Virulence-associated Proteins Research Articles

Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes

BackgroundThe Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa.MethodsUsing mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides.ResultsMetacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes.ConclusionsThe data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.

Elongation Factor Thermo Unstable (EF-Tu) Moonlights as an Adhesin on the Surface of Mycoplasma hyopneumoniae by Binding to Fibronectin.

Mycoplasma hyopneumoniae is a colonizing respiratory pathogen that can cause great economic losses to the pig industry worldwide. Although putative virulence factors have been reported, the pathogenesis of this species remains unclear. Here, we used the virulent M. hyopneumoniae strain 168 to infect swine tracheal epithelial cells (STEC) to identify the infection-associated factors by two-dimensional electrophoresis (2-DE). Whole proteins of M. hyopneumoniae were obtained and compared with samples cultured in broth. Six differentially expressed proteins with an increase in abundance of ≥1.5 in the cell infection group were successfully identified. A String network of virulence-associated proteins showed that all the six differential abundance proteins were involved in virulence of M. hyopneumoniae. One of the most important upregulated hubs in this network, elongation factor thermo unstable (EF-Tu), which showed a relatively higher expression in M. hyopneumoniae-infected STEC and obtained a higher score on mass spectrometry was successfully recombined. In addition to its canonical enzymatic activities in protein synthesis, EF-Tu was also reported to be located on the cell surface as an important adhesin in many other pathogens. The cell surface location of EF-Tu was then observed in M. hyopneumoniae with flow cytometry. Recombinant EF-Tu (rEF-Tu) was found to be able to adhere to STEC and anti-rEF-Tu antibody enclosed M. hyopneumoniae decreased adherence to STEC. In addition, surface plasmon resonance (SPR) analysis showed that rEF-Tu could bind to fibronectin with a specific and moderately strong interaction, a dissociation constant (KD) of 605 nM. Furthermore, the block of fibronectin in STEC also decreased the binding of M. hyopneumoniae to the cell surface. Collectively, these data imply EF-Tu as an important adhesin of M. hyopneumoniae and fibronectin as an indispensable receptor on STEC. The binding between EF-Tu with fibronectin contributes to the adhesion of M. hyopneumoniae to STEC.HIGHLIGHTS Elongation factor thermo unstable (EF-Tu) exists on the cell surface of M. hyopneumoniae.EF-Tu moonlights as an adhesin of M. hyopneumoniae.The adhesive effect of EF-Tu is partly meditated by fibronectin.

Identification of Mycobacterium species and Rhodococcus equi in peccary lymph nodes.

Mycobacterium species and the virulence-associated proteins (vapA, vapB, and vapN genes) of Rhodococcus equi isolated from 330 lymph nodes of collared peccaries (Tayassu tajacu) and white-lipped peccaries (Tayassu pecari) intended for human consumption were investigated. Thirty-six (10.9%) R. equi strains were isolated; 3.3% (n = 11/330) were from white-lipped peccary lymph nodes, and 7.6% (25/330) were from collared peccary lymph nodes. Among the 11 isolates of R. equi from the white-lipped peccaries, 90.9% (n = 10/11) were obtained from the mesenteric lymph nodes, and only 9.1% (n = 1/10) were obtained from the mediastinal lymph nodes. In the 25 isolates of R. equi obtained from the collared peccaries, 40.0% (n = 10/25) were recovered from the mesenteric lymph nodes, 36% (n = 9/25) from the submandibular lymph nodes, and 24.0% (n = 6/25) from the mediastinal lymph nodes. No vapA, vapB, or vapN genes (plasmidless) or three host-associated types (pVAPA, pVAPB, and pVAPN) were identified among the R. equi isolates. Mycobacterium species were isolated in 3.03% (n = 10/330) of all the lymph nodes analyzed. Among the 10 mycobacterial isolates, 60% (n = 6/10) were from the white-lipped peccary lymph nodes, and 40% (n = 4/10) were from the collared peccary lymph nodes. Ten Mycobacterium species were detected by PCR-PRA with a predominance of M. avium type 1. Sequencing of the hsp65 and rpob genes revealed mycobacteria that were saprophytic (M. sinense and M. kumamotonense) and potentially pathogenic (M. colombiense and M. intracellulare) to humans and animals. To our knowledge, this is the first description of R. equi and/or mycobacterial species identified in the lymph nodes of peccary specimens. R. equi (plasmidless) and the mycobacterial species described here have been reported as causes of pulmonary and extrapulmonary infections in both immunocompetent and immunocompromised humans.

