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The LuxS quorum sensing system is a widespread system employed by many bacteria for cell-to-cell communication. The luxS gene has been demonstrated to play a crucial role in intramacrophage survival of piscine Streptococcus agalactiae, but the underlying mechanism remains largely unknown. In this study, transcriptome analysis, followed by the luxS gene deletion and subsequent functional studies, confirmed that impaired bacterial survival inside macrophages due to the inactivation of luxS was associated with reduced transcription of the fruRKI operon, encoding the fructose-specific phosphotransferase system. Further, luxS was determined not to enhance the transcription of fruRKI operon by binding its promoter, but to upregulate the expression of this operon via affecting the binding ability of catabolite control protein A (CcpA) to the catabolite responsive element (cre) in the promoter of fruRKI. Collectively, our study identifies a novel and previously unappreciated role for luxS in bacterial intracellular survival, which may give a more thorough understanding of the immune evasion mechanism in S. agalactiae. [ABSTRACT FROM AUTHOR]

The LuxS quorum sensing system is a widespread system employed by many bacteria for cell-to-cell communication. The luxS gene has been demonstrated to play a crucial role in intramacrophage survival of piscine Streptococcus agalactiae, but the underlying mechanism remains largely unknown. In this study, transcriptome analysis, followed by the luxS gene deletion and subsequent functional studies, confirmed that impaired bacterial survival inside macrophages due to the inactivation of luxS was associated with reduced transcription of the fruRKI operon, encoding the fructose-specific phosphotransferase system. Further, luxS was determined not to enhance the transcription of fruRKI operon by binding its promoter, but to upregulate the expression of this operon via affecting the binding ability of catabolite control protein A (CcpA) to the catabolite responsive element (cre) in the promoter of fruRKI. Collectively, our study identifies a novel and previously unappreciated role for luxS in bacterial intracellular survival, which may give a more thorough understanding of the immune evasion mechanism in S. agalactiae. [ABSTRACT FROM AUTHOR]

Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen resulting in manifestations as pneumonia and septic shock. The upper respiratory tract is typically thought to be the main colonization and entry site of SS2 in pigs, but the mechanism through which it penetrates the respiratory barrier is still unclear. In this study, a mutant with low invasive potential to swine tracheal epithelial cells (STECs) was screened from the TnYLB-1 transposon insertion mutant library of SS2, and the interrupted gene was identified as autolysin (atl). Compared to wild-type (WT) SS2, Δatl mutant exhibited lower ability to penetrate the tracheal epithelial barrier in a mouse model. Purified Atl also enhanced SS2 translocation across STEC monolayers in Transwell inserts. Furthermore, Atl redistributed the tight junctions (TJs) in STECs through myosin light chain kinase (MLCK) signaling, which led to increased barrier permeability. Using mass spectrometry, co-immunoprecipitation (co-IP), pull-down, bacterial two-hybrid and saturation binding experiments, we showed that Atl binds directly to vimentin. CRISPR/Cas9-targeted deletion of vimentin in STECs (VIM KO STECs) abrogated the capacity of SS2 to translocate across the monolayers, SS2-induced phosphorylation of myosin II regulatory light chain (MLC) and MLCK transcription, indicating that vimentin is indispensable for MLCK activation. Consistently, vimentin null mice were protected from SS2 infection and exhibited reduced tracheal and lung injury. Thus, MLCK-mediated epithelial barrier opening caused by the Atl-vimentin interaction is found to be likely the key mechanism by which SS2 penetrates the tracheal epithelium. Author summary: Streptococcus suis serotype 2 (SS2), an emerging zoonotic agent, can breach the respiratory barrier and cause invasive disease in pigs. Here, we identified the novel role of autolysin Atl in penetration of the respiratory barrier by SS2 and its systemic dissemination and identified its binding partner, vimentin, a type III intermediate filament protein. Atl contributed to the MLCK-triggered redistribution of tight junctions to open the tracheal epithelial barrier. Knockout of vimentin abolished the ability of SS2 to penetrate the monolayer barrier and the activation of MLCK. Furthermore, vimentin null mice were protected from infection by intranasally administered SS2. This study is the first to demonstrate that the interaction between the GBS Bsp-like domain of Atl and vimentin promotes MLCK-mediated dysfunction of the epithelial barrier, which may provide theoretical information for prophylactic and/or therapeutic treatments against diseases caused by similar respiratory pathogens. [ABSTRACT FROM AUTHOR]

This study analyzed the difference between biofilm and planktonic Brucella abortus using metabolomics and proteomics. Brucella abortus was cultured in different media to induce Brucella abortus biofilm formation and planktonic cells, followed by metabolomics and proteomics analyses for these two samples. Significant differential metabolites were identified, followed by KEGG pathway analysis. Differentially expressed proteins were identified, followed by subcellular localization, GO annotation, and KEGG pathway enrichment. Additionally, a correlation analysis of metabolomics and proteomics was performed. Metabolomics analysis showed 7682 positive and 4433 negative metabolites, including 188 positive and 117 negative significant differential metabolites. These differential metabolites were enriched in fatty acid/unsaturated fatty acid biosynthesis and linoleic acid metabolism. Proteomics analysis revealed 1759 proteins, including 486 differentially expressed proteins, which were enriched in various metabolic and degradation-related pathways. Subcellular localization showed that 74.3% of the differential proteins were cytoplasmic proteins. Correlation analysis showed that 1-palmitoyl-2-oleoyl-phosphatidylglycerol had the most significant correlations with proteins, followed by cytosine. Both metabolites correlated with the protein Q57EI7 (RbsB-1, ribose ABC transporter). One common pathway, fatty acid biosynthesis, was identified by both proteomics and metabolomics analyses that involved the metabolites, oleic acid, and protein Q57DK3 (biotin carboxylase). There were metabolomic and proteomic differences between Brucella abortus biofilm and planktonic cells, and these results provide novel insights into the biofilm-forming process of Brucella abortus. [ABSTRACT FROM AUTHOR]

The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a significantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no effect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efflux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efflux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance afforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any influence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system significantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efflux. [ABSTRACT FROM AUTHOR]

