of iturins.

  9. Ca2+ dynamics in zebrafish morphogenesis

    PubMed Central

    Tsutsui, Kenta; Ogawa, Tomohisa

    2017-01-01

    Intracellular calcium ion (Ca2+) signaling is heavily involved in development, as illustrated by the use of a number of Ca2+ indicators. However, continuous Ca2+ patterns during morphogenesis have not yet been studied using fluorescence resonance energy transfer to track the Ca2+ sensor. In the present study, we monitored Ca2+ levels during zebrafish morphogenesis and differentiation with yellow cameleon, YC2.12. Our results show not only clear changes in Ca2+ levels but also continuous Ca2+ patterns at 24 hpf and later periods for the first time. Serial Ca2+dynamics during early pharyngula period (Prim-5-20; 24–33 hpf) was successfully observed with cameleon, which have not reported anywhere yet. In fact, high Ca2+ level occurred concurrently with hindbrain development in segmentation and pharyngula periods. Ca2+ patterns in the late gastrula through segmentation periods which were obtained with cameleon, were similar to those obtained previously with other Ca2+sensor. Our results suggested that the use of various Ca2+ sensors may lead to novel findings in studies of Ca2+ dynamics. We hope that these results will prove valuable for further research in Ca2+ signaling. PMID:28133572

  10. Homophilic Dscam interactions control complex dendrite morphogenesis

    PubMed Central

    Hughes, Michael E.; Bortnick, Rachel; Tsubouchi, Asako; Bäumer, Philipp; Kondo, Masahiro; Uemura, Tadashi; Schmucker, Dietmar

    2007-01-01

    Summary The morphogenesis of complex dendritic fields requires highly specific patterning and dendrite-dendrite recognition mechanisms. Alternative splicing of the Drosophila cell surface receptor Dscam results in up to 38,016 different receptor isoforms and in vitro binding studies suggested that sequence variability in immunoglobulin-like ecto-domains determines the specificity of strictly homophilic interactions. We report that diverse Dscam receptors play an important role in controlling cell-intrinsic aspects of dendrite guidance. We examined the function of Dscam during morphogenesis of dendrite arborization neurons (“da” neurons) and found that loss of Dscam in single neurons causes abnormal dendritic fasciculation and a strong increase in self-crossing of dendritic branches of da neurons. Restriction of dendritic fields of neighboring class III neurons appeared intact in Dscam deficient neurons suggesting that dendritic self-avoidance but not hetero-neuronal tiling may depend on Dscam function. Over-expression of the same Dscam isoforms in two da neurons with normally overlapping dendritic fields forced a spatial segregation of the two dendritic fields. Taken together, our results suggest that dendritic branches of all four classes of da neurons use isoform-specific homophilic interactions of Dscam to ensure minimal overlap of dendrites. The large pool of Dscam’s extracellular recognition domains may allow the same ‘core’ repulsion mechanism to be used in every da neuron without interfering with hetero-neuronal interactions. PMID:17481395

  11. Hyphal tip extension in Aspergillus nidulans requires the manA gene, which encodes phosphomannose isomerase.

    PubMed Central

    Smith, D J; Payton, M A

    1994-01-01

    A strain of Aspergillus nidulans carrying a temperature-sensitive mutation in the manA gene produces cell walls depleted of D-mannose and forms hyphal tip balloons at the restrictive temperature (B.P. Valentine and B.W. Bainbridge, J. Gen. Microbiol. 109:155-168, 1978). We have isolated and characterized the manA gene and physically located it between 3.5 and 5.5 kb centromere distal of the riboB locus on chromosome VIII. The manA gene contains four introns and encodes a 50.6-kDa protein which has significant sequence identity to type I phosphomannose isomerase proteins from other eukaryotes. We have constructed by integrative transformation a null mutation in the manA gene which can only be maintained in a heterokaryotic strain with wild-type manA+ nuclei. Thus, a manA null mutation is lethal in A. nidulans. The phenotype of the mutation was analyzed in germinating conidia. Such conidia are able to commence germination but swell abnormally, sometimes producing a misshapen germ tube, before growth ceases. The reason for the lethality is probably the lack of synthesis of mannose-containing cell wall polymers that must be required for normal cell wall development in growing hyphae. Images PMID:8065336

  12. Neurospora COP9 signalosome integrity plays major roles for hyphal growth, conidial development, and circadian function.

    PubMed

    Zhou, Zhipeng; Wang, Ying; Cai, Gaihong; He, Qun

    2012-01-01

    The COP9 signalosome (CSN) is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EX(n)HXHX(10)D) of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCF(FWD-1) complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ) in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa.

  13. Hyphal responses of Neurospora crassa to micron-sized beads with functional chemical surface groups

    NASA Astrophysics Data System (ADS)

    Held, Marie; Edwards, Clive; Nicolau, Dan V.

    2011-02-01

    Filamentous fungi include serious plant and animal pathogens that explore their environment efficiently in order to penetrate the host. This environment is physically and chemically heterogeneous and the fungi rely on specific physical and chemical signals to find the optimal point/s of attack. This study presents a methodology to introduce distinct structures with dimensions similar to the hyphal diameter and specific chemical surface groups into a controllable environment in order to study the fungal response. We introduced 3.3 μm polystyrene beads covered with Epoxy surface groups into microfluidic channels made from PDMS by rapid replica molding. The experimental setup resulted in different areas with low and high densities of beads as well as densely packed patches. The observations of the fungus exploring the areas long-term showed that the growth parameters were altered significantly, compared with the values measured on agar. The fungus responded to both, the physical and chemical parameters of the beads, including temporary directional changes, increased branching angles, decreased branching distances, decreased apical extension velocities and occasional cell wall lysis. The wealth and magnitude of the observed responses indicates that the microfluidic structures provide a powerful platform for the investigation of micron-sized features on filamentous fungi.

  14. Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici.

    PubMed

    Ma, W; Kilaru, S; Collins, C; Courbot, M; Steinberg, G

    2015-06-01

    Pathogenic fungi are constantly emerging resistance to anti-fungal treatments. Therefore, identification of new fungicide targets is important. Good candidates are essential fungal proteins and their regulators. An efficient way to reveal the molecular environment of an essential protein is the search for interacting factors. Here, we establish three yeast two-hybrid libraries, covering yeast and hyphal stages of the wheat pathogen Zymoseptoria tritici. No detectable genomic DNA was present in any of the 3 libraries. Random amplification revealed that the libraries include cDNA fragments of up to 2000bp, suggesting that small-to-medium sized proteins are represented therein. Indeed, full-length cDNAs of five proteins were found in all libraries. The full-length cDNA of large chitin synthase gene mcs1 (5742bp with introns; 5568bp without introns) could not be amplified, but its 5' and 3' regions were represented, suggesting that even larger genes are covered in all libraries. Finally, we tested for the expected interaction of the autophagy proteins ZtAtg4 and ZtAtg8 in Z. tritici, and then used ZtAtg4 to screen one of the two-hybrid libraries. Indeed, we found ZtAtg8 as a positive interaction partner, confirming that interacting proteins can be identified. Thus, these molecular tools promise to be useful in identifying novel fungicide target proteins.

  15. HOY1, a homeo gene required for hyphal formation in Yarrowia lipolytica.

    PubMed Central

    Torres-Guzmán, J C; Domínguez, A

    1997-01-01

    The dimorphic fungus Yarrowia lipolytica grows to form hyphae either in rich media or in media with GlcNAc as a carbon source. A visual screening, called FIL (filamentation minus), for Y. lipolytica yeast growth mutants has been developed. The FIL screen was used to identify three Y. lipolytica genes that abolish hypha formation in all media assayed. Y. lipolytica HOY1, a gene whose deletion prevents the yeast-hypha transition both in liquid and solid media, was characterized. HOY1 is predicted to encode a 509-amino-acid protein with a homeodomain homologous to that found in the chicken Hox4.8 gene. Analysis of the protein predicts a nuclear location. These observations suggest that Hoy1p may function as a transcriptional regulatory protein. In disrupted strains, reintroduction of HOY1 restored the capacity for hypha formation. Northern blot hybridization revealed the HOY1 transcript to be approximately 1.6 kb. Expression of this gene was detected when Y. lipolytica grew as a budding yeast, but an increase in its expression was observed by 1 h after cells had been induced to form hyphae. The possible functions of HOY1 in hyphal growth and the uses of the FIL screen to identify morphogenetic regulatory genes from heterologous organisms are discussed. PMID:9343389

  16. Virulence Attributes and Hyphal Growth of C. neoformans Are Quantitative Traits and the MATα Allele Enhances Filamentation

    PubMed Central

    Lin, Xiaorong; Huang, Johnny C; Mitchell, Thomas G; Heitman, Joseph

    2006-01-01

    Cryptococcus neoformans is a fungal human pathogen with a bipolar mating system. It undergoes a dimorphic transition from a unicellular yeast to hyphal filamentous growth during mating and monokaryotic fruiting. The traditional sexual cycle that leads to the production of infectious basidiospores involves cells of both α and a mating type. Monokaryotic fruiting is a modified form of sexual reproduction that involves cells of the same mating type, most commonly α, which is the predominant mating type in both the environment and clinical isolates. However, some a isolates can also undergo monokaryotic fruiting. To determine whether mating type and other genetic loci contribute to the differences in fruiting observed between α and a cells, we applied quantitative trait loci (QTL) mapping to an inbred population of F2 progeny. We discovered that variation in hyphal length produced during fruiting is a quantitative trait resulting from the combined effects of multiple genetic loci, including the mating type (MAT) locus. Importantly, the α allele of the MAT locus enhanced hyphal growth compared with the a allele. Other virulence traits, including melanization and growth at 39 °C, also are quantitative traits that share a common QTL with hyphal growth. The Mac1 transcription factor, encoded in this common QTL, regulates copper homeostasis. MAC1 allelic differences contribute to phenotypic variation, and mac1Δ mutants exhibit defects in filamentation, melanin production, and high temperature growth. Further characterization of these QTL regions will reveal additional quantitative trait genes controlling biological processes central to fungal development and pathogenicity. PMID:17112316

  17. Carlactone-type strigolactones and their synthetic analogues as inducers of hyphal branching in arbuscular mycorrhizal fungi.

    PubMed

    Mori, Narumi; Nishiuma, Kenta; Sugiyama, Takuya; Hayashi, Hideo; Akiyama, Kohki

    2016-10-01

    Hyphal branching in the vicinity of host roots is a host recognition response of arbuscular mycorrhizal fungi. This morphological event is elicited by strigolactones. Strigolactones are carotenoid-derived terpenoids that are synthesized from carlactone and its oxidized derivatives. To test the possibility that carlactone and its oxidized derivatives might act as host-derived precolonization signals in arbuscular mycorrhizal symbiosis, carlactone, carlactonoic acid, and methyl carlactonoate as well as monohydroxycarlactones, 4-, 18-, and 19-hydroxycarlactones, were synthesized chemically and evaluated for hyphal branching-inducing activity in germinating spores of the arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal branching activity was found to correlate with the degree of oxidation at C-19 methyl. Carlactone was only weakly active (100 ng/disc), whereas carlactonoic acid showed comparable activity to the natural canonical strigolactones such as strigol and sorgomol (100 pg/disc). Hydroxylation at either C-4 or C-18 did not significantly affect the activity. A series of carlactone analogues, named AD ester and AA'D diester, was synthesized by reacting formyl Meldrum's acid with benzyl, cyclohexylmethyl, and cyclogeranyl alcohols (the A-ring part), followed by coupling of the potassium enolates of the resulting formylacetic esters with the D-ring butenolide. AD ester analogues exhibited moderate activity (1 ng-100 pg/disc), while AA'D diester analogues having cyclohexylmethyl and cyclogeranyl groups were highly active on the AM fungus (10 pg/disc). These results indicate that the oxidation of methyl to carboxyl at C-19 in carlactone is a prerequisite but BC-ring formation is not essential to show hyphal branching activity comparable to that of canonical strigolactones.

  18. A Trypsin Inhibitor from Tecoma stans Leaves Inhibits Growth and Promotes ATP Depletion and Lipid Peroxidation in Candida albicans and Candida krusei

    PubMed Central

    Patriota, Leydianne L. S.; Procópio, Thamara F.; de Souza, Maria F. D.; de Oliveira, Ana Patrícia S.; Carvalho, Lidiane V. N.; Pitta, Maira G. R.; Rego, Moacyr J. B. M.; Paiva, Patrícia M. G.; Pontual, Emmanuel V.; Napoleão, Thiago H.

    2016-01-01

    Tecoma stans (yellow elder) has shown medicinal properties and antimicrobial activity. Previous reports on antifungal activity of T. stans preparations and presence of trypsin inhibitor activity from T. stans leaves stimulated the investigation reported here. In this work, we proceeded to the purification and characterization of a trypsin inhibitor (TesTI), which was investigated for anti-Candida activity. Finally, in order to determine the potential of TesTI as a new natural chemotherapeutic product, its cytotoxicity to human peripheral blood mononuclear cells (PBMCs) was evaluated. TesTI was isolated from saline extract by ammonium sulfate fractionation followed by ion exchange and gel filtration chromatographies. Antifungal activity was evaluated by determining the minimal inhibitory (MIC) and fungicide (MFC) concentrations using fungal cultures containing only yeast form or both yeast and hyphal forms. Candida cells treated with TesTI were evaluated for intracellular ATP levels and lipid peroxidation. Cytotoxicity of TesTI to PBMCs was evaluated by MTT assay. TesTI (39.8 kDa, pI 3.41, Ki 43 nM) inhibited similarly the growth of both C. albicans and C. krusei culture types at MIC of 100 μg/mL. The MFCs were 200 μg/mL for C. albicans and C. krusei. Time-response curves revealed that TesTI (at MIC) was more effective at inhibiting the replication of C. albicans cells. At MIC, TesTI promoted reduction of ATP levels and lipid peroxidation in the Candida cells, being not cytotoxic to PBMCs. In conclusion, TesTI is an antifungal agent against C. albicans and C. krusei, without toxicity to human cells. PMID:27199940

  19. A Gin4-Like Protein Kinase GIL1 Involvement in Hyphal Growth, Asexual Development, and Pathogenesis in Fusarium graminearum

    PubMed Central

    Yu, Dan; Zhang, Shijie; Li, Xiaoping; Xu, Jin-Rong; Schultzhaus, Zachary; Jin, Qiaojun

    2017-01-01

    Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) on wheat and barley. In a previous study, a GIN4-like protein kinase gene, GIL1, was found to be important for plant infection and sexual reproduction. In this study we further characterized the functions of GIL1 kinase in different developmental processes. The Δgil1 mutants were reduced in growth, conidiation, and virulence, and formed whitish and compact colonies. Although phialide formation was rarely observed in the mutants, deletion of GIL1 resulted in increased hyphal branching and increased tolerance to cell wall and cell membrane stresses. The Δgil1 mutants produced straight, elongated conidia lacking of distinct foot cells and being delayed in germination. Compared with the wild type, some compartments in the vegetative hyphae of Δgil1 mutants had longer septal distances and increased number of nuclei, suggesting GIL1 is related to cytokinesis and septation. Localization of the GIL1-GFP fusion proteins to the septum and hyphal branching and fusion sites further supported its roles in septation and branching. Overall, our results indicate that GIL1 plays a role in vegetative growth and plant infection in F. graminearum, and is involved in septation and hyphal branching. PMID:28212314

  20. Visualization of wound periderm and hyphal profiles in pine stems inoculated with the pitch canker fungus Fusarium circinatum.

    PubMed

    Kim, Ki Woo; Lee, In Jung; Thoungchaleun, Vilakon; Kim, Chang Soo; Lee, Don Koo; Park, Eun Woo

    2009-12-01

    Postpenetration behavior of Fusarium circinatum in stems of pine species was investigated with light and transmission electron microscopy. Two-year-old stems of Pinus rigida and P. densiflora were wound-inoculated with the fungal conidial suspension and subjected to 25 degrees C for up to 30 days. It was common to observe the formation of wound periderm on each pine species, recovering wounded sites with newly formed tissues. The outermost thick layer of wound periderm was pink to red colored with the phloroglucinol-EtOH staining, indicating heavy deposition of lignin in wound periderm. The cork layers in the wound periderm of the two pine species consisted of cells that were mostly devoid of cellular contents in cytoplasm. The cork cells showed convoluted cell walls with different electron density (lamellations), which was seemingly more prevalent in P. densiflora than P. rigida. Hyphae of F. circinatum appeared normal with typical eucaryotic cytoplasm in P. rigida on ultrathin sections. Meanwhile, hyphae in P. densiflora were found to possess highly vacuolated cytoplasm, implying hyphal weakening and disintegration. Hyphal cytoplasm appeared to be a thin layer between the vacuole and the plasma membrane surrounded by cell wall. In addition, intrahyphal hyphae and concentric bodies were observed in hyphal cytoplasm. These results suggest that the architecture of wound periderm may be responsible for different responses of pine species to the invasion of F. circinatum.

  1. Cytology and molecular phylogenetics of Monoblepharidomycetes provide evidence for multiple independent origins of the hyphal habit in the Fungi.

    PubMed

    Dee, Jaclyn M; Mollicone, Marilyn; Longcore, Joyce E; Roberson, Robert W; Berbee, Mary L

    2015-01-01

    The evolution of filamentous hyphae underlies an astounding diversity of fungal form and function. We studied the cellular structure and evolutionary origins of the filamentous form in the Monoblepharidomycetes (Chytridiomycota), an early-diverging fungal lineage that displays an exceptional range of body types, from crescent-shaped single cells to sprawling hyphae. To do so, we combined light and transmission electron microscopic analyses of hyphal cytoplasm with molecular phylogenetic reconstructions. Hyphae of Monoblepharidomycetes lack a complex aggregation of secretory vesicles at the hyphal apex (i.e. Spitzenkörper), have centrosomes as primary microtubule organizing centers and have stacked Golgi cisternae instead of tubular/fenestrated Golgi equivalents. The cytoplasmic distribution of actin in Monoblepharidomycetes is comparable to the arrangement observed previously in other filamentous fungi. To discern the origins of Monoblepharidomycetes hyphae, we inferred a phylogeny of the fungi based on 18S and 28S ribosomal DNA sequence data with maximum likelihood and Bayesian inference methods. We focused sampling on Monoblepharidomycetes to infer intergeneric relationships within the class and determined 78 new sequences. Analyses showed class Monoblepharidomycetes to be monophyletic and nested within Chytridiomycota. Hyphal Monoblepharidomycetes formed a clade sister to the genera without hyphae, Harpochytrium and Oedogoniomyces. A likelihood ancestral state reconstruction indicated that hyphae arose independently within the Monoblepharidomycetes lineage and in at least two other lineages. Cytological differences among monoblepharidalean and other fungal hyphae are consistent with these convergent origins.

  2. Kexin-Like Endoprotease KexB is Required for N-Glycan Processing, Morphogenesis and Virulence in Aspergillus fumigatus

    PubMed Central

    Wang, Jingyang; Zhou, Hui; Lu, Hua; Du, Ting; Luo, Yuanming; Wilson, Iain B. H.; Jin, Cheng

    2015-01-01

    Kexin-like proteins belong to the subtilisin-like family of the proteinases that cleave secretory proproteins to their active forms. Several fungal kexin-like proteins have been investigated. The mutants lacking of kexin-like protein display strong phenotypes such as cell wall defect, abnormal polarity, and, in case of Candida albicans, diminished virulence. However, only several proteins have been confirmed as the substrates of kexin-like proteases in these fungal species. It still remains unclear how kexin-like proteins contribute to the morphogenesis in these fungal species. In this study, a kexB-null mutant of the human opportunistic fungal pathogen Aspergillus fumigatus was constructed and analyzed. The ΔkexB mutant showed retarded growth, temperature-sensitive cell wall defect, reduced conidia formation, and abnormal polarity. Biochemical analyses revealed that deletion of the kexB gene resulted in impaired N-glycan processing, activation of the MpkA-dependent cell wall integrity signaling pathway, and ER-stress. Results from in vivo assays demonstrated that the mutant exhibited an attenuated virulence in immunecompromised mice. Based on our results, the kexin-like endoprotease KexB was involved in the N-glycan processing, which provides a novel insight to understand how kexin-like protein affects the cell-wall modifying enzymes and therefore morphogenesis in fungi. PMID:25687931

  3. Adherence and receptor relationships of Candida albicans.

    PubMed Central

    Calderone, R A; Braun, P C

    1991-01-01

    The cell surface of Candida albicans is composed of a variety of polysaccharides such as glucan, chitin, and mannan. The first two components primarily provide structure, while the mannan, often covalently linked to protein, constitutes the major antigen of the organism. Mannoproteins also have enzymatic activity (acid protease) and ligand-receptor functions. The complement receptors of C. albicans appear to be mannoproteins that are required for the adherence of the organism to endothelial cells. This is certainly true of the CR3-like protein of C. albicans. Proof that the CR3 is the Candida receptor for endothelial cells is derived from two observations. First, mutants lacking CR3 activity are less adherent in vitro and, in fact, less virulent. Second, the ligand recognized by the CR3 receptor (C3bi) as well as anti-CR3 antibodies blocks adherence of the organism to endothelial cells. The CR2 of C. albicans appears to promote the adherence of the organism to plastic substrates. Unlike the CR2 of mammalian cells, the Candida CR2 recognizes ligands containing the RGD sequence of amino acids in addition to the C3d ligand, which does not contain the RGD sequence. There is uncertainty as to whether the Candida CR2 and CR3 are, in fact, different proteins. A mannoprotein has also been described as the adhesin for epithelial cells. In this case, the receptor has a lectinlike activity and recognizes fucose- or glucosamine-containing glycoproteins of epithelial cells, depending on the strain of C. albicans. The oligosaccharide component of the receptor is probably not involved in ligand recognition and may serve to stabilize the receptor. However, the oligosaccharide factor 6 epitope of mannan may also provide adhesin activity in the recognition of epithelial cells. Mannoproteins can be extracted from cells by a number of reagents. Zymolyase, for instance, tends to remove structural mannoproteins, which contain relatively little protein and are linked to glucan. Reagents

  4. Morphogenesis in Belousov-Zhabotinsky microdroplets

    NASA Astrophysics Data System (ADS)

    Li, Ning; Tompkins, Nathan; Girabawe, Camille; Epstein, Irving; Fraden, Seth; Brandeis/Mrsec Team

    2013-03-01

    We present experimental evidence for the six cases Alan Turing predicted using linear stability analysis in his 1952 paper ``The chemical basis of morphogenesis'' in our reaction diffusion system. Our experimental system consists of a microfluidically generated microemulsion consisting of Ru(bipy)3 catalyzed light sensitive BZ aqueous droplets which are diffusively coupled through oil gaps. We observed that some droplets grow and others shrink due to the unequal consumption of chemicals in the droplets which leads to an osmotic pressure change, as Turing predicted in his paper. The initial and boundary conditions of our system were controlled by programmable illumination via the light sensitive catalyst Ru(bipy)3. Simulation and linear stability analysis were performed and compared with the experiments. Funded by MRSEC.

  5. Molecular and cellular mechanisms of dendritic morphogenesis

    PubMed Central

    Gao, Fen-Biao

    2008-01-01

    Summary Dendrites exhibit unique cell-type specific branching patterns and targeting specificity that are critically important for neuronal function and connectivity. Recent evidence indicates that highly complex transcriptional regulatory networks dictate various aspects of dendritic outgrowth, branching, and routing. In addition to other intrinsic molecular pathways such as membrane protein trafficking, interactions between neighboring dendritic branches also contribute to the final specification of dendritic morphology. Nonredundant coverage by dendrites of same type of neurons, known as tiling, requires the actions of the Tricornered/Furry (Sax-1/Sax-2) signaling pathway. However, the dendrites of a neuron do not cross over each other, a process called self-avoidance that is mediated by Down’s syndrome cell adhesion molecule (Dscam). Those exciting findings have enhanced significantly our understanding of dendritic morphogenesis and revealed the magnitude of complexity in the underlying molecular regulatory networks. PMID:17933513

  6. Morphogenesis of the human excretory lacrimal system

    PubMed Central

    de la Cuadra-Blanco, C; Peces-Peña, M D; Jáñez-Escalada, L; Mérida-Velasco, J R

    2006-01-01

    The aim of this study was to determine the principal developmental stages in the formation of the excretory lacrimal system in humans and to establish its morphogenetic period. The study was performed using light microscopy on serial sections of 51 human specimens: 33 embryos and 18 fetuses ranging from 8 to 137 mm crown–rump length (CR; 5–16 weeks of development). Three stages were identified in the morphogenesis of the excretory lacrimal system: (1) the formative stage of the lacrimal lamina (Carnegie stages 16–18); (2) the formative stage of the lacrimal cord (Carnegie stages 19–23); and (3) the maturative stage of the excretory lacrimal system, from the 9th week of development onward. A three-dimensional reconstruction of the excretory lacrimal system was performed from serial sections of an embryo at the end of the embryonic period (27 mm CR). PMID:16879594

  7. Palate Morphogenesis: Current Understanding and Future Directions

    PubMed Central

    Greene, Robert M.; Pisano, M. Michele

    2011-01-01

    In the past, most scientists conducted their inquiries of nature via inductivism, the patient accumulation of “pieces of information” in the pious hope that the sum of the parts would clarify the whole. Increasingly, modern biology employs the tools of bioinformatics and systems biology in attempts to reveal the “big picture.” Most successful laboratories engaged in the pursuit of the secrets of embryonic development, particularly those whose research focus is craniofacial development, pursue a middle road where research efforts embrace, rather than abandon, what some have called the “pedestrian” qualities of inductivism, while increasingly employing modern data mining technologies. The secondary palate has provided an excellent paradigm that has enabled examination of a wide variety of developmental processes. Examination of cellular signal transduction, as it directs embryogenesis, has proven exceptionally revealing with regard to clarification of the “facts” of palatal ontogeny—at least the facts as we currently understand them. Herein, we review the most basic fundamentals of orofacial embryology and discuss how functioning of TGFβ, BMP, Shh, and Wnt signal transduction pathways contributes to palatal morphogenesis. Our current understanding of palate medial edge epithelial differentiation is also examined. We conclude with a discussion of how the rapidly expanding field of epigenetics, particularly regulation of gene expression by miRNAs and DNA methylation, is critical to control of cell and tissue differentiation, and how examination of these epigenetic processes has already begun to provide a better understanding of, and greater appreciation for, the complexities of palatal morphogenesis. PMID:20544696

  8. Influence of Hyphal Inoculum potential on the Competitive Success of Fungi Colonizing Wood.

    PubMed

    Song, Zewei; Vail, Andrew; Sadowsky, Michael J; Schilling, Jonathan S

    2015-05-01

    The relative amounts of hyphal inoculum in forest soils may determine the capacity for fungi to compete with and replace early colonizers of wood in ground contact. Our aim in this study was to test the flexibility of priority effects (colonization timing) by varying the timing of inoculum introduction (i.e., precolonization) and amount of inoculum (i.e., inoculum potential). We controlled these variables in soil-block microcosms using fungi with known competitive outcomes in similar conditions, tracking isolate-specific fungal biomass, and residue physiochemistry over time. In the precolonization trial (experiment I), a brown rot fungus Gloeophyllum trabeum was given 1, 3, or 5 weeks to precolonize wood blocks (oak, birch, pine, and spruce) prior the introduction of a white rot fungus, Irpex lacteus, a more aggressive colonizer in this set-up. In the inoculum potential trial (experiment II), the fungi were inoculated simultaneously, but with eightfold higher brown rot inoculum than that of experiment I. As expected, longer precolonization duration increased the chance for the less-competitive brown rot fungus to outcompete its white rot opponent. Higher brown rot fungal inoculum outside of the wood matrix also resulted in competitive success for the brown rot isolate in most cases. These temporal shifts in fungal dominance were detectable in a 'community snapshot' as isolate-specific quantitative PCR, but also as functionally-relevant consequences of wood rot type, including carbohydrate depolymerization and pH. These results from a controlled system reinforce fungal-fungal interaction and suggest that relative inoculum availability beyond the wood matrix (i.e., soils) might regulate the duration of priority effects and shift the functional trajectory of wood decomposition.

  9. Identification of race-specific resistance in North American Vitis spp. limiting Erysiphe necator hyphal growth.

    PubMed

    Ramming, David W; Gabler, Franka; Smilanick, Joseph L; Margosan, Dennis A; Cadle-Davidson, Molly; Barba, Paola; Mahanil, Siraprapa; Frenkel, Omer; Milgroom, Michael G; Cadle-Davidson, Lance

    2012-01-01

    Race-specific resistance against powdery mildews is well documented in small grains but, in other crops such as grapevine, controlled analysis of host-pathogen interactions on resistant plants is uncommon. In the current study, we attempted to confirm powdery mildew resistance phenotypes through vineyard, greenhouse, and in vitro inoculations for test cross-mapping populations for two resistance sources: (i) a complex hybrid breeding line, 'Bloodworth 81-107-11', of at least Vitis rotundifolia, V. vinifera, V. berlandieri, V. rupestris, V. labrusca, and V. aestivalis background; and (ii) Vitis hybrid 'Tamiami' of V. aestivalis and V. vinifera origin. Statistical analysis of vineyard resistance data suggested the segregation of two and three race-specific resistance genes from the two sources, respectively. However, in each population, some resistant progeny were susceptible in greenhouse or in vitro screens, which suggested the presence of Erysiphe necator isolates virulent on progeny segregating for one or more resistance genes. Controlled inoculation of resistant and susceptible progeny with a diverse set of E. necator isolates clearly demonstrated the presence of fungal races differentially interacting with race-specific resistance genes, providing proof of race specificity in the grape powdery mildew pathosystem. Consistent with known race-specific resistance mechanisms, both resistance sources were characterized by programmed cell death of host epidermal cells under appressoria, which arrested or slowed hyphal growth; this response was also accompanied by collapse of conidia, germ tubes, appressoria, and secondary hyphae. The observation of prevalent isolates virulent on progeny with multiple race-specific resistance genes before resistance gene deployment has implications for grape breeding strategies. We suggest that grape breeders should characterize the mechanisms of resistance and pyramid multiple resistance genes with different mechanisms for improved

  10. Molecular Analysis of the Candida albicans Homolog of Saccharomyces cerevisiae MNN9, Required for Glycosylation of Cell Wall Mannoproteins

    PubMed Central

    Southard, Susan B.; Specht, Charles A.; Mishra, Chitra; Chen-Weiner, Joan; Robbins, Phillips W.