Type VII secretion system in Mycobacteria

Type Ⅶ secretion system (T7SS) is a novel and specialized secretion system discovered in recent years. It was first found in Mycobacterium tuberculosis. Type Ⅶ secretion system is involved in the secretion of virulence-associated proteins, the interaction between pathogens and hosts and the balance of zinc/iron ions. Moreover, it plays a critical role in the growth and pathogenesis of Mycobacteria. This review summarizes the components, substrates and translocation mechanisms of the type Ⅶ secretion system and its relation to the virulence of Mycobacteria aiming to provide references for developing novel strategies for diseases diagnosis, treatment and prevention. Key words: Mycobacteria; Type Ⅶ secretion system; Gene structure; Secretion mechanism; Pathogenicity

System-Wide Analysis Unravels the Differential Regulation and In Vivo Essentiality of Virulence-Associated Proteins B and C Toxin-Antitoxin Systems of Mycobacterium tuberculosis

Toxin-antitoxin (TA) systems are bicistronic genetic modules that are ubiquitously present in bacterial genomes. The Mycobacterium tuberculosis genome encodes 90 putative TA systems, and these are considered to be associated with maintenance of bacterial genomic stability or bacterial survival under unfavorable environmental conditions. The majority of these in M. tuberculosis have been annotated as belonging to the virulence-associated protein B and C (VapBC) family. However, their precise role in bacterial physiology has not been elucidated. Here, we functionally characterized VapC toxins from M. tuberculosis and show that overexpression of some homologs inhibits growth of Mycobacterium bovis bacillus Calmette-Guérin in a bacteriostatic manner. Expression profiling of messenger RNA revealed that these VapC toxins were differentially induced upon exposure of M. tuberculosis to stress conditions. We also unraveled that transcriptional cross-activation exists between TA systems in M. tuberculosis. This study provides the first evidence for the essentiality of VapBC3 and VapBC4 systems in M. tuberculosis virulence.

Characterization and functional analysis of PnuC that is involved in the oxidative stress tolerance and virulence of Streptococcus suis serotype 2

Streptococcus suis, an important swine pathogen and a major zoonotic agent, is responsible for severe financial losses in the global swine industry. Although a multitude of virulence factors have been reported, the pathogenesis of S. suis infections remains poorly understood. In our previous work, we identified a potential virulence-associated protein, named PnuC, unique to virulent strains of S. suis serotype 2 (S. suis 2). To investigate the functions of PnuC, the pnuC gene deletion mutant (ΔpnuC) was constructed in S. suis 2 strain ZY05719 to assess the phenotypic changes between ΔpnuC and the parental strain. The mutant strain ΔpnuC exhibited highly sensitive to H2O2 stress and reduced the growth ability under vigorous shaking, suggesting that PnuC contributes to the oxidative stress tolerance. Additionally, zebrafish infection model showed that the virulence of pnuC+ strains were significantly higher than pnuC- strains. Mouse infection experiments demonstrated that the abilities of ΔpnuC to colonize the tissues were significantly attenuated compared with the parental strain. Furthermore, the pnuC mutation decreased the virulence of S. suis 2 in both BALB/c mice and zebrafish infection models. Taken together, these results indicated for the first time that PnuC is involved in the oxidative stress tolerance and virulence of S. suis 2 during infection.

