Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

PubMed Central

Burnside, Kellie; Lembo, Annalisa; de los Reyes, Melissa; Iliuk, Anton; BinhTran, Nguyen-Thao; Connelly, James E.; Lin, Wan-Jung; Schmidt, Byron Z.; Richardson, Anthony R.; Fang, Ferric C.; Tao, Weiguo Andy; Rajagopal, Lakshmi

2010-01-01

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence. PMID:20552019

Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

PubMed

Ikigai, H; Ono, T; Nakae, T; Otsuru, H; Shimamura, T

1999-01-08

Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

Cell Vacuolation Caused by Vibrio cholerae Hemolysin

PubMed Central

Figueroa-Arredondo, Paula; Heuser, John E.; Akopyants, Natalia S.; Morisaki, J. Hiroshi; Giono-Cerezo, Silvia; Enríquez-Rincón, Fernando; Berg, Douglas E.

2001-01-01

Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (∼1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (∼16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses. PMID:11179335

Cell vacuolation caused by Vibrio cholerae hemolysin.

PubMed

Figueroa-Arredondo, P; Heuser, J E; Akopyants, N S; Morisaki, J H; Giono-Cerezo, S; Enríquez-Rincón, F; Berg, D E

2001-03-01

Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (approximately 1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (approximately 16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.

Characterization of a Vibrio alginolyticus Strain, Isolated from Alaskan Oysters, Carrying a Hemolysin Gene Similar to the Thermostable Direct Hemolysin-Related Hemolysin Gene (trh) of Vibrio parahaemolyticus▿

PubMed Central

González-Escalona, Narjol; Blackstone, George M.; DePaola, Angelo

2006-01-01

A Vibrio strain isolated from Alaskan oysters and classified by its biochemical characteristics as Vibrio alginolyticus possessed a thermostable direct hemolysin-related hemolysin (trh) gene previously reported only in Vibrio parahaemolyticus. This trh-like gene was cloned and sequenced and was 98% identical to the trh2 gene of V. parahaemolyticus. This gene seems to be functional since it was transcriptionally active in early-stationary-phase growing cells. To our knowledge, this is the first report of V. alginolyticus possessing a trh gene. PMID:17056701

Isolation and Characterization of Escherichia coli tolC Mutants Defective in Secreting Enzymatically Active Alpha-Hemolysin

PubMed Central

Vakharia, Hema; German, Greg J.; Misra, Rajeev

2001-01-01

This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed α-helical domain, while the remaining two mapped within the outer membrane-embedded β-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC. PMID:11698380

Enteroaggregative Escherichia coli strains secrete a heat-labile toxin antigenically related to E. coli hemolysin.

PubMed Central

Baldwin, T J; Knutton, S; Sellers, L; Hernandez, H A; Aitken, A; Williams, P H

1992-01-01

A protein toxin of approximately 120,000 Da secreted by nonhemolytic enteroaggregative Escherichia coli strains cross-reacted in Western blots (immunoblots) with antibodies raised against the C-terminal region of E. coli hemolysin. Treatment of HEp-2 cells with enteroaggregative E. coli or culture supernatants caused elevation of intracellular calcium and stimulated calcium-dependent protein phosphorylation. Images PMID:1563799

Bordetella Adenylate Cyclase-Hemolysin Toxins

PubMed Central

Guiso, Nicole

2017-01-01

Adenylate cyclase-hemolysin toxin is secreted and produced by three classical species of the genus Bordetella: Bordetella pertussis, B. parapertussis and B. bronchiseptica. This toxin has several properties such as: (i) adenylate cyclase activity, enhanced after interaction with the eukaryotic protein, calmodulin; (ii) a pore-forming activity; (iii) an invasive activity. It plays an important role in the pathogenesis of these Bordetella species responsible for whooping cough in humans or persistent respiratory infections in mammals, by modulating host immune responses. In contrast with other Bordetella toxins or adhesins, lack of (or very low polymorphism) is observed in the structural gene encoding this toxin, supporting its importance as well as a potential role as a vaccine antigen against whooping cough. In this article, an overview of the investigations undertaken on this toxin is presented. PMID:28892012

Water transport by the bacterial channel alpha-hemolysin

NASA Technical Reports Server (NTRS)

Paula, S.; Akeson, M.; Deamer, D.

1999-01-01

This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels.

Single-molecule study of thymidine glycol and i-motif through the alpha-hemolysin ion channel

NASA Astrophysics Data System (ADS)

He, Lidong

Nanopore-based devices have emerged as a single-molecule detection and analysis tool for a wide range of applications. Through electrophoretically driving DNA molecules across a nanosized pore, a lot of information can be received, including unfolding kinetics and DNA-protein interactions. This single-molecule method has the potential to sequence kilobase length DNA polymers without amplification or labeling, approaching "the third generation" genome sequencing for around $1000 within 24 hours. alpha-Hemolysin biological nanopores have the advantages of excellent stability, low-noise level, and precise site-directed mutagenesis for engineering this protein nanopore. The first work presented in this thesis established the current signal of the thymidine glycol lesion in DNA oligomers through an immobilization experiment. The thymidine glycol enantiomers were differentiated from each other by different current blockage levels. Also, the effect of bulky hydrophobic adducts to the current blockage was investigated. Secondly, the alpha-hemolysin nanopore was used to study the human telomere i-motif and RET oncogene i-motif at a single-molecule level. In Chapter 3, it was demonstrated that the alpha-hemolysin nanopore can differentiate an i-motif form and single-strand DNA form at different pH values based on the same sequence. In addition, it shows potential to differentiate the folding topologies generated from the same DNA sequence.

[Antibacterial and anti-hemolysin activities of tea catechins and their structural relatives].

PubMed

Toda, M; Okubo, S; Ikigai, H; Shimamura, T

1990-03-01

Among catechins tested, (-)epigallocatechin (EGC), (-)epicatechin gallate (ECg), (-) epigallocatechin gallate (EGCg) inhibited the growth of Staphylococcus aureus, Vibrio cholerae O1 classical Inaba 569B and El Tor Inaba V86. S. aureus was more sensitive than V. cholerae O1 to these compounds. EGCg showed also a bactericidal activity against V. cholerae O1 569B. Pyrogallol showed a stronger antibacterial activity against S. aureus and V. cholerae O1 than tannic and gallic acid. Rutin or caffein had no effect on them. ECg and EGCg showed the most potent anti-hemolysin activity against S. aureus alpha-toxin, Vibrio parahaemolyticus thermostable direct hemolysin (Vp-TDH) and cholera hemolysin. Among catechin relatives, only tannic acid had a potent anti-hemolysin activity against alpha-toxin. These results suggest that the catechol and pyrogallol groups are responsible for the antibacterial and bactericidal activities, while the conformation of catechins might play an important role in the anti-hemolysin activity.

HlyU Is a Positive Regulator of Hemolysin Expression in Vibrio anguillarum ▿

PubMed Central

Li, Ling; Mou, Xiangyu; Nelson, David R.

2011-01-01

The two hemolysin gene clusters previously identified in Vibrio anguillarum, the vah1 cluster and the rtxACHBDE cluster, are responsible for the hemolytic and cytotoxic activities of V. anguillarum in fish. In this study, we used degenerate PCR to identify a positive hemolysin regulatory gene, hlyU, from the unsequenced V. anguillarum genome. The hlyU gene of V. anguillarum encodes a 92-amino-acid protein and is highly homologous to other bacterial HlyU proteins. An hlyU mutant was constructed, which exhibited an ∼5-fold decrease in hemolytic activity on sheep blood agar with no statistically significant decrease in cytotoxicity of the wild-type strain. Complementation of the hlyU mutation restored both hemolytic activity and cytotoxic activity. Both semiquantitative reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR) were used to examine expression of the hemolysin genes under exponential and stationary-phase conditions in wild-type, hlyU mutant, and hlyU complemented strains. Compared to the wild-type strain, expression of rtx genes decreased in the hlyU mutant, while expression of vah1 and plp was not affected in the hlyU mutant. Complementation of the hlyU mutation restored expression of the rtx genes and increased vah1 and plp expression to levels higher than those in the wild type. The transcriptional start sites in both the vah1-plp and rtxH-rtxB genes' intergenic regions were determined using 5′ random amplification of cDNA ends (5′-RACE), and the binding sites for purified HlyU were discovered using DNA gel mobility shift experiments and DNase protection assays. PMID:21764937

Free-energy calculations reveal the subtle differences in the interactions of DNA bases with α-hemolysin.

PubMed

Manara, Richard M A; Guy, Andrew T; Wallace, E Jayne; Khalid, Syma

2015-02-10

Next generation DNA sequencing methods that utilize protein nanopores have the potential to revolutionize this area of biotechnology. While the technique is underpinned by simple physics, the wild-type protein pores do not have all of the desired properties for efficient and accurate DNA sequencing. Much of the research efforts have focused on protein nanopores, such as α-hemolysin from Staphylococcus aureus. However, the speed of DNA translocation has historically been an issue, hampered in part by incomplete knowledge of the energetics of translocation. Here we have utilized atomistic molecular dynamics simulations of nucleotide fragments in order to calculate the potential of mean force (PMF) through α-hemolysin. Our results reveal specific regions within the pore that play a key role in the interaction with DNA. In particular, charged residues such as D127 and K131 provide stabilizing interactions with the anionic DNA and therefore are likely to reduce the speed of translocation. These regions provide rational targets for pore optimization. Furthermore, we show that the energetic contributions to the protein-DNA interactions are a complex combination of electrostatics and short-range interactions, often mediated by water molecules.

[Research on the expression of hemolysin genes of Leptospira in vivo by genechip].

PubMed

Zhao, Hui; Bao, Lang

2012-07-01

To explore the expression of hemolysin genes of Leptospira in infected host. Amplified the gene segment of hemolysin genes from the genome of Leptospira by PCR for gene probe. Manufacture genechip by the VersArray Chipwriter systerm. The total RNAs of Leptospira before and after infection host were extracted, reversely transcribed to cDNA, after the random PCR, the products were marked with HEX and CY5 respectively, and hybridized to genechip to demonstrate the expression of hemolysin genes of Leptospira. The hemolysin genes LA1029 (Ratio = 0.65), LA1027 (Ratio = 0.53) were up-regulated after infection of host; LA3540 (Ratio = 1.88), LA3937 (Ratio = 5.58), LA1029 (Ratio = 3.00) were up-regulated and LA4004 (Ratio = 0.67) was down-regulated in live than in blood; LA3937 (Ratio = 2.28), LA1029 (Ratio = 2.20) were up-regulated in kidney than in blood. The expression level of hemolysin genes exist observable differences with inducement in vivo and in different organs. These suggested that these genes are probably involved in the pathogenesis and and disease progression.

Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar.

PubMed Central

Beecher, D J; Wong, A C

1994-01-01

Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin BL in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains. In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood. Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL. The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates. Images PMID:8017944

El Tor hemolysin of Vibrio cholerae O1 forms channels in planar lipid bilayer membranes.

PubMed

Ikigai, H; Ono, T; Iwata, M; Nakae, T; Shimamura, T

1997-05-15

We investigated the channel formation by El Tor hemolysin (molecular mass, 65 kDa) of Vibrio cholerae O1 biotype El Tor in planar lipid bilayers. The El Tor hemolysin channel exhibited asymmetric and hyperbolic membrane current with increasing membrane potential, meaning that the channel is voltage dependent. The zero-current membrane potential measured in KCI solution showed that permeability ratio PK+/PCl- was 0.16, indicating that the channel is 6-fold more anion selective over cation. The hemolysin channel frequently flickered in the presence of divalent cations, suggesting that the channel spontaneously opens and closes. These data imply that the El Tor hemolysin damages target cells by the formation of transmembrane channels and, consequently, is the cause of osmotic cytolysis.

CHARACTERIZATION OF THE HEMOLYSIN, FROM STACHYBOTRYS CHARTARUM

EPA Science Inventory

Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage and hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its apparent monomeric form has a molecular mass of 11,920
Da as determ...

Blocking of Single α-Hemolysin Pore by Rhodamine Derivatives.

PubMed

Rokitskaya, Tatyana I; Nazarov, Pavel A; Golovin, Andrey V; Antonenko, Yuri N

2017-06-06

Measurements of ion conductance through α-hemolysin pore in a bilayer lipid membrane revealed blocking of the ion channel by a series of rhodamine 19 and rhodamine B esters. The longest dwell closed time of the blocking was observed with rhodamine 19 butyl ester (C4R1), whereas the octyl ester (C8R1) was of poor effect. Voltage asymmetry in the binding kinetics indicated that rhodamine derivatives bound to the stem part of the aqueous pore lumen. The binding frequency was proportional to a quadratic function of rhodamine concentrations, thereby showing that the dominant binding species were rhodamine dimers. Two levels of the pore conductance and two dwell closed times of the pore were found. The dwell closed times lengthened as the voltage increased, suggesting impermeability of the channel for the ligands. Molecular docking analysis revealed two distinct binding sites within the lumen of the stem of the α-hemolysin pore for the C4R1 dimer, but only one binding site for the C8R1 dimer. The blocking of the α-hemolysin nanopore by rhodamines could be utilized in DNA sequencing as additional optical sensing owing to bright fluorescence of rhodamines if used for DNA labeling. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Hemolysin as a Virulence Factor for Systemic Infection with Isolates of Mycobacterium avium Complex

PubMed Central

Maslow, Joel N.; Dawson, David; Carlin, Elizabeth A.; Holland, Steven M.

1999-01-01

Isolates of the Mycobacterium avium complex were examined for hemolysin expression. Only invasive isolates of M. avium were observed to be hemolytic (P < 0.001), with activity the greatest for isolates of serovars 4 and 8. Thus, M. avium hemolysin appears to represent a virulence factor necessary for invasive disease. PMID:9889239

CHARACTERIZATION OF THE HEMOLYSIN, STACHYLYSIN, FROM STACHYBOTRYS CHARTARUM

EPA Science Inventory

Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage/hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its monomeric form, has a molecular wieght of 11,920 daltons as determined by m...

The β-hemolysin and intracellular survival of Streptococcus agalactiae in human macrophages.

PubMed

Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

2013-01-01

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.

The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

PubMed Central

Sagar, Anubha; Klemm, Carolin; Hartjes, Lara; Mauerer, Stefanie; van Zandbergen, Ger; Spellerberg, Barbara

2013-01-01

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches. PMID:23593170

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

DOE PAGES

Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; ...

2016-07-27

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different beta-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin alpha-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels aremore » consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. In conclusion, the obtained results demonstrate that the general concept of "polymers pushing polymers" is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores.« less

Size-dependent forced PEG partitioning into channels: VDAC, OmpC, and α-hemolysin

PubMed Central

Aksoyoglu, M. Alphan; Podgornik, Rudolf; Bezrukov, Sergey M.; Gurnev, Philip A.; Muthukumar, Murugappan; Parsegian, V. Adrian

2016-01-01

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different β-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of “polymers pushing polymers” is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores. PMID:27466408

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

PubMed Central

Maglia, Giovanni; Restrepo, Marcela Rincon; Mikhailova, Ellina; Bayley, Hagan

2008-01-01

Both protein and solid-state nanopores are under intense investigation for the analysis of nucleic acids. A crucial advantage of protein nanopores is that site-directed mutagenesis permits precise tuning of their properties. Here, by augmenting the internal positive charge within the α-hemolysin pore and varying its distribution, we increase the frequency of translocation of a 92-nt single-stranded DNA through the pore at +120 mV by ≈10-fold over the wild-type protein and dramatically lower the voltage threshold at which translocation occurs, e.g., by 50 mV for 1 event·s−1·μM−1. Further, events in which DNA enters the pore, but is not immediately translocated, are almost eliminated. These experiments provide a basis for improved nucleic acid analysis with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. PMID:19060213

Hybrid MD-Nernst Planck Model of Alpha-hemolysin Conductance Properties

NASA Technical Reports Server (NTRS)

Cozmuta, Ioana; O'Keefer, James T.; Bose, Deepak; Stolc, Viktor

2006-01-01

Motivated by experiments in which an applied electric field translocates polynucleotides through an alpha-hemolysin protein channel causing ionic current transient blockade, a hybrid simulation model is proposed to predict the conductance properties of the open channel. Time scales corresponding to ion permeation processes are reached using the Poisson-Nemst-Planck (PNP) electro-diffusion model in which both solvent and local ion concentrations are represented as a continuum. The diffusion coefficients of the ions (K(+) and Cl(-)) input in the PNP model are, however, calculated from all-atom molecular dynamics (MD). In the MD simulations, a reduced representation of the channel is used. The channel is solvated in a 1 M KCI solution, and an external electric field is applied. The pore specific diffusion coefficients for both ionic species are reduced 5-7 times in comparison to bulk values. Significant statistical variations (17-45%) of the pore-ions diffusivities are observed. Within the statistics, the ionic diffusivities remain invariable for a range of external applied voltages between 30 and 240mV. In the 2D-PNP calculations, the pore stem is approximated by a smooth cylinder of radius approx. 9A with two constriction blocks where the radius is reduced to approx. 6A. The electrostatic potential includes the contribution from the atomistic charges. The MD-PNP model shows that the atomic charges are responsible for the rectifying behaviour and for the slight anion selectivity of the a-hemolysin pore. Independent of the hierarchy between the anion and cation diffusivities, the anionic contribution to the total ionic current will dominate. The predictions of the MD-PNP model are in good agreement with experimental data and give confidence in the present approach of bridging time scales by combining a microscopic and macroscopic model.

Grafting synthetic transmembrane units to the engineered low-toxicity α-hemolysin to restore its hemolytic activity.

PubMed

Ui, Mihoko; Harima, Kousuke; Takei, Toshiaki; Tsumoto, Kouhei; Tabata, Kazuhito V; Noji, Hiroyuki; Endo, Sumire; Akiyama, Kimio; Muraoka, Takahiro; Kinbara, Kazushi

2014-12-01

The chemical modification of proteins to provide desirable functions and/or structures broadens their possibilities for use in various applications. Usually, proteins can acquire new functions and characteristics, in addition to their original ones, via the introduction of synthetic functional moieties. Here, we adopted a more radical approach to protein modification, i.e., the replacement of a functional domain of proteins with alternative chemical compounds to build "cyborg proteins." As a proof of concept model, we chose staphylococcal α-hemolysin (Hla), which is a well-studied, pore-forming toxin. The hemolytic activity of Hla mutants was dramatically decreased by truncation of the stem domain, which forms a β-barrel pore in the membrane. However, the impaired hemolytic activity was significantly restored by attaching a pyrenyl-maleimide unit to the cysteine residue that was introduced in the remaining stem domain. In contrast, negatively charged fluorescein-maleimide completely abolished the remaining activity of the mutants.

Evaluation of in vivo and in vitro biological activity of a Vibrio cholerae 01 hemolysin.

PubMed

Arellano Galindo, José; Rodriquez Angeles, María Guadalupe; Guadarrama, Norma Valázquez; Esteban, Enrique Santos; Cerezo, Silvia Giono

2007-01-01

To evaluate the hemolysin effect by ileal loop model produced by Vibrio cholerae O1 strains, compared with the cellular lysis or cytotoxic activity (CA) observed in cell culture. We studied nine V. cholerae O1 strains, obtained during the Mexican outbreak of cholera (1990-1993), which had CA in Vero and CHO cells. Hemolysin was monitored with the hemolysis test. Titers of CA were calculated by CD50, and the association between CA and cholera toxin (CT) production was discarded by means of neutralization tests using an anti-CT polyclonal antibody. The CT production was measured with ELISA test. The LAL assay was performed in order to study relationships between the CA and bacterial lipopolysaccharide. Strains with CA were evaluated in rabbit and rat ileal loop models; hemorrhagic fluid was also measured. Tissues from ileal loop were included in paraffin to detect intestinal epithelial damage. The hemolysin CA was not neutralized with the anti-CT polyclonal antibody. However, the associated factor of CA was heat labile. CA in cell cultures was not related to the bacterial lipopolysaccharide. The ileal loop test exhibited the presence of hemorrhagic tissue with inflammation. The V. cholerae O1 strains isolated were able to secrete hemolysin which, in turn, caused CA in cell cultures and produced the hemorrhagic and inflammatory effects observed in the ileal loop of rabbit and rat models.

Proteolysis of truncated hemolysin A yields a stable dimerization interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Novak, Walter R. P.; Bhattacharyya, Basudeb; Grilley, Daniel P.

2017-02-21

Wild-type and variant forms of HpmA265 (truncated hemolysin A) fromProteus mirabilisreveal a right-handed, parallel β-helix capped and flanked by segments of antiparallel β-strands. The low-salt crystal structures form a dimeric structureviathe implementation of on-edge main-chain hydrogen bonds donated by residues 243–263 of adjacent monomers. Surprisingly, in the high-salt structures of two variants, Y134A and Q125A-Y134A, a new dimeric interface is formedviamain-chain hydrogen bonds donated by residues 203–215 of adjacent monomers, and a previously unobserved tetramer is formed. In addition, an eight-stranded antiparallel β-sheet is formed from the flap regions of crystallographically related monomers in the high-salt structures. This new interfacemore » is possible owing to additional proteolysis of these variants after Tyr240. The interface formed in the high-salt crystal forms of hemolysin A variants may mimic the on-edge β-strand positioning used in template-assisted hemolytic activity.« less

Effect of Heat (Arrhenius Effect) on Crude Hemolysin of Vibrio parahaemolyticus

PubMed Central

Miwatani, Toshio; Takeda, Yoshifumi; Sakurai, Jun; Yoshihara, Akiko; Taga, Sekiko

1972-01-01

Crude hemolysins prepared from various strains of Vibrio parahaemolyticus, which give positive Kanagawa phenomenon, were partly inactivated by heating at 60 C, but not inactivated significantly by heating at 80 to 90 C. The similar phenomenon has been reported as the Arrhenius effect in staphylococcal alpha toxin. Images PMID:4638496

Detection and analysis of hemolysin genes in Aeromonas hydrophila isolated from Gouramy (Osphronemus gouramy) by polymerase chain reaction (PCR)

NASA Astrophysics Data System (ADS)

Rozi; Rahayu, K.; Daruti, D. N.

2018-04-01

The goal of this study was to detect of Aeromonas hydrophila carrying the hlyA gene in guramy by PCR assay. A total of 5 A. hydrophila strains were isolated from gouramy with different location and furthermore genotypic of all A. hydrophila strains havedetected by PCR assay for 16S rRNA gene. The primers used in the PCR targeted a 592-bp fragment of the hlyA gene coding for the hemolysin gene. Particularly hlyA genes are responsible for haemolysin toxins production in this genus. After gel electrophoresis, the amplicons from representative strains of the A. hydrophila were purified using extraction kit and were subjected to the DNA sequencing analysis. The results showed that: (i) the 592bp amplicon of the hlyA gene was detected in 5/6 of the A. hydrophila; (ii) the nucleotide blast results of hemolysin gene sequences of the strains of A. hydrophila revealed a high homology of 90-97 % with published sequences, and;(iii) the protein blast showed 95-98 % homology when compared to the published sequences. The PCR clearly identified the haemolysin-producing strains of A. hydrophila by detection in hlyA genes and may have application as a rapid species-specific virulence test.

Hyperexpression of α-hemolysin explains enhanced virulence of sequence type 93 community-associated methicillin-resistant Staphylococcus aureus

PubMed Central

2014-01-01

Background The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined. Results Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus. Conclusions These data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA. PMID:24512075

INITIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE FUNGAL HEMOLYSIN STACHYLYSIN FROM STACHYBOTRYS CHARTARUM

EPA Science Inventory

Stachybotrys chartarum is known to produce the hemolysin stachylysin and its detection in human serum has been proposed as a biomarker for exposure to the fungus. In this study we report the initial characterization of monoclonal antibodies (mAbs) against stachylysin and the dev...

Nanoscale Investigation of Generation 1 PAMAM Dendrimers Interaction with a Protein Nanopore.

PubMed

Asandei, Alina; Ciuca, Andrei; Apetrei, Aurelia; Schiopu, Irina; Mereuta, Loredana; Seo, Chang Ho; Park, Yoonkyung; Luchian, Tudor

2017-07-21

Herein, we describe at uni-molecular level the interactions between poly(amidoamine) (PAMAM) dendrimers of generation 1 and the α-hemolysin protein nanopore, at acidic and neutral pH, and ionic strengths of 0.5 M and 1 M KCl, via single-molecule electrical recordings. The results indicate that kinetics of dendrimer-α-hemolysin reversible interactions is faster at neutral as compared to acidic pH, and we propose as a putative explanation the fine interplay among conformational and rigidity changes on the dendrimer structure, and the ionization state of the dendrimer and the α-hemolysin. From the analysis of the dendrimer's residence time inside the nanopore, we posit that the pH- and salt-dependent, long-range electrostatic interactions experienced by the dendrimer inside the ion-selective α-hemolysin, induce a non-Stokesian diffusive behavior of the analyte inside the nanopore. We also show that the ability of dendrimer molecules to adapt their structure to nanoscopic spaces, and control the flow of matter through the α-hemolysin nanopore, depends non-trivially on the pH- and salt-induced conformational changes of the dendrimer.

Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI

PubMed Central

Petersen, Lauren M.; LaCourse, Kaitlyn; Schöner, Tim A.; Bode, Helge

2017-01-01

ABSTRACT Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens. The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA, which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli, swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA, these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI. IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential

Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI.

PubMed

Petersen, Lauren M; LaCourse, Kaitlyn; Schöner, Tim A; Bode, Helge; Tisa, Louis S

2017-11-01

Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA , which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli , swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA , these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI. IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential differences

Production of deoxyribonuclease, ribonuclease, coagulase, and hemolysins by anaerobic gram-positive cocci.

PubMed Central

Marshall, R; Kaufman, A K

1981-01-01

Clinical isolates of Peptococcus and Peptostreptococcus species and Streptococcus intermedius strains were obtained from local hospitals. After confirmed identification, each isolate was tested for the in vitro production of deoxyribonuclease, ribonuclease, coagulase, and hemolysins. Of the 60 strains studied, 18 had enzymatic activity. The variability of enzyme production suggests that such assays are not suitable as an aid to identification of these organisms. PMID:7229018

Epidemiological evidence of lesser role of thermostable direct hemolysin (TDH)-related hemolysin (TRH) than TDH on Vibrio parahaemolyticus pathogenicity.

PubMed

Saito, Shioko; Iwade, Yoshito; Tokuoka, Eisuke; Nishio, Tomohiro; Otomo, Yoshimitsu; Araki, Emiko; Konuma, Hirotaka; Nakagawa, Hiroshi; Tanaka, Hiroyuki; Sugiyama, Kanji; Hasegawa, Akio; Sugita-Konishi, Yoshiko; Hara-Kudo, Yukiko

2015-02-01

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus.

PubMed

Salles, Hévila Oliveira; Braga, Ana Carolina Linhares; Nascimento, Maria Thayana dos Santos Canuto do; Sousa, Ana Márjory Paiva; Lima, Adriano Rodrigues; Vieira, Luiz da Silva; Cavalcante, Antônio Cézar Rocha; Egito, Antonio Silvio do; Andrade, Lúcia Betânia da Silva

2014-01-01

Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (<12 kDa) for Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P<0.05, Bonferroni test). The two fractions of Crotalaria spectabilis showed the same ovicidal activity (P>0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

Soft Wall Ion Channel in Continuum Representation with Application to Modeling Ion Currents in α-Hemolysin

PubMed Central

Simakov, Nikolay A.

2010-01-01

A soft repulsion (SR) model of short range interactions between mobile ions and protein atoms is introduced in the framework of continuum representation of the protein and solvent. The Poisson-Nernst-Plank (PNP) theory of ion transport through biological channels is modified to incorporate this soft wall protein model. Two sets of SR parameters are introduced: the first is parameterized for all essential amino acid residues using all atom molecular dynamic simulations; the second is a truncated Lennard – Jones potential. We have further designed an energy based algorithm for the determination of the ion accessible volume, which is appropriate for a particular system discretization. The effects of these models of short-range interaction were tested by computing current-voltage characteristics of the α-hemolysin channel. The introduced SR potentials significantly improve prediction of channel selectivity. In addition, we studied the effect of choice of some space-dependent diffusion coefficient distributions on the predicted current-voltage properties. We conclude that the diffusion coefficient distributions largely affect total currents and have little effect on rectifications, selectivity or reversal potential. The PNP-SR algorithm is implemented in a new efficient parallel Poisson, Poisson-Boltzman and PNP equation solver, also incorporated in a graphical molecular modeling package HARLEM. PMID:21028776

Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

PubMed

Ikigai, H; Akatsuka, A; Tsujiyama, H; Nakae, T; Shimamura, T

1996-08-01

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.

NIGERLYSINTM, HEMOLYSIN PRODUCED BY ASPERGILLUS NIGER, CAUSES LETHALITY OF PRIMARY RAT CORTICAL NEURONAL CELLS IN VITRO

EPA Science Inventory

Aspergillus niger produced a proteinaceous hemolysin, nigerlysin^TM when incubated on sheep's blood agar at both 23° C and 37°C. Nigerlysin was purified from tryptic soy broth culture filtrate. Purified nigerlysin has a molecular weight of approximately 72 kDa, with an...

Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

PubMed Central

Ikigai, H; Akatsuka, A; Tsujiyama, H; Nakae, T; Shimamura, T

1996-01-01

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane. PMID:8757822

Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

PubMed

Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P N; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino; Ferlenghi, Ilaria; Bagnoli, Fabio

2016-06-01

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus. Copyright © 2016 Fiaschi et al.

Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin

PubMed Central

Fiaschi, Luigi; Di Palo, Benedetta; Scarselli, Maria; Pozzi, Clarissa; Tomaszewski, Kelly; Galletti, Bruno; Nardi-Dei, Vincenzo; Arcidiacono, Letizia; Mishra, Ravi P. N.; Mori, Elena; Pallaoro, Michele; Falugi, Fabiana; Torre, Antonina; Fontana, Maria Rita; Soriani, Marco; Bubeck Wardenburg, Juliane; Grandi, Guido; Rappuoli, Rino

2016-01-01

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus. PMID:27030589

T and B lymphocyte function in response to a protein-free diet.

PubMed Central

Carlomagno, M A; Alito, A E; Almiron, D I; Gimeno, A

1982-01-01

Groups of female adult rats were fed either isocaloric protein-free or 18% protein diets for various intervals. Four days before sacrifice, the animals were immunized either with sheep erythrocytes or with a trinitrophenyl-lipopolysaccharide (TNP-LPS) conjugate. Spleen lymphoid cell populations, spleen plaque-forming cells, and serum hemolysins were measured. A persistent diminution, proportional to the duration of protein deprivation, was observed in all parameters studied after immunization with the T-dependent antigen, sheep erythrocytes. The immune dysfunction was more pronounced for hemolysin titers, which became undetectable after 15 days of protein-free diet. The response of the protein-free group to the T-independent antigen (TNP-LPS) after 15 days of diet was only 34% of the control. When a T-cell lymphokine, macrophage migration inhibitory factor, was measured, a normal response was observed in the protein-free group. Feeding a normal diet rapidly restored the spleen plaque-forming cell populations to 60% of normal after 4 days and to 100% after 6 days. Protein starvation influenced the production of antibodies more than it did the number of antibody-forming cells. The nutritional impairment of immunoglobulin synthesis appears to be reversible. PMID:6216214

Evaluation of esterase and hemolysin activities of different Candida species isolated from vulvovaginitis cases in Lorestan Province, Iran.

PubMed

Noori, Maryam; Dakhili, Mohammad; Sepahvand, Asghar; Davari, Nader

2017-12-01

Annually affecting millions of women, vulvovaginal candidiasis (VVC) is commonly described by signs and symptoms of vulvovaginal inflammation in the presence of  Candida  species. Today, the detection of the virulence factors plays a major role in the understanding of pathogenesis of candidiasis and helps produce new anticandidial drugs to improve its treatment efficiency. Herein, we aimed to evaluate the esterase and hemolysin activities of the vaginal isolates of Candida and their relationship with the presence of VVC. One-hundred vaginal clinical specimens were randomly collected during September-December 2016. The target population consisted of married women suspected of VVC who presented to health centers in Lorestan Province, Iran. In this study, the esterase activity and hemolysin production of Candida clinical isolates were evaluated using the Tween 80 opacity test and the plate assay, respectively. The most frequent Candida species was C. albicans (66; 66%), followed by C. glabrata (11; 11%) and C. tropicalis (11; 11%). The highest esterase activity was found in C. krusei (75%), followed by C. albicans (68.2%) and C. glabrata (54.5%). The greater part of the positive esterase isolates had Pz 4+ scores. Among the Candida species, C. albicans (22.7 % ), C. glabrata (63.6%), and C. krusei (50%) were found to have the highest rates of alpha, beta, and gamma hemolysin production, respectively. The level of hemolytic activity in 51% of the Candida species was Pz 4+ scores. According to our results, the higher expression rates of both enzymes in C. albicans species relative to those of non-albicans Candidate species can partly reflect the role of the virulence factors involved in C. albicans pathogenicity.

Morin hydrate attenuates Staphylococcus aureus virulence by inhibiting the self-assembly of α-hemolysin.

PubMed

Wang, J; Zhou, X; Liu, S; Li, G; Shi, L; Dong, J; Li, W; Deng, X; Niu, X

2015-03-01

To investigate the mechanism by which morin hydrate inhibits the haemolytic activity of α-hemolysin (Hla), a channel-forming toxin that is important for the pathogenesis of disease in experimental animals, and its therapeutic effect against Staphylococcus aureus pneumonia in a mouse model. The results from the in vitro (haemolysis, western blot and cytotoxicity assays) and in vivo (mouse model of intranasal lung infection) experiments indicated that morin hydrate, a natural compound with little anti-Staph. aureus activity, could effectively antagonize the cytolytic activity of Hla, alleviate human lung cell injury, and protect against mortality of Staph. aureus pneumonia in a mouse model of infection. Molecular dynamics simulations, free energy calculations and mutagenesis assays were further employed to determine the catalytic mechanism of inhibition, which indicated that a direct binding of morin to the 'Stem' domain of Hla (residues I107 and T109) and the concomitant change in conformation led to the inhibition of the self-assembly of the heptameric transmembrane pore, thus inhibiting the biological activity of Hla for cell lysis. Morin inhibited Staph. aureus virulence via inhibiting the haemolytic activity of α-hemolysin. These findings suggested that morin is a promising candidate for the development of anti-virulence therapeutic agents for the treatment of Staph. aureus infections. © 2015 The Society for Applied Microbiology.

[Distribution of thermostable direct hemolysin (TDH)- and TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in coastal Shimane Prefecture and TDH and TRH V parahaemolyticus contamination of retail shellfish].

PubMed

Fukushima, Hiroshi

2007-03-01

We studied distribution of thermostable direct hemolysin (TDH)- and TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in coastal sea water, sediment, and shellfish and related retail shellfish contamination in Shimane Prefecture, Japan, between 2002 and 2004. V. parahaemolyticus was isolated from > 80% of sea water, sediment, and shellfish. The detection of TDH gene (tdh) and TRH gene (trh)-positive V parahaemolyticus in sea water was 11%, in sediment 16%, and in shellfish 26%. The number of genes and gene-related in seawater was 23 MPN/L, in sediment 29 MPN/100 g, and in shellfish 460 MPN/10 g. TDH- and TRH-producing V. parahaemolyticus detected in seawater was 5%, in sediment 11% and in shellfish 14%. The continuous distribution of TDH-producing O2:K28, O4:K88, O4:K37, and O4:KUT organisms on the western coast and TRH2-producing O5:k30, O5:K43, O10:K19, O10:KUT, O11:K40, O11:KUT, and OUT:KUT organisms on the Oki Island coast suggested the settlement of these organisms in these coastal environments. From 7 (12%) of 59 retail short-necked clam samples, we isolated TDH-producing O 1:KUT, O3:K6 (2 strains from 2 samples imported from Korea), O4:K12, OUT:K8, and TRH2-producing OUT:K40 and OUT:K51 organisms. These findings suggested that TDH- and TRH-producing V. parahaemolyticus are widely distributed along the coast of this prefecture and are transported by contaminated retail shellfish from other areas.

Evaluation of esterase and hemolysin activities of different Candida species isolated from vulvovaginitis cases in Lorestan Province, Iran

PubMed Central

Noori, Maryam; Dakhili, Mohammad; Sepahvand, Asghar; Davari, Nader

2017-01-01

Background and Purpose: Annually affecting millions of women, vulvovaginal candidiasis (VVC) is commonly described by signs and symptoms of vulvovaginal inflammation in the presence of Candida species. Today, the detection of the virulence factors plays a major role in the understanding of pathogenesis of candidiasis and helps produce new anticandidial drugs to improve its treatment efficiency. Herein, we aimed to evaluate the esterase and hemolysin activities of the vaginal isolates of Candida and their relationship with the presence of VVC. Materials and Methods: One-hundred vaginal clinical specimens were randomly collected during September-December 2016. The target population consisted of married women suspected of VVC who presented to health centers in Lorestan Province, Iran. In this study, the esterase activity and hemolysin production of Candida clinical isolates were evaluated using the Tween 80 opacity test and the plate assay, respectively. Results: The most frequent Candida species was C. albicans (66; 66%), followed by C. glabrata (11; 11%) and C. tropicalis (11; 11%). The highest esterase activity was found in C. krusei (75%), followed by C. albicans (68.2%) and C. glabrata (54.5%). The greater part of the positive esterase isolates had Pz 4+ scores. Among the Candida species, C. albicans (22.7%), C. glabrata (63.6%), and C. krusei (50%) were found to have the highest rates of alpha, beta, and gamma hemolysin production, respectively. The level of hemolytic activity in 51% of the Candida species was Pz 4+ scores. Conclusion: According to our results, the higher expression rates of both enzymes in C. albicans species relative to those of non-albicans Candidate species can partly reflect the role of the virulence factors involved in C. albicans pathogenicity. PMID:29707672

Structure and Function of Thermostable Direct Hemolysin (TDH) from Vibrio Parahaemolyticus

NASA Astrophysics Data System (ADS)

Hashimoto, Hiroshi; Yamane, Tsutomu; Ikeguchi, Mitsunori; Nakahira, Kumiko; Yanagihara, Itaru

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic food-borne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramer forms a central pore with dimensions of 23 Å in diameter and ∼50 Å in depth. π-cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside the pore. Molecular dynamics (MD) simulations suggested that water molecules permeate freely through the central and side channel pores. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.

Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties

PubMed Central

Wang, Yu-Kuo; Huang, Sheng-Cih; Wu, Yi-Fang; Chen, Yu-Ching; Lin, Yen-Ling; Nayak, Manoswini; Lin, Yan Ren; Chen, Wen-Hung; Chiu, Yi-Rong; Li, Thomas Tien-Hsiung; Yeh, Bo-Sou; Wu, Tung-Kung

2011-01-01

Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 oC but is reactivated by additional heating above 80 oC. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDHWT) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDHY53H/T59I and Gh-rTDHT59I/S63T double-mutants and the Gh-rTDHY53H/T59I/S63T triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDHWT. Circular dichroism measurements of Gh-rTDHWT and Gh-rTDHmut showed a conspicuous change from a β-sheet to α-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy. PMID:21494434

Site-directed mutations of thermostable direct hemolysin from Grimontia hollisae alter its arrhenius effect and biophysical properties.

PubMed

Wang, Yu-Kuo; Huang, Sheng-Cih; Wu, Yi-Fang; Chen, Yu-Ching; Lin, Yen-Ling; Nayak, Manoswini; Lin, Yan Ren; Chen, Wen-Hung; Chiu, Yi-Rong; Li, Thomas Tien-Hsiung; Yeh, Bo-Sou; Wu, Tung-Kung

2011-03-31

Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 °C but is reactivated by additional heating above 80 °C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDH(WT). Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a β-sheet to α-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.

Molecular detection of HpmA and HlyA hemolysin of uropathogenic Proteus mirabilis.

PubMed

Cestari, Silvia Emanoele; Ludovico, Marilucia Santos; Martins, Fernando Henrique; da Rocha, Sérgio Paulo Dejato; Elias, Waldir Pereira; Pelayo, Jacinta Sanchez

2013-12-01

Urinary tract infection (UTI) is one of the bacterial infections frequently documented in humans. Proteus mirabilis is associated with UTI mainly in individuals with urinary tract abnormality or related with vesicular catheterism and it can be difficult to treat because of the formation of stones in the bladder and kidneys. These stones are formed due to the presence of urease synthesized by the bacteria. Another important factor is that P. mirabilis produces hemolysin HpmA, used by the bacteria to damage the kidney tissues. Proteus spp. samples can also express HlyA hemolysin, similar to that found in Escherichia coli. A total of 211 uropathogenic P. mirabilis isolates were analyzed to detect the presence of the hpmA and hpmB genes by the techniques of polymerase chain reaction (PCR) and dot blot and hlyA by PCR. The hpmA and hpmB genes were expressed by the RT-PCR technique and two P. mirabilis isolates were sequenced for the hpmA and hpmB genes. The presence of the hpmA and hpmB genes was confirmed by PCR in 205 (97.15 %) of the 211 isolates. The dot blot confirmed the presence of the hpmA and hpmB genes in the isolates that did not amplify in the PCR. None of the isolates studied presented the hlyA gene. The hpmA and hpmB genes that were sequenced presented 98 % identity with the same genes of the HI4320 P. mirabilis sample. This study showed that the PCR technique has good sensitivity for detecting the hpmA and hpmB genes of P. mirabilis.

Structure and Functional Characterization of Vibrio parahaemolyticus Thermostable Direct Hemolysin*

PubMed Central

Yanagihara, Itaru; Nakahira, Kumiko; Yamane, Tsutomu; Kaieda, Shuji; Mayanagi, Kouta; Hamada, Daizo; Fukui, Takashi; Ohnishi, Kiyouhisa; Kajiyama, Shin'ichiro; Shimizu, Toshiyuki; Sato, Mamoru; Ikegami, Takahisa; Ikeguchi, Mitsunori; Honda, Takeshi; Hashimoto, Hiroshi

2010-01-01

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 Å resolution. The TDH tetramer forms a central pore with dimensions of 23 Å in diameter and ∼50 Å in depth. π-Cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin. PMID:20335168

Alpha Hemolysin Induces an Increase of Erythrocytes Calcium: A FLIM 2-Photon Phasor Analysis Approach

PubMed Central

Sanchez, Susana; Bakás, Laura; Gratton, Enrico; Herlax, Vanesa

2011-01-01

α-hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells such as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation, and fluorescence lifetime imaging microscopy (FLIM). Data were analyzed using the phasor representation. In this report, we present evidence that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s. Results are discussed in relation to the difficulties of measuring calcium concentrations in erythrocytes where hemoglobin is present, the contribution of the background and the heterogeneity of the response observed in individual cells. PMID:21698153

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity

PubMed Central

An, Na; Fleming, Aaron M.; Middleton, Eric G.; Burrows, Cynthia J.

2014-01-01

Human telomeric DNA consists of tandem repeats of the sequence 5′-TTAGGG-3′ that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structure-dependent electrical signatures through DNA–protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2′-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology. PMID:25225404

First report of an anti-tumor, anti-fungal, anti-yeast and anti-bacterial hemolysin from Albizia lebbeck seeds.

PubMed

Lam, Sze Kwan; Ng, Tzi Bun

2011-05-15

A monomeric 5.5-kDa protein with hemolytic activity toward rabbit erythrocytes was isolated from seeds of Albizia lebbeck by using a protocol that involved ion-exchange chromatography on Q-Sepharose and SP-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, and gel filtration on Superdex 75. It was unadsorbed on both Q-Sepharose and SP-Sepharose, but adsorbed on Phenyl-Sepharose. Its hemolytic activity was fully preserved in the pH range 0-14 and in the temperature range 0-100 °C, and unaffected in the presence of a variety of metal ions and carbohydrates. The hemolysin reduced viability of murine splenocytes and inhibited proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells with an IC₅₀ of 0.21, 0.97, and 1.37 μM, respectively. It impeded mycelial growth in the fungi Rhizoctonia solani with an IC₅₀ of 39 μM but there was no effect on a variety of other filamentous fungi, including Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Mycosphaerella arachidicola. Lebbeckalysin inhibited growth of Escherichia coli with an IC₅₀ of 0.52 μM. Copyright © 2010 Elsevier GmbH. All rights reserved.

Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes.

PubMed

Nallani, Madhavan; Andreasson-Ochsner, Mirjam; Tan, Cherng-Wen Darren; Sinner, Eva-Kathrin; Wisantoso, Yudi; Geifman-Shochat, Susana; Hunziker, Walter

2011-12-01

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

Shrimp pathogenicity, hemolysis, and the presence of hemolysin and TTSS genes in Vibrio harveyi isolated from Thailand.