    1999-01-01

    The fungal cell wall has generated interest as a potential target for developing antifungal drugs, and the genes encoding glucan and chitin in fungal pathogens have been studied to this end. Mannoproteins, the third major component of the cell wall, contain mannose in either O- or N-glycosidic linkages. Here we describe the molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, a gene required for the synthesis of N-linked outer-chain mannan in yeast, and the phenotypes associated with its disruption. CaMNN9 has significant homology with S. cerevisiae MNN9, including a putative N-terminal transmembrane domain, and represents a member of a similar gene family in Candida. CaMNN9 resides on chromosome 3 and is expressed at similar levels in both yeast and hyphal cells. Disruption of both copies of CaMNN9 leads to phenotypic effects characteristic of cell wall defects including poor growth in liquid media and on solid media, formation of aggregates in liquid culture, osmotic sensitivity, aberrant hyphal formation, and increased sensitivity to lysis after treatment with β-1,3-glucanase. Like all members of the S. cerevisiae MNN9 gene family the Camnn9Δ strain is resistant to sodium orthovanadate and sensitive to hygromycin B. Analysis of cell wall-associated carbohydrates showed the Camnn9Δ strain to contain half the amount of mannan present in cell walls derived from the wild-type parent strain. Reverse transcription-PCR and Northern analysis of the expression of MNN9 gene family members CaVAN1 and CaANP1 in the Camnn9Δ strain showed that transcription of those genes is not affected in the absence of CaMNN9 transcription. Our results suggest that, while the role MNN9 plays in glycosylation in both Candida and Saccharomyces is conserved, loss of MNN9 function in C. albicans leads to phenotypes that are inconsistent with the pathogenicity of the organism and thus identify CaMnn9p as a potential drug target. PMID:10601199

  11. The APSES transcription factor Efg1 is a global regulator that controls morphogenesis and biofilm formation in Candida parapsilosis

    PubMed Central

    Connolly, Leona A; Riccombeni, Alessandro; Grózer, Zsuzsana; Holland, Linda M; Lynch, Denise B; Andes, David R; Gácser, Attila; Butler, Geraldine

    2013-01-01

    Efg1 (a member of the APSES family) is an important regulator of hyphal growth and of the white-to-opaque transition in Candida albicans and very closely related species. We show that in Candida parapsilosis Efg1 is a major regulator of a different morphological switch at the colony level, from a concentric to smooth morphology. The rate of switching is at least 20-fold increased in an efg1 knockout relative to wild type. Efg1 deletion strains also have reduced biofilm formation, attenuated virulence in an insect model, and increased sensitivity to SDS and caspofungin. Biofilm reduction is more dramatic in in vitro than in in vivo models. An Efg1 paralogue (Efh1) is restricted to Candida species, and does not regulate concentric-smooth phenotype switching, biofilm formation or stress response. We used ChIP-seq to identify the Efg1 regulon. A total of 931 promoter regions bound by Efg1 are highly enriched for transcription factors and regulatory proteins. Efg1 also binds to its own promoter, and negatively regulates its expression. Efg1 targets are enriched in binding sites for 93 additional transcription factors, including Ndt80. Our analysis suggests that Efg1 has an ancient role as regulator of development in fungi, and is central to several regulatory networks. PMID:23895281

  12. Comparison of the hemolytic activity between C. albicans and non-albicans Candida species.

    PubMed

    Rossoni, Rodnei Dennis; Barbosa, Júnia Oliveira; Vilela, Simone Furgeri Godinho; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2013-01-01

    The ability to produce enzymes, such as hemolysins, is an important virulence factor for the genus Candida.The objective of this study was to compare the hemolytic activity between C. albicansand non-albicans Candida species. Fifty strains of Candida species, isolated from the oral cavity of patients infected with HIV were studied. The isolates included the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. dubliniensis, C. norvegensis, C. lusitaniae, and C. guilliermondii. Hemolysin production was evaluated on Sabouraud dextrose agar containing chloramphenicol, blood, and glucose. A loop-full of pure Candidaculture was spot-inoculated onto plates and incubated at 37 ºC for 24 h in a 5% CO2 atmosphere. Hemolytic activity was defined as the formation of a translucent halo around the colonies. All C. albicansstrains that were studied produced hemolysins. Among the non-albicans Candidaspecies, 86% exhibited hemolytic activity. Only C. guilliermondiiand some C. parapsilosis isolates were negative for this enzyme. In conclusion, most non-albicans Candidaspecies had a similar ability to produce hemolysins when compared to C. albicans.

  13. Mucosal Immunity and Candida albicans Infection

    PubMed Central

    Moyes, David L.; Naglik, Julian R.

    2011-01-01

    Interactions between mucosal surfaces and microbial microbiota are key to host defense, health, and disease. These surfaces are exposed to high numbers of microbes and must be capable of distinguishing between those that are beneficial or avirulent and those that will invade and cause disease. Our understanding of the mechanisms involved in these discriminatory processes has recently begun to expand as new studies bring to light the importance of epithelial cells and novel immune cell subsets such as Th17 T cells in these processes. Elucidating how these mechanisms function will improve our understanding of many diverse diseases and improve our ability to treat patients suffering from these conditions. In our voyage to discover these mechanisms, mucosal interactions with opportunistic commensal organisms such as the fungus Candida albicans provide insights that are invaluable. Here, we review current knowledge of the interactions between C. albicans and epithelial surfaces and how this may shape our understanding of microbial-mucosal interactions. PMID:21776285

  14. Development of DNA probes for Candida albicans

    SciTech Connect

    Cheung, L.L.; Hudson, J.B.

    1988-07-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both /sup 32/P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.

  15. Direct electrochemical determination of Candida albicans activity.

    PubMed

    Hassan, Rabeay Y A; Bilitewski, Ursula

    2013-11-15

    Despite advances made in the field, rapid detection methods for the human pathogen Candida albicans are still missing. In this regard, bio-electrochemical systems including electrochemical sensors and biosensors satisfy the increasing demand for rapid, reliable, and direct microbial analyses. In this study, the bioelectrochemical characteristics of C. albicans were investigated for use in an analytical system that determines the viability of the organisms. The electrochemical responses of viable and non-viable cells of C. albicans and Saccharomyces cerevisiae were monitored. Cyclic voltammograms (CV) showed an irreversible oxidation peak at about 750 mV that accounts for viable cells. The peak current increased at viable cell numbers ranging from 3 × 10(5) to 1.6 × 10(7)cells/ml, indicating that the amount of viable cells can be accurately quantified. To elucidate the underlying electron transfer processes, the influence of electron transfer chain (ETC) - inhibitors on the electrochemical behavior of the two organisms were investigated. Inhibition of the function of classical respiratory chain (CRC) led to a decrease in the electrochemical response, whereas the oxidation current increased when the alternative oxidase (AOX) pathway was blocked by salicylhydroxamic acid (SHA). Blocking the AOX pathway improved the electrochemical performance, suggesting an involvement in the CRC, with cytochrome c oxidase (COX) as a relevant protein complex. Mutants, in which components of COX were deleted, showed a lower electro-activity than the wild-type strain. Particularly, deletion of subunit COX5a almost completely abolished the electrochemical signal. We believe that this work can be utilized for the development of early detection assays and opens the door for new technological developments in the field of C. albicans.

  16. Candida albicans isolates from a Malaysian hospital exhibit more potent phospholipase and haemolysin activities than non-albicans Candida isolates.

    PubMed

    Chin, V K; Foong, K J; Maha, A; Rusliza, B; Norhafizah, M; Ng, K P; Chong, P P

    2013-12-01

    This study was aimed at determining the phospholipase and haemolysin activity of Candida isolates in Malaysia. A total of 37 Candida clinical isolates representing seven species, Candida albicans (12), Candida tropicalis (8), Candida glabrata (4), Candida parapsilosis (1), Candida krusei (4), Candida orthopsilosis (1) and Candida rugosa (7) were tested. In vitro phospholipase activity was determined by using egg yolk plate assay whereas in vitro haemolysin activity was tested by using blood plate assay on sheep blood Sabouraud's dextrose agar (SDA) enriched with glucose. Phospholipase activity was detected in 75% (9 out of 12) of the C. albicans isolates. Among the 25 non- C. albicans Candida isolates, phospholipase activity was detected in only 24% of these isolates. The phospholipase activity of C. albicans was significantly higher than that of the non- C. albicans Candida isolates (P=0.002). Haemolysin activity was detected in 100% of the C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, and C. orthopsilosis isolates while 75% of the C. krusei isolates and 12.3% of the C. rugosa isolates showed haemolysin activity. The haemolytic activity of C. albicans was significantly higher than that of the non- C. albicans Candida isolates (P=0.0001).The findings in this study indicate that C. albicans isolates in Malaysia may possess greater virulence potential than the non-albicans species.

  17. Epithelial Cell Innate Response to Candida albicans

    PubMed Central

    Naglik, J.R.; Moyes, D.

    2011-01-01

    With the advent of treatments and diseases such as AIDS resulting in increasing numbers of patients with suppressed immune systems, fungal diseases are an escalating problem. Candida albicans is the most common of these fungal pathogens, causing infections in many of these patients. It is therefore important to understand how immunity to this fungus is regulated and how it might be manipulated. Although work has been done to identify the receptors, fungal moieties, and responses involved in anti-Candida immunity, most studies have investigated interactions with myeloid or lymphoid cells. Given that the first site of contact of C. albicans with its host is the mucosal epithelial surface, recent studies have begun to focus on interactions of C. albicans with this site. The results are startling yet in retrospect obvious, indicating that epithelial cells play an important role in these interactions, initiating responses and even providing a level of protection. These findings have obvious implications, not just for fungal pathogens, but also for identifying how host organisms can distinguish between commensal and pathogenic microbes. This review highlights some of these recent findings and discusses their importance in the wider context of infection and immunity. PMID:21441481

  18. Characterization of Mucosal Candida albicans Biofilms

    PubMed Central

    Dongari-Bagtzoglou, Anna; Kashleva, Helena; Dwivedi, Prabhat; Diaz, Patricia; Vasilakos, John

    2009-01-01

    C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide β-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of β-glucan mediated signaling in the host response. PMID:19956771

  19. Killer system: a simple method for differentiating Candida albicans strains.

    PubMed Central

    Polonelli, L; Archibusacci, C; Sestito, M; Morace, G

    1983-01-01

    The killer effect of 37 species of Candida, Cryptococcus, Hansenula, Pichia, Rhodotorula, Saccharomyces, and Trichosporon on 100 Candida albicans isolates of human and animal origin was studied. All of the C. albicans cultures were sensitive to one or more killer yeasts. The factors affecting the killer phenomenon on C. albicans were investigated for realizing a simple system for the differentiation of the 100 C. albicans isolates. By using this system, it was possible to differentiate up to 512 isolates of C. albicans according to their susceptibility to the killer effect of nine selected killer yeasts. The use of this method as an epidemiological marker in the case of presumptive nosocomial infections due to C. albicans is also reported. Images PMID:6345575

  20. Extracellular matrix and growth factors in branching morphogenesis

    NASA Technical Reports Server (NTRS)

    Hardman, P.; Spooner, B. S.

    1993-01-01

    The unifying hypothesis of the NSCORT in gravitational biology postulates that the ECM and growth factors are key interrelated components of a macromolecular regulatory system. The ECM is known to be important in growth and branching morphogenesis of embryonic organs. Growth factors have been detected in the developing embryo, and often the pattern of localization is associated with areas undergoing epithelial-mesenchymal interactions. Causal relationships between these components may be of fundamental importance in control of branching morphogenesis.

  1. Baicalin prevents Candida albicans infections via increasing its apoptosis rate

    SciTech Connect

    Yang, Shulong; Fu, Yingyuan Wu, Xiuzhen; Zhou, Zhixing; Xu, Jing; Zeng, Xiaoping; Kuang, Nanzhen; Zeng, Yurong

    2014-08-15

    Highlights: • Baicalin increases the ratio of the G0/G1 stages and C. albicans apoptosis. • Baicalin decreases the proliferation index of C. albicans. • Baicalin inhibits the biosynthesis of DNA, RNA and protein in C. albicans. • Baicalin depresses Succinate Dehydrogenase and Ca{sup 2+}–Mg{sup 2+} ATPase in C. albicans. • Baicalin increases the endocytic free Ca{sup 2+} concentration in C. albicans. - Abstract: Background: These experiments were employed to explore the mechanisms underlying baicalin action on Candida albicans. Methodology and principal findings: We detected the baicalin inhibition effects on three isotope-labeled precursors of {sup 3}H-UdR, {sup 3}H-TdR and {sup 3}H-leucine incorporation into C. albicans using the isotope incorporation technology. The activities of Succinate Dehydrogenase (SDH), cytochrome oxidase (CCO) and Ca{sup 2+}–Mg{sup 2+} ATPase, cytosolic Ca{sup 2+} concentration, the cell cycle and apoptosis, as well as the ultrastructure of C.albicans were also tested. We found that baicalin inhibited {sup 3}H-UdR, {sup 3}H-TdR and {sup 3}H-leucine incorporation into C.albicans (P < 0.005). The activities of the SDH and Ca{sup 2+}–Mg{sup 2+} ATPase of C.albicans in baicalin groups were lower than those in control group (P < 0.05). Ca{sup 2+} concentrations of C. albicans in baicalin groups were much higher than those in control group (P < 0.05). The ratio of C.albicans at the G0/G1 stage increased in baicalin groups in dose dependent manner (P < 0.01). There were a significant differences in the apoptosis rate of C.albicans between baicalin and control groups (P < 0.01). After 12–48 h incubation with baicalin (1 mg/ml), C. albicans shown to be markedly damaged under transmission electron micrographs. Innovation and significance: Baicalin can increase the apoptosis rate of C. albicans. These effects of Baicalin may involved in its inhibiting the activities of the SDH and Ca{sup 2+}–Mg{sup 2+} ATPase, increasing

  2. Non-albicans Candida Infection: An Emerging Threat

    PubMed Central

    Deorukhkar, Sachin C.; Saini, Santosh

    2014-01-01

    The very nature of infectious diseases has undergone profound changes in the past few decades. Fungi once considered as nonpathogenic or less virulent are now recognized as a primary cause of morbidity and mortality in immunocompromised and severely ill patients. Candida spp. are among the most common fungal pathogens. Candida albicans was the predominant cause of candidiasis. However, a shift toward non-albicans Candida species has been recently observed. These non-albicans Candida species demonstrate reduced susceptibility to commonly used antifungal drugs. In the present study, we investigated the prevalence of non-albicans Candida spp. among Candida isolates from various clinical specimens and analysed their virulence factors and antifungal susceptibility profile. A total of 523 Candida spp. were isolated from various clinical specimens. Non-albicans Candida species were the predominant pathogens isolated. Non-albicans Candida species also demonstrated the production of virulence factors once attributed to Candida albicans. Non-albicans Candida demonstrated high resistance to azole group of antifungal agents. Therefore, it can be concluded that non-albicans Candida species have emerged as an important cause of infections. Their isolation from clinical specimen can no longer be ignored as a nonpathogenic isolate nor can it be dismissed as a contaminant. PMID:25404942

  3. A role for the rap GTPase YlRsr1 in cellular morphogenesis and the involvement of YlRsr1 and the ras GTPase YlRas2 in bud site selection in the dimorphic yeast Yarrowia lipolytica.

    PubMed

    Li, Yun-Qing; Li, Min; Zhao, Xiao-Feng; Gao, Xiang-Dong

    2014-05-01

    Yarrowia lipolytica is a dimorphic yeast species that can grow in the ovoid yeast form or in the elongated pseudohyphal or hyphal form depending on the growth conditions. Here, we show that the Rap GTPase Rsr1 of Y. lipolytica (YlRsr1) plays an important role in cellular morphogenesis in this microorganism. Cells deleted for YlRSR1 exhibited impaired polarized growth during yeast-form growth. Pseudohyphal and hyphal development were also abnormal. YlRsr1 is also important for cell growth, since the deletion of YlRSR1 in cells lacking the Ras GTPase YlRas2 caused lethality. Y. lipolytica cells bud in a bipolar pattern in which the cells produce the new buds at the two poles. YlRsr1 plays a prominent role in this bud site selection process. YlRsr1's function in bud site selection absolutely requires the cycling of YlRsr1 between the GTP- and GDP-bound states but its function in cellular morphogenesis does not, suggesting that the two processes are differentially regulated. Interestingly, the Ras GTPase YlRas2 is also involved in the control of bud site selection, as Ylras2Δ cells were severely impaired in bipolar bud site selection. The GTP/GDP cycling and the plasma membrane localization of YlRas2 are important for YlRas2's function in bud site selection. However, they are not essential for this process, suggesting that the mechanism by which YlRas2 acts is different from that of YlRsr1. Our results suggest that YlRsr1 is regulated by the GTPase-activating protein (GAP) YlBud2 and partially by YlCdc25, the potential guanine nucleotide exchange factor (GEF) for YlRas2.

  4. Candida albicans virulence and drug-resistance requires the O-acyltransferase Gup1p

    PubMed Central

    2010-01-01

    Background GUP1 gene was primarily identified in Saccharomyces cerevisiae being connected with glycerol uptake defects in association with osmotic stress response. Soon after, Gup1p was implicated in a complex and extensive series of phenotypes involving major cellular processes. These include membrane and wall maintenance, lipid composition, bud-site selection, cytoskeleton orientation, vacuole morphology, secretory/endocytic pathway, GPI anchors remodelling, and lipid-ordered domains assembly, which is compatible with their inclusion in the Membrane Bound O-acyl transferases (MBOAT) family. In mammals, it has been described as a negative regulator of the Sonic hedgehog pathway involved in morphogenesis, differentiation, proliferation, among other processes. Results We show that Candida albicans Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors. Furthermore, the mutant colonies exhibited an aberrant morphology/differentiation pattern. Identically to S. cerevisiae, Cagup1Δ null mutant was more resistant to antifungals like fluconazole, ketoconazole, and clotrimazole, and displayed an abnormal even sterol distribution at the plasma membrane. Conclusions This work is the first study in the opportunistic yeast Candida albicans, showing a role for the GUP1 gene in virulence as well as in the mechanisms underlying antifungal resistance. Moreover, its impact is even more significant since these results, taken together with all the knowledge about GUP1 gene (from S. cerevisiae and mammals) give consistence to the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog. PMID:20843317

  5. Induction of germ tube formation by N-acetyl-D-glucosamine in Candida albicans: uptake of inducer and germinative response.

    PubMed Central

    Mattia, E; Carruba, G; Angiolella, L; Cassone, A

    1982-01-01

    A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur. PMID:6752114

  6. An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans.

    PubMed

    Byrd, Angel S; O'Brien, Xian M; Johnson, Courtney M; Lavigne, Liz M; Reichner, Jonathan S

    2013-04-15

    The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as neutrophil extracellular traps (NETs). The receptor/ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Because the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrix, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤ 30 min) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn with β-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on β-glucan recognition by complement receptor 3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by complement receptor 3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid antifungal response.

  7. Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

    SciTech Connect

    Delorme, Caroline; Joshi, Monika; Allingham, John S.

    2012-11-30

    Highlights: Black-Right-Pointing-Pointer The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. Black-Right-Pointing-Pointer The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. Black-Right-Pointing-Pointer The MBP-Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance, we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the 'ATP state' of the mechanochemical cycle. This site differs from the Kar3 neck-core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.

  8. The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans

    PubMed Central

    2011-01-01

    Background The MP65 gene of Candida albicans (orf19.1779) encodes a putative β-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. Results The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of β-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. Conclusions We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus. PMID:21575184

  9. The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

    PubMed Central

    Alvarez, Francisco J.; Douglas, Lois M.; Rosebrock, Adam

    2008-01-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains. PMID:18799621

  10. Characterization of a putative thioredoxin peroxidase prx1 of Candida albicans.

    PubMed

    Srinivasa, Kavitha; Kim, Na-Rae; Kim, Jiwon; Kim, Minsun; Bae, Ju Yun; Jeong, Woojin; Kim, Wankee; Choi, Wonja

    2012-03-01

    In this study, we characterized a putative peroxidase Prx1 of Candida albicans by: 1) demonstrating the thioredoxin-linked peroxidase activity with purified proteins, 2) examining the sensitivity to several oxidants and the accumulation of intracellular reactive oxygen species with a null mutant (prx1Δ), a mutant (C69S) with a point mutation at Cys69, and a revertant, and 3) subcelluar localization. Enzymatic assays showed that Prx1 is a thioredoxin-linked peroxidase which reduces both hydrogen peroxide (H(2)O(2)) and tert-butyl hydroperoxide (t-BOOH). Compared with two other strong H(2)O(2) scavenger mutants for TSA1 and CAT1, prx1Δ and C69S were less sensitive to H(2)O(2), menadione and diamide at all concentrations tested, but were more sensitive to low concentration of t-BOOH. Intracellular reactive oxygen species accumulated in prx1Δ and C69S cells treated with t-BOOH but not H(2)O(2). These results suggest that peroxidase activity of Prx1 is specified to t-BOOH in cells. In both biochemical and physiological cases, the evolutionarily conserved Cys69 was found to be essential for the function. Immunocytochemical staining revealed Prx1 is localized in the cytosol of yeast cells, but is translocated to the nucleus during the hyphal transition, though the significances of this observation are unclear. Our data suggest that PRX1 has a thioredoxin peroxidase activity reducing both t-BOOH and H(2)O(2), but its cellular function is specified to t-BOOH.

  11. Multiscale information modelling for heart morphogenesis

    NASA Astrophysics Data System (ADS)

    Abdulla, T.; Imms, R.; Schleich, J. M.; Summers, R.

    2010-07-01

    Science is made feasible by the adoption of common systems of units. As research has become more data intensive, especially in the biomedical domain, it requires the adoption of a common system of information models, to make explicit the relationship between one set of data and another, regardless of format. This is being realised through the OBO Foundry to develop a suite of reference ontologies, and NCBO Bioportal to provide services to integrate biomedical resources and functionality to visualise and create mappings between ontology terms. Biomedical experts tend to be focused at one level of spatial scale, be it biochemistry, cell biology, or anatomy. Likewise, the ontologies they use tend to be focused at a particular level of scale. There is increasing interest in a multiscale systems approach, which attempts to integrate between different levels of scale to gain understanding of emergent effects. This is a return to physiological medicine with a computational emphasis, exemplified by the worldwide Physiome initiative, and the European Union funded Network of Excellence in the Virtual Physiological Human. However, little work has been done on how information modelling itself may be tailored to a multiscale systems approach. We demonstrate how this can be done for the complex process of heart morphogenesis, which requires multiscale understanding in both time and spatial domains. Such an effort enables the integration of multiscale metrology.

  12. Morphogenesis of the human lacrimal gland

    PubMed Central

    de la Cuadra-Blanco, C; Peces-Peña, M D; Mérida-Velasco, J R

    2003-01-01

    The aim of this study was to determine the main stages of the lacrimal gland's developmental process in humans and to establish its precise morphogenetic timetable. Its onset is generally assumed to take place at O'Rahilly's stage 21, arising from an epithelial thickening of the superior extreme of the temporary conjunctival fornix. However, the present study points to a prior stage in the process: the presence of epithelial–mesenchymal changes in embryos at O'Rahilly's stage 19. The study was performed using light microscopy on serial sections of 37 human specimens: 23 embryos and 14 fetuses ranging from 15 to 137 mm crown–rump length (7–116 weeks of development). Three stages in lacrimal gland morphogenesis were identified: (1) the presumptive glandular stage, O'Rahilly's stages 19–20, characterized by a thickening of the superior fornix epithelium together with surrounding mesenchymal condensation; (2) the bud stage, generally assumed to be the first manifestation of glandular origin, characterized initially by the appearance of nodular formations in the region of the superior conjunctival fornix and concluding with the appearance of lumina within the epithelial buds; and (3) the glandular maturity stage, weeks 9–16, the period in which the gland begins to take on the morphology of adulthood. PMID:14635806

  13. Heparin activates Wnt signaling for neuronal morphogenesis.

    PubMed

    Colombres, Marcela; Henríquez, Juan Pablo; Reig, Germán F; Scheu, Jessica; Calderón, Rosario; Alvarez, Alejandra; Brandan, Enrique; Inestrosa, Nibaldo C

    2008-09-01

    Wnt factors are secreted ligands that affect different aspects of the nervous system behavior like neurodevelopment, synaptogenesis and neurodegeneration. In different model systems, Wnt signaling has been demonstrated to be regulated by heparan sulfate proteoglycans (HSPGs). Whether HSPGs modulate Wnt signaling in the context of neuronal behavior is currently unknown. Here we demonstrate that activation of Wnt signaling with the endogenous ligand Wnt-7a results in an increased of neurite outgrowth in the neuroblastoma N2a cell line. Interestingly, heparin induces glycogen synthase kinase-3beta (GSK-3beta) inhibition, beta-catenin stabilization and morphological differentiation in both N2a cells and in rat primary hippocampal neuronal cultures. We also show that heparin modulates Wnt-3a-induced stabilization of beta-catenin. Several extracellular matrix and membrane-attached HSPGs were found to be expressed in both in vitro neuronal models. Changes in the expression of specific HSPGs were observed upon differentiation of N2a cells. Taken together, our findings suggest that HSPGs may modulate canonical Wnt signaling for neuronal morphogenesis.

  14. Cellular Mechanisms of Drosophila Heart Morphogenesis

    PubMed Central

    Vogler, Georg; Bodmer, Rolf

    2015-01-01

    Many of the major discoveries in the fields of genetics and developmental biology have been made using the fruit fly, Drosophila melanogaster. With regard to heart development, the conserved network of core cardiac transcription factors that underlies cardiogenesis has been studied in great detail in the fly, and the importance of several signaling pathways that regulate heart morphogenesis, such as Slit/Robo, was first shown in the fly model. Recent technological advances have led to a large increase in the genomic data available from patients with congenital heart disease (CHD). This has highlighted a number of candidate genes and gene networks that are potentially involved in CHD. To validate genes and genetic interactions among candidate CHD-causing alleles and to better understand heart formation in general are major tasks. The specific limitations of the various cardiac model systems currently employed (mammalian and fish models) provide a niche for the fly model, despite its evolutionary distance to vertebrates and humans. Here, we review recent advances made using the Drosophila embryo that identify factors relevant for heart formation. These underline how this model organism still is invaluable for a better understanding of CHD. PMID:26236710

  15. Mechanical feedback stabilizes budding yeast morphogenesis

    NASA Astrophysics Data System (ADS)

    Banavar, Samhita; Trogdon, Michael; Petzold, Linda; Campas, Otger

    Walled cells have the ability to remodel their shape while sustaining an internal turgor pressure that can reach values up to 10 atmospheres. This requires a tight and simultaneous regulation of cell wall assembly and mechanochemistry, but the underlying mechanisms by which this is achieved remain unclear. Using the growth of mating projections in budding yeast (S. cerevisiae) as a motivating example, we have developed a theoretical description that couples the mechanics of cell wall expansion and assembly via a mechanical feedback. In the absence of a mechanical feedback, cell morphogenesis is inherently unstable. The presence of a mechanical feedback stabilizes changes in cell shape and growth, and provides a mechanism to prevent cell lysis in a wide range of conditions. We solve for the dynamics of the system and obtain the different dynamical regimes. In particular, we show that several parameters affect the stability of growth, including the strength of mechanical feedback in the system. Finally, we compare our results to existing experimental data.

  16. Genetic regulation of dentate gyrus morphogenesis.

    PubMed

    Li, Guangnan; Pleasure, Samuel J

    2007-01-01

    The dentate gyrus is one of the small number of forebrain areas that have continued adult neurogenesis. During development the dentate gyrus acquires the capacity for neurogenesis by generating a new neurogenic stem cell niche at the border between the hilus and dentate granule cell layer. This is in distinction to the other prominent zone of continued neurogenesis in the subventricular zone where neurons are born in a structure directly descended from the mid-gestation subventricular zone. The ability to generate this newly formed dentate neurogenic niche is controlled by the action of a number of genes during prenatal and early postnatal development that regulate the fate, survival, migration, expansion, and differentiation of the cellular components of the dentate neurogenic niche. In this review, we provide an updated framework discussing the molecular steps and genes involved in these early stages of dentate gyrus formation. We previously described a molecular framework for dentate gyrus morphogenesis that can be associated with specific gene defects (Li, G., Pleasure, S.J. (2005). Dev. Neurosci., 27, 93-99), and here we add additional recently described molecular players and discuss this framework.

