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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]

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]

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]

The capsular polysaccharide ( CPS) synthesis locus of 13 S treptococcus suis serotypes (serotype 1, 3, 4, 5, 7, 8, 9, 10, 14, 19, 23, 25 and 1/2) was sequenced and compared with that of serotype 2 and 16. The CPS synthesis locus of these 15 serotypes falls into two genetic groups. The locus is located on the chromosome between orfZ and aroA. All the translated proteins in the CPS synthesis locus were clustered into 127 homology groups using the tribemcl algorithm. The general organization of the locus suggested that the CPS of S . suis could be synthesized by the Wzy-dependent pathway. The capsule of serotypes 3, 4, 5, 7, 9, 10, 19 and 23 was predicted to be amino-polysaccharide. Sialic acid was predicted to be present in the capsule of serotypes 1, 2, 14, 16 and 1/2. The characteristics of the CPS synthesis locus suggest that some genes may have been imported into S . suis (or their ancestors) on multiple occasions from different and unknown sources. [ABSTRACT FROM AUTHOR]

A proteomic approach combining two-dimensional electrophoresis, Western blot and matrix-assisted laser desorption tandem time-of-flight mass spectrometry has been used to map the extracellular proteins of Streptococcus equi ssp. zooepidemicus ( S. zooepidemicus) strain ATCC 35246. These bioinformatic technologies facilitated the identification of novel S. zooepidemicus vaccine candidate antigens and therapeutic agents. Despite the limitations posed by the unavailability of complete genome and proteome data for S. zooepidemicus, seven of 15 chosen immunogenic spots were successfully identified as streptococcal proteins (AE1 and AE4 c. 10) from homologous Streptococcus species. Among these, AE6 and AE7 were identified as S. zooepidemicus UDP- N-acetyl-glucosamine pyrophosphorylase and UDP-glucose pyrophosphorylase proteins. In addition, AE4 was determined to be glyceraldehyde-3-phosphate dehydrogenase from Enterococcus faecalis. Followingsignalip 3.0 ( ) prediction, data suggested that AE5, AE7 and AE9 contained signal peptides.blast ( ) results found that nucleotide sequences of all identified proteins shared high homology (≥65%) with S. zooepidemicus. The majority of proteins identified in our study remain formally unreported in S. zooepidemicus. However, these proteins serve a vital role in the immune system and reproduction of host species. Therefore, we further evaluated the proteins as vaccine candidates in this study. [ABSTRACT FROM AUTHOR]

The transmission of a recently identified Theileria species pathogenic for sheep and goats in northern China is described. Haemaphysalis qinghaiensis nymphs which had been collected from grass in epidemic areas were able to transmit this Theileria species to sheep. The pathogen was also transmitted to sheep and goats by three batches of adult ticks collected from grass, ticks collected when moving about on sheep and ticks which were found partially engorged on sheep or goats. [ABSTRACT FROM AUTHOR]

NorV has been known to be an anaerobic nitric oxide reductase associated with nitric oxide (NO) detoxification. Recently, we showed that the norV gene of Aeromonas hydrophila was highly upregulated after co-culturing with Tetrahymena thermophila. Here, we demonstrated that the transcription and expression levels of norV were upregulated in a dose-dependent manner after exposure to NO under aerobic and anaerobic conditions. To investigate the roles of norV in resisting predatory protists and virulence of A. hydrophila, we constructed the norV gene-deletion mutant (ΔnorV). Compared to the wild type, the ΔnorV mutant showed no significant difference in growth at various NO concentrations under aerobic conditions but significantly stronger NO-mediated growth inhibition under anaerobic conditions. The deletion of norV exhibited markedly decreased cytotoxicity, hemolytic and protease activities under aerobic and anaerobic conditions. Also, the hemolysin co-regulated protein (Hcp) in the ΔnorV mutant showed increased secretion under aerobic conditions but decreased secretion under anaerobic conditions as compared to the wild-type. Moreover, the inactivation of norV led to reduced resistance to predation by T. thermophila, decreased survival within macrophages and highly attenuated virulence in zebrafish. Our data indicate a diverse role for norV in the expression of A. hydrophila virulence-associated traits that is not completely dependent on its function as a nitric oxide reductase. This study provides insights into an unexplored area of NorV, which will contribute to our understanding of bacterial pathogenesis and the development of new control strategies for A. hydrophila infection. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is an important aquatic microorganism that can cause fish hemorrhagic septicemia. In this study, we identified a novel LysR family transcriptional regulator (LahS) in the A. hydrophila Chinese epidemic strain NJ-35 from a library of 947 mutant strains. The deletion of lahS caused bacteria to exhibit significantly decreased hemolytic activity, motility, biofilm formation, protease production, and anti-bacterial competition ability when compared to the wild-type strain. In addition, the determination of the fifty percent lethal dose (LD50) in zebrafish demonstrated that the lahS deletion mutant (ΔlahS) was highly attenuated in virulence, with an approximately 200-fold increase in LD50 observed as compared with that of the wild-type strain. However, the ΔlahS strain exhibited significantly increased antioxidant activity (six-fold). Label-free quantitative proteome analysis resulted in the identification of 34 differentially expressed proteins in the ΔlahS strain. The differentially expressed proteins were involved in flagellum assembly, metabolism, redox reactions, and cell density induction. The data indicated that LahS might act as a global regulator to directly or indirectly regulate various biological processes in A. hydrophila NJ-35, contributing to a greater understanding the pathogenic mechanisms of A. hydrophila. [ABSTRACT FROM AUTHOR]