Background: Aeromonas hydrophila is an important water-borne pathogen that leads to a great economic loss in aquaculture. Along with the abuse of antibiotics, drug-resistant strains rise rapidly. In addition, the biofilms formed by this bacterium limited the antibacterial effect of antibiotics. Bacteriophages have been attracting increasing attention as a potential alternative to antibiotics against bacterial infections. Results: Five phages against pathogenic A. hydrophila, named N21, W3, G65, Y71 and Y81, were isolated. Morphological analysis by transmission electron microscopy revealed that phages N21, W3 and G65 belong to the family Myoviridae, while Y71 and Y81 belong to the Podoviridae. These phages were found to have broad host spectra, short latent periods and normal burst sizes. They were sensitive to high temperature but had a wide adaptability to the pH. In addition, the phages G65 and Y81 showed considerable bacterial killing effect and potential in preventing formation of A. hydrophila biofilm; and the phages G65, W3 and N21 were able to scavenge mature biofilm effectively. Phage treatments applied to the pathogenic A. hydrophila in mice model resulted in a significantly decreased bacterial loads in tissues. Conclusions: Five A. hydrophila phages were isolated with broad host ranges, low latent periods, and wide pH and thermal tolerance. And the phages exhibited varying abilities in controlling A. hydrophila infection. This work presents promising data supporting the future use of phage therapy. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a multiprotein weapon that kills eukaryotic predators or prokaryotic competitors by delivering toxic effectors. Despite the importance of T6SS in bacterial environmental adaptation, it is still challenging to systematically identify T6SS effectors because of their high diversity and lack of conserved domains. In this report, we discovered a putative effector gene, U876-17730, in the whole genome of Aeromonas hydrophila NJ-35 based on the reported conservative domain DUF4123 (domain of unknown function), with two cognate immunity proteins encoded downstream. Phylogenetic tree analysis of amino acids indicates that AH17730 belongs to the Tle1 (type VI lipase effector) family, and therefore was named Tle1AH. The deletion of tle1AH resulted in significantly decreased biofilm formation, antibacterial competition ability and virulence in zebrafish (Danio rerio) when compared to the wild-type strain. Only when the two immunity proteins coexist can bacteria protect themselves from the toxicity of Tle1AH. Further study shows that Tle1AH is a kind of phospholipase that possesses a conserved lipase motif, Gly-X-Ser-X-Gly (X is for any amino acid). Tle1AH is secreted by T6SS, and this secretion requires its interaction with an associated VgrG (valine-glycine repeat protein G). In conclusion, we identified a T6SS effector-immunity pair and verified its function, which lays the foundation for future research on the role of T6SS in the pathogenic mechanism of A. hydrophila. [ABSTRACT FROM AUTHOR]

The present study aimed to explore the differences in protein and gene expression of Brucella abortus cultured under biofilm and planktonic conditions. The proteins unique to biofilms and planktonic B. abortus were separated by two-dimensional (2-D) electrophoresis and then identified by matrix-assisted laser desorption/ionization-tandem time of flight-mass spectrometry (MALDI-TOF/TOF-MS). High-throughput sequencing and bioinformatic analyses were performed to identify differentially expressed genes between B. abortus cultured under biofilm and planktonic conditions. The proteins and genes identified by proteomic and genomic analyses were further evaluated via western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. 2-D electrophoresis identified 20 differentially expressed protein spots between biofilms and planktonic cells, which corresponded to 18 individual proteins (12 downregulated and 6 upregulated) after MALDI-TOF/TOF-MS analysis, including elongation factor Tu and enolase. RT-qPCR analysis revealed that all of the 18 genes were downregulated in biofilms compared with planktonic cells. Western blot analysis identified 9 downregulated and 3 upregulated proteins. High-throughput sequencing and bioinformatic analyses identified 14 function and pathway-associated genes (e.g., BAbS19_I14970). RT-qPCR analysis of the 14 genes showed that they were upregulated in biofilm compared with in planktonic state. In conclusion, these differentially expressed genes may play important roles in bacterial defense, colonization, invasion, and virulence. [ABSTRACT FROM AUTHOR]

The TonB system functions in iron transport and has been identified in certain Gram-negative bacteria. Recently, we reported three TonB systems in the Aeromonas hydrophila Chinese epidemic strain NJ-35, but the functions of these systems have not been thoroughly elucidated to date. In this study, we investigated the role of these TonB systems in A. hydrophila iron utilization and virulence. We found that tonB1 and tonB2 were preferentially transcribed in iron-chelated conditions, where gene expression levels were approximately 8- and 68-fold higher compared with iron-rich conditions, respectively; tonB3 was consistently transcribed at a low level under iron-repleted and iron-depleted conditions. Only the TonB2 system was required to utilize iron-binding proteins. The tonB123 mutant showed increased susceptibility to erythromycin and roxithromycin. In addition, all three tonB genes were involved in A. hydrophila virulence in zebrafish, and various phenotypes associated with environmental survival were changed with varying degrees in each tonB mutant. TonB2 plays a relatively major role in adhesion, motility, and biofilm formation, while TonB3 is more involved in the anti-phagocytosis of A. hydrophila. In each observed phenotype, no significant difference was found between the single- and double-deletion mutants, whereas the triple-deletion mutant exhibited the most serious defects, indicating that all three TonB systems of A. hydrophila coordinately complement one another. In conclusion, this study elucidates the importance of TonB in iron acquisition and virulence of A. hydrophila, which lays the foundation for future studies regarding the survival mechanisms of this bacterium in iron-restricted environments. [ABSTRACT FROM AUTHOR]

Streptococcus equi subsp. zooepidemicus (SEZ) is a zoonotic pathogen capable of causing meningitis in humans. The mechanisms that enable pathogens to traverse the blood-brain barrier (BBB) are incompletely understood. Here, we investigated the role of a newly identified Fic domain-containing protein, BifA, in SEZ virulence. BifA was required for SEZ to cross the BBB and to cause meningitis in mice. BifA also enhanced SEZ translocation across human Brain Microvascular Endothelial Cell (hBMEC) monolayers. Purified BifA or its Fic domain-containing C-terminus alone were able to enter into hBMECs, leading to disruption of monolayer barrier integrity. A SILAC-based proteomic screen revealed that BifA binds moesin. BifA’s Fic domain was required for its binding to this regulator of host cell cytoskeletal processes. BifA treatment of hBMECs led to moesin phosphorylation and downstream RhoA activation. Inhibition of moesin activation or moesin depletion in hBMEC monolayers abrogated BifA-mediated increases in barrier permeability and SEZ’s capacity to translocate across monolayers. Thus, BifA activation of moesin appears to constitute a key mechanism by which SEZ disrupts endothelial monolayer integrity to penetrate the BBB. [ABSTRACT FROM AUTHOR]