Mucosal Infections and Invasive Potential of Nonencapsulated Streptococcus pneumoniae Are Enhanced by Oligopeptide Binding Proteins AliC and AliD.

ABSTRACTNonencapsulated Streptococcus pneumoniae (NESp) is an emerging human pathogen that colonizes the nasopharynx and is associated with noninvasive diseases such as otitis media (OM), conjunctivitis, and nonbacteremic pneumonia. Since capsule expression was previously thought to be necessary for establishment of invasive pneumococcal disease (IPD), serotype-specific polysaccharide capsules are targeted by currently licensed pneumococcal vaccines. Yet, NESp expressing oligopeptide binding proteins AliC and AliD have been isolated during IPD. Thus, we hypothesize AliC and AliD are major NESp virulence determinants that facilitate persistence and development of IPD. Our study reveals that NESp expressing AliC and AliD have intensified virulence compared to isogenic mutants. Specifically, we demonstrate AliC and AliD enhance murine nasopharyngeal colonization and pulmonary infection and are required for OM in a chinchilla model. Furthermore, AliC and AliD increase pneumococcal survival in chinchilla whole blood and aid in resistance to killing by human leukocytes. Comparative proteome analysis revealed significant alterations in protein levels when AliC and AliD were absent. Virulence-associated proteins, including a pneumococcal surface protein C variant (CbpAC), were significantly downregulated, while starvation response indicators were upregulated in the double mutant relative to wild-type levels. We also reveal that differentially expressed CbpAC was essential for NESp adherence to epithelial cells, virulence during OM, reduction of C3b deposition on the NESp surface, and binding to nonspecific IgA. Altogether, the rise in NESp prevalence urges the need to understand how NESp establishes disease and persists in a host. This study highlights the roles of AliC, AliD, and CbpAC in the pathogenesis of NESp.

B-Cell Epitope Mapping Using a Library of Overlapping Synthetic Peptides in an Enzyme-Linked Immunosorbent Assay.

This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identified which corresponded to a 20mer sequence of the VapA protein.

The bile salt sodium taurocholate induces Campylobacter jejuni outer membrane vesicle production and increases OMV-associated proteolytic activity.

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence-associated proteins within outer membrane vesicles (OMVs). C.jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E-cadherin, promoting enhanced C.jejuni adhesion to and invasion of IECs. C.jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence-associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C.jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C.jejuni growth until the early stationary phase. Coculture of C.jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C.jejuni ST-OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST-OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs-associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs-induced cleavage of E-cadherin and occludin. C.jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C.jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence-associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.

Novel bovine-associated pVAPN plasmid type in Rhodococcus equi identified from lymph nodes of slaughtered cattle and lungs of people living with HIV/AIDS.

Rhodococcus equi is a well-recognized Gram-positive intracellular facultative bacterium that is opportunistic in nature, which causes pyogranulomatous infections in humans and multiple host animals. The pathogenicity of the microorganism has been attributed to the presence of plasmid-encoded virulence-associated proteins (Vap). To date, three host-associated virulence plasmid types of R.equi have been identified as follows: the circular pVAPA and pVAPB, related, respectively, to equine and porcine isolates, and a recently described linear pVAPN plasmid associated with bovine strains, although these three types are found in human isolates. Recent phylogenomic studies support the evidence that human R.equi infection is zoonotically acquired. Nevertheless, data regarding distribution and prevalence of the host-adapted virulence plasmid types of R.equi isolated from meat animals are scarce or unnoticed. Here, the three host-associated virulence plasmid types (pVAPA, pVAPB, and pVAPN) were investigated in 154 R.equi isolates recovered from lymph nodes of cattle with lymphadenitis (n=31), faeces of cattle without enteric signs (n=49), as well as different clinical specimens from human patients (n=74). The analysis of virulence profile of 74 R.equi from humans revealed six (8.1%) isolates pVAPB (type 8), two (2.7%) pVAPN, and one (1.3%) pVAPB (type 11), all of which were from lung samples from people living with HIV/AIDS. From the lymph node samples of cattle, 41.9% (13 of 31) isolates revealed pVAPN type, whereas all isolates from faecal samples were negative for three host-associated types. Here, recently described bovine-associated pVAPN type was detected in R.equi isolates recovered from the lungs of people living with HIV/AIDS and lymph nodes from slaughtered cattle intended for human consumption; a finding that represents a public health concern, mainly in countries where undercooked or raw meat are traditionally consumed.