  17. FGF10 signaling controls stomach morphogenesis

    PubMed Central

    Nyeng, Pia; Norgaard, Gitte Anker; Kobberup, Sune; Jensen, Jan

    2007-01-01

    Maintenance of progenitor cell properties in development is required for proper organogenesis of most organs, including those derived from the endoderm. FGF10 has been shown to play a role in both lung and pancreatic development. Here we find that FGF10 signaling controls stomach progenitor maintenance, morphogenesis and cellular differentiation. Through a characterization of the initiation of terminal differentiation of the three major gastric regions in the mouse, forestomach, corpus and antrum, we first describe the existence of a “secondary transition” event occurring in mouse stomach between E15.5-E16.5. This includes the formation of terminally differentiated squamous cells, parietal, chief a nd gastric endocrine cells from a pre-patterned gastric progenitor epithelium. Expression analysis of both FGF and Notch signaling components suggested a role of these networks in such progenitors, which was tested through ectopically expressing FGF10 in the developing posterior stomach. These data provide evidence that gastric gland specification and progenitor cell maintenance is controlled by FGF10. The glandular proliferative niche was disrupted in pPDX-FGF10FLAG mice leading to aberrant gland formation, and endocrine and parietal cell differentiation was attenuated. These effects were paralleled by changes in Hes1, Shh, and Wnt6 expression, suggesting that FGF10 acts in concert with multiple morphogenetic signaling systems during gastric development. PMID:17196193

  18. SUMO enhances vestigial function during wing morphogenesis.

    PubMed

    Takanaka, Yoko; Courey, Albert J

    2005-10-01

    The conjugation of the ubiquitin-like protein SUMO to lysine side chains plays widespread roles in the regulation of nuclear protein function. Since little information is available about the roles of SUMO in development, we have screened a collection of chromosomal deficiencies to identify developmental processes regulated by SUMO. We found that flies heterozygous for a deficiency uncovering vestigial (vg) and mutations in any of several genes encoding components of the SUMO conjugation machinery exhibit severe wing notching. This phenotype is due to an interaction between sumo and vg since it is suppressed by expression of Vg from a transgene, and is also observed in flies doubly heterozygous for vg hypomorphic alleles and sumo. In addition, the ability of Vg to direct the formation of ectopic wings when misexpressed in the eye field is enhanced by simultaneous misexpression of SUMO. In S2 cell transient transfection assays, overexpression of SUMO and the SUMO conjugating enzyme Ubc9, but not a catalytically inactive form of Ubc9, results in sumoylation of Vg and augments the activation of a Vg-responsive reporter. These findings are consistent with the idea that sumoylation stimulates Vg function during wing morphogenesis.

  19. Microtubules, polarity and vertebrate neural tube morphogenesis.

    PubMed

    Cearns, Michael D; Escuin, Sarah; Alexandre, Paula; Greene, Nicholas D E; Copp, Andrew J

    2016-07-01

    Microtubules (MTs) are key cellular components, long known to participate in morphogenetic events that shape the developing embryo. However, the links between the cellular functions of MTs, their effects on cell shape and polarity, and their role in large-scale morphogenesis remain poorly understood. Here, these relationships were examined with respect to two strategies for generating the vertebrate neural tube: bending and closure of the mammalian neural plate; and cavitation of the teleost neural rod. The latter process has been compared with 'secondary' neurulation that generates the caudal spinal cord in mammals. MTs align along the apico-basal axis of the mammalian neuroepithelium early in neural tube closure, participating functionally in interkinetic nuclear migration, which indirectly impacts on cell shape. Whether MTs play other functional roles in mammalian neurulation remains unclear. In the zebrafish, MTs are important for defining the neural rod midline prior to its cavitation, both by localizing apical proteins at the tissue midline and by orienting cell division through a mirror-symmetric MT apparatus that helps to further define the medial localization of apical polarity proteins. Par proteins have been implicated in centrosome positioning in neuroepithelia as well as in the control of polarized morphogenetic movements in the neural rod. Understanding of MT functions during early nervous system development has so far been limited, partly by techniques that fail to distinguish 'cause' from 'effect'. Future developments will likely rely on novel ways to selectively impair MT function in order to investigate the roles they play.

  20. Mechanical growth and morphogenesis of seashells.

    PubMed

    Moulton, D E; Goriely, A; Chirat, R

    2012-10-21

    Seashells grow through the local deposition of mass along the aperture. Many mathematical descriptions of the shapes of shells have been provided over the years, and the basic logarithmic coiling seen in mollusks can be simulated with few parameters. However, the developmental mechanisms underlying shell coiling are largely not understood and the ubiquitous presence of ornamentation such as ribs, tubercles, or spines presents yet another level of difficulty. Here we develop a general model for shell growth based entirely on the local geometry and mechanics of the aperture and mantle. This local description enables us to efficiently describe both arbitrary growth velocities and the evolution of the shell aperture itself. We demonstrate how most shells can be simulated within this framework. We then turn to the mechanics underlying the shell morphogenesis, and develop models for the evolution of the aperture. We demonstrate that the elastic response of the mantle during shell deposition provides a natural mechanism for the formation of three-dimensional ornamentation in shells.

  1. Ultrastructural morphogenesis of salmonid alphavirus 1.

    PubMed

    Herath, T K; Ferguson, H W; Thompson, K D; Adams, A; Richards, R H

    2012-11-01

    Studies on the ultrastructural morphogenesis of viruses give an insight into how the host cell mechanisms are utilized for new virion synthesis. A time course examining salmonid alphavirus 1 (SAV 1) assembly was performed by culturing the virus on Chinook salmon embryo cells (CHSE-214). Different stages of viral replication were observed under electron microscopy. Virus-like particles were observed inside membrane-bound vesicles as early as 1 h following contact of the virus with the cells. Membrane-dependent replication complexes were observed in the cytoplasm of the cells, with spherules found at the periphery of late endosome-like vacuoles. The use of intracellular membranes for RNA replication is similar to other positive-sense single-stranded RNA (+ssRNA) viruses. The number of Golgi apparatus and associated vacuoles characterized by 'fuzzy'-coated membranes was greater in virus-infected cells. The mature enveloped virions started to bud out from the cells at approximately 24 h post-infection. These observations suggest that the pathway used by SAV 1 for the generation of new virus particles in vitro is comparable to viral replication observed with mammalian alphaviruses but with some interesting differences.

  2. Otite externe maligne à Candida Albicans

    PubMed Central

    Elayoubi, Fahd; Lachkar, Azeddine; Aabach, Ahmed; Chouai, Mohamed; Ghailan, Mohamed Rachid

    2016-01-01

    L’otite externe maligne est une ostéomyélite de la base du crane. Le Pseudomonas aeruginosa est le germe le plus incriminé. Cependant l’origine fongique n’est pas rare. Patiente âgée de 80 ans avait présenté une otalgie gauche persistante depuis deux mois malgré un traitement bien conduit. L’examen otologique mettait en évidence des signes inflammatoires au niveau du pavillon, une sténose du conduit avec des granulomes, et otorrhée d’allure purulente. Le scanner montrait un comblement otomastoïdien, un processus inflammatoire extensif des tissus pré et rétro-auriculaire et une lyse du tympanal. Vu l’absence d’amélioration un examen mycologique a été réalisé et qui a révélé la présence de Candida Albicans. Les cas d’otite externe maligne à Candida Albicans sont rarement rapportés. L’origine fongique doit être suspecté devant la négativité des prélèvements bactériologiques et la non amélioration malgré un traitement antibiotique bien conduit, et confirmée par des prélèvements mycologiques parfois multiples. L’otite externe maligne à Candida Albicans est une infection rare potentiellement mortelle. PMID:28154677

  3. Melittin induces apoptotic features in Candida albicans

    SciTech Connect

    Park, Cana; Lee, Dong Gun

    2010-03-26

    Melittin is a well-known antimicrobial peptide with membrane-active mechanisms. In this study, it was found that Melittin exerted its antifungal effect via apoptosis. Candida albicans exposed to Melittin showed the increased reactive oxygen species (ROS) production, measured by DHR-123 staining. Fluorescence microscopy staining with FITC-annexin V, TUNEL and DAPI further confirmed diagnostic markers of yeast apoptosis including phosphatidylserine externalization, and DNA and nuclear fragmentation. The current study suggests that Melittin possesses an antifungal effect with another mechanism promoting apoptosis.

  4. Electron Microscopy of Young Candida albicans Chlamydospores

    PubMed Central

    Miller, Sara E.; Spurlock, Ben O.; Michaels, G. E.

    1974-01-01

    One- to three-day-old cultures of Candida albicans bearing chlamydospores were grown and harvested by a special technique, free of agar, and prepared for ultramicrotomy and electron microscopy. These young chlamydospores exhibited a subcellular structure similar to that of the yeast phase, e.g., cytoplasmic membrane, ribosomes, and mitochondria. Other structural characteristics unique to chlamydospores were a very thick, layered cell wall, the outer layer of which was continuous with the outer layer of the suspensor cell wall and was covered by hair-like projections; membrane bound organelles; and large lipoid inclusions. Only young chlamydospores less than 3 to 4 days old exhibited these ultrastructural characteristics. Images PMID:4368664

  5. Accumulation of acyclic polyols and trehalose as related to growth form and carbohydrate source in the dimorphic fungi Mucor rouxii and Candida albicans.

    PubMed

    Pfyffer, G E; Rast, D M

    1989-01-01

    Yeast (Y) and hyphal (H) cells of Mucor rouxii and Candida albicans were cultivated in liquid media containing different carbon nutrient sources (glucose, fructose, ribose) and their free acyclic polyol and trehalose contents determined using capillary gas liquid chromatography (TMS- and OAc-derivatization). Irrespective of growth form and C-source, the fraction of the water-soluble neutral components of the cellular mass of the cultures - highly homogeneous with regard to the respective cell form produced - contained glycerol, ribitol and arabitol, in addition to trehalose. The polyols contributed 0.5-2% to the biomass of M. rouxii and 1.5-6% to that of C. albicans; the values for trehalose ranged from 0.2-11% in the former and 1-3.5% in the latter species. Mucor contained higher amounts of ribitol and arabitol in H cells and larger quantities of trehalose and glycerol in Y cells. In Candida, too, hyphae always exhibited higher ribitol contents, whereas arabitol attained higher levels in yeasts under almost any conditions - regardless of the type of medium (synthetic vs. complex), stage of culture (early vs. late log-phase) and strain used. Glycerol concentration was not correlated with the growth form; trehalose contents tended to be higher in Y cells. Taking into account the facts that C. albicans and certain Mucor species are agents of opportunistic infections and are invasive mainly in the filamentous form, and that the prospective hosts do not accumulate either of these carbohydrates, the possibility is considered of using trehalose- and polyol-metabolizing enzymes as targets for designing antifungal drugs.

  6. Epithelial Cell Secretions from the Human Female Reproductive Tract Inhibit Sexually Transmitted Pathogens and Candida albicans but not Lactobacillus

    PubMed Central

    Wira, CR; Ghosh, M; Smith, JM; Shen, L; Connor, RI; Sundstrom, P; Frechette, Gregory M.; Hill, EM; Fahey, JV

    2011-01-01

    Female reproductive tract (FRT) epithelial cells protect against potential pathogens and sexually transmitted infections. The purpose of this study was to determine if epithelial cells from the upper FRT secrete antimicrobials that inhibit reproductive tract pathogens which threaten women's health. Apical secretions from primary cultures of Fallopian tube, uterine, cervical and ectocervical epithelial cells were incubated with Neisseria gonorrhoeae, Candida albicans (yeast and hyphal forms), HIV-1, and Lactobacillus crispatus, prior to being tested for their ability to grow and/or infect target cells. Epithelial cell secretions from the upper FRT inhibit N. gonorrhoeae and both forms of Candida, as well as reduce HIV-1 (R5) infection of target cells. In contrast, none had an inhibitory effect on L. crispatus. Cytokines and chemokines analysis in uterine secretions revealed several molecules that could account for pathogen inhibition. These findings provide definitive evidence for the critical role of epithelial cells in protecting the FRT from infections, without comprising the beneficial presence of L. crispatus, which is part of the normal vaginal microflora of humans. PMID:21048705

  7. The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger.

    PubMed

    Priegnitz, Bert-Ewald; Brandt, Ulrike; Pahirulzaman, Khomaizon A K; Dickschat, Jeroen S; Fleißner, André

    2015-06-01

    Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.

  8. Pathogenicity and virulence of Candida dubliniensis: comparison with C. albicans.

    PubMed

    Vilela, M M S; Kamei, K; Sano, A; Tanaka, R; Uno, J; Takahashi, I; Ito, J; Yarita, K; Miyaji, M

    2002-06-01

    Candida dubliniensis is a newly described fungus that is frequently isolated from the oral cavities of HIV-positive patients. Although extensive studies have been performed on the phylogeny of C. dubliniensis, little is known about the pathogenic ecology of this yeast. Here we examined aspects related to C. dubliniensis in comparison with those of C. albicans. When injected intravenously into mice, C. dubliniensis had a higher survival rate than C. albicans. Histopathological analysis disclosed that C. dubliniensis remained mostly in the yeast form in the infected organs, whereas C. albicans changed into the mycelial form. The host inflammatory reaction was aggressive with C. dubliniensis infection and mild with C. albicans infection. Co-culture of the yeasts with human polymorphonuclear leukocytes disclosed that C. dubliniensis is more vulnerable to the fungicidal activity of leukocytes than C. albicans. C. dubliniensis was also more susceptible to the toxic effect of hydrogen peroxide. When cultured in vitro, C. dubliniensis grew more slowly than C. albicans, but the formation of germ tubes was faster. When the fungi were cultured in RPMI 1640, a fetal bovine serum supplement suppressed the growth of C. dubliniensis but enhanced that of C. albicans. These results clearly indicated that C. dubliniensis is less virulence than C. albicans.

  9. Sensitization of Candida albicans to terbinafine by berberine and berberrubine

    PubMed Central

    LAM, PIKLING; KOK, STANTON HON LUNG; LEE, KENNETH KA HO; LAM, KIM HUNG; HAU, DESMOND KWOK PO; WONG, WAI YEUNG; BIAN, ZHAOXIANG; GAMBARI, ROBERTO; CHUI, CHUNG HIN

    2016-01-01

    Candida albicans (C. albicans) is an opportunistic fungal pathogen, particularly observed in immunocompromised patients. C. albicans accounts for 50–70% of cases of invasive candidiasis in the majority of clinical settings. Terbinafine, an allylamine antifungal drug, has been used to treat fungal infections previously. It has fungistatic activity against C. albicans. Traditional Chinese medicines can be used as complementary medicines to conventional drugs to treat a variety of ailments and diseases. Berberine is a quaternary alkaloid isolated from the traditional Chinese herb, Coptidis Rhizoma, while berberrubine is isolated from the medicinal plant Berberis vulgaris, but is also readily derived from berberine by pyrolysis. The present study demonstrates the possible complementary use of berberine and berberrubine with terbinafine against C. albicans. The experimental findings assume that the potential application of these alkaloids together with reduced dosage of the standard drug would enhance the resulting antifungal potency. PMID:27073630

  10. Quantitative methods to study epithelial morphogenesis and polarity.

    PubMed

    Aigouy, B; Collinet, C; Merkel, M; Sagner, A

    2017-01-01

    Morphogenesis of an epithelial tissue emerges from the behavior of its constituent cells, including changes in shape, rearrangements, and divisions. In many instances the directionality of these cellular events is controlled by the polarized distribution of specific molecular components. In recent years, our understanding of morphogenesis and polarity highly benefited from advances in genetics, microscopy, and image analysis. They now make it possible to measure cellular dynamics and polarity with unprecedented precision for entire tissues throughout their development. Here we review recent approaches to visualize and measure cell polarity and tissue morphogenesis. The chapter is organized like an experiment. We first discuss the choice of cell and polarity reporters and describe the use of mosaics to reveal hidden cell polarities or local morphogenetic events. Then, we outline application-specific advantages and disadvantages of different microscopy techniques and image projection algorithms. Next, we present methods to extract cell outlines to measure cell polarity and detect cellular events underlying morphogenesis. Finally, we bridge scales by presenting approaches to quantify the specific contribution of each cellular event to global tissue deformation. Taken together, we provide an in-depth description of available tools and theoretical concepts to quantitatively study cell polarity and tissue morphogenesis over multiple scales.

  11. Heterotypic Control of Basement Membrane Dynamics During Branching Morphogenesis

    PubMed Central

    Nelson, Deirdre A.; Larsen, Melinda

    2015-01-01

    Many mammalian organs undergo branching morphogenesis to create highly arborized structures with maximized surface area for specialized organ function. Cooperative cell-cell and cell-matrix adhesions that sculpt the emerging tissue architecture are guided by dynamic basement membranes. Properties of the basement membrane are reciprocally controlled by the interacting epithelial and mesenchymal cell populations. Here we discuss how basement membrane remodeling is required for branching morphogenesis to regulate cell-matrix and cell-cell adhesions that are required for cell patterning during morphogenesis and how basement membrane impacts morphogenesis by stimulation of cell patterning, force generation, and mechanotransduction. We suggest that in addition to creating mature epithelial architecture, remodeling of the epithelial basement membrane during branching morphogenesis is also essential to promote maturation of the stromal mesenchyme to create mature organ structure. Recapitulation of developmental cell-matrix and cell-cell interactions are of critical importance in tissue engineering and regeneration strategies that seek to restore organ function. PMID:25527075

  12. Myosin II Dynamics during Embryo Morphogenesis

    NASA Astrophysics Data System (ADS)

    Kasza, Karen

    2013-03-01

    During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

  13. Microtubules, polarity and vertebrate neural tube morphogenesis

    PubMed Central

    Cearns, Michael D.; Escuin, Sarah; Alexandre, Paula; Greene, Nicholas D. E.; Copp, Andrew J.

    2016-01-01

    Microtubules are key cellular components, long known to participate in morphogenetic events that shape the developing embryo. However, the links between the cellular functions of microtubules, their effects on cell shape and polarity and their role in large-scale morphogenesis remain poorly understood. Here, we examine these relationships with respect to two strategies for generating the vertebrate neural tube: bending and closure of the mammalian neural plate, and cavitation of the teleost neural rod. The latter process has been compared to ‘secondary’ neurulation that generates the caudal spinal cord in mammals. Microtubules align along the apico-basal axis of the mammalian neuroepithelium early in neural tube closure, participating functionally in interkinetic nuclear migration which indirectly impacts on cell shape. Whether microtubules play other functional roles in mammalian neurulation remains unclear. In the zebrafish, microtubules are important for defining the neural rod midline prior to its cavitation, both by localizing apical proteins at the tissue midline and by orienting cell division through a mirror-symmetric microtubule apparatus that helps to further define the medial localization of apical polarity proteins. Par proteins have been implicated in centrosome positioning in neuroepithelia as well as in the control of polarized morphogenetic movements in the neural rod. Understanding of microtubule functions during early nervous system development has so far been limited, partly by techniques that fail to distinguish ‘cause’ from ‘effect’. Future developments will likely rely on novel ways to selectively impair microtubule function in order to investigate the roles they play. PMID:27025884

  14. Ultrastructure and morphogenesis of human immunodeficiency virus.

    PubMed

    Nakai, M; Goto, T

    1996-08-01

    The ultrastructure and morphogenesis of human immunodeficiency virus (HIV) were elucidated by observation with several techniques including immunoelectron microscopy and cryo-microscopy. The virus particle consists of an envelope, a core and matrix. The virus particles were observed extracellularly as having one of three profiles: (1) a centric or an eccentric electron-dense core, (2) rod-shaped electron-dense core, and (3) doughnut-shaped. HIV-1 particles in the hydrated state were observed by high resolution electron cryo-microscopy to be globular, and the lipid membrane was clearly resolved as a bilayer. Many projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially head parts, were found to vary in each projection. By isolation with Nonidet P40 and glutaraldehyde, HIV-1 cores were confirmed to consist of p24 protein by immunogold labeling. When the virus enters the cell, two entry modes were found: membrane fusion and endocytosis. No structures resembling virus particles could be seen in the cytoplasm after viral entry. In HIV-1-infected cells, positive reactions by immuno-labeling suggest that HIV-1 Gag may be produced in membrane-bound structures and transported to the cell surface by cytoskeletons. Then a crescent electron-dense layer was first formed underneath the cell membrane. Finally, the virus particle was released from the cell surface. Several cell clones producing defective particles were isolated from MT-4/HIV-1 cells. Among them, doughnut-shaped or teardrop-shaped particles were seen to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins faced each other against the inner electron dense ring, suggesting that the inner ring consists of a precursor Gag protein.

  15. Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity

    PubMed Central

    2014-01-01

    Background Candida albicans infections have become increasingly recognised as being biofilm related. Recent studies have shown that there is a relationship between biofilm formation and poor clinical outcomes in patients infected with biofilm proficient strains. Here we have investigated a panel of clinical isolates in an attempt to evaluate their phenotypic and transcriptional properties in an attempt to differentiate and define levels of biofilm formation. Results Biofilm formation was shown to be heterogeneous; with isolates being defined as either high or low biofilm formers (LBF and HBF) based on different biomass quantification. These categories could also be differentiated using a cell surface hydrophobicity assay with 24 h biofilms. HBF isolates were more resistance to amphotericin B (AMB) treatment than LBF, but not voriconazole (VRZ). In a Galleria mellonella model of infection HBF mortality was significantly increased in comparison to LBF. Histological analysis of the HBF showed hyphal elements intertwined indicative of the biofilm phenotype. Transcriptional analysis of 23 genes implicated in biofilm formation showed no significant differential expression profiles between LBF and HBF, except for Cdr1 at 4 and 24 h. Cluster analysis showed similar patterns of expression for different functional classes of genes, though correlation analysis of the 4 h biofilms with overall biomass at 24 h showed that 7 genes were correlated with high levels of biofilm, including Als3, Eap1, Cph1, Sap5, Plb1, Cdr1 and Zap1. Conclusions Our findings show that biofilm formation is variable amongst C. albicans isolates, and categorising isolates depending on this can be used to predict how pathogenic the isolate will behave clinically. We have shown that looking at individual genes in less informative than looking at multiple genes when trying to categorise isolates at LBF or HBF. These findings are important when developing biofilm-specific diagnostics as these could be

  16. Neuropilin-2 promotes branching morphogenesis in the mouse mammary gland.

    PubMed

    Goel, Hira Lal; Bae, Donggoo; Pursell, Bryan; Gouvin, Lindsey M; Lu, Shaolei; Mercurio, Arthur M

    2011-07-01

    Although the neuropilins were characterized as semaphorin receptors that regulate axon guidance, they also function as vascular endothelial growth factor (VEGF) receptors and contribute to the development of other tissues. Here, we assessed the role of NRP2 in mouse mammary gland development based on our observation that NRP2 is expressed preferentially in the terminal end buds of developing glands. A floxed NRP2 mouse was bred with an MMTV-Cre strain to generate a mammary gland-specific knockout of NRP2. MMTV-Cre;NRP2(loxP/loxP) mice exhibited significant defects in branching morphogenesis and ductal outgrowth compared with either littermate MMTV-Cre;NRP2(+/loxP) or MMTV-Cre mice. Mechanistic insight into this morphological defect was obtained from a mouse mammary cell line in which we observed that VEGF(165), an NRP2 ligand, induces branching morphogenesis in 3D cultures and that branching is dependent upon NRP2 as shown using shRNAs and a function-blocking antibody. Epithelial cells in the mouse mammary gland express VEGF, supporting the hypothesis that this NRP2 ligand contributes to mammary gland morphogenesis. Importantly, we demonstrate that VEGF and NRP2 activate focal adhesion kinase (FAK) and promote FAK-dependent branching morphogenesis in vitro. The significance of this mechanism is substantiated by our finding that FAK activation is diminished significantly in developing MMTV-Cre;NRP2(loxP/loxP) mammary glands compared with control glands. Together, our data reveal a VEGF/NRP2/FAK signaling axis that is important for branching morphogenesis and mammary gland development. In a broader context, our data support an emerging hypothesis that directional outgrowth and branching morphogenesis in a variety of tissues are influenced by signals that were identified initially for their role in axon guidance.

  17. Azole Antifungal Resistance in Candida albicans and Emerging Non-albicans Candida Species

    PubMed Central

    Whaley, Sarah G.; Berkow, Elizabeth L.; Rybak, Jeffrey M.; Nishimoto, Andrew T.; Barker, Katherine S.; Rogers, P. David

    2017-01-01

    Within the limited antifungal armamentarium, the azole antifungals are the most frequent class used to treat Candida infections. Azole antifungals such as fluconazole are often preferred treatment for many Candida infections as they are inexpensive, exhibit limited toxicity, and are available for oral administration. There is, however, extensive documentation of intrinsic and developed resistance to azole antifungals among several Candida species. As the frequency of azole resistant Candida isolates in the clinical setting increases, it is essential to elucidate the mechanisms of such resistance in order to both preserve and improve upon the azole class of antifungals for the treatment of Candida infections. This review examines azole resistance in infections caused by C. albicans as well as the emerging non-albicans Candida species C. parapsilosis, C. tropicalis, C. krusei, and C. glabrata and in particular, describes the current understanding of molecular basis of azole resistance in these fungal species. PMID:28127295

  18. PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

    PubMed

    LaFayette, Shantelle L; Collins, Cathy; Zaas, Aimee K; Schell, Wiley A; Betancourt-Quiroz, Marisol; Gunatilaka, A A Leslie; Perfect, John R; Cowen, Leah E

    2010-08-26

    Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug

  19. AI-2 of Aggregatibacter actinomycetemcomitans inhibits Candida albicans biofilm formation.

    PubMed

    Bachtiar, Endang W; Bachtiar, Boy M; Jarosz, Lucja M; Amir, Lisa R; Sunarto, Hari; Ganin, Hadas; Meijler, Michael M; Krom, Bastiaan P

    2014-01-01

    Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, and Candida albicans, a polymorphic fungus, are both commensals of the oral cavity but both are opportunistic pathogens that can cause oral diseases. A. actinomycetemcomitans produces a quorum-sensing molecule called autoinducer-2 (AI-2), synthesized by LuxS, that plays an important role in expression of virulence factors, in intra- but also in interspecies communication. The aim of this study was to investigate the role of AI-2 based signaling in the interactions between C. albicans and A. actinomycetemcomitans. A. actinomycetemcomitans adhered to C. albicans and inhibited biofilm formation by means of a molecule that was secreted during growth. C. albicans biofilm formation increased significantly when co-cultured with A. actinomycetemcomitans luxS, lacking AI-2 production. Addition of wild-type-derived spent medium or synthetic AI-2 to spent medium of the luxS strain, restored inhibition of C. albicans biofilm formation to wild-type levels. Addition of synthetic AI-2 significantly inhibited hypha formation of C. albicans possibly explaining the inhibition of biofilm formation. AI-2 of A. actinomycetemcomitans is synthesized by LuxS, accumulates during growth and inhibits C. albicans hypha- and biofilm formation. Identifying the molecular mechanisms underlying the interaction between bacteria and fungi may provide important insight into the balance within complex oral microbial communities.

  20. Survival of Candida albicans in tropical marine and fresh waters.

    PubMed Central

    Valdes-Collazo, L; Schultz, A J; Hazen, T C

    1987-01-01

    A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard. Images PMID:3310885

  1. Global Identification of Biofilm-Specific Proteolysis in Candida albicans

    PubMed Central

    Winter, Michael B.; Salcedo, Eugenia C.; Lohse, Matthew B.; Hartooni, Nairi; Gulati, Megha; Sanchez, Hiram; Takagi, Julie; Hube, Bernhard; Andes, David R.

    2016-01-01

    ABSTRACT Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans. Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation. PMID:27624133

  2. Candida albicans and non-Candida albicans fungemia in an institutional hospital during a decade.

    PubMed

    Parmeland, Laurence; Gazon, Mathieu; Guerin, Claude; Argaud, Laurent; Lehot, Jean-Jacques; Bastien, Olivier; Allaouchiche, Bernard; Michallet, Mauricette; Picot, Stephane; Bienvenu, Anne-Lise

    2013-01-01

    Since the outcomes of patients with candidemia is poor and Candida spp. with increased resistance to antifungal therapy may be associated with these results, the emergence of these blood infections caused by non-C. albicans Candida spp. was explored prospectively over a two-year period (2009-2010). Candidemia was defined as the recovery of Candida spp. in culture from a patient's blood sample. The in vitro susceptibility of each isolate to amphotericin B, caspofungin, fluconazole and voriconazole was determined. In addition, characteristics of patients and outcomes were investigated in real-time. The Candida distribution was compared to that observed in a similar study 10 years earlier in the same hospital. A total of 182 patients with candidemia were included in the study. While C. albicans was the most frequently isolated species (n = 102), non-C. albicans Candida spp. included; C. glabrata (n = 32), C. parapsilosis (n = 21), C. tropicalis (n = 13), C. krusei (n = 8), C. kefyr (n = 3), C. lusitaniae (n = 2), C. lipolytica (n = 2), C. famata (n = 1), C. guilliermondii (n = 1), C. inconspicua (n = 1), C. dubliniensis (n = 1), C. sake (n = 1) and C. nivariensis (n = 1). In seven patients, C. albicans was associated with another Candida spp. Surprisingly, this prospective study demonstrated that regardless of the department (intensive care unit or hematological department), Candida spp. distribution was no different from that found in the 1998-2001 survey, except for C. krusei. A reduction in the proportion of C. krusei isolates was observed from 2000-2010 (P = 0.028) as a result of its decreased recovery in the hematological department.

  3. Molecular epidemiology, phylogeny and evolution of Candida albicans.

    PubMed

    McManus, Brenda A; Coleman, David C

    2014-01-01

    A small number of Candida species form part of the normal microbial flora of mucosal surfaces in humans and may give rise to opportunistic infections when host defences are impaired. Candida albicans is by far the most prevalent commensal and pathogenic Candida species. Several different molecular typing approaches including multilocus sequence typing, multilocus microsatellite typing and DNA fingerprinting using C. albicans-specific repetitive sequence-containing DNA probes have yielded a wealth of information regarding the epidemiology and population structure of this species. Such studies revealed that the C. albicans population structure consists of multiple major and minor clades, some of which exhibit geographical or phenotypic enrichment and that C. albicans reproduction is predominantly clonal. Despite this, losses of heterozygosity by recombination, the existence of a parasexual cycle, toleration of a wide range of aneuploidies and the recent description of viable haploid strains have all demonstrated the extensive plasticity of the C. albicans genome. Recombination and gross chromosomal rearrangements are more common under stressful environmental conditions, and have played a significant role in the evolution of this opportunistic pathogen. Surprisingly, Candida dubliniensis, the closest relative of C. albicans exhibits more karyotype variability than C. albicans, but is significantly less adaptable to unfavourable environments. This disparity most likely reflects the evolutionary processes that occurred during or soon after the divergence of both species from their common ancestor. Whilst C. dubliniensis underwent significant gene loss and pseudogenisation, C. albicans expanded gene families considered to be important in virulence. It is likely that technological developments in whole genome sequencing and data analysis in coming years will facilitate its routine use for population structure, epidemiological investigations, and phylogenetic analyses of

  4. Changes in hyphal morphology and activity of phenoloxidases during interactions between selected ectomycorrhizal fungi and two species of Trichoderma.