A correction to the article "Selective capture of transcribed sequences in the functional gene analysis of microbial pathogens" that was published in the August 27, 2015 issue is presented.

One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by 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]

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]

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]

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]

luxS-mediated autoinducer 2 (AI-2)-dependent quorum sensing (QS) has been demonstrated to affect many bacterial phenotypes, including virulence. Streptococcus agalactiae harbors a functional luxS gene required for the biosynthesis of AI-2. In this study, we investigated the regulation effect and mechanism of the luxS /AI-2 QS system in the pathogenicity of the piscine S. agalactiae strain GD201008-001. We found that inactivation of luxS caused a marked decrease in biofilm formation, hemolytic activity, antiphagocytosis and intracellular survival of S. agalactiae. Except for hemolytic activity, the altered phenotypes due to the luxS deletion were AI-2-independent. Further investigation indicated that high levels of the proinflammatory cytokines IL-1β and IL-6 could be induced in macrophages co-incubated with the luxS deletion mutant and synthetic AI-2, single or combined. Also, the results of tilapia infection showed that inactivation of luxS significantly decreased the virulence of S. agalactiae but upregulated the expression of cytokines in spleens and brains. Increased proinflammatory effects of the luxS mutant were restored in the luxS complemented strain but could not be restored by AI-2 addition. All the findings suggest that luxS is involved in virulence-associated phenotypes and immunological evasion of S. agalactiae , and furthermore, this involvement is mostly AI-2-independent. This study will provide valuable insights into our understanding of the role of the LuxS/AI-2 QS system in the pathogenesis of S. agalactiae. • Inactivation of luxS caused virulence decrease in the piscine S. agalactiae strain GD201008-001. • Both luxS deletion and AI-2 addition induced high expression levels of the proinflammatory cytokines in macrophages. • Most of altered phenotypes due to the luxS deletion were AI-2-independent. [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]

Graphical abstract Highlights • 13 novel potential sRNAs were found firstly in E. tarda ET13 strain, based on RNA sequencing and bioinformatic analyses. • The expression, conservation, direction and structure of EsR240 were found by RT-PCR, Northern blotting, RACE and bioinformatic analyses. • EsR240 may positively regulate its target genes in E. tarda to maintain intracellular survival within host macrophages and virulence. Abstract Bacterial small non-coding RNAs (sRNAs) are gene expression modulators that respond to environmental changes and pathogenic conditions. In this study, 13 novel sRNAs were identified in the intracellular pathogen, Edwardsiella tarda (E. tarda) ET13 strain, based on RNA sequencing and bioinformatic analyses. Eight of the 13 putative sRNAs from the ET13 strain were transcribed (as indicated by RT-PCR) following exposure to different stresses. The transcription levels of three sRNAs (EsR128, EsR139 and EsR240) were all highly induced under these stress conditions. Northern blot hybridization was employed to verify that EsR240 was expressed in the ET13 strain under both logarithmic and stationary growth phases, and that it formed a single copy transcript in the chromosomes of the ET13 strain. The precise start and end points of EsR240 were determined using 5'and 3' RACE. The conservation of EsR240 was in agreement with the characteristics of sRNA, as indicated by a BLAST analysis. Furthermore, the survival rates of EsR240 mutant were lower than the rates of the wild type ET13 under stress conditions. When the infection time was extended 4 or 6 h, the CFUs of the wild type bacteria increased more significantly within macrophages compared to the mutant. When the intra-peritoneal (i.p.) route of infection was used in mice, the bacterial loads of the tissues in the mice infected with the wild type bacteria were significantly higher than in the mice infected with the mutants. The virulence of the EsR240 mutant was 6.79-fold lower than the wild type bacterium based on the LD 50. In addition, the IntaRNA program was used to predict the target genes of EsR240. Out of the top 10 predicted target genes, 9 genes were regulated by EsR240. These target genes may encode FtsH protease modulator YccA, Na+ and H+ antiporters, FtsX-like permease family protein, glycoside hydrolases or various other proteins. Therefore, EsR240 may positively regulate its target genes in E. tarda to maintain intracellular survival within host macrophages and to increase its virulence. [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]