The virulence-associated protein HsvA from the fire blight pathogen Erwinia amylovora is a polyamine amidinotransferase

Studies of virulence determinants in the bacterial phytopathogen Erwinia amylovora, the cause of devastating fire blight disease in apple and pear, have shown that HsvA, a putative amidinotransferase enzyme located in the Hrp pathogenicity island, is required for systemic infection in apple. However, the mechanism by which HsvA contributes to virulence is unclear. To investigate the role of HsvA in virulence, we carried out a series of biochemical and structural studies to characterize the amidinotransferase activity of HsvA. We found that HsvA displays a preference for linear aliphatic polyamines as the amidino acceptor substrate, especially for spermidine and putrescine (Km values of 33 μm and 3.9 mm, respectively). The three-dimensional structure, determined at 2.30 Å resolution using X-ray crystallography, revealed that the overall architecture of HsvA is similar to that of the human arginine-glycine amidinotransferase in the creatine biosynthesis pathway. The active site is located in the core of the protein at the base of a long, narrow substrate access channel. Specific amino acids near the entrance of the channel may serve as major determinants of the substrate specificity, including a glutamate residue at the rim of the channel entrance that appears to be positioned to interact with the distal primary amine in the putrescine substrate as well as the internal and distal amines in the spermidine substrate. These results suggest potential in vivo functions for HsvA as a virulence factor in fire blight and may also provide a basis for strategies to control fire blight by inhibiting HsvA activity.

Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome.

Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer's clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs. P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics.

Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome

Background: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. Methods: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate that had been adapted to in vitro culture for a relatively shorter period, and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). Results: We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer’s clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that PfEVs may play a role in parasite-host interactions. Comparison of this dataset with previously published datasets helps to define a core secretome present in PfEVs. Conclusions: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify new targets for interventions or diagnostics.

Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome

Background: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. Methods: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). Results: We show that P. falciparum extracellular vesicles (PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer’s clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs. Conclusions: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics.

ITRAQ-based quantitative proteomic analysis reveals multiple effects of Emodin to Haemophilus parasuis

Haemophilus parasuis, a symbiotic bacteria of upper respiratory tract of swine, is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis. Emodin, exhibits antibacterial activity against H. parasuis, yet the action mode has not been fully understood. In present study, isobaric tag for relative and absolute quantification (iTRAQ) method was applied to analyze the global protein alteration of H. parasuis in response to 16μg/mL Emodin. In total, 338 proteins exhibiting significant differential expressions were identified. It was speculated that, through application of bioinformatics analysis to theses differentially expressed proteins, Emodin mainly inhibited some key proteins expression of ABC transport system, carbohydrate metabolism pathway and bacterial cell division by inhibiting the ribosome synthesis, resulting in the growth inhibition of H. parasuis. Remarkably, nine virulence-associated proteins were detected differently expressed, further experiments revealed that after treatment with Emodin, H. parasuis could be inhibited to adhere to and invade into porcine kidney epithelial cells (PK-15 line) and exhibited increased sensitivity to serum complement in a concentration-dependent manner. Phagocytosis assay showed Emodin also could enhance phagocytic activity of porcine alveolar macrophages PAM to H. parasuis. These results indicated that Emodin also can attenuate virulence of H. parasuis and reduce infection. Biological significanceThe Glässer's disease caused by H. parasuis has become a typical bacterial disease and cause serious economic loss to the swine industry around the world. Antibiotics are extensively used to control the infection, but increasing antibiotic resistance has been a severe problem. Hence, novel treatment agents are needed. So far, few antibacterial agents were reported that could control H. parasuis infection. In the present study, the state-of-the-art quantitative proteomic technology was applied to uncover underlying action mechanism of Emodin. This study extends understanding of antibacterial effect of Emodin to H. parasuis at molecular level and provides useful information for further investigations. Moreover, our results provide theoretical foundation for the practical application of Emodin.