    PubMed

    Mucha, Joanna

    2011-06-01

    Patterns of phenoloxidase activity can be used to characterize fungi of different life styles, and changes in phenoloxidase synthesis were suspected to play a role in the interaction between ectomycorrhizal and two species of Trichoderma. Confrontation between the ectomycorrhizal fungi Amanita muscaria and Laccaria laccata with species of Trichoderma resulted in induction of laccase synthesis, and the laccase enzyme was bound to mycelia of ectomycorrhizal fungi. Tyrosinase release was noted only during interaction of L. laccata strains with Trichoderma harzianum and T. virens. Ectomycorrhizal fungi, especially strains of Suillus bovinus and S. luteus, inhibited growth of Trichoderma species and caused morphological changes in its colonies in the zone of interaction. In contrast, hyphal changes occurred less often in the ectomycorrhizal fungi tested. Species of Suillus are suggested to present a different mechanism in their interaction with other fungi than A. muscaria and L. laccata.

  5. The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger

    PubMed Central

    Priegnitz, Bert-Ewald; Brandt, Ulrike; Pahirulzaman, Khomaizon A. K.; Dickschat, Jeroen S.

    2015-01-01

    Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi. PMID:25888553

  6. Development of Protective Inflammation and Cell-Mediated Immunity against Cryptococcus neoformans after Exposure to Hyphal Mutants

    PubMed Central

    Zhai, Bing; Wozniak, Karen L.; Masso-Silva, Jorge; Upadhyay, Srijana; Hole, Camaron; Rivera, Amariliz; Wormley, Floyd L.

    2015-01-01

    ABSTRACT Morphological switch is tightly coupled with the pathogenesis of many dimorphic fungal pathogens. Cryptococcus neoformans, the major causative agent of cryptococcal meningitis, mostly presents as the yeast form but is capable of switching to the hyphal form. The filamentous form has long been associated with attenuated virulence, yet the underlying mechanism remains elusive. We previously identified the master regulator Znf2 that controls the yeast-to-hypha transition in Cryptococcus. Activation of Znf2 promotes hyphal formation and abolishes fungal virulence in vivo. Here we demonstrated that the cryptococcal strain overexpressing ZNF2 elicited strong and yet temporally confined proinflammatory responses in the early stage of infection. In contrast, exacerbated inflammation in mice infected with the wild-type (WT) strain showed that they were unable to control the infection. Animals inoculated with this filamentous Cryptococcus strain had fewer pulmonary eosinophils and CD11c+ CD11b+ cells than animals inoculated with WT yeast. Moreover, mice infected with this strain developed protective Th1- or Th17-type T cell responses. These findings suggest that the virulence attenuation of the filamentous form is likely due to its elicitation of protective host responses. The antivirulence effect of Znf2 was independent of two previously identified factors downstream of Znf2. Interestingly, mucosal immunizations with high doses of ZNF2-overexpressing cells, either in the live or heat-killed form, offered 100% protection to the host from a subsequent challenge with the otherwise lethal clinical strain H99. Our results demonstrate that heat-resistant cellular components presented in cryptococcal cells with activated ZNF2 elicit protective host immune responses. These findings could facilitate future research on novel immunological therapies. PMID:26443458

  7. Metal Chelation as a Powerful Strategy to Probe Cellular Circuitry Governing Fungal Drug Resistance and Morphogenesis

    PubMed Central

    Averette, Anna F.; Lee, Soo Chan; Kim, Taeyup; Bahn, Yong-Sun; Robbins, Nicole; Heitman, Joseph; Cowen, Leah E.

    2016-01-01

    Fungal pathogens have evolved diverse strategies to sense host-relevant cues and coordinate cellular responses, which enable virulence and drug resistance. Defining circuitry controlling these traits opens new opportunities for chemical diversity in therapeutics, as the cognate inhibitors are rarely explored by conventional screening approaches. This has great potential to address the pressing need for new therapeutic strategies for invasive fungal infections, which have a staggering impact on human health. To explore this approach, we focused on a leading human fungal pathogen, Candida albicans, and screened 1,280 pharmacologically active compounds to identify those that potentiate the activity of echinocandins, which are front-line therapeutics that target fungal cell wall synthesis. We identified 19 compounds that enhance activity of the echinocandin caspofungin against an echinocandin-resistant clinical isolate, with the broad-spectrum chelator DTPA demonstrating the greatest synergistic activity. We found that DTPA increases susceptibility to echinocandins via chelation of magnesium. Whole genome sequencing of mutants resistant to the combination of DTPA and caspofungin identified mutations in the histidine kinase gene NIK1 that confer resistance to the combination. Functional analyses demonstrated that DTPA activates the mitogen-activated protein kinase Hog1, and that NIK1 mutations block Hog1 activation in response to both caspofungin and DTPA. The combination has therapeutic relevance as DTPA enhanced the efficacy of caspofungin in a mouse model of echinocandin-resistant candidiasis. We found that DTPA not only reduces drug resistance but also modulates morphogenesis, a key virulence trait that is normally regulated by environmental cues. DTPA induced filamentation via depletion of zinc, in a manner that is contingent upon Ras1-PKA signaling, as well as the transcription factors Brg1 and Rob1. Thus, we establish a new mechanism by which metal chelation

  8. An Optimized Lock Solution Containing Micafungin, Ethanol and Doxycycline Inhibits Candida albicans and Mixed C. albicans – Staphyloccoccus aureus Biofilms

    PubMed Central

    Lown, Livia; Peters, Brian M.; Walraven, Carla J.; Noverr, Mairi C.; Lee, Samuel A.

    2016-01-01

    Candida albicans is a major cause of catheter-related bloodstream infections and is associated with high morbidity and mortality. Due to the propensity of C. albicans to form drug-resistant biofilms, the current standard of care includes catheter removal; however, reinsertion may be technically challenging or risky. Prolonged exposure of an antifungal lock solution within the catheter in conjunction with systemic therapy has been experimentally attempted for catheter salvage. Previously, we demonstrated excellent in vitro activity of micafungin, ethanol, and high-dose doxycycline as single agents for prevention and treatment of C. albicans biofilms. Thus, we sought to investigate optimal combinations of micafungin, ethanol, and/or doxycycline as a lock solution. We performed two- and three-drug checkerboard assays to determine the in vitro activity of pairwise or three agents in combination for prevention or treatment of C. albicans biofilms. Optimal lock solutions were tested for activity against C. albicans clinical isolates, reference strains and polymicrobial C. albicans-S. aureus biofilms. A solution containing 20% (v/v) ethanol, 0.01565 μg/mL micafungin, and 800 μg/mL doxycycline demonstrated a reduction of 98% metabolic activity and no fungal regrowth when used to prevent fungal biofilm formation; however there was no advantage over 20% ethanol alone. This solution was also successful in inhibiting the regrowth of C. albicans from mature polymicrobial biofilms, although it was not fully bactericidal. Solutions containing 5% ethanol with low concentrations of micafungin and doxycycline demonstrated synergistic activity when used to prevent monomicrobial C. albicans biofilm formation. A combined solution of micafungin, ethanol and doxycycline is highly effective for the prevention of C. albicans biofilm formation but did not demonstrate an advantage over 20% ethanol alone in these studies. PMID:27428310

  9. Comparison of the clinical risk factors between Candida albicans and Candida non-albicans species for bloodstream infection.

    PubMed

    Shigemura, Katsumi; Osawa, Kayo; Jikimoto, Takumi; Yoshida, Hiroyuki; Hayama, Brian; Ohji, Goh; Iwata, Kentaro; Fujisawa, Masato; Arakawa, Soichi

    2014-04-01

    The purpose of this study is to investigate the risk factors and susceptibilities to antifungal agents of Candida albicans and Candida non-albicans species (spp.) in candidemia cases in Kobe University Hospital. We investigated all consecutive patients with candida bloodstream infection (BSI) from 2008-2013 for whose full data were available for analyses, examining clinical factors such as gender, general complications, postoperative status or susceptibilities to antifungal agents. These factors were also compared between Candida albicans spp. and Candida non-albicans by univariate and multivariate analyses. Univariate analyses showed a significantly higher rate of Candida non-albicans species BSI patients cancer (odds ratio (OR) (95% confidence interval (CI))=2.29 (1.04-5.06) and P=0.040), chemotherapy (OR=4.35 (1.11-17.1) and P=0.035), fluconazole (FLCZ) resistance (OR=77.3 (4.51-1324) and P=0.003), and itraconazole (ITCZ) resistance (OR=15.6 (5.39-45.1) and P<0.001) and lower rate of underlying cardiovascular diseases (OR=0.27 (0.09-0.80) and P=0.018) and postoperative status (OR=0.35 (0.16-0.77) and P=0.035) in than Candida albicans. Multivariate analyses demonstrated that Candida non-albicans spp. had significantly higher rate of chemotherapy (OR=4.44 (1.04-19.0) and P=0.045), FLCZ resistance (OR=5.87 (2.01-17.1) and P=0.001), and ITCZ resistance (OR=18.7(5.77-60.4) and P<0.001) and lower rate of underlying cardiovascular diseases (OR=0.25 (0.08-0.82) and P=0.022) than Candida albicans. In conclusion, this study revealed several risk factors for BSI with Candida albicans (underlying cardiovascular diseases and postoperative status) and Candida non-albicans spp. (cancer and chemotherapy), and demonstrated that Candida non-albicans spp. were more resistant to FLCZ and ITCZ than Candida albicans.

  10. Unique Function of the Bacterial Chromosome Segregation Machinery in Apically Growing Streptomyces - Targeting the Chromosome to New Hyphal Tubes and its Anchorage at the Tips

    PubMed Central

    Lipietta, Natalia; Tilley, Emma; Zakrzewska-Czerwińska, Jolanta; Herron, Paul

    2016-01-01

    The coordination of chromosome segregation with cell growth is fundamental to the proliferation of any organism. In most unicellular bacteria, chromosome segregation is strictly coordinated with cell division and involves ParA that moves the ParB nucleoprotein complexes bi- or unidirectionally toward the cell pole(s). However, the chromosome organization in multiploid, apically extending and branching Streptomyces hyphae challenges the known mechanisms of bacterial chromosome segregation. The complex Streptomyces life cycle involves two stages: vegetative growth and sporulation. In the latter stage, multiple cell divisions accompanied by chromosome compaction and ParAB assisted segregation turn multigenomic hyphal cell into a chain of unigenomic spores. However, the requirement for active chromosome segregation is unclear in the absence of canonical cell division during vegetative growth except in the process of branch formation. The mechanism by which chromosomes are targeted to new hyphae in streptomycete vegetative growth has remained unknown until now. Here, we address the question of whether active chromosome segregation occurs at this stage. Applied for the first time in Streptomyces, labelling of the chromosomal replication initiation region (oriC) and time-lapse microscopy, revealed that in vegetative hyphae every copy of the chromosome is complexed with ParB, whereas ParA, through interaction with the apical protein complex (polarisome), tightly anchors only one chromosome at the hyphal tip. The anchor is maintained during replication, when ParA captures one of the daughter oriCs. During spore germination and branching, ParA targets one of the multiple chromosomal copies to the new hyphal tip, enabling efficient elongation of hyphal tube. Thus, our studies reveal a novel role for ParAB proteins during hyphal tip establishment and extension. PMID:27977672

  11. Subtractive transcriptomics : establishing polarity drives human endothelial morphogenesis

    SciTech Connect

    Glesne, D. A.; Zhang, W.; Mandava, S.; Ursos, L.; Buell, M. E.; Makowski, L.; Rodi, D. J.; Biosciences Division

    2006-04-15

    Although investigations of mature normal and tumor-derived capillaries have resulted in characterization of these structures at the phenotypic level, less is known regarding the initial molecular cues for cellular assembly of endothelial cells into human capillaries. Here, we employ a novel combination of microenvironmental manipulation and microarray data filtration over narrowly delineated temporal data series to identify the morphogenesis component apart from the proliferation component, as pooled human microvascular-derived endothelial cells are induced to form capillary-like structures in vitro in a murine tumor-derived matrix. The 217 morphogenesis-specific genes identified using this subtractive transcriptomics approach are mostly independent of the angiogenic proteins currently used as therapeutic targets for aberrant angiogenesis. Quantitative real-time PCR was used to validate 20% of these transcripts. Immunofluorescent analysis of proliferating and tube-forming cells validates at the protein level the morphogenesis-specific expression pattern of 16 of the 217 gene products identified. The transcripts that are selectively up-regulated in tube-forming endothelial cells reveal a temporal expression pattern of genes primarily associated with intracellular trafficking, guided migration, cytoskeletal reorganization, cellular adhesion, and proliferation inhibition. These data show that a sequential upregulation of genes that establish and maintain polarity occurs during migration and morphogenesis of in vitro human endothelial cells undergoing tubulogenesis; some of which may well be effective as novel antiangiogenic drug targets.

  12. Autophagy is essential for cardiac morphogenesis during vertebrate development.

    PubMed

    Lee, Eunmyong; Koo, Yeon; Ng, Aylwin; Wei, Yongjie; Luby-Phelps, Kate; Juraszek, Amy; Xavier, Ramnik J; Cleaver, Ondine; Levine, Beth; Amatruda, James F

    2014-04-01

    Genetic analyses indicate that autophagy, an evolutionarily conserved lysosomal degradation pathway, is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryogenesis. To address this question, we examined the role of autophagy in the early development of zebrafish, a model organism for studying vertebrate tissue and organ morphogenesis. Using zebrafish that transgenically express the fluorescent autophagy reporter protein, GFP-LC3, we found that autophagy is active in multiple tissues, including the heart, during the embryonic period. Inhibition of autophagy by morpholino knockdown of essential autophagy genes (including atg5, atg7, and becn1) resulted in defects in morphogenesis, increased numbers of dead cells, abnormal heart structure, and reduced organismal survival. Further analyses of cardiac development in autophagy-deficient zebrafish revealed defects in cardiac looping, abnormal chamber morphology, aberrant valve development, and ectopic expression of critical transcription factors including foxn4, tbx5, and tbx2. Consistent with these results, Atg5-deficient mice displayed abnormal Tbx2 expression and defects in valve development and chamber septation. Thus, autophagy plays an essential, conserved role in cardiac morphogenesis during vertebrate development.

  13. Cuticle morphogenesis in crustacean embryonic and postembryonic stages.

    PubMed

    Mrak, Polona; Bogataj, Urban; Štrus, Jasna; Žnidaršič, Nada

    2017-01-01

    The crustacean cuticle is a chitin-based extracellular matrix, produced in general by epidermal cells and ectodermally derived epithelial cells of the digestive tract. Cuticle morphogenesis is an integrative part of embryonic and postembryonic development and it was studied in several groups of crustaceans, but mainly with a focus on one selected aspect of morphogenesis. Early studies were focused mainly on in vivo or histological observations of embryonic or larval molt cycles and more recently, some ultrastructural studies of the cuticle differentiation during development were performed. The aim of this paper is to review data on exoskeletal and gut cuticle formation during embryonic and postembryonic development in crustaceans, obtained in different developmental stages of different species and to bring together and discuss different aspects of cuticle morphogenesis, namely data on the morphology, ultrastructure, composition, connections to muscles and molt cycles in relation to cuticle differentiation. Based on the comparative evaluation of microscopic analyses of cuticle in crustacean embryonic and postembryonic stages, common principles of cuticle morphogenesis during development are discussed. Additional studies are suggested to further clarify this topic and to connect the new knowledge to related fields.

  14. Epithelial morphogenesis: the mouse eye as a model system.

    PubMed

    Chauhan, Bharesh; Plageman, Timothy; Lou, Ming; Lang, Richard

    2015-01-01

    Morphogenesis is the developmental process by which tissues and organs acquire the shape that is critical to their function. Here, we review recent advances in our understanding of the mechanisms that drive morphogenesis in the developing eye. These investigations have shown that regulation of the actin cytoskeleton is central to shaping the presumptive lens and retinal epithelia that are the major components of the eye. Regulation of the actin cytoskeleton is mediated by Rho family GTPases, by signaling pathways and indirectly, by transcription factors that govern the expression of critical genes. Changes in the actin cytoskeleton can shape cells through the generation of filopodia (that, in the eye, connect adjacent epithelia) or through apical constriction, a process that produces a wedge-shaped cell. We have also learned that one tissue can influence the shape of an adjacent one, probably by direct force transmission, in a process we term inductive morphogenesis. Though these mechanisms of morphogenesis have been identified using the eye as a model system, they are likely to apply broadly where epithelia influence the shape of organs during development.

  15. Slit and Robo control cardiac cell polarity and morphogenesis.

    PubMed

    Qian, Li; Liu, Jiandong; Bodmer, Rolf

    2005-12-20

    Basic aspects of heart morphogenesis involving migration, cell polarization, tissue alignment, and lumen formation may be conserved between Drosophila and humans, but little is known about the mechanisms that orchestrate the assembly of the heart tube in either organism. The extracellular-matrix molecule Slit and its Robo-family receptors are conserved regulators of axonal guidance. Here, we report a novel role of the Drosophila slit, robo, and robo2 genes in heart morphogenesis. Slit and Robo proteins specifically accumulate at the dorsal midline between the bilateral myocardial progenitors forming a linear tube. Manipulation of Slit localization or its overexpression causes disruption in heart tube alignment and assembly, and slit-deficient hearts show disruptions in cell-polarity marker localization within the myocardium. Similar phenotypes are observed when Robo and Robo2 are manipulated. Rescue experiments suggest that Slit is secreted from the myocardial progenitors and that Robo and Robo2 act in myocardial and pericardial cells, respectively. Genetic interactions suggest a cardiac morphogenesis network involving Slit/Robo, cell-polarity proteins, and other membrane-associated proteins. We conclude that Slit and Robo proteins contribute significantly to Drosophila heart morphogenesis by guiding heart cell alignment and adhesion and/or by inhibiting cell mixing between the bilateral compartments of heart cell progenitors and ensuring proper polarity of the myocardial epithelium.

  16. Phenotypic identification of Candida albicans by growth on chocolate agar.

    PubMed

    Sheth, Chirag C; Johnson, Elizabeth; Baker, Mark E; Haynes, Ken; Mühlschlegel, Fritz A

    2005-12-01

    In this study, we describe a simple method for the identification of Candida albicans in clinical samples. A total of 383 clinical isolates of Candida species were streaked onto chocolate agar and incubated for 48 h at 37 degrees C in the presence of an atmosphere of 6% CO2. All 208 of the C. albicans isolates tested, developed an easy to identify filamentous colony morphology. Of 175 other Candida species tested, 172 (98.3%) were distinguishable from C. albicans by their smooth colony morphology. Three isolates (1.7%) exhibited weak filamentation after prolonged incubation. Although not a routine medium in medical mycology a significant advantage of using chocolate agar lies in its use in clinical bacteriology laboratories for the isolation of fastidious bacteria. Implementation of the proposed method is applicable across a range of specimen types, thus allowing the direct identification of C. albicans in clinical samples. This simple method may allow a quicker entry into directed treatment.

  17. Murine model of concurrent oral and vaginal Candida albicans colonisation.

    PubMed

    Rahman, Durdana; Mistry, Mukesh; Thavaraj, Selvam; Naglik, Julian R; Challacombe, Stephen J

    2012-01-01

    Investigations into the complex interaction between the fungal pathogen Candida albicans and its human host require the use of animals as in vivo models. A major advance is the creation of a low-oestrogen murine model of concurrent oral and vaginal C. albicans colonisation that resembles human candidal carriage at both mucosal sites. Weekly intramuscular (5 μg) and subcutaneous (5 μg) oestrogen administration was determined as optimal, enhancing oral colonisation but essential for vaginal colonisation. Using a clinical C. albicans oral isolate, persistent colonisation for up to 6 weeks can be achieved at both sites in two strains of mice (BALB/c and C57BL/6). This concurrent model of mucosal colonisation reduces the numbers of experimental mice by half, and opens up new avenues of research in assessing potential mucosal vaccine candidates and in studying delicate host-pathogen interactions during the most natural state of C. albicans epithelial colonisation.

  18. Budding off: bringing functional genomics to Candida albicans.

    PubMed

    Anderson, Matthew Z; Bennett, Richard J

    2016-03-01

    Candida species are the most prevalent human fungal pathogens, with Candida albicans being the most clinically relevant species. Candida albicans resides as a commensal of the human gastrointestinal tract but is a frequent cause of opportunistic mucosal and systemic infections. Investigation of C. albicans virulence has traditionally relied on candidate gene approaches, but recent advances in functional genomics have now facilitated global, unbiased studies of gene function. Such studies include comparative genomics (both between and within Candida species), analysis of total RNA expression, and regulation and delineation of protein-DNA interactions. Additionally, large collections of mutant strains have begun to aid systematic screening of clinically relevant phenotypes. Here, we will highlight the development of functional genomics in C. albicans and discuss the use of these approaches to addressing both commensalism and pathogenesis in this species.

  19. Innate immune cell response upon Candida albicans infection.

    PubMed

    Qin, Yulin; Zhang, Lulu; Xu, Zheng; Zhang, Jinyu; Jiang, Yuan-Ying; Cao, Yongbing; Yan, Tianhua

    2016-07-03

    Candida albicans is a polymorphic fungus which is the predominant cause of superficial and deep tissue fungal infections. This microorganism has developed efficient strategies to invade the host and evade host defense systems. However, the host immune system will be prepared for defense against the microbe by recognition of receptors, activation of signal transduction pathways and cooperation of immune cells. As a consequence, C. albicans could either be eliminated by immune cells rapidly or disseminate hematogenously, leading to life-threatening systemic infections. The interplay between Candida albicans and the host is complex, requiring recognition of the invaded pathogens, activation of intricate pathways and collaboration of various immune cells. In this review, we will focus on the effects of innate immunity that emphasize the first line protection of host defense against invaded C. albicans including the basis of receptor-mediated recognition and the mechanisms of cell-mediated immunity.

  20. Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis.

    PubMed

    Young, Li-Sen; Harrison, Benjamin R; Narayana Murthy, U M; Moffatt, Barbara A; Gilroy, Simon; Masson, Patrick H

    2006-10-01

    Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.

  1. Serum factors involved in human microvascular endothelial cell morphogenesis.

    PubMed

    Harvey, Kevin; Siddiqui, Rafat A; Sliva, Daniel; Garcia, Joe G N; English, Denis

    2002-09-01

    Our previous studies have demonstrated that lipid and protein angiogenic factors operate in tandem to induce optimal angiogenic responses in vivo. This study was undertaken to clarify the nature of the substances in human serum that are responsible for its remarkable ability to promote capillary morphogenesis in vitro. The ability of dilute (2%) human serum to promote the morphogenic differentiation of human dermal microvascular endothelial cells on Matrigel supports was depleted by more than 50% by treatment of the serum with activated charcoal, a procedure that effectively removes biologically active lipid growth factors. The remainder of the activity within serum was lost on heating to 60 degrees C for 60 minutes, indicating the involvement of a protein in the response. The ability of charcoal-treated serum to promote capillary morphogenesis was completely restored by the addition of sphingosine 1-phosphate (SPP, 500 nmol/L), but other lipids thought to be released into serum during clotting were ineffective. In addition, basic fibroblast growth factor (bFGF) effectively restored the ability of heat-treated serum to promote endothelial cell morphogenesis, but other protein growth factors, including vascular endothelial growth factor and platelet-derived growth factor, were ineffective. Together, SPP and bFGF were as effective as whole serum in promoting capillary morphogenesis. Responses to purified SPP were entirely sensitive to the effects of preexposure of the cells to pertussis toxin, whereas responses to bFGF were entirely pertussis toxin-resistant. Consistent with our hypothesis that two distinct factors in serum play a role in promoting capillary morphogenesis, responses induced by serum were inhibited approximately 50% by preexposure of endothelial cells to pertussis toxin. We conclude that platelet-released SPP acts in conjunction with circulating bFGF to promote capillary formation by microvascular endothelial cells. Lipid and protein growth factors

  2. Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis.

    PubMed

    Hielscher, Abigail; Ellis, Kim; Qiu, Connie; Porterfield, Josh; Gerecht, Sharon

    2016-01-01

    The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5β1 and αvβ3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis.

  3. Mechanism underlying renal failure caused by pathogenic Candida albicans infection.

    PubMed

    Jae-Chen, Shin; Young-Joo, Jeon; Seon-Min, Park; Kang Seok, Seo; Jung-Hyun, Shim; Jung-Il, Chae

    2015-03-01

    Candida albicans (C. albicans) is an opportunistic fungal pathogen that commonly causes nosocomial infections. Systemic candidiasis is encountered with increasing frequency in immunocompromised hosts, leading to renal failure that results in severe morbidity and mortality. The present study investigated the mechanisms underlying kidney susceptibility following infection with several C. albicans strains, such as B311 and SC5314. Fungal growth of the highly virulent SC5314 strain was 10(3)-fold higher compared to the nonpathogenic B311 strain in the kidneys. An intravenous challenge of SC5314 in mice, elevated blood urea nitrogen (BUN) and creatine levels, which resulted in mortality at 8 or 35 days after infection in a dose- and time-dependent manner, whereas all the B311-infected mice had BUN and creatinine levels in the normal range and survived. Whether virulent C. albicans may escape clearance by activating signaling pathways that lead to the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, was investigated. B311 infections significantly elevated TNF-α and IL-1β mRNA expression in the kidneys, whereas the expression in SC5314-infected mice remained unchanged. Furthermore, B311 infection significantly elevated the plasma levels of TNF-α and IL-1β. These results indicated that the less virulent strains of C. albicans induced pro-inflammatory cytokines in mice. These results determined that an impairment of the protective mechanisms occurred in the kidneys with virulent C. albicans infection.

  4. Effectiveness of magnetic fluid hyperthermia against Candida albicans cells.

    PubMed

    Chudzik, Barbara; Miaskowski, Arkadiusz; Surowiec, Zbigniew; Czernel, Grzegorz; Duluk, Tomasz; Marczuk, Andrzej; Gagoś, Mariusz

    2016-12-01

    Candida albicans is one of the most frequently isolated fungal pathogens causing opportunistic infections in humans. Targeted magnetic fluid hyperthermia (MFH) is a promising method in thermal therapy facilitating selective heating of pathogen cells like C. albicans. In the paper, we used meso-2,3-dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles (MNPs) and functionalised anti-C. albicans immunomagnetic nanoparticles (IMNPs) to investigate the potential of MFH in combating C. albicans cells in vitro. Using Mössbauer spectroscopy it was found that synthesised MNPs exhibited superparamagnetic phenomena. On the basis of calorimetric experiments, the maximum SAR (specific absorption rate) was found and a proper concentration of MNPs was established to control the temperature. MFH based on both DMSA-coated MNPs and functionalised anti-C. albicans IMNPs was more effective in combating C. albicans cells in vitro than thermostat hyperthermia. Especially promising results were obtained using functionalised IMNPs, which eradicated most of the pathogen colonies at the temperature of 43 °C.

  5. In vitro damage of Candida albicans biofilms by chitosan

    PubMed Central

    PU, YU; LIU, AIBO; ZHENG, YUQIANG; YE, BIN

    2014-01-01

    With the increasing usage of indwelling medical devices in clinical practice, the frequency of fungal infections has increased, such as that of Candida albicans (C. albicans). Biofilms, a protected niche for microorganisms, are resistant to a range of current antifungal agents. Chitosan is a polyatomic biopolymer with advantageous biocompatibility, biodegradation, nontoxicity and antibacterial properties. To investigate the inhibitory effect of chitosan on biofilms formed by C. albicans, cell viability, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-caboxanilide reduction, and morphological assays, including fluorescence microscopy and scanning electron microscopy (SEM), were employed. As assessed by cell viability assay, chitosan showed significant inhibitory effects on the planktonic cells and the biofilm of C. albicans in a dose-dependent manner. Fluorescence microscopy and SEM assays confirmed that the chitosan-treated group showed delayed C. albicans biofilm formation with defect morphological features, due to the inhibitory effects of the vast majority of fungal cell growth. In conclusion, C. albicans biofilms were compromised by the treatment with chitosan, providing an alternative therapeutic strategy against the fungal biofilms in the medical devices. PMID:25120626

  6. Anti-biofilm Properties of Peganum harmala against Candida albicans

    PubMed Central

    Aboualigalehdari, Elham; Sadeghifard, Nourkhoda; Taherikalani, Morovat; Zargoush, Zaynab; Tahmasebi, Zahra; Badakhsh, Behzad; Rostamzad, Arman; Ghafourian, Sobhan; Pakzad, Iraj

    2016-01-01

    Objectives Vaginitis still remains as a health issue in women. It is notable that Candida albicans producing biofilm is considered a microorganism responsible for vaginitis with hard to treat. Also, Peganum harmala was applied as an anti fungal in treatment for many infections in Iran. Therefore, this study goal to investigate the role of P. harmala in inhibition of biofilm formation in C. albicans. Methods So, 27 C. albicans collected from women with Vaginitis, then subjected for biofilm formation assay. P. harmala was applied as antibiofilm formation in C. albicans. Results Our results demonstrated that P. harmala in concentration of 12 μg/ml easily inhibited strong biofilm formation; while the concentrations of 10 and 6 μg/ml inhibited biofilm formation in moderate and weak biofilm formation C. albicans strains, respectively. Conclusion Hence, the current study presented P. harmala as antibiofilm herbal medicine for C. albicans; but in vivo study suggested to be performed to confirm its effectiveness. PMID:27169010

  7. Sur7 Promotes Plasma Membrane Organization and Is Needed for Resistance to Stressful Conditions and to the Invasive Growth and Virulence of Candida albicans

    PubMed Central

    Douglas, Lois M.; Wang, Hong X.; Keppler-Ross, Sabine; Dean, Neta; Konopka, James B.