PubMed

Rattanama, Pimonsri; Srinitiwarawong, Kanchana; Thompson, Janelle R; Pomwised, Rattanaruji; Supamattaya, Kidchakarn; Vuddhakul, Varaporn

2009-09-23

The virulence factors of Vibrio harveyi, the causative agent of luminous vibriosis, are not completely understood. We investigated the correlations between shrimp mortality, hemolysis, the presence of a hemolysin gene (vhh), and a gene involved in the type III secretion system (the Vibrio calcium response gene vcrD). V harveyi HY01 was isolated from a shrimp that died from vibriosis, and 36 other V. harveyi isolates were obtained from fish and shellfish in Hat Yai city, Thailand. An ocean isolate of V. harveyi BAA-1116 was also included. Thirteen isolates including V harveyi HYO1 caused shrimp death 12 h after injection. Most V harveyi isolates in this group (designated as Group A) caused hemolysis on prawn blood agar. None of the shrimp died after injection with V harveyi BAA-1116. Molecular analysis of all V harveyi isolates revealed the presence of vcrD in both pathogenic and non-pathogenic strains. Although vhh was detected in all V harveyi isolates, some isolates did not cause hemolysis, indicating that vhh gene expression might be regulated. Analysis of the V harveyi HYO1 genome revealed a V cholerae like-hemolysin gene, hlyA (designated as hhl). Specific primers designed for hhl detected this gene in 3 additional V harveyi isolates but the presence of this gene was not correlated with pathogenicity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity in all V harveyi isolates, and there were no correlations among the hhl-positive isolates or the pathogenic strains.

Simulation of polymer translocation through protein channels

PubMed Central

Muthukumar, M.; Kong, C. Y.

2006-01-01

A modeling algorithm is presented to compute simultaneously polymer conformations and ionic current, as single polymer molecules undergo translocation through protein channels. The method is based on a combination of Langevin dynamics for coarse-grained models of polymers and the Poisson–Nernst–Planck formalism for ionic current. For the illustrative example of ssDNA passing through the α-hemolysin pore, vivid details of conformational fluctuations of the polymer inside the vestibule and β-barrel compartments of the protein pore, and their consequent effects on the translocation time and extent of blocked ionic current are presented. In addition to yielding insights into several experimentally reported puzzles, our simulations offer experimental strategies to sequence polymers more efficiently. PMID:16567657

Temperature and electrolyte optimization of the α-hemolysin latch sensing zone for detection of base modification in double-stranded DNA.

PubMed

Johnson, Robert P; Fleming, Aaron M; Jin, Qian; Burrows, Cynthia J; White, Henry S

2014-08-19

The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12-35°C) and KCl concentration (0.15-1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼ 8 kJ mol(-1) decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼ 2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Molecular Bases of cyclodextrin Adapter Interactions with Engineered Protein Nanopores

DOE Office of Scientific and Technical Information (OSTI.GOV)

Banerjee, A.; Mikhailova, E; Cheley, S

2010-01-01

Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe single-molecule chemistry, and in the construction of nano- and micro-devices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the {alpha}-hemolysin ({alpha}HL) pore that bind the adapter {beta}-cyclodextrin ({beta}CD) {approx}10{sup 4} times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and high-resolution x-ray crystallography to provide definitive structural information on these engineered protein nanoporesmore » in unparalleled detail.« less

Internal vs Fishhook Hairpin DNA: Unzipping Locations and Mechanisms in the α-Hemolysin Nanopore

PubMed Central

2015-01-01

Studies on the interaction of hairpin DNA with the α-hemolysin (α-HL) nanopore have determined hairpin unzipping kinetics, thermodynamics, and sequence-dependent DNA/protein interactions. Missing from these results is a systematic study comparing the unzipping process for fishhook (one-tail) vs internal (two-tail) hairpins when they are electrophoretically driven from the cis to the trans side of α-HL via a 30-mer single-stranded tail. In the current studies, fishhook hairpins showed long unzipping times with one deep blockage current level. In contrast, the internal hairpins demonstrated relatively fast unzipping and a characteristic pulse-like current pattern. These differences were further explored with respect to stem length and sequence context. Further, a series of internal hairpins with asymmetric tails were studied, for which it was determined that a second tail longer than 12 nucleotides results in internal hairpin unzipping behavior, while tail lengths of 6 nucleotides behaved like fishhook hairpins. Interestingly, these studies were able to resolve a current difference of ∼6% between hairpin DNA immobilized in the nanopore waiting to unzip vs the translocating unzipped DNA, with the latter showing a deeper current blockage level. This demonstration of different currents for immobilized and translocating DNA has not been described previously. These results were interpreted as fishhook hairpins unzipping inside the vestibule, while the internal hairpins unzip outside the vestibule of α-HL. Lastly, we used this knowledge to study the unzipping of a long double-stranded DNA (>50 base pairs) outside the vestibule of α-HL. The conclusions drawn from these studies are anticipated to be beneficial in future application of nanopore analysis of nucleic acids. PMID:25333648

Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

PubMed

Bando, Hiroki; Hisada, Hiromoto; Ishida, Hiroki; Hata, Yoji; Katakura, Yoshio; Kondo, Akihiko

2011-11-01

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

NASA Astrophysics Data System (ADS)

Zhao, Yonggang; Tang, Xiaoqian; Zhan, Wenbin

2011-09-01

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea, Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors. They are thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR). We ligated the three genes into prokaryotic expression vector pET-28a (+), and transformed the recombinant plasmids into Escherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins, TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis. The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.

DNA-assisted oligomerization of pore-forming toxin monomers into precisely-controlled protein channels

PubMed Central

Knechtel, Johann

2017-01-01

Abstract We have developed a novel approach for creating membrane-spanning protein-based pores. The construction principle is based on using well-defined, circular DNA nanostructures to arrange a precise number of pore-forming protein toxin monomers. We can thereby obtain, for the first time, protein pores with specifically set diameters. We demonstrate this principle by constructing artificial alpha-hemolysin (αHL) pores. The DNA/αHL hybrid nanopores composed of twelve, twenty or twenty-six monomers show stable insertions into lipid bilayers during electrical recordings, along with steady, pore size-dependent current levels. Our approach successfully advances the applicability of nanopores, in particular towards label-free studies of single molecules in large nanoscaled biological structures. PMID:29088457

Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin activity from a case of vulvovaginitis.

PubMed

Kumar, Dharmendra; Banerjee, Tuhina; Pratap, Chandra Bhan; Tilak, Ragini

2015-04-15

Since the emergence of pathogenic non-albicans Candida species, a number of new isolates have been added to the list. One such unusual species is Candida auris (C. auris), recently isolated and studied in few reports. In this study, a case of vulvovaginitis caused by Candida auris incidentally identified by molecular methods using internal transcribed spacer polymerase chain reaction (ITS PCR) is described. Antifungal susceptibility testing revealed the isolate to be resistant to itraconazole (MIC ≥ 2 µg/ml) and expressed important virulence factors including phospholipase, proteinase and hemolysin activity. The patient was successfully treated with oral fluconazole and did not have any invasive fungemia. Very few cases of this emerging pathogen have been reported. However, its isolation from clinical specimens reveals the significance of non-albicans candida species over C. albicans and the diversity of Candida spp causing infections.

Fluctuating bottleneck model studies on kinetics of DNA escape from α-hemolysin nanopores

NASA Astrophysics Data System (ADS)

Bian, Yukun; Wang, Zilin; Chen, Anpu; Zhao, Nanrong

2015-11-01

We have proposed a fluctuation bottleneck (FB) model to investigate the non-exponential kinetics of DNA escape from nanometer-scale pores. The basic idea is that the escape rate is proportional to the fluctuating cross-sectional area of DNA escape channel, the radius r of which undergoes a subdiffusion dynamics subjected to fractional Gaussian noise with power-law memory kernel. Such a FB model facilitates us to obtain the analytical result of the averaged survival probability as a function of time, which can be directly compared to experimental results. Particularly, we have applied our theory to address the escape kinetics of DNA through α-hemolysin nanopores. We find that our theoretical framework can reproduce the experimental results very well in the whole time range with quite reasonable estimation for the intrinsic parameters of the kinetics processes. We believe that FB model has caught some key features regarding the long time kinetics of DNA escape through a nanopore and it might provide a sound starting point to study much wider problems involving anomalous dynamics in confined fluctuating channels.

Iron regulates expression of Bacillus cereus hemolysin II via global regulator Fur.

PubMed

Sineva, Elena; Shadrin, Andrey; Rodikova, Ekaterina A; Andreeva-Kovalevskaya, Zhanna I; Protsenko, Alexey S; Mayorov, Sergey G; Galaktionova, Darya Yu; Magelky, Erica; Solonin, Alexander S

2012-07-01

The capacity of pathogens to respond to environmental signals, such as iron concentration, is key to bacterial survival and establishment of a successful infection. Bacillus cereus is a widely distributed bacterium with distinct pathogenic properties. Hemolysin II (HlyII) is one of its pore-forming cytotoxins and has been shown to be involved in bacterial pathogenicity in a number of cell and animal models. Unlike many other B. cereus pathogenicity factors, HlyII is not regulated by pleiotropic transcriptional regulator PlcR but is controlled by its own regulator, HlyIIR. Using a combination of in vivo and in vitro techniques, we show that hlyII expression is also negatively regulated by iron by the global regulator Fur via direct interaction with the hlyII promoter. DNase I footprinting and in vitro transcription experiments indicate that Fur prevents RNA polymerase binding to the hlyII promoter. HlyII expression profiles demonstrate that both HlyIIR and Fur regulate HlyII expression in a concerted fashion, with the effect of Fur being maximal in the early stages of bacterial growth. In sum, these results show that Fur serves as a transcriptional repressor for hlyII expression.

Exploring transmembrane transport through alpha-hemolysin with grid-steered molecular dynamics.

PubMed

Wells, David B; Abramkina, Volha; Aksimentiev, Aleksei

2007-09-28

The transport of biomolecules across cell boundaries is central to cellular function. While structures of many membrane channels are known, the permeation mechanism is known only for a select few. Molecular dynamics (MD) is a computational method that can provide an accurate description of permeation events at the atomic level, which is required for understanding the transport mechanism. However, due to the relatively short time scales accessible to this method, it is of limited utility. Here, we present a method for all-atom simulation of electric field-driven transport of large solutes through membrane channels, which in tens of nanoseconds can provide a realistic account of a permeation event that would require a millisecond simulation using conventional MD. In this method, the average distribution of the electrostatic potential in a membrane channel under a transmembrane bias of interest is determined first from an all-atom MD simulation. This electrostatic potential, defined on a grid, is subsequently applied to a charged solute to steer its permeation through the membrane channel. We apply this method to investigate permeation of DNA strands, DNA hairpins, and alpha-helical peptides through alpha-hemolysin. To test the accuracy of the method, we computed the relative permeation rates of DNA strands having different sequences and global orientations. The results of the G-SMD simulations were found to be in good agreement in experiment.

Interaction of DNA and Proteins with Single Nanopores

NASA Astrophysics Data System (ADS)

Kasianowicz, J. J.

2006-03-01

The bacterial toxins Staphylococcus aureus alpha-hemolysin and Bacillus anthracis protective antigen kill cells in part by forming ion channels in target membranes. We are using electrophysiology, molecular biology/protein biochemistry and computer modeling to study how biopolymers (e.g., single-stranded DNA and proteins) bind to and transport through these nanometer-scale pores. The results provide insight into the mechanism by which these toxins work and are the basis for several potential nanobiotechnology applications including ultra-rapid DNA sequencing, the sensitive and selective detection of a wide range of analytes and high throughput screening of therapeutic agents against several anthrax toxins. In collaboration with V.M. Stanford, M. Misakian, B. Nablo, S.E. Henrickson, NIST, EEEL, Gaithersburg, MD; T. Nguyen, R. Gussio, NCI, Ft. Detrick, MD; and K.M. Halverson, S. Bavari, R.G. Panchal, USAMRIID, Ft. Detrick, MD.

Serodiagnosis of Acute Typhoid Fever in Nigerian Pediatric Cases by Detection of Serum IgA and IgG Against Hemolysin E and Lipopolysaccharide.

PubMed

Davies, D Huw; Jain, Aarti; Nakajima, Rie; Liang, Li; Jasinskis, Algis; Supnet, Medalyn; Felgner, Philip L; Teng, Andy; Pablo, Jozelyn; Molina, Douglas M; Obaro, Stephen K

2016-08-03

Inexpensive, easy-to-use, and highly sensitive diagnostic tests are currently unavailable for typhoid fever. To identify candidate serodiagnostic markers, we have probed microarrays displaying the full Salmonella enterica serovar Typhi (S. Typhi) proteome of 4,352 different proteins + lipopolysaccharides (LPSs), with sera from Nigerian pediatric typhoid and other febrile cases, Nigerian healthy controls, and healthy U.S. adults. Nigerian antibody profiles were broad (∼500 seropositive antigens) and mainly low level, with a small number of stronger "hits," whereas the profile in U.S. adults was < 1/5 as broad, consistent with endemic exposure in Nigeria. Nigerian profiles were largely unaffected by clinical diagnosis, although the response against t1477 (hemolysin E) consistently emerged as stronger in typhoid cases. The response to LPS was also a strong discriminator of healthy controls and typhoid, although LPS did not discriminate between typhoid and nontyphoidal Salmonella (NTS) disease. As a first step toward the development of a point-of-care diagnostic, t1477 and LPS were evaluated on immunostrips. Both provided good discrimination between healthy controls and typhoid/NTS disease. Such a test could provide a useful screen for salmonellosis (typhoid and NTS disease) in suspected pediatric cases that present with undefined febrile disease. © The American Society of Tropical Medicine and Hygiene.

Serodiagnosis of Acute Typhoid Fever in Nigerian Pediatric Cases by Detection of Serum IgA and IgG against Hemolysin E and Lipopolysaccharide

PubMed Central

Huw Davies, D.; Jain, Aarti; Nakajima, Rie; Liang, Li; Jasinskis, Algis; Supnet, Medalyn; Felgner, Philip L.; Teng, Andy; Pablo, Jozelyn; Molina, Douglas M.; Obaro, Stephen K.

2016-01-01

Inexpensive, easy-to-use, and highly sensitive diagnostic tests are currently unavailable for typhoid fever. To identify candidate serodiagnostic markers, we have probed microarrays displaying the full Salmonella enterica serovar Typhi (S. Typhi) proteome of 4,352 different proteins + lipopolysaccharides (LPSs), with sera from Nigerian pediatric typhoid and other febrile cases, Nigerian healthy controls, and healthy U.S. adults. Nigerian antibody profiles were broad (∼500 seropositive antigens) and mainly low level, with a small number of stronger “hits,” whereas the profile in U.S. adults was < 1/5 as broad, consistent with endemic exposure in Nigeria. Nigerian profiles were largely unaffected by clinical diagnosis, although the response against t1477 (hemolysin E) consistently emerged as stronger in typhoid cases. The response to LPS was also a strong discriminator of healthy controls and typhoid, although LPS did not discriminate between typhoid and nontyphoidal Salmonella (NTS) disease. As a first step toward the development of a point-of-care diagnostic, t1477 and LPS were evaluated on immunostrips. Both provided good discrimination between healthy controls and typhoid/NTS disease. Such a test could provide a useful screen for salmonellosis (typhoid and NTS disease) in suspected pediatric cases that present with undefined febrile disease. PMID:27215295

Two-step processing for activation of the cytolysin/hemolysin of Vibrio cholerae O1 biotype El Tor: nucleotide sequence of the structural gene (hlyA) and characterization of the processed products.

PubMed

Yamamoto, K; Ichinose, Y; Shinagawa, H; Makino, K; Nakata, A; Iwanaga, M; Honda, T; Miwatani, T

1990-12-01

Vibrio cholerae O1 biotype El Tor produces and secretes a 65-kDa cytolysin/hemolysin into the culture medium. We cloned the structural gene (hlyA) for the cytolysin from the total DNA of a V. cholerae O1 El Tor strain, N86. Nucleotide sequence analysis of hlyA revealed an open reading frame consisting of 2,223 bp which can code for a protein of 741 amino acids with a molecular weight of 81,961. Consistent with this, a 79-kDa protein was identified as the product of hlyA by maxicell analysis in Escherichia coli. N-terminal amino acids of this 79-kDa HlyA protein and those of a 65-kDa El Tor cytolysin purified from V. cholerae were Asn-26 and Asn-158, respectively. The 82- and 79-kDa precursors of the 65-kDa mature cytolysin were found in V. cholerae by pulse-chase labeling and Western blot (immunoblot) analysis of hlyA products. Hemolytic activity of the 79-kDa HlyA protein from E. coli was less than 5% that for the 65-kDa cytolysin from V. cholerae. Our results suggest that in V. cholerae, the 82-kDa preprotoxin synthesized in the cytoplasm is secreted through the membranes into the culture medium as the 79-kDa inactive protoxin after cleavage of the signal peptide and is then further processed into the 65-kDa active cytolysin by release of the N-terminal 15-kDa fragment.

Structural requirements of cholesterol for binding to Vibrio cholerae hemolysin.

PubMed

Ikigai, Hajime; Otsuru, Hiroshi; Yamamoto, Koichiro; Shimamura, Tadakatsu

2006-01-01

Cholesterol is necessary for the conversion of Vibrio cholerae hemolysin (VCH) monomers into oligomers in liposome membranes. Using different sterols, we determined the stereochemical structures of the VCH-binding active groups present in cholesterol. The VCH monomers are bound to cholesterol, diosgenin, campesterol, and ergosterol, which have a hydroxyl group at position C-3 (3betaOH) in the A ring and a C-C double bond between positions C-5 and C-6 (C-C Delta(5)) in the B ring. They are not bound to epicholesterol and dihydrocholesterol, which form a covalent link with a 3alphaOH group and a C-C single bond between positions C-5 and C-6, respectively. This result suggests that the 3betaOH group and the C-CDelta(5) bond in cholesterol are required for VCH monomer binding. We further examined VCH oligomer binding to cholesterol. However, this oligomer did not bind to cholesterol, suggesting that the disappearance of the cholesterol-binding potential of the VCH oligomer might be a result of the conformational change caused by the conversion of the monomer into the oligomer. VCH oligomer formation was observed in liposomes containing sterols with the 3betaOH group and the C-C Delta(5) bond, and it correlated with the binding affinity of the monomer to each sterol. Therefore, it seems likely that monomer binding to membrane sterol leads to the assembly of the monomer. However, since oligomer formation was induced by liposomes containing either epicholesterol or dihydrocholesterol, the 3betaOH group and the C-C Delta(5) bond were not essential for conversion into the oligomer.

Python erythrocytes are resistant to α-hemolysin from Escherichia coli.

PubMed

Larsen, Casper K; Skals, Marianne; Wang, Tobias; Cheema, Muhammad U; Leipziger, Jens; Praetorius, Helle A

2011-12-01

α-Hemolysin (HlyA) from Escherichia coli lyses mammalian erythrocytes by creating nonselective cation pores in the membrane. Pore insertion triggers ATP release and subsequent P2X receptor and pannexin channel activation. Blockage of either P2X receptors or pannexin channels reduces HlyA-induced hemolysis. We found that erythrocytes from Python regius and Python molurus are remarkably resistant to HlyA-induced hemolysis compared to human and Trachemys scripta erythrocytes. HlyA concentrations that induced maximal hemolysis of human erythrocytes did not affect python erythrocytes, but increasing the HlyA concentration 40-fold did induce hemolysis. Python erythrocytes were more resistant to osmotic stress than human erythrocytes, but osmotic stress tolerance per se did not confer HlyA resistance. Erythrocytes from T. scripta, which showed higher osmotic resistance than python erythrocytes, were as susceptible to HlyA as human erythrocytes. Therefore, we tested whether python erythrocytes lack the purinergic signalling known to amplify HlyA-induced hemolysis in human erythrocytes. P. regius erythrocytes increased intracellular Ca²⁺ concentration and reduced cell volume when exposed to 3 mM ATP, indicating the presence of a P2X₇-like receptor. In addition, scavenging extracellular ATP or blocking P2 receptors or pannexin channels reduced the HlyA-induced hemolysis. We tested whether the low HlyA sensitivity resulted from low affinity of HlyA to the python erythrocyte membrane. We found comparable incorporation of HlyA into human and python erythrocyte membranes. Taken together, the remarkable HlyA resistance of python erythrocytes was not explained by increased osmotic resistance, lack of purinergic hemolysis amplification, or differences in HlyA affinity.

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

PubMed Central

Skopova, Karolina; Tomalova, Barbora; Kanchev, Ivan; Rossmann, Pavel; Svedova, Martina; Adkins, Irena; Bibova, Ilona; Tomala, Jakub; Masin, Jiri; Guiso, Nicole; Osicka, Radim; Sedlacek, Radislav; Kovar, Marek

2017-01-01

ABSTRACT The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b− cells. The nonhemolytic AC+ Hly− bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly− mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b− cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent. PMID:28396322

Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

NASA Astrophysics Data System (ADS)

Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

2015-02-01

The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2‧-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5‧ or 3‧ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

Interactions of neuropathogenic Escherichia coli K1 (RS218) and its derivatives lacking genomic islands with phagocytic Acanthamoeba castellanii and nonphagocytic brain endothelial cells.

PubMed

Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

2014-01-01

Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α -hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity.

Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

PubMed Central

Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

2014-01-01

Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. PMID:24818136

Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

PubMed Central

Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter

2012-01-01

The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742

Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Armstrong, S.K.

1986-01-01

Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and /sup 125/I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulatedmore » strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25/sup 0/C instead of 100/sup 0/C.« less

Analysis of Receptor for Vibrio cholerae El Tor Hemolysin with a Monoclonal Antibody That Recognizes Glycophorin B of Human Erythrocyte Membrane

PubMed Central

Zhang, Dongyan; Takahashi, Junko; Seno, Taiko; Tani, Yoshihiko; Honda, Takeshi

1999-01-01

El Tor hemolysin (ETH), a pore-forming toxin secreted by Vibrio cholerae O1 biotype El Tor and most Vibrio cholerae non-O1 isolates, is able to lyse erythrocytes and other mammalian cells. To study the receptor for this toxin or the related molecule(s) on erythrocyte, we first isolated a monoclonal antibody, B1, against human erythrocyte membrane, which not only blocks the binding of ETH to human erythrocyte but also inhibits the hemolytic activity of ETH. Biochemical characterization and immunoblotting revealed that this antibody recognized an epitope on the extracellular domain of glycophorin B, a sialoglycoprotein of erythrocyte membrane. Erythrocytes lacking glycophorin B but not glycophorin A were less sensitive to the toxin than were normal human erythrocytes. These results indicate that glycophorin B is a receptor for ETH or at least an associated molecule of the receptor for ETH on human erythrocytes. PMID:10496913

Host Response Signature to Staphylococcus aureus Alpha-Hemolysin Implicates Pulmonary Th17 Response

PubMed Central

Zhou, Tong; Moreno-Vinasco, Liliana; Hollett, Brian; Garcia, Joe G. N.

2012-01-01

Staphylococcus aureus pneumonia causes significant morbidity and mortality. Alpha-hemolysin (Hla), a pore-forming cytotoxin of S. aureus, has been identified through animal models of pneumonia as a critical virulence factor that induces lung injury. In spite of considerable molecular knowledge of how this cytotoxin injures the host, the precise host response to Hla in the context of infection remains poorly understood. We employed whole-genome expression profiling of infected lungs to define the host response to wild-type S. aureus compared with the response to an Hla-deficient isogenic mutant in experimental pneumonia. These data provide a complete expression profile at 4 and at 24 h postinfection, revealing a unique response to the toxin-expressing strain. Gene ontogeny analysis revealed significant differences in the extracellular matrix and cardiomyopathy pathways, both of which govern cellular interactions in the tissue microenvironment. Evaluation of individual transcript responses to Hla-secreting staphylococci was notable for upregulation of host cytokine and chemokine genes, including the p19 subunit of interleukin-23. Consistent with this observation, the cellular immune response to infection was characterized by a prominent Th17 response to the wild-type pathogen. These findings define specific host mRNA responses to Hla-producing S. aureus, coupling the pulmonary Th17 response to the secretion of this cytotoxin. Expression profiling to define the host response to a single virulence factor proved to be a valuable tool in identifying pathways for further investigation in S. aureus pneumonia. This approach may be broadly applicable to the study of bacterial toxins, defining host pathways that can be targeted to mitigate toxin-induced disease. PMID:22733574

Antibodies against Stachybotrys chartarum extract and its antigenic components, Stachyhemolysin and Stachyrase-A: a new clinical biomarker.

PubMed

Vojdani, Aristo

2005-05-01

IgG and IgE antibodies against Stachybotrys extract have been reported in allergic patients and residents of water-damaged buildings. Detection of these antibodies in blood was partially attributed to cross-reacting proteins from other fungi. There is a need for a specific method to detect antibodies against characteristic components of S. chartarum. We measured IgG and IgE antibodies against Stachybotrys hemolysin and proteinase-Stachyrase-A by ELISA and ELISA-inhibition techniques. Of 50 reference sera with IgE greater than 500 IU ml and positive against different mold extracts used in this study, significant elevation in IgG or IgE antibodies against S. chartarum extract was present in 25 and 21 specimens. Of these specimens 20 (80%) and 10 (40%) were positive for IgG anti-Stachybotrys hemolysin and anti-Stachyrase-A, while 8 out of 21 sera (38%) and 17 out of 21 (81%) specimens were positive for IgE anti-Stachybotrys hemolysin and anti-Stachyrase-A respectively. Inhibition studies using Stachybotrys hemolysin and Stachyrase-A at a concentration of 50 microg/ml prevented binding of anti-Stachybotrys to S. chartarum extract. Detection of IgG as well as IgE antibodies against Stachybotrys hemolysin and Stachyrase-A and inhibition of anti-Stachybotrys binding to Stachybotrys antigens indicate that Stachybotrys hemolysin and Stachyrase-A are two major antigenic components of S. chartarum extract, which can be used in antibody assays. Measurement of antibodies against these characteristic components of S. chartarum may be considered for demonstration of exposure and possibly allergy to the fungus.

Phloretin derived from apple can reduce alpha-hemolysin expression in methicillin-resistant Staphylococcus aureus USA300.

PubMed

Zhou, Xuan; Liu, Shui; Li, Wenhua; Zhang, Bing; Liu, Bowen; Liu, Yan; Deng, Xuming; Peng, Liping

2015-08-01

Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly important because it is the most common cause of hospital-acquired infections, which have become globally epidemic. Our study specifically focused on the MRSA strain USA300, which was shown in 2014 to be responsible for the most current pandemic of highly virulent MRSA in the United States. We aimed to evaluate the in vitro effect of phloretin on USA300. Susceptibility testing, western blotting assays, hemolysis assays and real-time RT-PCR were employed to examine the in vitro effects of phloretin on alpha-hemolysin (Hla) production when the bacterium was co-cultured with phloretin. The protective effect of phloretin against the USA300-mediated injury of human alveolar epithelial cells (A549) was tested using the live/dead analysis and cytotoxicity assays. We showed that sub-inhibitory concentrations of phloretin have no effect on bacterial viability; however, they can markedly inhibit the production of Hla in culture supernatants and the transcriptional levels of hla (the gene encoding Hla) and agrA (the accessory gene regulator). Phloretin, at a final concentration of 16 µg/ml, could protect A549 cells from injury caused by USA300 in the co-culture system. Our study suggests that phloretin might have a potential application in the development of treatment for MRSA infections.

A Protein Nanopore-Based Approach for Bacteria Sensing

NASA Astrophysics Data System (ADS)

Apetrei, Aurelia; Ciuca, Andrei; Lee, Jong-kook; Seo, Chang Ho; Park, Yoonkyung; Luchian, Tudor

2016-11-01

We present herein a first proof of concept demonstrating the potential of a protein nanopore-based technique for real-time detection of selected Gram-negative bacteria ( Pseudomonas aeruginosa or Escherichia coli) at a concentration of 1.2 × 108 cfu/mL. The anionic charge on the bacterial outer membrane promotes the electrophoretically driven migration of bacteria towards a single α-hemolysin nanopore isolated in a lipid bilayer, clamped at a negative electric potential, and followed by capture at the nanopore's mouth, which we found to be described according to the classical Kramers' theory. By using a specific antimicrobial peptide as a putative molecular biorecognition element for the bacteria used herein, we suggest that the detection system can combine the natural sensitivity of the nanopore-based sensing techniques with selective biological recognition, in aqueous samples, and highlight the feasibility of the nanopore-based platform to provide portable, sensitive analysis and monitoring of bacterial pathogens.

Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques

PubMed Central

Letchumanan, Vengadesh; Chan, Kok-Gan; Lee, Learn-Han

2014-01-01

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that is found in estuarine, marine and coastal environments. V. parahaemolyticus is the leading causal agent of human acute gastroenteritis following the consumption of raw, undercooked, or mishandled marine products. In rare cases, V. parahaemolyticus causes wound infection, ear infection or septicaemia in individuals with pre-existing medical conditions. V. parahaemolyticus has two hemolysins virulence factors that are thermostable direct hemolysin (tdh)-a pore-forming protein that contributes to the invasiveness of the bacterium in humans, and TDH-related hemolysin (trh), which plays a similar role as tdh in the disease pathogenesis. In addition, the bacterium is also encodes for adhesions and type III secretion systems (T3SS1 and T3SS2) to ensure its survival in the environment. This review aims at discussing the V. parahaemolyticus growth and characteristics, pathogenesis, prevalence and advances in molecular identification techniques. PMID:25566219

The Extracellular Metalloprotease of Vibrio tubiashii Is a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae▿

PubMed Central

Hasegawa, Hiroaki; Lind, Erin J.; Boin, Markus A.; Häse, Claudia C.

2008-01-01

Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae. PMID:18456850

Semisynthetic protein nanoreactor for single-molecule chemistry

PubMed Central

Lee, Joongoo; Bayley, Hagan

2015-01-01

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach. PMID:26504203

Sword and shield: linked group B streptococcal beta-hemolysin/cytolysin and carotenoid pigment function to subvert host phagocyte defense.

PubMed

Liu, George Y; Doran, Kelly S; Lawrence, Toby; Turkson, Nicole; Puliti, Manuela; Tissi, Luciana; Nizet, Victor

2004-10-05

Group B Streptococcus (GBS) is a major cause of pneumonia, bacteremia, and meningitis in neonates and has been found to persist inside host phagocytic cells. The pore-forming GBS beta-hemolysin/cytolysin (betaH/C) encoded by cylE is an important virulence factor as demonstrated in several in vivo models. Interestingly, cylE deletion results not only in the loss of betaH/C activity, but also in the loss of a carotenoid pigment of unknown function. In this study, we sought to define the mechanism(s) by which cylE may contribute to GBS phagocyte resistance and increased virulence potential. We found that cylE-deficient GBS was more readily cleared from a mouse's bloodstream, human whole blood, and isolated macrophage and neutrophil cultures. Survival was linked to the ability of betaH/C to induce cytolysis and apoptosis of the phagocytes. At a lower bacterial inoculum, cylE also contributed to enhanced survival within phagocytes that was attributed to the ability of carotenoid to shield GBS from oxidative damage. In oxidant killing assays, cylE mutants were shown to be more susceptible to hydrogen peroxide, hypochlorite, superoxide, and singlet oxygen. Together, these data suggest a mechanism by which the linked cylE-encoded phenotypes, betaH/C (sword) and carotenoid (shield), act in partnership to thwart the immune phagocytic defenses.

Unfolding Kinetics of the Human Telomere i-Motif Under a 10 pN Force Imposed by the α-Hemolysin Nanopore Identify Transient Folded-State Lifetimes at Physiological pH.

PubMed

Ding, Yun; Fleming, Aaron M; He, Lidong; Burrows, Cynthia J

2015-07-22

Cytosine (C)-rich DNA can adopt i-motif folds under acidic conditions, with the human telomere i-motif providing a well-studied example. The dimensions of this i-motif are appropriate for capture in the nanocavity of the α-hemolysin (α-HL) protein pore under an electrophoretic force. Interrogation of the current vs time (i-t) traces when the i-motif interacts with α-HL identified characteristic signals that were pH dependent. These features were evaluated from pH 5.0 to 7.2, a region surrounding the transition pH of the i-motif (6.1). When the i-motif without polynucleotide tails was studied at pH 5.0, the folded structure entered the nanocavity of α-HL from either the top or bottom face to yield characteristic current patterns. Addition of a 5' 25-mer poly-2'-deoxyadensosine tail allowed capture of the i-motif from the unfolded terminus, and this was used to analyze the pH dependency of unfolding. At pH values below the transition point, only folded strands were observed, and when the pH was increased above the transition pH, the number of folded events decreased, while the unfolded events increased. At pH 6.8 and 7.2 4% and 2% of the strands were still folded, respectively. The lifetimes for the folded states at pH 6.8 and 7.2 were 21 and 9 ms, respectively, at 160 mV electrophoretic force. These lifetimes are sufficiently long to affect enzymes operating on DNA. Furthermore, these transient lifetimes are readily obtained using the α-HL nanopore, a feature that is not easily achievable by other methods.

Group B Streptococcus β-hemolysin/Cytolysin Breaches Maternal-Fetal Barriers to Cause Preterm Birth and Intrauterine Fetal Demise in Vivo

PubMed Central

Randis, Tara M.; Gelber, Shari E.; Hooven, Thomas A.; Abellar, Rosanna G.; Akabas, Leor H.; Lewis, Emma L.; Walker, Lindsay B.; Byland, Leah M.; Nizet, Victor; Ratner, Adam J.

2014-01-01

Background. Maternal vaginal colonization with Streptococcus agalactiae (Group B Streptococcus [GBS]) is a precursor to chorioamnionitis, fetal infection, and neonatal sepsis, but the understanding of specific factors in the pathogenesis of ascending infection remains limited. Methods. We used a new murine model to evaluate the contribution of the pore-forming GBS β-hemolysin/cytolysin (βH/C) to vaginal colonization, ascension, and fetal infection. Results. Competition assays demonstrated a marked advantage to βH/C-expressing GBS during colonization. Intrauterine fetal demise and/or preterm birth were observed in 54% of pregnant mice colonized with wild-type (WT) GBS and 0% of those colonized with the toxin-deficient cylE knockout strain, despite efficient colonization and ascension by both strains. Robust placental inflammation, disruption of maternal-fetal barriers, and fetal infection were more frequent in animals colonized with WT bacteria. Histopathologic examination revealed bacterial tropism for fetal lung and liver. Conclusions. Preterm birth and fetal demise are likely the direct result of toxin-induced damage and inflammation rather than differences in efficiency of ascension into the upper genital tract. These data demonstrate a distinct contribution of βH/C to GBS chorioamnionitis and subsequent fetal infection in vivo and showcase a model for this most proximal step in GBS pathogenesis. PMID:24474814

Slowing down single-molecule trafficking through a protein nanopore reveals intermediates for peptide translocation

NASA Astrophysics Data System (ADS)

Mereuta, Loredana; Roy, Mahua; Asandei, Alina; Lee, Jong Kook; Park, Yoonkyung; Andricioaei, Ioan; Luchian, Tudor

2014-01-01

The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.

Dysregulation of Escherichia coli α-hemolysin expression alters the course of acute and persistent urinary tract infection.

PubMed

Nagamatsu, Kanna; Hannan, Thomas J; Guest, Randi L; Kostakioti, Maria; Hadjifrangiskou, Maria; Binkley, Jana; Dodson, Karen; Raivio, Tracy L; Hultgren, Scott J

2015-02-24

Urinary tract infections (UTIs) are among the most common bacterial infections, causing considerable morbidity in females. Infection is highly recurrent despite appropriate antibiotic treatment. Uropathogenic Escherichia coli (UPEC), the most common causative agent of UTIs, invades bladder epithelial cells (BECs) and develops into clonal intracellular bacterial communities (IBCs). Upon maturation, IBCs disperse, with bacteria spreading to neighboring BECs to repeat this cycle. This process allows UPEC to gain a foothold in the face of innate defense mechanisms, including micturition, epithelial exfoliation, and the influx of polymorphonuclear leukocytes. Here, we investigated the mechanism and dynamics of urothelial exfoliation in the early acute stages of infection. We show that UPEC α-hemolysin (HlyA) induces Caspase-1/Caspase-4-dependent inflammatory cell death in human urothelial cells, and we demonstrate that the response regulator (CpxR)-sensor kinase (CpxA) two-component system (CpxRA), which regulates virulence gene expression in response to environmental signals, is critical for fine-tuning HlyA cytotoxicity. Deletion of the cpxR transcriptional response regulator derepresses hlyA expression, leading to enhanced Caspase-1/Caspase-4- and NOD-like receptor family, pyrin domain containing 3-dependent inflammatory cell death in human urothelial cells. In vivo, overexpression of HlyA during acute bladder infection induces more rapid and extensive exfoliation and reduced bladder bacterial burdens. Bladder fitness is restored fully by inhibition of Caspase-1 and Caspase-11, the murine homolog of Caspase-4. Thus, we have discovered that fine-tuning of HlyA expression by the CpxRA system is critical for enhancing UPEC fitness in the urinary bladder. These results have significant implications for our understanding of how UPEC establishes persistent colonization.

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions.

PubMed

Chifiriuc, Mariana Carmen; Bleotu, Coralia; Pîrcălăbioru, Gratiela; Israil, Anca Michaela; Dinu, Sorin; Rută, Simona Maria; Grancea, Camelia; Lazăr, Veronica

2010-01-01

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.

A single residue mutation in Hha preserving structure and binding to H-NS results in loss of H-NS mediated gene repression properties.

PubMed

Cordeiro, Tiago N; Garcia, Jesús; Pons, José-Ignacio; Aznar, Sonia; Juárez, Antonio; Pons, Miquel

2008-09-03

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.

Dysregulation of Escherichia coli α-hemolysin expression alters the course of acute and persistent urinary tract infection

PubMed Central

Nagamatsu, Kanna; Hannan, Thomas J.; Guest, Randi L.; Kostakioti, Maria; Hadjifrangiskou, Maria; Binkley, Jana; Dodson, Karen; Raivio, Tracy L.; Hultgren, Scott J.

2015-01-01

Urinary tract infections (UTIs) are among the most common bacterial infections, causing considerable morbidity in females. Infection is highly recurrent despite appropriate antibiotic treatment. Uropathogenic Escherichia coli (UPEC), the most common causative agent of UTIs, invades bladder epithelial cells (BECs) and develops into clonal intracellular bacterial communities (IBCs). Upon maturation, IBCs disperse, with bacteria spreading to neighboring BECs to repeat this cycle. This process allows UPEC to gain a foothold in the face of innate defense mechanisms, including micturition, epithelial exfoliation, and the influx of polymorphonuclear leukocytes. Here, we investigated the mechanism and dynamics of urothelial exfoliation in the early acute stages of infection. We show that UPEC α-hemolysin (HlyA) induces Caspase-1/Caspase-4–dependent inflammatory cell death in human urothelial cells, and we demonstrate that the response regulator (CpxR)-sensor kinase (CpxA) two-component system (CpxRA), which regulates virulence gene expression in response to environmental signals, is critical for fine-tuning HlyA cytotoxicity. Deletion of the cpxR transcriptional response regulator derepresses hlyA expression, leading to enhanced Caspase-1/Caspase-4– and NOD-like receptor family, pyrin domain containing 3-dependent inflammatory cell death in human urothelial cells. In vivo, overexpression of HlyA during acute bladder infection induces more rapid and extensive exfoliation and reduced bladder bacterial burdens. Bladder fitness is restored fully by inhibition of Caspase-1 and Caspase-11, the murine homolog of Caspase-4. Thus, we have discovered that fine-tuning of HlyA expression by the CpxRA system is critical for enhancing UPEC fitness in the urinary bladder. These results have significant implications for our understanding of how UPEC establishes persistent colonization. PMID:25675528

Infection and cellular defense dynamics in a novel 17β-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation.

PubMed

Carey, Alison J; Tan, Chee Keong; Mirza, Shaper; Irving-Rodgers, Helen; Webb, Richard I; Lam, Alfred; Ulett, Glen C

2014-02-15

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (10(6)-10(7) CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

In-vivo-induced antigenic determinants of Fusobacterium nucleatum subsp. nucleatum.

PubMed

Lee, H-R; Rhyu, I-C; Kim, H-D; Jun, H-K; Min, B-M; Lee, S-H; Choi, B-K

2011-04-01

Fusobacterium nucleatum plays a pivotal role in dental plaque biofilm formation and is known to be involved in chronic inflammatory systemic disease. However, limited knowledge of F. nucleatum genes expressed in vivo interferes with our understanding of pathogenesis. In this study, we identified F. nucleatum genes induced in vivo using in-vivo-induced antigen technology (IVIAT). Among 30,000 recombinant clones screened, 87 reacted reproducibly with pooled sera from 10 patients with periodontitis. The clones encoded for 32 different proteins, of which 28 could be assigned to their functions, which were categorized in translation, transcription, transport, energy metabolism, cell envelope, cellular process, fatty acid and phospholipid metabolism, transposition, cofactor biosynthesis, amino acid biosynthesis, and DNA replication. Putative virulence factors detected were ABC transporter, butyrate-acetoacetate CoA-transferase, hemin receptor, hemolysin, hemolysin-related protein, LysR family transcriptional regulator, serine protease, and transposase. Analysis of immune responses to the in-vivo-induced (ivi) antigens in five patients demonstrated that most were reactive to these proteins, confirming results with pooled sera. IVIAT-identified F. nucleatum genes in this study may accelerate the elucidation of F. nucleatum-mediated molecular pathogenesis. © 2011 John Wiley & Sons A/S.

Escherichia coli α-Hemolysin Triggers Shrinkage of Erythrocytes via KCa3.1 and TMEM16A Channels with Subsequent Phosphatidylserine Exposure*

PubMed Central

Skals, Marianne; Jensen, Uffe B.; Ousingsawat, Jiraporn; Kunzelmann, Karl; Leipziger, Jens; Praetorius, Helle A.

2010-01-01

α-Hemolysin from Escherichia coli (HlyA) readily lyse erythrocytes from various species. We have recently demonstrated that this pore-forming toxin provokes distinct shrinkage and crenation before it finally leads to swelling and lysis of erythrocytes. The present study documents the underlying mechanism for this severe volume reduction. We show that HlyA-induced shrinkage and crenation of human erythrocytes occur subsequent to a significant rise in [Ca2+]i. The Ca2+-activated K+ channel KCa3.1 (or Gardos channel) is essential for the initial shrinkage, because both clotrimazole and TRAM-34 prevent the shrinkage and potentiate hemolysis produced by HlyA. Notably, the recently described Ca2+-activated Cl− channel TMEM16A contributes substantially to HlyA-induced cell volume reduction. Erythrocytes isolated from TMEM16A−/− mice showed significantly attenuated crenation and increased lysis compared with controls. Additionally, we found that HlyA leads to acute exposure of phosphatidylserine in the outer leaflet of the plasma membrane. This exposure was considerably reduced by KCa3.1 antagonists. In conclusion, this study shows that HlyA triggers acute erythrocyte shrinkage, which depends on Ca2+-activated efflux of K+ via KCa3.1 and Cl− via TMEM16A, with subsequent phosphatidylserine exposure. This mechanism might potentially allow HlyA-damaged erythrocytes to be removed from the bloodstream by macrophages and thereby reduce the risk of intravascular hemolysis. PMID:20231275

Protective efficacy of a novel alpha hemolysin subunit vaccine (AT62) against Staphylococcus aureus skin and soft tissue infections.

PubMed

Adhikari, Rajan P; Thompson, Christopher D; Aman, M Javad; Lee, Jean C

2016-12-07

Alpha hemolysin (Hla) is a pore-forming toxin produced by most Staphylococcus aureus isolates. Hla is reported to play a key role in the pathogenesis of staphylococcal infections, such as skin and soft tissue infection, pneumonia, and lethal peritonitis. This study makes use of a novel recombinant subunit vaccine candidate (AT62) that was rationally designed based on the Hla heptameric crystal structure. AT62 comprises a critical structural domain at the N terminus of Hla, and it has no inherent toxic properties. We evaluated the efficacy of AT62 in protection against surgical wound infection and skin and soft tissue infection. Mice were vaccinated on days 0, 14, and 28 with 20μg AT62 or bovine serum albumin (BSA) mixed with Sigma adjuvant system®. Mice immunized with AT62 produced a robust antibody response against native Hla. In the surgical wound infection model, mice immunized with AT62 and challenged with a USA300 S. aureus strain showed a significantly reduced bacterial burden in the infected tissue compared to animals given BSA. Similarly, mice passively immunized with rabbit IgG to AT62 showed reduced wound infection and tissue damage. Subcutaneous abscess formation was not prevented by immunization with AT62. However, in a skin necrosis infection model, immunization with the AT62 vaccine resulted in smaller lesions and reduced mouse weight loss compared to controls. Although AT62 immunization reduced tissue necrosis, it did not reduce the bacterial burdens in the lesions compared to controls. Our data indicate that AT62 may be a valuable component of a multivalent vaccine against S. aureus. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain.

PubMed

Pader, Vera; James, Ellen H; Painter, Kimberley L; Wigneshweraraj, Sivaramesh; Edwards, Andrew M

2014-10-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Agr Quorum-Sensing System Regulates Fibronectin Binding but Not Hemolysis in the Absence of a Functional Electron Transport Chain

PubMed Central

Pader, Vera; James, Ellen H.; Painter, Kimberley L.; Wigneshweraraj, Sivaramesh

2014-01-01

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain. PMID:25092909

Escherichia coli alpha-hemolysin triggers shrinkage of erythrocytes via K(Ca)3.1 and TMEM16A channels with subsequent phosphatidylserine exposure.