Differential distribution of vapA-positive Rhodococcus equi in affected and unaffected horse-breeding farms

Rhodococcus equi causes pulmonary and extrapulmonary infections in animals and humans, with endemic situations and significant young foal mortality in horse-breeding farms worldwide (von Bargen and Haas 2009, Muscatello 2012,...

Vaccination of Mice with Virulence-Associated Protein G (VapG) Antigen Confers Partial Protection against Rhodococcus equi Infection through Induced Humoral Immunity.

Rhodococcus equi is a facultative intracellular bacterium causing severe pyogranulomatous pneumonia, ulcerative enterocolitis, and mesenteric lymphadenopathy in foals aged less than 6 months. Less frequently, this pathogen affects various other species, such as pigs, cattle, cats, and even humans. Although rhodococcosis is treated with a combination of antimicrobial agents, resistance is developed in some cases, and thus, antimicrobial susceptibility must be monitored and managed. Considering these limitations of the current therapy and unavailability of a vaccine to prevent the disease, research is particularly focused on the development of an effective vaccine against rhodococcosis. Most vaccines undergoing development utilize the virulence-associated protein (Vap) A antigen, which was identified previously as a key virulence factor of R. equi. Nevertheless, other proteins, such as VapG, present in most virulent R. equi strains, are also encoded by vap genes located on the R. equi bacterial virulence plasmid. In the present study, we evaluated the effect of VapG immunization on the survival of R. equi-challenged mice. We used attenuated Salmonella as a carrier for VapG (Salmonella-vapG+), a procedure previously adopted to develop a VapA-based vaccine. We observed that vaccination with Salmonella-vapG+ induced both an increased IFN-γ, IL-12, and TNF-α production, and a decreased bacterial burden in organs of the R. equi-challenged mice. Nevertheless, Salmonella-vapG+ vaccination protected only 50% of the mice challenged with a lethal dose of R. equi. Interestingly, we observed an increased frequency of B cells in the spleen of Salmonella-vapG+-vaccinated mice and showed that Salmonella-vapG+-vaccinated R. equi-challenged B-cell-knockout mice did not reduce the bacterial burden. Given these results, we discussed the potential role of the humoral immune response induced by Salmonella-vapG+ vaccination in conferring protection against R. equi infection, as well as the employment of VapG antigen for obtaining hyperimmune plasma to prevent rhodoccocosis in young foals.

Epigallocatechin gallate inhibits Streptococcus pneumoniae virulence by simultaneously targeting pneumolysin and sortaseA.

Streptococcus pneumoniae (pneumococcus), the causative agent of several human diseases, possesses numerous virulence factors associated with pneumococcal infection and pathogenesis. Pneumolysin (PLY), an important virulence factor, is a member of the cholesterol‐dependent cytolysin family and has cytolytic activity. Sortase A (SrtA), another crucial pneumococcal virulence determinate, contributes greatly to the anchoring of many virulence‐associated surface proteins to the cell wall. In this study, epigallocatechin gallate (EGCG), a natural compound with little known antipneumococcal activity, was shown to directly inhibit PLY‐mediated haemolysis and cytolysis by blocking the oligomerization of PLY and simultaneously reduce the peptidase activity of SrtA. The biofilm formation, production of neuraminidase A (NanA, the pneumococcal surface protein anchored by SrtA), and bacterial adhesion to human epithelial cells (Hep2) were inhibited effectively when S. pneumoniae D39 was cocultured with EGCG. The results from molecular dynamics simulations and mutational analysis confirmed the interaction of EGCG with PLY and SrtA, and EGCG binds to Glu277, Tyr358, and Arg359 in PLY and Thr169, Lys171, and Phe239 in SrtA. In vivo studies further demonstrated that EGCG protected mice against S. pneumoniae pneumonia. Our results imply that EGCG is an effective inhibitor of both PLY and SrtA and that an antivirulence strategy that directly targets PLY and SrtA using EGCG is a promising therapeutic option for S. pneumoniae pneumonia.