    2011-01-01

    ABSTRACT The human fungal pathogen Candida albicans causes lethal systemic infections because of its ability to grow and disseminate in a host. The C. albicans plasma membrane is essential for virulence by acting as a protective barrier and through its key roles in interfacing with the environment, secretion of virulence factors, morphogenesis, and cell wall synthesis. Difficulties in studying hydrophobic membranes have limited the understanding of how plasma membrane organization contributes to its function and to the actions of antifungal drugs. Therefore, the role of the recently discovered plasma membrane subdomains termed the membrane compartment containing Can1 (MCC) was analyzed by assessing the virulence of a sur7Δ mutant. Sur7 is an integral membrane protein component of the MCC that is needed for proper localization of actin, morphogenesis, cell wall synthesis, and responding to cell wall stress. MCC domains are stable 300-nm-sized punctate patches that associate with a complex of cytoplasmic proteins known as an eisosome. Analysis of virulence-related properties of a sur7Δ mutant revealed defects in intraphagosomal growth in macrophages that correlate with increased sensitivity to oxidation and copper. The sur7Δ mutant was also strongly defective in pathogenesis in a mouse model of systemic candidiasis. The mutant cells showed a decreased ability to initiate an infection and greatly diminished invasive growth into kidney tissues. These studies on Sur7 demonstrate that the plasma membrane MCC domains are critical for virulence and represent an important new target for the development of novel therapeutic strategies. PMID:22202230

  8. High-frequency switching in Candida albicans.

    PubMed Central

    Soll, D R

    1992-01-01

    Most strains of Candida albicans are capable of switching frequently and reversibly between a number of phenotypes distinguishable by colony morphology. A number of different switching systems have been defined according to the limited set of phenotypes in each switching repertoire, and each strain appears to possess a single system. Switching can affect many aspects of cellular physiology and morphology and appears to be a second level of phenotypic variability superimposed upon the bud-hypha transition. The most dramatic switching system so far identified is the "white-opaque transition." This system dramatizes the extraordinary effects switching can have on the budding cell phenotype, including the synthesis of opaque-specific antigens, the expression of white-specific and opaque-specific genes, and the genesis of unique cell wall structures. Switching has been demonstrated to occur at sites of infection and between episodes of recurrent vaginitis, and it may function to generate variability in commensal and infecting populations for adaptive reasons. Although the molecular mechanisms involved in the switch event are not understood, recent approaches to its elucidation are discussed and an epigenetic mechanism is proposed. Images PMID:1576587

  9. Spaceflight enhances cell aggregation and random budding in Candida albicans.

    PubMed

    Crabbé, Aurélie; Nielsen-Preiss, Sheila M; Woolley, Christine M; Barrila, Jennifer; Buchanan, Kent; McCracken, James; Inglis, Diane O; Searles, Stephen C; Nelman-Gonzalez, Mayra A; Ott, C Mark; Wilson, James W; Pierson, Duane L; Stefanyshyn-Piper, Heidemarie M; Hyman, Linda E; Nickerson, Cheryl A

    2013-01-01

    This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans-induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid

  10. Cellular Components Mediating Coadherence of Candida albicans and Fusobacterium nucleatum

    PubMed Central

    Wu, T.; Cen, L.; Kaplan, C.; Zhou, X.; Lux, R.; Shi, W.; He, X.

    2015-01-01

    Candida albicans is an opportunistic fungal pathogen found as part of the normal oral flora. It can be coisolated with Fusobacterium nucleatum, an opportunistic bacterial pathogen, from oral disease sites, such as those involved in refractory periodontitis and pulp necrosis. The physical coadherence between these 2 clinically important microbes has been well documented and suggested to play a role in facilitating their oral colonization and colocalization and contributing to polymicrobial pathogenesis. Previous studies indicated that the physical interaction between C. albicans and F. nucleatum was mediated by the carbohydrate components on the surface of C. albicans and the protein components on the Fusobaterium cell surface. However, the identities of the components involved still remain elusive. This study was aimed at identifying the genetic determinants involved in coaggregation between the 2 species. By screening a C. albicans SN152 mutant library and a panel of F. nucleatum 23726 outer membrane protein mutants, we identified FLO9, which encodes a putative adhesin-like cell wall mannoprotein of C. albicans and radD, an arginine-inhibitable adhesin-encoding gene in F. nucleatum that is involved in interspecies coadherence. Consistent with these findings, we demonstrated that the strong coaggregation between wild-type F. nucleatum 23726 and C. albicans SN152 in an in vitro assay could be greatly inhibited by arginine and mannose. Our study also suggested a complex multifaceted mechanism underlying physical interaction between C. albicans and F. nucleatum and for the first time revealed the identity of major genetic components involved in mediating the coaggregation. These observations provide useful knowledge for developing new targeted treatments for disrupting interactions between these 2 clinically relevant pathogens. PMID:26152186

  11. A Monoclonal Antibody Directed against a Candida albicans Cell Wall Mannoprotein Exerts Three Anti-C. albicans Activities

    PubMed Central

    Moragues, María D.; Omaetxebarria, Miren J.; Elguezabal, Natalia; Sevilla, María J.; Conti, Stefania; Polonelli, Luciano; Pontón, José

    2003-01-01

    Antibodies are believed to play a role in the protection against Candida albicans infections by a number of mechanisms, including the inhibition of adhesion or germ tube formation, opsonization, neutralization of virulence-related enzymes, and direct candidacidal activity. Although some of these biological activities have been demonstrated individually in monoclonal antibodies (MAbs), it is not clear if all these anti-C. albicans activities can be displayed by a single antibody. In this report, we characterized a monoclonal antibody raised against the main target of salivary secretory immunoglobulin A in the cell wall of C. albicans, which exerts three anti-C. albicans activities: (i) inhibition of adherence to HEp-2 cells, (ii) inhibition of germination, and (iii) direct candidacidal activity. MAb C7 reacted with a proteinic epitope from a mannoprotein with a molecular mass of >200 kDa predominantly expressed on the C. albicans germ tube cell wall surface as well as with a number of antigens from Candida lusitaniae, Cryptococcus neoformans, Aspergillus fumigatus, and Scedosporium prolificans. MAb C7 caused a 31.1% inhibition in the adhesion of C. albicans to HEp-2 monolayers and a 55.3% inhibition in the adhesion of C. albicans to buccal epithelial cells, produced a 38.5% decrease in the filamentation of C. albicans, and exhibited a potent fungicidal effect against C. albicans, C. lusitaniae, Cryptococcus neoformans, A. fumigatus, and S. prolificans, showing reductions in fungal growth ranging from 34.2 to 88.7%. The fungicidal activity showed by MAb C7 seems to be related to that reported by antibodies mimicking the activity of a killer toxin produced by the yeast Pichia anomala, since one of these MAbs also reacted with the C. albicans mannoprotein with a molecular mass of >200 kDa. Results presented in this study support the concept of a family of microbicidal antibodies that could be useful in the treatment of a wide range of microbial infections when used

  12. Fusarium and Candida albicans Biofilms on Soft Contact Lenses: Model Development, Influence of Lens Type, and Susceptibility to Lens Care Solutions▿

    PubMed Central

    Imamura, Yoshifumi; Chandra, Jyotsna; Mukherjee, Pranab K.; Lattif, Ali Abdul; Szczotka-Flynn, Loretta B.; Pearlman, Eric; Lass, Jonathan H.; O'Donnell, Kerry; Ghannoum, Mahmoud A.

    2008-01-01

    Fungal keratitis is commonly caused by Fusarium species and less commonly by Candida species. Recent outbreaks of Fusarium keratitis were associated with contact lens wear and with ReNu with MoistureLoc contact lens care solution, and biofilm formation on contact lens/lens cases was proposed to play a role in this outbreak. However, no in vitro model for contact lens-associated fungal biofilm has been developed. In this study, we developed and characterized in vitro models of biofilm formation on various soft contact lenses using three species of Fusarium and Candida albicans. The contact lenses tested were etafilcon A, galyfilcon A, lotrafilcon A, balafilcon A, alphafilcon A, and polymacon. Our results showed that clinical isolates of Fusarium and C. albicans formed biofilms on all types of lenses tested and that the biofilm architecture varied with the lens type. Moreover, differences in hyphal content and architecture were found between the biofilms formed by these fungi. We also found that two recently isolated keratitis-associated fusaria formed robust biofilms, while the reference ATCC 36031 strain (recommended by the International Organization for Standardization guidelines for testing of disinfectants) failed to form biofilm. Furthermore, using the developed in vitro biofilm model, we showed that phylogenetically diverse planktonic fusaria and Candida were susceptible to MoistureLoc and MultiPlus. However, Fusarium biofilms exhibited reduced susceptibility against these solutions in a species- and time-dependent manner. This in vitro model should provide a better understanding of the biology and pathogenesis of lens-related fungal keratitis. PMID:17999966

  13. Extracellular matrix bioscaffolds in tissue remodeling and morphogenesis

    PubMed Central

    Swinehart, Ilea T.; Badylak, Stephen F.

    2016-01-01

    During normal morphogenesis the extracellular matrix (ECM) influences cell motility, proliferation, apoptosis, and differentiation. Tissue engineers have attempted to harness the cell signaling potential of ECM to promote the functional reconstruction, if not regeneration, of injured or missing adult tissues that otherwise heal by the formation of scar tissue. ECM bioscaffolds, derived from decellularized tissues, have been used to promote the formation of site appropriate, functional tissues in many clinical applications including skeletal muscle, fibrocartilage, lower urinary tract, and esophageal reconstruction, among others. These scaffolds function by the release or exposure of growth factors and cryptic peptides, modulation of the immune response, and recruitment of progenitor cells. Herein, we describe this process of ECM induced constructive remodeling and examine similarities to normal tissue morphogenesis. PMID:26699796

  14. MicroRNAs regulate brain morphogenesis in zebrafish.

    PubMed

    Giraldez, Antonio J; Cinalli, Ryan M; Glasner, Margaret E; Enright, Anton J; Thomson, J Michael; Baskerville, Scott; Hammond, Scott M; Bartel, David P; Schier, Alexander F

    2005-05-06

    MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis.

  15. Polarized protein transport and lumen formation during epithelial tissue morphogenesis.

    PubMed

    Blasky, Alex J; Mangan, Anthony; Prekeris, Rytis

    2015-01-01

    One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form the multicellular epithelial lumen during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. Some epithelial lumens form from preexisting polarized epithelial structures. However, de novo lumen formation from nonpolarized cells has recently emerged as an important driver of epithelial tissue morphogenesis, especially during the formation of small epithelial tubule networks. In this review, we discuss the latest findings regarding the mechanisms and regulation of de novo lumen formation in vitro and in vivo.

  16. Polarized Protein Transport and Lumen Formation During Epithelial Tissue Morphogenesis

    PubMed Central

    Blasky, Alex J.; Mangan, Anthony; Prekeris, Rytis

    2015-01-01

    One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form the multicellular epithelial lumen during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. Some epithelial lumens form from preexisting polarized epithelial structures. However, de novo lumen formation from nonpolarized cells has recently emerged as an important driver of epithelial tissue morphogenesis, especially during the formation of small epithelial tubule networks. In this review, we discuss the latest findings regarding the mechanisms and regulation of de novo lumen formation in vitro and in vivo. PMID:26359775

  17. Multi-scale mechanics from molecules to morphogenesis

    PubMed Central

    Davidson, Lance; von Dassow, Michelangelo; Zhou, Jian

    2009-01-01

    Dynamic mechanical processes shape the embryo and organs during development. Little is understood about the basic physics of these processes, what forces are generated, or how tissues resist or guide those forces during morphogenesis. This review offers an outline of some of the basic principles of biomechanics, provides working examples of biomechanical analyses of developing embryos, and reviews the role of structural proteins in establishing and maintaining the mechanical properties of embryonic tissues. Drawing on examples we highlight the importance of investigating mechanics at multiple scales from milliseconds to hours and from individual molecules to whole embryos. Lastly, we pose a series of questions that will need to be addressed if we are to understand the larger integration of molecular and physical mechanical processes during morphogenesis and organogenesis. PMID:19394436

  18. The unfolded protein response is required for dendrite morphogenesis.

    PubMed

    Wei, Xing; Howell, Audrey S; Dong, Xintong; Taylor, Caitlin A; Cooper, Roshni C; Zhang, Jianqi; Zou, Wei; Sherwood, David R; Shen, Kang

    2015-06-08

    Precise patterning of dendritic fields is essential for the formation and function of neuronal circuits. During development, dendrites acquire their morphology by exuberant branching. How neurons cope with the increased load of protein production required for this rapid growth is poorly understood. Here we show that the physiological unfolded protein response (UPR) is induced in the highly branched Caenorhabditis elegans sensory neuron PVD during dendrite morphogenesis. Perturbation of the IRE1 arm of the UPR pathway causes loss of dendritic branches, a phenotype that can be rescued by overexpression of the ER chaperone HSP-4 (a homolog of mammalian BiP/grp78). Surprisingly, a single transmembrane leucine-rich repeat protein, DMA-1, plays a major role in the induction of the UPR and the dendritic phenotype in the UPR mutants. These findings reveal a significant role for the physiological UPR in the maintenance of ER homeostasis during morphogenesis of large dendritic arbors.

  19. The evolution of fungal morphogenesis, a personal account.

    PubMed

    Bartnicki-Garcia, Salomon

    2016-01-01

    This article describes the evolution of the field of fungal morphogenesis, its beginning at the end of the 19th century and its exponential growth during the second half of the 20th century, continuing until the present day. The main theme correlates biological progress with the advent of new technologies. Accordingly the article describes the discovery of apical growth, the fibrillar nature of the fungal wall, the chemistry of the cell wall, the search for biochemical pathways in morphogenesis, the discovery of the Spitzenkörper, the apical gradient of wall synthesis, key highlights in ultrastructural research, the development of mathematical models particularly the vesicle supply center (VSC) model, the revolution brought about by molecular biology and unique discoveries such as the hydrophobins and γ-tubulin and some the latest triumphs of the marriage between molecular genetics and confocal microscopy. Credit is given to the investigators responsible for all the advances.

  20. Three-dimensional vertex model for simulating multicellular morphogenesis

    PubMed Central

    Okuda, Satoru; Inoue, Yasuhiro; Adachi, Taiji

    2015-01-01

    During morphogenesis, various cellular activities are spatiotemporally coordinated on the protein regulatory background to construct the complicated, three-dimensional (3D) structures of organs. Computational simulations using 3D vertex models have been the focus of efforts to approach the mechanisms underlying 3D multicellular constructions, such as dynamics of the 3D monolayer or multilayer cell sheet like epithelia as well as the 3D compacted cell aggregate, including dynamic changes in layer structures. 3D vertex models enable the quantitative simulation of multicellular morphogenesis on the basis of single-cell mechanics, with complete control of various cellular activities such as cell contraction, growth, rearrangement, division, and death. This review describes the general use of the 3D vertex model, along with its applications to several simplified problems of developmental phenomena. PMID:27493850

  1. Oscarella lobularis (Homoscleromorpha, Porifera) Regeneration: Epithelial Morphogenesis and Metaplasia.

    PubMed

    Ereskovsky, Alexander V; Borisenko, Ilya E; Lapébie, Pascal; Gazave, Eve; Tokina, Daria B; Borchiellini, Carole

    2015-01-01

    Sponges are known to possess remarkable reconstitutive and regenerative abilities ranging from common wounding or body part regeneration to more impressive re-building of a functional body from dissociated cells. Among the four sponge classes, Homoscleromorpha is notably the only sponge group to possess morphologically distinct basement membrane and specialized cell-junctions, and is therefore considered to possess true epithelia. The consequence of this peculiar organization is the predominance of epithelial morphogenesis during ontogenesis of these sponges. In this work we reveal the underlying cellular mechanisms used during morphogenesis accompanying ectosome regeneration in the homoscleromorph sponge model: Oscarella lobularis. We identified three main sources of novel exopinacoderm during the processes of its regeneration and the restoration of functional peripheral parts of the aquiferous system in O. lobularis: (1) intact exopinacoderm surrounding the wound surface, (2) the endopinacoderm from peripheral exhalant and inhalant canals, and (3) the intact choanoderm found on the wound surface. The basic morphogenetic processes during regeneration are the spreading and fusion of epithelial sheets that merge into one continuous epithelium. Transdifferentiation of choanocytes into exopinacocytes is also present. Epithelial-mesenchymal transition is absent during regeneration. Moreover, we cannot reveal any other morphologically distinct pluripotent cells. In Oscarella, neither blastema formation nor local dedifferentiation and proliferation have been detected, which is probably due to the high morphogenetic plasticity of the tissue. Regeneration in O. lobularis goes through cell transdifferentiation and through the processes, when lost body parts are replaced by the remodeling of the remaining tissue. Morphogenesis during ectosome regeneration in O. lobularis is correlated with its true epithelial organization. Knowledge of the morphological basis of

  2. Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis.

    PubMed

    Simpson, Cory L; Patel, Dipal M; Green, Kathleen J

    2011-08-23

    To provide a stable environmental barrier, the epidermis requires an integrated network of cytoskeletal elements and cellular junctions. Nevertheless, the epidermis ranks among the body's most dynamic tissues, continually regenerating itself and responding to cutaneous insults. As keratinocytes journey from the basal compartment towards the cornified layers, they completely reorganize their adhesive junctions and cytoskeleton. These architectural components are more than just rivets and scaffolds - they are active participants in epidermal morphogenesis that regulate epidermal polarization, signalling and barrier formation.

  3. Oscarella lobularis (Homoscleromorpha, Porifera) Regeneration: Epithelial Morphogenesis and Metaplasia

    PubMed Central

    Ereskovsky, Alexander V.; Borisenko, Ilya E.; Lapébie, Pascal; Gazave, Eve; Tokina, Daria B.; Borchiellini, Carole

    2015-01-01

    Sponges are known to possess remarkable reconstitutive and regenerative abilities ranging from common wounding or body part regeneration to more impressive re-building of a functional body from dissociated cells. Among the four sponge classes, Homoscleromorpha is notably the only sponge group to possess morphologically distinct basement membrane and specialized cell-junctions, and is therefore considered to possess true epithelia. The consequence of this peculiar organization is the predominance of epithelial morphogenesis during ontogenesis of these sponges. In this work we reveal the underlying cellular mechanisms used during morphogenesis accompanying ectosome regeneration in the homoscleromorph sponge model: Oscarella lobularis. We identified three main sources of novel exopinacoderm during the processes of its regeneration and the restoration of functional peripheral parts of the aquiferous system in O. lobularis: (1) intact exopinacoderm surrounding the wound surface, (2) the endopinacoderm from peripheral exhalant and inhalant canals, and (3) the intact choanoderm found on the wound surface. The basic morphogenetic processes during regeneration are the spreading and fusion of epithelial sheets that merge into one continuous epithelium. Transdifferentiation of choanocytes into exopinacocytes is also present. Epithelial-mesenchymal transition is absent during regeneration. Moreover, we cannot reveal any other morphologically distinct pluripotent cells. In Oscarella, neither blastema formation nor local dedifferentiation and proliferation have been detected, which is probably due to the high morphogenetic plasticity of the tissue. Regeneration in O. lobularis goes through cell transdifferentiation and through the processes, when lost body parts are replaced by the remodeling of the remaining tissue. Morphogenesis during ectosome regeneration in O. lobularis is correlated with its true epithelial organization. Knowledge of the morphological basis of

  4. The Role of Isocitrate Lyase (ICL1) in the Metabolic Adaptation of Candida albicans Biofilms

    PubMed Central

    Ishola, Oluwaseun Ayodeji; Ting, Seng Yeat; Tabana, Yasser M; Ahmed, Mowaffaq Adam; Yunus, Muhammad Amir; Mohamed, Rafeezul; Lung Than, Leslie Thian; Sandai, Doblin

    2016-01-01

    Background A major characteristic of Candida biofilm cells that differentiates them from free-floating cells is their high tolerance to antifungal drugs. This high resistance is attributed to particular biofilm properties, including the accumulation of extrapolymeric substances, morphogenetic switching, and metabolic flexibility. Objectives This study evaluated the roles of metabolic processes (in particular the glyoxylate cycle) on biofilm formation, antifungal drug resistance, morphology, and cell wall components. Methods Growth, adhesion, biofilm formation, and cell wall carbohydrate composition were quantified for isogenic Candida albicans ICL1/ICL1, ICL1/icl1, and icl1/icl1 strains. The morphology and topography of these strains were compared by light microscopy and scanning electron microscopy. FKS1 (glucan synthase), ERG11 (14-α-demethylase), and CDR2 (efflux pump) mRNA levels were quantified using qRT-PCR. Results The ICL1/icl1 and icl1/icl1 strains formed similar biofilms and exhibited analogous drug-tolerance levels to the control ICL1/ICL1 strains. Furthermore, the drug sequestration ability of β-1, 3-glucan, a major carbohydrate component of the extracellular matrix, was not impaired. However, the inactivation of ICL1 did impair morphogenesis. ICL1 deletion also had a considerable effect on the expression of the FKS1, ERG11, and CDR2 genes. FKS1 and ERG11 were upregulated in ICL1/icl1 and icl1/icl1 cells throughout the biofilm developmental stages, and CDR2 was upregulated at the early phase. However, their expression was downregulated compared to the control ICL1/ICL1 strain. Conclusions We conclude that the glyoxylate cycle is not a specific determinant of biofilm drug resistance. PMID:27800147

  5. Bypass of Candida albicans Filamentation/Biofilm Regulators through Diminished Expression of Protein Kinase Cak1

    PubMed Central

    Xu, Wenjie; Aleynikov, Tatyana; Cullen, Paul J.; Lanni, Frederick; Andes, David R.

    2016-01-01

    Biofilm formation on implanted medical devices is a major source of lethal invasive infection by Candida albicans. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for surface adherence. Cell cycle or cell growth defects can induce filamentation, but we have limited information about the coupling between filamentation and filamentation-associated gene expression after cell cycle/cell growth inhibition. Here we identified the CDK activating protein kinase Cak1 as a determinant of filamentation and filamentation-associated gene expression through a screen of mutations that diminish expression of protein kinase-related genes implicated in cell cycle/cell growth control. A cak1 diminished expression (DX) strain displays filamentous growth and expresses filamentation-associated genes in the absence of typical inducing signals. In a wild-type background, expression of filamentation-associated genes depends upon the transcription factors Bcr1, Brg1, Efg1, Tec1, and Ume6. In the cak1 DX background, the dependence of filamentation-associated gene expression on each transcription factor is substantially relieved. The unexpected bypass of filamentation-associated gene expression activators has the functional consequence of enabling biofilm formation in the absence of Bcr1, Brg1, Tec1, Ume6, or in the absence of both Brg1 and Ume6. It also enables filamentous cell morphogenesis, though not biofilm formation, in the absence of Efg1. Because these transcription factors are known to have shared target genes, we suggest that cell cycle/cell growth limitation leads to activation of several transcription factors, thus relieving dependence on any one. PMID:27935965

  6. UvHOG1 is important for hyphal growth and stress responses in the rice false smut fungus Ustilaginoidea virens

    PubMed Central

    Zheng, Dawei; Wang, Yi; Han, Yu; Xu, Jin-Rong; Wang, Chenfang

    2016-01-01

    Rice false smut caused by Ustilaginoidea virens is one of the most important diseases of rice worldwide. Although its genome has been sequenced, to date there is no report on targeted gene deletion in U. virens and no molecular studies on genetic mechanisms regulating the infection processes of this destructive pathogen. In this study, we attempted to generate knockout mutants of the ortholog of yeast HOG1 MAP kinase gene in U. virens. One Uvhog1 deletion mutant was identified after screening over 600 hygromycin-resistant transformants generated by Agrobacterium tumefaciens mediated transformation. The Uvhog1 mutant was reduced in growth rate and conidiation but had increased sensitivities to SDS, Congo red, and hyperosmotic stress. Deletion of UvHOG1 resulted in reduced expression of the stress response-related genes UvATF1 and UvSKN7. In the Uvhog1 mutant, NaCl treatment failed to stimulate the accumulation of sorbitol and glycerol. In addition, the Uvhog1 mutant had reduced toxicity on shoot growth in rice seed germination assays. Overall, as the first report of targeted gene deletion mutant in U. virens, our results showed that UvHOG1 likely has conserved roles in regulating stress responses, hyphal growth, and possibly secondary metabolism. PMID:27095476

  7. A Genome-Wide Transcriptional Analysis of Yeast-Hyphal Transition in Candida tropicalis by RNA-Seq

    PubMed Central

    Yu, Shuan-bao; Li, Wen-ge; Liu, Xiao-shu; Zhao, Lei; Lu, Jin-xing

    2016-01-01

    Candida tropicalis is considered as the leading pathogen in nosocomial fungemia and hepatosplenic fungal infections in patients with cancer, particularly in leukemia. The yeast-filament transition is required for virulent infection by Candida. Several studies have explored the genome-wide transcription profile of Candida, however, no report on the transcriptional profile of C. tropicalis under yeast-filament transition has been published. In this study, the transcriptomes of three C. tropicalis isolates with different adhesion and biofilm formation abilities, identified in our previous studies, were analyzed in both the yeast and filament states using RNA-Seq. Differentially expressed genes were found for each isolate during the transition. A total of 115 genes were up- or down- regulated in the two hyphal-producing isolates (ZRCT 4 and ZRCT 45). Among these differentially expressed genes, only two were down-regulated during the yeast-filament transition. Furthermore, six filament-associated genes were up-regulated in the hyphae-producing isolates. According to Candida Hypha Growth Database established in this study, 331 hyphae- related genes were discovered in C. tropicalis. ALS1 and ALS3 were down-regulated and up-regulated, respectively, during filamentous growth of C. tropicalis. These findings proved a better understanding of gene expression dynamics during the yeast-filament transition in C. tropicalis. PMID:27851809

  8. FgFlbD regulates hyphal differentiation required for sexual and asexual reproduction in the ascomycete fungus Fusarium graminearum.

    PubMed

    Son, Hokyoung; Kim, Myung-Gu; Chae, Suhn-Kee; Lee, Yin-Won

    2014-11-01

    Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.

  9. Enhanced hyphal growth of arbuscular mycorrhizae by root exudates derived from high R/FR treated Lotus japonicus.

    PubMed

    Nagata, Maki; Yamamoto, Naoya; Miyamoto, Taro; Shimomura, Aya; Arima, Susumu; Hirsch, Ann M; Suzuki, Akihiro

    2016-06-02

    Red/Far Red (R/FR) sensing positively influences the arbuscular mycorrhizal (AM) symbiosis of both legume and nonlegume plants through jasmonic acid (JA) and strigolactone signaling. We previously reported that root exudates obtained from high R/FR-grown plants contained more strigolactone than low R/FR-grown plants. To determine whether JA and JA derivatives were secreted from roots, we investigated the expression levels of JA-responsive genes in L. japonicus Miyakojima MG20 plants treated with root exudates prepared from either high or low R/FR light-treated plants. The root exudates from high R/FR light-treated plants were found to enhance the expression levels of JA-responsive genes significantly. Moreover, exogenous JA increased AM fungal hyphal elongation as did the root exudates derived from high R/FR-grown L. japonicus plants. We conclude that increased JA accumulation and secretion into root exudates from high R/FR light-grown plants is the best explanation for increased colonization and enhanced mycorrhization under these conditions.

  10. Molecular cloning and characterization of the Candida albicans enolase gene.

    PubMed Central

    Mason, A B; Buckley, H R; Gorman, J A

    1993-01-01

    A DNA clone containing the putative Candida albicans enolase gene (ENO1) was isolated from a genomic DNA library. The sequenced insert contained a continuous open reading frame of 1,320 bp. The predicted 440-amino-acid protein is 78 and 76% identical, respectively, to Saccharomyces cerevisiae enolase proteins 1 and 2. Only one enolase gene could be detected in C. albicans genomic DNA by Southern analysis with a homologous probe. Northern (RNA) analysis detected a single, abundant C. albicans ENO1 transcript of approximately 1,600 nucleotides. When cells were grown on glucose, levels of ENO1 mRNA were markedly increased by comparison with ENO1 mRNA levels in cells grown on ethanol, a gluconeogenic carbon source. In contrast to this glucose-mediated transcriptional induction, the carbon source had no dramatic effect on the levels of enolase protein or enzyme activity in the C. albicans strains tested. These results suggest that posttranscriptional mechanisms are responsible for modulating expression of the C. albicans enolase gene. Images PMID:8478328

  11. Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis

    PubMed Central

    Naglik, Julian R.; Challacombe, Stephen J.; Hube, Bernhard

    2003-01-01

    Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis. PMID:12966142

  12. Nicotine Enhances Interspecies Relationship between Streptococcus mutans and Candida albicans.

    PubMed

    Liu, Shiyu; Qiu, Wei; Zhang, Keke; Zhou, Xuedong; Ren, Biao; He, Jinzhi; Xu, Xin; Cheng, Lei; Li, Mingyun

    2017-01-01

    Streptococcus mutans and Candida albicans are common microorganisms in the human oral cavity. The synergistic relationship between these two species has been deeply explored in many studies. In the present study, the effect of alkaloid nicotine on the interspecies between S. mutans and C. albicans is explored. We developed a dual-species biofilm model and studied biofilm biomass, biofilm structure, synthesis of extracellular polysaccharides (EPS), and expression of glucosyltransferases (Gtfs). Biofilm formation and bacterial and fungal cell numbers in dual-species biofilms increased in the presence of nicotine. More C. albicans cells were present in the dual-species biofilms in the nicotine-treated groups as determined by scanning electron microscopy. The synthesis of EPS was increased by 1 mg/ml of nicotine as detected by confocal laser scanning microscopy. The result of qRT-PCR showed gtfs expression was upregulated when 1 mg/ml of nicotine was used. We speculate that nicotine promoted the growth of S. mutans, and more S. mutans cells attracted more C. albicans cells due to the interaction between two species. Since S. mutans and C. albicans are putative pathogens for dental caries, the enhancement of the synergistic relationship by nicotine may contribute to caries development in smokers.