PubMed

Skals, Marianne; Jensen, Uffe B; Ousingsawat, Jiraporn; Kunzelmann, Karl; Leipziger, Jens; Praetorius, Helle A

2010-05-14

alpha-Hemolysin from Escherichia coli (HlyA) readily lyse erythrocytes from various species. We have recently demonstrated that this pore-forming toxin provokes distinct shrinkage and crenation before it finally leads to swelling and lysis of erythrocytes. The present study documents the underlying mechanism for this severe volume reduction. We show that HlyA-induced shrinkage and crenation of human erythrocytes occur subsequent to a significant rise in [Ca(2+)](i). The Ca(2+)-activated K(+) channel K(Ca)3.1 (or Gardos channel) is essential for the initial shrinkage, because both clotrimazole and TRAM-34 prevent the shrinkage and potentiate hemolysis produced by HlyA. Notably, the recently described Ca(2+)-activated Cl(-) channel TMEM16A contributes substantially to HlyA-induced cell volume reduction. Erythrocytes isolated from TMEM16A(-/-) mice showed significantly attenuated crenation and increased lysis compared with controls. Additionally, we found that HlyA leads to acute exposure of phosphatidylserine in the outer leaflet of the plasma membrane. This exposure was considerably reduced by K(Ca)3.1 antagonists. In conclusion, this study shows that HlyA triggers acute erythrocyte shrinkage, which depends on Ca(2+)-activated efflux of K(+) via K(Ca)3.1 and Cl(-) via TMEM16A, with subsequent phosphatidylserine exposure. This mechanism might potentially allow HlyA-damaged erythrocytes to be removed from the bloodstream by macrophages and thereby reduce the risk of intravascular hemolysis.

Effect of sodium chloride and citric acid on growth and toxin production by A. caviae and A. sobria at moderate and low temperatures.

PubMed

Abu-Ghazaleh, B M

2000-10-01

The effect of sodium chloride and citric acid on hemolysin and caseinase production by Aeromonas caviae and Aeromonas sobria at 32 degrees C and 5 degrees C was investigated. At 32 degrees C, although both strains were tolerant to 3% NaCl in TSB, the production of caseinase was decreased in the presence of 1-3% NaCl, and the production of hemolysin was abolished by 2-3% NaCl. Citric acid (0.03%) was less effective than NaCl in reducing hemolysin and caseinase production by both strains at 32 degrees C. A combination of low temperature (5 degrees C) and citric acid treatment reduced hemolysin and caseinase production by both strains. A combination of low temperature (5 degrees C) and NaCl (3%) treatment was the most effective procedure in reducing growth and hemolysin and caseinase production by the tested strains.

The toxR Gene of Vibrio (Listonella) anguillarum Controls Expression of the Major Outer Membrane Proteins but Not Virulence in a Natural Host Model

PubMed Central

Okuda, Jun; Nakai, Toshihiro; Chang, Park Se; Oh, Takanori; Nishino, Takeshi; Koitabashi, Tsutomu; Nishibuchi, Mitsuaki

2001-01-01

To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae. Distribution of the Va-toxR gene sequence in V. anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic toxR mutant of V. anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant. For the toxR mutant, the MICs of two β-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a

Insertion and self-diffusion of a monotopic protein, the Aquifex aeolicus sulfide quinone reductase, in supported lipid bilayers.

PubMed

Harb, Frédéric; Prunetti, Laurence; Giudici-Orticoni, Marie-Thérèse; Guiral, Marianne; Tinland, Bernard

2015-10-01

Monotopic proteins constitute a class of membrane proteins that bind tightly to cell membranes, but do not span them. We present a FRAPP (Fluorescence Recovery After Patterned Photobleaching) study of the dynamics of a bacterial monotopic protein, SQR (sulfide quinone oxidoreductase) from the thermophilic bacteria Aquifex aeolicus, inserted into two different types of lipid bilayers (EggPC: L-α-phosphatidylcholine (Egg, Chicken) and DMPC: 1,2-dimyristoyl-sn-glycero-3-phosphocholine) supported on two different types of support (mica or glass). It sheds light on the behavior of a monotopic protein inside the bilayer. The insertion of SQR is more efficient when the bilayer is in the fluid phase than in the gel phase. We observed diffusion of the protein, with no immobile fraction, and deduced from the diffusion coefficient measurements that the resulting inserted object is the same whatever the incubation conditions, i.e. homogeneous in terms of oligomerization state. As expected, the diffusion coefficient of the SQR is smaller in the gel phase than in the fluid phase. In the supported lipid bilayer, the diffusion coefficient of the SQR is smaller than the diffusion coefficient of phospholipids in both gel and fluid phase. SQR shows a diffusion behavior different from the transmembrane protein α-hemolysin, and consistent with its monotopic character. Preliminary experiments in the presence of the substrate of SQR, DecylUbiquinone, an analogue of quinone, component of transmembrane electrons transport systems of eukaryotic and prokaryotic organisms, have been carried out. Finally, we studied the behavior of SQR, in terms of insertion and diffusion, in bilayers formed with lipids from Aquifex aeolicus. All the conclusions that we have found in the biomimetic systems applied to the biological system.

Characteristics of hemolytic activity induced by the aqueous extract of the Mexican fire coral Millepora complanata.

PubMed

García-Arredondo, Alejandro; Murillo-Esquivel, Luis J; Rojas, Alejandra; Sanchez-Rodriguez, Judith

2014-01-01

Millepora complanata is a plate-like fire coral common throughout the Caribbean. Contact with this species usually provokes burning pain, erythema and urticariform lesions. Our previous study suggested that the aqueous extract of M. complanata contains non-protein hemolysins that are soluble in water and ethanol. In general, the local damage induced by cnidarian venoms has been associated with hemolysins. The characterization of the effects of these components is important for the understanding of the defense mechanisms of fire corals. In addition, this information could lead to better care for victims of envenomation accidents. An ethanolic extract from the lyophilized aqueous extract was prepared and its hemolytic activity was compared with the hemolysis induced by the denatured aqueous extract. Based on the finding that ethanol failed to induce nematocyst discharge, ethanolic extracts were prepared from artificially bleached and normal M. complanata fragments and their hemolytic activity was tested in order to obtain information about the source of the heat-stable hemolysins. Rodent erythrocytes were more susceptible to the aqueous extract than chicken and human erythrocytes. Hemolytic activity started at ten minutes of incubation and was relatively stable within the range of 28-50°C. When the aqueous extract was preincubated at temperatures over 60°C, hemolytic activity was significantly reduced. The denatured extract induced a slow hemolytic activity (HU50 = 1,050.00 ± 45.85 μg/mL), detectable four hours after incubation, which was similar to that induced by the ethanolic extract prepared from the aqueous extract (HU50 = 1,167.00 ± 54.95 μg/mL). No significant differences were observed between hemolysis induced by ethanolic extracts from bleached and normal fragments, although both activities were more potent than hemolysis induced by the denatured extract. The results showed that the aqueous extract of M. complanata possesses one or

Quorum Sensing Inhibitors for Staphylococcus aureus from Italian Medicinal Plants

PubMed Central

Quave, Cassandra L.; Plano, Lisa R.W.; Bennett, Bradley C.

2010-01-01

Morbidity and mortality estimates due to methicillin-resistant Staphylococcus aureus (MRSA) infections continue to rise. Therapeutic options are limited by antibiotic resistance. Anti-pathogenic compounds, which inhibit quorum sensing (QS) pathways, may be a useful alternative to antibiotics. Staphylococcal QS is encoded by the agr locus and is responsible for the production of δ-hemolysin. Quantification of δ-hemolysin found in culture supernatants permits the analysis of agr activity at the translational, rather than transcriptional, level. We employed RP-HPLC techniques to investigate the anti-QS activity of 168 extracts from 104 Italian plants through quantification of δ-hemolysin. Extracts from three medicinal plants (Ballota nigra, Castanea sativa, and Sambucus ebulus) exhibited a dose-dependent response in the production of δ-hemolysin, indicating strong anti-QS activity in a pathogenic MRSA isolate. PMID:20645243

[Pathogenic factors of vibrios with special emphasis on Vibrio vulnificus].

PubMed

Shinoda, Sumio

2005-07-01

Bacteria of the genus Vibrio are normal habitants of the aquatic environment and play roles for biocontrole of aquatic ecosystem, but some species are believed to be human pathogens. These species can be classified into two groups according to the types of diseases they cause: the gastrointestinal infections and the extraintestinal infections. The pathogenic species produce various pathogenic factors including enterotoxin, hemolysin, cytotoxin, protease, siderophore, adhesive factor, and hemagglutinin. We studied various pathogenic factors of vibrios with special emphasis on protease and hemolysin of V. vulnificus. V. vulnificus is now recognized as being among the most rapidly fatal of human pathogens, although the infection is appeared in patients having underlying disease(s) such as liver dysfunction, alcoholic cirrhosis or haemochromatosis. V. vulnificus protease (VVP) is thought to be a major toxic factor causing skin damage in the patients having septicemia. VVP is a metalloprotease and degrades a number of biologically important proteins including elastin, fibrinogen, and plasma proteinase inhibitors of complement components. VVP causes skin damages through activation of the Factor XII-plasma kallikrein-kinin cascade and/or exocytotic histamine release from mast cells, and a haemorrhagic lesion through digestion of the vascular basement membrane. Thus, the protease is the most probable candidate for tissue damage and bacterial invasion during an infection. Pathogenic roles and functional mechanism of other factors including hemolysins of V. vulnificus and V. mimicus are also shown in this review article.

Loss of Regulatory Protein RfaH Attenuates Virulence of Uropathogenic Escherichia coli

PubMed Central

Nagy, Gábor; Dobrindt, Ulrich; Schneider, György; Khan, A. Salam; Hacker, Jörg; Emödy, Levente

2002-01-01

RfaH is a regulatory protein in Escherichia coli and Salmonella enterica serovar Typhimurium. Although it enhances expression of different factors that are proposed to play a role in bacterial virulence, a direct effect of RfaH on virulence has not been investigated so far. We report that inactivation of rfaH dramatically decreases the virulence of uropathogenic E. coli strain 536 in an ascending mouse model of urinary tract infection. The mortality rate caused by the wild-type strain in this assay is 100%, whereas that of its isogenic rfaH mutant does not exceed 18%. In the case of coinfection, the wild-type strain 536 shows higher potential to colonize the urinary tract even when it is outnumbered 100-fold by its rfaH mutant in the inoculum. In contrast to the wild-type strain, serum resistance of strain 536rfaH::cat is fully abolished. Furthermore, we give evidence that, besides a major decrease in the amount of hemin receptor ChuA (G. Nagy, U. Dobrindt, M. Kupfer, L. Emody, H. Karch, and J. Hacker, Infect. Immun. 69:1924-1928, 2001), loss of the RfaH protein results in an altered lipopolysaccharide phenotype as well as decreased expression of K15 capsule and alpha-hemolysin, whereas levels of other pathogenicity factors such as siderophores, flagella, Prf, and S fimbriae appear to be unaltered in strain 536rfaH::cat in comparison to the wild-type strain. trans complementation of the mutant strain with the rfaH gene restores wild-type levels of the affected virulence factors and consequently restitutes virulence in the mouse model of ascending urinary tract infection. PMID:12117951

Stochastic detection of enantiomers.

PubMed

Kang, Xiao-Feng; Cheley, Stephen; Guan, Xiyun; Bayley, Hagan

2006-08-23

The rapid quantification of the enantiomers of small chiral molecules is very important, notably in pharmacology. Here, we show that the enantiomers of drug molecules can be distinguished by stochastic sensing, a single-molecule detection technique. The sensing element is an engineered alpha-hemolysin protein pore, fitted with a beta-cyclodextrin adapter. By using the approach, the enantiomeric composition of samples of ibuprofen and thalidomide can be determined in less than 1 s.

Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli

PubMed Central

Skals, Marianne

2016-01-01

α-Hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans are important virulence factors in ascending urinary tract infections and aggressive periodontitis, respectively. The extracellular signaling molecule ATP is released immediately after insertion of the toxins into plasma membranes and, via P2X receptors, is essential for the erythrocyte damage inflicted by these toxins. Moreover, ATP signaling is required for the ensuing recognition and phagocytosis of damaged erythrocytes by the monocytic cell line THP-1. Here, we investigate how these toxins affect THP-1 monocyte function. We demonstrate that both toxins trigger early ATP release and a following increase in the intracellular Ca2+ concentration ([Ca2+]i) in THP-1 monocytes. The HlyA- and LtxA-induced [Ca2+]i response is diminished by the P2 receptor antagonist in a pattern that fits the functional P2 receptor expression in these cells. Both toxins are capable of lysing THP-1 cells, with LtxA being more aggressive. Either desensitization or blockage of P2X1, P2X4, or P2X7 receptors markedly reduces toxin-induced cytolysis. This pattern is paralleled in freshly isolated human monocytes from healthy volunteers. Interestingly, only a minor fraction of the toxin-damaged THP-1 monocytes eventually lyse. P2X7 receptor inhibition generally prevents cell damage, except from a distinct cell shrinkage that prevails in response to the toxins. Moreover, we find that preexposure to HlyA preserves the capacity of THP-1 monocytes to phagocytose damaged erythrocytes and may induce readiness to discriminate between damaged and healthy erythrocytes. These findings suggest a new pharmacological target for protecting monocytes during exposure to pore-forming cytolysins during infection or injury. PMID:27528275

Computational discovery of putative quorum sensing inhibitors against LasR and RhlR receptor proteins of Pseudomonas aeruginosa

NASA Astrophysics Data System (ADS)

Annapoorani, Angusamy; Umamageswaran, Venugopal; Parameswari, Radhakrishnan; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

2012-09-01

Drugs have been discovered in the past mainly either by identification of active components from traditional remedies or by unpredicted discovery. A key motivation for the study of structure based virtual screening is the exploitation of such information to design targeted drugs. In this study, structure based virtual screening was used in search for putative quorum sensing inhibitors (QSI) of Pseudomonas aeruginosa. The virtual screening programme Glide version 5.5 was applied to screen 1,920 natural compounds/drugs against LasR and RhlR receptor proteins of P. aeruginosa. Based on the results of in silico docking analysis, five top ranking compounds namely rosmarinic acid, naringin, chlorogenic acid, morin and mangiferin were subjected to in vitro bioassays against laboratory strain PAO1 and two more antibiotic resistant clinical isolates, P. aeruginosa AS1 (GU447237) and P. aeruginosa AS2 (GU447238). Among the five compounds studied, except mangiferin other four compounds showed significant inhibition in the production of protease, elastase and hemolysin. Further, all the five compounds potentially inhibited the biofilm related behaviours. This interaction study provided promising ligands to inhibit the quorum sensing (QS) mediated virulence factors production in P. aeruginosa.

Prevalence and molecular typing of Vibrio parahaemolyticus isolated from seafood in Shanghai using multilocus sequence typing (MLST)

USDA-ARS?s Scientific Manuscript database

Vibrio parahaemolyticus is a gram-negative bacterium that inhabits coastal and marine environments. Thermostable direct hemolysin (tdh), tdh-related hemolysin (trh) and the type III secretion system are considered the potential virulent factors of pathogenic V. parahaemolyticus. The frequency of str...

Web Interface for Brownian Dynamics Simulation of Ion Transport and Its Applications to Beta-Barrel Pores

PubMed Central

Lee, Kyu Il; Jo, Sunhwan; Rui, Huan; Egwolf, Bernhard; Roux, Benoît; Pastor, Richard W.; Im, Wonpil

2011-01-01

Brownian dynamics (BD) in a suitably constructed potential of mean force is an efficient and accurate method for simulating ion transport through wide ion channels. Here, a web-based graphical user interface (GUI) is presented for grand canonical Monte Carlo (GCMC) BD simulations of channel proteins: http://www.charmm-gui.org/input/gcmcbd. The webserver is designed to help users avoid most of the technical difficulties and issues encountered in setting up and simulating complex pore systems. GCMC/BD simulation results for three proteins, the voltage dependent anion channel (VDAC), α-Hemolysin, and the protective antigen pore of the anthrax toxin (PA), are presented to illustrate system setup, input preparation, and typical output (conductance, ion density profile, ion selectivity, and ion asymmetry). Two models for the input diffusion constants for potassium and chloride ions in the pore are compared: scaling of the bulk diffusion constants by 0.5, as deduced from previous all-atom molecular dynamics simulations of VDAC; and a hydrodynamics based model (HD) of diffusion through a tube. The HD model yields excellent agreement with experimental conductances for VDAC and α-Hemolysin, while scaling bulk diffusion constants by 0.5 leads to underestimates of 10–20%. For PA, simulated ion conduction values overestimate experimental values by a factor of 1.5 to 7 (depending on His protonation state and the transmembrane potential), implying that the currently available computational model of this protein requires further structural refinement. PMID:22102176

Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean.

PubMed

Dreesen, Rozemarijn; Capt, Annabelle; Oberdoerfer, Regina; Coats, Isabelle; Pallett, Kenneth Edward

2018-06-09

By transgenic expression technology, a modified 4-hydroxyphenylpyruvate dioxygenase enzyme (HPPD W336) originating from Pseudomonas fluorescens is expressed in MST-FGØ72-2 soybean to confer tolerance to 4-benzoyl isoxazole and triketone type of herbicides1F. Characterization and safety assessment of HPPD W336 were performed. No relevant sequence homologies were found with known allergens or toxins. Although sequence identity to known toxins showed identity to HPPD proteins annotated as hemolysins, the absence of hemolytic activity of HPPD W336 was demonstrated in vitro. HPPD W336 degrades rapidly in simulated gastric fluid. The absence of toxicity and hemolytic potential of HPPD W336 was confirmed by in vivo studies. The substrate spectrum of HPPD W336 was compared with wild type HPPD proteins, demonstrating that its expression is unlikely to induce any metabolic shifts in soybean. The potential effect of expression of HPPD W336 on metabolic pathways related to tyrosine was investigated by comparing seed composition of MST-FGØ72-2 soybean with non-genetically modified varieties, demonstrating that expression of HPPD W336 does not change aromatic amino acid, homogentisate and tocochromanol levels. In conclusion, HPPD W336 was demonstrated to be as safe as other food proteins. No adverse metabolic effects were identified related to HPPD W336 expression in MST-FGØ72-2 soybean. Copyright © 2018. Published by Elsevier Inc.

What controls open-pore and residual currents in the first sensing zone of alpha-hemolysin nanopore? Combined experimental and theoretical study

NASA Astrophysics Data System (ADS)

de Biase, Pablo M.; Ervin, Eric N.; Pal, Prithwish; Samoylova, Olga; Markosyan, Suren; Keehan, Michael G.; Barrall, Geoffrey A.; Noskov, Sergei Yu.

2016-06-01

The electrophoretic transport of single-stranded DNA through biological nanopores such as alpha-hemolysin (αHL) is a promising and cost-effective technology with the potential to revolutionize genomics. The rational design of pores with the controlled polymer translocation rates and high contrast between different nucleotides could improve significantly nanopore sequencing applications. Here, we apply a combination of theoretical and experimental methods in an attempt to elucidate several selective modifications in the pore which were proposed to be central for the effective discrimination between purines and pyrimidines. Our nanopore test set includes the wild type αHL and six mutants (E111N/M113X/K147N) in which the cross-section and chemical functionality of the first constriction zone of the pore are modified. Electrophysiological recordings were combined with all-atom Molecular Dynamics simulations (MD) and a recently developed Brownian Dynamics (BROMOC) protocol to investigate residual ion currents and pore-DNA interactions for two homo-polymers e.g. poly(dA)40 or poly(dC)40 blocking the pore. The calculated residual currents and contrast in the poly(dA)40/poly(dC)40 blocked pore are in qualitative agreement with the experimental recordings. We showed that a simple structural metric allows rationalization of key elements in the emergent contrast between purines and pyrimidines in the modified αHL mutants. The shape of the pore and its capacity for hydrogen bonding to a translocated polynucleotide are two essential parameters for contrast optimization. To further probe the impact of these two factors in the ssDNA sensing, we eliminated the effect of the primary constriction using serine substitutions (i.e. E111S/M113S/T145S/K147S) and increased the hydrophobic volume of the central residue in the secondary constriction (L135I). This pore modification sharply increased the contrast between Adenine (A) and Cytosine (C).The electrophoretic transport of single

The β-Prism Lectin Domain of Vibrio cholerae Hemolysin Promotes Self-assembly of the β-Pore-forming Toxin by a Carbohydrate-independent Mechanism*

PubMed Central

Ganguly, Sreerupa; Mukherjee, Amarshi; Mazumdar, Budhaditya; Ghosh, Amar N.; Banerjee, Kalyan K.

2014-01-01

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC50) without the lectin domain, and mutant VCCD617A with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 107 m−1. However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC50 was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer. PMID:24356964

Massively Parallel, Molecular Analysis Platform Developed Using a CMOS Integrated Circuit With Biological Nanopores

PubMed Central

Roever, Stefan

2012-01-01

A massively parallel, low cost molecular analysis platform will dramatically change the nature of protein, molecular and genomics research, DNA sequencing, and ultimately, molecular diagnostics. An integrated circuit (IC) with 264 sensors was fabricated using standard CMOS semiconductor processing technology. Each of these sensors is individually controlled with precision analog circuitry and is capable of single molecule measurements. Under electronic and software control, the IC was used to demonstrate the feasibility of creating and detecting lipid bilayers and biological nanopores using wild type α-hemolysin. The ability to dynamically create bilayers over each of the sensors will greatly accelerate pore development and pore mutation analysis. In addition, the noise performance of the IC was measured to be 30fA(rms). With this noise performance, single base detection of DNA was demonstrated using α-hemolysin. The data shows that a single molecule, electrical detection platform using biological nanopores can be operationalized and can ultimately scale to millions of sensors. Such a massively parallel platform will revolutionize molecular analysis and will completely change the field of molecular diagnostics in the future.

A hemolytic pigment of Group B Streptococcus allows bacterial penetration of human placenta

PubMed Central

Whidbey, Christopher; Harrell, Maria Isabel; Burnside, Kellie; Ngo, Lisa; Becraft, Alexis K.; Iyer, Lakshminarayan M.; Aravind, L.; Hitti, Jane

2013-01-01

Microbial infection of the amniotic fluid is a significant cause of fetal injury, preterm birth, and newborn infections. Group B Streptococcus (GBS) is an important human bacterial pathogen associated with preterm birth, fetal injury, and neonatal mortality. Although GBS has been isolated from amniotic fluid of women in preterm labor, mechanisms of in utero infection remain unknown. Previous studies indicated that GBS are unable to invade human amniotic epithelial cells (hAECs), which represent the last barrier to the amniotic cavity and fetus. We show that GBS invades hAECs and strains lacking the hemolysin repressor CovR/S accelerate amniotic barrier failure and penetrate chorioamniotic membranes in a hemolysin-dependent manner. Clinical GBS isolates obtained from women in preterm labor are hyperhemolytic and some are associated with covR/S mutations. We demonstrate for the first time that hemolytic and cytolytic activity of GBS is due to the ornithine rhamnolipid pigment and not due to a pore-forming protein toxin. Our studies emphasize the importance of the hemolytic GBS pigment in ascending infection and fetal injury. PMID:23712433

Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics.

PubMed

Artursson, Karin; Söderlund, Robert; Liu, Lihong; Monecke, Stefan; Schelin, Jenny

2016-09-25

Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype. Copyright © 2016 Elsevier B.V. All rights reserved.

Plasma and Mucosal Immunoglobulin M, Immunoglobulin A, and Immunoglobulin G Responses to the Vibrio cholerae O1 Protein Immunome in Adults With Cholera in Bangladesh.

PubMed

Charles, Richelle C; Nakajima, Rie; Liang, Li; Jasinskas, Al; Berger, Amanda; Leung, Daniel T; Kelly, Meagan; Xu, Peng; Kovác, Pavol; Giffen, Samantha R; Harbison, James D; Chowdhury, Fahima; Khan, Ashraful I; Calderwood, Stephen B; Bhuiyan, Taufiqur Rahman; Harris, Jason B; Felgner, Philip L; Qadri, Firdausi; Ryan, Edward T

2017-07-01

Cholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera. We probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay. Overall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase. This study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

The Xylella fastidosa RTX operons: evidence for the evolution of protein mosaics through novel genetic exchanges.

PubMed

Gambetta, Gregory A; Matthews, Mark A; Syvanen, Michael

2018-05-04

Xylella fastidiosa (Xf) is a gram negative bacterium inhabiting the plant vascular system. In most species this bacterium lives as a benign symbiote, but in several agriculturally important plants (e.g. coffee, citrus, grapevine) Xf is pathogenic. Xf has four loci encoding homologues to hemolysin RTX proteins, virulence factors involved in a wide range of plant pathogen interactions. We show that all four genes are expressed during pathogenesis in grapevine. The sequences from these four genes have a complex repetitive structure. At the C-termini, sequence diversity between strains is what would be expected from orthologous genes. However, within strains there is no N-terminal homology, indicating these loci encode RTXs of different functions and/or specificities. More striking is that many of the orthologous loci between strains share this extreme variation at the N-termini. Thus these RTX orthologues are most easily visualized as fusions between the orthologous C-termini and different N-termini. Further, the four genes are found in operons having a peculiar structure with an extensively duplicated module encoding a small protein with homology to the N-terminal region of the full length RTX. Surprisingly, some of these small peptides are most similar not to their corresponding full length RTX, but to the N-termini of RTXs from other Xf strains, and even other remotely related species. These results demonstrate that these genes are expressed in planta during pathogenesis. Their structure suggests extensive evolutionary restructuring through horizontal gene transfers and heterologous recombination mechanisms. The sum of the evidence suggests these repetitive modules are a novel kind of mobile genetic element.

Effect of solar irradiation on extracellular enzymes of Aeromonas proteolytica

NASA Technical Reports Server (NTRS)

Foster, B. G.

1973-01-01

The bacterium Aeromonas proteolytica was selected for studying the effects of solar irradiation on extracellular enzymes because it produces an endopeptidase that is capable of degrading proteins and a hemolysin that is active in lysing human erythrocytes. Possible alterations in the rate of enzyme production in response to the test conditions are currently underway and are not available for this preliminary report. Completed viability studies are indicative that little difference exists among the survival curves derived for cells exposed to various components of ultraviolet irradiation in space.

Ehrlichia chaffeensis Tandem Repeat Proteins and Ank200 are Type 1 Secretion System Substrates Related to the Repeats-in-Toxin Exoprotein Family

PubMed Central

Wakeel, Abdul; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; McBride, Jere W.

2011-01-01

Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host–pathogen interactions including DNA binding and a network of protein–protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host–pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria. PMID:22919588

Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

PubMed

Santamaría-Hernando, Saray; Krell, Tino; Ramos-González, María-Isabel

2012-01-01

Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca(2+) coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+) binding with a K(D) of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.

Virulence-associated characteristics of Escherichia coli in urinary tract infection: a statistical analysis with special attention to type 1C fimbriation.

PubMed

Siitonen, A; Martikainen, R; Ikäheimo, R; Palmgren, J; Mäkelä, P H

1993-07-01

The relative virulence (defined as odds ratio) associated with different O and K antigens, adhesins and hemolysin production of Escherichia coli strains was assessed by separate and multivariate logistic regression analyses comparing 383 strains isolated from urine of adults with a urinary tract infection with 287 fecal strains from healthy adults; special interest was paid to evaluating the role of type 1C fimbriation. Type 1C fimbriae, found on 14% of UTI and 7% of fecal strains, were associated with O groups O2, O6, O18, and O75, with capsular type K5, with mannose-resistant (both P and non-P) adhesins, and with hemolysin production. In separate analyses, O8 (odds ratio 5.9) and O75 (9.2), capsular types other than K1 (1.9-2.1), P (2.9) and non-P mannose-resistant (17.4) adhesins, and hemolysin production (3.1) were each associated with high relative virulence compared to O1, Rough, and K1 phenotypes or lack of mannose-resistant adhesins or hemolysin. All these virulence effects were independent of type 1C fimbriation. In multivariate analysis, joint variation between factors decreased the apparent virulence-promoting effect of type 1C fimbriae, O6 antigen and hemolysin but increased that of other adhesins. Especially high relative virulence (odds ratio 404.2) was associated with the combination of O75:K5:non-P mannose-resistant adhesin identified on seven UTI but no fecal strains.

Nanopore analysis of polymers in solution.

NASA Astrophysics Data System (ADS)

Deamer, David

2002-03-01

Nanopores represent a novel approach for investigating macromolecules in solution. Polymers that have been analyzed by this technique include polyethylene glycol (PEG), certain proteins and nucleic acids. The a-hemolysin pore inserted into lipid bilayers provides continuous non-gated ion current through a pore diameter of approximately 1.5 - 2 nm. Nucleic acid molecules can be driven through the pore by imposing a voltage across the supporting membrane. Single stranded, but not double stranded nucleic acids pass through in strict linear sequence from one end of the molecule to the other. While in the pore, the molecule reduces ionic current, and properties of the ionic current blockade such as duration, mean amplitude and modulations of amplitude provide information about structure and composition of the nucleic acid. For a given molecular species, the duration of the blockade is a function of chain length, and the rate of blockades is linearly related to concentration. More recent studies have shown that the a-hemolysin nanopore can discriminate between synthetic DNA molecules differing by a single base pair or even a single nucleotide. These results indicate that a nanopore may have the resolution required for nucleic acid sequencing applications.

Relatedness of Streptococcus suis Isolates of Various Serotypes and Clinical Backgrounds as Evaluated by Macrorestriction Analysis and Expression of Potential Virulence Traits

PubMed Central

Allgaier, Achim; Goethe, Ralph; Wisselink, Henk J.; Smith, Hilde E.; Valentin-Weigand, Peter

2001-01-01

We evaluated the genetic diversity of Streptococcus suis isolates of different serotypes by macrorestriction analysis and elucidated possible relationships between the genetic background, expression of potential virulence traits, and source of isolation. Virulence traits included expression of serotype-specific polysaccharides, muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin activity, and adherence to epithelial cells. Macrorestriction analysis of streptococcal DNA digested with restriction enzymes SmaI and ApaI allowed differentiation of single isolates that could be assigned to four major clusters, named A1, A2, B1, and B2. Comparison of the genotypic and phenotypic features of the isolates with their source of isolation showed that (i) the S. suis population examined, which originated mainly from German pigs, exhibited a genetic diversity and phenotypic patterns comparable to those found for isolates from other European countries; (ii) certain phenotypic features, such as the presence of capsular antigens of serotypes 2, 1, and 9, expression of MRP and EF, and hemolysin activity (and in particular, combinations of these features), were strongly associated with the clinical background of meningitis and septicemia; and (iii) isolates from pigs with meningitis and septicemia showed a significantly higher degree of genetic homogeneity compared to that for isolates from pigs with pneumonia and healthy pigs. Since the former isolates are considered highly virulent, this supports the theory of a clonal relationship among highly virulent strains. PMID:11158088

Improved Experimental Techniques for Analyzing Nucleic Acid Transport Through Protein Nanopores in Planar Lipid Bilayers

NASA Astrophysics Data System (ADS)

Costa, Justin A.

The translocation of nucleic acid polymers across cell membranes is a fundamental requirement for complex life and has greatly contributed to genomic molecular evolution. The diversity of pathways that have evolved to transport DNA and RNA across membranes include protein receptors, active and passive transporters, endocytic and pinocytic processes, and various types of nucleic acid conducting channels known as nanopores. We have developed a series of experimental techniques, collectively known as "Wicking", that greatly improves the biophysical analysis of nucleic acid transport through protein nanopores in planar lipid bilayers. We have verified the Wicking method using numerous types of classical ion channels including the well-studied chloride selective channel, CLIC1. We used the Wicking technique to reconstitute α-hemolysin and found that DNA translocation events of types A and B could be routinely observed using this method. Furthermore, measurable differences were observed in the duration of blockade events as DNA length and composition was varied, consistent with previous reports. Finally, we tested the ability of the Wicking technology to reconstitute the dsRNA transporter Sid-1. Exposure to dsRNAs of increasing length and complexity showed measurable differences in the current transitions suggesting that the charge carrier was dsRNA. However, the translocation events occurred so infrequently that a meaningful electrophysiological analysis was not possible. Alterations in the lipid composition of the bilayer had a minor effect on the frequency of translocation events but not to such a degree as to permit rigorous statistical analysis. We conclude that in many instances the Wicking method is a significant improvement to the lipid bilayer technique, but is not an optimal method for analyzing transport through Sid-1. Further refinements to the Wicking method might have future applications in high throughput DNA sequencing, DNA computation, and

Ion flux through membrane channels--an enhanced algorithm for the Poisson-Nernst-Planck model.

PubMed

Dyrka, Witold; Augousti, Andy T; Kotulska, Malgorzata

2008-09-01

A novel algorithmic scheme for numerical solution of the 3D Poisson-Nernst-Planck model is proposed. The algorithmic improvements are universal and independent of the detailed physical model. They include three major steps: an adjustable gradient-based step value, an adjustable relaxation coefficient, and an optimized segmentation of the modeled space. The enhanced algorithm significantly accelerates the speed of computation and reduces the computational demands. The theoretical model was tested on a regular artificial channel and validated on a real protein channel-alpha-hemolysin, proving its efficiency. (c) 2008 Wiley Periodicals, Inc.

ABSENCE OF LECITHIN FROM THE STROMATA OF THE RED CELLS OF CERTAIN ANIMALS (RUMINANTS), AND ITS RELATION TO VENOM HEMOLYSIS

PubMed Central

Turner, Joseph C.

1957-01-01

Lipide extracts of the red cells of several animal species have been analyzed chromatographically. Genetically determined differences in phospholipide composition were found. Lecithin is absent from the cells of ox, sheep, and goat. Cells containing lecithin are susceptible to the direct hemolysin of cobra venom while cells not containing lecithin are resistant. The facts indicate that the direct hemolysin is a lecithinase. PMID:13406178

Identification of the Staphylococcus aureus vfrAB operon, a novel virulence factor regulatory locus.

PubMed

Bose, Jeffrey L; Daly, Seth M; Hall, Pamela R; Bayles, Kenneth W

2014-05-01

During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis.

The role of genomic islands in Escherichia coli K1 interactions with intestinal and kidney epithelial cells.

PubMed

Yousuf, Farzana Abubakar; Rafiq, Sahar; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

2016-04-01

The completion of Escherichia coli K1 genome has identified several genomic islands that are present in meningitis-causing E. coli RS218 but absent in the non-pathogenic E. coli MG1655. In this study, the role of various genomic islands in E. coli K1 interactions with intestinal epithelial cells (Caco-2) and kidney epithelial cells (MA104) was determined. Using association assays, invasion assays, and intracellular survival assays, the findings revealed that the genomic island deletion mutants of RS218 related to P fimbriae, S fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, protein secretion system (T1SS for hemolysin; T2SS; T5SS for antigen 43), Iro system and hmu system), invasins (CNF1, IbeA), toxins (α-hemolysin), K1 capsule biosynthesis, metabolism (d-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism), prophage genes, showed reduced interactions with both cell types. Next, we determined the role of various genomic islands in E. coli K1 resistance to serum. When exposed to the normal human serum, the viability of the genomic island deletion mutants related to adhesins such as S fimbriae, P fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, antigen 43 and T5SS for antigen 43, T2SS, and T1SS for hemolysin, Iro system and hmu system, prophage genes, metabolism (sugar metabolism and d-serine catabolism), K1 capsule biosynthesis, and invasins such as CNF1 was affected, suggesting their role in bacteremia. The characterization of these genomic islands should reveal mechanisms of E. coli K1 pathogenicity that could be of value as therapeutic targets. Copyright © 2016 Elsevier Ltd. All rights reserved.

A porin-like protein from oral secretions of Spodoptera littoralis larvae induces defense-related early events in plant leaves.

PubMed

Guo, Huijuan; Wielsch, Natalie; Hafke, Jens B; Svatoš, Aleš; Mithöfer, Axel; Boland, Wilhelm

2013-09-01

Insect herbivory on plants is a complex incident consisting of at least two different aspects, namely mechanical damage and chemical challenge, as feeding insects introduce oral secretions (OS) into the wounded tissue of the attacked plant. Mechanical wounding alone is sufficient to induce a set of defense-related reactions in host plants, but some early events such as membrane potential (Vm) changes and cytosolic Ca²⁺-elevations can be triggered only by herbivores suggesting that OS-derived molecules are involved in those processes. Following an assay-guided purification based on planar lipid bilayer membrane technique in combination with proteomic analysis, a porin-like protein (PLP) of most likely bacterial origin was determined from collected OS of Spodoptera littoralis larvae. PLP exhibited channel-forming activity. Further, early defense-related events in plant-insect interaction were evaluated by using a purified fraction and α-hemolysin (α-HL) as a commercial pore-forming compound. Both up-regulated the calmodulin-like CML42 in Arabidopsis thaliana, which only responds to oral secretion and not to wounding. An elevation of in vivo [Ca²⁺](cyt) was not observed. Because membrane channel formation is a widespread phenomenon in plant-insect interactions, this PLP might represent an example for microbial compounds from the insect gut which are initially involved in plant-insect interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

PubMed

Jeon, Hyejin; Oh, Man Hwan; Jun, So Hyun; Kim, Seung Il; Choi, Chi Won; Kwon, Hyo Il; Na, Seok Hyeon; Kim, Yoo Jeong; Nicholas, Asiimwe; Selasi, Gati Noble; Lee, Je Chul

2016-04-01

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Staphylococcus aureus α-Toxin Induces Actin Filament Remodeling in Human Airway Epithelial Model Cells.

PubMed

Ziesemer, Sabine; Eiffler, Ina; Schönberg, Alfrun; Müller, Christian; Hochgräfe, Falko; Beule, Achim G; Hildebrandt, Jan-Peter

2018-04-01

Exposure of cultured human airway epithelial model cells (16HBE14o-, S9) to Staphylococcus aureus α-toxin (hemolysin A, Hla) induces changes in cell morphology and cell layer integrity that are due to the inability of the cells to maintain stable cell-cell or focal contacts and to properly organize their actin cytoskeletons. The aim of this study was to identify Hla-activated signaling pathways involved in regulating the phosphorylation level of the actin-depolymerizing factor cofilin. We used recombinant wild-type hemolysin A (rHla) and a variant of Hla (rHla-H35L) that is unable to form functional transmembrane pores to treat immortalized human airway epithelial cells (16HBE14o-, S9) as well as freshly isolated human nasal tissue. Our results indicate that rHla-mediated changes in cofilin phosphorylation require the formation of functional Hla pores in the host cell membrane. Formation of functional transmembrane pores induced hypophosphorylation of cofilin at Ser3, which was mediated by rHla-induced attenuation of p21-activated protein kinase and LIM kinase activities. Because dephosphorylation of pSer3-cofilin results in activation of this actin-depolymerizing factor, treatment of cells with rHla resulted in loss of actin stress fibers from the cells and destabilization of cell shape followed by the appearance of paracellular gaps in the cell layers. Activation of protein kinase A or activation of small GTPases (Rho, Rac, Cdc42) do not seem to be involved in this response.

The genome of Brucella melitensis.

PubMed

DelVecchio, Vito G; Kapatral, Vinayak; Elzer, Philip; Patra, Guy; Mujer, Cesar V

2002-12-20

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3,198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other alpha-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.

Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip.

PubMed

Diederichs, Tim; Nguyen, Quoc Hung; Urban, Michael; Tampé, Robert; Tornow, Marc

2018-06-13

Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10 -3 s -1 . Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution.

Hemolytic activity of Fusobacterium necrophorum culture supernatants due to presence of phospholipase A and lysophospholipase.

PubMed

Abe, P M; Kendall, C J; Stauffer, L R; Holland, J W

1979-01-01

Culture supernatants of Fusobacterium necrophorum demonstrated hemolytic activity. The hemolysin(s), which was partially purified by ammonium sulfate precipitation, was temperature-dependent and heat labile. The spectrum of hemolytic activity against various erythrocytes included rabbit, human, and dog erythrocytes. Goats, sheep, and bovine erythrocytes showed only trace hemolysis. According to results of thin-layer chromatography, the hemolysin hydrolyzed rabbit erythrocyte phosphatidyl choline, phosphatidyl ethanolamine, lysophosphatidyl choline, and bovine phosphatidyl choline. Hydrolysis of egg yolk phosphatidyl choline, bovine phosphatidyl ethanolamine, cholesterol, 1,2-dipalmitin, 1,3-dipalmitin, sphingomyelin, or triolein was not detected by thin layer chromatography. A more sensitive procedure utilizing gas-liquid chromatography revealed that, of the substrates tested, the following were bein hydrolyzed: bovine and egg yolk phosphatidyl choline, lysophosphatidyl choline, alpha-palmito-beta-eleoyl-L-alpha lecithin and alpha-oleoyl-betal-palmitoyl-L-alpha lecithin. Substrates which were weakly hydrolyzed were bovine phosphatidyl ethanolamine, DL-alpha-hosphatidyl ethanolamine dipalmitoyl, 1,2-dipalmitin, 1,3-dipalmitin, and triolein.

Pathogenicity of Vibrio parahaemolyticus in Different Food Matrices.

PubMed

Wang, Rundong; Sun, Lijun; Wang, Yaling; Deng, Yijia; Liu, Ying; Xu, Defeng; Liu, Huanming; Ye, Riying; Gooneratne, Ravi

2016-02-01

The pathogenicity and virulence factors of Vibrio parahaemolyticus in four food matrices--shrimp, freshwater fish, pork, and egg-fried rice--were compared by measuring the thermostable direct hemolysin activity and total hemolytic titer. Significantly high thermostable direct hemolysin and also hemolytic titers (P < 0.05) were produced by V. parahaemolyticus in egg-fried rice > shrimp > freshwater fish > pork. Filtrates of V. parahaemolyticus in shrimp given intraperitoneally induced marked liver and kidney damage and were highly lethal to adult mice compared with filtrates of V. parahaemolyticus in freshwater fish > egg-fried rice > pork. From in vitro and in vivo pathogenicity tests, it seems the type of food matrix has a significant impact on the virulence of V. parahaemolyticus. These results suggest that hemolysin may not necessarily be the only virulence factor for pathogenicity of V. parahaemolyticus. This is the first report that shows that virulence factors produced by V. parahaemolyticus in seafood such as shrimp are more toxic in vivo than in nonseafood.

Membrane Fusion Proteins of Type I Secretion System and Tripartite Efflux Pumps Share a Binding Motif for TolC in Gram-Negative Bacteria

PubMed Central

Yoon, Bo-Young; Song, Saemee; Lee, Kangseok; Ha, Nam-Chul

2012-01-01

The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA). In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette)-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed. PMID:22792337

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana, Maryland, Mississippi, and Washington (United States)

PubMed Central

Bowers, John C.; Griffitt, Kimberly J.; Molina, Vanessa; Clostio, Rachel W.; Pei, Shaofeng; Laws, Edward; Paranjpye, Rohinee N.; Strom, Mark S.; Chen, Arlene; Hasan, Nur A.; Huq, Anwar; Noriea, Nicholas F.; Grimes, D. Jay; Colwell, Rita R.

2012-01-01

Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST. PMID:22865080

The type II secretion system is essential for erythrocyte lysis and gut colonization by the leech digestive tract symbiont Aeromonas veronii.

PubMed

Maltz, Michele; Graf, Joerg

2011-01-01

Hemolysin and the type II secretion system (T2SS) have been shown to be important for virulence in many pathogens, but very few studies have shown their importance in beneficial microbes. Here, we investigated the importance of the type II secretion pathway in the beneficial digestive-tract association of Aeromonas veronii and the medicinal leech Hirudo verbana and revealed a critical role for the hemolysis of erythrocytes. A mutant with a miniTn5 insertion in exeM, which is involved in forming the inner membrane platform in the T2SS, was isolated by screening mutants for loss of hemolysis on blood agar plates. A hemolysis assay was used to quantify the mutant's deficiency in lysing sheep erythrocytes and revealed a 99.9% decrease compared to the parent strain. The importance of the T2SS in the colonization of the symbiotic host was assessed. Colonization assays revealed that the T2SS is critical for initial colonization of the leech gut. The defect was tied to the loss of hemolysin production by performing a colonization assay with blood containing lysed erythrocytes. This restored the colonization defect in the mutant. Complementation of the mutant using the promoter region and exeMN revealed that the T2SS is responsible for secreting hemolysin into the extracellular space and that both the T2SS and hemolysin export by the T2SS are critical for initial establishment of A. veronii in the leech gut.

Bifactorial versus monofactorial molecular status of Staphylococcus aureus gamma-toxin.

PubMed

Guidi-Rontani, C; Fouque, F; Alouf, J E

1994-01-01

The extracellular Staphylococcus aureus gamma-toxin (hemolysin) released by the Smith 5R strain has been purified (M(r) 38 kDa, pl 9.55). We established that this cytolysin is a single polypeptide fully lytic on rabbit erythrocytes. In contrast, this toxin alone was unable to lyse other cells and was required to act jointly with an accessory 58 kDa protein released by the same strain. This protein, named sensitizing protein (SP), was required in order to damage the cytoplasmic membranes of other red blood cells including human erythrocytes as well as that of other eukaryotic cells (Jurkat and Hep-2). The lytic process can be referred to as conditional synergistic or cooperative lysis. gamma-toxin, and SP were also found able to disrupt phospholipid/cholesterol containing liposomes. We demonstrated that a minor membrane phospholipid, phosphatidylinositol, is crucial for gamma-toxin binding to cells and/or channel formation through membrane lipid bilayer.