Streptococcus suis is a major swine pathogen and an important zoonotic agent worldwide. At least nine serotypes can infect human so far. Although 29 serotypes (1–19, 21, 23–25, 27–31 and 1/2) strains are considered as authentic S. suis, a novel variant serotype Chz and strains carrying 20 novel capsular polysaccharide loci (NCL) have been identified recently. However, information about pathogenic and antimicrobial resistance characteristics of strains carrying NCLs is still unavailable. In this study, we identified six new NCLs (designated as NCL21‐26) from 35 non‐typeable S. suis strains by agglutination tests and whole genome sequencing analysis. Further analysis of the genetic context of NCL25 and NCL26 showed a mosaic structure of the capsular polysaccharide loci. NCL25 exhibited considerable similarity to that of serotypes 10 and 11, and NCL26 shared similarity to that of serotype 9 and NCL4. Antimicrobial susceptibility testing demonstrated that strains carrying NCL21‐26 were all resistant to clindamycin, lincomycin, erythromycin, tilmicosin and tetracycline. Animal infection experiments showed that the virulence of NCL26 strain NJ1112 isolated from a disease pig was similar to that of S. suis serotype 2 virulent strain SC070731 in both zebrafish and mouse infection models, highlighting the necessity for surveillance of strains belonging to NCL26. We also developed a multiplex PCR assay to detect NCL21‐26 strains. Our findings expand the views of genetic diversity of S. suis capsular polysaccharide loci and S. suis pathogenic characteristic. [ABSTRACT FROM AUTHOR]

Background: Aeromonas hydrophila is a potential zoonotic pathogen and primary fish pathogen. With overlapping characteristics, multiple isolates are often mislabelled and misclassified. Moreover, the potential pathogenic factors among the publicly available genomes in A. hydrophila strains of different origins have not yet been investigated. Results: To identify the valid strains of A. hydrophila and their pathogenic factors, we performed a pan-genomic study. It revealed that there were 13 mislabelled strains and 49 valid strains that were further verified by Average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and in silico multiple locus strain typing (MLST). Multiple numbers of phages were detected among the strains and among them Aeromonas phi 018 was frequently present. The diversity in type III secretion system (T3SS) and conservation of type II and type VI secretion systems (T2SS and T6SS, respectively) among all the strains are important to study for designing future strategies. The most prevalent antibiotic resistances were found to be beta-lactamase, polymyxin and colistin resistances. The comparative analyses of sequence type (ST) 251 and other ST groups revealed that there were higher numbers of virulence factors in ST-251 than in other STs group. Conclusion: Publicly available genomes have 13 mislabelled organisms, and there are only 49 valid A. hydrophila strains. This valid pan-genome identifies multiple prophages that can be further utilized. Different A. hydrophila strains harbour multiple virulence factors and antibiotic resistance genes. Identification of such factors is important for designing future treatment regimes. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) has been considered as a crucial factor in bacterial competition and virulence. The hemolysin co-regulated protein (Hcp) is the hallmark of T6SS. The secretion of Hcp in Aeromonas hydrophila Chinese epidemic strain NJ-35 indicated a functional T6SS. In this study, three copies of the hcp gene were identified in the genome of strain NJ-35. We targeted these Hcp family proteins for generating deletion mutants. These mutants showed varying levels in Hcp production, the interaction with other bacteria or eukaryotic cells, and bacterial virulence. Hcp1 was necessary for T6SS assembly and played a predominant role in the bacterial competition; Hcp2 negatively functioned in the biofilm formation and bacterial adhesion and was more involved in the A. hydrophila virulence in zebrafish and survival against the predation of Tetrahymena, and Hcp3 positively influenced the biofilm formation and bacterial adhesion. These findings illustrate that the T6SS of A. hydrophila NJ-35 is active, and the three Hcp family proteins take part in different processes in environmental adaptation and virulence of this bacterium. This study will provide valuable insights into our understanding of microbial interactions and thus contribute to a broader effort to manipulate these interactions for therapeutic or environmental benefit. [ABSTRACT FROM AUTHOR]

Persistence of Aeromonas hydrophila in aquatic environments is the principle cause of fish hemorrhagic septicemia. Protistan predation has been considered to be a strong driving force for the evolution of bacterial defense strategies. In this study, we investigated the adaptive traits of A. hydrophila NJ-35, a carp pathogenic strain, in response to Tetrahymena thermophila predation. After subculturing with Tetrahymena, over 70% of A. hydrophila colonies were small colony variants (SCVs). The SCVs displayed enhanced biofilm formation, adhesion, fitness, and resistance to bacteriophage infection and oxidative stress as compared to the non-Tetrahymena-exposed strains. In contrast, the SCVs exhibited decreased intracellular bacterial number in RAW264.7 macrophages and were highly attenuated for virulence in zebrafish. Considering the outer membrane proteins (OMPs) are directly involved in bacterial interaction with the external surroundings, we investigated the roles of OMPs in the antipredator fitness behaviors of A. hydrophila. A total of 38 differentially expressed proteins were identified in the SCVs by quantitative proteomics. Among them, three lipoproteins including SurA, Slp, and LpoB, and a serine/threonine protein kinase (Stpk) were evidenced to be associated with environmental adaptation of the SCVs. Also, the three lipoproteins were involved in attenuated virulence of SCVs through the proinflammatory immune response mediated by TLR2. This study provides an important contribution to the understanding of the defensive traits of A. hydrophila against protistan predators. [ABSTRACT FROM AUTHOR]

Streptococcus suis is a major porcine bacterial pathogen and emerging zoonotic agent. S. suis 5ʹ-nucleotidase is able to convert adenosine monophosphate to adenosine, resulting in inhibiting neutrophil functions in vitro and it is an important virulence factor. Here, we show that S. suis 5ʹ-nucleotidase not only enables producing 2ʹ-deoxyadenosine from 2ʹ-deoxyadenosine monophosphate by the enzymatic assay and reversed-phase high performance liquid chromatography (RP-HPLC) analysis in vitro, but also synthesizes both 2ʹ-deoxyadenosine and adenosine in mouse blood in vivo by RP-HPLC and liquid chromatography with tandem mass spectrometry analyses. Cellular cytotoxicity assay and Western blot analysis indicated that the production of 2ʹ-deoxyadenosine by 5ʹ-nucleotidase triggered the death of mouse macrophages RAW 264.7 in a caspase-3-dependent way. The in vivo infection experiment showed that 2ʹ-deoxyadenosine synthesized by 5ʹ-nucleotidase caused monocytopenia in mouse blood. The in vivo transcriptome analysis in mouse blood showed the inhibitory effect of 5ʹ-nucleotidase on neutrophil functions and immune responses probably mediated through the generation of adenosine. Taken together, these findings indicate that S. suis synthesizes 2ʹ-deoxyadenosine and adenosine by 5ʹ-nucleotidase to dampen host immune responses, which represents a new mechanism of S. suis pathogenesis. [ABSTRACT FROM AUTHOR]