  13. Nicotine Enhances Interspecies Relationship between Streptococcus mutans and Candida albicans

    PubMed Central

    Qiu, Wei; Zhang, Keke; Zhou, Xuedong; Ren, Biao; He, Jinzhi; Xu, Xin

    2017-01-01

    Streptococcus mutans and Candida albicans are common microorganisms in the human oral cavity. The synergistic relationship between these two species has been deeply explored in many studies. In the present study, the effect of alkaloid nicotine on the interspecies between S. mutans and C. albicans is explored. We developed a dual-species biofilm model and studied biofilm biomass, biofilm structure, synthesis of extracellular polysaccharides (EPS), and expression of glucosyltransferases (Gtfs). Biofilm formation and bacterial and fungal cell numbers in dual-species biofilms increased in the presence of nicotine. More C. albicans cells were present in the dual-species biofilms in the nicotine-treated groups as determined by scanning electron microscopy. The synthesis of EPS was increased by 1 mg/ml of nicotine as detected by confocal laser scanning microscopy. The result of qRT-PCR showed gtfs expression was upregulated when 1 mg/ml of nicotine was used. We speculate that nicotine promoted the growth of S. mutans, and more S. mutans cells attracted more C. albicans cells due to the interaction between two species. Since S. mutans and C. albicans are putative pathogens for dental caries, the enhancement of the synergistic relationship by nicotine may contribute to caries development in smokers. PMID:28280743

  14. A Photonic Crystal Protein Hydrogel Sensor for Candida albicans.

    PubMed

    Cai, Zhongyu; Kwak, Daniel H; Punihaole, David; Hong, Zhenmin; Velankar, Sachin S; Liu, Xinyu; Asher, Sanford A

    2015-10-26

    We report two-dimensional (2D) photonic crystal (PC) sensing materials that selectively detect Candida albicans (C. albicans). These sensors utilize Concanavalin A (Con A) protein hydrogels with a 2D PC embedded on the Con A protein hydrogel surface, that multivalently and selectively bind to mannan on the C. albicans cell surface to form crosslinks. The resulting crosslinks shrink the Con A protein hydrogel, reduce the 2D PC particle spacing, and blue-shift the light diffracted from the PC. The diffraction shifts can be visually monitored, measured with a spectrometer, or determined from the Debye diffraction ring diameter. Our unoptimized hydrogel sensor has a detection limit of around 32 CFU/mL for C. albicans. This sensor distinguishes between C. albicans and those microbes devoid of cell-surface mannan such as the gram-negative bacterium E. coli. This sensor provides a proof-of-concept for utilizing recognition between lectins and microbial cell surface carbohydrates to detect microorganisms in aqueous environments.

  15. Candida albicans-induced inflammatory response in human keratinocytes.

    PubMed

    Wollina, U; Künkel, W; Bulling, L; Fünfstück, C; Knöll, B; Vennewald, I; Hipler, U-C

    2004-06-01

    Candida albicans strains 3153a, ATCC 48867, CBS 2730, DSM 70014, and Vir 13 were cultivated and sterile C. albicans filtrates were produced. The interaction of soluble Candida factors of these infiltrates with human HaCaT keratinocytes was assayed in vitro. The following parameters were analyzed: cell proliferation, protein synthesis, nuclear matrix protein (NMP) 41 release, cytokine release (IL-1beta, soluble IL-2 receptor, IL-6, and IL-8), and reactive oxygen species (ROS). Cell counts at 1, 12, and 24 h were significantly lower for C. albicans strains CBS 2730 and VIR 13 (P < 0.05). There was no significant change for the remaining strains. Neither the protein synthesis nor the NMP-41 release was significantly affected. IL-6 and IL-8 were stimulated by C. albicans filtrates to different amounts with higher levels in strains of low virulence. There was no effect on the other cytokines. The production of ROS by HaCaT keratinocytes was suppressed. The induction of an inflammatory keratinocyte response by soluble C. albicans factors may play a role among the host-yeast interactions.

  16. Candida albicans Is Resistant to Polyglutamine Aggregation and Toxicity

    PubMed Central

    Leach, Michelle D.; Kim, TaeHyung; DiGregorio, Sonja E.; Collins, Cathy; Zhang, Zhaolei; Duennwald, Martin L.; Cowen, Leah E.

    2016-01-01

    Disruption of protein quality control can be detrimental, having toxic effects on single cell organisms and contributing to neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s in humans. Here, we examined the effects of polyglutamine (polyQ) aggregation in a major fungal pathogen of humans, Candida albicans, with the goal of identifying new approaches to disable this fungus. However, we discovered that expression of polyQ stretches up to 230Q had no effect on C. albicans ability to grow and withstand proteotoxic stress. Bioinformatics analysis demonstrates that C. albicans has a similarly glutamine-rich proteome to the unicellular fungus Saccharomyces cerevisiae, which exhibits polyQ toxicity with as few as 72Q. Surprisingly, global transcriptional profiles indicated no significant change upon induction of up to 230Q. Proteomic analysis highlighted two key interactors of 230Q, Sis1 and Sgt2; however, loss of either protein had no additional effect on C. albicans toxicity. Our data suggest that C. albicans has evolved powerful mechanisms to overcome the toxicity associated with aggregation-prone proteins, providing a unique model for studying polyQ-associated diseases. PMID:27807047

  17. Embryo mechanics: balancing force production with elastic resistance during morphogenesis.

    PubMed

    Davidson, Lance A

    2011-01-01

    Morphogenesis requires the spatial and temporal control of embryo mechanics, including force production and mechanical resistance to those forces, to coordinate tissue deformation and large-scale movements. Thus, biomechanical processes play a key role in directly shaping the embryo. Additional roles for embryo mechanics during development may include the patterning of positional information and to provide feedback to ensure the success of morphogenetic movements in shaping the larval body and organs. To understand the multiple roles of mechanics during development requires familiarity with engineering principles of the mechanics of structures, the viscoelastic properties of biomaterials, and the integration of force and stress within embryonic structures as morphogenesis progresses. In this chapter, we review the basic engineering principles of biomechanics as they relate to morphogenesis, introduce methods for quantifying embryo mechanics and the limitations of these methods, and outline a formalism for investigating the role of embryo mechanics in birth defects. We encourage the nascent field of embryo mechanics to adopt standard engineering terms and test methods so that studies of diverse organisms can be compared and universal biomechanical principles can be revealed.

  18. Testing Turing's Theory of Morphogenesis in Chemical Cells

    NASA Astrophysics Data System (ADS)

    Tompkins, Nathan; Li, Ning; Girabawe, Camille; Heymann, Michael; Ermentrout, G. Bard; Epstein, Irving; Fraden, Seth

    2015-03-01

    Alan Turing's 1952 paper ``The Chemical Basis of Morphogenesis'' described how reaction-diffusion dynamics could create six spatiotemporal patterns including a stationary pattern that could lead to physical morphogenesis (which now bears his name). This stationary ``Turing pattern'' has been observed in continuous media of various chemical systems but never in diffusively coupled discrete reactors as Turing theorized. We have created a system of microfluidically produced chemical compartments containing the Belousov-Zhabotinsky reaction that are designed to fulfill the assumptions of Turing's theoretical system. This system demonstrates all six spatiotemporal patterns that Turing predicted. In particular, we observe the stationary case that bears Turing's name where the cells create a pattern of oxidized and reduced states. As Turing predicted, this chemical heterogeneity gives rise to physical heterogeneity by driving an osmotic flow, swelling the reduced cells and shrinking the oxidized cells. In addition to the six patterns and physical morphogenesis predicted by Turing we observe a seventh pattern of mixed stationary/oscillatory states that is not predicted by Turing. This seventh pattern requires modifying Turing's theory to include slight heterogeneity to match experiments.

  19. Morphogenesis, Dictyostelium, and the search for shared developmental processes.

    PubMed

    Sunderland, Mary Evelyn

    2011-12-01

    In the 1930s John Tyler Bonner began studying the slime mold, Dictyostelium discoideum, as a way to investigate how organisms develop. With a life cycle that includes periods of unicellularity and multicellularity, Dictyostelium raises questions fundamental to development and evolution. In Morphogenesis: An Essay on Development (1952), Bonner built on his work with Dictyostelium to inform developmental theory and practice. By exploring how Bonner's early work with Dictyostelium motivated his synthetic approach in Morphogenesis, this paper presents an example of how those who studied development sought ways to gain traction in the rapidly changing life sciences. While a biochemical viewpoint of development became dominant, morphogenesis provided a way to reintroduce and emphasize biological organization at the organismal level. Bonner's early work offers a window to mid-twentieth century studies of development, an understudied area in the history of science, and shows that it was a time when growing experimental evidence enabled new ways of thinking about the relationship between ontogeny and evolution, and more broadly, about how the parts of nature might fit together.

  20. Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection

    PubMed Central

    Clark, Heather L.; Jhingran, Anupam; Sun, Yan; Vareechon, Chairut; Carrion, Steven de Jesus; Skaar, Eric P.; Chazin, Walter J.; Calera, Jose Antonio; Hohl, Tobias M.; Pearlman, Eric

    2015-01-01

    Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein which possesses anti-microbial activity primarily due to its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9 −/− mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro, and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9−/− mice by injecting recombinant calprotectin. Further, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin’s anti-hyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro, or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ΔzafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared to wild-type. Collectively, these studies demonstrate a novel stage - specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin. PMID:26582948

  1. The regulation of methyl jasmonate on hyphal branching and GA biosynthesis in Ganoderma lucidum partly via ROS generated by NADPH oxidase.

    PubMed

    Shi, Liang; Gong, Li; Zhang, Xiangyang; Ren, Ang; Gao, Tan; Zhao, Mingwen

    2015-08-01

    Ganoderma lucidum is one of the best known medicinal basidiomycetes because it produces many pharmacologically active compounds, and methyl jasmonate (MeJA) was previously reported to induce the biosynthesis of ganoderic acids (GA) in G. lucidum. In this study, we found that MeJA not only increased the amount of GA but also increased the distance between hyphal branches by approximately 1.2-fold. Further analysis showed that MeJA could increase the intracellular ROS (reactive oxygen species) content by approximately 2.2-2.7-fold. Furthermore, the hyphal branching and GA biosynthesis regulated by MeJA treatment could be abolished by ROS scavengers to a level similar to or lower than that of the control group. These results indicated that the regulation of hyphal branching and GA biosynthesis by MeJA might occur via a ROS signaling pathway. Further analysis revealed that NADPH oxidase (NOX) plays an important role in MeJA-regulated ROS generation. Importantly, our results highlight that NOX functions in signaling cross-talk between ROS and MeJA. In addition, these findings provide an excellent opportunity to identify potential pathways linking ROS networks to MeJA signaling in fungi and suggest that plants and fungi share a conserved signaling-crosstalk mechanism.

  2. Multi-species biofilm of Candida albicans and non-Candida albicans Candida species on acrylic substrate

    PubMed Central

    K PATHAK, Apurva; SHARMA, Sanjay; SHRIVASTVA, Pallavi

    2012-01-01

    Objective In polymicrobial biofilms bacteria extensively interact with Candida species, but the interaction among the different species of the Candida is yet to be completely evaluated. In the present study, the difference in biofilm formation ability of clinical isolates of four species of Candida in both single-species and multi-species combinations on the surface of dental acrylic resin strips was evaluated. Material and Methods The species of Candida, isolated from multiple species oral candidiasis of the neutropenic patients, were used for the experiment. Organisms were cultured on Sabouraud dextrose broth with 8% glucose (SDB). Biofilm production on the acrylic resins strips was determined by crystal violet assay. Student's t-test and ANOVA were used to compare in vitro biofilm formation for the individual species of Candida and its different multi-species combinations. Results In the present study, differences between the mean values of the biofilm-forming ability of individual species (C. glabrata>C. krusei>C. tropicalis>C. albicans) and in its multi-species' combinations (the highest for C. albicans with C. glabrata and the lowest for all the four species combination) were reported. Conclusions The findings of this study showed that biofilm-forming ability was found greater for non-Candida albicans Candida species (NCAC) than for C. albicans species with intra-species variation. Presence of C. albicans in multi-species biofilms increased, whereas; C. tropicalis decreased the biofilm production with all other NCAC species. PMID:22437681

  3. Polymicrobial biofilm formation by Candida albicans, Actinomyces naeslundii, and Streptococcus mutans is Candida albicans strain and medium dependent.

    PubMed

    Arzmi, Mohd Hafiz; Alnuaimi, Ali D; Dashper, Stuart; Cirillo, Nicola; Reynolds, Eric C; McCullough, Michael

    2016-11-01

    Oral biofilms comprise of extracellular polysaccharides and polymicrobial microorganisms. The objective of this study was to determine the effect of polymicrobial interactions of Candida albicans, Actinomyces naeslundii, and Streptococcus mutans on biofilm formation with the hypotheses that biofilm biomass and metabolic activity are both C. albicans strain and growth medium dependent. To study monospecific biofilms, C. albicans, A. naeslundii, and S. mutans were inoculated into artificial saliva medium (ASM) and RPMI-1640 in separate vials, whereas to study polymicrobial biofilm formation, the inoculum containing microorganisms was prepared in the same vial prior inoculation into a 96-well plate followed by 72 hours incubation. Finally, biofilm biomass and metabolic activity were measured using crystal violet and XTT assays, respectively. Our results showed variability of monospecies and polymicrobial biofilm biomass between C. albicans strains and growth medium. Based on cut-offs, out of 32, seven RPMI-grown biofilms had high biofilm biomass (HBB), whereas, in ASM-grown biofilms, 14 out of 32 were HBB. Of the 32 biofilms grown in RPMI-1640, 21 were high metabolic activity (HMA), whereas in ASM, there was no biofilm had HMA. Significant differences were observed between ASM and RPMI-grown biofilms with respect to metabolic activity (P <01). In conclusion, biofilm biomass and metabolic activity were both C. albicans strain and growth medium dependent.

  4. Genome-wide functional analysis in Candida albicans.

    PubMed

    Motaung, Thabiso E; Ells, Ruan; Pohl, Carolina H; Albertyn, Jacobus; Tsilo, Toi J

    2017-02-08

    Candida albicans is an important etiological agent of superficial and life-threatening infections in individuals with compromised immune systems. To date, we know of several overlapping genetic networks that govern virulence attributes in this fungal pathogen. Classical use of deletion mutants has led to the discovery of numerous virulence factors over the years, and genome-wide functional analysis has propelled gene discovery at an even faster pace. Indeed, a number of recent studies using large-scale genetic screens followed by genome-wide functional analysis has allowed for the unbiased discovery of many new genes involved in C. albicans biology. Here we share our perspectives on the role of these studies in analyzing fundamental aspects of C. albicans virulence properties.

  5. Two unlike cousins: Candida albicans and C. glabrata infection strategies

    PubMed Central

    Brunke, Sascha; Hube, Bernhard

    2013-01-01

    Candida albicans and C. glabrata are the two most common pathogenic yeasts of humans, yet they are phylogenetically, genetically and phenotypically very different. In this review, we compare and contrast the strategies of C. albicans and C. glabrata to attach to and invade into the host, obtain nutrients and evade the host immune response. Although their strategies share some basic concepts, they differ greatly in their outcome. While C. albicans follows an aggressive strategy to subvert the host response and to obtain nutrients for its survival, C. glabrata seems to have evolved a strategy which is based on stealth, evasion and persistence, without causing severe damage in murine models. However, both fungi are successful as commensals and as pathogens of humans. Understanding these strategies will help in finding novel ways to fight Candida, and fungal infections in general. PMID:23253282

  6. Rapid characterisation of Candida albicans by pyrolysis mass spectrometry.

    PubMed

    White, G C; Sisson, P R; Freeman, R; Cookson, B D

    1994-08-01

    Clinical isolates (41) of Candida spp. from three possible outbreaks of nosocomially-acquired infection were compared by pyrolysis mass spectrometry (PMS) and by a combined morphotyping and resistotyping (M-R typing) method. Both systems characterised all the isolates and distinguished one isolate of C. tropicalis and another of C. glabrata from the 39 isolates of C. albicans. Results from both systems suggested that cross-infection with a single strain contributed to two of the outbreaks. In several instances, more than one strain of C. albicans was found amongst multiple isolates from the same patient. PMS is a simple, rapid and objective technique capable of characterising C. albicans isolates; discrimination was similar to M-R typing.

  7. Ocimum sanctum essential oil inhibits virulence attributes in Candida albicans.

    PubMed

    Khan, Amber; Ahmad, Aijaz; Xess, Immaculata; Khan, Luqman A; Manzoor, Nikhat

    2014-03-15

    Candida albicans is an opportunistic human fungal pathogen which causes disease mainly in immunocompromised patients. Activity of hydrolytic enzymes is essential for virulence of C. albicans and so is the capacity of these cells to undergo transition from yeast to mycelial form of growth. Ocimum sanctum is cultivated worldwide for its essential oil which exhibits medicinal properties. This work evaluates the anti-virulence activity of O. sanctum essential oil (OSEO) on 22 strains of C. albicans (including a standard strain ATCC 90028) isolated from both HIV positive and HIV negative patients. Candida isolates were exposed to sub-MICs of OSEO. In vitro secretion of proteinases and phospholipases was evaluated by plate assay containing BSA and egg yolk respectively. Morphological transition from yeast to filamentous form was monitored microscopically in LSM. For genetic analysis, respective genes associated with morphological transition (HWP1), proteinase (SAP1) and phospholipase (PLB2) were also investigated by Real Time PCR (qRT-PCR). Results were analyzed using Student's t-test. OSEO inhibits morphological transition in C. albicans and had a significant inhibitory effect on extracellular secretion of proteinases and phospholipases. Expression profile of respective selected genes associated with C. albicans virulence by qRT-PCR showed a reduced expression of HWP1, SAP1 and PLB2 genes in cells treated with sub-inhibitory concentrations of OSEO. This work suggests that OSEO inhibits morphological transition in C. albicans and decreases the secretion of hydrolytic enzymes involved in the early stage of infection as well as down regulates the associated genes. Further studies will assess the clinical application of OSEO and its constituents in the treatment of fungal infections.

  8. Acid production by oral strains of Candida albicans and lactobacilli.

    PubMed

    Klinke, T; Kneist, S; de Soet, J J; Kuhlisch, E; Mauersberger, S; Forster, A; Klimm, W

    2009-01-01

    Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42-66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important.

  9. Candida albicans specializations for iron homeostasis: from commensalism to virulence.

    PubMed

    Noble, Suzanne M

    2013-12-01

    Candida albicans is a fungal commensal-pathogen that persistently associates with its mammalian hosts. Between the commensal and pathogenic lifestyles, this microorganism inhabits host niches that differ markedly in the levels of bioavailable iron. A number of recent studies have exposed C. albicans specializations for acquiring iron from specific host molecules in regions where iron is scarce, while also defending against iron-related toxicity in regions where iron occurs in surfeit. Together, these results point to a central role for iron homeostasis in the evolution of this important human pathogen.

  10. Modulation of Morphogenesis by Egfr during Dorsal Closure in Drosophila

    PubMed Central

    Cormier, Olga; Cheng, David Chung-Pei; Reed, Bruce; Harden, Nicholas

    2013-01-01

    During Drosophila embryogenesis the process of dorsal closure (DC) results in continuity of the embryonic epidermis, and DC is well recognized as a model system for the analysis of epithelial morphogenesis as well as wound healing. During DC the flanking lateral epidermal sheets stretch, align, and fuse along the dorsal midline, thereby sealing a hole in the epidermis occupied by an extra-embryonic tissue known as the amnioserosa (AS). Successful DC requires the regulation of cell shape change via actomyosin contractility in both the epidermis and the AS, and this involves bidirectional communication between these two tissues. We previously demonstrated that transcriptional regulation of myosin from the zipper (zip) locus in both the epidermis and the AS involves the expression of Ack family tyrosine kinases in the AS in conjunction with Dpp secreted from the epidermis. A major function of Ack in other species, however, involves the negative regulation of Egfr. We have, therefore, asked what role Egfr might play in the regulation of DC. Our studies demonstrate that Egfr is required to negatively regulate epidermal expression of dpp during DC. Interestingly, we also find that Egfr signaling in the AS is required to repress zip expression in both the AS and the epidermis, and this may be generally restrictive to the progression of morphogenesis in these tissues. Consistent with this theme of restricting morphogenesis, it has previously been shown that programmed cell death of the AS is essential for proper DC, and we show that Egfr signaling also functions to inhibit or delay AS programmed cell death. Finally, we present evidence that Ack regulates zip expression by promoting the endocytosis of Egfr in the AS. We propose that the general role of Egfr signaling during DC is that of a braking mechanism on the overall progression of DC. PMID:23579691

  11. A Chick Embryo in-Vitro Model of Knee Morphogenesis

    PubMed Central

    Rodriguez, Edward K.; Munasinghe, Jeeva

    2016-01-01

    Background: In this feasibility study, a mechanically loaded in-vitro tissue culture model of joint morphogenesis using the isolated lower extremity of the 8 day old chick embryo was developed to assess the effects of mechanical loading on joint morphogenesis. Methods: The developed in-vitro system allows controlled flexion and extension of the chick embryonic knee with a range of motion of 20 degrees from a resting position of 90-100 degrees of flexion. Joint morphogenesis at 2, 3, 4 and 7 days of culture was assessed by histology and micro MRI in 4 specimen types: undisturbed in-ovo control embryos, in-ovo paralyzed embryos, in-vitro unloaded limb cultures, and in-vitro loaded limb cultures. Relative glycosaminoglycan (GAG) concentration across the joint was assessed with an MRI technique referred to as dGEMRIC (delayed gadolinium enhanced MRI of cartilage) where T1 is proportional to glycosaminoglycan concentration. Results: Average T1 over the entire tissue image for the normal control (IC) knee was 480 msec; for the 4 day loaded specimen average T1 was 354 msec; and for the 7 day loaded specimens T1 was 393 msec. The 4 day unloaded specimen had an average T1 of 279 msec while the 7 day unloaded specimen had an average T1 of 224 msec. The higher T1 values in loaded than unloaded specimens suggest that more glycosaminoglycan is produced in the loaded culture than in the unloaded preparation. Conclusion: Isolated limb tissue cultures under flexion-extension load can be viable and exhibit more progression of joint differentiation and glycosaminoglycan production than similarly cultured but unloaded specimens. However, when compared with controls consisting of intact undisturbed embryos in-ovo, the isolated loaded limbs in culture do not demonstrate equivalent amounts of absolute growth or joint differentiation. PMID:27200386

  12. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

    PubMed

    Tafesse, Fikadu G; Rashidfarrokhi, Ali; Schmidt, Florian I; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L

    2015-10-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

  13. Candida albicans in oral biofilms could prevent caries.

    PubMed

    Willems, Hubertine Marjoleine; Kos, Kevin; Jabra-Rizk, Mary Ann; Krom, Bastiaan P

    2016-07-01

    Streptococcus mutans is a Gram-positive bacterium involved in development to caries, the most common infectious disease of our time. Streptococcus mutans interacts with other microbes, like the fungus Candida albicans and both are commonly isolated from patients with caries. Since the role of C. albicans in caries remains unknown, our aim was to unravel this using an in vitro dual-species cariogenic oral biofilm model. Biofilms were grown for 24-72 h on glass cover slips or hydroxyapatite (HA) disks to mimic the surface of teeth. Medium pH, lactic acid production capacity and calcium release from HA disks were determined. All 24-h biofilms had external pH values below the critical pH of 5.5 where enamel dissolves. In contrast, 72-h dual-species biofilms had significantly higher pH (above the critical pH) and consequently decreased calcium release compared to single-species S. mutans biofilms. Counter intuitively, lactic acid production and growth of S. mutans were increased in 72-h dual-species biofilms. Candida albicans modulates the pH in dual-species biofilms to values above the critical pH where enamel dissolves. Our results suggest that C. albicans is not by definition a cariogenic microorganism; it could prevent caries by actively increasing pH preventing mineral loss.

  14. Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae

    SciTech Connect

    Yannai, S.; Berdicevsky, I.; Duek, L. )

    1991-01-01

    Saccharomyces cerevisiae and Candida albicans were incubated with 0.25, 0.5, or 0.75 {mu}g of Hg (as HgCl{sub 2}) per ml of Nelson's medium in the presence of trace amounts of oxygen at 28{degree}C for 12 days. Two control media were used, one without added Hg and one without yeast inoculum. Yeast cell growth was estimated after 1, 2, 3, and 8 days of incubation. The contents of organomercury in the system and of elemental mercury released from the media and collected in traps were determined at the end of the experiments. The results were as follows: (1) C. albicans was the more mercury-resistant species, but both yeast species failed to grown in the media containing 0.75 {mu}g of Hg per ml.; (2) The amounts of organomercury produced by the two species were proportional to the amount of HgCl{sub 2} added to the medium. In all cases C. albicans produced considerably larger amounts of methylmercury than S. cerevisiae; (3) The amounts of elemental Hg produced were inversely proportional to the HgCl{sub 2} level added in the case of S. cerevisiae but were all similar in the case of C. albicans;and (4) Neither organomercury nor elemental Hg was produced in any of the control media.

  15. Effects of ambroxol on Candida albicans growth and biofilm formation.

    PubMed

    Rene, Hernandez-Delgadillo; José, Martínez-Sanmiguel Juan; Isela, Sánchez-Nájera Rosa; Claudio, Cabral-Romero

    2014-04-01

    Typically, the onset of candidiasis is characterised by the appearance of a biofilm of Candida albicans, which is associated with several diseases including oral candidiasis in young and elderly people. The objective of this work was to investigate the in vitro fungicidal activity as well as the antibiofilm activity of ambroxol (AMB) against C. albicans growth. In the present investigation, the fungicidal activity of AMB was established using the cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Also the minimum inhibitory concentration (MIC) of AMB required to inhibit the fungal growth was determined. Simultaneously, the antibiofilm activity of AMB was evaluated using fluorescence microscopy. The study revealed that 2 mg ml(-1) of AMB exhibited higher fungicidal activity than 3.3 mg ml(-1) of terbinafine, one of most common commercial antifungals. A MIC of 1 mg ml(-1) was determined for AMB to interfere with C. albicans growth. Furthermore, AMB was found to be effective in inhibiting the biofilm formation of C. albicans and exerted its fungicidal activity against the fungal cells interspersed in the preformed biofilm. The study suggests a potential role of the mucolytic agent, AMB, as an interesting therapeutic alternative in the treatment of oral candidiasis.

  16. Lipidomics and in Vitro Azole Resistance in Candida albicans

    PubMed Central

    Singh, Ashutosh; Mahto, Kaushal Kumar

    2013-01-01

    Abstract We have shown earlier that fluconazole (FLC) stress induces global changes in the lipidome of Candida albicans in clinically adapted isolates. However, several laboratories have developed adapted in vitro FLC resistant strains of C. albicans to study azole resistance mechanisms. This study aimed to identify the lipid changes associated with FLC resistance in these in vitro adapted isolates. Using comparative lipidomics and principal component and discriminant analyses, we observed gradual changes in several lipid classes and molecular species upon FLC exposure of in vitro resistant C. albicans strains. Although the lipid imprint of FLC in vitro resistant isolates was very distinct from that of clinical isolates of C. albicans, the overall changes in lipid class compositions were similar in both cases. For example, an increased sterol content and depleted sphingolipid levels were the salient features of FLC resistance in both conditions. Taken together, it appears that the overall cellular lipid homeostasis is a critical factor in the observed FLC resistance and in handling FLC stress in both clinical and laboratory situations. The new observations reported herein have implications for more efficacious antifungal drug development as well as understanding host–infectious agent interactions in postgenomics microbiology practice. PMID:23374108

  17. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

    PubMed Central

    Schmidt, Florian I.; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L.

    2015-01-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. PMID:26431038

  18. [Suppression of activity of Candida albicans proteinases by cobalt chloride].

    PubMed

    Kutyreva, M P; Mukhametzianova, A R; Ulakhovich, N A

    2012-01-01

    Influence of cobalt (II) chloride on the system of Candida albicans proteinase (SAP C. alb.) (both in solution and immobilized on a surface of nitrocellulose membranes) has been investigated. In solution cobalt chloride inactivated inducible but not constitute enzyme. In the heterogenous sytem proteolitical effect of the cobalt ion on inductible proteinase was also observed.

  19. Interaction of Candida albicans with Human Leukocytes and Serum1

    PubMed Central

    Lehrer, Robert I.; Cline, Martin J.