Biomarkers of Aspergillus spores

NASA Astrophysics Data System (ADS)

Sulc, Miroslav; Peslova, Katerina; Zabka, Martin; Hajduch, Marian; Havlicek, Vladimir

2009-02-01

We applied both matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric and 1D sodium dodecylsulfate polyacrylamide gel electrophoretic (1D-PAGE) approaches for direct analysis of intact fungal spores of twenty four Aspergillus species. In parallel, we optimized various protocols for protein extraction from Aspergillus spores using acidic conditions, step organic gradient and variable sonication treatment. The MALDI-TOF mass spectra obtained from optimally prepared samples provided a reproducible fingerprint demonstrating the capability of the MALDI-TOF approach to type and characterize different fungal strains within the Aspergillus genus. Mass spectra of intact fungal spores provided signals mostly below 20 kDa. The minimum material amount represented 0.3 [mu]g (10,000 spores). Proteins with higher molecular weight were detected by 1D-PAGEE Eleven proteins were identified from three selected strains in the range 5-25 kDa by the proteomic approach. Hemolysin and hydrophobin have the highest relevance in host-pathogen interactions.

Insights from the draft genome into the pathogenicity of a clinical isolate of Elizabethkingia meningoseptica Em3.

PubMed

Chen, Shicheng; Soehnlen, Marty; Downes, Frances P; Walker, Edward D

2017-01-01

Elizabethkingia meningoseptica is an emerging, healthcare-associated pathogen causing a high mortality rate in immunocompromised patients. We report the draft genome sequence of E. meningoseptica Em3, isolated from sputum from a patient with multiple underlying diseases. The genome has a length of 4,037,922 bp, a GC-content 36.4%, and 3673 predicted protein-coding sequences. Average nucleotide identity analysis (>95%) assigned the bacterium to the species E. meningoseptica. Genome analysis showed presence of the curli formation and assembly operon and a gene encoding hemagglutinins, indicating ability to form biofilm. In vitro biofilm assays demonstrated that E. meningoseptica Em3 formed more biofilm than E. anophelis Ag1 and E. miricola Emi3, both lacking the curli operon. A gene encoding thiol-activated cholesterol-dependent cytolysin in E. meningoseptica Em3 (potentially involved in lysing host immune cells) was also absent in E. anophelis Ag1 and E. miricola Emi3. Strain Em3 showed α-hemolysin activity on blood agar medium, congruent with presence of hemolysin and cytolysin genes. Furthermore, presence of heme uptake and utilization genes demonstrated adaptations for bloodstream infections. Strain Em3 contained 12 genes conferring resistance to β-lactams, including β-lactamases class A, class B, and metallo-β-lactamases. Results of comparative genomic analysis here provide insights into the evolution of E. meningoseptica Em3 as a pathogen.

A heating-superfusion platform technology for the investigation of protein function in single cells.

PubMed

Xu, Shijun; Ainla, Alar; Jardemark, Kent; Jesorka, Aldo; Jeffries, Gavin D M

2015-01-06

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans.

PubMed

Narayanavari, Suneel A; Lourdault, Kristel; Sritharan, Manjula; Haake, David A; Matsunaga, James

2015-01-01

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity

Conversion of commensal Escherichia coli K-12 to an invasive form via expression of a mutant histone-like protein.

PubMed

Koli, Preeti; Sudan, Sudhanshu; Fitzgerald, David; Adhya, Sankar; Kar, Sudeshna

2011-01-01

The HUα(E38K, V42L) mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397-16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUα(E38K, V42L) was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins Bim(EL) and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUα(E38K, V42L) variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes. Escherichia coli K-12 is well established as a benign laboratory strain and a human intestinal commensal. Recent evidences, however, indicate that the typical noninvasive nature of resident E. coli can be reversed under specific circumstances even in the absence of any major genomic flux. We previously engineered an E. coli strain with a mutant histone-like protein, HU, which exhibited significant changes in nucleoid organization and global transcription. Here we showed that the changes induced by the mutant HU have critical functional consequences: from a strict extracellular existence, the mutant E. coli adopts an almost obligate intracellular lifestyle. The internalized E. coli exhibits many of the prototypical characteristics of traditional intracellular bacteria, like phagosomal

TTSS2-deficient hha mutant of Salmonella Typhimurium exhibits significant systemic attenuation in immunocompromised hosts

PubMed Central

Vishwakarma, Vikalp; Pati, Niladri Bhusan; Ray, Shilpa; Das, Susmita; Suar, Mrutyunjay

2014-01-01

Non-typhoidal Salmonella (NTS) infections are emerging as leading problem worldwide and the variations in host immune status append to the concern of NTS. Salmonella enterica serovar Typhimurium is one of the causative agents of NTS infections and has been extensively studied. The inactivation of Salmonella pathogenicity island 2 (SPI2) encoded type-III secretion system 2 (TTSS2) has been reported rendering the strain incapable for systemic dissemination to host sites and has also been proposed as live-attenuated vaccine. However, infections from TTSS2-deficient Salmonella have also been reported. In this study, mutant strain MT15 was developed by inactivation of the hemolysin expression modulating protein (hha) in TTSS2-deficient S. Typhimurium background. The MT15 strain showed significant level of attenuation in immune-deprived murine colitis model when tested in iNos−/−, IL10−/−, and CD40L−/− mice groups in C57BL/6 background. Further, the mutation in hha does not implicate any defect in bacterial colonization to the host gut. The long-term infection of developed mutant strain conferred protective immune responses to suitably immunized streptomycin pre-treated C57BL/6 mice. The immunization enhanced the CD4+ and CD8+ cell types involved in bacterial clearance. The serum IgG and luminal secretory IgA (sIgA) was also found to be elevated after the due course of infection. Additionally, the immunized C57BL/6 mice were protected from the subsequent lethal infection of Salmonella Typhimurium. Collectively, these findings implicate the involvement of hemolysin expression modulating protein (Hha) in establishment of bacterial infection. In light of the observed attenuation of the developed mutant strain, this study proposes the possible significance of SPI2-deficient hha mutant as an alternative live-attenuated vaccine strain for use against lethal Salmonella infections. PMID:24401482

Nanoscale Bio-engineering Solutions for Space Exploration: The Nanopore Sequencer

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Cozmuta, Ioana

2004-01-01

Characterization of biological systems at the molecular level and extraction of essential information for nano-engineering design to guide the nano-fabrication of solid-state sensors and molecular identification devices is a computational challenge. The alpha hemolysin protein ion channel is used as a model system for structural analysis of nucleic acids like DNA. Applied voltage draws a DNA strand and surrounding ionic solution through the biological nanopore. The subunits in the DNA strand block ion flow by differing amounts. Atomistic scale simulations are employed using NASA supercomputers to study DNA translocation, with the aim to enhance single DNA subunit identification. Compared to protein channels, solid-state nanopores offer a better temporal control of the translocation of DNA and the possibility to easily tune its chemistry to increase the signal resolution. Potential applications for NASA missions, besides real-time genome sequencing include astronaut health, life detection and decoding of various genomes.

Analyte-Triggered DNA-Probe Release from a Triplex Molecular Beacon for Nanopore Sensing.

PubMed

Guo, Bingyuan; Sheng, Yingying; Zhou, Ke; Liu, Quansheng; Liu, Lei; Wu, Hai-Chen

2018-03-26

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex-forming molecular beacon and a stem-forming DNA component that is modified with a host-guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α-hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point-of-care diagnostics with a portable nanopore kit in the future. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Profiling of Virulence Determinants in Cronobacter sakazakii Isolates from Different Plant and Environmental Commodities.

PubMed

Singh, Niharika; Raghav, Mamta; Narula, Shifa; Tandon, Simran; Goel, Gunjan

2017-05-01

Cronobacter sakazakii is an emerging pathogen causing meningitis, sepsis and necrotizing enterocolitis in neonates and immune-compromised adults. The present study describes the profiling of different virulence factors associated with C. sakazakii isolates derived from plant-based materials and environmental samples (soil, water, and vacuum dust). All the isolates exhibited β-hemolysis and chitinase activity, and were able to utilize inositol. Among the nine virulence-associated genes, hly gene coding for hemolysin was detected in all the isolates followed by ompA (outer membrane protein); however, plasmid-borne genes were detected at a level of 60% for both cpa (cronobacter plasminogen activator) and eitA (Ferric ion transporter protein) gene, respectively. Furthermore, the isolate C. sakazakii N81 showed cytotoxicity for Caco-2 cells. The presence of the virulence determinants investigated in this study indicates the pathogenic potential of C. sakazakii with their plausible connection with clinical manifestations.

Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

PubMed

Fatahinia, M; Halvaeezadeh, M; Rezaei-Matehkolaei, A

2017-06-01

Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-β, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-β=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and β among all studied isolates were almost similar. In the present study, the prevalence

Relationships between environmental factors and pathogenic Vibrios in the Northern Gulf of Mexico.

PubMed

Johnson, C N; Flowers, A R; Noriea, N F; Zimmerman, A M; Bowers, J C; DePaola, A; Grimes, D J

2010-11-01

Although autochthonous vibrio densities are known to be influenced by water temperature and salinity, little is understood about other environmental factors associated with their abundance and distribution. Densities of culturable Vibrio vulnificus containing vvh (V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh (thermostable direct hemolysin gene, V. parahaemolyticus pathogenicity factor), and trh (tdh-related hemolysin gene, V. parahaemolyticus pathogenicity factor) were measured in coastal waters of Mississippi and Alabama. Over a 19-month sampling period, vibrio densities in water, oysters, and sediment varied significantly with sea surface temperature (SST). On average, tdh-to-tlh ratios were significantly higher than trh-to-tlh ratios in water and oysters but not in sediment. Although tlh densities were lower than vvh densities in water and in oysters, the opposite was true in sediment. Regression analysis indicated that SST had a significant association with vvh and tlh densities in water and oysters, while salinity was significantly related to vibrio densities in the water column. Chlorophyll a levels in the water were correlated significantly with vvh in sediment and oysters and with pathogenic V. parahaemolyticus (tdh and trh) in the water column. Furthermore, turbidity was a significant predictor of V. parahaemolyticus density in all sample types (water, oyster, and sediment), and its role in predicting the risk of V. parahaemolyticus illness may be more important than previously realized. This study identified (i) culturable vibrios in winter sediment samples, (ii) niche-based differences in the abundance of vibrios, and (iii) predictive signatures resulting from correlations between environmental parameters and vibrio densities.

Relationships between Environmental Factors and Pathogenic Vibrios in the Northern Gulf of Mexico ▿ †

PubMed Central

Johnson, C. N.; Flowers, A. R.; Noriea, N. F.; Zimmerman, A. M.; Bowers, J. C.; DePaola, A.; Grimes, D. J.

2010-01-01

Although autochthonous vibrio densities are known to be influenced by water temperature and salinity, little is understood about other environmental factors associated with their abundance and distribution. Densities of culturable Vibrio vulnificus containing vvh (V. vulnificus hemolysin gene) and V. parahaemolyticus containing tlh (thermolabile hemolysin gene, ubiquitous in V. parahaemolyticus), tdh (thermostable direct hemolysin gene, V. parahaemolyticus pathogenicity factor), and trh (tdh-related hemolysin gene, V. parahaemolyticus pathogenicity factor) were measured in coastal waters of Mississippi and Alabama. Over a 19-month sampling period, vibrio densities in water, oysters, and sediment varied significantly with sea surface temperature (SST). On average, tdh-to-tlh ratios were significantly higher than trh-to-tlh ratios in water and oysters but not in sediment. Although tlh densities were lower than vvh densities in water and in oysters, the opposite was true in sediment. Regression analysis indicated that SST had a significant association with vvh and tlh densities in water and oysters, while salinity was significantly related to vibrio densities in the water column. Chlorophyll a levels in the water were correlated significantly with vvh in sediment and oysters and with pathogenic V. parahaemolyticus (tdh and trh) in the water column. Furthermore, turbidity was a significant predictor of V. parahaemolyticus density in all sample types (water, oyster, and sediment), and its role in predicting the risk of V. parahaemolyticus illness may be more important than previously realized. This study identified (i) culturable vibrios in winter sediment samples, (ii) niche-based differences in the abundance of vibrios, and (iii) predictive signatures resulting from correlations between environmental parameters and vibrio densities. PMID:20817802

Genotypic intraspecies heterogeneity of Enterococcus italicus: data from dairy environments.

PubMed

Borgo, Francesca; Ferrario, Chiara; Ricci, Giovanni; Fortina, Maria Grazia

2013-01-01

The diversity of a collection of 19 Enterococcus italicus strains isolated from different dairy sources was explored using a molecular polyphasic approach, comprising random amplification of polymorphic DNA (RAPD-PCR), repetitive element PCR (REP-PCR), plasmid profiling and ribotyping. The data obtained showed a high-level of biodiversity, not always correlated to the niche of isolation. Particularly, REP-PCR with primer BOXA1R and plasmid profiling allowed the best discrimination at strain level. Exploiting the genome shotgun sequence of the type strain of the species, available in public database, genes related to insertion sequences present on enterococcal Pathogenic Islands (ISEf1, IS905), determinants related to virulence factors (codifying for hemolysin and cell wall surface proteins), exogenously DNA (conjugal transfer protein, replication plasmid protein, pheromone shutdown protein, phage integrase/recombinase) and penicillin binding proteins system were detected. The presence of most of these genes seemed a common genetic trait in the Enterococcus genus, sur gene (cell wall surface protein) was only detected in strains of E. italicus. To our knowledge, this is the first time that specific primers, with the expection of the species-specific probe targeted to 16S rRNA gene, have been designed for this species. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Resveratrol Improved the Progression of Chronic Prostatitis via the Downregulation of c-kit/SCF by Activating Sirt1.

PubMed

He, Yi; Zeng, Huizhi; Yu, Yang; Zhang, Jiashu; Zeng, Xiaona; Gong, Fengtao; Duan, Xingping; Liu, Qi; Yang, Bo

2017-07-19

The regulation mechanism of inflammation inducing prostate carcinogenesis remains largely unknown. Therefore, we investigated the role of the c-kit/SCF pathway, which has been associated with the control of prostate carcinogenesis, in chronic prostatitis (CP) rats and evaluated the anti-prostatitis effect of resveratrol. We performed hemolysin and eosin staining to evaluate the histopathological changes in prostates. Multiple approaches evaluated the expression levels of c-kit, stem cell factor (SCF), Sirt1, and carcinogenesis-associated proteins. The CP group exhibited severe diffuse chronic inflammation. Meanwhile, the prostate cells appeared atypia; the activity of c-kit/SCF was upregulated, and carcinogenesis-associated proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. In summary, CP could further cause prostate carcinogenesis, which may be associated with activated c-kit/SCF signaling. Resveratrol treatment could improve the progression of CP via the downregulation of c-kit/SCF by activating Sirt1.

Nanoscale Bioengineering Solutions for Space Exploration the Nanopore Sequencer

NASA Technical Reports Server (NTRS)

Ioana, Cozmuta; Viktor, Stoic

2005-01-01

Characterization of biological systems at the molecular level and extraction of essential information for nano-engineering design to guide the nano-fabrication of solid-state sensors and molecular identification devices is a computational challenge. The alpha hemolysin protein ion channel is used as a model system for structural analysis of nucleic acids like DNA. Applied voltage draws a DNA strand and surrounding ionic solution through the biological nanopore. The subunits in the DNA strand block ion flow by differing amounts. Atomistic scale simulations are employed using NASA supercomputers to study DNA translocation. with the aim to enhance single DNA subunit identification. Compared to protein channels, solid-state nanopores offer a better temporal control of the translocation of DNA and the possibility to easily tune its chemistry to increase the signal resolution. Potential applications for NASA missions, besides real-time genome sequencing include astronaut health, life detection and decoding of various genomes. http://phenomrph.arc.nasa.gov/index.php

Identification and Characterization of Staphylococcus aureus Strains with an Incomplete Hemolytic Phenotype.

PubMed

Zhang, Haifang; Zheng, Yi; Gao, Huasheng; Xu, Ping; Wang, Min; Li, Aiqing; Miao, Minhui; Xie, Xiaofang; Deng, Yimai; Zhou, Huiqin; Du, Hong

2016-01-01

Staphylococcus aureus is a common pathogen causing both hospital and community-acquired infections. Hemolysin is one of the important virulence factors for S. aureus and causes the typical β-hemolytic phenotype which is called complete hemolytic phenotype as well. Recently, S. aureus with an incomplete hemolytic phenotype (SIHP) was isolated from clinical samples. To study the microbiologic characteristics of SIHP, the special hemolytic phenotype of SIHP was verified on the sheep blood agar plates supplied by different manufacturers. Expression of hemolysin genes hla, hlb, hlgC , and hld of SIHP was detected by qRT-PCR and it was showed that expression of hlb in SIHP was obviously increased compared to the control S. aureus strains with complete hemolytic phenotype (SCHP), while the expression of hla, hlgC , and hld in SIHP was significantly decreased. In addition, the α-hemolysin encoded by gene hla was decreased obviously in SIHP compared to SCHP by western blot. All 60 SIHP strains were identified to be the methicillin resistant S. aureus (MRSA), and moreover these SIHP strains all contains mecA gene. The virulence gene tst were all present in SIHP, and the intracellular survival ability of SIHP was much greater than that of the gene tst negative S. aureus . We also found that IL-2, IL-6, and IL-17A secreted in the supernatant of SIHP infected macrophages increased significantly compared to tst negative control strains infected ones. MLST analysis showed that all of SIHP strains were classified into ST5 clone. To our knowledge, this study firstly showed that SIHP strains are a kind of methicillin resistant strains which express β-hemolysin highly and possess a potential high virulence, and it was suggested that SIHP should be paid more attention in hospital.

Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

NASA Technical Reports Server (NTRS)

Vercoutere, Wenonah; DeGuzman, Veronica

2003-01-01

The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

Eugenol reduces the expression of virulence-related exoproteins in Staphylococcus aureus.

PubMed

Qiu, Jiazhang; Feng, Haihua; Lu, Jing; Xiang, Hua; Wang, Dacheng; Dong, Jing; Wang, Jianfeng; Wang, Xiaoliang; Liu, Juxiong; Deng, Xuming

2010-09-01

Eugenol, an essential oil component in plants, has been demonstrated to possess activity against both gram-positive and gram-negative bacteria. This study examined the influence that subinhibitory concentrations of eugenol may have on the expression of the major exotoxins produced by Staphylococcus aureus. The results from a tumor necrosis factor (TNF) release assay and a hemolysin assay indicated that S. aureus cultured with graded subinhibitory concentrations of eugenol (16 to 128 microg/ml) dose dependently decreased the TNF-inducing and hemolytic activities of culture supernatants. Western blot analysis showed that eugenol significantly reduced the production of staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1 (the key exotoxins to induce TNF release), as well as the expression of alpha-hemolysin (the major hemolysin to cause hemolysis). In addition, this suppression was also evaluated at the transcriptional level via real-time reverse transcription (RT)-PCR analysis. The transcriptional analysis indicated that 128 microg/ml of eugenol remarkably repressed the transcription of the S. aureus sea, seb, tst, and hla genes. According to these results, eugenol has the potential to be rationally applied on food products as a novel food antimicrobial agent both to inhibit the growth of bacteria and to suppress the production of exotoxins by S. aureus.

Detection of tlh and tdh genes in Vibrio Parahaemolyticus inhabiting farmed water ecosystem used for L. Vannamei aquaculture

NASA Astrophysics Data System (ADS)

Nawan Hasrimi, Adila; Budiharjo, Anto; Nur Jannah, Siti

2018-05-01

Vibrio parahaemolyticus is hallophilic gram-negative bacteria that live as natural inhabitant in aquatic environment. All Vibrio parahaemolyticus strain known to have thermolabile hemolysin encoded by tlh gene as species marker. Thermostable direct hemolysin encoded by tdh gene is responsible for regulating virulence factor in Vibrio parhaemolyticus. Aim of this research is to detect tlh and tdh gene from water of L. vannamei aquaculture in Rembang regency. Colonies of green-blueish bacteria grew from isolation of L. vannamei aquaculture water in CD-VP media which was identified as Vibrio parahaemolyticus. Colonies of V. parahaemolyticus grew to be small and green-blueish bacteria colonies in TCBS agar. Result of molecular analysis showed that bacteria isolated from water sample are specifically identified as Vibrio parahaemolyticus bacteria by the detection of tlh gene. Vibrio parahaemolyticus isolated from water of L. vannamei aquaculture detected as tdh negative that indicates tdh gene is not present in isolated bacteria. Vibrio parahaemolyticus isolate were cultured in Wagatsuma agar for tdh gene confirmation test that showed Kanagawa negative result, which indicated that V. parahaemolyticus did not produce thermostable direct hemolysin. These results showed that Vibrio parahaemolyticus isolated from aquatic environment of L. vannamei aquaculture in Rembang regency did not show virulence factors.

Evaluation of CAMP-Like Effect, Biofilm Formation, and Discrimination of Candida africana from Vaginal Candida albicans Species

PubMed Central

Bordbar, Mahboubeh; Nouraei, Hasti; Khodadadi, Hossein

2017-01-01

Candida africana as a species recovered from female genital specimens is highly close to C. albicans. The present study was conducted to discriminate C. africana from presumptive vaginal C. albicans strains by molecular assay and evaluate their hemolysin activity, biofilm formation, and cohemolytic effect (CAMP) with vaginal bacterial flora. A total of 110 stock vaginal C. albicans isolates were examined by HWP1 gene amplification. Hemolysin activity and the ability of biofilm formation were evaluated by blood plate assay and visual detection methods, respectively. Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus agalactiae were used to evaluate the CAMP-like effects in Sabouraud blood agar media. Based on the size of the amplicons (941 bp), all isolates were identified as C. albicans. All samples were able to produce beta-hemolysin. Moreover, 69 out of 110 of the isolates (62.7%) were biofilm-positive, 54 out of 110 Candida isolates (49%) demonstrated cohemolytic effects with S. agalactiae, and 48 out of 110 showed this effect with S. aureus (43.6%). All isolates were CAMP-negative with S. epidermidis. We detected all isolates as Candida albicans and almost half of the isolates were CAMP-positive with S. aureus and S. agalactiae, suggesting that these bacteria increase the pathogenicity of Candida in vaginal candidiasis. PMID:29318048

Virulence characteristics of Escherichia coli in nosocomial urinary tract infection.

PubMed

Ikäheimo, R; Siitonen, A; Kärkkäinen, U; Mäkelä, P H

1993-06-01

We examined 148 strains of Escherichia coli isolated from the urine from patients with nosocomial urinary tract infection (UTI). The prevalence of P fimbriation was only 11.5%. Of the strains, 17.6% expressed non-P M(R) adhesins (defined as strains expressing mannose-resistant but not P-specific hemagglutination); 33.1% produced hemolysin, and 15.2% expressed type 1C fimbriae. O6 was the most common group of O antigens (12.2%), closely followed by O75 (9.5%); both of these groups are relatively uncommon (4.5% and 1%, respectively) in fecal strains isolated from healthy adults. Of the strains with O6 and O75 antigens, 78.8% and 79% produced hemolysin, but of all other strains causing UTI, only 21% produced hemolysin. Of the strains with O6 antigens, 61% expressed non-P M(R) adhesins, but only 12% of all other strains causing UTI expressed non-P M(R) adhesins. There were no significant differences in the prevalence of virulence properties between strains isolated from patients with or without an underlying medical illness or between strains causing different clinical categories of UTI. We conclude that the prevalence of bacterial virulence factors is low among patients with nosocomial UTI.

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shrimps in Malaysia

PubMed Central

Letchumanan, Vengadesh; Yin, Wai-Fong; Lee, Learn-Han; Chan, Kok-Gan

2015-01-01

Vibrio parahaemolyticus is a marine and estuarine bacterium that has been the leading cause of foodborne outbreaks which leads to a significant threat to human health worldwide. Consumption of seafood contaminated with V. parahaemolyticus causes acute gastroenteritis in individuals. The bacterium poses two main virulence factor including the thermostable direct hemolysin (tdh) which is a pore-forming protein that contributes to the invasiveness of the bacterium in humans and TDH-related hemolysin (trh), which plays a similar role as tdh in the disease pathogenesis. This study aimed to investigate the antimicrobial resistance V. parahaemolyticus strains in shrimps purchased from wetmarkets and supermarkets. The toxR-based PCR assay indicated that a total of 57.8% (185/320) isolates were positive for V. parahaemolyticus. Only 10% (19/185) toxR-positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The MAR index was measured for 14 common antimicrobial agents. The results indicated 98% of the isolates were highly susceptible to imipenem, ampicillin sulbactam (96%), chloramphenicol (95%), trimethoprim-sulfamethoxazole (93%), gentamicin (85%), levofloxacin (83%), and tetracycline (82%). The chloramphenicol (catA2) and kanamycin (aphA-3) resistance genes were detected in the resistant V. parahaemolyticus isolates. Our results demonstrate that shrimps are contaminated with V. parahaemolyticus, some of which carry the trh-gene thus being potential to cause food borne illness. The occurrence of multidrug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields. PMID:25688239

Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

PubMed Central

Huyet, Jessica; Naylor, Claire E.; Savva, Christos G.; Gibert, Maryse; Popoff, Michel R.; Basak, Ajit K.

2013-01-01

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins. PMID:23805259

Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors

PubMed Central

Ceccarelli, Daniela; Hasan, Nur A.; Huq, Anwar; Colwell, Rita R.

2013-01-01

Vibrio parahaemolyticus, autochthonous to estuarine, marine, and coastal environments throughout the world, is the causative agent of food-borne gastroenteritis. More than 80 serotypes have been described worldwide, based on antigenic properties of the somatic (O) and capsular (K) antigens. Serovar O3:K6 emerged in India in 1996 and subsequently was isolated worldwide, leading to the conclusion that the first V. parahaemolyticus pandemic had taken place. Most strains of V. parahaemolyticus isolated from the environment or seafood, in contrast to clinical strains, do not produce a thermostable direct hemolysin (TDH) and/or a TDH-related hemolysin (TRH). Type 3 secretion systems (T3SSs), needle-like apparatuses able to deliver bacterial effectors into host cytoplasm, were identified as triggering cytotoxicity and enterotoxicity. Type 6 secretion systems (T6SS) predicted to be involved in intracellular trafficking and vesicular transport appear to play a role in V. parahaemolyticus virulence. Recent advances in V. parahaemolyticus genomics identified several pathogenicity islands (VpaIs) located on either chromosome in both epidemic and pandemic strains and comprising additional colonization factors, such as restriction-modification complexes, chemotaxis proteins, classical bacterial surface virulence factors, and putative colicins. Furthermore, studies indicate strains lacking toxins and genomic regions associated with pathogenicity may also be pathogenic, suggesting other important virulence factors remain to be identified. The unique repertoire of virulence factors identified to date, their occurrence and distribution in both epidemic and pandemic strains worldwide are described, with the aim of highlighting the complexity of V. parahaemolyticus pathogenicity as well as its dynamic genome. PMID:24377090

Genomic analysis of two emergent Vibrio parahaemolyticus ecotypes

NASA Astrophysics Data System (ADS)

Moreno, E.; Parks, M. C.; Pinnell, L. J.; Turner, J.

2016-02-01

Vibrio parahaemolyticus [Vp] is a Gram-negative bacterium indigenous to marine coastal waters. Vp is also the causative agent of a mild to severe gastroenteritis associated with the consumption of raw or undercooked seafood. The majority of infections are caused by a genetically distinct ecotype commonly referred to as the pandemic clonal complex. However, localized outbreaks associated with non-pandemic ecotypes are frequently reported. In the East Pacific, two such ecotypes, identified as ST65 and ST417 by multilocus sequence typing, have been associated with outbreaks in Peru, Chile and the United States. In this study, we sequenced and assembled draft genomes from 4 clinical isolates (ST65: 3328, 3355; ST417: 3646, 3631) that were positive for both the thermostable direct hemolysin (tdh) and thermostable direct-related hemolysin (trh). When compared with the pandemic type strain (V. parahaemolyticus RIMD2210633), each of these isolates harbored more than 400 Kb of novel genetic material. Proteins encoded by this novel genetic material include CcdA-CcdB toxin-antitoxin systems, an efflux pump belonging to the multidrug and toxic efflux (MATE) family, and a repeats-in-toxin (RTX) gene cluster. These features share significant homology and synteny with virulence-associated features found in clinical V. vulnificus and Escherichia coli strains. We hypothesize that these features contribute to a pathogenic phenotype. The identification and characterization of multiple clinical ecotypes could improve efforts aimed at preventing V. parahaemolyticus infections. Further, a greater understanding of the species' biogeography may lead to a more effective public health response.

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shrimps in Malaysia.

PubMed

Letchumanan, Vengadesh; Yin, Wai-Fong; Lee, Learn-Han; Chan, Kok-Gan

2015-01-01

Vibrio parahaemolyticus is a marine and estuarine bacterium that has been the leading cause of foodborne outbreaks which leads to a significant threat to human health worldwide. Consumption of seafood contaminated with V. parahaemolyticus causes acute gastroenteritis in individuals. The bacterium poses two main virulence factor including the thermostable direct hemolysin (tdh) which is a pore-forming protein that contributes to the invasiveness of the bacterium in humans and TDH-related hemolysin (trh), which plays a similar role as tdh in the disease pathogenesis. This study aimed to investigate the antimicrobial resistance V. parahaemolyticus strains in shrimps purchased from wetmarkets and supermarkets. The toxR-based PCR assay indicated that a total of 57.8% (185/320) isolates were positive for V. parahaemolyticus. Only 10% (19/185) toxR-positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The MAR index was measured for 14 common antimicrobial agents. The results indicated 98% of the isolates were highly susceptible to imipenem, ampicillin sulbactam (96%), chloramphenicol (95%), trimethoprim-sulfamethoxazole (93%), gentamicin (85%), levofloxacin (83%), and tetracycline (82%). The chloramphenicol (catA2) and kanamycin (aphA-3) resistance genes were detected in the resistant V. parahaemolyticus isolates. Our results demonstrate that shrimps are contaminated with V. parahaemolyticus, some of which carry the trh-gene thus being potential to cause food borne illness. The occurrence of multidrug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields.

Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

PubMed

Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

2018-05-01

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

PubMed

Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

2002-11-01

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

Ham test

MedlinePlus

Acid hemolysin test; Paroxysmal nocturnal hemoglobinuria - Ham test; PNH - Ham test ... BJ. In: Chernecky CC, Berger BJ, eds. Laboratory Tests and Diagnostic Procedures . 6th ed. Philadelphia, PA: Elsevier ...

Entérite nécrotique chez le poulet de gril II. Caractères des souches de Clostridium perfringens isolées

PubMed Central

Bernier, G.; Filion, R.; Malo, R.; Phaneuf, J.-B.

1974-01-01

A Gram positive bacillus, strictly anaerobic, was isolated from the viscera of all diseased birds showing lesions of necrotic enteritis. Its morphology and biochemical reactions, the presence of alpha and thêta hemolysins and the production of a lecithinase-C in vitro, all these characteristics indicated a similarity to those belonging to the group of Clostridium perfringens. The two hemolysins were neutralized in vitro only by the antitoxin A. Broiler chickens injected I.V. with a Viande-Foie (VF) broth culture of Clostridium perfringens together with the antitoxin A survived, whereas those receiving antitoxin C died. These results seem to indicate that this organism belongs to the type A. This bacillus was sensitive to a great variety of antibiotics, except neomycin. PMID:4368193

Protein docking prediction using predicted protein-protein interface.

PubMed

Li, Bin; Kihara, Daisuke

2012-01-10

Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm), is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

PubMed

Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

2014-10-01

Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

PubMed Central

Gurnev, Philip A.; Nestorovich, Ekaterina M.

2014-01-01

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications. PMID:25153255

In silico Analysis of Toxins of Staphylococcus aureus for Validating Putative Drug Targets.

PubMed

Mohana, Ramadevi; Venugopal, Subhashree

2017-01-01

Toxins are one among the numerous virulence factors produced by the bacteria. These are powerful poisonous substances enabling the bacteria to encounter the defense mechanism of human body. The pathogenic system of Staphylococcus aureus is evolved with various exotoxins that cause detrimental effects on human immune system. Four toxins namely enterotoxin A, exfoliative toxin A, TSST-1 and γ-hemolysin were downloaded from Uniprot database and were analyzed to understand the nature of the toxins and for drug target validation. The results inferred that the toxins were found to interact with many protein partners and no homologous sequences for human proteome were found, and based on similarity search in Drugbank, the targets were identified as novel drug targets. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A novel input-parasitic compensation technique for a nanopore-based CMOS DNA detection sensor

NASA Astrophysics Data System (ADS)

Kim, Jungsuk

2016-12-01

This paper presents a novel input-parasitic compensation (IPC) technique for a nanopore-based complementary metal-oxide-semiconductor (CMOS) DNA detection sensor. A resistive-feedback transimpedance amplifier is typically adopted as the headstage of a DNA detection sensor to amplify the minute ionic currents generated from a nanopore and convert them to a readable voltage range for digitization. But, parasitic capacitances arising from the headstage input and the nanopore often cause headstage saturation during nanopore sensing, thereby resulting in significant DNA data loss. To compensate for the unwanted saturation, in this work, we propose an area-efficient and automated IPC technique, customized for a low-noise DNA detection sensor, fabricated using a 0.35- μm CMOS process; we demonstrated this prototype in a benchtop test using an α-hemolysin ( α-HL) protein nanopore.

FACTORS INFLUENCING IN VITRO KILLING OF BACTERIA BY HEMOCYTES OF THE EASTERN OYSTER (CRASSOSTREA VIRGINICA)

EPA Science Inventory

Vibrio parahaemolyticus strains altered in motility or colonial morphology (opaque versus translucent), Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, or phospholipase activities, and Vibrio vulnificus strains, possessing and lacking capsules we...

Complete genome sequence of Vibrio parahaemolyticus strain FORC_008, a foodborne pathogen from a flounder fish in South Korea.

PubMed

Kim, Suyeon; Chung, Han Young; Lee, Dong-Hoon; Lim, Jong Gyu; Kim, Se Keun; Ku, Hye-Jin; Kim, You-Tae; Kim, Heebal; Ryu, Sangryeol; Lee, Ju-Hoon; Choi, Sang Ho

2016-07-01

Vibrio parahaemolyticus is a Gram-negative, motile, nonspore-forming pathogen that causes foodborne illness associated with the consumption of contaminated seafoods. Although many cases of foodborne outbreaks caused by V. parahaemolyticus have been reported, the genomes of only five strains have been completely sequenced and analyzed using bioinformatics. In order to characterize overall virulence factors and pathogenesis of V. parahaemolyticus associated with foodborne outbreak in South Korea, a new strain FORC_008 was isolated from flounder fish and its genome was completely sequenced. The genomic analysis revealed that the genome of FORC_008 consists of two circular DNA chromosomes of 3266 132 bp (chromosome I) and 1772 036 bp (chromosome II) with a GC content of 45.36% and 45.53%, respectively. The entire genome contains 4494 predicted open reading frames, 129 tRNAs and 31 rRNA genes. While the strain FORC_008 does not have genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), its genome encodes many other virulence factors including hemolysins, pathogenesis-associated secretion systems and iron acquisition systems, suggesting that it may be a potential pathogen. This report provides an extended understanding on V. parahaemolyticus in genomic level and would be helpful for rapid detection, epidemiological investigation and prevention of foodborne outbreak in South Korea. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Presence of Genes Encoding Panton-Valentine Leukocidin Is Not the Primary Determinant of Outcome in Patients with Hospital-Acquired Pneumonia Due to Staphylococcus aureus

PubMed Central

Sharma-Kuinkel, Batu K.; Ahn, Sun H.; Rude, Thomas H.; Zhang, Yurong; Tong, Steven Y. C.; Ruffin, Felicia; Genter, Fredric C.; Braughton, Kevin R.; DeLeo, Frank R.; Barriere, Steven L.

2012-01-01

The impact of Panton-Valentine leukocidin (PVL) on the outcome in Staphylococcus aureus pneumonia is controversial. We genotyped S. aureus isolates from patients with hospital-acquired pneumonia (HAP) enrolled in two registrational multinational clinical trials for the genetic elements carrying pvl and 30 other virulence genes. A total of 287 isolates (173 methicillin-resistant S. aureus [MRSA] and 114 methicillin-susceptible S. aureus [MSSA] isolates) from patients from 127 centers in 34 countries for whom clinical outcomes of cure or failure were available underwent genotyping. Of these, pvl was detected by PCR and its product confirmed in 23 isolates (8.0%) (MRSA, 18/173 isolates [10.4%]; MSSA, 5/114 isolates [4.4%]). The presence of pvl was not associated with a higher risk for clinical failure (4/23 [17.4%] versus 48/264 [18.2%]; P = 1.00) or mortality. These findings persisted after adjustment for multiple potential confounding variables. No significant associations between clinical outcome and (i) presence of any of the 30 other virulence genes tested, (ii) presence of specific bacterial clone, (iii) levels of alpha-hemolysin, or (iv) delta-hemolysin production were identified. This study suggests that neither pvl presence nor in vitro level of alpha-hemolysin production is the primary determinant of outcome among patients with HAP caused by S. aureus. PMID:22205797

Interaction entropy for protein-protein binding

NASA Astrophysics Data System (ADS)

Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

2017-03-01

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Interaction entropy for protein-protein binding.

PubMed

Sun, Zhaoxi; Yan, Yu N; Yang, Maoyou; Zhang, John Z H

2017-03-28

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interactionentropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interactionentropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Serologic and Molecular Characterization of Vibrio parahaemolyticus Strains Isolated from Seawater and Fish Products of the Gulf of Mexico

PubMed Central

Cabrera-García, María Eugenia; Vázquez-Salinas, Carlos; Quiñones-Ramírez, Elsa Irma

2004-01-01

The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the main virulence factors of Vibrio parahaemolyticus. We isolated V. parahaemolyticus from seawater, fish, and oysters obtained from the Pueblo Viejo Lagoon in Veracruz, determined the serogroups, phenotypically and genotypically characterized TDH and TRH, and investigated the presence of the toxR gene. A total of 46 V. parahaemolyticus strains were isolated, and all of them amplified the 368-bp toxR gene fragment. The trh gene was not identified in any of the strains; 4 of the 46 strains were Kanagawa phenomenon (KP) positive and amplified the 251-bp tdh gene fragment. The most frequent serogroup was serogroup O3. This is the first report of the presence of KP-positive tdh-positive environmental V. parahaemolyticus strains in Mexico. PMID:15528498

Penicillinase Studies on L-Phase Variants, G-Phase Variants, and Reverted Strains of Staphylococcus aureus

PubMed Central

Simon, Harold J.; Yin, Elaine Jong

1970-01-01

L-phase variants and small colony (G-phase) variants derived from penicillinase-producing Staphylococcus aureus strains were tested for penicillinase (beta lactamase) production. A refined variation of the modified Gots test for penicillinase was used to demonstrate penicillinase synthesis. Penicillinase synthesis was reduced in L-phase variants and G-phase variants when compared to parental strains. After reversion of variants to vegetative stages had been induced, revertants were tested for production of penicillinase, coagulase, and alpha hemolysin, mannitol fermentation, and pigment production, and comparisons were made between parent and reverted vegetative forms. All revertants of G-phase variants retained penicillinase activity. Most revertants of L-phase variants showed reduction or loss of penicillinase activity. Retention of coagulase activity, alpha hemolysin production, mannitol fermentation, pigmentation, and phage type varied among revertants. Images PMID:16557890

Optimization of protein-protein docking for predicting Fc-protein interactions.

PubMed

Agostino, Mark; Mancera, Ricardo L; Ramsland, Paul A; Fernández-Recio, Juan

2016-11-01

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc-protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc-binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc-protein complexes are available, numerous others have not yet been determined. Protein-protein docking could be used to investigate Fc-protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking-based structural bioinformatics approach is developed for predicting the structures of Fc-protein complexes. Based on the available set of X-ray structures of Fc-protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc-protein complexes, as well as in the design of peptides and proteins that target Fc. Copyright © 2016 John Wiley & Sons, Ltd.

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

PubMed

Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

Theoretical studies of protein-protein and protein-DNA binding rates

NASA Astrophysics Data System (ADS)

Alsallaq, Ramzi A.

Proteins are folded chains of amino acids. Some of the amino acids (e.g. Lys, Arg, His, Asp, and Glu) carry charges under physiological conditions. Proteins almost always function through binding to other proteins or ligands, for example barnase is a ribonuclease protein, found in the bacterium Bacillus amyloliquefaceus. Barnase degrades RNA by hydrolysis. For the bacterium to inhibit the potentially lethal action of Barnase within its own cell it co-produces another protein called barstar which binds quickly, and tightly, to barnase. The biological function of this binding is to block the active site of barnase. The speeds (rates) at which proteins associate are vital to many biological processes. They span a wide range (from less than 103 to 108 M-1s-1 ). Rates greater than ˜ 106 M -1s-1 are typically found to be manifestations of enhancements by long-range electrostatic interactions between the associating proteins. A different paradigm appears in the case of protein binding to DNA. The rate in this case is enhanced through attractive surface potential that effectively reduces the dimensionality of the available search space for the diffusing protein. This thesis presents computational and theoretical models on the rate of association of ligands/proteins to other proteins or DNA. For protein-protein association we present a general strategy for computing protein-protein rates of association. The main achievements of this strategy is the ability to obtain a stringent reaction criteria based on the landscape of short-range interactions between the associating proteins, and the ability to compute the effect of the electrostatic interactions on the rates of association accurately using the best known solvers for Poisson-Boltzmann equation presently available. For protein-DNA association we present a mathematical model for proteins targeting specific sites on a circular DNA topology. The main achievements are the realization that a linear DNA with reflecting ends

Electrostatic complementarity at protein/protein interfaces.

PubMed

McCoy, A J; Chandana Epa, V; Colman, P M

1997-05-02

Calculation of the electrostatic potential of protein-protein complexes has led to the general assertion that protein-protein interfaces display "charge complementarity" and "electrostatic complementarity". In this study, quantitative measures for these two terms are developed and used to investigate protein-protein interfaces in a rigorous manner. Charge complementarity (CC) was defined using the correlation of charges on nearest neighbour atoms at the interface. All 12 protein-protein interfaces studied had insignificantly small CC values. Therefore, the term charge complementarity is not appropriate for the description of protein-protein interfaces when used in the sense measured by CC. Electrostatic complementarity (EC) was defined using the correlation of surface electrostatic potential at protein-protein interfaces. All twelve protein-protein interfaces studied had significant EC values, and thus the assertion that protein-protein association involves surfaces with complementary electrostatic potential was substantially confirmed. The term electrostatic complementarity can therefore be used to describe protein-protein interfaces when used in the sense measured by EC. Taken together, the results for CC and EC demonstrate the relevance of the long-range effects of charges, as described by the electrostatic potential at the binding interface. The EC value did not partition the complexes by type such as antigen-antibody and proteinase-inhibitor, as measures of the geometrical complementarity at protein-protein interfaces have done. The EC value was also not directly related to the number of salt bridges in the interface, and neutralisation of these salt bridges showed that other charges also contributed significantly to electrostatic complementarity and electrostatic interactions between the proteins. Electrostatic complementarity as defined by EC was extended to investigate the electrostatic similarity at the surface of influenza virus neuraminidase where the

Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

PubMed

Kim, Sanggil; Ko, Wooseok; Sung, Bong Hyun; Kim, Sun Chang; Lee, Hyun Soo

2016-11-15

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance. Copyright © 2016. Published by Elsevier Ltd.

Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions.

PubMed

Hentz, N G; Daunert, S

1996-11-15

An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

Molecular modelling of protein-protein/protein-solvent interactions

NASA Astrophysics Data System (ADS)

Luchko, Tyler

The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule

Formation of droplet interface bilayers in a Teflon tube

NASA Astrophysics Data System (ADS)

Walsh, Edmond; Feuerborn, Alexander; Cook, Peter R.

2016-09-01

Droplet-interface bilayers (DIBs) have applications in disciplines ranging from biology to computing. We present a method for forming them manually using a Teflon tube attached to a syringe pump; this method is simple enough it should be accessible to those without expertise in microfluidics. It exploits the properties of interfaces between three immiscible liquids, and uses fluid flow through the tube to pack together drops coated with lipid monolayers to create bilayers at points of contact. It is used to create functional nanopores in DIBs composed of phosphocholine using the protein α-hemolysin (αHL), to demonstrate osmotically-driven mass transfer of fluid across surfactant-based DIBs, and to create arrays of DIBs. The approach is scalable, and thousands of DIBs can be prepared using a robot in one hour; therefore, it is feasible to use it for high throughput applications.

Characterization of a new phage, termed ϕA318, which is specific for Vibrio alginolyticus.