Muramidase-released protein (MRP) of Streptococcus suis serotype 2 (SS2) is an important epidemic virulence marker with an unclear role in bacterial infection. To investigate the biologic functions of MRP, 3 mutants named Dmrp, Dmrp domain 1 (Δmrp-d1), and Δmrp domain 2 (Δmrp-d2) were constructed to assess the phenotypic changes between the parental strain and the mutant strains. The results indicated that MRP domain 1 (MRP-D1, the non-conserved region of MRP from a virulent strain, a.a. 242-596) played a critical role in adherence of SS2 to host cells, compared with MRP domain 1 (MRP-D1, the non-conserved region of MRP from a low virulent strain, a.a. 239-598) or MRP domain 2 (MRP-D2, the conserved region of MRP, a.a. 848-1222). We found that MRP-D1 but not MRP-D2, could bind specifically to fibronectin (FN), factor H (FH), fibrinogen (FG), and immunoglobulin G (IgG). Additionally, we confirmed that mrp-d1 mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1 mutation also attenuated the intracellular survival of SS2 in RAW246.7 macrophages, shortened the growth ability in pig blood and decreased the virulence of SS2 in BALB/c mice. Furthermore, antiserum against MRP-D1 was found to dramatically impede SS2 survival in pig blood. Finally, immunization with recombinant MRP-D1 efficiently enhanced murine viability after SS2 challenge, indicating its potential use in vaccination strategies. Collectively, these results indicated that MRP-D1 is involved in SS2 virulence and eloquently demonstrate the function of MRP in pathogenesis of infection. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3-4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4O1) genes caused by a terminationcodon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors. [ABSTRACT FROM AUTHOR]

Streptococcus suis is a major pathogen of pigs and also an important zoonotic agent for humans. A S. suis protein containing Mac-1 domain (designated Mac) is a protective antigen, exclusively cleaves porcine IgM, and contributes to complement evasion with the presence of high titers of specific porcine anti- S. suis IgM, but its role in S. suis virulence has not been investigated in natural healthy host without specific IgM. In this study, a mac deletion mutant was constructed by homologous recombination in S. suis serotype 2 virulent reference strain P1/7. Deletion of mac did not significantly influence phagocytosis or intracellular survival within murine macrophages RAW264.7, or the oxidative-burst induction of RAW264.7 and murine neutrophils. Furthermore, the mutant is as virulent as the wild-type strain in pig, mouse, and zebrafish infection models. Our data suggest that Mac is not essential for S. suis virulence in strain P1/7 in natural healthy host without specific IgM, and the immunogenicity of Mac does not appear to correlate with its significance for virulence. [ABSTRACT FROM AUTHOR]

The type VI secretion system (T6SS) of bacteria plays a key role in competing for specific niches by the contact-dependent killing of competitors. Recently, Rhs proteins with polymorphic C-terminal toxin-domains that inhibit or kill neighboring cells were identified. In this report, we identified a novel Rhs with an MPTase4 (Metallopeptidase-4) domain (designated as Rhs-CT1) that showed an antibacterial effect via T6SS in Escherichia coli. We managed to develop a specific strategy by matching the diagnostic domain-architecture of Rhs-CT1 (Rhs with an N-terminal PAAR-motif and a C-terminal toxin domain) for effector retrieval and discovered a series of Rhs-CTs in E. coli. Indeed, the screened Rhs-CT3 with a REase-3 (Restriction endonuclease-3) domain also mediated interbacterial antagonism. Further analysis revealed that vgrGO1 and eagR/DUF1795 (upstream of rhs-ct) were required for the delivery of Rhs-CTs, suggesting eagR as a potential T6SS chaperone. In addition to chaperoned Rhs-CTs, neighborless Rhs-CTs could be classified into a distinct family (Rhs-Nb) sharing close evolutionary relationship with T6SS2-Rhs (encoded in the T6SS2 cluster of E. coli). Notably, the Rhs-Nb-CT5 was confirmed bioinformatically and experimentally to mediate interbacterial antagonism via Hcp2B-VgrG2 module. In a further retrieval analysis, we discovered various toxin/immunity pairs in extensive bacterial species that could be systematically classified into eight referential clans, suggesting that Rhs-CTs greatly diversify the antibacterial pathways of T6SS. [ABSTRACT FROM AUTHOR]

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, resulting in severe economic losses worldwide. Coliphages represent alternative antibacterial substitutes based on high lytic efficiency and few side-effects. However, the complete genome sequences information of APEC phages are limited, and knowledge of undesired genes and the narrow host range restrict their applications. In this study, we isolated a virulent phage QL01, with a relatively broad lytic spectrum (41 of 78 APEC strains). Transmission electron micrography showed it belonged to the family Myoviridae with an elongated head and a contractile tail. Whole genome sequencing revealed a linear double-stranded DNA (170,527 kb; GC content, 39.6%) with 275 possible ORFs. Comparative genome analysis revealed high homology between QL01 and other T4-like phages. However, it also showed some unique features, for example, ORF142 and ORF143, which encode IP9 and IP8, respectively, and may counteract host resistance only exist in a few T4-like phages such as IME08 and vB_EcoM_VR5. Furthermore, phage therapy in artificially infected ducks showed a 26.67% decrease in mortality compared with the untreated group. Our study indicates the potential antibacterial function of phage QL01 against APEC infections and highlights unique molecular features underlying the relatively broad host range. [ABSTRACT FROM AUTHOR]