    1969-01-01

    A quantitative assay of candidacidal activity based on differential staining of non-viable Candida albicans by methylene blue was developed and applied to studies of leukocytes from normal individuals and patients with fungal and other infections. Serum factors were necessary for optimal phagocytosis of C. albicans but lacked direct candidacidal activity. Normal human neutrophils (38 studies) killed 29.0 ± 7.4% of ingested C. albicans in 1 hr. Eosinophils and monocytes killed a smaller percentage. Neutrophil candidacidal activity did not require protein or ribonucleic acid synthesis by the leukocyte but was inhibited by anaerobic conditions, potassium cyanide, and colchicine. Leukocytes of a patient with hereditary myeloperoxidase deficiency and of three children with chronic granulomatous disease phagocytized C. albicans normally, yet failed to kill them. Our data suggest that the neutrophil can play an important role in resistance to Candida infection and that the lysosomal enzyme myeloperoxidase and its oxidant substrate hydrogen peroxide are the major participants in neutrophil candidacidal activity. Images PMID:4182532

  20. Hydrophobic polyoxins are resistant to intracellular degradation in Candida albicans.

    PubMed Central

    Smith, H A; Shenbagamurthi, P; Naider, F; Kundu, B; Becker, J M

    1986-01-01

    Two novel polyoxins, N-epsilon-(octanoyl)-lysyl-uracil polyoxin C (Oct-Lys-UPOC) and N-gamma-(octyl)-glutaminyluracil polyoxin C (Oct-Gln-UPOC), were synthesized by reacting uracil polyoxin C with the appropriate amino acid p-nitrophenyl ester. Oct-Lys-UPOC and Oct-Gln-UPOC were strong inhibitors (Kis = 1.7 X 10(-6)M) of chitin synthetase from Candida albicans membrane preparations. In a permeabilized-cell assay, Oct-Gln-UPOC had a 10-fold-lower inhibitory activity toward chitin synthetase than did the Oct-Lys-UPOC analog. Both compounds were resistant to hydrolysis by a cell extract of C. albicans H317; however, Oct-Gln-UPOC was hydrolyzed with a half-life of 23 min by a permeabilized-cell preparation. Oct-Lys-UPOC was resistant to hydrolysis by permeabilized cells. Oct-Gln-UPOC and Oct-Lys-UPOC did not compete with the transport of peptides or uridine into the cell. At concentrations up to 2 mM these two new polyoxins were ineffective in the inhibition of cell growth or reduction of cell viability, but they induced aberrant morphologies in C. albicans at a concentration of 0.25 mM. These data suggest that polyoxins containing hydrophobic amino acids retain strong chitin synthetase inhibitory activity and are resistant to cellular hydrolysis. They provide the first example of effective synthetic chitin synthetase inhibitors which are stable inside C. albicans. PMID:3524423

  1. Two FgLEU2 Genes with Different Roles in Leucine Biosynthesis and Infection-Related Morphogenesis in Fusarium graminearum

    PubMed Central

    Liu, Xin; Han, Qi; Wang, Jian; Wang, Xin; Xu, Jianhong; Shi, Jianrong

    2016-01-01

    3-isopropylmalate dehydrogenase (IPMD) encoded by LEU2 is a key enzyme in leucine (Leu) biosynthetic pathway. Analysis of the genome sequence of Fusarium graminearum revealed two paralogous LEU2 genes (designated as FgLEU2A and FgLEU2B) in this fungus and the deduced amino acid sequences of FgLeu2A and FgLeu2B share 45% identity. Targeted disruption of individual FgLEU2A/B gene in F. graminearum assigned a more crucial role of FgLeu2A in Leu biosynthesis as disruption of FgLEU2A resulted in mutant (ΔFgLeu2A-10) that was Leu-auxotrophic and could not grow in minimal medium limited for amino acids, whereas FgLEU2B deletion mutant ΔFgLeu2B-2 was morphologically indistinguishable from the wild type strain PH-1. The growth defects of ΔFgLeu2A-10 could be overcome by exogenous addition of Leu at 0.25 mM. Double deletion of FgLEU2A and FgLEU2B (ΔFgLeu2AB-8) caused a more severe Leu-auxotrophic phenotype as the concentration of Leu exogenously added to medium to rescue the growth defect of ΔFgLeu2AB-8 should be raised to 1.25 mM, indicating a less important but nonnegligible role of FgLeu2B in Leu biosynthesis. Disturb of Leu biosynthesis caused by FgLEU2A deletion leads to slower growth rate, reduced aerial hyphal formation and red pigmentation on PDA plates and completely blocked conidial production and germination. All of the defects above could be overcome by Leu addition or complementation of the full-length FgLEU2A gene. ΔFgLeu2A-10 also showed significantly increased sensitivity to osmotic and oxidative stresses. Pathogenicity assay results showed that virulence of mutants lacking FgLEU2A were dramatically impaired on wheat heads and non-host cherry tomatoes. Additionally, a low level of deoxynivalenol (DON) production of ΔFgLeu2A-10 and ΔFgLeu2AB-8 in wheat kernels was also detected. Taken together, results of this study indicated a crucial role of FgLeu2A and a less important role of FgLeu2B in Leu biosynthesis and fungal infection-related morphogenesis in

  2. Cortical forces in cell shape changes and tissue morphogenesis.

    PubMed

    Rauzi, Matteo; Lenne, Pierre-François

    2011-01-01

    Cortical forces drive a variety of cell shape changes and cell movements during tissue morphogenesis. While the molecular components underlying these forces have been largely identified, how they assemble and spatially and temporally organize at cell surfaces to promote cell shape changes in developing tissues are open questions. We present here different key aspects of cortical forces: their physical nature, some rules governing their emergence, and how their deployment at cell surfaces drives important morphogenetic movements in epithelia. We review a wide range of literature combining genetic/molecular, biophysical and modeling approaches, which explore essential features of cortical force generation and transmission in tissues.

  3. Isolation of an algal morphogenesis inducer from a marine bacterium.

    PubMed

    Matsuo, Yoshihide; Imagawa, Hiroshi; Nishizawa, Mugio; Shizuri, Yoshikazu

    2005-03-11

    Ulva and Enteromorpha are cosmopolitan and familiar marine algal genera. It is well known that these green macroalgae lose their natural morphology during short-term cultivation under aseptic conditions and during long-term cultivation in nutrient-added seawater and adopt an unusual form instead. These phenomena led to the belief that undefined morphogenetic factors that were indispensable to the foliaceous morphology of macroalgae exist throughout the oceans. We characterize a causative factor, named thallusin, isolated from an epiphytic marine bacterium. Thallusin induces normal germination and morphogenesis of green macroalgae.

  4. Morphogenesis and morphology of HIV. Structure-function relations.

    PubMed

    Gelderblom, H R; Ozel, M; Pauli, G

    1989-01-01

    Fine structure and antigenic make-up analysis of HIV were combined in a 2D model, from which functional aspects can be deduced. On the envelope 72 probably trimeric surface knobs (gp120) are connected to the virion via the transmembrane protein gp41. Gp120 is shed during ageing of the virion, but host cell antigens stay firmly anchored to the envelope. Underneath the envelope, p17 forms the matrix protein layer, while the capsid of the double cone shaped core is built up of p24. The relation between biochemical findings and morphogenesis and maturation of HIV as well as aspects of pathogenesis and vaccination are discussed.

  5. Salivary Gland Branching Morphogenesis — Recent Progress and Future Opportunities

    PubMed Central

    Hsu, Jeff Chi-feng; Yamada, Kenneth M

    2010-01-01

    Salivary glands provide saliva to maintain oral health, and a loss of salivary gland function substantially decreases quality-of-life. Understanding the biological mechanisms that generate salivary glands during embryonic development may identify novel ways to regenerate function or design artificial salivary glands. This review article summarizes current research on the process of branching morphogenesis of salivary glands, which creates gland structure during development. We highlight exciting new advances and opportunities in studies of cell-cell interactions, mechanical forces, growth factors, and gene expression patterns to improve our understanding of this important process. PMID:21125789

  6. A piglet model for studying Candida albicans colonization of the human oro-gastrointestinal tract.

    PubMed

    Hoeflinger, Jennifer L; Coleman, David A; Oh, Soon-Hwan; Miller, Michael J; Hoyer, Lois L

    2014-08-01

    Pigs from a variety of sources were surveyed for oro-gastrointestinal (oro-GIT) carriage of Candida albicans. Candida albicans-positive animals were readily located, but we also identified C. albicans-free pigs. We hypothesized that pigs could be stably colonized with a C. albicans strain of choice, simply by feeding yeast cells. Piglets were farrowed routinely and remained with the sow for 4 days to acquire a normal microbiota. Piglets were then placed in an artificial rearing environment and fed sow milk replacer. Piglets were inoculated orally with one of three different C. albicans strains. Piglets were weighed daily, and culture swabs were collected to detect C. albicans orally, rectally and in the piglet's environment. Stable C. albicans colonization over the course of the study did not affect piglet growth. Necropsy revealed mucosally associated C. albicans throughout the oro-GIT with the highest abundance in the esophagus. Uninoculated control piglets remained C. albicans-negative. These data establish the piglet as a model to study C. albicans colonization of the human oro-GIT. Similarities between oro-GIT colonization in humans and pigs, as well as the ease of working with the piglet model, suggest its adaptability for use among investigators interested in understanding C. albicans-host commensal interactions.

  7. The Pea Seedling as a Model of Normal and Abnormal Morphogenesis

    ERIC Educational Resources Information Center

    Kurkdjian, Armen; And Others

    1974-01-01

    Describes several simple and inexpensive experiments designed to facilitate the study of normal and abnormal morphogenesis in the biology laboratory. Seedlings of the common garden pea are used in the experiments, and abnormal morphogenesis (tumors) are induced by a virulent strain of the crown-gall organism, Agrobacterium tumefaciens. (JR)

  8. Un(MaSC)ing Stem Cell Dynamics in Mammary Branching Morphogenesis.

    PubMed

    Greenwood, Erin; Wrenn, Emma D; Cheung, Kevin J

    2017-02-27

    The properties of stem cells that participate in mammary gland branching morphogenesis remain contested. Reporting in Nature, Scheele et al. (2017) establish a model for post-pubertal mammary branching morphogenesis in which position-dependent, lineage-restricted stem cells undergo cell mixing in order to contribute to long-term growth.

  9. Glucose modulates antimicrobial photodynamic inactivation of Candida albicans in biofilms.

    PubMed

    Suzuki, Luis Cláudio; Kato, Ilka Tiemy; Prates, Renato Araujo; Sabino, Caetano Padial; Yoshimura, Tania Mateus; Silva, Tamires Oliveira; Ribeiro, Martha Simões

    2017-03-01

    Candida albicans biofilm is a main cause of infections associated with medical devices such as catheters, contact lens and artificial joint prosthesis. The current treatment comprises antifungal chemotherapy that presents low success rates. Photodynamic inactivation (PDI) involves the combination of a photosensitizing compound (PS) and light to generate oxidative stress that has demonstrated effective antimicrobial activity against a broad-spectrum of pathogens, including C. albicans. This fungus senses glucose inducing an upregulation of membrane transporters that can facilitate PS uptake into the cell. The aim of this study was to evaluate the effects of glucose on methylene blue (MB) uptake and its influence on PDI efficiency when combined to a red LED with central wavelength at λ=660nm. C. albicans biofilms were grown on hydrogel disks. Prior to PDI assays, MB uptake tests were performed with and without glucose-sensitization. In this system, the optimum PS administration was determined as 500μM of MB in contact with the biofilm during 30min before irradiation. Irradiation was performed during 3, 6, 9, 12, 15 and 18min with irradiance of 127.3mW/cm(2). Our results showed that glucose was able to increase MB uptake in C. albicans cells. In addition, PDI without glucose showed a higher viability reduction until 6min; after 9min, glucose group demonstrated a significant decrease in cell viability when compared to glucose-free group. Taken together, our data suggest that glucose is capable to enhance MB uptake and modulate photodynamic inactivation of C. albicans biofilm.

  10. ECM Signaling Regulates Collective Cellular Dynamics to Control Pancreas Branching Morphogenesis.

    PubMed

    Shih, Hung Ping; Panlasigui, Devin; Cirulli, Vincenzo; Sander, Maike

    2016-01-12

    During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.

  11. Eye morphogenesis and patterning of the optic vesicle.

    PubMed

    Fuhrmann, Sabine

    2010-01-01

    Organogenesis of the eye is a multistep process that starts with the formation of optic vesicles followed by invagination of the distal domain of the vesicles and the overlying lens placode resulting in morphogenesis of the optic cup. The late optic vesicle becomes patterned into distinct ocular tissues: the neural retina, retinal pigment epithelium (RPE), and optic stalk. Multiple congenital eye disorders, including anophthalmia or microphthalmia, aniridia, coloboma, and retinal dysplasia, stem from disruptions in embryonic eye development. Thus, it is critical to understand the mechanisms that lead to initial specification and differentiation of ocular tissues. An accumulating number of studies demonstrate that a complex interplay between inductive signals provided by tissue-tissue interactions and cell-intrinsic factors is critical to ensuring proper specification of ocular tissues as well as maintenance of RPE cell fate. While several of the extrinsic and intrinsic determinants have been identified, we are just at the beginning in understanding how these signals are integrated. In addition, we know very little about the actual output of these interactions. In this chapter, we provide an update of the mechanisms controlling the early steps of eye development in vertebrates, with emphasis on optic vesicle evagination, specification of neural retina and RPE at the optic vesicle stage, the process of invagination during morphogenesis of the optic cup, and maintenance of the RPE cell fate.

  12. Genomic Regions Required for Morphogenesis of the Drosophila Embryonic Midgut

    PubMed Central

    Bilder, D.; Scott, M. P.

    1995-01-01

    The Drosophila midgut is an excellent system for studying the cell migration, cell-cell communication, and morphogenetic events that occur in organ formation. Genes representative of regulatory gene families common to all animals, including homeotic, TGFβ, and Wnt genes, play roles in midgut development. To find additional regulators of midgut morphogenesis, we screened a set of genomic deficiencies for midgut phenotypes. Fifteen genomic intervals necessary for proper midgut morphogenesis were identified; three contain genes already known to act in the midgut. Three other genomic regions are required for formation of the endoderm or visceral mesoderm components of the midgut. Nine regions are required for proper formation of the midgut constrictions. The E75 ecdysone-induced gene, which encodes a nuclear receptor superfamily member, is the relevant gene in one region and is essential for proper formation of midgut constrictions. E75 acts downstream of the previously known constriction regulators or in parallel. Temporal hormonal control may therefore work in conjunction with spatial regulation by the homeotic genes in midgut development. Another genomic region is required to activate transcription of the homeotic genes Antp and Scr specifically in visceral mesoderm. The genomic regions identified by this screen provide a map to novel midgut development regulators. PMID:8582615

  13. Morphogenesis of callosal arbors in the parietal cortex of hamsters.

    PubMed

    Hedin-Pereira, C; Lent, R; Jhaveri, S

    1999-01-01

    The morphogenesis of callosal axons originating in the parietal cortex was studied by anterograde labeling with Phaseolus lectin or biocytin injected in postnatal (P) hamsters aged 7-25 days. Some labeled fibers were serially reconstructed. At P7, some callosal fibers extended as far as the contralateral rhinal fissure, with simple arbors located in the homotopic region of the opposite cortical gray matter, and two or three unbranched sprouts along their trajectory. From P7 to P13, the homotopic arbors became more complex, with branches focused predominantly, but not exclusively, in the supra- and infragranular layers of the homotopic region. Simultaneously, the lateral extension of the trunk axon in the white matter became shorter, finally disappearing by P25. Arbors in the gray matter were either bilaminar (layers 2/3 and 5) or supragranular. A heterotopic projection to the lateral cortex was consistently seen at all ages; the heterotopic arbors follow a similar sequence of events to that seen in homotopic regions. These observations document that callosal axons undergo regressive tangential remodeling during the first postnatal month, as the lateral extension of the trunk fiber gets eliminated. Radially, however, significant arborization occurs in layer-specific locations. The protracted period of morphogenesis suggests a correspondingly long plastic period for this system of cortical fibers.

  14. PARP6 is a Regulator of Hippocampal Dendritic Morphogenesis

    PubMed Central

    Huang, Jeffrey Y.; Wang, Kang; Vermehren-Schmaedick, Anke; Adelman, John P.; Cohen, Michael S.

    2016-01-01

    Mono-ADP-ribosylation (MARylation) of mammalian proteins was first described as a post-translational modification catalyzed by bacterial toxins. It is now known that endogenous MARylation occurs in mammalian cells and is catalyzed by 11 members of the poly-ADP-ribose polymerase (PARP) family of proteins (17 in humans). The physiological roles of these PARPs remain largely unknown. Here we demonstrate that PARP6, a neuronally enriched PARP that catalyzes MARylation, regulates hippocampal dendrite morphogenesis, a process that is critical for proper neural circuit formation during development. Knockdown of PARP6 significantly decreased dendritic complexity in embryonic rat hippocampal neurons in culture and in vivo. Expression of wild-type PARP6 increased dendritic complexity; conversely, expression of a catalytically inactive PARP6 mutant, or a cysteine-rich domain deletion mutant that has significantly reduced catalytic activity, decreased dendritic complexity. The identification of PARP6 as a regulator of dendrite morphogenesis supports a role for MARylation in neurons during development. PMID:26725726

  15. Evolutionary stasis in pollen morphogenesis due to natural selection.

    PubMed

    Matamoro-Vidal, Alexis; Prieu, Charlotte; Furness, Carol A; Albert, Béatrice; Gouyon, Pierre-Henri

    2016-01-01

    The contribution of developmental constraints and selective forces to the determination of evolutionary patterns is an important and unsolved question. We test whether the long-term evolutionary stasis observed for pollen morphogenesis (microsporogenesis) in eudicots is due to developmental constraints or to selection on a morphological trait shaped by microsporogenesis: the equatorial aperture pattern. Most eudicots have three equatorial apertures but several taxa have independently lost the equatorial pattern and have microsporogenesis decoupled from aperture pattern determination. If selection on the equatorial pattern limits variation, we expect to see increased variation in microsporogenesis in the nonequatorial clades. Variation of microsporogenesis was studied using phylogenetic comparative analyses in 83 species dispersed throughout eudicots including species with and without equatorial apertures. The species that have lost the equatorial pattern have highly variable microsporogenesis at the intra-individual and inter-specific levels regardless of their pollen morphology, whereas microsporogenesis remains stable in species with the equatorial pattern. The observed burst of variation upon loss of equatorial apertures shows that there are no strong developmental constraints precluding variation in microsporogenesis, and that the stasis is likely to be due principally to selective pressure acting on pollen morphogenesis because of its implication in the determination of the equatorial aperture pattern.

  16. Dkk1 regulates ventral midbrain dopaminergic differentiation and morphogenesis.

    PubMed

    Ribeiro, Diogo; Ellwanger, Kristina; Glagow, Désirée; Theofilopoulos, Spyridon; Corsini, Nina S; Martin-Villalba, Ana; Niehrs, Christof; Arenas, Ernest

    2011-02-11

    Dickkopf1 (Dkk1) is a Wnt/β-catenin inhibitor that participates in many processes during embryonic development. One of its roles during embryogenesis is to induce head formation, since Dkk1-null mice lack head structures anterior to midbrain. The Wnt/β-catenin pathway is also known to regulate different aspects of ventral midbrain (VM) dopaminergic (DA) neuron development and, in vitro, Dkk1-mediated inhibition of the Wnt/β-catenin pathway improves the DA differentiation in mouse embryonic stem cells (mESC). However, the in vivo function of Dkk1 on the development of midbrain DA neurons remains to be elucidated. Here we examined Dkk1(+/-) embryos and found that Dkk1 is required for the differentiation of DA precursors/neuroblasts into DA neurons at E13.5. This deficit persisted until E17.5, when a defect in the number and distribution of VM DA neurons was detected. Furthermore, analysis of the few Dkk1(-/-) embryos that survived until E17.5 revealed a more severe loss of midbrain DA neurons and morphogenesis defects. Our results thus show that Dkk1 is required for midbrain DA differentiation and morphogenesis.

  17. Size-dependent symmetry breaking in models for morphogenesis

    NASA Astrophysics Data System (ADS)

    Barrio, R. A.; Maini, P. K.; Aragón, J. L.; Torres, M.

    2002-08-01

    A general property of dynamical systems is the appearance of spatial and temporal patterns due to a change of stability of a homogeneous steady state. Such spontaneous symmetry breaking is observed very frequently in all kinds of real systems, including the development of shape in living organisms. Many nonlinear dynamical systems present a wide variety of patterns with different shapes and symmetries. This fact restricts the applicability of these models to morphogenesis, since one often finds a surprisingly small variation in the shapes of living organisms. For instance, all individuals in the Phylum Echinodermata share a persistent radial fivefold symmetry. In this paper, we investigate in detail the symmetry-breaking properties of a Turing reaction-diffusion system confined in a small disk in two dimensions. It is shown that the symmetry of the resulting pattern depends only on the size of the disk, regardless of the boundary conditions and of the differences in the parameters that differentiate the interior of the domain from the outer space. This study suggests that additional regulatory mechanisms to control the size of the system are of crucial importance in morphogenesis.

  18. Early epithelial signaling center governs tooth budding morphogenesis

    PubMed Central

    Thesleff, Irma

    2016-01-01

    During organogenesis, cell fate specification and patterning are regulated by signaling centers, specialized clusters of morphogen-expressing cells. In many organs, initiation of development is marked by bud formation, but the cellular mechanisms involved are ill defined. Here, we use the mouse incisor tooth as a model to study budding morphogenesis. We show that a group of nonproliferative epithelial cells emerges in the early tooth primordium and identify these cells as a signaling center. Confocal live imaging of tissue explants revealed that although these cells reorganize dynamically, they do not reenter the cell cycle or contribute to the growing tooth bud. Instead, budding is driven by proliferation of the neighboring cells. We demonstrate that the activity of the ectodysplasin/Edar/nuclear factor κB pathway is restricted to the signaling center, and its inactivation leads to fewer quiescent cells and a smaller bud. These data functionally link the signaling center size to organ size and imply that the early signaling center is a prerequisite for budding morphogenesis. PMID:27621364

  19. A simple probabilistic model of submicroscopic diatom morphogenesis

    PubMed Central

    Willis, L.; Cox, E. J.; Duke, T.

    2013-01-01

    Unicellular algae called diatoms morph biomineral compounds into tough exoskeletons via complex intracellular processes about which there is much to be learned. These exoskeletons feature a rich variety of structures from submicroscale to milliscale, many that have not been reproduced in vitro. In order to help understand this complex miniature morphogenesis, here we introduce and analyse a simple model of biomineral kinetics, focusing on the exoskeleton's submicroscopic patterned planar structures called pore occlusions. The model reproduces most features of these pore occlusions by retuning just one parameter, thereby indicating what physio-biochemical mechanisms could sufficiently explain morphogenesis at the submicroscopic scale: it is sufficient to identify a mechanism of lateral negative feedback on the biomineral reaction kinetics. The model is nonlinear and stochastic; it is an extended version of the threshold voter model. Its mean-field equation provides a simple and, as far as the authors are aware, new way of mapping out the spatial patterns produced by lateral inhibition and variants thereof. PMID:23554345

  20. Ultrastructural and biochemical basis for hepatitis C virus morphogenesis.

    PubMed

    Falcón, Viviana; Acosta-Rivero, Nelson; González, Sirenia; Dueñas-Carrera, Santiago; Martinez-Donato, Gillian; Menéndez, Ivon; Garateix, Rocio; Silva, José A; Acosta, Emilio; Kourı, Juan

    2017-04-01

    Chronic infection with HCV is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. One of the least understood steps in the HCV life cycle is the morphogenesis of new viral particles. HCV infection alters the lipid metabolism and generates a variety of microenvironments in the cell cytoplasm that protect viral proteins and RNA promoting viral replication and assembly. Lipid droplets (LDs) have been proposed to link viral RNA synthesis and virion assembly by physically associating these viral processes. HCV assembly, envelopment, and maturation have been shown to take place at specialized detergent-resistant membranes in the ER, rich in cholesterol and sphingolipids, supporting the synthesis of luminal LDs-containing ApoE. HCV assembly involves a regulated allocation of viral and host factors to viral assembly sites. Then, virus budding takes place through encapsidation of the HCV genome and viral envelopment in the ER. Interaction of ApoE with envelope proteins supports the viral particle acquisition of lipids and maturation. HCV secretion has been suggested to entail the ion channel activity of viral p7, several components of the classical trafficking and autophagy pathways, ESCRT, and exosome-mediated export of viral RNA. Here, we review the most recent advances in virus morphogenesis and the interplay between viral and host factors required for the formation of HCV virions.

  1. Tribolium embryo morphogenesis: may the force be with you.

    PubMed

    Benton, Matthew A; Pavlopoulos, Anastasios

    2014-01-01

    Development of multicellular organisms depends on patterning and growth mechanisms encoded in the genome, but also on the physical properties and mechanical interactions of the constituent cells that interpret these genetic cues. This fundamental biological problem requires integrated studies at multiple levels of biological organization: from genes, to cell behaviors, to tissue morphogenesis. We have recently combined functional genetics with live imaging approaches in embryos of the insect Tribolium castaneum, in order to understand their remarkable transformation from a uniform single-layered blastoderm into a condensed multi-layered embryo covered by extensive extra-embryonic tissues. We first developed a quick and reliable methodology to fluorescently label various cell components in entire Tribolium embryos. Live imaging of labeled embryos at single cell resolution provided detailed descriptions of cell behaviors and tissue movements during normal embryogenesis. We then compared cell and tissue dynamics between wild-type and genetically perturbed embryos that exhibited altered relative proportions of constituent tissues. This systematic comparison led to a qualitative model of the molecular, cellular and tissue interactions that orchestrate the observed epithelial rearrangements. We expect this work to establish the Tribolium embryo as a powerful and attractive model system for biologists and biophysicists interested in the molecular, cellular and mechanical control of tissue morphogenesis.

  2. Involvement of MoVMA11, a Putative Vacuolar ATPase c’ Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae

    PubMed Central

    Chen, Guoqing; Liu, Xiaohong; Zhang, Lilin; Cao, Huijuan; Lu, Jianping; Lin, Fucheng

    2013-01-01

    Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaportheoryzae, subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c’ subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M. oryzae. PMID:23826342

  3. Photoinactivation of single and mixed biofilms of Candida albicans and non-albicans Candida species using Phorodithazine(®).

    PubMed

    Carmello, Juliana Cabrini; Alves, Fernanda; Mima, Ewerton Garcia de Oliveira; Jorge, Janaina Habib; Bagnato, Vanderlei Salvador; Pavarina, Ana Cláudia

    2017-03-01

    This study evaluated the effectiveness of antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine(®) (PDZ) formulated in hydrogel, in the inactivation of mono and duo-species biofilms of Candida albicans, Candida glabrata and Candida tropicalis. Standardized suspensions of each strain were prepared and after biofilm formation, mono-species were treated with 150 and 175mg/L of PDZ for 20min (pre-irradiation time), and exposed to LED light at a dose of 37.5J/cm(2) (660nm). The duo-species biofilms (C. albicans+C. glabrata and C. albicans+C. tropicalis) were treated with 150mg/L of PDZ and light. Additional samples were treated with PDZ or light only, and the control did not receive any treatment. Next, microbiological evaluation was performed by spreading the cells on Sabouraud Dextrose Agar and CHROMagar Candida for colony forming units (CFU/mL). Moreover, the total biomass of biofilm was verified using the crystal violet staining assay (CV). The data were submitted to ANOVA and Tukey post-hoc (α=0.05). The use of PDZ 150mg/L promoted a reduction of 1.0, 1.2, 1.5 log10 in the viability of C. glabrata, C. albicans and C. tropicalis, respectively. The same concentration reduced in 1.0 log10 the viability of each species grown as duo-species biofilms. The crystal violet assay showed that the use of 150mg/L reduced 24.4%, 39.2% and 43.7% of the total biomass of C. albicans, C. tropicalis and C. glabrata, respectively. aPDT did not reduce the total biomass to the duo-species biofilms. Thus, PDZ-mediated aPDT was more effective in the inactivation of mono-species biofilms of Candida spp. compared with duo-species biofilm.

  4. Cross-membranes orchestrate compartmentalization and morphogenesis in Streptomyces

    PubMed Central

    Celler, Katherine; Koning, Roman I.; Willemse, Joost; Koster, Abraham J.; van Wezel, Gilles P.

    2016-01-01

    Far from being simple unicellular entities, bacteria have complex social behaviour and organization, living in large populations, and some even as coherent, multicellular entities. The filamentous streptomycetes epitomize such multicellularity, growing as a syncytial mycelium with physiologically distinct hyphal compartments separated by infrequent cross-walls. The viability of mutants devoid of cell division, which can be propagated from fragments, suggests the presence of a different form of compartmentalization in the mycelium. Here we show that complex membranes, visualized by cryo-correlative light microscopy and electron tomography, fulfil this role. Membranes form small assemblies between the cell wall and cytoplasmic membrane, or, as evidenced by FRAP experiments, large protein-impermeable cross-membrane structures, which compartmentalize the multinucleoid mycelium. All areas containing cross-membrane structures are nucleoid-restricted zones, suggesting that the membrane assemblies may also act to protect nucleoids from cell-wall restructuring events. Our work reveals a novel mechanism of controlling compartmentalization and development in multicellular bacteria. PMID:27291257

  5. Reconstruction of signaling networks regulating fungal morphogenesis by transcriptomics.

    PubMed

    Meyer, Vera; Arentshorst, Mark; Flitter, Simon J; Nitsche, Benjamin M; Kwon, Min Jin; Reynaga-Peña, Cristina G; Bartnicki-Garcia, Salomon; van den Hondel, Cees A M J J; Ram, Arthur F J

    2009-11-01

    Coordinated control of hyphal elongation and branching is essential for sustaining mycelial growth of filamentous fungi. In order to study the molecular machinery ensuring polarity control in the industrial fungus Aspergillus niger, we took advantage of the temperature-sensitive (ts) apical-branching ramosa-1 mutant. We show here that this strain serves as an excellent model system to study critical steps of polar growth control during mycelial development and report for the first time a transcriptomic fingerprint of apical branching for a filamentous fungus. This fingerprint indicates that several signal transduction pathways, including TORC2, phospholipid, calcium, and cell wall integrity signaling, concertedly act to control apical branching. We furthermore identified the genetic locus affected in the ramosa-1 mutant by complementation of the ts phenotype. Sequence analyses demonstrated that a single amino acid exchange in the RmsA protein is responsible for induced apical branching of the ramosa-1 mutant. Deletion experiments showed that the corresponding rmsA gene is essential for the growth of A. niger, and complementation analyses with Saccharomyces cerevisiae evidenced that RmsA serves as a functional equivalent of the TORC2 component Avo1p. TORC2 signaling is required for actin polarization and cell wall integrity in S. cerevisiae. Congruently, our microscopic investigations showed that polarized actin organization and chitin deposition are disturbed in the ramosa-1 mutant. The integration of the transcriptomic, genetic, and phenotypic data obtained in this study allowed us to reconstruct a model for cellular events involved in apical branching.