PubMed

Lin, Ying-Rong; Chiu, Chi-Wen; Chang, Feng-Yi; Lin, Chan-Shing

2012-05-01

Vibrio alginolyticus is an opportunistic pathogen of animals and humans; its related strains can also produce tetrodotoxin and hemolysins. A new phage, ϕA318, which lysed its host V. alginolyticus with high efficiency, was characterized. The burst size of ϕA318 in V. alginolyticus was 72 PFU/bacterium at an MOI of 1 at room temperature; the plaque size was as large as 5 mm in diameter. Electron microscopy (EM) of the phage particles revealed a 50- to 55-nm isomorphous icosahedral head with a 12-nm non-contractile tail, similar to the T7-like phages of the family Podoviridae. Phylogenetic analysis based on complete sequences of the DNA-directed RNA polymerase gene revealed that ϕA318 had 28-47% amino acid identity to enterobacteria phages T7 and SP6, and other Vibrio phages, and the phylogenetic distance suggested that ϕA318 could be classified as a new T7-like bacteriophage. Nevertheless, several motifs in the ϕA318 phage RNA polymerase were highly conserved, including DFRGR (T7-421 motif), DG (T7-537 motif), PSEKPQDIYGAVS (T7-563 motif), RSMTKKPVMTL PYGS (T7-627 motif), and HDS (T7-811 motif). Genetic analysis indicated that phage ϕA318 is not a thermostable direct hemolysin producer. The results suggest that the MOI should be higher than 0.1 to prevent the chance of hemolysin production by the bacteria before they are lysed by the phage.

Conformational Heterogeneity of Unbound Proteins Enhances Recognition in Protein-Protein Encounters.

PubMed

Pallara, Chiara; Rueda, Manuel; Abagyan, Ruben; Fernández-Recio, Juan

2016-07-12

To understand cellular processes at the molecular level we need to improve our knowledge of protein-protein interactions, from a structural, mechanistic, and energetic point of view. Current theoretical studies and computational docking simulations show that protein dynamics plays a key role in protein association and support the need for including protein flexibility in modeling protein interactions. Assuming the conformational selection binding mechanism, in which the unbound state can sample bound conformers, one possible strategy to include flexibility in docking predictions would be the use of conformational ensembles originated from unbound protein structures. Here we present an exhaustive computational study about the use of precomputed unbound ensembles in the context of protein docking, performed on a set of 124 cases of the Protein-Protein Docking Benchmark 3.0. Conformational ensembles were generated by conformational optimization and refinement with MODELLER and by short molecular dynamics trajectories with AMBER. We identified those conformers providing optimal binding and investigated the role of protein conformational heterogeneity in protein-protein recognition. Our results show that a restricted conformational refinement can generate conformers with better binding properties and improve docking encounters in medium-flexible cases. For more flexible cases, a more extended conformational sampling based on Normal Mode Analysis was proven helpful. We found that successful conformers provide better energetic complementarity to the docking partners, which is compatible with recent views of binding association. In addition to the mechanistic considerations, these findings could be exploited for practical docking predictions of improved efficiency.

Protein- protein interaction detection system using fluorescent protein microdomains

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2010-02-23

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

Long-term human serum antibody responses after immunization with whole-cell pertussis vaccine in France.

PubMed Central

Grimprel, E; Bégué, P; Anjak, I; Njamkepo, E; François, P; Guiso, N

1996-01-01

Three hundred sixty children were tested for pertussis serology 0.5 to 1.58 months after complete whole-cell pertussis vaccination. An immunoblot assay was used to detect serum antibodies to pertussis toxin, filamentous hemagglutinin, adenylate cyclase-hemolysin, and pertactin, and agglutination was used for detection of anti-agglutinogen antibodies. Antibodies against pertussis toxin, pertactin, and agglutinogens decreased rapidly after vaccination but increased secondarily, suggesting exposure to infected persons. In contrast, anti-filamentous hemagglutinin antibodies persisted and anti-adenylate cyclase-hemolysin antibodies increased continuously, suggesting either cross-reaction with non-Bordetella antigens or exposure to Bordetella isolates expressing these two antigens, including Bordetella pertussis. These data suggest that unrecognized pertussis is common in France despite massive and sustained immunization in infants and that vaccinated children become susceptible to infection more than 6 years after their last vaccination. PMID:8770511

UDoNC: An Algorithm for Identifying Essential Proteins Based on Protein Domains and Protein-Protein Interaction Networks.

PubMed

Peng, Wei; Wang, Jianxin; Cheng, Yingjiao; Lu, Yu; Wu, Fangxiang; Pan, Yi

2015-01-01

Prediction of essential proteins which are crucial to an organism's survival is important for disease analysis and drug design, as well as the understanding of cellular life. The majority of prediction methods infer the possibility of proteins to be essential by using the network topology. However, these methods are limited to the completeness of available protein-protein interaction (PPI) data and depend on the network accuracy. To overcome these limitations, some computational methods have been proposed. However, seldom of them solve this problem by taking consideration of protein domains. In this work, we first analyze the correlation between the essentiality of proteins and their domain features based on data of 13 species. We find that the proteins containing more protein domain types which rarely occur in other proteins tend to be essential. Accordingly, we propose a new prediction method, named UDoNC, by combining the domain features of proteins with their topological properties in PPI network. In UDoNC, the essentiality of proteins is decided by the number and the frequency of their protein domain types, as well as the essentiality of their adjacent edges measured by edge clustering coefficient. The experimental results on S. cerevisiae data show that UDoNC outperforms other existing methods in terms of area under the curve (AUC). Additionally, UDoNC can also perform well in predicting essential proteins on data of E. coli.

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

Protein-protein interaction network-based detection of functionally similar proteins within species.

PubMed

Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

2012-07-01

Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

Protein-Protein Docking in Drug Design and Discovery.

PubMed

Kaczor, Agnieszka A; Bartuzi, Damian; Stępniewski, Tomasz Maciej; Matosiuk, Dariusz; Selent, Jana

2018-01-01

Protein-protein interactions (PPIs) are responsible for a number of key physiological processes in the living cells and underlie the pathomechanism of many diseases. Nowadays, along with the concept of so-called "hot spots" in protein-protein interactions, which are well-defined interface regions responsible for most of the binding energy, these interfaces can be targeted with modulators. In order to apply structure-based design techniques to design PPIs modulators, a three-dimensional structure of protein complex has to be available. In this context in silico approaches, in particular protein-protein docking, are a valuable complement to experimental methods for elucidating 3D structure of protein complexes. Protein-protein docking is easy to use and does not require significant computer resources and time (in contrast to molecular dynamics) and it results in 3D structure of a protein complex (in contrast to sequence-based methods of predicting binding interfaces). However, protein-protein docking cannot address all the aspects of protein dynamics, in particular the global conformational changes during protein complex formation. In spite of this fact, protein-protein docking is widely used to model complexes of water-soluble proteins and less commonly to predict structures of transmembrane protein assemblies, including dimers and oligomers of G protein-coupled receptors (GPCRs). In this chapter we review the principles of protein-protein docking, available algorithms and software and discuss the recent examples, benefits, and drawbacks of protein-protein docking application to water-soluble proteins, membrane anchoring and transmembrane proteins, including GPCRs.

Genome-wide protein-protein interactions and protein function exploration in cyanobacteria

PubMed Central

Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

2015-01-01

Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and “interologs” in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria. PMID:26490033

Aeromonas Caviae Strain Induces Th1 Cytokine Response in Mouse Intestinal Tract

EPA Science Inventory

Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus. Microarray profiling of murine small i...

Aeromonas caviae strain induces Th1 cytokine response in mouse intestinal tract

EPA Science Inventory

Aeromonas caviae has been associated with human gastrointestinal disease. Strains of this species typically lack virulence factors (VFs) such as enterotoxins and hemolysins that are produced by other human pathogens of the Aeromonas genus,. Microarray profiling of...

Protein function prediction using neighbor relativity in protein-protein interaction network.

PubMed

Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

2013-04-01

There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

Protein Structure Prediction by Protein Threading

NASA Astrophysics Data System (ADS)

Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

NanoPad: an integrated platform for bacterial production of camel nanobodies aimed at detecting environmental biomarkers.

PubMed

Fraile, Sofía; Jiménez, Jose I; Gutiérrez, Carlos; de Lorenzo, Víctor

2013-10-01

The presence of given antigens in environmental samples (e.g. biodegradative enzymes) reports the quality and catalytic vigor of particular soils or aquatic ecosystems. In this context, we have developed the NanoPad system consisting of a complete platform for isolation, amplification, and extracellular production of specific antibodies against antigens that diagnose the occurrence of protein markers in crude environmental samples. The workflow starts with the inoculation of camels (Camelus dromedarius) with various proteins (e.g. catabolic enzymes) for generating a phage display library of variable heavy-chain antibody H fragment (VHH ) domains that bind the different antigens. Instead of being subjected to a conventional panning, such a library is then probed with a Western-panning technique that allows direct isolation of specific binders of proteins blotted on membranes from polyacrylamide gels. Finally, VHH s are fused to the C-domain of hemolysin for secretion to the culture media as virtually pure dimeric proteins that can be used as a primary antibody without further processing. The value of NanoPad is shown with the selection of nanobodies for detection of biphenyl 2,3-dioxygenase, a key enzyme for biodegradation of polychlorinated biphenyls. The thereby generated anti-biphenyl 2,3-dioxygenase VHH s revealed the presence of this enzyme in the metaproteome of an oil refinery waste treatment plant. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Broussard, Tyler C.; Bose, Jeffrey L.; Rosch, Jason W.; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O.

2014-01-01

Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus. PMID:25002480

Predicting protein-protein interactions from protein domains using a set cover approach.

PubMed

Huang, Chengbang; Morcos, Faruck; Kanaan, Simon P; Wuchty, Stefan; Chen, Danny Z; Izaguirre, Jesús A

2007-01-01

One goal of contemporary proteome research is the elucidation of cellular protein interactions. Based on currently available protein-protein interaction and domain data, we introduce a novel method, Maximum Specificity Set Cover (MSSC), for the prediction of protein-protein interactions. In our approach, we map the relationship between interactions of proteins and their corresponding domain architectures to a generalized weighted set cover problem. The application of a greedy algorithm provides sets of domain interactions which explain the presence of protein interactions to the largest degree of specificity. Utilizing domain and protein interaction data of S. cerevisiae, MSSC enables prediction of previously unknown protein interactions, links that are well supported by a high tendency of coexpression and functional homogeneity of the corresponding proteins. Focusing on concrete examples, we show that MSSC reliably predicts protein interactions in well-studied molecular systems, such as the 26S proteasome and RNA polymerase II of S. cerevisiae. We also show that the quality of the predictions is comparable to the Maximum Likelihood Estimation while MSSC is faster. This new algorithm and all data sets used are accessible through a Web portal at http://ppi.cse.nd.edu.

Imaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopy

PubMed Central

Sugimoto, Shinya; Okuda, Ken-ichi; Miyakawa, Reina; Sato, Mari; Arita-Morioka, Ken-ichi; Chiba, Akio; Yamanaka, Kunitoshi; Ogura, Teru; Mizunoe, Yoshimitsu; Sato, Chikara

2016-01-01

Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liquid is challenging. Here, we develop atmospheric scanning electron microscopy (ASEM) to visualise Gram-positive and -negative bacterial biofilms immersed in aqueous solution. Biofilms cultured on electron-transparent film were directly imaged from below using the inverted SEM, allowing the formation of the region near the substrate to be studied at high resolution. We visualised intercellular nanostructures and the exocytosis of membrane vesicles, and linked the latter to the trafficking of cargos, including cytoplasmic proteins and the toxins hemolysin and coagulase. A thick dendritic nanotube network was observed between microbes, suggesting multicellular communication in biofilms. A universal immuno-labelling system was developed for biofilms and tested on various examples, including S. aureus biofilms. In the ECM, fine DNA and protein networks were visualised and the precise distribution of protein complexes was determined (e.g., straight curli, flagella, and excreted cytoplasmic molecular chaperones). Our observations provide structural insights into bacteria-substratum interactions, biofilm development and the internal microbe community. PMID:27180609

Ion Move Brownian Dynamics (IMBD)--simulations of ion transport.

PubMed

Kurczynska, Monika; Kotulska, Malgorzata

2014-01-01

Comparison of the computed characteristics and physiological measurement of ion transport through transmembrane proteins could be a useful method to assess the quality of protein structures. Simulations of ion transport should be detailed but also timeefficient. The most accurate method could be Molecular Dynamics (MD), which is very time-consuming, hence is not used for this purpose. The model which includes ion-ion interactions and reduces the simulation time by excluding water, protein and lipid molecules is Brownian Dynamics (BD). In this paper a new computer program for BD simulation of the ion transport is presented. We evaluate two methods for calculating the pore accessibility (round and irregular shape) and two representations of ion sizes (van der Waals diameter and one voxel). Ion Move Brownian Dynamics (IMBD) was tested with two nanopores: alpha-hemolysin and potassium channel KcsA. In both cases during the simulation an ion passed through the pore in less than 32 ns. Although two types of ions were in solution (potassium and chloride), only ions which agreed with the selectivity properties of the channels passed through the pores. IMBD is a new tool for the ion transport modelling, which can be used in the simulations of wide and narrow pores.

Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

PubMed

den Reijer, P M; Haisma, E M; Lemmens-den Toom, N A; Willemse, J; Koning, R I; Koning, R A; Demmers, J A A; Dekkers, D H W; Rijkers, E; El Ghalbzouri, A; Nibbering, P H; van Wamel, W

2016-01-01

The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

Do cancer proteins really interact strongly in the human protein-protein interaction network?

PubMed Central

Xia, Junfeng; Sun, Jingchun; Jia, Peilin; Zhao, Zhongming

2011-01-01

Protein-protein interaction (PPI) network analysis has been widely applied in the investigation of the mechanisms of diseases, especially cancer. Recent studies revealed that cancer proteins tend to interact more strongly than other categories of proteins, even essential proteins, in the human interactome. However, it remains unclear whether this observation was introduced by the bias towards more cancer studies in humans. Here, we examined this important issue by uniquely comparing network characteristics of cancer proteins with three other sets of proteins in four organisms, three of which (fly, worm, and yeast) whose interactomes are essentially not biased towards cancer or other diseases. We confirmed that cancer proteins had stronger connectivity, shorter distance, and larger betweenness centrality than non-cancer disease proteins, essential proteins, and control proteins. Our statistical evaluation indicated that such observations were overall unlikely attributed to random events. Considering the large size and high quality of the PPI data in the four organisms, the conclusion that cancer proteins interact strongly in the PPI networks is reliable and robust. This conclusion suggests that perturbation of cancer proteins might cause major changes of cellular systems and result in abnormal cell function leading to cancer. PMID:21666777

Detection of protein complex from protein-protein interaction network using Markov clustering

NASA Astrophysics Data System (ADS)

Ochieng, P. J.; Kusuma, W. A.; Haryanto, T.

2017-05-01

Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks.

Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity

PubMed Central

Wei, Yang; Thyparambil, Aby A.; Latour, Robert A.

2013-01-01

While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2 h to saturate the surface, followed by immersion in pure buffer solution for 15 h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein’s secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL’s structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL’s bioactivity on this surface, such as the accessibility of HEWL’s bioactive site being blocked by neighboring proteins or the surface

Do cancer proteins really interact strongly in the human protein-protein interaction network?

PubMed

Xia, Junfeng; Sun, Jingchun; Jia, Peilin; Zhao, Zhongming

2011-06-01

Protein-protein interaction (PPI) network analysis has been widely applied in the investigation of the mechanisms of diseases, especially cancer. Recent studies revealed that cancer proteins tend to interact more strongly than other categories of proteins, even essential proteins, in the human interactome. However, it remains unclear whether this observation was introduced by the bias towards more cancer studies in humans. Here, we examined this important issue by uniquely comparing network characteristics of cancer proteins with three other sets of proteins in four organisms, three of which (fly, worm, and yeast) whose interactomes are essentially not biased towards cancer or other diseases. We confirmed that cancer proteins had stronger connectivity, shorter distance, and larger betweenness centrality than non-cancer disease proteins, essential proteins, and control proteins. Our statistical evaluation indicated that such observations were overall unlikely attributed to random events. Considering the large size and high quality of the PPI data in the four organisms, the conclusion that cancer proteins interact strongly in the PPI networks is reliable and robust. This conclusion suggests that perturbation of cancer proteins might cause major changes of cellular systems and result in abnormal cell function leading to cancer. © 2011 Elsevier Ltd. All rights reserved.

Fatty acid composition modulates sensitivity of Legionella pneumophila to warnericin RK, an antimicrobial peptide.

PubMed

Verdon, Julien; Labanowski, Jérome; Sahr, Tobias; Ferreira, Thierry; Lacombe, Christian; Buchrieser, Carmen; Berjeaud, Jean-Marc; Héchard, Yann

2011-04-01

Warnericin RK is an antimicrobial peptide, produced by a Staphyloccocus warneri strain, described to be specifically active against Legionella, the pathogenic bacteria responsible for Legionnaires' disease. Warnericin RK is an amphiphilic alpha-helical peptide, which possesses a detergent-like mode of action. Two others peptides, δ-hemolysin I and II, produced by the same S. warneri strain, are highly similar to S. aureus δ-hemolysin and also display anti-Legionella activity. It has been recently reported that S. aureus δ-hemolysin activity on vesicles is likewise related to phospholipid acyl-chain structure, such as chain length and saturation. As staphylococcal δ-hemolysins were highly similar, we thus hypothesized that fatty acid composition of Legionella's membrane might influence the sensitivity of the bacteria to warnericin RK. Relationship between sensitivity to the peptide and fatty acid composition was then followed in various conditions. Cells in stationary phase, which were already described as less resistant than cells in exponential phase, displayed higher amounts of branched-chain fatty acids (BCFA) and short chain fatty acids. An adapted strain, able to grow at a concentration 33 fold higher than minimal inhibitory concentration of the wild type (i.e. 1μM), was isolated after repeated transfers of L. pneumophila in the presence of increased concentrations of warnericin RK. The amount of BCFA was significantly higher in the adapted strain than in the wild type strain. Also, a transcriptomic analysis of the wild type and adapted strains showed that two genes involved in fatty acid biosynthesis were repressed in the adapted strain. These genes encode enzymes involved in desaturation and elongation of fatty acids respectively. Their repression was in agreement with the decrease of unsaturated fatty acids and fatty acid chain length in the adapted strain. Conclusively, our results indicate that the increase of BCFA and the decrease of fatty acid

Involvement of polyphosphate kinase in virulence and stress tolerance of uropathogenic Proteus mirabilis.

PubMed

Peng, Liang; Jiang, Qiao; Pan, Jia-Yun; Deng, Cong; Yu, Jing-Yi; Wu, Xiao-Man; Huang, Sheng-He; Deng, Xiao-Yan

2016-04-01

Proteus mirabilis (P. mirabilis), a gram-negative enteric bacterium, frequently causes urinary tract infections. Many virulence factors of uropathogenic P. mirabilis have been identified, including urease, flagella, hemolysin and fimbriae. However, the functions of polyphosphate kinase (PPK), which are related to the pathogenicity of many bacteria, remain entirely unknown in P. mirabilis. In this study, a ppk gene encoding the PPK insertional mutant in P. mirabilis strain HI4320 was constructed, and its biological functions were examined. The results of survival studies demonstrated that the ppk mutant was deficient in resistance to oxidative, hyperosmotic and heat stress. The swarming and biofilm formation abilities of P. mirabilis were also attenuated after the ppk interruption. In vitro and in vivo experiments suggested that ppk was required for P. mirabilis to invade the bladder. The negative phenotypes of the ppk mutant could be restored by ppk gene complementation. Furthermore, two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry were used to analyze the proteomes of the wild-type strain and the ppk mutant. Compared with the wild-type strain, seven proteins including TonB-dependent receptor, universal stress protein G, major mannose-resistant/Proteus-like fimbrial protein (MR/P fimbriae), heat shock protein, flagellar capping protein, putative membrane protein and multidrug efflux protein were down-regulated, and four proteins including exported peptidase, repressor protein for FtsI, FKBP-type peptidyl-prolyl cis-trans isomerase and phosphotransferase were up-regulated in the ppk mutant. As a whole, these results indicate that PPK is an important regulator and plays a crucial role in stress tolerance and virulence in uropathogenic P. mirabilis.

Quantifying protein-protein interactions in high throughput using protein domain microarrays.

PubMed

Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

2010-04-01

Protein microarrays provide an efficient way to identify and quantify protein-protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain-peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (K(D)s) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein-ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein-protein interaction networks.

Assessment of the reliability of protein-protein interactions and protein function prediction.

PubMed

Deng, Minghua; Sun, Fengzhu; Chen, Ting

2003-01-01

As more and more high-throughput protein-protein interaction data are collected, the task of estimating the reliability of different data sets becomes increasingly important. In this paper, we present our study of two groups of protein-protein interaction data, the physical interaction data and the protein complex data, and estimate the reliability of these data sets using three different measurements: (1) the distribution of gene expression correlation coefficients, (2) the reliability based on gene expression correlation coefficients, and (3) the accuracy of protein function predictions. We develop a maximum likelihood method to estimate the reliability of protein interaction data sets according to the distribution of correlation coefficients of gene expression profiles of putative interacting protein pairs. The results of the three measurements are consistent with each other. The MIPS protein complex data have the highest mean gene expression correlation coefficients (0.256) and the highest accuracy in predicting protein functions (70% sensitivity and specificity), while Ito's Yeast two-hybrid data have the lowest mean (0.041) and the lowest accuracy (15% sensitivity and specificity). Uetz's data are more reliable than Ito's data in all three measurements, and the TAP protein complex data are more reliable than the HMS-PCI data in all three measurements as well. The complex data sets generally perform better in function predictions than do the physical interaction data sets. Proteins in complexes are shown to be more highly correlated in gene expression. The results confirm that the components of a protein complex can be assigned to functions that the complex carries out within a cell. There are three interaction data sets different from the above two groups: the genetic interaction data, the in-silico data and the syn-express data. Their capability of predicting protein functions generally falls between that of the Y2H data and that of the MIPS protein complex

Building protein-protein interaction networks for Leishmania species through protein structural information.

PubMed

Dos Santos Vasconcelos, Crhisllane Rafaele; de Lima Campos, Túlio; Rezende, Antonio Mauro

2018-03-06

Systematic analysis of a parasite interactome is a key approach to understand different biological processes. It makes possible to elucidate disease mechanisms, to predict protein functions and to select promising targets for drug development. Currently, several approaches for protein interaction prediction for non-model species incorporate only small fractions of the entire proteomes and their interactions. Based on this perspective, this study presents an integration of computational methodologies, protein network predictions and comparative analysis of the protozoan species Leishmania braziliensis and Leishmania infantum. These parasites cause Leishmaniasis, a worldwide distributed and neglected disease, with limited treatment options using currently available drugs. The predicted interactions were obtained from a meta-approach, applying rigid body docking tests and template-based docking on protein structures predicted by different comparative modeling techniques. In addition, we trained a machine-learning algorithm (Gradient Boosting) using docking information performed on a curated set of positive and negative protein interaction data. Our final model obtained an AUC = 0.88, with recall = 0.69, specificity = 0.88 and precision = 0.83. Using this approach, it was possible to confidently predict 681 protein structures and 6198 protein interactions for L. braziliensis, and 708 protein structures and 7391 protein interactions for L. infantum. The predicted networks were integrated to protein interaction data already available, analyzed using several topological features and used to classify proteins as essential for network stability. The present study allowed to demonstrate the importance of integrating different methodologies of interaction prediction to increase the coverage of the protein interaction of the studied protocols, besides it made available protein structures and interactions not previously reported.

Prevalence and Molecular Typing of Vibrio parahaemolyticus (tdh+) isolated from seafood using PCR-based methods

USDA-ARS?s Scientific Manuscript database

Vibrio parahaemolyticus is a pathogen most frequently implicated in foodborne outbreaks linked to the consumption of seafood in the coastal cities of China. The pathogenicity of environmental V. parahaemolyticus is mostly correlated with the production of thermostable direct hemolysin (TDH). In orde...

Detection of protein-protein interactions by ribosome display and protein in situ immobilisation.

PubMed

He, Mingyue; Liu, Hong; Turner, Martin; Taussig, Michael J

2009-12-31

We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

PubMed

Svedova, Martina; Masin, Jiri; Fiser, Radovan; Cerny, Ondrej; Tomala, Jakub; Freudenberg, Marina; Tuckova, Ludmila; Kovar, Marek; Dadaglio, Gilles; Adkins, Irena; Sebo, Peter

2016-04-01

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

PREFACE: Protein protein interactions: principles and predictions

NASA Astrophysics Data System (ADS)

Nussinov, Ruth; Tsai, Chung-Jung

2005-06-01

Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

Coevolution study of mitochondria respiratory chain proteins: toward the understanding of protein--protein interaction.

PubMed

Yang, Ming; Ge, Yan; Wu, Jiayan; Xiao, Jingfa; Yu, Jun

2011-05-20

Coevolution can be seen as the interdependency between evolutionary histories. In the context of protein evolution, functional correlation proteins are ever-present coordinated evolutionary characters without disruption of organismal integrity. As to complex system, there are two forms of protein--protein interactions in vivo, which refer to inter-complex interaction and intra-complex interaction. In this paper, we studied the difference of coevolution characters between inter-complex interaction and intra-complex interaction using "Mirror tree" method on the respiratory chain (RC) proteins. We divided the correlation coefficients of every pairwise RC proteins into two groups corresponding to the binary protein--protein interaction in intra-complex and the binary protein--protein interaction in inter-complex, respectively. A dramatical discrepancy is detected between the coevolution characters of the two sets of protein interactions (Wilcoxon test, p-value = 4.4 × 10(-6)). Our finding reveals some critical information on coevolutionary study and assists the mechanical investigation of protein--protein interaction. Furthermore, the results also provide some unique clue for supramolecular organization of protein complexes in the mitochondrial inner membrane. More detailed binding sites map and genome information of nuclear encoded RC proteins will be extraordinary valuable for the further mitochondria dynamics study. Copyright © 2011. Published by Elsevier Ltd.

Antimicrobial resistance and production of toxins in Escherichia coli strains from wild ruminants and the alpine marmot.

PubMed

Caprioli, A; Donelli, G; Falbo, V; Passi, C; Pagano, A; Mantovani, A

1991-04-01

Escherichia coli strains isolated from 81 fecal samples from red deer (Cervus elaphus), roe deer (Capreoulus capreoulus), chamois (Rupicapra rupicapra) and alpine marmot (Marmota marmota) living in the Stelvio National Park, Italy, were examined for antimicrobial resistance and production of toxic factors. Direct plating of specimens on media containing antimicrobial drugs allowed us to isolate resistant strains of E. coli from 10 of 59 (17%) specimens examined by this technique. Nine of 31 specimens from red deer (29%) contained resistant strains. Different animals were likely colonized by the same resistant strain of E. coli. Conjugative R plasmids were found in four strains isolated from the marmot, roe deer and chamois. A strain from red deer produced heat-stable enterotoxin and another strain produced both hemolysin and cytotoxic necrotizing factor. A marmot isolate produced hemolysin alone. No strains were found to produce heat-labile enterotoxin or verotoxins.

Protein complex prediction in large ontology attributed protein-protein interaction networks.

PubMed

Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian; Li, Yanpeng; Xu, Bo

2013-01-01

Protein complexes are important for unraveling the secrets of cellular organization and function. Many computational approaches have been developed to predict protein complexes in protein-protein interaction (PPI) networks. However, most existing approaches focus mainly on the topological structure of PPI networks, and largely ignore the gene ontology (GO) annotation information. In this paper, we constructed ontology attributed PPI networks with PPI data and GO resource. After constructing ontology attributed networks, we proposed a novel approach called CSO (clustering based on network structure and ontology attribute similarity). Structural information and GO attribute information are complementary in ontology attributed networks. CSO can effectively take advantage of the correlation between frequent GO annotation sets and the dense subgraph for protein complex prediction. Our proposed CSO approach was applied to four different yeast PPI data sets and predicted many well-known protein complexes. The experimental results showed that CSO was valuable in predicting protein complexes and achieved state-of-the-art performance.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

C-Myc Protein-Protein and Protein-DNA Interactions: Targets for Therapeutic Intervention.

DTIC Science & Technology

1997-09-01

including those of the Myc family. In fact, members of different bHLH protein subgroups, including the Myc proteins, are characterized by conserved BR...important functional consequences, and they provide insights into how different bHLH proteins can act on different targets. The zinc finger protein...roles for a number of BR residues which do not contact bases, yet are conserved within different bHLH protein sub- families (Benezra et al. 1990), and

Protein Solubility and Protein Homeostasis: A Generic View of Protein Misfolding Disorders

PubMed Central

Vendruscolo, Michele; Knowles, Tuomas P.J.; Dobson, Christopher M.

2011-01-01

According to the “generic view” of protein aggregation, the ability to self-assemble into stable and highly organized structures such as amyloid fibrils is not an unusual feature exhibited by a small group of peptides and proteins with special sequence or structural properties, but rather a property shared by most proteins. At the same time, through a wide variety of techniques, many of which were originally devised for applications in other disciplines, it has also been established that the maintenance of proteins in a soluble state is a fundamental aspect of protein homeostasis. Taken together, these advances offer a unified framework for understanding the molecular basis of protein aggregation and for the rational development of therapeutic strategies based on the biological and chemical regulation of protein solubility. PMID:21825020

Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

PubMed

Yang, Lei; Zhao, Xudong; Tang, Xianglong

2014-01-01

Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases.

Risk of Vibrio transmission linked to the consumption of crustaceans in coastal towns of Côte d'Ivoire.

PubMed

Traoré, S G; Bonfoh, B; Krabi, R; Odermatt, P; Utzinger, J; Rose, K-N; Tanner, M; Frey, J; Quilici, M-L; Koussémon, M

2012-06-01

The purpose of this study was to assess the risk of Vibrio spp. transmission from crustaceans to humans in two coastal towns of Côte d'Ivoire. Bacteriologic analysis was performed on 322 crustacean samples obtained from six markets in Abidjan and one in Dabou. Suspected Vibrio colonies were identified by morphological, cultural, biochemical, and molecular tests and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. PCR assays were used to further characterize Vibrio strains. A survey on consumption of crustaceans was conducted among 120 randomly selected households in Abidjan. Overall, Vibrio spp. were isolated from 7.8% of the crustacean samples studied, at levels as high as 6.3 log CFU/g. Of the Vibrio strains identified, 40% were V. alginolyticus, 36% were V. parahaemolyticus, and 24% were nontoxigenic V. cholerae; the latter two species can cause mild to severe forms of seafood-associated gastroenteritis. Among interviewed households, 11.7% reported daily consumption of crustaceans, confirming the high probability of exposure of human population to Vibrio spp., and 7.5% reported symptoms of food poisoning after consumption of crustaceans. The absence of genes encoding major virulence factors in the studied strains, i.e., cholera toxin (ctxA and ctxB) in V. cholerae and thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh) in V. parahaemolyticus, does not exclude the possibility of exposure to pathogenic strains. However, human infections are not common because most households (96.7%) boil crustaceans, usually for at least 45 min (85.9% of households) before consumption.

Regulation of protein turnover by heat shock proteins.

PubMed

Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

2014-12-01

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system. Copyright © 2014 Elsevier Inc. All rights reserved.

Principles of Protein Recognition and Properties of Protein-protein Interfaces

NASA Astrophysics Data System (ADS)

Keskin, Ozlem; Gursoy, Attila; Nussinov, Ruth

In this chapter we address two aspects - the static physical interactions which allow the information transfer for the function to be performed; and the dynamic, i.e. how the information is transmitted between the binding sites in the single protein molecule and in the network. We describe the single protein molecules and their complexes; and the analogy between protein folding and protein binding. Eventually, to fully understand the interactome and how it performs the essential cellular functions, we have to put all together - and hierarchically progress through the system.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

PubMed

Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

2015-02-23

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions

PubMed Central

Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

2015-01-01

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

Immunomodulatory effects of Hericium erinaceus derived polysaccharides are mediated by intestinal immunology.

PubMed

Sheng, Xiaotong; Yan, Jingmin; Meng, Yue; Kang, Yuying; Han, Zhen; Tai, Guihua; Zhou, Yifa; Cheng, Hairong

2017-03-22

This study was aimed at investigating the immunomodulating activity of Hericium erinaceus polysaccharide (HEP) in mice, by assessing splenic lymphocyte proliferation (cell-mediated immunity), serum hemolysin levels (humoral immunity), phagocytic capacity of peritoneal cavity phagocytes (macrophage phagocytosis), and NK cell activity. ELISA of immunoglobulin A (SIgA) in the lamina propria, and western blotting of small intestinal proteins were also performed to gain insight into the mechanism by which HEP affects the intestinal immune system. Here, we report that HEP improves immune function by functionally enhancing cell-mediated and humoral immunity, macrophage phagocytosis, and NK cell activity. In addition, HEP was found to upregulate the secretion of SIgA and activate the MAPK and AKT cellular signaling pathways in the intestine. In conclusion, all these results allow us to postulate that the immunomodulatory effects of HEP are most likely attributed to the effective regulation of intestinal mucosal immune activity.

Predicting permanent and transient protein-protein interfaces.

PubMed

La, David; Kong, Misun; Hoffman, William; Choi, Youn Im; Kihara, Daisuke

2013-05-01

Protein-protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. Copyright © 2013 Wiley Periodicals, Inc.

Molecular simulations of lipid-mediated protein-protein interactions.

PubMed

de Meyer, Frédérick Jean-Marie; Venturoli, Maddalena; Smit, Berend

2008-08-01

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.

Global differential gene expression in response to growth temperature alteration in group A Streptococcus.

PubMed

Smoot, L M; Smoot, J C; Graham, M R; Somerville, G A; Sturdevant, D E; Migliaccio, C A; Sylva, G L; Musser, J M

2001-08-28

Pathogens are exposed to different temperatures during an infection cycle and must regulate gene expression accordingly. However, the extent to which virulent bacteria alter gene expression in response to temperatures encountered in the host is unknown. Group A Streptococcus (GAS) is a human-specific pathogen that is responsible for illnesses ranging from superficial skin infections and pharyngitis to severe invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS survives and multiplies at different temperatures during human infection. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in a serotype M1 strain grown to exponential phase at 29 degrees C and 37 degrees C. Approximately 9% of genes were differentially expressed by at least 1.5-fold at 29 degrees C relative to 37 degrees C, including genes encoding transporter proteins, proteins involved in iron homeostasis, transcriptional regulators, phage-associated proteins, and proteins with no known homologue. Relatively few known virulence genes were differentially expressed at this threshold. However, transcription of 28 genes encoding proteins with predicted secretion signal sequences was altered, indicating that growth temperature substantially influences the extracellular proteome. TaqMan real-time reverse transcription-PCR assays confirmed the microarray data. We also discovered that transcription of genes encoding hemolysins, and proteins with inferred roles in iron regulation, transport, and homeostasis, was influenced by growth at 40 degrees C. Thus, GAS profoundly alters gene expression in response to temperature. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen lines of pathogenesis investigation.

Nature and consequences of protein-protein interactions in high protein concentration solutions.

PubMed

Saluja, Atul; Kalonia, Devendra S

2008-06-24

High protein concentration solutions are becoming increasingly important in the pharmaceutical industry. The solution behavior of proteins at high concentrations can markedly differ from that predicted based on dilute solution analysis due to thermodynamic non-ideality in these solutions. The non-ideality observed in these systems is related to the protein-protein interactions (PPI). Different types of forces play a key role in determining the overall nature and extent of these PPI and their relative contributions are affected by solute and solvent properties. However, individual contributions of these forces to the solution properties of concentrated protein solutions are not fully understood. The role of PPI, driven by these intermolecular forces, in governing solution rheology and physical stability of high protein concentration solutions is discussed from the point of view of pharmaceutical product development. Investigation of protein self-association and aggregation in concentrated protein solutions is crucial for ensuring the safety and efficacy of the final product for the duration of the desired product shelf life. Understanding rheology of high concentration protein solutions is critical for addressing issues during product manufacture and administration of final formulation to the patient. To this end, analysis of solution viscoelastic character can also provide an insight into the nature of PPI affecting solution rheology.

Computer Simulation of Protein-Protein and Protein-Peptide Interactions

DTIC Science & Technology

1983-12-08

a full molecular dynamic z simulation is performed, with resulting dipolar re - laxation. However, this is prohibitive when a large . number of...1993 Dr. Mike Marron Program Manager Molecular Biology Office of Naval Research 800 N. Quincy Street Arlington, VA 22217 Dear Mike, Here is the...rztnbutior is unLi--ited. , 93-30630 98 12 � 12/08/93 01/0/92-;03/31/93: Final Report, Computer Simulation of Protein-Protein and Protein-Peptide

Prediction of physical protein protein interactions

NASA Astrophysics Data System (ADS)

Szilágyi, András; Grimm, Vera; Arakaki, Adrián K.; Skolnick, Jeffrey

2005-06-01

Many essential cellular processes such as signal transduction, transport, cellular motion and most regulatory mechanisms are mediated by protein-protein interactions. In recent years, new experimental techniques have been developed to discover the protein-protein interaction networks of several organisms. However, the accuracy and coverage of these techniques have proven to be limited, and computational approaches remain essential both to assist in the design and validation of experimental studies and for the prediction of interaction partners and detailed structures of protein complexes. Here, we provide a critical overview of existing structure-independent and structure-based computational methods. Although these techniques have significantly advanced in the past few years, we find that most of them are still in their infancy. We also provide an overview of experimental techniques for the detection of protein-protein interactions. Although the developments are promising, false positive and false negative results are common, and reliable detection is possible only by taking a consensus of different experimental approaches. The shortcomings of experimental techniques affect both the further development and the fair evaluation of computational prediction methods. For an adequate comparative evaluation of prediction and high-throughput experimental methods, an appropriately large benchmark set of biophysically characterized protein complexes would be needed, but is sorely lacking.

Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.

PubMed

Paulmurugan, R; Gambhir, S S

2003-04-01

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.

Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu,P.

2007-01-01

The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behaviormore » and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.« less

CONVERSION OF PLASMA PROTEIN TO TISSUE PROTEIN WITHOUT EVIDENCE OF PROTEIN BREAKDOWN

PubMed Central

Yuile, C. L.; Lamson, B. G.; Miller, L. L.; Whipple, G. H.

1951-01-01

Labeled plasma proteins obtained from donor dogs, previously fed ε-C14-dl-lysine, have been given intravenously to recipient dogs. The disappearance of labeled globulin from the plasma at a rate considerably faster than albumin has been confirmed. Evidence suggesting that the mass of protein in solution in the extravascular, extracellular fluid is approximately equal to the plasma proteins in circulation has been derived from a study of the dilution of labeled plasma protein by repeated injections of non-labeled plasma protein. In a period of 7 days the transfer of C14 from plasma to tissue proteins amounted to between 30 and 40 per cent of the activity in the labeled plasma protein injected intravenously. The conversion was accompanied by a very small loss of activity in the urine and expired air and the activity remained in the lysine residue of the liver and probably of other tissues. The data presented favor the view that plasma proteins are utilized in the body economy after partial catabolism within the cell area and provide no evidence of complete breakdown to the amino acid level. PMID:14832401

Benchmarking protein-protein interface predictions: why you should care about protein size.

PubMed

Martin, Juliette

2014-07-01

A number of predictive methods have been developed to predict protein-protein binding sites. Each new method is traditionally benchmarked using sets of protein structures of various sizes, and global statistics are used to assess the quality of the prediction. Little attention has been paid to the potential bias due to protein size on these statistics. Indeed, small proteins involve proportionally more residues at interfaces than large ones. If a predictive method is biased toward small proteins, this can lead to an over-estimation of its performance. Here, we investigate the bias due to the size effect when benchmarking protein-protein interface prediction on the widely used docking benchmark 4.0. First, we simulate random scores that favor small proteins over large ones. Instead of the 0.5 AUC (Area Under the Curve) value expected by chance, these biased scores result in an AUC equal to 0.6 using hypergeometric distributions, and up to 0.65 using constant scores. We then use real prediction results to illustrate how to detect the size bias by shuffling, and subsequently correct it using a simple conversion of the scores into normalized ranks. In addition, we investigate the scores produced by eight published methods and show that they are all affected by the size effect, which can change their relative ranking. The size effect also has an impact on linear combination scores by modifying the relative contributions of each method. In the future, systematic corrections should be applied when benchmarking predictive methods using data sets with mixed protein sizes. © 2014 Wiley Periodicals, Inc.

Rigid-Docking Approaches to Explore Protein-Protein Interaction Space.

PubMed

Matsuzaki, Yuri; Uchikoga, Nobuyuki; Ohue, Masahito; Akiyama, Yutaka

Protein-protein interactions play core roles in living cells, especially in the regulatory systems. As information on proteins has rapidly accumulated on publicly available databases, much effort has been made to obtain a better picture of protein-protein interaction networks using protein tertiary structure data. Predicting relevant interacting partners from their tertiary structure is a challenging task and computer science methods have the potential to assist with this. Protein-protein rigid docking has been utilized by several projects, docking-based approaches having the advantages that they can suggest binding poses of predicted binding partners which would help in understanding the interaction mechanisms and that comparing docking results of both non-binders and binders can lead to understanding the specificity of protein-protein interactions from structural viewpoints. In this review we focus on explaining current computational prediction methods to predict pairwise direct protein-protein interactions that form protein complexes.

Reverse Nearest Neighbor Search on a Protein-Protein Interaction Network to Infer Protein-Disease Associations.

PubMed

Suratanee, Apichat; Plaimas, Kitiporn

2017-01-01

The associations between proteins and diseases are crucial information for investigating pathological mechanisms. However, the number of known and reliable protein-disease associations is quite small. In this study, an analysis framework to infer associations between proteins and diseases was developed based on a large data set of a human protein-protein interaction network integrating an effective network search, namely, the reverse k -nearest neighbor (R k NN) search. The R k NN search was used to identify an impact of a protein on other proteins. Then, associations between proteins and diseases were inferred statistically. The method using the R k NN search yielded a much higher precision than a random selection, standard nearest neighbor search, or when applying the method to a random protein-protein interaction network. All protein-disease pair candidates were verified by a literature search. Supporting evidence for 596 pairs was identified. In addition, cluster analysis of these candidates revealed 10 promising groups of diseases to be further investigated experimentally. This method can be used to identify novel associations to better understand complex relationships between proteins and diseases.

Purification of Proteins Fused to Maltose-Binding Protein.

PubMed

Lebendiker, Mario; Danieli, Tsafi

2017-01-01

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

Characterization of Vibrio parahaemolyticus isolated from oysters in Korea: Resistance to various antibiotics and prevalence of virulence genes.

PubMed

Kang, Chang-Ho; Shin, YuJin; Jang, SeokCheol; Yu, HongSik; Kim, SuKyung; An, Sera; Park, Kunbawui; So, Jae-Seong

2017-05-15

Vibrio parahaemolyticus, found frequently in oysters, is the most prevalent gastroenteritis-causing pathogen in Korea and in several other Asian countries. This study monitored changes in the environmental parameters and occurrence of V. parahaemolyticus in oyster aquaculture sites. Of the 44 presumed V. parahaemolyticus isolates obtained, when tested against 16 antibiotics, 90.9, 86.4, and 75.0% of the 44 isolates exhibited resistance to vancomycin, ampicillin, and streptomycin, respectively. PCR analysis for the presence of the toxR gene confirmed 31 of the 44 isolates as being positive V. parahaemolyticus strains. The toxR positive isolates were tested for the presence of thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) virulence genes. Only 9.1% toxR positive isolate exhibit the trh gene and none of the isolates were tested positive for tdh. The occurrence of multi drug resistance strains in the environment could be an indication of excessive usage of antibiotics in agriculture and aquaculture fields. Copyright © 2017 Elsevier Ltd. All rights reserved.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

PubMed

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-X(L), IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

Manipulating Protein-Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins.

PubMed

Jaremko, Matt J; Lee, D John; Patel, Ashay; Winslow, Victoria; Opella, Stanley J; McCammon, J Andrew; Burkart, Michael D

2017-10-10

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein-protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

NASA Astrophysics Data System (ADS)

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

Protein-Protein Interface and Disease: Perspective from Biomolecular Networks.

PubMed

Hu, Guang; Xiao, Fei; Li, Yuqian; Li, Yuan; Vongsangnak, Wanwipa

Protein-protein interactions are involved in many important biological processes and molecular mechanisms of disease association. Structural studies of interfacial residues in protein complexes provide information on protein-protein interactions. Characterizing protein-protein interfaces, including binding sites and allosteric changes, thus pose an imminent challenge. With special focus on protein complexes, approaches based on network theory are proposed to meet this challenge. In this review we pay attention to protein-protein interfaces from the perspective of biomolecular networks and their roles in disease. We first describe the different roles of protein complexes in disease through several structural aspects of interfaces. We then discuss some recent advances in predicting hot spots and communication pathway analysis in terms of amino acid networks. Finally, we highlight possible future aspects of this area with respect to both methodology development and applications for disease treatment.

Protein-protein recognition control by modulating electrostatic interactions.