Dogs are susceptible to infectious diseases that occur primarily in the respiratory tract. The airway epithelium acts as a first line of defense and is constantly exposed to microorganisms present in the environment. Respiratory epithelial cells have recently gained wide use as a cell model for studying the pathogenesis of human, murine or swine respiratory pathogen infections. However, studies of the pathogenic mechanisms of canine pathogens have been hindered by the lack of reliable respiratory cell lines. Here, we cultured primary canine bronchiolar epithelial cells (CBECs), whose characteristics were confirmed by their expression of the epithelial cell-specific marker cytokeratin 18, and have provided protocols for their isolation and ex vivo expansion. Further, we established immortalized CBECs containing the human telomerase reverse transcriptase (hTERT) gene via transfection of primary CBECs with the recombinant plasmid pEGFP-hTERT. Immortalized bronchiolar epithelial cells (hTERT-CBECs) retain the morphological and functional features of primary CBECs, as indicated by reverse transcriptase polymerase chain reaction, proliferation assays, karyotype analysis, telomerase activity assay, and Western blotting, which demonstrate that hTERT-CBECs have higher telomerase activity, an extended proliferative lifespan, and a diploid complement of chromosomes, even after Passage 50. Moreover, this cell line is not transformed, as evaluated using soft agar assays and tumorigenicity analysis in nude mice, and can therefore be safely used in future studies. The isolation and establishment of stable hTERT-CBECs is of great importance for use as an in vitro model for mechanistic studies of canine pathogenic infections. [ABSTRACT FROM AUTHOR]

Streptococcus suis has emerged as an important zoonotic pathogen that causes meningitis, arthritis, septicemia and even sudden death in pigs and humans. Quorum sensing is the signaling network for cell-to-cell communication that bacterial cells can use to monitor their own population density through production and exchange of signal molecules. S-Ribosylhomocysteinase (LuxS) is the key enzyme involved in the activated methyl cycle. Autoinducer 2 (AI-2) is the adduct of borate and a ribose derivative and is produced from S-adenosylhomocysteine (SAH). AI-2 can mediate interspecies communication and in some species facilitate the bacterial behavior regulation such as biofilm formation and virulence in both Gram-positive and Gram-negative bacteria. Here, we reported the overexpression, purification and crystallographic structure of LuxS from S. suis. Our results showed the catalytically active LuxS exists as a homodimer in solution. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed the presence of Zn2+ in LuxS. Although the core structure shares the similar topology with LuxS proteins from other bacterial species, structural analyses and comparative amino acid sequence alignments identified two key amino acid differences in S. suis LuxS, Phe80 and His87, which are located near the substrate binding site. The results of site-directed mutagenesis and enzymology studies confirmed that these two residues affect the catalytic activity of the enzyme. These in vitro results were corroborated in vivo by expression of the LuxS variants in a S. suis ΔluxS strain. The single and two amino acid of LuxS variant decreased AI-2 production and biofilm formation significantly compared to that of the parent strain. Our findings highlight the importance of key LuxS residues that influence the AI-2 production and biofilm formation in S.suis. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a primary pathogen that causes bovine mastitis resulting in serious economic losses and herd management problems in dairy cows. A novel bacteriophage, JS01, specifically infecting bovine S. aureus, was isolated from milk of mastitis-affected cattle. TEM observation showed that it belonged to the family Siphovirus. The JS01 strain demonstrated a broad host range. The prediction result of PHACTS suggested that the JS01 strain was temperate phage. The JS01 genome is 43,458 bp long, with a GC content of 33.32 % and no tRNAs. Annotation and functional analysis of the predicted ORFs revealed six functional groups: structure and morphology, DNA replication and regulation, packaging, lysogeny, lysis, and pathogenicity. Comparative analysis between JS01, S. aureus MSSA476, and S. aureus prophage PVL was also performed. The characterization and genomic analysis of JS01 provide a better understanding of S. aureus-targeting bacteriophages and useful information for the development of phage-based biocontrol agents against S. aureus. [ABSTRACT FROM AUTHOR]

Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. IbeR, located on genomic island GimA, was shown to serve as an RpoS-like regulator in rpoS gene mutation neonatal meningitis E. coli (NMEC) RS218. However, the role of IbeR in pathogenicity of APEC carrying active RpoS has not yet been investigated. We showed that the APEC IbeR could elicit antibodies in infected ducks, suggesting that IbeR might be involved in APEC pathogenicity. To investigate the function of IbeR in APEC pathogenesis, mutant and complementation strains were constructed and characterized. Inactivation of ibeR led to attenuated virulence and reduced invasion capacity towards DF-1 cells, brains and cerebrospinal fluid (CSF) in vitro and in vivo. Bactericidal assays demonstrated that the mutant strain had impaired resistance to environmental stress and specific pathogen-free (SPF) chicken serum. These virulence-related phenotypes were restored by genetic complementation. Quantitative real-time reverse transcription PCR revealed that IbeR controlled expression of stress-resistance genes and virulence genes, which might led to the associated virulence phenotype. [ABSTRACT FROM AUTHOR]

Haemophilus parasuis ( H. parasuis) is associated with meningitis, polyserositis, polyarthritis and bacterial pneumonia. At present, its prevention and control is difficult because of the lack of suitable subunit vaccines. Nowadays, high-throughput methods, immunoproteomics, are available to screen for more vaccine candidates. A protein extraction method for H. parasuis and two-dimensional electrophoresis (2-DE) were optimized to provide high-resolution profiles covering pH 3 to 10. Twenty immunoreactive spots were excised from gels after strict comparison between 2-DE Western blot membranes and the relevant gels. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-TOF-MS successfully identified 16 different proteins. Fifteen of them were reported as immunoreactive proteins in H. parasuis for the first time. In addition, recombinant HP5-7 (ABC transporter, periplasmic-binding protein) showed immunoreactivity both with hyperimmune rabbit serum and convalescent swine serum. Four recombinants of the 14 successfully expressed genes showed immunoreactivity with hyperimmune rabbit serum. [ABSTRACT FROM AUTHOR]

Selective capture of transcribed sequences (SCOTS) is an effective method to identify bacterial genes differentially expressed during different biological processes, including pathogenic interactions with a host species. The method can be used to elucidate molecular mechanisms driving and maintaining such interactions. The method is a powerful genetic tool that overcomes limitations found in other methods, by working with small amounts of mRNA and allowing for the separation of bacterial cDNA from host cDNA. It has been increasingly used in the discovery of genes involved in the bacterium-host interaction. In this review, we briefly introduce the SCOTS method, outline the technical advances offered in the method, and focus on the method's applications in several microbial pathogens. [ABSTRACT FROM AUTHOR]