  6. Candida/Candida biofilms. First description of dual-species Candida albicans/C. rugosa biofilm.

    PubMed

    Martins, Carlos Henrique Gomes; Pires, Regina Helena; Cunha, Aline Oliveira; Pereira, Cristiane Aparecida Martins; Singulani, Junya de Lacorte; Abrão, Fariza; Moraes, Thais de; Mendes-Giannini, Maria José Soares

    2016-04-01

    Denture liners have physical properties that favour plaque accumulation and colonization by Candida species, irritating oral tissues and causing denture stomatitis. To isolate and determine the incidence of oral Candida species in dental prostheses, oral swabs were collected from the dental prostheses of 66 patients. All the strains were screened for their ability to form biofilms; both monospecies and dual-species combinations were tested. Candida albicans (63 %) was the most frequently isolated microorganism; Candida tropicalis (14 %), Candida glabrata (13 %), Candida rugosa (5 %), Candida parapsilosis (3 %), and Candida krusei (2 %) were also detected. The XTT assay showed that C. albicans SC5314 possessed a biofilm-forming ability significantly higher (p < 0.001) than non-albicans Candida strains, after 6 h 37 °C. The total C. albicans CFU from a dual-species biofilm was less than the total CFU of a monospecies C. albicans biofilm. In contrast to the profuse hyphae verified in monospecies C. albicans biofilms, micrographies showed that the C. albicans/non-albicans Candida biofilms consisted of sparse yeast forms and profuse budding yeast cells that generated a network. These results suggested that C. albicans and the tested Candida species could co-exist in biofilms displaying apparent antagonism. The study provide the first description of C. albicans/C. rugosa mixed biofilm.

  7. The ER-mitochondria encounter structure contributes to hyphal growth, mitochondrial morphology and virulence of the pathogenic mold Aspergillus fumigatus.

    PubMed

    Geißel, Bernadette; Penka, Mirjam; Neubauer, Michael; Wagener, Johannes

    2017-01-01

    Aspergillus fumigatus is an opportunistic fungal pathogen and the primary causative species of invasive aspergillosis, a systemic disease associated with high mortality rates. Treatment of invasive fungal infection relies on a very limited number of antifungal drug classes. In order to extend the spectrum of antifungal drugs novel target structures have to be identified. The ER-mitochondria encounter structure (ERMES), a recently discovered tether that links mitochondria and endoplasmic reticulum, is a potential drug target based on its absence in Metazoa. Very recently, it was shown that ERMES is important for the fitness and immune evasion of the pathogenic yeast Candida albicans. We studied the role of the four ERMES core components Mdm10, Mdm12, Mdm34 and Mmm1 in the pathogenic mold A. fumigatus. By construction and characterizing conditional mutants of all four core components and deletion mutants of mdm10 and mdm12, we show that each component is of significant importance for growth of the fungal pathogen. While markedness of the individual mutant phenotypes differed slightly, all components are important for maintenance of the mitochondrial morphology and the intra-organellar distribution of nucleoids. Characterization of the Mmm1 ERMES mutant in a Galleria mellonella infection model indicates that ERMES contributes to virulence of A. fumigatus. Our results demonstrate that pharmacologic inhibition of ERMES could exert antifungal activity against this important pathogen.

  8. Cadherins and catenins in dendrite and synapse morphogenesis

    PubMed Central

    Seong, Eunju; Yuan, Li; Arikkath, Jyothi

    2015-01-01

    Neurons are highly polarized specialized cells. Neuronal integrity and functional roles are critically dependent on dendritic architecture and synaptic structure, function and plasticity. The cadherins are glycosylated transmembrane proteins that form cell adhesion complexes in various tissues. They are associated with a group of cytosolic proteins, the catenins. While the functional roles of the complex have been extensively investigates in non-neuronal cells, it is becoming increasingly clear that components of the complex have critical roles in regulating dendritic and synaptic architecture, function and plasticity in neurons. Consistent with these functional roles, aberrations in components of the complex have been implicated in a variety of neurodevelopmental disorders. In this review, we discuss the roles of the classical cadherins and catenins in various aspects of dendrite and synapse architecture and function and their relevance to human neurological disorders. Cadherins are glycosylated transmembrane proteins that were initially identified as Ca2+-dependent cell adhesion molecules. They are present on plasma membrane of a variety of cell types from primitive metazoans to humans. In the past several years, it has become clear that in addition to providing mechanical adhesion between cells, cadherins play integral roles in tissue morphogenesis and homeostasis. The cadherin family is composed of more than 100 members and classified into several subfamilies, including classical cadherins and protocadherins. Several of these cadherin family members have been implicated in various aspects of neuronal development and function.1-3 The classical cadherins are associated with a group of cytosolic proteins, collectively called the catenins. While the functional roles of the cadherin-catenin cell adhesion complex have been extensively investigated in epithelial cells, it is now clear that components of the complex are well expressed in central neurons at different

  9. Candida albicans and non-albicans species as etiological agent of vaginitis in pregnant and non-pregnant women.

    PubMed

    Babic, Mirela; Hukic, Mirsada

    2010-02-01

    Pregnancy represents a risk factor in the occurrence of vaginal candidosis. The objectives of our study were: to make determination of the microscopic findings of vaginal swab, frequency of Candida species in the culture of pregnant women and patients who are not pregnant, determine the Candida species in all cultures, and to determine the frequency and differences in the frequency of C. albicans and other non-albicans species. In one year study performed during 2006 year, we tested patients of Gynaecology and Obstetrics clinic of the Clinical Centre in Sarajevo and Gynaecology department of the General hospital in Sarajevo. 447 woman included in the study were separated in two groups: 203 pregnant (in the last trimester of pregnancy), and 244 non-pregnant woman in period of fertility. Each vaginal swab was examined microscopically. The yeast, number of colonies, and the species of Candida were determined on Sabouraud dextrose agar with presence of antibiotics. For determination of Candida species, we used germ tube test for detection of C. albicans, and cultivation on the selective medium and assimilation tests for detection of non-albicans species. The results indicated positive microscopic findings in the test group (40,9%), as well as greater number of positive cultures (46,8%). The most commonly detected species for both groups was C. albicans ( test group 40.9% and control group 23,0%). The most commonly detected non-albicans species for the test group were C. glabrata (4,2 %) and C. krusei (3,2%), and for the control group were C. glabrata (3,2%) and C. parapsilosis (3,2%). The microscopic findings correlated with the number of colonies in positive cultures. In the test group, we found an increased number of yeasts (64,3%), and the pseudopyphae and blastopores by microscopic examination as an indication of infection. In the control group, we found a small number of yeasts (64,6%) , in the form of blastopores, as an indication of the candida colonisation. Our

  10. Molecular methods for strain typing of Candida albicans: a review.

    PubMed

    Saghrouni, F; Ben Abdeljelil, J; Boukadida, J; Ben Said, M

    2013-06-01

    Candida albicans is one of the most medically important fungi because of its high frequency as a commensal and pathogenic microorganism causing superficial as well as invasive infections. Strain typing and delineation of the species are essential for understanding its biology, epidemiology and population structure. A wide range of molecular techniques have been used for this purpose including non-DNA-based methods (multi-locus enzyme electrophoresis), conventional DNA-based methods (electrophoretic karyotyping, random amplified polymorphic DNA, amplified fragment length polymorphism, restriction enzyme analysis with and without hybridization, rep-PCR) and DNA-based methods called exact typing methods because they generate unambiguous and highly reproducible typing data (including microsatellite length polymorphism and multi-locus sequence typing). In this review, the main molecular methods used for C. albicans strain typing are summarized, and their advantages and limitations are discussed with regard to their discriminatory power, reproducibility, cost and ease of performance.

  11. Adaptation of Candida albicans to commensalism in the gut.

    PubMed

    Prieto, Daniel; Correia, Inês; Pla, Jesús; Román, Elvira

    2016-01-01

    Candida albicans is a common resident of the oral cavity, GI tract and vagina in healthy humans where it establishes a commensal relationship with the host. Colonization of the gut, which is an important niche for the microbe, may lead to systemic dissemination and disease upon alteration of host defences. Understanding the mechanisms responsible for the adaptation of C. albicans to the gut is therefore important for the design of new ways of combating fungal diseases. In this review we discuss the available models to study commensalism of this yeast, the main mechanisms controlling the establishment of the fungus, such as microbiota, mucus layer and antimicrobial peptides, and the gene regulatory circuits that ensure its survival in this niche.

  12. An immunological link between Candida albicans colonization and Crohn's disease.

    PubMed

    Gerard, Romain; Sendid, Boualem; Colombel, Jean-Frederic; Poulain, Daniel; Jouault, Thierry

    2015-06-01

    The etiology of Crohn's disease (CD), an autoimmune, inflammatory bowel disease (IBD) which affects approximately one million people in Europe, is still unclear. Nevertheless, it is widely accepted that CD could result from an inappropriate inflammatory response to intestinal microorganisms in a genetically susceptible host. Most studies to date have concerned the involvement of bacteria in disease progression. In addition to bacteria, there appears to be a possible link between the commensal yeast Candida albicans and disease development. In this review, in an attempt to link the gut colonization process and the development of CD, we describe the different pathways that are involved in the progression of CD and in the host response to C. albicans, making the yeast a possible initiator of the inflammatory process observed in this IBD.

  13. Recent advances on Candida albicans biology and virulence

    PubMed Central

    Sellam, Adnane; Whiteway, Malcolm

    2016-01-01

    Candida albicans is an important human fungal pathogen, in terms of both its clinical significance and its use as an experimental model for scientific investigation. Although this opportunistic pathogen is a natural component of the human flora, it can cause life-threatening infections in immunosuppressed patients. There are currently a limited number of antifungal molecules and drug targets, and increasing resistance to the front-line therapeutics, demonstrating a clear need for new antifungal drugs. Understanding the biology of this pathogen is an important prerequisite for identifying new drug targets for antifungal therapeutics. In this review, we highlight some recent developments that help us to understand how virulence traits are regulated at the molecular level, in addition to technical advances that improve the ability of genome editing in C. albicans. PMID:27853524

  14. Growing Candida albicans Biofilms on Paper Support and Dynamic Conditions.

    PubMed

    Selow, Marcela Lima Cardoso; Rymovicz, Alinne Ulbrich Mores; Ribas, Cristina Rauen; Saad, Renata Simão; Rosa, Rosimeire Takaki; Rosa, Edvaldo Antonio Ribeiro

    2015-08-01

    A stainless steel paper-embedded biofilm reactor (PEBR) was developed for Candida spp. growth, permitting confluent distribution of nutrients by capillary diffusion through ordinary laboratory filter paper. Antibiogram disks were distributed along the filter paper rim, and the PEBR received 0.1 or 0.01 % crystal violet (CV) at 200 μL min(-1) and at 37 °C, for 48 h. CV was recovered from the disks and measured at 540 nm. Candida albicans SC5314 cells were applied onto antibiogram disks. The bioreactor was assembled, and YEPD broth was admitted (200 μL min(-1)) at 37 °C, for 72 h. Biofilm growth was estimated via the MTT reduction test. Controls were disks that received the same treatments, except for the fungus. The PEBR was considered high-throughput table, low-cost, and feasible to grow C. albicans biofilms.

  15. Starvation survival of Candida albicans in various water microcosms.

    PubMed

    Chaieb, Kamel; Kouidhi, Bochra; Zmantar, Tarek; Mahdouani, Kacem; Bakhrouf, Amina

    2011-08-01

    Candida is a major Human pathogen causing a variety of infections and can survive for extended period of time in aquatic environment including marine and fresh water. In this study we compared a colorimetric XTT assay to colony forming units (CFU) count to evaluate the survival potential of Candida albicans incubated in water microcosms. Our results showed that cells maintain cultivability within a long period followed by a decline in cultivability and a drop of plate counts to less than 20 cell ml(-1) after 150 days in tap water, 190 days in rain water and 200 days in seawater. In addition we noted that 10% of cells viability was reached after 150 days in seawater, 180 days in rain water and 210 days in tap water. Molecular method confirms the persistence of C. albicans cells in water during long time starvation period.

  16. Host response to Candida albicans bloodstream infection and sepsis

    PubMed Central

    Duggan, Seána; Leonhardt, Ines; Hünniger, Kerstin; Kurzai, Oliver

    2015-01-01

    Candida albicans is a major cause of bloodstream infection which may present as sepsis and septic shock - major causes of morbidity and mortality world-wide. After invasion of the pathogen, innate mechanisms govern the early response. Here, we outline the models used to study these mechanisms and summarize our current understanding of innate immune responses during Candida bloodstream infection. This includes protective immunity as well as harmful responses resulting in Candida induced sepsis. Neutrophilic granulocytes are considered principal effector cells conferring protection and recognize C. albicans mainly via complement receptor 3. They possess a range of effector mechanisms, contributing to elimination of the pathogen. Neutrophil activation is closely linked to complement and modulated by activated mononuclear cells. A thorough understanding of these mechanisms will help in creating an individualized approach to patients suffering from systemic candidiasis and aid in optimizing clinical management. PMID:25785541

  17. Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection.

    PubMed

    Clark, Heather L; Jhingran, Anupam; Sun, Yan; Vareechon, Chairut; de Jesus Carrion, Steven; Skaar, Eric P; Chazin, Walter J; Calera, José Antonio; Hohl, Tobias M; Pearlman, Eric

    2016-01-01

    Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein that possesses antimicrobial activity primarily because of its ability to chelate zinc and manganese. In the current study, we showed that neutrophils from calprotectin-deficient S100A9(-/-) mice have an impaired ability to inhibit Aspergillus fumigatus hyphal growth in vitro and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9(-/-) mice by injecting recombinant calprotectin. Furthermore, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both zinc and manganese binding are necessary for calprotectin's antihyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ∆zafA mutant, which demonstrates deficient zinc transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared with wild-type. Collectively, these studies demonstrate a novel stage-specific susceptibility of A. fumigatus to zinc and manganese chelation by neutrophil-derived calprotectin.

  18. Calibration of quantitative real-time TaqMan PCR by correlation with hyphal biomass and ITS copies in mycelia of Piloderma croceum.

    PubMed

    Raidl, S; Bonfigli, R; Agerer, R

    2005-11-01

    DNA-based quantification methods such as real-time TaqMan PCR allow a rapid and highly sensitive species-specific quantification of isolated fungal DNA material, but most quantification systems are only able to measure relative amounts of biomass or biomass changes during different treatments. In this experiment, an already established DNA quantification system for the ectomycorrhizal fungus Piloderma croceum, based on the ITS region of ribosomal DNA, was calibrated to absolute biomass to obtain a direct correlation between mycelial biomass and isolated ITS copies. Thin layers of sterile mycelia were cultured on slides. The mycelial biomass was calculated from measurements of the total hyphal length using image analysis, followed by determination of the mycelial volume, and multiplied by the specific weight of hyphae obtained from literature data. Using the very same mycelium, the number of ITS copies was quantified by TaqMan PCR. The mean value of 1047 (+/- 185) copies per mm hypha results in possible data for a direct conversion: one billion (10 (9)) ITS copies corresponded to 0.79 mg hyphal dry weight. For the ribosomal ITS multi-copy genes, the number of ITS copies could be calculated to approx. 152 (+/- 26) copies per dikaryotic cell. These conversion data now allow determination of the mycelial biomass of Piloderma croceum using real-time TaqMan PCR, a prerequisite for competition experiments with Piloderma croceum.

  19. Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants

    SciTech Connect

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel; McDonald, Bradon R.; Nicora, Carrie D.; Wu, Si; Moore, Ronald J.; Orton, Daniel J.; Monroe, Matthew E.; Piehowski, Paul D.; Purvine, Samuel O.; Smith, Richard D.; Lipton, Mary S.; Burnum-Johnson, Kristin E.; Currie, Cameron R.

    2015-08-28

    Leaf-cutter ants are prolific and conspicuous Neotropical herbivores that derive energy from specialized fungus gardens they cultivate using foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain lignocellulases that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as it is foraged by the ants. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous lignocellulases likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three-quarters of all lignocellulases identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 23 lignocellulases enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.

  20. mrflbA, encoding a putative FlbA, is involved in aerial hyphal development and secondary metabolite production in Monascus ruber M-7.

    PubMed

    Yang, Yishan; Li, Li; Li, Xin; Shao, Yanchun; Chen, Fusheng

    2012-02-01

    FlbA (fluffy low brlA expression), a regulator of the G protein signalling (RGS) pathway, has been implicated in the control of hyphal development, sporulation, mycotoxin/pigment production in many kinds of filamentous fungi and yeasts. In the current study, a FlbA-like protein gene mrflbA (Monascus ruber flbA) was isolated, sequenced, and disrupted in order to investigate the RGS function in M. ruber. The results revealed that the derived protein of the mrflbA gene consisted of 734 amino acids and had the conserved RGS domain at the C-terminus and two DEP (dishevelled, Egl-10, pleckstrin) domains at the N-terminus similar to the structure of RGS proteins in other filamentous fungi. Deletion of the mrflbA gene resulted in the formation of an abnormal colony phenotype with fluffy aerial hyphae that autolyzed as the colony grew on potato dextrose agar (PDA) at 28°C. Additionally, mrflbA deletion could repress conidial germination and pigment/citrinin production in M. ruber M-7. Real-time RT-PCR analysis demonstrated that the transcription level of the G protein α subunit (Gα) was remarkably increased in the mrflbA deletion strain. These results suggest that mrflbA is involved in the modulation of aerial hyphal development and secondary metabolism, as well as, negative regulation of Gα subunit expression in M. ruber M-7.

  1. A class V chitin synthase gene, chsA is essential for conidial and hyphal wall strength in the fungus Colletotrichum graminicola (Glomerella graminicola).

    PubMed

    Amnuaykanjanasin, Alongkorn; Epstein, Lynn

    2003-04-01

    The Colletotrichum graminicola tagged mutant T30 has conidia that burst and hyphal tips that swell in media with low osmotic pressure. The disrupted gene in T30 was identified as a class V chitin synthase (CSV) "chsA," which has an open reading frame of 1783 amino acids and two introns that are 52 and 54 bp. C. graminicola has one copy of chsA and no other highly homologous class V CHSs. Reverse transcriptase PCR indicated that the T30 mutant does not express the chsA transcript fragment in the conserved region in CSVs. Complementation of the mutant with chsA indicates that the encoded protein is responsible for approximately 29% of the chitin in conidial walls, is essential for conidial wall strength in media with high water potential and contributes to strength of hyphal tips. Analysis of the aligned deduced amino acid sequences of the 10 fully sequenced CSVs suggests that they are in two subgroups.

  2. Identification and characterization of a novel gene, TrCCD1, and its possible function in hyphal growth and conidiospore development of Trichoderma reesei.

    PubMed

    Zhong, Yao Hua; Wang, Tian Hong; Wang, Xiao Li; Zhang, Guang Tao; Yu, Hai Na

    2009-03-01

    To investigate genes with essential functions during hyphal growth or sporulation in the asexual filamentous fungus Trichoderma reesei, we screened a collection of T-DNA insertion mutants and identified the genomic integration events. Two mutants with abnormal phenotypes, named as ccdO and ccdP, were found to have independent T-DNA insertions into a putative TrCCD1 gene locus, the product of which has significant homology to carotenoid cleavage dioxygenases (CCDs). Compared to the parental strain, both mutants tended to produce slow-growing hyphae and had a more than 50% reduction in colony growth rate. Simultaneously, the hyphae of the growing mutants formed wilting tip while the parental strain elongated straightly. To the effect of the TrCCD1 mutation on the conidiospore development, less spores were formed in the mutants than in the parental strain. In addition, disruption of TrCCD1 resulted in another phenotype characterized by a remarkable enhancement in the total carotenoid content. When the wild-type TrCCD1 gene was reintroduced into the ccd mutants, the abnormal phenotypes were rescued. These results suggest that TrCCD1 is involved in carotenoid metabolism and likely required for hyphal growth and conidiospore development in filamentous fungi T. reesei.

  3. Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells.

    PubMed

    Sowa-Jasiłek, Aneta; Zdybicka-Barabas, Agnieszka; Stączek, Sylwia; Wydrych, Jerzy; Skrzypiec, Krzysztof; Mak, Paweł; Deryło, Kamil; Tchórzewski, Marek; Cytryńska, Małgorzata

    2016-12-01

    The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and β-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation.

  4. Candida albicans repetitive elements display epigenetic diversity and plasticity

    PubMed Central

    Freire-Benéitez, Verónica; Price, R. Jordan; Tarrant, Daniel; Berman, Judith; Buscaino, Alessia

    2016-01-01

    Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30 °C, while robust heterochromatin is assembled over these regions at 39 °C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation. PMID:26971880

  5. Potent Synergy between Spirocyclic Pyrrolidinoindolinones and Fluconazole against Candida albicans.

    PubMed

    Premachandra, Ilandari Dewage Udara Anulal; Scott, Kevin A; Shen, Chengtian; Wang, Fuqiang; Lane, Shelley; Liu, Haoping; Van Vranken, David L

    2015-10-01

    A spiroindolinone, (1S,3R,3aR,6aS)-1-benzyl-6'-chloro-5-(4-fluorophenyl)-7'-methylspiro[1,2,3a,6a-tetrahydropyrrolo[3,4-c]pyrrole-3,3'-1H-indole]-2',4,6-trione, was previously reported to enhance the antifungal effect of fluconazole against Candida albicans. A diastereomer of this compound was synthesized, along with various analogues. Many of the compounds were shown to enhance the antifungal effect of fluconazole against C. albicans, some with exquisite potency. One spirocyclic piperazine derivative, which we have named synazo-1, was found to enhance the effect of fluconazole with an EC50 value of 300 pM against a susceptible strain of C. albicans and going as low as 2 nM against some resistant strains. Synazo-1 exhibits true synergy with fluconazole, with an FIC index below 0.5 in the strains tested. Synazo-1 exhibited low toxicity in mammalian cells relative to the concentrations required for antifungal synergy.

  6. Genetic and phenotypic intra-species variation in Candida albicans.

    PubMed

    Hirakawa, Matthew P; Martinez, Diego A; Sakthikumar, Sharadha; Anderson, Matthew Z; Berlin, Aaron; Gujja, Sharvari; Zeng, Qiandong; Zisson, Ethan; Wang, Joshua M; Greenberg, Joshua M; Berman, Judith; Bennett, Richard J; Cuomo, Christina A

    2015-03-01

    Candida albicans is a commensal fungus of the human gastrointestinal tract and a prevalent opportunistic pathogen. To examine diversity within this species, extensive genomic and phenotypic analyses were performed on 21 clinical C. albicans isolates. Genomic variation was evident in the form of polymorphisms, copy number variations, chromosomal inversions, subtelomeric hypervariation, loss of heterozygosity (LOH), and whole or partial chromosome aneuploidies. All 21 strains were diploid, although karyotypic changes were present in eight of the 21 isolates, with multiple strains being trisomic for Chromosome 4 or Chromosome 7. Aneuploid strains exhibited a general fitness defect relative to euploid strains when grown under replete conditions. All strains were also heterozygous, yet multiple, distinct LOH tracts were present in each isolate. Higher overall levels of genome heterozygosity correlated with faster growth rates, consistent with increased overall fitness. Genes with the highest rates of amino acid substitutions included many cell wall proteins, implicating fast evolving changes in cell adhesion and host interactions. One clinical isolate, P94015, presented several striking properties including a novel cellular phenotype, an inability to filament, drug resistance, and decreased virulence. Several of these properties were shown to be due to a homozygous nonsense mutation in the EFG1 gene. Furthermore, loss of EFG1 function resulted in increased fitness of P94015 in a commensal model of infection. Our analysis therefore reveals intra-species genetic and phenotypic differences in C. albicans and delineates a natural mutation that alters the balance between commensalism and pathogenicity.

  7. Inhibition of human natural killer (NK) cytotoxicity by Candida albicans

    SciTech Connect

    Zunino, S.; Hudig, D.

    1986-03-01

    Experiments were initiated to determine whether human NK cells are cytotoxic to C. albicans with similar activity observed for mouse NK cells against the yeast Paracoccidiodes brasiliensis. In 48 hour assays using limiting dilutions of C. albicans, strain 3153A, mononuclear leukocytes with NK activity had only marginal effects on yeast outgrowth, whereas granulocytes killed most of the yeast. However, these yeast were able to block NK activity in 4 hr /sup 51/Cr release assays with K562 cells, at yeast to K562 ratios of 10:1 and 100:1. Yeast pretreated with the serum of the majority of donors blocked the NK activity more than untreated yeast. Two of the 7 donors did not enhance NK inhibition after pretreatment of the yeast with their serum. Serum antibody to C. albicans and complement consumption by the yeast correlated with the relative efficiency of NK inhibition for most donors. This report suggests that there may be in vivo interactions between NK cells of the immune system and opportunistic fungal pathogens, which may compromise NK cell function.

  8. Genetic and phenotypic intra-species variation in Candida albicans

    PubMed Central

    Hirakawa, Matthew P.; Martinez, Diego A.; Sakthikumar, Sharadha; Anderson, Matthew Z.; Berlin, Aaron; Gujja, Sharvari; Zeng, Qiandong; Zisson, Ethan; Wang, Joshua M.; Greenberg, Joshua M.; Berman, Judith

    2015-01-01

    Candida albicans is a commensal fungus of the human gastrointestinal tract and a prevalent opportunistic pathogen. To examine diversity within this species, extensive genomic and phenotypic analyses were performed on 21 clinical C. albicans isolates. Genomic variation was evident in the form of polymorphisms, copy number variations, chromosomal inversions, subtelomeric hypervariation, loss of heterozygosity (LOH), and whole or partial chromosome aneuploidies. All 21 strains were diploid, although karyotypic changes were present in eight of the 21 isolates, with multiple strains being trisomic for Chromosome 4 or Chromosome 7. Aneuploid strains exhibited a general fitness defect relative to euploid strains when grown under replete conditions. All strains were also heterozygous, yet multiple, distinct LOH tracts were present in each isolate. Higher overall levels of genome heterozygosity correlated with faster growth rates, consistent with increased overall fitness. Genes with the highest rates of amino acid substitutions included many cell wall proteins, implicating fast evolving changes in cell adhesion and host interactions. One clinical isolate, P94015, presented several striking properties including a novel cellular phenotype, an inability to filament, drug resistance, and decreased virulence. Several of these properties were shown to be due to a homozygous nonsense mutation in the EFG1 gene. Furthermore, loss of EFG1 function resulted in increased fitness of P94015 in a commensal model of infection. Our analysis therefore reveals intra-species genetic and phenotypic differences in C. albicans and delineates a natural mutation that alters the balance between commensalism and pathogenicity. PMID:25504520

  9. Nanocapsules with glycerol monolaurate: Effects on Candida albicans biofilms.

    PubMed

    Lopes, Leonardo Quintana Soares; Santos, Cayane Genro; Vaucher, Rodrigo de Almeida; Raffin, Renata Platcheck; Santos, Roberto Christ Vianna

    2016-08-01

    Candida albicans does not only occur in the free living planktonic form but also grows in surface-attached biofilm communities. Moreover, these biofilms appear to be the most common lifestyle and are involved in the majority of human Candida infections. Nanoparticles can be used as an alternative to conventional antimicrobial agents and can also act as carriers for antibiotics and other drugs. In view of this, the aim of the study was develop, characterize and verify the anti-biofilm potential of GML Nanocapsules against C. albicans. The GML Nanocapsules showed mean diameter of 193.2 nm, polydispersion index of 0.044, zeta potential of -23.3 mV and pH 6.32. The microdilution assay showed MIC of 15.5 μg mL(-1) to GML Nanocapsules and 31.25 μg mL(-1) to GML. The anti-biofilm assay showed the significantly reduction of biomass of C. albicans biofilm treated with GML Nanocapsules while the GML does not exhibit effect. The kinetic assay demonstrated that at 48 h, the GML Nanocapsules reduce 94% of formed biofilm. The positive results suggest the promisor alternative for this public health problem that is biofilm infections.

  10. Potent Synergy between Spirocyclic Pyrrolidinoindolinones and Fluconazole against Candida albicans

    PubMed Central

    Premachandra, Ilandari Dewage Udara Anulal; Scott, Kevin A.; Shen, Chengtian; Wang, Fuqiang; Lane, Shelley; Liu, Haoping

    2015-01-01

    A spiroindolinone (1S,3R,3aR,6aS)-1-benzyl-6′-chloro-5-(4-fluorophenyl)-7′-methylspiro[1,2,3a,6a-tetrahydropyrrolo[3,4-c]pyrrole-3,3′-1H-indole]-2′,4,6-trione was previously reported to enhance the antifungal effect of fluconazole against C. albicans. A diastereomer of that compound was synthesized, along with various analogues. Many of the compounds were shown to enhance the antifungal effect of fluconazole against C. albicans, some with exquisite potency. One spirocyclic piperazine derivative, which we have named synazo-1, enhanced the effect of fluconazole with EC50 of 300 pM against a susceptible strain of C. albicans and as low as 2 nM against some resistant strains. Synazo-1 exhibits true synergy with fluconazole with an FIC index below 0.5 in the strains tested. Synazo-1 exhibited low toxicity in mammalian cells relative to the concentrations required for the antifungal synergy. PMID:26263912

  11. Candida albicans biofilm on titanium: effect of peroxidase precoating

    PubMed Central

    Ahariz, Mohamed; Courtois, Philippe

    2010-01-01

    The present study aimed to document Candida albicans biofilm development on titanium and its modulation by a peroxidase-precoated material which can generate antimicrobials, such as hypoiodite or hypothiocyanite, from hydrogen peroxide, iodide, or thiocyanate. For this purpose, titanium (powder or foil) was suspended in Sabouraud liquid medium inoculated with C. albicans ATCC10231. After continuous stirring for 2–21 days at room temperature, the supernatant was monitored by turbidimetry at 600 nm and titanium washed three times in sterile Sabouraud broth. Using the tetrazolium salt MTT-formazan assay, the titanium-adherent fungal biomass was measured as 7.50 ± 0.60 × 106 blastoconidia per gram of titanium powder (n = 30) and 0.50 ± 0.04 × 106 blastoconidia per cm2 of titanium foil (n = 12). The presence of yeast on the surface of titanium was confirmed by microscopy both on fresh prep