PubMed

Han, Song; Yin, Shijin; Yi, Hong; Mouhat, Stéphanie; Qiu, Su; Cao, Zhijian; Sabatier, Jean-Marc; Wu, Yingliang; Li, Wenxin

2010-06-04

Protein-protein control recognition remains a huge challenge, and its development depends on understanding the chemical and biological mechanisms by which these interactions occur. Here we describe a protein-protein control recognition technique based on the dominant electrostatic interactions occurring between the proteins. We designed a potassium channel inhibitor, BmP05-T, that was 90.32% identical to wild-type BmP05. Negatively charged residues were translocated from the nonbinding interface to the binding interface of BmP05 inhibitor, such that BmP05-T now used BmP05 nonbinding interface as the binding interface. This switch demonstrated that nonbinding interfaces were able to control the orientation of protein binding interfaces in the process of protein-protein recognition. The novel function findings of BmP05-T peptide suggested that the control recognition technique described here had the potential for use in designing and utilizing functional proteins in many biological scenarios.

Biophysics of protein evolution and evolutionary protein biophysics

PubMed Central

Sikosek, Tobias; Chan, Hue Sun

2014-01-01

The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence–structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by ‘hidden’ conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution. PMID:25165599

Protein and protein hydrolysates in sports nutrition.

PubMed

van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

2007-08-01

With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

A modified Lowry protein test for dilute protein solutions

Treesearch

Garold F. Gregory; Keith F. Jensen

1971-01-01

A modified Lowry protein test for dilute protein solutions modified Lowry protein test was compared with the standard Lowry protein test. The modified test was found to give estimates of protein concentration that were as good as the standard test and has the advange that proteins can be measured in very dilute solutions.

The Role of Shape Complementarity in the Protein-Protein Interactions

NASA Astrophysics Data System (ADS)

Li, Ye; Zhang, Xianren; Cao, Dapeng

2013-11-01

We use a dissipative particle dynamic simulation to investigate the effects of shape complementarity on the protein-protein interactions. By monitoring different kinds of protein shape-complementarity modes, we gave a clear mechanism to reveal the role of the shape complementarity in the protein-protein interactions, i.e., when the two proteins with shape complementarity approach each other, the conformation of lipid chains between two proteins would be restricted significantly. The lipid molecules tend to leave the gap formed by two proteins to maximize the configuration entropy, and therefore yield an effective entropy-induced protein-protein attraction, which enhances the protein aggregation. In short, this work provides an insight into understanding the importance of the shape complementarity in the protein-protein interactions especially for protein aggregation and antibody-antigen complexes. Definitely, the shape complementarity is the third key factor affecting protein aggregation and complex, besides the electrostatic-complementarity and hydrophobic complementarity.

Electrostatic design of protein-protein association rates.

PubMed

Schreiber, Gideon; Shaul, Yossi; Gottschalk, Kay E

2006-01-01

De novo design and redesign of proteins and protein complexes have made promising progress in recent years. Here, we give an overview of how to use available computer-based tools to design proteins to bind faster and tighter to their protein-complex partner by electrostatic optimization between the two proteins. Electrostatic optimization is possible because of the simple relation between the Debye-Huckel energy of interaction between a pair of proteins and their rate of association. This can be used for rapid, structure-based calculations of the electrostatic attraction between the two proteins in the complex. Using these principles, we developed two computer programs that predict the change in k(on), and as such the affinity, on introducing charged mutations. The two programs have a web interface that is available at www.weizmann.ac.il/home/bcges/PARE.html and http://bip.weizmann.ac.il/hypare. When mutations leading to charge optimization are introduced outside the physical binding site, the rate of dissociation is unchanged and therefore the change in k(on) parallels that of the affinity. This design method was evaluated on a number of different protein complexes resulting in binding rates and affinities of hundreds of fold faster and tighter compared to wild type. In this chapter, we demonstrate the procedure and go step by step over the methodology of using these programs for protein-association design. Finally, the way to easily implement the principle of electrostatic design for any protein complex of choice is shown.

Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

PubMed

Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

2018-01-15

The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

A Method for Predicting Protein Complexes from Dynamic Weighted Protein-Protein Interaction Networks.

PubMed

Liu, Lizhen; Sun, Xiaowu; Song, Wei; Du, Chao

2018-06-01

Predicting protein complexes from protein-protein interaction (PPI) network is of great significance to recognize the structure and function of cells. A protein may interact with different proteins under different time or conditions. Existing approaches only utilize static PPI network data that may lose much temporal biological information. First, this article proposed a novel method that combines gene expression data at different time points with traditional static PPI network to construct different dynamic subnetworks. Second, to further filter out the data noise, the semantic similarity based on gene ontology is regarded as the network weight together with the principal component analysis, which is introduced to deal with the weight computing by three traditional methods. Third, after building a dynamic PPI network, a predicting protein complexes algorithm based on "core-attachment" structural feature is applied to detect complexes from each dynamic subnetworks. Finally, it is revealed from the experimental results that our method proposed in this article performs well on detecting protein complexes from dynamic weighted PPI networks.

A protein interaction network analysis for yeast integral membrane protein.

PubMed

Shi, Ming-Guang; Huang, De-Shuang; Li, Xue-Ling

2008-01-01

Although the yeast Saccharomyces cerevisiae is the best exemplified single-celled eukaryote, the vast number of protein-protein interactions of integral membrane proteins of Saccharomyces cerevisiae have not been characterized by experiments. Here, based on the kernel method of Greedy Kernel Principal Component analysis plus Linear Discriminant Analysis, we identify 300 protein-protein interactions involving 189 membrane proteins and get the outcome of a highly connected protein-protein interactions network. Furthermore, we study the global topological features of integral membrane proteins network of Saccharomyces cerevisiae. These results give the comprehensive description of protein-protein interactions of integral membrane proteins and reveal global topological and robustness of the interactome network at a system level. This work represents an important step towards a comprehensive understanding of yeast protein interactions.

Protein kinesis: The dynamics of protein trafficking and stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

The Role of Shape Complementarity in the Protein-Protein Interactions

PubMed Central

Li, Ye; Zhang, Xianren; Cao, Dapeng

2013-01-01

We use a dissipative particle dynamic simulation to investigate the effects of shape complementarity on the protein-protein interactions. By monitoring different kinds of protein shape-complementarity modes, we gave a clear mechanism to reveal the role of the shape complementarity in the protein-protein interactions, i.e., when the two proteins with shape complementarity approach each other, the conformation of lipid chains between two proteins would be restricted significantly. The lipid molecules tend to leave the gap formed by two proteins to maximize the configuration entropy, and therefore yield an effective entropy-induced protein-protein attraction, which enhances the protein aggregation. In short, this work provides an insight into understanding the importance of the shape complementarity in the protein-protein interactions especially for protein aggregation and antibody–antigen complexes. Definitely, the shape complementarity is the third key factor affecting protein aggregation and complex, besides the electrostatic-complementarity and hydrophobic complementarity. PMID:24253561

FRODOCK 2.0: fast protein-protein docking server.

PubMed

Ramírez-Aportela, Erney; López-Blanco, José Ramón; Chacón, Pablo

2016-08-01

The prediction of protein-protein complexes from the structures of unbound components is a challenging and powerful strategy to decipher the mechanism of many essential biological processes. We present a user-friendly protein-protein docking server based on an improved version of FRODOCK that includes a complementary knowledge-based potential. The web interface provides a very effective tool to explore and select protein-protein models and interactively screen them against experimental distance constraints. The competitive success rates and efficiency achieved allow the retrieval of reliable potential protein-protein binding conformations that can be further refined with more computationally demanding strategies. The server is free and open to all users with no login requirement at http://frodock.chaconlab.org pablo@chaconlab.org Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Computational prediction of host-pathogen protein-protein interactions.

PubMed

Dyer, Matthew D; Murali, T M; Sobral, Bruno W

2007-07-01

Infectious diseases such as malaria result in millions of deaths each year. An important aspect of any host-pathogen system is the mechanism by which a pathogen can infect its host. One method of infection is via protein-protein interactions (PPIs) where pathogen proteins target host proteins. Developing computational methods that identify which PPIs enable a pathogen to infect a host has great implications in identifying potential targets for therapeutics. We present a method that integrates known intra-species PPIs with protein-domain profiles to predict PPIs between host and pathogen proteins. Given a set of intra-species PPIs, we identify the functional domains in each of the interacting proteins. For every pair of functional domains, we use Bayesian statistics to assess the probability that two proteins with that pair of domains will interact. We apply our method to the Homo sapiens-Plasmodium falciparum host-pathogen system. Our system predicts 516 PPIs between proteins from these two organisms. We show that pairs of human proteins we predict to interact with the same Plasmodium protein are close to each other in the human PPI network and that Plasmodium pairs predicted to interact with same human protein are co-expressed in DNA microarray datasets measured during various stages of the Plasmodium life cycle. Finally, we identify functionally enriched sub-networks spanned by the predicted interactions and discuss the plausibility of our predictions. Supplementary data are available at http://staff.vbi.vt.edu/dyermd/publications/dyer2007a.html. Supplementary data are available at Bioinformatics online.

Investigation of whether the acute hemolysis associated with Rho(D) immune globulin intravenous (human) administration for treatment of immune thrombocytopenic purpura is consistent with the acute hemolytic transfusion reaction model

PubMed Central

Gaines, Ann Reed; Lee-Stroka, Hallie; Byrne, Karen; Scott, Dorothy E.; Uhl, Lynne; Lazarus, Ellen; Stroncek, David F.

2012-01-01

BACKGROUND Immune thrombocytopenic purpura and secondary thrombocytopenia patients treated with Rho(D) immune globulin intravenous (human; anti-D IGIV) have experienced acute hemolysis, which is inconsistent with the typical presentation of extravascular hemolysis—the presumed mechanism of action of anti-D IGIV. Although the mechanism of anti-D-IGIV–associated acute hemolysis has not been established, the onset, signs/symptoms, and complications appear consistent with the intravascular hemolysis of acute hemolytic transfusion reactions (AHTRs). In transfusion medicine, the red blood cell (RBC) antigen-antibody incompatibility(-ies) that precipitate AHTRs can be detected in vitro with compatibility testing. Under the premise that anti-D-IGIV–associated acute hemolysis results from RBC antigen-antibody–mediated complement activation, this study evaluated whether the incompatibility(-ies) could be detected in vitro with a hemolysin assay, which would support the AHTR model as the hemolytic mechanism. STUDY DESIGN AND METHODS Seven anti-D IGIV lots were tested to determine the RBC antibody identities in those lots, including four lots that had been implicated in acute hemolytic episodes. Hemolysin assays were performed that tested each of 73 RBC specimens against each lot, including the RBCs of one patient who had experienced acute hemolysis after anti-D IGIV administration. RESULTS Only two anti-D IGIV lots contained RBC antibodies beyond those expected. No hemolysis endpoint was observed in any of the hemolysin assays. CONCLUSION Although the findings did not support the AHTR model, the results are reported to contribute knowledge about the mechanism of anti-D-IGIV–associated acute hemolysis and to prompt continued investigation into cause(s), prediction, and prevention of this potentially serious adverse event. PMID:19220820

Contribution of hly homologs to the hemolytic activity of Prevotella intermedia.

PubMed

Suzuki, Naoko; Fukamachi, Haruka; Arimoto, Takafumi; Yamamoto, Matsuo; Igarashi, Takeshi

2012-06-01

Prevotella intermedia is a periodontal pathogen that requires iron for its growth. Although this organism has hemolytic activity, the precise nature of its hemolytic substances and their associated hemolytic actions are yet to be fully determined. In the present study, we identified and characterized several putative hly genes in P. intermedia ATCC25611 which appear to encode hemolysins. Six hly genes (hlyA, B, C, D, E, and hlyI) of P. intermedia were identified by comparing their nucleotide sequences to those of known hly genes of Bacteroides fragilis NCTC9343. The hlyA-E, and hlyI genes were overexpressed individually in the non-hemolytic Escherichia coli strain JW5181 and examined its contribution to the hemolytic activity on sheep blood agar plates. E. coli cells expressing the hlyA and hlyI genes exhibited hemolytic activity under anaerobic conditions. On the other hand, only E. coli cells stably expressing the hlyA gene were able to lyse the red blood cells when cultured under aerobic conditions. In addition, expression of the hlyA and hlyI genes was significantly upregulated in the presence of red blood cells. Furthermore, we found that the growth of P. intermedia was similar in an iron-limited medium supplemented with either red blood cells or heme. Taken together, our results indicate that the hlyA and hlyI genes of P. intermedia encode putative hemolysins that appear to be involved in the lysis of red blood cells, and suggest that these hemolysins might play important roles in the iron-dependent growth of this organism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structurally designed attenuated subunit vaccines for S. aureus LukS-PV and LukF-PV confer protection in a mouse bacteremia model.

PubMed

Karauzum, Hatice; Adhikari, Rajan P; Sarwar, Jawad; Devi, V Sathya; Abaandou, Laura; Haudenschild, Christian; Mahmoudieh, Mahta; Boroun, Atefeh R; Vu, Hong; Nguyen, Tam; Warfield, Kelly L; Shulenin, Sergey; Aman, M Javad

2013-01-01

Previous efforts towards S. aureus vaccine development have largely focused on cell surface antigens to induce opsonophagocytic killing aimed at providing sterile immunity, a concept successfully applied to other Gram-positive pathogens such as Streptococcus pneumoniae. However, these approaches have largely failed, possibly in part due to the remarkable diversity of the staphylococcal virulence factors such as secreted immunosuppressive and tissue destructive toxins. S. aureus produces several pore-forming toxins including the single subunit alpha hemolysin as well as bicomponent leukotoxins such as Panton-Valentine leukocidin (PVL), gamma hemolysins (Hlg), and LukED. Here we report the generation of highly attenuated mutants of PVL subunits LukS-PV and LukF-PV that were rationally designed, based on an octameric structural model of the toxin, to be deficient in oligomerization. The attenuated subunit vaccines were highly immunogenic and showed significant protection in a mouse model of S. aureus USA300 sepsis. Protection against sepsis was also demonstrated by passive transfer of rabbit immunoglobulin raised against LukS-PV. Antibodies to LukS-PV inhibited the homologous oligomerization of LukS-PV with LukF-PV as well heterologous oligomerization with HlgB. Importantly, immune sera from mice vaccinated with the LukS mutant not only inhibited the PMN lytic activity produced by the PVL-positive USA300 but also blocked PMN lysis induced by supernatants of PVL-negative strains suggesting a broad protective activity towards other bicomponent toxins. These findings strongly support the novel concept of an anti-virulence, toxin-based vaccine intended for prevention of clinical S. aureus invasive disease, rather than achieving sterile immunity. Such a multivalent vaccine may include attenuated leukotoxins, alpha hemolysin, and superantigens.

Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters▿

PubMed Central

Nordstrom, Jessica L.; Vickery, Michael C. L.; Blackstone, George M.; Murray, Shelley L.; DePaola, Angelo

2007-01-01

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh+ and trh+ strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus. PMID:17644647

The De Novo Design of Protein-Protein Interfaces

DTIC Science & Technology

it was our intention to add to this body by engineering de novo (from scratch) protein/protein complexes. Using this inverse approach we have furthered...key physical features needed to drive specific protein/protein interactions. It is considered inverse because, instead of studying natural complexes

Influence of Protein Abundance on High-Throughput Protein-Protein Interaction Detection

DTIC Science & Technology

2009-06-05

the interaction data sets we determined, via comparisons with strict randomized simulations , the propensity for essential proteins to selectively...and analysis of high- quality PPI data sets. Materials and Methods We analyzed protein interaction networks for yeast and E. coli determined from Y2H...we reinvestigated the centrality-lethality rule, which implies that proteins having more interactions are more likely to be essential. From analysis

ProtPhylo: identification of protein-phenotype and protein-protein functional associations via phylogenetic profiling.

PubMed

Cheng, Yiming; Perocchi, Fabiana

2015-07-01

ProtPhylo is a web-based tool to identify proteins that are functionally linked to either a phenotype or a protein of interest based on co-evolution. ProtPhylo infers functional associations by comparing protein phylogenetic profiles (co-occurrence patterns of orthology relationships) for more than 9.7 million non-redundant protein sequences from all three domains of life. Users can query any of 2048 fully sequenced organisms, including 1678 bacteria, 255 eukaryotes and 115 archaea. In addition, they can tailor ProtPhylo to a particular kind of biological question by choosing among four main orthology inference methods based either on pair-wise sequence comparisons (One-way Best Hits and Best Reciprocal Hits) or clustering of orthologous proteins across multiple species (OrthoMCL and eggNOG). Next, ProtPhylo ranks phylogenetic neighbors of query proteins or phenotypic properties using the Hamming distance as a measure of similarity between pairs of phylogenetic profiles. Candidate hits can be easily and flexibly prioritized by complementary clues on subcellular localization, known protein-protein interactions, membrane spanning regions and protein domains. The resulting protein list can be quickly exported into a csv text file for further analyses. ProtPhylo is freely available at http://www.protphylo.org. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The NMR contribution to protein-protein networking in Fe-S protein maturation.

PubMed

Banci, Lucia; Camponeschi, Francesca; Ciofi-Baffoni, Simone; Piccioli, Mario

2018-03-22

Iron-sulfur proteins were among the first class of metalloproteins that were actively studied using NMR spectroscopy tailored to paramagnetic systems. The hyperfine shifts, their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues are an efficient fingerprint of the nature and the oxidation state of the Fe-S cluster. NMR significantly contributed to the analysis of the magnetic coupling patterns and to the understanding of the electronic structure occurring in [2Fe-2S], [3Fe-4S] and [4Fe-4S] clusters bound to proteins. After the first NMR structure of a paramagnetic protein was obtained for the reduced E. halophila HiPIP I, many NMR structures were determined for several Fe-S proteins in different oxidation states. It was found that differences in chemical shifts, in patterns of unobserved residues, in internal mobility and in thermodynamic stability are suitable data to map subtle changes between the two different oxidation states of the protein. Recently, the interaction networks responsible for maturing human mitochondrial and cytosolic Fe-S proteins have been largely characterized by combining solution NMR standard experiments with those tailored to paramagnetic systems. We show here the contribution of solution NMR in providing a detailed molecular view of "Fe-S interactomics". This contribution was particularly effective when protein-protein interactions are weak and transient, and thus difficult to be characterized at high resolution with other methodologies.

Molecular tweezers modulate 14-3-3 protein-protein interactions

NASA Astrophysics Data System (ADS)

Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

2013-03-01

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

Predicting Functions of Proteins in Mouse Based on Weighted Protein-Protein Interaction Network and Protein Hybrid Properties

PubMed Central

Shi, Xiaohe; Lu, Wen-Cong; Cai, Yu-Dong; Chou, Kuo-Chen

2011-01-01

Background With the huge amount of uncharacterized protein sequences generated in the post-genomic age, it is highly desirable to develop effective computational methods for quickly and accurately predicting their functions. The information thus obtained would be very useful for both basic research and drug development in a timely manner. Methodology/Principal Findings Although many efforts have been made in this regard, most of them were based on either sequence similarity or protein-protein interaction (PPI) information. However, the former often fails to work if a query protein has no or very little sequence similarity to any function-known proteins, while the latter had similar problem if the relevant PPI information is not available. In view of this, a new approach is proposed by hybridizing the PPI information and the biochemical/physicochemical features of protein sequences. The overall first-order success rates by the new predictor for the functions of mouse proteins on training set and test set were 69.1% and 70.2%, respectively, and the success rate covered by the results of the top-4 order from a total of 24 orders was 65.2%. Conclusions/Significance The results indicate that the new approach is quite promising that may open a new avenue or direction for addressing the difficult and complicated problem. PMID:21283518

TGF-beta signaling proteins and the Protein Ontology.

PubMed

Arighi, Cecilia N; Liu, Hongfang; Natale, Darren A; Barker, Winona C; Drabkin, Harold; Blake, Judith A; Smith, Barry; Wu, Cathy H

2009-05-06

The Protein Ontology (PRO) is designed as a formal and principled Open Biomedical Ontologies (OBO) Foundry ontology for proteins. The components of PRO extend from a classification of proteins on the basis of evolutionary relationships at the homeomorphic level to the representation of the multiple protein forms of a gene, including those resulting from alternative splicing, cleavage and/or post-translational modifications. Focusing specifically on the TGF-beta signaling proteins, we describe the building, curation, usage and dissemination of PRO. PRO is manually curated on the basis of PrePRO, an automatically generated file with content derived from standard protein data sources. Manual curation ensures that the treatment of the protein classes and the internal and external relationships conform to the PRO framework. The current release of PRO is based upon experimental data from mouse and human proteins wherein equivalent protein forms are represented by single terms. In addition to the PRO ontology, the annotation of PRO terms is released as a separate PRO association file, which contains, for each given PRO term, an annotation from the experimentally characterized sub-types as well as the corresponding database identifiers and sequence coordinates. The annotations are added in the form of relationship to other ontologies. Whenever possible, equivalent forms in other species are listed to facilitate cross-species comparison. Splice and allelic variants, gene fusion products and modified protein forms are all represented as entities in the ontology. Therefore, PRO provides for the representation of protein entities and a resource for describing the associated data. This makes PRO useful both for proteomics studies where isoforms and modified forms must be differentiated, and for studies of biological pathways, where representations need to take account of the different ways in which the cascade of events may depend on specific protein modifications. PRO provides

Energy Landscape and Transition State of Protein-Protein Association

NASA Astrophysics Data System (ADS)

Alsallaq, Ramzi; Zhou, Huan-Xiang

2006-11-01

Formation of a stereospecific protein complex is favored by specific interactions between two proteins but disfavored by the loss of translational and rotational freedom. Echoing the protein folding process, we have previously proposed a transition state for protein-protein association. Here we clarify the specification of the transition state by working with two toy models for protein association. The models demonstrate that a sharp transition between the bound state with numerous short-range interactions but restricted translation and rotational freedom and the unbound state with at most a small number of interactions but expanded configurational freedom. This transition sets the outer boundary of the bound state as well as the transition state for association. The energy landscape is funnel-like, with the deep well of the bound state surrounded by a broad shallow basin. This formalism of protein-protein association is applied to four protein-protein complexes, and is found to give accurate predictions for the effects of charge mutations and ionic strength on the association rates.

An ontology-based search engine for protein-protein interactions.

PubMed

Park, Byungkyu; Han, Kyungsook

2010-01-18

Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology.

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

PubMed Central

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Prediction of Heterodimeric Protein Complexes from Weighted Protein-Protein Interaction Networks Using Novel Features and Kernel Functions

PubMed Central

Ruan, Peiying; Hayashida, Morihiro; Maruyama, Osamu; Akutsu, Tatsuya

2013-01-01

Since many proteins express their functional activity by interacting with other proteins and forming protein complexes, it is very useful to identify sets of proteins that form complexes. For that purpose, many prediction methods for protein complexes from protein-protein interactions have been developed such as MCL, MCODE, RNSC, PCP, RRW, and NWE. These methods have dealt with only complexes with size of more than three because the methods often are based on some density of subgraphs. However, heterodimeric protein complexes that consist of two distinct proteins occupy a large part according to several comprehensive databases of known complexes. In this paper, we propose several feature space mappings from protein-protein interaction data, in which each interaction is weighted based on reliability. Furthermore, we make use of prior knowledge on protein domains to develop feature space mappings, domain composition kernel and its combination kernel with our proposed features. We perform ten-fold cross-validation computational experiments. These results suggest that our proposed kernel considerably outperforms the naive Bayes-based method, which is the best existing method for predicting heterodimeric protein complexes. PMID:23776458

Light-scattering studies of protein solutions: role of hydration in weak protein-protein interactions.

PubMed

Paliwal, A; Asthagiri, D; Abras, D; Lenhoff, A M; Paulaitis, M E

2005-09-01

We model the hydration contribution to short-range electrostatic/dispersion protein interactions embodied in the osmotic second virial coefficient, B(2), by adopting a quasi-chemical description in which water molecules associated with the protein are identified through explicit molecular dynamics simulations. These water molecules reduce the surface complementarity of highly favorable short-range interactions, and therefore can play an important role in mediating protein-protein interactions. Here we examine this quasi-chemical view of hydration by predicting the interaction part of B(2) and comparing our results with those derived from light-scattering measurements of B(2) for staphylococcal nuclease, lysozyme, and chymotrypsinogen at 25 degrees C as a function of solution pH and ionic strength. We find that short-range protein interactions are influenced by water molecules strongly associated with a relatively small fraction of the protein surface. However, the effect of these strongly associated water molecules on the surface complementarity of short-range protein interactions is significant, and must be taken into account for an accurate description of B(2). We also observe remarkably similar hydration behavior for these proteins despite substantial differences in their three-dimensional structures and spatial charge distributions, suggesting a general characterization of protein hydration.

Computational Prediction of Protein-Protein Interactions

PubMed Central

Ehrenberger, Tobias; Cantley, Lewis C.; Yaffe, Michael B.

2015-01-01

The prediction of protein-protein interactions and kinase-specific phosphorylation sites on individual proteins is critical for correctly placing proteins within signaling pathways and networks. The importance of this type of annotation continues to increase with the continued explosion of genomic and proteomic data, particularly with emerging data categorizing posttranslational modifications on a large scale. A variety of computational tools are available for this purpose. In this chapter, we review the general methodologies for these types of computational predictions and present a detailed user-focused tutorial of one such method and computational tool, Scansite, which is freely available to the entire scientific community over the Internet. PMID:25859943

Protein Condensation

NASA Astrophysics Data System (ADS)

Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

2007-09-01

Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

Protein Condensation

NASA Astrophysics Data System (ADS)

Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

2014-07-01

Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed Central

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-01-01

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions. Images PMID:2556266

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-12-20

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

PubMed

Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

2017-08-01

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

PubMed Central

Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

2012-01-01

Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with

Attributes of Stachybotrys chartarum and its association with human disease.

PubMed

Hossain, Mohammad Ashraf; Ahmed, Mohamed Sotohy; Ghannoum, Mahmoud Afif

2004-02-01

Mold contamination and toxicities are not limited to crops and animals; they are also a concern in human health. Molds occur in outdoor and indoor environments, and water-damaged buildings harbor and provide substrate for several mold species. Of these, Stachybotrys chartarum poses a particular threat to occupants. Patients with building-related symptoms and infant idiopathic pulmonary hemorrhage often have histories of living in moldy, water-damaged buildings. Although a causal connection is far from being unequivocally proven, S. chartarum has been associated with such clinical conditions. These illnesses could be attributed in part to mycotoxins released by S. chartarum. Recently, a hemolysin released by this mold was found to be hemolytic in vitro and in vivo. In addition, allergenic proteins have been characterized from S. chartarum. The exact mechanism of S. chartarum pathogenesis has not yet been defined. Moreover, a causality-effect relation is not yet established. This review summarizes available information on the pathogenic attributes of S. chartarum and calls for well-controlled objective studies.

Detection of single ion channel activity with carbon nanotubes

NASA Astrophysics Data System (ADS)

Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J.

2015-03-01

Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level.

Protein subcellular localization assays using split fluorescent proteins

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2009-09-08

The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

An ontology-based search engine for protein-protein interactions

PubMed Central

2010-01-01

Background Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. Results We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Conclusion Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology. PMID:20122195

Hot-spot analysis for drug discovery targeting protein-protein interactions.

PubMed

Rosell, Mireia; Fernández-Recio, Juan

2018-04-01

Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

Protein-induced satiety: effects and mechanisms of different proteins.

PubMed

Veldhorst, M; Smeets, A; Soenen, S; Hochstenbach-Waelen, A; Hursel, R; Diepvens, K; Lejeune, M; Luscombe-Marsh, N; Westerterp-Plantenga, M

2008-05-23

Relatively high protein diets, i.e. diets that maintain the absolute number of grams of protein ingested as compared to before dieting, are a popular strategy for weight loss and weight maintenance. Research into multiple mechanisms regulating body weight has focused on the effects of different quantities and types of dietary protein. Satiety and energy expenditure are important in protein-enhanced weight loss and weight maintenance. Protein-induced satiety has been shown acutely, with single meals, with contents of 25% to 81% of energy from protein in general or from specific proteins, while subsequent energy intake reduction was significant. Protein-induced satiety has been shown with high protein ad libitum diets, lasting from 1 to 6 days, up to 6 months. Also significantly greater weight loss has been observed in comparison with control. Mechanisms explaining protein-induced satiety are nutrient-specific, and consist mainly of synchronization with elevated amino acid concentrations. Different proteins cause different nutrient related responses of (an)orexigenic hormones. Protein-induced satiety coincides with a relatively high GLP-1 release, stimulated by the carbohydrate content of the diet, PYY release, while ghrelin does not seem to be especially affected, and little information is available on CCK. Protein-induced satiety is related to protein-induced energy expenditure. Finally, protein-induced satiety appears to be of vital importance for weight loss and weight maintenance. With respect to possible adverse events, chronic ingestion of large amounts of sulphur-containing amino acids may have an indirect effect on blood pressure by induction of renal subtle structural damage, ultimately leading to loss of nephron mass, and a secondary increase in blood pressure. The established synergy between obesity and low nephron number on induction of high blood pressure and further decline of renal function identifies subjects with obesity, metabolic syndrome and

A Discontinuous Potential Model for Protein-Protein Interactions.

PubMed

Shao, Qing; Hall, Carol K

2016-01-01

Protein-protein interactions play an important role in many biologic and industrial processes. In this work, we develop a two-bead-per-residue model that enables us to account for protein-protein interactions in a multi-protein system using discontinuous molecular dynamics simulations. This model deploys discontinuous potentials to describe the non-bonded interactions and virtual bonds to keep proteins in their native state. The geometric and energetic parameters are derived from the potentials of mean force between sidechain-sidechain, sidechain-backbone, and backbone-backbone pairs. The energetic parameters are scaled with the aim of matching the second virial coefficient of lysozyme reported in experiment. We also investigate the performance of several bond-building strategies.

Protein Charge and Mass Contribute to the Spatio-temporal Dynamics of Protein-Protein Interactions in a Minimal Proteome

PubMed Central

Xu, Yu; Wang, Hong; Nussinov, Ruth; Ma, Buyong

2013-01-01

We constructed and simulated a ‘minimal proteome’ model using Langevin dynamics. It contains 206 essential protein types which were compiled from the literature. For comparison, we generated six proteomes with randomized concentrations. We found that the net charges and molecular weights of the proteins in the minimal genome are not random. The net charge of a protein decreases linearly with molecular weight, with small proteins being mostly positively charged and large proteins negatively charged. The protein copy numbers in the minimal genome have the tendency to maximize the number of protein-protein interactions in the network. Negatively charged proteins which tend to have larger sizes can provide large collision cross-section allowing them to interact with other proteins; on the other hand, the smaller positively charged proteins could have higher diffusion speed and are more likely to collide with other proteins. Proteomes with random charge/mass populations form less stable clusters than those with experimental protein copy numbers. Our study suggests that ‘proper’ populations of negatively and positively charged proteins are important for maintaining a protein-protein interaction network in a proteome. It is interesting to note that the minimal genome model based on the charge and mass of E. Coli may have a larger protein-protein interaction network than that based on the lower organism M. pneumoniae. PMID:23420643

NMR Studies of Protein Hydration and Protein-Ligand Interactions

NASA Astrophysics Data System (ADS)

Chong, Yuan

Water on the surface of a protein is called hydration water. Hydration water is known to play a crucial role in a variety of biological processes including protein folding, enzymatic activation, and drug binding. Although the significance of hydration water has been recognized, the underlying mechanism remains far from being understood. This dissertation employs a unique in-situ nuclear magnetic resonance (NMR) technique to study the mechanism of protein hydration and the role of hydration in alcohol-protein interactions. Water isotherms in proteins are measured at different temperatures via the in-situ NMR technique. Water is found to interact differently with hydrophilic and hydrophobic groups on the protein. Water adsorption on hydrophilic groups is hardly affected by the temperature, while water adsorption on hydrophobic groups strongly depends on the temperature around 10 C, below which the adsorption is substantially reduced. This effect is induced by the dramatic decrease in the protein flexibility below 10 C. Furthermore, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are studied as a function of hydration level and temperature. A crossover at 10 C in protein dynamics and thermodynamics is revealed. The effect of water at hydrophilic groups on protein dynamics and thermodynamics shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 C. In addition, I investigate the role of water in alcohol binding to the protein using the in-situ NMR detection. The isotherms of alcohols are first measured on dry proteins, then on proteins with a series of controlled hydration levels. The free energy, enthalpy, and entropy of alcohol binding are also determined. Two distinct types of alcohol binding are identified. On the one hand, alcohols can directly bind to a few specific sites on the protein. This type of binding is independent of temperature and can be

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

PubMed Central

Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

2009-01-01

Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254

DiffSLC: A graph centrality method to detect essential proteins of a protein-protein interaction network.

PubMed

Mistry, Divya; Wise, Roger P; Dickerson, Julie A

2017-01-01

Identification of central genes and proteins in biomolecular networks provides credible candidates for pathway analysis, functional analysis, and essentiality prediction. The DiffSLC centrality measure predicts central and essential genes and proteins using a protein-protein interaction network. Network centrality measures prioritize nodes and edges based on their importance to the network topology. These measures helped identify critical genes and proteins in biomolecular networks. The proposed centrality measure, DiffSLC, combines the number of interactions of a protein and the gene coexpression values of genes from which those proteins were translated, as a weighting factor to bias the identification of essential proteins in a protein interaction network. Potentially essential proteins with low node degree are promoted through eigenvector centrality. Thus, the gene coexpression values are used in conjunction with the eigenvector of the network's adjacency matrix and edge clustering coefficient to improve essentiality prediction. The outcome of this prediction is shown using three variations: (1) inclusion or exclusion of gene co-expression data, (2) impact of different coexpression measures, and (3) impact of different gene expression data sets. For a total of seven networks, DiffSLC is compared to other centrality measures using Saccharomyces cerevisiae protein interaction networks and gene expression data. Comparisons are also performed for the top ranked proteins against the known essential genes from the Saccharomyces Gene Deletion Project, which show that DiffSLC detects more essential proteins and has a higher area under the ROC curve than other compared methods. This makes DiffSLC a stronger alternative to other centrality methods for detecting essential genes using a protein-protein interaction network that obeys centrality-lethality principle. DiffSLC is implemented using the igraph package in R, and networkx package in Python. The python package can be

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

PubMed

Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

2016-10-20

RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

PubMed

Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

2016-02-01

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. © 2016 Schütze et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

A credit-card library approach for disrupting protein-protein interactions.

PubMed

Xu, Yang; Shi, Jin; Yamamoto, Noboru; Moss, Jason A; Vogt, Peter K; Janda, Kim D

2006-04-15

Protein-protein interfaces are prominent in many therapeutically important targets. Using small organic molecules to disrupt protein-protein interactions is a current challenge in chemical biology. An important example of protein-protein interactions is provided by the Myc protein, which is frequently deregulated in human cancers. Myc belongs to the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors. It is biologically active only as heterodimer with the bHLH-ZIP protein Max. Herein, we report a new strategy for the disruption of protein-protein interactions that has been corroborated through the design and synthesis of a small parallel library composed of 'credit-card' compounds. These compounds are derived from a planar, aromatic scaffold and functionalized with four points of diversity. From a 285 membered library, several hits were obtained that disrupted the c-Myc-Max interaction and cellular functions of c-Myc. The IC50 values determined for this small focused library for the disruption of Myc-Max dimerization are quite potent, especially since small molecule antagonists of protein-protein interactions are notoriously difficult to find. Furthermore, several of the compounds were active at the cellular level as shown by their biological effects on Myc action in chicken embryo fibroblast assays. In light of our findings, this approach is considered a valuable addition to the armamentarium of new molecules being developed to interact with protein-protein interfaces. Finally, this strategy for disrupting protein-protein interactions should prove applicable to other families of proteins.

Noninvasive imaging of protein-protein interactions in living animals

NASA Astrophysics Data System (ADS)

Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

2002-05-01

Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

NOXclass: prediction of protein-protein interaction types.

PubMed

Zhu, Hongbo; Domingues, Francisco S; Sommer, Ingolf; Lengauer, Thomas

2006-01-19

Structural models determined by X-ray crystallography play a central role in understanding protein-protein interactions at the molecular level. Interpretation of these models requires the distinction between non-specific crystal packing contacts and biologically relevant interactions. This has been investigated previously and classification approaches have been proposed. However, less attention has been devoted to distinguishing different types of biological interactions. These interactions are classified as obligate and non-obligate according to the effect of the complex formation on the stability of the protomers. So far no automatic classification methods for distinguishing obligate, non-obligate and crystal packing interactions have been made available. Six interface properties have been investigated on a dataset of 243 protein interactions. The six properties have been combined using a support vector machine algorithm, resulting in NOXclass, a classifier for distinguishing obligate, non-obligate and crystal packing interactions. We achieve an accuracy of 91.8% for the classification of these three types of interactions using a leave-one-out cross-validation procedure. NOXclass allows the interpretation and analysis of protein quaternary structures. In particular, it generates testable hypotheses regarding the nature of protein-protein interactions, when experimental results are not available. We expect this server will benefit the users of protein structural models, as well as protein crystallographers and NMR spectroscopists. A web server based on the method and the datasets used in this study are available at http://noxclass.bioinf.mpi-inf.mpg.de/.

Modelling of DNA-Mediated of Two- and -Three dimensional Protein-Protein and Protein-Nanoparticle Self-Assembly

NASA Astrophysics Data System (ADS)

Millan, Jaime; McMillan, Janet; Brodin, Jeff; Lee, Byeongdu; Mirkin, Chad; Olvera de La Cruz, Monica

Programmable DNA interactions represent a robust scheme to self-assemble a rich variety of tunable superlattices, where intrinsic and in some cases non-desirable nano-scale building blocks interactions are substituted for DNA hybridization events. Recent advances in synthesis has allowed the extension of this successful scheme to proteins, where DNA distribution can be tuned independently of protein shape by selectively addressing surface residues, giving rise to assembly properties in three dimensional protein-nanoparticle superlattices dependent on DNA distribution. In parallel to this advances, we introduced a scalable coarse-grained model that faithfully reproduces the previously observed co-assemblies from nanoparticles and proteins conjugates. Herein, we implement this numerical model to explain the stability of complex protein-nanoparticle binary superlattices and to elucidate experimentally inaccessible features such as protein orientation. Also, we will discuss systematic studies that highlight the role of DNA distribution and sequence on two-dimensional protein-protein and protein-nanoparticle superlattices.

Prediction of Protein Aggregation in High Concentration Protein Solutions Utilizing Protein-Protein Interactions Determined by Low Volume Static Light Scattering.

PubMed

Hofmann, Melanie; Winzer, Matthias; Weber, Christian; Gieseler, Henning

2016-06-01

The development of highly concentrated protein formulations is more demanding than for conventional concentrations due to an elevated protein aggregation tendency. Predictive protein-protein interaction parameters, such as the second virial coefficient B22 or the interaction parameter kD, have already been used to predict aggregation tendency and optimize protein formulations. However, these parameters can only be determined in diluted solutions, up to 20 mg/mL. And their validity at high concentrations is currently controversially discussed. This work presents a μ-scale screening approach which has been adapted to early industrial project needs. The procedure is based on static light scattering to directly determine protein-protein interactions at concentrations up to 100 mg/mL. Three different therapeutic molecules were formulated, varying in pH, salt content, and addition of excipients (e.g., sugars, amino acids, polysorbates, or other macromolecules). Validity of the predicted aggregation tendency was confirmed by stability data of selected formulations. Based on the results obtained, the new prediction method is a promising screening tool for fast and easy formulation development of highly concentrated protein solutions, consuming only microliter of sample volumes. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Anomalous Protein-Protein Interactions in Multivalent Salt Solution.

PubMed

Pasquier, Coralie; Vazdar, Mario; Forsman, Jan; Jungwirth, Pavel; Lund, Mikael

2017-04-13

The stability of aqueous protein solutions is strongly affected by multivalent ions, which induce ion-ion correlations beyond the scope of classical mean-field theory. Using all-atom molecular dynamics (MD) and coarse grained Monte Carlo (MC) simulations, we investigate the interaction between a pair of protein molecules in 3:1 electrolyte solution. In agreement with available experimental findings of "reentrant protein condensation", we observe an anomalous trend in the protein-protein potential of mean force with increasing electrolyte concentration in the order: (i) double-layer repulsion, (ii) ion-ion correlation attraction, (iii) overcharge repulsion, and in excess of 1:1 salt, (iv) non Coulombic attraction. To efficiently sample configurational space we explore hybrid continuum solvent models, applicable to many-protein systems, where weakly coupled ions are treated implicitly, while strongly coupled ones are treated explicitly. Good agreement is found with the primitive model of electrolytes, as well as with atomic models of protein and solvent.

Emerging Role of Protein-Protein Transnitrosylation in Cell Signaling Pathways

PubMed Central

2013-01-01

Abstract Significance: Protein S-nitrosylation, a covalent reaction of a nitric oxide (NO) group with a critical protein thiol (or more properly thiolate anion), mediates an important form of redox-related signaling as well as aberrant signaling in disease states. Recent Advances: A growing literature suggests that over 3000 proteins are S-nitrosylated in cell systems. Our laboratory and several others have demonstrated that protein S-nitrosylation can regulate protein function by directly inhibiting catalytically active cysteines, by reacting with allosteric sites, or via influencing protein-protein interaction. For example, S-nitrosylation of critical cysteine thiols in protein-disulfide isomerase and in parkin alters their activity, thus contributing to protein misfolding in Parkinson's disease. Critical Issues: However, the mechanism by which specific protein S-nitrosylation occurs in cell signaling pathways is less well investigated. Interestingly, the recent discovery of protein-protein transnitrosylation reactions (transfer of an NO group from one protein to another) has revealed a unique mechanism whereby NO can S-nitrosylate a particular set of protein thiols, and represents a major class of nitrosylating/denitrosylating enzymes in mammalian systems. In this review, we will discuss recent evidence for transnitrosylation reactions between (i) hemoglobin/anion exchanger 1, (ii) thioredoxin/caspase-3, (iii) X-linked inhibitor of apoptosis/caspase-3, (iv) GAPDH-HDAC2/SIRT1/DNA-PK, and (v) Cdk5/dynamin related protein 1 (Drp1). This review also discusses experimental techniques useful in characterizing protein-protein transnitrosylations. Future Directions: Elucidation of additional transnitrosylation cascades will further our understanding of the enzymes that catalyze nitrosation, thereby contributing to NO-mediated signaling pathways. Antioxid. Redox Signal. 18, 239–249. PMID:22657837

Modular protein switches derived from antibody mimetic proteins.

PubMed

Nicholes, N; Date, A; Beaujean, P; Hauk, P; Kanwar, M; Ostermeier, M

2016-02-01

Protein switches have potential applications as biosensors and selective protein therapeutics. Protein switches built by fusion of proteins with the prerequisite input and output functions are currently developed using an ad hoc process. A modular switch platform in which existing switches could be readily adapted to respond to any ligand would be advantageous. We investigated the feasibility of a modular protein switch platform based on fusions of the enzyme TEM-1 β-lactamase (BLA) with two different antibody mimetic proteins: designed ankyrin repeat proteins (DARPins) and monobodies. We created libraries of random insertions of the gene encoding BLA into genes encoding a DARPin or a monobody designed to bind maltose-binding protein (MBP). From these libraries, we used a genetic selection system for β-lactamase activity to identify genes that conferred MBP-dependent ampicillin resistance to Escherichia coli. Some of these selected genes encoded switch proteins whose enzymatic activity increased up to 14-fold in the presence of MBP. We next introduced mutations into the antibody mimetic domain of these switches that were known to cause binding to different ligands. To different degrees, introduction of the mutations resulted in switches with the desired specificity, illustrating the potential modularity of these platforms. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Optimizing energy functions for protein-protein interface design.

PubMed

Sharabi, Oz; Yanover, Chen; Dekel, Ayelet; Shifman, Julia M

2011-01-15

Protein design methods have been originally developed for the design of monomeric proteins. When applied to the more challenging task of protein–protein complex design, these methods yield suboptimal results. In particular, they often fail to recapitulate favorable hydrogen bonds and electrostatic interactions across the interface. In this work, we aim to improve the energy function of the protein design program ORBIT to better account for binding interactions between proteins. By using the advanced machine learning framework of conditional random fields, we optimize the relative importance of all the terms in the energy function, attempting to reproduce the native side-chain conformations in protein–protein interfaces. We evaluate the performance of several optimized energy functions, each describes the van der Waals interactions using a different potential. In comparison with the original energy function, our best energy function (a) incorporates a much “softer” repulsive van der Waals potential, suitable for the discrete rotameric representation of amino acid side chains; (b) does not penalize burial of polar atoms, reflecting the frequent occurrence of polar buried residues in protein–protein interfaces; and (c) significantly up-weights the electrostatic term, attesting to the high importance of these interactions for protein–protein complex formation. Using this energy function considerably improves side chain placement accuracy for interface residues in a large test set of protein–protein complexes. Moreover, the optimized energy function recovers the native sequences of protein–protein interface at a higher rate than the default function and performs substantially better in predicting changes in free energy of binding due to mutations.

Conserved Cysteine Residues Provide a Protein-Protein Interaction Surface in Dual Oxidase (DUOX) Proteins*

PubMed Central

Meitzler, Jennifer L.; Hinde, Sara; Bánfi, Botond; Nauseef, William M.; Ortiz de Montellano, Paul R.