Streptococcus suis, a major swine pathogen, is an emerging zoonotic agent that causes meningitis and septic shock. Bacterial cell wall and secreted proteins are often involved in interactions with extracellular matrix proteins (ECMs), which play important roles in the initial steps of pathogenesis. In this study, 2D SDS-PAGE, western blotting-based binding affinity measurements, and microtiter plate binding assays were used to identify cell wall and secreted proteins from S. suis that interact with fibronectin and collagen type I. We identified six proteins from S. suis, including three proteins (translation elongation factor G, oligopeptide-binding protein OppA precursor, and phosphoglycerate mutase) that show both fibronectin and collagen type I binding activity. To the best of our knowledge, these three newly identified proteins had no previously reported fibronectin or collagen type I binding activity. Overall, the aim in this study was to identify proteins with ECM binding activity from S. suis and it represents the first report of six new proteins from S. suis that interact with fibronectin or collagen type I. [ABSTRACT FROM AUTHOR]

Streptococcus suis serotype 2 (SS2) is an emerging zoonotic agent responsible for a number of infections in pigs and humans. Pili have been proposed as virulence factors in Gram-positive bacteria. However, due to the abolition of pili production, the function of the srtBCD pilus cluster, especially the truncated major pilin subunit Sbp2 (Sbp2′, Sbp2″), has not been explored. In this study, isogenic mutants (Δsbp2′, Δsbp2″) were constructed by homologous replacement in SS2 strain P1/7. Deletion of sbp2′ attenuated the virulence in a zebrafish model as shown by more than an eightfold increase in the LD of Δsbp2′, compared with that of the parent strain. In addition, the adhesion of Δsbp2′ to HEp-2 cell monolayers decreased significantly. Compared with the parent strain, no obvious differences in virulence and adherence efficiency were observed for Δsbp2″. Our data suggest that Sbp2′ could be involved in SS2 pathogenesis despite absence of its pilus shaft. [ABSTRACT FROM AUTHOR]

Autoinducer-2 (AI-2) is a universal signal molecule mediating intra- and interspecies communication among bacteria. AI-2 is a byproduct of the LuxS enzyme during the catabolism of S-adenosylhomocysteine and plays critical roles in regulating various behaviors of bacteria. In our previous study, the function of LuxS in AI-2 production was verified in Streptococcus suis (SS). Decreased levels of SS biofilm formation and host-cell adherence as well as the inability to produce AI-2 were observed in SS having a luxS mutant gene. In this study, exogenous addition of a low concentration of AI-2 synthesized in vitro was found to promote biofilm formation and host-cell adherence. However, higher concentrations of AI-2 inhibited SS biofilm formation and host-cell adherence. Real-time PCR results showed that the mRNA level of virulence factors of SS biofilm, gdh, cps2, sly, and mrp increased and ef, fbps, and gapdh decreased with increasing AI-2 concentrations. These findings demonstrated that AI-2 supplemented exogenously acted as a concentration-dependent signaling molecule to regulate SS biofilm formation, host-cell adherence, and transcription levels of many virulence genes. [ABSTRACT FROM AUTHOR]

Hemolysis causes major symptoms such as the reddening skin and systemic hemorrhagic septicemia of diseased fish infected by Edwardsiella tarda. Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene in Escherichia coli K-12. In this study, we observed that the heterologous expression of the eha gene from E. tarda could confer hemolytic activity upon a hemolytic-silent E. coli strain. The transcription of clyA is positively controlled by the eha gene in E. tarda by RT-PCR. We cloned and purified Eha protein which had shown preferential binding ability to the clyA sequences in its promoter region, as evidenced by gel shift assay. The eha controls the transcriptional start predominantly at 72 bp upstream in the clyA promoter region, as determined by primer extension assays. We suggest that Eha protein is a new positive regulator found in E. tarda. In addition, we constructed the eha mutant and complementary strains of E. tarda. The hemolytic activity of the eha mutant was found to be attenuated compared with the wild-type strain. The complementary strains restored the hemolytic activity to levels between those of the wild type and the eha mutation. Our results indicate that the Eha protein is an important positive regulator in the hemolytic properties of E. tarda. [ABSTRACT FROM AUTHOR]

Streptococcus suis serotype 2 (SS2) is an important swine and human pathogen responsible for septicemia and meningitis. The bacterial homologues of eukaryotic-type serine/threonine kinases (ESTKs) have been reported to play critical roles in various cellular processes. To investigate the role of STK in SS2, an isogenic stk mutant strain (Δstk) and a complemented strain (CΔstk) were constructed. The Δstk showed a significant decrease in adherence to HEp-2 cells, compared with the wild-type strain, and a reduced survival ratio in whole blood. In addition, the Δstk exhibited a notable reduced tolerance of environmental stresses including high temperature, acidic pH, oxidative stress, and high osmolarity. More importantly, the Δstk was attenuated in both the CD1 mouse and piglet models of infection. The results of quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that the expressions of a few genes involving in adherence, stress response and virulence were clearly decreased in the Δstk mutant strain. Our data suggest that SsSTK is required for virulence and stress response in SS2. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae ( Group B streptococcus, GBS) is an important zoonotic pathogen that can cause lethal infections in humans and animals, including aquatic species. Immunoreactive proteins of the S. agalactiae strain, GD201008-001, isolated from cultured tilapia in China, were screened by immunoproteomics using hyperimmune sera, convalescent guinea pig sera and GD201008-001-infected tilapia antisera as primary detection antibodies. A total of 16 different proteins were identified including 13 novel immunoreactive proteins of S. agalactiae. Four proteins, serine-rich repeat glycoprotein 1, branched-chain alpha-keto acid dehydrogenase ( BKD) subunit E2, 5′-nucleotidase family protein and ornithine carbamoyltransferase, were shown to react with the three types of sera and thus were considered to represent novel S. agalactiae vaccine candidate antigens. Our findings represent the basis for vaccine development for piscine S. agalactiae and are necessary for understanding virulence factors and immunogenicity of S. agalactiae with different hosts. [ABSTRACT FROM AUTHOR]