Protective efficacy of six immunogenic recombinant proteins of Vibrio anguillarum and evaluation them as vaccine candidate for flounder (Paralichthys olivaceus).

Vibrio anguillarum is a severe bacterium that causes terminal haemorrhagic septicaemia in freshwater and marine fish. Virulence-associated proteins play an important role in bacterial pathogenicity and could be applied for immunoprophylaxis. In this study, six antigenic proteins from V.anguillarum were selected and the immune protective efficacy of their recombinant proteins was investigated. VirA, CheR, FlaC, OmpK, OmpR and Hsp33 were recombinantly produced and the reactions of recombinant proteins to flounder-anti-V.anguillarum antibodies (fV-ab) were detected, respectively. Then the recombinant proteins were injected to fish, after immunization, the percentages of surface membrane immunoglobulin-positive (sIg+) cell in lymphocytes, total antibodies, antibodies against V.anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were analyzed, respectively. The results showed that all the recombinant proteins could react to fV-ab, proliferate sIg+cells in lymphocytes and induce production of total antibodies, specific antibodies against V.anguillarum or the recombinant proteins; the RPS of rVirA, rCheR, rFlaC, rOmpK, rOmpR and rHsp33 against V.anguillarum was 70.27%, 27.03%, 16.22%, 62.16%, 45.95% and 81.08%, respectively. The results revealed that rHsp33, rVirA and rOmpK have good protections against V.anguillarum and could be vaccine candidates against V.anguillarum.

Type IV Secretion and Signal Transduction of Helicobacter pylori CagA through Interactions with Host Cell Receptors.

Helicobacter pylori is a highly successful human bacterium, which is exceptionally equipped to persistently inhabit the human stomach. Colonization by this pathogen is associated with gastric disorders ranging from chronic gastritis and peptic ulcers to cancer. Highly virulent H. pylori strains express the well-established adhesins BabA/B, SabA, AlpA/B, OipA, and HopQ, and a type IV secretion system (T4SS) encoded by the cag pathogenicity island (PAI). The adhesins ascertain intimate bacterial contact to gastric epithelial cells, while the T4SS represents an extracellular pilus-like structure for the translocation of the effector protein CagA. Numerous T4SS components including CagI, CagL, CagY, and CagA have been shown to target the integrin-β1 receptor followed by translocation of CagA across the host cell membrane. The interaction of CagA with membrane-anchored phosphatidylserine and CagA-containing outer membrane vesicles may also play a role in the delivery process. Translocated CagA undergoes tyrosine phosphorylation in C-terminal EPIYA-repeat motifs by oncogenic Src and Abl kinases. CagA then interacts with an array of host signaling proteins followed by their activation or inactivation in phosphorylation-dependent and phosphorylation-independent fashions. We now count about 25 host cell binding partners of intracellular CagA, which represent the highest quantity of all currently known virulence-associated effector proteins in the microbial world. Here we review the research progress in characterizing interactions of CagA with multiple host cell receptors in the gastric epithelium, including integrin-β1, EGFR, c-Met, CD44, E-cadherin, and gp130. The contribution of these interactions to H. pylori colonization, signal transduction, and gastric pathogenesis is discussed.