2013-01-01

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1. PMID:23362256

Structure and Protein-Protein Interaction Studies on Chlamydia trachomatis Protein CT670 (YscO Homolog)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lorenzini, Emily; Singer, Alexander; Singh, Bhag

2010-07-28

Comparative genomic studies have identified many proteins that are found only in various Chlamydiae species and exhibit no significant sequence similarity to any protein in organisms that do not belong to this group. The CT670 protein of Chlamydia trachomatis is one of the proteins whose genes are in one of the type III secretion gene clusters but whose cellular functions are not known. CT670 shares several characteristics with the YscO protein of Yersinia pestis, including the neighboring genes, size, charge, and secondary structure, but the structures and/or functions of these proteins remain to be determined. Although a BLAST search withmore » CT670 did not identify YscO as a related protein, our analysis indicated that these two proteins exhibit significant sequence similarity. In this paper, we report that the CT670 crystal, solved at a resolution of 2 {angstrom}, consists of a single coiled coil containing just two long helices. Gel filtration and analytical ultracentrifugation studies showed that in solution CT670 exists in both monomeric and dimeric forms and that the monomer predominates at lower protein concentrations. We examined the interaction of CT670 with many type III secretion system-related proteins (viz., CT091, CT665, CT666, CT667, CT668, CT669, CT671, CT672, and CT673) by performing bacterial two-hybrid assays. In these experiments, CT670 was found to interact only with the CT671 protein (YscP homolog), whose gene is immediately downstream of ct670. A specific interaction between CT670 and CT671 was also observed when affinity chromatography pull-down experiments were performed. These results suggest that CT670 and CT671 are putative homologs of the YcoO and YscP proteins, respectively, and that they likely form a chaperone-effector pair.« less

[Chromosomal proteins: histones and acid proteins].

PubMed

Salvini, M; Gabrielli, F

1976-01-01

Experimental data about the chemistry and the biology of chromosomal proteins are reviewed. Paragraphs include: aminoacid sequential data and post-translational covalent modications of histones, histone chemical differences in different tissues of the same species and in homologous organs of different species, histone synthesis subcellular localization and its association with DNA synthesis, histone synthesis transcriptional and translational control, histone synthesis during meiosis, oogenesis and early embryogenesis. The possible role of histones as controllers of gene expression is discussed and a model of primary structure of chromatine is proposed. The "acidic proteins" data concern the high tissue eterogenity of these proteins and their role in the steroid-hormon-controlled gene expression. The possible role of acidic proteins as general controllers of gene expression in eucariotic cells is discussed.

Protein-protein interactions and cancer: targeting the central dogma.

PubMed

Garner, Amanda L; Janda, Kim D

2011-01-01

Between 40,000 and 200,000 protein-protein interactions have been predicted to exist within the human interactome. As these interactions are of a critical nature in many important cellular functions and their dysregulation is causal of disease, the modulation of these binding events has emerged as a leading, yet difficult therapeutic arena. In particular, the targeting of protein-protein interactions relevant to cancer is of fundamental importance as the tumor-promoting function of several aberrantly expressed proteins in the cancerous state is directly resultant of its ability to interact with a protein-binding partner. Of significance, these protein complexes play a crucial role in each of the steps of the central dogma of molecular biology, the fundamental processes of genetic transmission. With the many important discoveries being made regarding the mechanisms of these genetic process, the identification of new chemical probes are needed to better understand and validate the druggability of protein-protein interactions related to the central dogma. In this review, we provide an overview of current small molecule-based protein-protein interaction inhibitors for each stage of the central dogma: transcription, mRNA splicing and translation. Importantly, through our analysis we have uncovered a lack of necessary probes targeting mRNA splicing and translation, thus, opening up the possibility for expansion of these fields.

Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

PubMed

He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

2012-07-01

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

Dynamic imaging of protein-protein interactions by MP-FLIM

NASA Astrophysics Data System (ADS)

Ameer-Beg, Simon M.; Peter, Marion; Keppler, Melanie D.; Prag, Soren; Barber, Paul R.; Ng, Tony C.; Vojnovic, Borivoj

2005-03-01

The spatio-temporal localization of molecular interactions within cells in situ is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localization of protein complexes may be achieved by the detection of fluorescence (or Forster) resonance energy transfer (FRET) between protein-conjugated fluorophores. We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localization of protein-protein interactions in live and fixed cell populations. Intramolecular interactions between protein hetero-dimers are investigated using green fluorescent protein variants. We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET. The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) α in carcinoma cells for both live and fixed cell experiments.

Non-interacting surface solvation and dynamics in protein-protein interactions.

PubMed

Visscher, Koen M; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

2015-03-01

Protein-protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure-based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein-protein binding, even for simple lock-and-key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein-protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein-protein surfaces. We compare properties of the interface, rim, and non-interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non-interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non-interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein-protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein-protein complexes from unintended protein-protein interactions. © 2014 Wiley Periodicals, Inc.

NDR proteins

PubMed Central

Jones, Alan M

2010-01-01

N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins. PMID:20724844

The interface of protein structure, protein biophysics, and molecular evolution

PubMed Central

Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

2012-01-01

Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

Evolutionary diversification of protein-protein interactions by interface add-ons.

PubMed

Plach, Maximilian G; Semmelmann, Florian; Busch, Florian; Busch, Markus; Heizinger, Leonhard; Wysocki, Vicki H; Merkl, Rainer; Sterner, Reinhard

2017-10-03

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein-protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein-protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein-protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein-protein interactions.

Identification of PDC-109-like protein(s) in buffalo seminal plasma.

PubMed

Harshan, Hiron M; Sankar, Surya; Singh, L P; Singh, Manish Kumar; Sudharani, S; Ansari, M R; Singh, S K; Majumdar, A C; Joshi, P

2009-10-01

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.

Racemic & quasi-racemic protein crystallography enabled by chemical protein synthesis.

PubMed

Kent, Stephen Bh

2018-04-04

A racemic protein mixture can be used to form centrosymmetric crystals for structure determination by X-ray diffraction. Both the unnatural d-protein and the corresponding natural l-protein are made by total chemical synthesis based on native chemical ligation-chemoselective condensation of unprotected synthetic peptide segments. Racemic protein crystallography is important for structure determination of the many natural protein molecules that are refractory to crystallization. Racemic mixtures facilitate the crystallization of recalcitrant proteins, and give diffraction-quality crystals. Quasi-racemic crystallization, using a single d-protein molecule, can facilitate the determination of the structures of a series of l-protein analog molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

General protein-protein cross-linking.

PubMed

Alegria-Schaffer, Alice

2014-01-01

This protocol describes a general protein-to-protein cross-linking procedure using the water-soluble amine-reactive homobifunctional BS(3) (bis[sulfosuccinimidyl] suberate); however, the protocol can be easily adapted using other cross-linkers of similar properties. BS(3) is composed of two sulfo-NHS ester groups and an 11.4 Å linker. Sulfo-NHS ester groups react with primary amines in slightly alkaline conditions (pH 7.2-8.5) and yield stable amide bonds. The reaction releases N-hydroxysuccinimide (see an application of NHS esters on Labeling a protein with fluorophores using NHS ester derivitization). © 2014 Elsevier Inc. All rights reserved.

Surface energetics and protein-protein interactions: analysis and mechanistic implications

PubMed Central

Peri, Claudio; Morra, Giulia; Colombo, Giorgio

2016-01-01

Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions. PMID:27050828

Quality control methodology for high-throughput protein-protein interaction screening.

PubMed

Vazquez, Alexei; Rual, Jean-François; Venkatesan, Kavitha

2011-01-01

Protein-protein interactions are key to many aspects of the cell, including its cytoskeletal structure, the signaling processes in which it is involved, or its metabolism. Failure to form protein complexes or signaling cascades may sometimes translate into pathologic conditions such as cancer or neurodegenerative diseases. The set of all protein interactions between the proteins encoded by an organism constitutes its protein interaction network, representing a scaffold for biological function. Knowing the protein interaction network of an organism, combined with other sources of biological information, can unravel fundamental biological circuits and may help better understand the molecular basics of human diseases. The protein interaction network of an organism can be mapped by combining data obtained from both low-throughput screens, i.e., "one gene at a time" experiments and high-throughput screens, i.e., screens designed to interrogate large sets of proteins at once. In either case, quality controls are required to deal with the inherent imperfect nature of experimental assays. In this chapter, we discuss experimental and statistical methodologies to quantify error rates in high-throughput protein-protein interactions screens.

Purine inhibitors of protein kinases, G proteins and polymerases

DOEpatents

Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

2001-07-03

The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

Mapping monomeric threading to protein-protein structure prediction.

PubMed

Guerler, Aysam; Govindarajoo, Brandon; Zhang, Yang

2013-03-25

The key step of template-based protein-protein structure prediction is the recognition of complexes from experimental structure libraries that have similar quaternary fold. Maintaining two monomer and dimer structure libraries is however laborious, and inappropriate library construction can degrade template recognition coverage. We propose a novel strategy SPRING to identify complexes by mapping monomeric threading alignments to protein-protein interactions based on the original oligomer entries in the PDB, which does not rely on library construction and increases the efficiency and quality of complex template recognitions. SPRING is tested on 1838 nonhomologous protein complexes which can recognize correct quaternary template structures with a TM score >0.5 in 1115 cases after excluding homologous proteins. The average TM score of the first model is 60% and 17% higher than that by HHsearch and COTH, respectively, while the number of targets with an interface RMSD <2.5 Å by SPRING is 134% and 167% higher than these competing methods. SPRING is controlled with ZDOCK on 77 docking benchmark proteins. Although the relative performance of SPRING and ZDOCK depends on the level of homology filters, a combination of the two methods can result in a significantly higher model quality than ZDOCK at all homology thresholds. These data demonstrate a new efficient approach to quaternary structure recognition that is ready to use for genome-scale modeling of protein-protein interactions due to the high speed and accuracy.

Quantifying the Molecular Origins of Opposite Solvent Effects on Protein-Protein Interactions

PubMed Central

Vagenende, Vincent; Han, Alvin X.; Pek, Han B.; Loo, Bernard L. W.

2013-01-01

Although the nature of solvent-protein interactions is generally weak and non-specific, addition of cosolvents such as denaturants and osmolytes strengthens protein-protein interactions for some proteins, whereas it weakens protein-protein interactions for others. This is exemplified by the puzzling observation that addition of glycerol oppositely affects the association constants of two antibodies, D1.3 and D44.1, with lysozyme. To resolve this conundrum, we develop a methodology based on the thermodynamic principles of preferential interaction theory and the quantitative characterization of local protein solvation from molecular dynamics simulations. We find that changes of preferential solvent interactions at the protein-protein interface quantitatively account for the opposite effects of glycerol on the antibody-antigen association constants. Detailed characterization of local protein solvation in the free and associated protein states reveals how opposite solvent effects on protein-protein interactions depend on the extent of dewetting of the protein-protein contact region and on structural changes that alter cooperative solvent-protein interactions at the periphery of the protein-protein interface. These results demonstrate the direct relationship between macroscopic solvent effects on protein-protein interactions and atom-scale solvent-protein interactions, and establish a general methodology for predicting and understanding solvent effects on protein-protein interactions in diverse biological environments. PMID:23696727

Quantifying the molecular origins of opposite solvent effects on protein-protein interactions.

PubMed

Vagenende, Vincent; Han, Alvin X; Pek, Han B; Loo, Bernard L W

2013-01-01

Although the nature of solvent-protein interactions is generally weak and non-specific, addition of cosolvents such as denaturants and osmolytes strengthens protein-protein interactions for some proteins, whereas it weakens protein-protein interactions for others. This is exemplified by the puzzling observation that addition of glycerol oppositely affects the association constants of two antibodies, D1.3 and D44.1, with lysozyme. To resolve this conundrum, we develop a methodology based on the thermodynamic principles of preferential interaction theory and the quantitative characterization of local protein solvation from molecular dynamics simulations. We find that changes of preferential solvent interactions at the protein-protein interface quantitatively account for the opposite effects of glycerol on the antibody-antigen association constants. Detailed characterization of local protein solvation in the free and associated protein states reveals how opposite solvent effects on protein-protein interactions depend on the extent of dewetting of the protein-protein contact region and on structural changes that alter cooperative solvent-protein interactions at the periphery of the protein-protein interface. These results demonstrate the direct relationship between macroscopic solvent effects on protein-protein interactions and atom-scale solvent-protein interactions, and establish a general methodology for predicting and understanding solvent effects on protein-protein interactions in diverse biological environments.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

NASA Astrophysics Data System (ADS)

Dong, Jinlan; Bruening, Merlin L.

2015-07-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

PubMed

Dong, Jinlan; Bruening, Merlin L

2015-01-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

PubMed Central

Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

2015-01-01

Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

PubMed

Sultana, Azmiri; Lee, Jeffrey E

2015-02-02

Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. Copyright © 2015 John Wiley & Sons, Inc.

[Detection of protein-protein interactions by FRET and BRET methods].

PubMed

Matoulková, E; Vojtěšek, B

2014-01-01

Nowadays, in vivo protein-protein interaction studies have become preferable detecting meth-ods that enable to show or specify (already known) protein interactions and discover their inhibitors. They also facilitate detection of protein conformational changes and discovery or specification of signaling pathways in living cells. One group of in vivo methods enabling these findings is based on fluorescent resonance energy transfer (FRET) and its bio-luminescent modification (BRET). They are based on visualization of protein-protein interactions via light or enzymatic excitation of fluorescent or bio-luminescent proteins. These methods allow not only protein localization within the cell or its organelles (or small animals) but they also allow us to quantify fluorescent signals and to discover weak or strong interaction partners. In this review, we explain the principles of FRET and BRET, their applications in the characterization of protein-protein interactions and we describe several findings using these two methods that clarify molecular and cellular mechanisms and signals related to cancer biology.

New paradigm in ankyrin repeats: Beyond protein-protein interaction module.

PubMed

Islam, Zeyaul; Nagampalli, Raghavendra Sashi Krishna; Fatima, Munazza Tamkeen; Ashraf, Ghulam Md

2018-04-01

Classically, ankyrin repeat (ANK) proteins are built from tandems of two or more repeats and form curved solenoid structures that are associated with protein-protein interactions. These are short, widespread structural motif of around 33 amino acids repeats in tandem, having a canonical helix-loop-helix fold, found individually or in combination with other domains. The multiplicity of structural pattern enables it to form assemblies of diverse sizes, required for their abilities to confer multiple binding and structural roles of proteins. Three-dimensional structures of these repeats determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. Recent work on the ANK has proposed novel structural information, especially protein-lipid, protein-sugar and protein-protein interaction. Self-assembly of these repeats was also shown to prevent the associated protein in forming filaments. In this review, we summarize the latest findings and how the new structural information has increased our understanding of the structural determinants of ANK proteins. We discussed latest findings on how these proteins participate in various interactions to diversify the ANK roles in numerous biological processes, and explored the emerging and evolving field of designer ankyrins and its framework for protein engineering emphasizing on biotechnological applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Correction to: The NMR contribution to protein-protein networking in Fe-S protein maturation.

PubMed

Banci, Lucia; Camponeschi, Francesca; Ciofi-Baffoni, Simone; Piccioli, Mario

2018-05-31

The article "The NMR contribution to protein-protein networking in Fe-S protein maturation", written by Lucia Banci, Francesca Camponeschi, Simone Ciofi‑Baffoni, Mario Piccioli was originally published electronically on the publisher's internet portal (currently SpringerLink) on 22 March, 2018 without open access.

Protein supplementation with sports protein bars in renal patients.

PubMed

Meade, Anthony

2007-05-01

Malnutrition prevalence in patients on dialysis is well established. The protein requirements for both hemodialysis and peritoneal dialysis have been documented elsewhere, including the Kidney Disease Outcomes Quality Initiative Clinical Practice Guidelines for Nutrition in Chronic Renal Failure. The clinical challenge is to assist patients in meeting these targets, especially in those with anorexia. Traditional supplements have included fluid, which is an issue for patients who are fluid restricted. The study objectives were to (1) investigate the range of sports protein supplements that may be suitable for patients on hemodialysis to use and (2) trial nonfluid protein supplements in patients on hemodialysis. Known manufacturers of sports protein bars and other sports supplements available in Australia were contacted for the nutrient breakdown of high-protein products, specifically potassium, protein, and phosphorus contents. As a result, selected high-protein sports bars (Protein FX, Aussie Bodies, Port Melbourne, Victoria, Australia) were used as an alternative to the more commonly used renal-specific fluid supplements (Nepro, Abbott Laboratories, Abbott Park, IL; Novasource Renal, Novartis Nutrition Corporation, Fremont, MI; and Renilon, Nutricia, Wiltshire, UK) in patients with poor nutritional status requiring supplementation. Patient satisfaction and clinical nutrition markers were investigated. The study took place at inpatient, in-center, and satellite hemodialysis settings in Adelaide, South Australia. A total of 32 patients (16 females and 16 males) with an average age of 62.9 years (range 32-86 years) undergoing hemodialysis (acute and maintenance) were included. Subjects were selected by the author as part of routine clinical nutrition care. Patients trialed sports protein bars as a protein supplement alone or in conjunction with other supplementary products. All patients were in favor of the trial, with 22 of 32 patients continuing with the protein

Protein enriched pasta: structure and digestibility of its protein network.

PubMed

Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

2016-02-01

Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest.

Transmembrane proteins in the Protein Data Bank: identification and classification.

PubMed

Tusnády, Gábor E; Dosztányi, Zsuzsanna; Simon, István

2004-11-22

Integral membrane proteins play important roles in living cells. Although these proteins are estimated to constitute 25% of proteins at a genomic scale, the Protein Data Bank (PDB) contains only a few hundred membrane proteins due to the difficulties with experimental techniques. The presence of transmembrane proteins in the structure data bank, however, is quite invisible, as the annotation of these entries is rather poor. Even if a protein is identified as a transmembrane one, the possible location of the lipid bilayer is not indicated in the PDB because these proteins are crystallized without their natural lipid bilayer, and currently no method is publicly available to detect the possible membrane plane using the atomic coordinates of membrane proteins. Here, we present a new geometrical approach to distinguish between transmembrane and globular proteins using structural information only and to locate the most likely position of the lipid bilayer. An automated algorithm (TMDET) is given to determine the membrane planes relative to the position of atomic coordinates, together with a discrimination function which is able to separate transmembrane and globular proteins even in cases of low resolution or incomplete structures such as fragments or parts of large multi chain complexes. This method can be used for the proper annotation of protein structures containing transmembrane segments and paves the way to an up-to-date database containing the structure of all known transmembrane proteins and fragments (PDB_TM) which can be automatically updated. The algorithm is equally important for the purpose of constructing databases purely of globular proteins.

Predicting protein crystallization propensity from protein sequence

PubMed Central

2011-01-01

The high-throughput structure determination pipelines developed by structural genomics programs offer a unique opportunity for data mining. One important question is how protein properties derived from a primary sequence correlate with the protein’s propensity to yield X-ray quality crystals (crystallizability) and 3D X-ray structures. A set of protein properties were computed for over 1,300 proteins that expressed well but were insoluble, and for ~720 unique proteins that resulted in X-ray structures. The correlation of the protein’s iso-electric point and grand average hydropathy (GRAVY) with crystallizability was analyzed for full length and domain constructs of protein targets. In a second step, several additional properties that can be calculated from the protein sequence were added and evaluated. Using statistical analyses we have identified a set of the attributes correlating with a protein’s propensity to crystallize and implemented a Support Vector Machine (SVM) classifier based on these. We have created applications to analyze and provide optimal boundary information for query sequences and to visualize the data. These tools are available via the web site http://bioinformatics.anl.gov/cgi-bin/tools/pdpredictor. PMID:20177794

Personalizing Protein Nourishment

PubMed Central

DALLAS, DAVID C.; SANCTUARY, MEGAN R.; QU, YUNYAO; KHAJAVI, SHABNAM HAGHIGHAT; VAN ZANDT, ALEXANDRIA E.; DYANDRA, MELISSA; FRESE, STEVEN A.; BARILE, DANIELA; GERMAN, J. BRUCE

2016-01-01

Proteins are not equally digestible—their proteolytic susceptibility varies by their source and processing method. Incomplete digestion increases colonic microbial protein fermentation (putrefaction), which produces toxic metabolites that can induce inflammation in vitro and have been associated with inflammation in vivo. Individual humans differ in protein digestive capacity based on phenotypes, particularly disease states. To avoid putrefaction-induced intestinal inflammation, protein sources and processing methods must be tailored to the consumer’s digestive capacity. This review explores how food processing techniques alter protein digestibility and examines how physiological conditions alter digestive capacity. Possible solutions to improving digestive function or matching low digestive capacity with more digestible protein sources are explored. Beyond the ileal digestibility measurements of protein digestibility, less invasive, quicker and cheaper techniques for monitoring the extent of protein digestion and fermentation are needed to personalize protein nourishment. Biomarkers of protein digestive capacity and efficiency can be identified with the toolsets of peptidomics, metabolomics, microbial sequencing and multiplexed protein analysis of fecal and urine samples. By monitoring individual protein digestive function, the protein component of diets can be tailored via protein source and processing selection to match individual needs to minimize colonic putrefaction and, thus, optimize gut health. PMID:26713355

Energy design for protein-protein interactions

PubMed Central

Ravikant, D. V. S.; Elber, Ron

2011-01-01

Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions. PMID:21842951

General introduction: recombinant protein production and purification of insoluble proteins.

PubMed

Ferrer-Miralles, Neus; Saccardo, Paolo; Corchero, José Luis; Xu, Zhikun; García-Fruitós, Elena

2015-01-01

Proteins are synthesized in heterologous systems because of the impossibility to obtain satisfactory yields from natural sources. The production of soluble and functional recombinant proteins is among the main goals in the biotechnological field. In this context, it is important to point out that under stress conditions, protein folding machinery is saturated and this promotes protein misfolding and, consequently, protein aggregation. Thus, the selection of the optimal expression organism and the most appropriate growth conditions to minimize the formation of insoluble proteins should be done according to the protein characteristics and downstream requirements. Escherichia coli is the most popular recombinant protein expression system despite the great development achieved so far by eukaryotic expression systems. Besides, other prokaryotic expression systems, such as lactic acid bacteria and psychrophilic bacteria, are gaining interest in this field. However, it is worth mentioning that prokaryotic expression system poses, in many cases, severe restrictions for a successful heterologous protein production. Thus, eukaryotic systems such as mammalian cells, insect cells, yeast, filamentous fungus, and microalgae are an interesting alternative for the production of these difficult-to-express proteins.

10′(Z),13′(E)-Heptadecadienylhydroquinone Inhibits Swarming and Virulence Factors and Increases Polymyxin B Susceptibility in Proteus mirabilis

PubMed Central

Wang, Won-Bo; Yuan, Yu-Han; Hsueh, Po-Ren; Liaw, Shwu-Jen

2012-01-01

In this study, we demonstrated that 10′(Z), 13′(E)-heptadecadienylhydroquinone (HQ17-2), isolated from the lacquer tree, could decrease swarming motility and hemolysin activity but increase polymyxin B (PB) susceptibilityof Proteus mirabilis which is intrinsically highly-resistant to PB. The increased PB susceptibility induced by HQ17-2 was also observed in clinical isolates and biofilm-grown cells. HQ17-2 could inhibit swarming in the wild-type and rppA mutant but not in the rcsB mutant, indicating that HQ17-2 inhibits swarming through the RcsB-dependent pathway, a two-component signaling pathway negatively regulating swarming and virulence factor expression. The inhibition of hemolysin activity by HQ17-2 is also mediated through the RcsB-dependent pathway, because HQ17-2 could not inhibit hemolysin activity in the rcsB mutant. Moreover, the finding that HQ17-2 inhibits the expression of flhDC gene in the wild-type and rcsB-complemented strain but not in the rcsB mutant supports the notion. By contrast, HQ17-2 could increase PB susceptibility in the wild-type and rcsB mutant but not in the rppA mutant, indicating that HQ17-2 increases PB susceptibility through the RppA-dependent pathway, a signaling pathway positively regulating PB resistance. In addition, HQ17-2 could inhibit the promoter activities of rppA and pmrI, a gene positively regulated by RppA and involved in PB resistance, in the wild-type but not in the rppA mutant. The inhibition of rppA and pmrI expression caused lipopolysaccharide purified from HQ17-2-treated cells to have higher affinity for PB. Altogether, this study uncovers new biological effects of HQ17-2 and provides evidence for the potential of HQ17-2 in clinical applications. PMID:23029100

Differences in Purinergic Amplification of Osmotic Cell Lysis by the Pore-Forming RTX Toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the Role of Pore Size

PubMed Central

Fiser, Radovan; Linhartova, Irena; Osicka, Radim; Bumba, Ladislav; Hewlett, Erik L.; Benz, Roland; Sebo, Peter

2013-01-01

A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins. PMID:24082076

Genetic diversity of clinical and environmental Vibrio parahaemolyticus strains from the Pacific Northwest.

PubMed

Paranjpye, Rohinee; Hamel, Owen S; Stojanovski, Asta; Liermann, Martin

2012-12-01

Since 1997, cases of Vibrio parahaemolyticus-related gastroenteritis from the consumption of raw oysters harvested in Washington State have been higher than historical levels. These cases have shown little or no correlation with concentrations of potentially pathogenic V. parahaemolyticus (positive for the thermostable direct hemolysin gene, tdh) in oysters, although significant concentrations of tdh(+) V. parahaemolyticus strains were isolated from shellfish-growing areas in the Pacific Northwest (PNW). We compared clinical and environmental strains isolated from the PNW to those from other geographic regions within the United States and Asia for the presence of virulence-associated genes, including the thermostable direct hemolysin (tdh), the thermostable-related hemolysin (trh), urease (ureR), the pandemic group specific markers orf8 and toxRS, and genes encoding both type 3 secretion systems (T3SS1 and T3SS2). The majority of clinical strains from the PNW were positive for tdh, trh, and ureR genes, while a significant proportion of environmental isolates were tdh(+) but trh negative. Hierarchical clustering grouped the majority of these clinical isolates into a cluster distinct from that including the pandemic strain RIMD2210633, clinical isolates from other geographical regions, and tdh(+), trh-negative environmental isolates from the PNW. We detected T3SS2-related genes (T3SS2β) in environmental strains that were tdh and trh negative. The presence of significant concentrations of tdh(+), trh-negative environmental strains in the PNW that have not been responsible for illness and T3SS2β in tdh- and trh-negative strains emphasizes the diversity in this species and the need to identify additional virulence markers for this bacterium to improve risk assessment tools for the detection of this pathogen.

Serratia marcescens internalization and replication in human bladder epithelial cells

PubMed Central

Hertle, Ralf; Schwarz, Heinz

2004-01-01

Background Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. Methods Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. Results We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. Conclusion The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections. PMID:15189566

Prediction of scaffold proteins based on protein interaction and domain architectures.

PubMed

Oh, Kimin; Yi, Gwan-Su

2016-07-28

Scaffold proteins are known for being crucial regulators of various cellular functions by assembling multiple proteins involved in signaling and metabolic pathways. Identification of scaffold proteins and the study of their molecular mechanisms can open a new aspect of cellular systemic regulation and the results can be applied in the field of medicine and engineering. Despite being highlighted as the regulatory roles of dozens of scaffold proteins, there was only one known computational approach carried out so far to find scaffold proteins from interactomes. However, there were limitations in finding diverse types of scaffold proteins because their criteria were restricted to the classical scaffold proteins. In this paper, we will suggest a systematic approach to predict massive scaffold proteins from interactomes and to characterize the roles of scaffold proteins comprehensively. From a total of 10,419 basic scaffold protein candidates in protein interactomes, we classified them into three classes according to the structural evidences for scaffolding, such as domain architectures, domain interactions and protein complexes. Finally, we could define 2716 highly reliable scaffold protein candidates and their characterized functional features. To assess the accuracy of our prediction, the gold standard positive and negative data sets were constructed. We prepared 158 gold standard positive data and 844 gold standard negative data based on the functional information from Gene Ontology consortium. The precision, sensitivity and specificity of our testing was 80.3, 51.0, and 98.5 % respectively. Through the function enrichment analysis of highly reliable scaffold proteins, we could confirm the significantly enriched functions that are related to scaffold protein binding. We also identified functional association between scaffold proteins and their recruited proteins. Furthermore, we checked that the disease association of scaffold proteins is higher than kinases. In

Purine inhibitors of protein kinases, G proteins and polymerases

DOEpatents

Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

2004-10-12

The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

Mirror Image Proteins

PubMed Central

Zhao, Le; Lu, Wuyuan

2017-01-01

Proteins composed entirely of unnatural D-amino acids and the achiral amino acid glycine are mirror image forms of their native L-protein counterparts. Recent advances in chemical protein synthesis afford unique and facile synthetic access to domain-sized mirror image D-proteins, enabling protein research to be conducted through “the looking glass” and in a way previously unattainable. D-proteins can facilitate structure determination of their native L-forms that are difficult to crystallize (racemic X-ray crystallography); D-proteins can serve as the bait for library screening to ultimately yield pharmacologically superior D-peptide/D-protein therapeutics (mirror image phage display); D-proteins can also be used as a powerful mechanistic tool for probing molecular events in biology. This review examines recent progress in the application of mirror image proteins to structural biology, drug discovery, and immunology. PMID:25282524

Protein-Protein Interface Predictions by Data-Driven Methods: A Review

PubMed Central

Xue, Li C; Dobbs, Drena; Bonvin, Alexandre M.J.J.; Honavar, Vasant

2015-01-01

Reliably pinpointing which specific amino acid residues form the interface(s) between a protein and its binding partner(s) is critical for understanding the structural and physicochemical determinants of protein recognition and binding affinity, and has wide applications in modeling and validating protein interactions predicted by high-throughput methods, in engineering proteins, and in prioritizing drug targets. Here, we review the basic concepts, principles and recent advances in computational approaches to the analysis and prediction of protein-protein interfaces. We point out caveats for objectively evaluating interface predictors, and discuss various applications of data-driven interface predictors for improving energy model-driven protein-protein docking. Finally, we stress the importance of exploiting binding partner information in reliably predicting interfaces and highlight recent advances in this emerging direction. PMID:26460190

The Proteins API: accessing key integrated protein and genome information

PubMed Central

Antunes, Ricardo; Alpi, Emanuele; Gonzales, Leonardo; Liu, Wudong; Luo, Jie; Qi, Guoying; Turner, Edd

2017-01-01

Abstract The Proteins API provides searching and programmatic access to protein and associated genomics data such as curated protein sequence positional annotations from UniProtKB, as well as mapped variation and proteomics data from large scale data sources (LSS). Using the coordinates service, researchers are able to retrieve the genomic sequence coordinates for proteins in UniProtKB. This, the LSS genomics and proteomics data for UniProt proteins is programmatically only available through this service. A Swagger UI has been implemented to provide documentation, an interface for users, with little or no programming experience, to ‘talk’ to the services to quickly and easily formulate queries with the services and obtain dynamically generated source code for popular programming languages, such as Java, Perl, Python and Ruby. Search results are returned as standard JSON, XML or GFF data objects. The Proteins API is a scalable, reliable, fast, easy to use RESTful services that provides a broad protein information resource for users to ask questions based upon their field of expertise and allowing them to gain an integrated overview of protein annotations available to aid their knowledge gain on proteins in biological processes. The Proteins API is available at (http://www.ebi.ac.uk/proteins/api/doc). PMID:28383659

Polyamines: naturally occurring small molecule modulators of electrostatic protein-protein interactions.

PubMed

Berwanger, Anja; Eyrisch, Susanne; Schuster, Inge; Helms, Volkhard; Bernhardt, Rita

2010-02-01

Modulations of protein-protein interactions are a key step in regulating protein function, especially in networks. Modulators of these interactions are supposed to be candidates for the development of novel drugs. Here, we describe the role of the small, polycationic and highly abundant natural polyamines that could efficiently bind to charged spots at protein interfaces as modulators of such protein-protein interactions. Using the mitochondrial cytochrome P45011A1 (CYP11A1) electron transfer system as a model, we have analyzed the capability of putrescine, spermidine, and spermine at physiologically relevant concentrations to affect the protein-protein interactions between adrenodoxin reductase (AdR), adrenodoxin (Adx), and CYP11A1. The actions of polyamines on the individual components, on their association/dissociation, on electron transfer, and on substrate conversion were examined. These studies revealed modulating effects of polyamines on distinct interactions and on the entire system in a complex way. Modulation via changed protein-protein interactions appeared plausible from docking experiments that suggested favourable high-affinity binding sites of polyamines (spermine>spermidine>putrescine) at the AdR-Adx interface. Our findings imply for the first time that small endogenous compounds are capable of interfering with distinct components of transient protein complexes and might control protein functions by modulating electrostatic protein-protein interactions.

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

PubMed

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System.

PubMed

Krute, Christina N; Rice, Kelly C; Bose, Jeffrey L

2017-03-01

In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of α-hemolysin and other virulence factors in Staphylococcus aureus In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB , saeR , and saeS mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I ( coa ) and class II ( hla ) target genes were downregulated during the exponential growth phase in the vfrB mutant, compared to the wild-type strain. In addition, saePQRS expression was decreased in the vfrB mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, hla expression was decreased in the vfrB mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated α-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in S. aureus IMPORTANCE The SaeRS two-component system is a key regulator of virulence determinant production in Staphylococcus aureus Although the regulon of this two-component system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase

Integrated web visualizations for protein-protein interaction databases.

PubMed

Jeanquartier, Fleur; Jean-Quartier, Claire; Holzinger, Andreas

2015-06-16

Understanding living systems is crucial for curing diseases. To achieve this task we have to understand biological networks based on protein-protein interactions. Bioinformatics has come up with a great amount of databases and tools that support analysts in exploring protein-protein interactions on an integrated level for knowledge discovery. They provide predictions and correlations, indicate possibilities for future experimental research and fill the gaps to complete the picture of biochemical processes. There are numerous and huge databases of protein-protein interactions used to gain insights into answering some of the many questions of systems biology. Many computational resources integrate interaction data with additional information on molecular background. However, the vast number of diverse Bioinformatics resources poses an obstacle to the goal of understanding. We present a survey of databases that enable the visual analysis of protein networks. We selected M=10 out of N=53 resources supporting visualization, and we tested against the following set of criteria: interoperability, data integration, quantity of possible interactions, data visualization quality and data coverage. The study reveals differences in usability, visualization features and quality as well as the quantity of interactions. StringDB is the recommended first choice. CPDB presents a comprehensive dataset and IntAct lets the user change the network layout. A comprehensive comparison table is available via web. The supplementary table can be accessed on http://tinyurl.com/PPI-DB-Comparison-2015. Only some web resources featuring graph visualization can be successfully applied to interactive visual analysis of protein-protein interaction. Study results underline the necessity for further enhancements of visualization integration in biochemical analysis tools. Identified challenges are data comprehensiveness, confidence, interactive feature and visualization maturing.

Vaccine Protection of Leukopenic Mice against Staphylococcus aureus Bloodstream Infection

PubMed Central

Rauch, Sabine; Gough, Portia; Kim, Hwan Keun; Schneewind, Olaf

2014-01-01

The risk for Staphylococcus aureus bloodstream infection (BSI) is increased in immunocompromised individuals, including patients with hematologic malignancy and/or chemotherapy. Due to the emergence of antibiotic-resistant strains, designated methicillin-resistant S. aureus (MRSA), staphylococcal BSI in cancer patients is associated with high mortality; however, neither a protective vaccine nor pathogen-specific immunotherapy is currently available. Here, we modeled staphylococcal BSI in leukopenic CD-1 mice that had been treated with cyclophosphamide, a drug for leukemia and lymphoma patients. Cyclophosphamide-treated mice were highly sensitive to S. aureus BSI and developed infectious lesions lacking immune cell infiltrates. Virulence factors of S. aureus that are key for disease establishment in immunocompetent hosts—α-hemolysin (Hla), iron-regulated surface determinants (IsdA and IsdB), coagulase (Coa), and von Willebrand factor binding protein (vWbp)—are dispensable for the pathogenesis of BSI in leukopenic mice. In contrast, sortase A mutants, which cannot assemble surface proteins, display delayed time to death and increased survival in this model. A vaccine with four surface antigens (ClfA, FnBPB, SdrD, and SpAKKAA), which was identified by genetic vaccinology using sortase A mutants, raised antigen-specific immune responses that protected leukopenic mice against staphylococcal BSI. PMID:25183728

RNA polymerase II conserved protein domains as platforms for protein-protein interactions

PubMed Central

García-López, M Carmen

2011-01-01

RNA polymerase II establishes many protein-protein interactions with transcriptional regulators to coordinate gene expression, but little is known about protein domains involved in the contact with them. We use a new approach to look for conserved regions of the RNA pol II of S. cerevisiae located at the surface of the structure of the complex, hypothesizing that they might be involved in the interaction with transcriptional regulators. We defined five different conserved domains and demonstrate that all of them make contact with transcriptional regulators. PMID:21922063

Mechanisms of protein stabilization and prevention of protein aggregation by glycerol.

PubMed

Vagenende, Vincent; Yap, Miranda G S; Trout, Bernhardt L

2009-11-24

The stability of proteins in aqueous solution is routinely enhanced by cosolvents such as glycerol. Glycerol is known to shift the native protein ensemble to more compact states. Glycerol also inhibits protein aggregation during the refolding of many proteins. However, mechanistic insight into protein stabilization and prevention of protein aggregation by glycerol is still lacking. In this study, we derive mechanisms of glycerol-induced protein stabilization by combining the thermodynamic framework of preferential interactions with molecular-level insight into solvent-protein interactions gained from molecular simulations. Contrary to the common conception that preferential hydration of proteins in polyol/water mixtures is determined by the molecular size of the polyol and the surface area of the protein, we present evidence that preferential hydration of proteins in glycerol/water mixtures mainly originates from electrostatic interactions that induce orientations of glycerol molecules at the protein surface such that glycerol is further excluded. These interactions shift the native protein toward more compact conformations. Moreover, glycerol preferentially interacts with large patches of contiguous hydrophobicity where glycerol acts as an amphiphilic interface between the hydrophobic surface and the polar solvent. Accordingly, we propose that glycerol prevents protein aggregation by inhibiting protein unfolding and by stabilizing aggregation-prone intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol. These mechanisms agree well with experimental data available in the literature, and we discuss the extent to which these mechanisms apply to other cosolvents, including polyols, arginine, and urea.

Protein leverage affects energy intake of high-protein diets in humans.

PubMed

Martens, Eveline A; Lemmens, Sofie G; Westerterp-Plantenga, Margriet S

2013-01-01

The protein leverage hypothesis requires specific evidence that protein intake is regulated more strongly than energy intake. The objective was to determine ad libitum energy intake, body weight changes, and appetite profile in response to protein-to-carbohydrate + fat ratio over 12 consecutive days and in relation to age, sex, BMI, and type of protein. A 12-d randomized crossover study was performed in 40 men and 39 women [mean ± SD age: 34.0 ± 17.6 y; BMI (in kg/m(2)): 23.7 ± 3.4] with the use of diets containing 5%, 15%, and 30% of energy from protein from a milk or plant source. Protein-content effects did not differ by age, sex, BMI, or type of protein. Total energy intake was significantly lower in the high-protein (7.21 ± 3.08 MJ/d) condition than in the low-protein (9.33 ± 3.52 MJ/d) and normal-protein (9.62 ± 3.51 MJ/d) conditions (P = 0.001), which was predominantly the result of a lower energy intake from meals (P = 0.001). Protein intake varied directly according to the amount of protein in the diet (P = 0.001). The AUC of visual analog scale appetite ratings did not differ significantly, yet fluctuations in hunger (P = 0.019) and desire to eat (P = 0.026) over the day were attenuated in the high-protein condition compared with the normal-protein condition. We found evidence to support the protein leverage hypothesis in that individuals underate relative to energy balance from diets containing a higher protein-to-carbohydrate + fat ratio. No evidence for protein leverage effects from diets containing a lower ratio of protein to carbohydrate + fat was obtained. It remains to be shown whether a relatively low protein intake would cause overeating or would be the effect of overeating of carbohydrate and fat. The study was registered at clinicaltrials.gov as NCT01320189.

Exploring the repeat protein universe through computational protein design

DOE PAGES

Brunette, TJ; Parmeggiani, Fabio; Huang, Po-Ssu; ...

2015-12-16

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. In this paper, we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix–loop–helix–loop structural motif. Eighty-three designs with sequences unrelatedmore » to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Finally, our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.« less

Protein profiling of water and alkali soluble cottonseed protein isolates.

PubMed

He, Zhongqi; Zhang, Dunhua; Cao, Heping

2018-06-18

Currently, there is only limited knowledge on the protein types and structures of the cottonseed proteins. In this work, water-soluble cottonseed proteins (CSPw) and alkali-soluble cottonseed proteins (CSPa) were sequentially extracted from defatted cottonseed meal. Proteins of the two fractions were separated by 4-20% gradient polyacrylamide gel electrophoresis (SDS-PAGE); There were 7 and 12 polypeptide bands on SDS-PAGE of CSPa and CSPw, respectively. These individual bands were then excised from the gel and subjected to mass spectrometric analysis. There were total 70 polypeptides identified from the proteins of the two cottonseed preparations, with molecular weights ranging from 10 to 381 kDa. While many proteins or their fragments were found in multiple bands, 18 proteins appeared only in one SDS-PAGE band (6 in CSPa, 12 in CSPw). Putative functions of these proteins include storage, transcription/translation, synthesis, energy metabolism, antimicrobial activity, and embryogenesis. Among the most abundant are legumin A (58 kDa), legumin B (59 kDa), vicilin C72 (70 kDa), vicilin GC72-A (71 kDa), and vicilin-like antimicrobial peptides (62 kDa). This work enriched the fundamental knowledge on cottonseed protein composition, and would help in better understanding of the functional and physicochemical properties of cottonseed protein and for enhancing its biotechnological utilization.

The Proteins API: accessing key integrated protein and genome information.

PubMed

Nightingale, Andrew; Antunes, Ricardo; Alpi, Emanuele; Bursteinas, Borisas; Gonzales, Leonardo; Liu, Wudong; Luo, Jie; Qi, Guoying; Turner, Edd; Martin, Maria

2017-07-03

The Proteins API provides searching and programmatic access to protein and associated genomics data such as curated protein sequence positional annotations from UniProtKB, as well as mapped variation and proteomics data from large scale data sources (LSS). Using the coordinates service, researchers are able to retrieve the genomic sequence coordinates for proteins in UniProtKB. This, the LSS genomics and proteomics data for UniProt proteins is programmatically only available through this service. A Swagger UI has been implemented to provide documentation, an interface for users, with little or no programming experience, to 'talk' to the services to quickly and easily formulate queries with the services and obtain dynamically generated source code for popular programming languages, such as Java, Perl, Python and Ruby. Search results are returned as standard JSON, XML or GFF data objects. The Proteins API is a scalable, reliable, fast, easy to use RESTful services that provides a broad protein information resource for users to ask questions based upon their field of expertise and allowing them to gain an integrated overview of protein annotations available to aid their knowledge gain on proteins in biological processes. The Proteins API is available at (http://www.ebi.ac.uk/proteins/api/doc). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Local Geometry and Evolutionary Conservation of Protein Surfaces Reveal the Multiple Recognition Patches in Protein-Protein Interactions

PubMed Central

Laine, Elodie; Carbone, Alessandra

2015-01-01

Protein-protein interactions (PPIs) are essential to all biological processes and they represent increasingly important therapeutic targets. Here, we present a new method for accurately predicting protein-protein interfaces, understanding their properties, origins and binding to multiple partners. Contrary to machine learning approaches, our method combines in a rational and very straightforward way three sequence- and structure-based descriptors of protein residues: evolutionary conservation, physico-chemical properties and local geometry. The implemented strategy yields very precise predictions for a wide range of protein-protein interfaces and discriminates them from small-molecule binding sites. Beyond its predictive power, the approach permits to dissect interaction surfaces and unravel their complexity. We show how the analysis of the predicted patches can foster new strategies for PPIs modulation and interaction surface redesign. The approach is implemented in JET2, an automated tool based on the Joint Evolutionary Trees (JET) method for sequence-based protein interface prediction. JET2 is freely available at www.lcqb.upmc.fr/JET2. PMID:26690684

Predicting Human Protein Subcellular Locations by the Ensemble of Multiple Predictors via Protein-Protein Interaction Network with Edge Clustering Coefficients

PubMed Central

Du, Pufeng; Wang, Lusheng

2014-01-01

One of the fundamental tasks in biology is to identify the functions of all proteins to reveal the primary machinery of a cell. Knowledge of the subcellular locations of proteins will provide key hints to reveal their functions and to understand the intricate pathways that regulate biological processes at the cellular level. Protein subcellular location prediction has been extensively studied in the past two decades. A lot of methods have been developed based on protein primary sequences as well as protein-protein interaction network. In this paper, we propose to use the protein-protein interaction network as an infrastructure to integrate existing sequence based predictors. When predicting the subcellular locations of a given protein, not only the protein itself, but also all its interacting partners were considered. Unlike existing methods, our method requires neither the comprehensive knowledge of the protein-protein interaction network nor the experimentally annotated subcellular locations of most proteins in the protein-protein interaction network. Besides, our method can be used as a framework to integrate multiple predictors. Our method achieved 56% on human proteome in absolute-true rate, which is higher than the state-of-the-art methods. PMID:24466278

HDOCK: a web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy.