A novel bacteriophage, JX01, specifically infecting bovine Streptococcus agalactiae was isolated from milk of mastitis-affected cattle. The phage morphology showed that JX01 belongs to the family Siphoviridae, and this phage demonstrated a broad host range. Microbiological characterization demonstrated that nearly 90 % of JX01 phage particles were adsorbed after 2.5 min of incubation, that the burst size was 20 virions released per infected host cell, and that there was a latent period of 30 min. JX01 was thermal sensitive and showed acid and alkaline resistance (pH 3-11). The genome of JX01 was found to consist of a linear, double-stranded 43,028-bp DNA molecule with a GC content of 36.81 % and 70 putative open reading frames (ORFs) plus one tRNA. Comparative genome analysis revealed high similarity between JX01 and the prophage 315.2 of Streptococcus pyogenes. [ABSTRACT FROM AUTHOR]

Bacterial ghost is a novel vaccine platform, and its safe and efficient production depends largely upon a suitable and functional vector. In this study, a series of temperature-inducible plasmids, carrying Phix174 lysis gene E and/or staphylococcal nuclease A (SNA) gene, were constructed and evaluated in Escherichia coli. The results showed that the direct product of SNA (pBV220-SNA) could degrade the plasmid and genomic DNA of E. coli while the fusion product of gene E and partial Cro gene (pKF396M-2) lost the ability to lyse the host strain. The insertion of enhancer T7g10 elements and Shine-Dalgarno box (ESD) between them (pKF396M-3) could resume the function of gene E. Using plasmid pKF396M-4 with gene E and SNA, respectively, under the immediate control of promoter pR and pL, the remnant plasmids and genomic DNA of E. coli were eliminated, and the rates of inactivation increased by two orders of magnitude over that obtained with the exclusive use of E-mediated lysis plasmid. By substituting these two genes with customized multiple cloning sites sequences, the plasmid could be modified to a dual expression vector (pKF396M-5). [ABSTRACT FROM AUTHOR]

The characterization of 61 Streptococcus suis strains isolated from Chinese slaughter pigs was investigated. S. suis serotypes 1, 2, 3, 4, 7, 9, 10, 11, 12, 13, 14, 15, 16, 22, 23, 25, 26, 28, 29, and 1/2 were found in the isolates by serum agglutination. Of all the prevalent serotypes, S. suis serotype 7 is the most predominant circulating in Chinese slaughter pigs. The virulence-associated genes profile and multilocus sequence typing scheme of the isolates were analyzed. The mrp-/ epf-/ sly- virulence-associated genes type is the most prevalent in the isolates from slaughter pigs. It is the first time to find S. suis serotypes 7 and 9 isolates with epf. The serotypes 7 and 9 isolates with mrp and/or epf genes did not express MRP and/or EF in the present research. Thirteen new ST types were identified for the first time. ST1 complex and ST27 complex of S. suis are prevalent in China. This paper supplied information to understand the characteristics, such as capsular serotypes, virulence factors, and gene backgrounds of S. suis carried by slaughter pigs. [ABSTRACT FROM AUTHOR]

Swine influenza (SI) is an acute respiratory infectious disease of swine caused by swine influenza virus (SIV). SIV is not only an important respiratory pathogen in pigs but also a potent threat to human health. Here, we report the construction of a recombinant swinepox virus (rSPV/H3-2A-H1) co-expressing hemagglutinin (HA1) of SIV subtypes H1N1 and H3N2. Immune responses and protection efficacy of the rSPV/H3-2A-H1 were evaluated in guinea pigs. Inoculation of rSPV/H3-2A-H1 yielded neutralizing antibodies against SIV H1N1 and H3N2. The IFN-γ and IL-4 concentrations in the supernatant of lymphocytes stimulated with purified SIV HA1 antigen were significantly higher ( P < 0.01) than those of the control groups. Complete protection of guinea pigs against SIV H1N1 or H3N2 challenge was observed. No SIV shedding was detected from guinea pigs vaccinated with rSPV/H3-2A-H1 after challenge. Most importantly, the guinea pigs immunized with rSPV/H3-2A-H1 did not show gross and micrographic lung lesions. However, the control guinea pigs experienced distinct gross and micrographic lung lesions at 7 days post-challenge. Our data suggest that the recombinant swinepox virus encoding HA1 of SIV H1N1 and H3N2 might serve as a promising candidate vaccine for protection against SIV H1N1 and H3N2 infections. [ABSTRACT FROM AUTHOR]

Torque teno sus virus 1 (TTSuV1) is a novel virus that has been found widely distributed in the swine population in recent years. Analysis of codon usage can reveal much about the molecular evolution of TTSuV1. In this study, synonymous codon usage patterns and the key determinants in the coding region of 29 available complete TTSuV1 genome sequences were examined. By calculating the nucleotide content and relative synonymous codon usage (RSCU) of TTSuV1 coding sequences, we found that the preferentially used codons were mostly those ending with A or C nucleotides; less-used codons were mostly codons ending with U or G nucleotides, and these were mainly affected by composition constraints. Although there was a variation in codon usage bias among different TTSuV1 genomes, the codon usage bias and GC content in the TTSuV1 coding region was lower, which was mainly determined by the base composition in the third codon position and the effective number of codons (ENC) value. Moreover, the results of correspondence analysis (COA) indicated that the codon usage patterns of TTSuV1 isolated from different countries varied greatly and had significant differences. In addition, Spearman's rank correlation analysis and an ENC plot revealed that apart from mutation pressure, which was critical in determining the codon usage pattern, other factors were involved in shaping the evolution of codon usage bias in TTSuV1, such as natural selection. Those results suggested that synonymous codon usage patterns of TTSuV1 genomes were the result of interaction between mutation pressure and natural selection. The information from this study not only provides important insights into the synonymous codon usage pattern of TTSuV1, but also helps to identify the main factors affecting codon usage by this virus. [ABSTRACT FROM AUTHOR]

The ibeA gene, one of the important invasion-associated genes in neonatal meningitis Escherichia coli (NMEC), has been recently detected in avian pathogenic E. coli (APEC). Thus, it is necessary to close monitor the possible contamination of the poultry farms and its products to people. Here, a dot blot method for detecting the ibeA gene in E. coli was developed and validated. For the present study, probe sequence was designed and optimized for the specificity of dot blot. A 342-bp conserved fragment of ibeA gene was selected and labeled with digoxigenin (DIG)-dUTP according to the manufacturer's guidelines, which indicated that this probe hybridizes with ibeA. In our established method, the bacteria culture samples were directly spotted on the membrane, following simple lyses on the membrane. Hence, the extraction of genomic DNA is not required, which reduces the workload and shortens the time. Furthermore, this assay was very sensitive, which could detect as few as 2.5 × 10 CFU bacteria. The diagnostic reliability of this dot blot was evaluated on 467 APEC bacteria samples by using PCR analysis. Both methods showed that the result was in complete concordance. The dot blot assay was proved to be a simple, rapid, highly accurate, and cost-effective method to identify invasion-associated genes ibeA, which could be applied for initial screening of a large number of clinical samples or direct detection of bacteria culture. [ABSTRACT FROM AUTHOR]

Development of novel antibacterial agents is required to control infection with multidrug-resistant Streptococcus suis. HolSMP and LySMP, the holin and lysin of S. suis serotype 2 bacteriophage, named SMP, are responsible for lysis of host cells and release of progeny phage. HolSMP and LySMP expressed in Escherichia coli BL21(DE3) exerted efficient activity at 37 °C, pH 5.2, with addition of 0.8 % β-mercaptoethanol. Lytic spectra of purified HolSMP, LySMP or HolSMP + LySMP mixture were investigated. HolSMP, exhibiting a narrow lytic spectrum, was effective against Staphylococcus aureus and Bacillus subtilis, which were insensitive to LySMP. Moreover, HolSMP was identified as a promising antibacterial agent which was able to extend the spectrum of LySMP. The data suggest that combined use of holin and lysin could be a candidate strategy for resolution of drug resistance. [ABSTRACT FROM AUTHOR]

Riemerella anatipestifer ( RA) is one of the most important bacterial pathogens of ducks and other avian species worldwide. Twenty-one serotypes of RA have been identified, with RA serotype two ( RA2) being reported as one of the most predominant serotypes underlying infections in China. Current approaches to the control of RA are hindered by the absence of effective vaccines, particularly those that exhibit cross-protection between different serotypes. In this study, a combination of two-dimensional electrophoresis, Western blot analysis and mass spectrometry were used to identify the antigenic proteins of RA2. A total of 16 immunoreactive proteins, representing 12 distinct proteins, were identified. These included OmpA, a known immunogenic protein of RA, as well as novel immunogens. PCR analysis also indicated that genes corresponding to each of the 12 distinct proteins were conserved among different RA serotypes. Eleven genes encoding these proteins were cloned and expressed in Escherichia coli BL21 ( DE3). Eight of the 11 expressed proteins were able to react with hyperimmune rabbit serum against RAf153. One of these, recombinant elongation factor G, responded to RA2 sera but not RA1, whereas recombinant OmpA responded to both RA1 and RA2 sera. These data form a basis for the development of vaccine for both homologous and heterogeneous RA serotypes in addition to the production of target antigens for the development of diagnostic antibodies with the potential to distinguish between RA serotypes. [ABSTRACT FROM AUTHOR]

The NS1 protein of classical swine H1N1 influenza A virus evolved dynamically during the past 80 years, most notable changes happened in the four C-terminal sequences and the C-terminal truncation of 11 amino acids. However, the role of these changes on the virulence of classical swine H1N1 influenza A virus remains unknown. Using reverse genetics, three NS1 mutant viruses (RSEV, GSEI, and EPEV) and a wild-type virus (PEQK) were generated from A/Swine/Shanghai/1/2005 virus and the pathogenicity of the viruses was determined in mice. The results showed that RSEV and PEQK viruses could not infect the mice. By contrast, GSEI and EPEV viruses could replicate in the lungs of mice without prior adaptation. The viral titers in lungs from GSEI and EPEV virus-infected mice were 2,300 and 7 pfu/g at fourth-day post-infection, respectively. Mild-to-moderate alveolitis was observed in the histopathological test of lungs from GSEI and EPEV virus-infected mice. The results indicated that C-terminal GSEI and EPEV motifs of NS1 protein involved in viral virulence and facilitated the A/Swine/Shanghai/1/2005 virus crossing the species barrier from swine to mice. [ABSTRACT FROM AUTHOR]

To evaluate the effect of natural infection with TTSuV1 on the antibody response to vaccination with PRRS vaccine and clinical signs when co-infected with virulent PRRSV, 15 4-week-old TTSuV1-positive piglets and 20 TTSuV1-negative piglets were selected by PCR from two pig farms in Jiangsu province. TTSuV1-negative pigs were divided into four groups, and TTSuV1-positive pigs were divided into three groups. Experimental pigs were vaccinated with a PRRSV modified live virus (MLV) at 6 weeks of age and subsequently challenged with a virulent strain of PRRSV at 10 weeks of age. A TTSuV1-negative control group and an unvaccinated PRRS MLV control group were tested at the same time. The levels of antibody/cytokine and protective efficiency against PRRS MLV vaccine were evaluated. TTSuV1-infected/PRRSV-vaccinated pigs had lower levels of PRRSV antibody, as well as IFN-γ, IL-10 and T lymphocyte proliferation, than the TTSuV1-uninfected/PRRSV-vaccinated group ( P < 0.05, except IL-10) after vaccination at only one time point. TTSuV1-infected/PRRS MLV-vaccinated/PRRSV-challenged pigs had more severe clinical signs ( P > 0.05), more macroscopic lung lesions ( P < 0.05 ) and lower levels of PRRSV antibody ( P < 0.05 at 7 to 14 days post-PRRSV-challenge) than TTSuV1-uninfected/PRRSV-vaccinated/PRRSV-challenged pigs. These data indicate that TTSuV1 natural infection has an adverse effect on the development of host immune responses, suppresses immunization by the PRRS MLV vaccine, and exacerbates PRRS to a certain extent in pigs. [ABSTRACT FROM AUTHOR]

Systemic infections in avian species caused by avian pathogenic E scherichia coli ( APEC) are economically devastating to poultry industries worldwide. To unravel factors possibly involved in APEC pathogenicity, suppression subtractive hybridization was applied, leading to the identification of a putative APEC autotransporter adhesin gene aatA in our previous study. In this study, pathogenic mechanism of AatA was further determined. A deletion mutant of aatA was constructed in the APEC DE205 B, which results in the reduced capacity to adhere to DF-1 cells, defective virulence in vivo, and decreased colonization capacity in lung during the systemic infection compared with the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatA makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, aggregation assays for strain AAEC189 expressing aatA indicated that AatA mediates cell aggregation and settling of cells. However, this cell aggregation is blocked by Type I fimbriae. This study illustrates the first examination of the role of AatA in aggregation and systemic infection. [ABSTRACT FROM AUTHOR]