PubMed

Yan, Yumeng; Zhang, Di; Zhou, Pei; Li, Botong; Huang, Sheng-You

2017-07-03

Protein-protein and protein-DNA/RNA interactions play a fundamental role in a variety of biological processes. Determining the complex structures of these interactions is valuable, in which molecular docking has played an important role. To automatically make use of the binding information from the PDB in docking, here we have presented HDOCK, a novel web server of our hybrid docking algorithm of template-based modeling and free docking, in which cases with misleading templates can be rescued by the free docking protocol. The server supports protein-protein and protein-DNA/RNA docking and accepts both sequence and structure inputs for proteins. The docking process is fast and consumes about 10-20 min for a docking run. Tested on the cases with weakly homologous complexes of <30% sequence identity from five docking benchmarks, the HDOCK pipeline tied with template-based modeling on the protein-protein and protein-DNA benchmarks and performed better than template-based modeling on the three protein-RNA benchmarks when the top 10 predictions were considered. The performance of HDOCK became better when more predictions were considered. Combining the results of HDOCK and template-based modeling by ranking first of the template-based model further improved the predictive power of the server. The HDOCK web server is available at http://hdock.phys.hust.edu.cn/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Druggable orthosteric and allosteric hot spots to target protein-protein interactions.

PubMed

Ma, Buyong; Nussinov, Ruth

2014-01-01

Drug designing targeting protein-protein interactions is challenging. Because structural elucidation and computational analysis have revealed the importance of hot spot residues in stabilizing these interactions, there have been on-going efforts to develop drugs which bind the hot spots and out-compete the native protein partners. The question arises as to what are the key 'druggable' properties of hot spots in protein-protein interactions and whether these mimic the general hot spot definition. Identification of orthosteric (at the protein- protein interaction site) and allosteric (elsewhere) druggable hot spots is expected to help in discovering compounds that can more effectively modulate protein-protein interactions. For example, are there any other significant features beyond their location in pockets in the interface? The interactions of protein-protein hot spots are coupled with conformational dynamics of protein complexes. Currently increasing efforts focus on the allosteric drug discovery. Allosteric drugs bind away from the native binding site and can modulate the native interactions. We propose that identification of allosteric hot spots could similarly help in more effective allosteric drug discovery. While detection of allosteric hot spots is challenging, targeting drugs to these residues has the potential of greatly increasing the hot spot and protein druggability.

Picornavirus proteins share antigenic determinants with heat shock proteins 60/65.

PubMed

Härkönen, T; Puolakkainen, M; Sarvas, M; Airaksinen, U; Hovi, T; Roivainen, M

2000-11-01

Immunological cross-reactions between enteroviruses and islet cell autoantigens have been suggested to play a role in the etiopathogenesis of insulin dependent diabetes mellitus (IDDM). In the nonobese diabetic mouse, an autoimmune model of IDDM, one of the reactive beta cell autoantigens is the heat shock protein 60 (HSP60). These studies were prompted by sequence homology discovered between the immunogenic region in HSP60 and two regions in enterovirus capsid proteins, one in the VP1 protein and the other in the VP0, the precursor of VP2 and VP4 proteins. Possible immunological cross-reactions between enterovirus proteins and heat shock proteins were studied by EIA and immunoblotting by using purified virus preparations, viral expression proteins VP1 and VP0, and recombinant HSP60/65 proteins, and corresponding polyclonal antisera. The HSP60/65 family of proteins is highly conserved and there is a striking degree of homology between bacterial and human heat shock proteins. Rabbit antibodies to HSP65 of Mycobacterium bovis that reacted with human HSP60 were also found to recognise capsid protein VP1 of coxsackievirus A9, VP1, and/or VP2 of coxsackievirus B4. Both viruses were also recognised by antisera raised against HSP60 of Chlamydia pneumoniae. In addition to the capsid proteins derived from native virions, antisera to both bacterial HSP proteins recognised expression protein VP1 of coxsackievirus A9. The cross-reactivity was also demonstrated the other way around; antisera to purified virus particles reacted with the HSP 60/65 proteins to some extent. These results suggest that apart from the well-documented sequence homology between the 2C protein of coxsackieviruses and the beta-cell autoantigen glutamic acid decarboxylase, there are other motifs in picornavirus proteins homologous to islet cell autoantigens, which might induce cross-reacting immune responses during picornavirus infections.

The essential and downstream common proteins of amyotrophic lateral sclerosis: A protein-protein interaction network analysis.

PubMed

Mao, Yimin; Kuo, Su-Wei; Chen, Le; Heckman, C J; Jiang, M C

2017-01-01

Amyotrophic Lateral Sclerosis (ALS) is a devastative neurodegenerative disease characterized by selective loss of motoneurons. While several breakthroughs have been made in identifying ALS genetic defects, the detailed molecular mechanisms are still unclear. These genetic defects involve in numerous biological processes, which converge to a common destiny: motoneuron degeneration. In addition, the common comorbid Frontotemporal Dementia (FTD) further complicates the investigation of ALS etiology. In this study, we aimed to explore the protein-protein interaction network built on known ALS-causative genes to identify essential proteins and common downstream proteins between classical ALS and ALS+FTD (classical ALS + ALS/FTD) groups. The results suggest that classical ALS and ALS+FTD share similar essential protein set (VCP, FUS, TDP-43 and hnRNPA1) but have distinctive functional enrichment profiles. Thus, disruptions to these essential proteins might cause motoneuron susceptible to cellular stresses and eventually vulnerable to proteinopathies. Moreover, we identified a common downstream protein, ubiquitin-C, extensively interconnected with ALS-causative proteins (22 out of 24) which was not linked to ALS previously. Our in silico approach provides the computational background for identifying ALS therapeutic targets, and points out the potential downstream common ground of ALS-causative mutations.

Interplay between binding affinity and kinetics in protein-protein interactions.

PubMed

Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

2016-07-01

To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Protein annotation from protein interaction networks and Gene Ontology.

PubMed

Nguyen, Cao D; Gardiner, Katheleen J; Cios, Krzysztof J

2011-10-01

We introduce a novel method for annotating protein function that combines Naïve Bayes and association rules, and takes advantage of the underlying topology in protein interaction networks and the structure of graphs in the Gene Ontology. We apply our method to proteins from the Human Protein Reference Database (HPRD) and show that, in comparison with other approaches, it predicts protein functions with significantly higher recall with no loss of precision. Specifically, it achieves 51% precision and 60% recall versus 45% and 26% for Majority and 24% and 61% for χ²-statistics, respectively. Copyright © 2011 Elsevier Inc. All rights reserved.

Interplay Between Protein Homeostasis Networks in Protein Aggregation and Proteotoxicity

PubMed Central

Douglas, Peter M.; Cyr, Douglas M.

2010-01-01

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self-association of disease proteins and determine whether they elicit a toxic or benign outcome. PMID:19768782

Modifications in nanoparticle-protein interactions by varying the protein conformation

NASA Astrophysics Data System (ADS)

Kumar, Sugam; Yadav, I.; Aswal, V. K.; Kohlbrecher, J.

2017-05-01

Small-angle neutron scattering has been used to study the interaction of silica nanoparticle with Bovine Serum Albumin (BSA) protein without and with a protein denaturing agent urea. The measurements have been carried out at pH 7 where both the components (nanoparticle and protein) are similarly charged. We show that the interactions in nanoparticle-protein system can be modified by changing the conformation of protein through the presence of urea. In the absence of urea, the strong electrostatic repulsion between the nanoparticle and protein prevents protein adsorption on nanoparticle surface. This non-adsorption, in turn gives rise to depletion attraction between nanoparticles. However, with addition of urea the depletion attraction is completely suppressed. Urea driven denaturation of protein is utilized to expose the positively charged patched of the BSA molecules which eventually leads to adsorption of BSA on nanoparticles eliminating the depletion interaction.

Dietary Protein Intake in Dutch Elderly People: A Focus on Protein Sources.

PubMed

Tieland, Michael; Borgonjen-Van den Berg, Karin J; Van Loon, Luc J C; de Groot, Lisette C P G M

2015-11-25

Sufficient high quality dietary protein intake is required to prevent or treat sarcopenia in elderly people. Therefore, the intake of specific protein sources as well as their timing of intake are important to improve dietary protein intake in elderly people. to assess the consumption of protein sources as well as the distribution of protein sources over the day in community-dwelling, frail and institutionalized elderly people. Habitual dietary intake was evaluated using 2- and 3-day food records collected from various studies involving 739 community-dwelling, 321 frail and 219 institutionalized elderly people. Daily protein intake averaged 71 ± 18 g/day in community-dwelling, 71 ± 20 g/day in frail and 58 ± 16 g/day in institutionalized elderly people and accounted for 16% ± 3%, 16% ± 3% and 17% ± 3% of their energy intake, respectively. Dietary protein intake ranged from 10 to 12 g at breakfast, 15 to 23 g at lunch and 24 to 31 g at dinner contributing together over 80% of daily protein intake. The majority of dietary protein consumed originated from animal sources (≥60%) with meat and dairy as dominant sources. Thus, 40% of the protein intake in community-dwelling, 37% in frail and 29% in institutionalized elderly originated from plant based protein sources with bread as the principle source. Plant based proteins contributed for >50% of protein intake at breakfast and between 34% and 37% at lunch, with bread as the main source. During dinner, >70% of the protein intake originated from animal protein, with meat as the dominant source. Daily protein intake in these older populations is mainly (>80%) provided by the three main meals, with most protein consumed during dinner. More than 60% of daily protein intake consumed is of animal origin, with plant based protein sources representing nearly 40% of total protein consumed. During dinner, >70% of the protein intake originated from animal protein, while during breakfast and lunch a large proportion of

Thioredoxin-interacting protein regulates protein disulfide isomerases and endoplasmic reticulum stress.

PubMed

Lee, Samuel; Min Kim, Soo; Dotimas, James; Li, Letitia; Feener, Edward P; Baldus, Stephan; Myers, Ronald B; Chutkow, William A; Patwari, Parth; Yoshioka, Jun; Lee, Richard T

2014-06-01

The endoplasmic reticulum (ER) is responsible for protein folding, modification, and trafficking. Accumulation of unfolded or misfolded proteins represents the condition of ER stress and triggers the unfolded protein response (UPR), a key mechanism linking supply of excess nutrients to insulin resistance and type 2 diabetes in obesity. The ER harbors proteins that participate in protein folding including protein disulfide isomerases (PDIs). Changes in PDI activity are associated with protein misfolding and ER stress. Here, we show that thioredoxin-interacting protein (Txnip), a member of the arrestin protein superfamily and one of the most strongly induced proteins in diabetic patients, regulates PDI activity and UPR signaling. We found that Txnip binds to PDIs and increases their enzymatic activity. Genetic deletion of Txnip in cells and mice led to increased protein ubiquitination and splicing of the UPR regulated transcription factor X-box-binding protein 1 (Xbp1s) at baseline as well as under ER stress. Our results reveal Txnip as a novel direct regulator of PDI activity and a feedback mechanism of UPR signaling to decrease ER stress. © 2014 Brigham and Women's Hospital. Published under the terms of the CC BY 4.0 license.

Introduction to current and future protein therapeutics: a protein engineering perspective.

PubMed

Carter, Paul J

2011-05-15

Protein therapeutics and its enabling sister discipline, protein engineering, have emerged since the early 1980s. The first protein therapeutics were recombinant versions of natural proteins. Proteins purposefully modified to increase their clinical potential soon followed with enhancements derived from protein or glycoengineering, Fc fusion or conjugation to polyethylene glycol. Antibody-based drugs subsequently arose as the largest and fastest growing class of protein therapeutics. The rationale for developing better protein therapeutics with enhanced efficacy, greater safety, reduced immunogenicity or improved delivery comes from the convergence of clinical, scientific, technological and commercial drivers that have identified unmet needs and provided strategies to address them. Future protein drugs seem likely to be more extensively engineered to improve their performance, e.g., antibodies and Fc fusion proteins with enhanced effector functions or extended half-life. Two old concepts for improving antibodies, namely antibody-drug conjugates and bispecific antibodies, have advanced to the cusp of clinical success. As for newer protein therapeutic platform technologies, several engineered protein scaffolds are in early clinical development and offer differences and some potential advantages over antibodies. Copyright © 2011 Elsevier Inc. All rights reserved.

Introduction to current and future protein therapeutics: A protein engineering perspective

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carter, Paul J., E-mail: pjc@gene.com

2011-05-15

Protein therapeutics and its enabling sister discipline, protein engineering, have emerged since the early 1980s. The first protein therapeutics were recombinant versions of natural proteins. Proteins purposefully modified to increase their clinical potential soon followed with enhancements derived from protein or glycoengineering, Fc fusion or conjugation to polyethylene glycol. Antibody-based drugs subsequently arose as the largest and fastest growing class of protein therapeutics. The rationale for developing better protein therapeutics with enhanced efficacy, greater safety, reduced immunogenicity or improved delivery comes from the convergence of clinical, scientific, technological and commercial drivers that have identified unmet needs and provided strategies tomore » address them. Future protein drugs seem likely to be more extensively engineered to improve their performance, e.g., antibodies and Fc fusion proteins with enhanced effector functions or extended half-life. Two old concepts for improving antibodies, namely antibody-drug conjugates and bispecific antibodies, have advanced to the cusp of clinical success. As for newer protein therapeutic platform technologies, several engineered protein scaffolds are in early clinical development and offer differences and some potential advantages over antibodies.« less

Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins.

PubMed

Cortajarena, Aitziber L; Kajander, Tommi; Pan, Weilan; Cocco, Melanie J; Regan, Lynne

2004-04-01

Protein design aims to understand the fundamentals of protein structure by creating novel proteins with pre-specified folds. An equally important goal is to understand protein function by creating novel proteins with pre-specified activities. Here we describe the design and characterization of a tetratricopeptide (TPR) protein, which binds to the C-terminal peptide of the eukaryotic chaperone Hsp90. The design emphasizes the importance of both direct, short-range protein-peptide interactions and of long-range electrostatic optimization. We demonstrate that the designed protein binds specifically to the desired peptide and discriminates between it and the similar C-terminal peptide of Hsp70.

Two potato proteins, including a novel RING finger protein (HIP1), interact with the potyviral multifunctional protein HCpro.

PubMed

Guo, Deyin; Spetz, Carl; Saarma, Mart; Valkonen, Jari P T

2003-05-01

Potyviral helper-component proteinase (HCpro) is a multifunctional protein exerting its cellular functions in interaction with putative host proteins. In this study, cellular protein partners of the HCpro encoded by Potato virus A (PVA) (genus Potyvirus) were screened in a potato leaf cDNA library using a yeast two-hybrid system. Two cellular proteins were obtained that interact specifically with PVA HCpro in yeast and in the two in vitro binding assays used. Both proteins are encoded by single-copy genes in the potato genome. Analysis of the deduced amino acid sequences revealed that one (HIP1) of the two HCpro interactors is a novel RING finger protein. The sequence of the other protein (HIP2) showed no resemblance to the protein sequences available from databanks and has known biological functions.

Texturized dairy proteins.

PubMed

Onwulata, Charles I; Phillips, John G; Tunick, Michael H; Qi, Phoebi X; Cooke, Peter H

2010-03-01

Dairy proteins are amenable to structural modifications induced by high temperature, shear, and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey protein concentrate (WPC), and whey protein isolate (WPI) were modified using a twin-screw extruder at melt temperatures of 50, 75, and 100 degrees C, and moistures ranging from 20 to 70 wt%. Viscoelasticity and solubility measurements showed that extrusion temperature was a more significant (P < 0.05) change factor than moisture content. The degree of texturization, or change in protein state, was characterized by solubility (R(2)= 0.98). The consistency of the extruded dairy protein ranged from rigid (2500 N) to soft (2.7 N). Extruding at or above 75 degrees C resulted in increased peak force for WPC (138 to 2500 N) and WPI (2.7 to 147.1 N). NDM was marginally texturized; the presence of lactose interfered with its texturization. WPI products extruded at 50 degrees C were not texturized; their solubility values ranged from 71.8% to 92.6%. A wide possibility exists for creating new foods with texturized dairy proteins due to the extensive range of states achievable. Dairy proteins can be used to boost the protein content in puffed snacks made from corn meal, but unmodified, they bind water and form doughy pastes with starch. To minimize the water binding property of dairy proteins, WPI, or WPC, or NDM were modified by extrusion processing. Extrusion temperature conditions were adjusted to 50, 75, or 100 degrees C, sufficient to change the structure of the dairy proteins, but not destroy them. Extrusion modified the structures of these dairy proteins for ease of use in starchy foods to boost nutrient levels. Dairy proteins can be used to boost the protein content in puffed snacks made from corn meal, but unmodified, they bind water and form doughy pastes with starch. To

Rapid discovery of protein interactions by cell-free protein technologies.

PubMed

He, M; Taussig, M J

2007-11-01

Cell-free transcription and translation provides an open, controllable environment for production of correctly folded, soluble proteins and allows the rapid generation of proteins from DNA without the need for cloning. Thus it is becoming an increasingly attractive alternative to conventional in vivo expression systems, especially when parallel expression of multiple proteins is required. Through novel design and exploitation, powerful cell-free technologies of ribosome display and protein in situ arrays have been developed for in vitro production and isolation of protein-binding molecules from large libraries. These technologies can be combined for rapid detection of protein interactions.

Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

NASA Astrophysics Data System (ADS)

Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

2016-04-01

Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems.

Jellyfish venomics and venom gland transcriptomics analysis of Stomolophus meleagris to reveal the toxins associated with sting.

PubMed

Li, Rongfeng; Yu, Huahua; Xue, Wei; Yue, Yang; Liu, Song; Xing, Ronge; Li, Pengcheng

2014-06-25

Jellyfish Stomolophus meleagris is a very dangerous animal because of its strong toxicity. However, the composition of the venom is still unclear. Both proteomics and transcriptomics approaches were applied in present study to investigate the major components and their possible relationships to the sting. The proteomics of the venom from S. meleagris was conducted by tryptic digestion of the crude venom followed by RP-HPLC separation and MS/MS analysis of the tryptic peptides. The venom gland transcriptome was analyzed using a high-throughput Illumina sequencing platform HiSeq 2000 with de novo assembly. A total of 218 toxins were identified including C-type lectin, phospholipase A₂ (PLA₂), potassium channel inhibitor, protease inhibitor, metalloprotease, hemolysin and other toxins, most of which should be responsible for the sting. Among them, serine protease inhibitor, PLA₂, potassium channel inhibitor and metalloprotease are predominant, representing 28.44%, 21.56%, 16.06% and 15.14% of the identified venom proteins, respectively. Overall, our combined proteomics and transcriptomics approach provides a systematic overview of the toxins in the venom of jellyfish S. meleagris and it will be significant to understand the mechanism of the sting. Jellyfish Stomolophus meleagris is a very dangerous animal because of its strong toxicity. It often bloomed in the coast of China in recent years and caused thousands of people stung and even deaths every year. However, the components which caused sting are still unknown yet. In addition, no study about the venomics of jellyfish S. meleagris has been reported. In the present study, both proteomics and transcriptomics approaches were applied to investigate the major components related to the sting. The result showed that major component included C-type lectin, phospholipase A₂, potassium channel inhibitor, protease inhibitor, metalloprotease, hemolysin and other toxins, which should be responsible for the effect of

Topology-function conservation in protein-protein interaction networks.

PubMed

Davis, Darren; Yaveroğlu, Ömer Nebil; Malod-Dognin, Noël; Stojmirovic, Aleksandar; Pržulj, Nataša

2015-05-15

Proteins underlay the functioning of a cell and the wiring of proteins in protein-protein interaction network (PIN) relates to their biological functions. Proteins with similar wiring in the PIN (topology around them) have been shown to have similar functions. This property has been successfully exploited for predicting protein functions. Topological similarity is also used to guide network alignment algorithms that find similarly wired proteins between PINs of different species; these similarities are used to transfer annotation across PINs, e.g. from model organisms to human. To refine these functional predictions and annotation transfers, we need to gain insight into the variability of the topology-function relationships. For example, a function may be significantly associated with specific topologies, while another function may be weakly associated with several different topologies. Also, the topology-function relationships may differ between different species. To improve our understanding of topology-function relationships and of their conservation among species, we develop a statistical framework that is built upon canonical correlation analysis. Using the graphlet degrees to represent the wiring around proteins in PINs and gene ontology (GO) annotations to describe their functions, our framework: (i) characterizes statistically significant topology-function relationships in a given species, and (ii) uncovers the functions that have conserved topology in PINs of different species, which we term topologically orthologous functions. We apply our framework to PINs of yeast and human, identifying seven biological process and two cellular component GO terms to be topologically orthologous for the two organisms. © The Author 2015. Published by Oxford University Press.

Protein space: a natural method for realizing the nature of protein universe.

PubMed

Yu, Chenglong; Deng, Mo; Cheng, Shiu-Yuen; Yau, Shek-Chung; He, Rong L; Yau, Stephen S-T

2013-02-07

Current methods cannot tell us what the nature of the protein universe is concretely. They are based on different models of amino acid substitution and multiple sequence alignment which is an NP-hard problem and requires manual intervention. Protein structural analysis also gives a direction for mapping the protein universe. Unfortunately, now only a minuscule fraction of proteins' 3-dimensional structures are known. Furthermore, the phylogenetic tree representations are not unique for any existing tree construction methods. Here we develop a novel method to realize the nature of protein universe. We show the protein universe can be realized as a protein space in 60-dimensional Euclidean space using a distance based on a normalized distribution of amino acids. Every protein is in one-to-one correspondence with a point in protein space, where proteins with similar properties stay close together. Thus the distance between two points in protein space represents the biological distance of the corresponding two proteins. We also propose a natural graphical representation for inferring phylogenies. The representation is natural and unique based on the biological distances of proteins in protein space. This will solve the fundamental question of how proteins are distributed in the protein universe. Copyright © 2012 Elsevier Ltd. All rights reserved.

Protein interactions and ligand binding: from protein subfamilies to functional specificity.

PubMed

Rausell, Antonio; Juan, David; Pazos, Florencio; Valencia, Alfonso

2010-02-02

The divergence accumulated during the evolution of protein families translates into their internal organization as subfamilies, and it is directly reflected in the characteristic patterns of differentially conserved residues. These specifically conserved positions in protein subfamilies are known as "specificity determining positions" (SDPs). Previous studies have limited their analysis to the study of the relationship between these positions and ligand-binding specificity, demonstrating significant yet limited predictive capacity. We have systematically extended this observation to include the role of differential protein interactions in the segregation of protein subfamilies and explored in detail the structural distribution of SDPs at protein interfaces. Our results show the extensive influence of protein interactions in the evolution of protein families and the widespread association of SDPs with protein interfaces. The combined analysis of SDPs in interfaces and ligand-binding sites provides a more complete picture of the organization of protein families, constituting the necessary framework for a large scale analysis of the evolution of protein function.

R4 RGS Proteins: Regulation of G Protein Signaling and Beyond

PubMed Central

Bansal, Geetanjali; Druey, Kirk M.; Xie, Zhihui

2007-01-01

The Regulators of G protein Signaling (RGS) proteins were initially characterized as inhibitors of signal transduction cascades initiated by G-protein-coupled receptors (GPCRs) because of their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins. This GTPase accelerating (GAP) activity enhances G protein deactivation and promotes desensitization. However, in addition to this signature trait, emerging data have revealed an expanding network of proteins, lipids, and ions that interact with RGS proteins and confer additional regulatory functions. This review highlights recent advances in our understanding of the physiological functions of one subfamily of RGS proteins with a high degree of homology (B/R4) gleaned from recent studies of knockout mice or cells with reduced RGS expression. We also discuss some of the newly-appreciated interactions of RGS proteins with cellular factors that suggest RGS control of several components of G-protein-mediated pathways as well as a diverse array of non-GPCR-mediated biological responses. PMID:18006065

Optimization of non-denaturing protein extraction conditions for plant PPR proteins.

PubMed

Andrés-Colás, Nuria; Van Der Straeten, Dominique

2017-01-01

Pentatricopeptide repeat proteins are one of the major protein families in flowering plants, containing around 450 members. They participate in RNA editing and are related to plant growth, development and reproduction, as well as to responses to ABA and abiotic stresses. Their characteristics have been described in silico; however, relatively little is known about their biochemical properties. Different types of PPR proteins, with different tasks in RNA editing, have been suggested to interact in an editosome to complete RNA editing. Other non-PPR editing factors, such as the multiple organellar RNA editing factors and the organelle RNA recognition motif-containing protein family, for example, have also been described in plants. However, while evidence on protein interactions between non-PPR RNA editing proteins is accumulating, very few PPR protein interactions have been reported; possibly due to their high instability. In this manuscript, we aimed to optimize the conditions for non-denaturing protein extraction of PPR proteins allowing in vivo protein analyses, such as interaction assays by co-immunoprecipitation. The unusually high protein degradation rate, the aggregation properties and the high pI, as well as the ATP-dependence of some PPR proteins, are key aspects to be considered when extracting PPR proteins in a non-denatured state. During extraction of PPR proteins, the use of proteasome and phosphatase inhibitors is critical. The use of the ATP-cofactor reduces considerably the degradation of PPR proteins. A short centrifugation step to discard cell debris is essential to avoid PPR precipitation; while in some cases, addition of a reductant is needed, probably caused by the pI/pH context. This work provides an easy and rapid optimized non-denaturing total protein extraction protocol from plant tissue, suitable for polypeptides of the PPR family.

Molecular simulation of the effect of cholesterol on lipid-mediated protein-protein interactions.

PubMed

de Meyer, Frédérick J-M; Rodgers, Jocelyn M; Willems, Thomas F; Smit, Berend

2010-12-01

Experiments and molecular simulations have shown that the hydrophobic mismatch between proteins and membranes contributes significantly to lipid-mediated protein-protein interactions. In this article, we discuss the effect of cholesterol on lipid-mediated protein-protein interactions as function of hydrophobic mismatch, protein diameter and protein cluster size, lipid tail length, and temperature. To do so, we study a mesoscopic model of a hydrated bilayer containing lipids and cholesterol in which proteins are embedded, with a hybrid dissipative particle dynamics-Monte Carlo method. We propose a mechanism by which cholesterol affects protein interactions: protein-induced, cholesterol-enriched, or cholesterol-depleted lipid shells surrounding the proteins affect the lipid-mediated protein-protein interactions. Our calculations of the potential of mean force between proteins and protein clusters show that the addition of cholesterol dramatically reduces repulsive lipid-mediated interactions between proteins (protein clusters) with positive mismatch, but does not affect attractive interactions between proteins with negative mismatch. Cholesterol has only a modest effect on the repulsive interactions between proteins with different mismatch. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Protein-protein interactions within late pre-40S ribosomes.

PubMed

Campbell, Melody G; Karbstein, Katrin

2011-01-20

Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

Validation of Biomarker Proteins Using Reverse Capture Protein Microarrays.

PubMed

Jozwik, Catherine; Eidelman, Ofer; Starr, Joshua; Pollard, Harvey B; Srivastava, Meera

2017-01-01

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody's titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

PubMed

Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina

2014-11-01

Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.

Protein-protein interaction predictions using text mining methods.

PubMed

Papanikolaou, Nikolas; Pavlopoulos, Georgios A; Theodosiou, Theodosios; Iliopoulos, Ioannis

2015-03-01

It is beyond any doubt that proteins and their interactions play an essential role in most complex biological processes. The understanding of their function individually, but also in the form of protein complexes is of a great importance. Nowadays, despite the plethora of various high-throughput experimental approaches for detecting protein-protein interactions, many computational methods aiming to predict new interactions have appeared and gained interest. In this review, we focus on text-mining based computational methodologies, aiming to extract information for proteins and their interactions from public repositories such as literature and various biological databases. We discuss their strengths, their weaknesses and how they complement existing experimental techniques by simultaneously commenting on the biological databases which hold such information and the benchmark datasets that can be used for evaluating new tools. Copyright © 2014 Elsevier Inc. All rights reserved.

Probing Protein-Protein Interactions by Dynamic Force Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Barsegov, V.; Thirumalai, D.

2005-10-01

We develop a formalism for single molecule dynamic force spectroscopy to map the energy landscape of protein-protein complex (P1P2). The joint distribution P(τ1,τ2) of unbinding lifetimes τ1 and τ2, measurable in a compression-tension cycle, which accounts for the internal relaxation dynamics of the proteins under tension, shows that the histogram of τ1 is not Poissonian. The theory is applied to the forced unbinding of protein P1, modeled as a wormlike chain, from P1P2. We propose a new class of experiments which can resolve the effect of internal protein dynamics on the unbinding lifetimes.

MetaGO: Predicting Gene Ontology of Non-homologous Proteins Through Low-Resolution Protein Structure Prediction and Protein-Protein Network Mapping.

PubMed

Zhang, Chengxin; Zheng, Wei; Freddolino, Peter L; Zhang, Yang

2018-03-10

Homology-based transferal remains the major approach to computational protein function annotations, but it becomes increasingly unreliable when the sequence identity between query and template decreases below 30%. We propose a novel pipeline, MetaGO, to deduce Gene Ontology attributes of proteins by combining sequence homology-based annotation with low-resolution structure prediction and comparison, and partner's homology-based protein-protein network mapping. The pipeline was tested on a large-scale set of 1000 non-redundant proteins from the CAFA3 experiment. Under the stringent benchmark conditions where templates with >30% sequence identity to the query are excluded, MetaGO achieves average F-measures of 0.487, 0.408, and 0.598, for Molecular Function, Biological Process, and Cellular Component, respectively, which are significantly higher than those achieved by other state-of-the-art function annotations methods. Detailed data analysis shows that the major advantage of the MetaGO lies in the new functional homolog detections from partner's homology-based network mapping and structure-based local and global structure alignments, the confidence scores of which can be optimally combined through logistic regression. These data demonstrate the power of using a hybrid model incorporating protein structure and interaction networks to deduce new functional insights beyond traditional sequence homology-based referrals, especially for proteins that lack homologous function templates. The MetaGO pipeline is available at http://zhanglab.ccmb.med.umich.edu/MetaGO/. Copyright © 2018. Published by Elsevier Ltd.

Anomalous Dynamics of Water Confined in Protein-Protein and Protein-DNA Interfaces.

PubMed

Chong, Song-Ho; Ham, Sihyun

2016-10-06

Confined water often exhibits anomalous properties not observable in the bulk phase. Although water in hydrophobic confinement has been the focus of intense investigation, the behavior of water confined between hydrophilic surfaces, which are more frequently found in biological systems, has not been fully explored. Here, we investigate using molecular dynamics simulations dynamical properties of the water confined in hydrophilic protein-protein and protein-DNA interfaces. We find that the interfacial water exhibits glassy slow relaxations even at 300 K. In particular, the rotational dynamics show a logarithmic decay that was observed in glass-forming liquids at deeply supercooled states. We argue that such slow water dynamics are indeed induced by the hydrophilic binding surfaces, which is in opposition to the picture that the hydration water slaves protein motions. Our results will significantly impact the view on the role of water in biomolecular interactions.

Protein particulates: another generic form of protein aggregation?

PubMed

Krebs, Mark R H; Devlin, Glyn L; Donald, A M

2007-02-15

Protein aggregation is a problem with a multitude of consequences, ranging from affecting protein expression to its implication in many diseases. Of recent interest is the specific form of aggregation leading to the formation of amyloid fibrils, structures associated with diseases such as Alzheimer's disease. The ability to form amyloid fibrils is now regarded as a property generic to all polypeptide chains. Here we show that around the isoelectric point a different generic form of aggregation can also occur by studying seven widely different, nonrelated proteins that are also all known to form amyloid fibrils. Under these conditions gels consisting of relatively monodisperse spherical particulates are formed. Although these gels have been described before for beta-lactoglobulin, our results suggest that the formation of particulates in the regime where charge on the molecules is minimal is a common property of all proteins. Because the proteins used here also form amyloid fibrils, we further propose that protein misfolding into clearly defined aggregates is a generic process whose outcome depends solely on the general properties of the state the protein is in when aggregation occurs, rather than the specific amino acid sequence. Thus under conditions of high net charge, amyloid fibrils form, whereas under conditions of low net charge, particulates form. This observation furthermore suggests that the rules of soft matter physics apply to these systems.

Viscosity Analysis of Dual Variable Domain Immunoglobulin Protein Solutions: Role of Size, Electroviscous Effect and Protein-Protein Interactions.

PubMed

Raut, Ashlesha S; Kalonia, Devendra S

2016-01-01

Increased solution viscosity results in difficulties in manufacturing and delivery of therapeutic protein formulations, increasing both the time and production costs, and leading to patient inconvenience. The solution viscosity is affected by the molecular properties of both the solute and the solvent. The purpose of this work was to investigate the effect of size, charge and protein-protein interactions on the viscosity of Dual Variable Domain Immunoglobulin (DVD-Ig(TM)) protein solutions. The effect of size of the protein molecule on solution viscosity was investigated by measuring intrinsic viscosity and excluded volume calculations for monoclonal antibody (mAb) and DVD-Ig(TM) protein solutions. The role of the electrostatic charge resulting in electroviscous effects for DVD-Ig(TM) protein was assessed by measuring zeta potential. Light scattering measurements were performed to detect protein-protein interactions affecting solution viscosity. DVD-Ig(TM) protein exhibited significantly higher viscosity compared to mAb. Intrinsic viscosity and excluded volume calculations indicated that the size of the molecule affects viscosity significantly at higher concentrations, while the effect was minimal at intermediate concentrations. Electroviscous contribution to the viscosity of DVD-Ig(TM) protein varied depending on the presence or absence of ions in the solution. In buffered solutions, negative k D and B 2 values indicated the presence of attractive interactions which resulted in high viscosity for DVD-Ig(TM) protein at certain pH and ionic strength conditions. Results show that more than one factor contributes to the increased viscosity of DVD-Ig(TM) protein and interplay of these factors modulates the overall viscosity behavior of the solution, especially at higher concentrations.

Moonlighting Proteins and Protein–Protein Interactions as Neurotherapeutic Targets in the G Protein-Coupled Receptor Field

PubMed Central

Fuxe, Kjell; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Palkovits, Miklós; Tarakanov, Alexander O; Ciruela, Francisco; Agnati, Luigi F

2014-01-01

There is serious interest in understanding the dynamics of the receptor–receptor and receptor–protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor–receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor–receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology. PMID:24105074

[Chemical libraries dedicated to protein-protein interactions].

PubMed

Sperandio, Olivier; Villoutreix, Bruno O; Morelli, Xavier; Roche, Philippe

2015-03-01

The identification of complete networks of protein-protein interactions (PPI) within a cell has contributed to major breakthroughs in understanding biological pathways, host-pathogen interactions and cancer development. As a consequence, PPI have emerged as a new class of promising therapeutic targets. However, they are still considered as a challenging class of targets for drug discovery programs. Recent successes have allowed the characterization of structural and physicochemical properties of protein-protein interfaces leading to a better understanding of how they can be disrupted with small molecule compounds. In addition, characterization of the profiles of PPI inhibitors has allowed the development of PPI-focused libraries. In this review, we present the current efforts at developing chemical libraries dedicated to these innovative targets. © 2015 médecine/sciences – Inserm.

A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition.

PubMed

Bourgeois, Michael; Jacquin, Françoise; Cassecuelle, Florence; Savois, Vincent; Belghazi, Maya; Aubert, Grégoire; Quillien, Laurence; Huart, Myriam; Marget, Pascal; Burstin, Judith

2011-05-01

Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using modified soy protein to enhance foaming of egg white protein.

PubMed

Wang, Guang; Troendle, Molly; Reitmeier, Cheryll A; Wang, Tong

2012-08-15

It is well known that the foaming properties of egg white protein are significantly reduced when a small amount of yolk is mixed in the white. To improve foaming properties of yolk-contaminated egg white protein, soy protein isolate (SPI) and egg proteins were modified to make basic proteins, and effects of these modified proteins on egg white foaming were evaluated in a model and an angel cake system. SPI and egg yolk proteins were modified to have an isoelectric point of 10, and sonication was used to increase protein dispersibility after the ethyl esterification reaction. However, only the addition of sonicated and modified SPI (SMSPI) showed improvement of foaming in the 5% egg protein model system with 0.4% yolk addition. SMSPI was then used in making angel food cake to examine whether the cake performance reduction due to yolk contamination of the white would be restored by such alkaline protein. Cake performance was improved when cream of tartar was used together with SMSPI. Basic soy protein can be made and used to improve egg white foaming properties and cake performance. Copyright © 2012 Society of Chemical Industry.

Resolubilization of Protein from Water-Insoluble Phlorotannin–Protein Complexes upon Acidification

PubMed Central

2017-01-01

Marine phlorotannins (PhT) from Laminaria digitata might protect feed proteins from ruminal digestion by formation of insoluble non-covalent tannin–protein complexes at rumen pH (6–7). Formation and disintegration of PhT–protein complexes was studied with β-casein (random coil) and bovine serum albumin (BSA, globular) at various pH. PhT had similar binding affinity for β-casein and BSA as pentagalloyl glucose, as studied by fluorescence quenching. The affinity of PhT for both proteins was independent of pH (3.0, 6.0, and 8.0). In the presence of PhT, the pH range for precipitation of tannin–protein complexes widened to 0.5–1.5 pH units around the isoelectric point (pI) of the protein. Complete protein resolubilization from insoluble PhT–protein complexes was achieved at pH 7 and 2 for β-casein and BSA, respectively. It was demonstrated that PhT modulate the solubility of proteins at neutral pH and that resolubilization of PhT–protein complexes at pH deviating from pI is mainly governed by the charge state of the protein. PMID:29058916

Specificity of molecular interactions in transient protein-protein interaction interfaces.

PubMed

Cho, Kyu-il; Lee, KiYoung; Lee, Kwang H; Kim, Dongsup; Lee, Doheon

2006-11-15

In this study, we investigate what types of interactions are specific to their biological function, and what types of interactions are persistent regardless of their functional category in transient protein-protein heterocomplexes. This is the first approach to analyze protein-protein interfaces systematically at the molecular interaction level in the context of protein functions. We perform systematic analysis at the molecular interaction level using classification and feature subset selection technique prevalent in the field of pattern recognition. To represent the physicochemical properties of protein-protein interfaces, we design 18 molecular interaction types using canonical and noncanonical interactions. Then, we construct input vector using the frequency of each interaction type in protein-protein interface. We analyze the 131 interfaces of transient protein-protein heterocomplexes in PDB: 33 protease-inhibitors, 52 antibody-antigens, 46 signaling proteins including 4 cyclin dependent kinase and 26 G-protein. Using kNN classification and feature subset selection technique, we show that there are specific interaction types based on their functional category, and such interaction types are conserved through the common binding mechanism, rather than through the sequence or structure conservation. The extracted interaction types are C(alpha)-- H...O==C interaction, cation...anion interaction, amine...amine interaction, and amine...cation interaction. With these four interaction types, we achieve the classification success rate up to 83.2% with leave-one-out cross-validation at k = 15. Of these four interaction types, C(alpha)--H...O==C shows binding specificity for protease-inhibitor complexes, while cation-anion interaction is predominant in signaling complexes. The amine ... amine and amine...cation interaction give a minor contribution to the classification accuracy. When combined with these two interactions, they increase the accuracy by 3.8%. In the case of

Protein-protein interactions in the regulation of WRKY transcription factors.

PubMed

Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

2013-03-01

It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

Functional structural motifs for protein-ligand, protein-protein, and protein-nucleic acid interactions and their connection to supersecondary structures.

PubMed

Kinjo, Akira R; Nakamura, Haruki

2013-01-01

Protein functions are mediated by interactions between proteins and other molecules. One useful approach to analyze protein functions is to compare and classify the structures of interaction interfaces of proteins. Here, we describe the procedures for compiling a database of interface structures and efficiently comparing the interface structures. To do so requires a good understanding of the data structures of the Protein Data Bank (PDB). Therefore, we also provide a detailed account of the PDB exchange dictionary necessary for extracting data that are relevant for analyzing interaction interfaces and secondary structures. We identify recurring structural motifs by classifying similar interface structures, and we define a coarse-grained representation of supersecondary structures (SSS) which represents a sequence of two or three secondary structure elements including their relative orientations as a string of four to seven letters. By examining the correspondence between structural motifs and SSS strings, we show that no SSS string has particularly high propensity to be found interaction interfaces in general, indicating any SSS can be used as a binding interface. When individual structural motifs are examined, there are some SSS strings that have high propensity for particular groups of structural motifs. In addition, it is shown that while the SSS strings found in particular structural motifs for nonpolymer and protein interfaces are as abundant as in other structural motifs that belong to the same subunit, structural motifs for nucleic acid interfaces exhibit somewhat stronger preference for SSS strings. In regard to protein folds, many motif-specific SSS strings were found across many folds, suggesting that SSS may be a useful description to investigate the universality of ligand binding modes.

Construction of reliable protein-protein interaction networks with a new interaction generality measure.

PubMed

Saito, Rintaro; Suzuki, Harukazu; Hayashizaki, Yoshihide

2003-04-12

Recent screening techniques have made large amounts of protein-protein interaction data available, from which biologically important information such as the function of uncharacterized proteins, the existence of novel protein complexes, and novel signal-transduction pathways can be discovered. However, experimental data on protein interactions contain many false positives, making these discoveries difficult. Therefore computational methods of assessing the reliability of each candidate protein-protein interaction are urgently needed. We developed a new 'interaction generality' measure (IG2) to assess the reliability of protein-protein interactions using only the topological properties of their interaction-network structure. Using yeast protein-protein interaction data, we showed that reliable protein-protein interactions had significantly lower IG2 values than less-reliable interactions, suggesting that IG2 values can be used to evaluate and filter interaction data to enable the construction of reliable protein-protein interaction networks.

Enteral delivery of proteins enhances the expression of proteins involved in the cytoskeleton and protein biosynthesis in human duodenal mucosa.

PubMed

Goichon, Alexis; Bertrand, Julien; Chan, Philippe; Lecleire, Stéphane; Coquard, Aude; Cailleux, Anne-Françoise; Vaudry, David; Déchelotte, Pierre; Coëffier, Moïse

2015-08-01

Amino acids are well known to be key effectors of gut protein turnover. We recently reported that enteral delivery of proteins markedly stimulated global duodenal protein synthesis in carbohydrate-fed healthy humans, but specifically affected proteins remain unknown. We aimed to assess the influence of an enteral protein supply on the duodenal mucosal proteome in carbohydrate-fed humans. Six healthy volunteers received for 5 h, on 2 occasions and in random order, either an enteral infusion of maltodextrins alone (0.25 g · kg⁻¹ · h⁻¹) mimicking the fed state or maltodextrins with a protein powder (0.14 g proteins · kg⁻¹ · h⁻¹). Endoscopic duodenal biopsy specimens were then collected and frozen until analysis. A 2-dimensional polyacrylamide gel electrophoresis-based comparative proteomics analysis was then performed, and differentially expressed proteins (at least ±1.5-fold change; Student's t test, P < 0.05) were identified by mass spectrometry. Protein expression changes were confirmed by Western blot analysis. Thirty-two protein spots were differentially expressed after protein delivery compared with maltodextrins alone: 28 and 4 spots were up- or downregulated, respectively. Among the 22 identified proteins, 11 upregulated proteins were involved either in the cytoskeleton (ezrin, moesin, plastin 1, lamin B1, vimentin, and β-actin) or in protein biosynthesis (glutamyl-prolyl-transfer RNA synthetase, glutaminyl-transfer RNA synthetase, elongation factor 2, elongation factor 1δ, and eukaryotic translation and initiation factor 3 subunit f). Enteral delivery of proteins altered the duodenal mucosal proteome and mainly stimulated the expression of proteins involved in cytoskeleton and protein biosynthesis. These results suggest that protein supply may affect intestinal morphology by stimulating actin cytoskeleton remodeling. © 2015 American Society for Nutrition.

Prediction of protein-protein interaction sites using electrostatic desolvation profiles.

PubMed

Fiorucci, Sébastien; Zacharias, Martin

2010-05-19

Protein-protein complex formation involves removal of water from the interface region. Surface regions with a small free energy penalty for water removal or desolvation may correspond to preferred interaction sites. A method to calculate the electrostatic free energy of placing a neutral low-dielectric probe at various protein surface positions has been designed and applied to characterize putative interaction sites. Based on solutions of the finite-difference Poisson equation, this method also includes long-range electrostatic contributions and the protein solvent boundary shape in contrast to accessible-surface-area-based solvation energies. Calculations on a large set of proteins indicate that in many cases (>90%), the known binding site overlaps with one of the six regions of lowest electrostatic desolvation penalty (overlap with the lowest desolvation region for 48% of proteins). Since the onset of electrostatic desolvation occurs even before direct protein-protein contact formation, it may help guide proteins toward the binding region in the final stage of complex formation. It is interesting that the probe desolvation properties associated with residue types were found to depend to some degree on whether the residue was outside of or part of a binding site. The probe desolvation penalty was on average smaller if the residue was part of a binding site compared to other surface locations. Applications to several antigen-antibody complexes demonstrated that the approach might be useful not only to predict protein interaction sites in general but to map potential antigenic epitopes on protein surfaces. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.