Abstract This study explored the immunomodulatory effect and inhibition effects of the candidate probiotic Lactococcus lactis 16-7, which was isolated from crucian carp, on Aeromonas hydrophila infection in crucian carp. The experimental fish were divided into two groups; one was fed a diet supplemented with L. lactis , while the other was fed the control probiotic-free diet. After feeding for 42 d with the experimental diets, the fish that received the diet supplemented with probiotics exhibited a significantly enhanced serum superoxide dismutase activity, phagocytic activities of innate immune cells, and the expression levels of immune-related genes [interferon-γ (INF-γ), interleukin-1β (IL-1β), interleukin-11 (IL-11), tumour necrosis factor α (TNF-α) and myeloid differentiation factor 88 (MyD88)], indicating that L. lactis 16-7 could activate the non-specific immune system of crucian carp. At the end of the feeding trial, the crucian carps in each group were orally infected with A. hydrophila NJ-35. The results show that L. lactis 16-7 could prevent the increase in d -lactic acid concentration and inflammatory response caused by A. hydrophila in crucian carp. Compared with A. hydrophila group, L. lactis 16-7 preserved the integrity of intestinal villi and mitigated A. hydrophila -induced reduce in the transcriptional levels of tight junction (TJ) proteins zonula occludens-1 (ZO-1) and occludin, indicating that L. lactis 16-7 could reduce intestinal mucosal barrier damage and inflammation induced by A. hydrophila in crucian carp. In addition, L. lactis 16-7 could effectively antagonize the colonization of A. hydrophila in the intestine. Overall, these data clearly indicate that L. lactis 16-7 has the potential to be developed as a probiotic agent against A. hydrophila infection in aquaculture. Highlights • Lactococcus lactis 16-7 could activate the non-specific immune system of crucian carp. • L. lactis 16-7 could reduce A. hydrophila -induced intestinal mucosal barrier function damage and inflammation. • L. lactis 16-7 could antagonize the colonization of A. hydrophila in the intestine. [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]

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that infects swine and humans with high mortality and morbidity. Although a number of virulence-associated factors have been reported, the understanding of the molecular mechanism underlying SS2 pathogenicity remains limited. Our previous studies revealed that srt BCD-associated protein 2’ (SBP2′) contributed to the pathogenesis of SS2, but the function of another member in the srt BCD cluster, srt BCD-associated protein 1 (SBP1) was still unknown. Here, we found that sbp 1 was widely distributed among high virulent SS2 strains, suggesting that sbp 1 may be involved in the pathogenesis of SS2. To investigate the function of SBP1, we firstly conducted Western blotting analyses to confirm that SBP1 was expressed in the high virulent SS2 strain ZY05719 both in vivo and in vitro , then constructed the deletion mutant of sbp 1 by homologous recombination. Bacterial adhesion assay, indirect immunofluorescence assay and protein binding assay all demonstrated that SBP1 was associated with adhesion of SS2 to HEp-2 cells. However, SBP1 did not influence the invasion, phagocytosis or intracellular survival of SS2. Furthermore, infection assays in vivo showed that inactivation of sbp 1 failed to impair the ability of SS2 to cause zebrafish and mouse mortality. Overall, these results indicate that SBP1 is an adhesion-associated factor without the involvement of virulence in Streptococcus suis serotype 2. [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]

The type VI secretion system (T6SS) is considered as one of the key competition strategies by injecting toxic effectors for intestinal pathogens to acquire optimal colonization in host gut, a microenviroment with high-density polymicrobial community where bacteria compete for niches and resources. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea in human and animals, widely encode T6SS clusters in their genomes. In this report, we first identified VT1, a novel amidase effector in ETEC, significantly hydrolyzed D-lactyl-L-Ala crosslinks between N -acetylmuramoyl and L-Ala in peptidoglycan. Further study showed that the VT1/VTI1 effector/immunity pair is encoded within a typical vgrG island, and plays a critical role for the successful establishment of ETEC in host gut. Numerous putative effectors with diverse toxin domains were found by retrieving vgrG islands in pathogenic E. coli , and designated as VT modules. Therein, VT5, a lysozyme-like effector widely encoded in ETEC, was confirmed to effectively kill adjacent cells, suggesting that VT toxin modules may be critical for pathogenic E. coli to seize a significantly competitive advantage for optimal intestinal colonization. To expand our analyses for large-scale search of VT antibacterial effectors based on vgrG island, >200 predicted effectors from 20 bacterial species were found and classified into 11 predicted toxins. This work reports a new retrieval strategy for screening T6SS effectors, and provides an example how pathogenic bacteria antagonize and displace commensal microbiome to successfully colonize in the host niches through a T6SS-dependent manner. [ABSTRACT FROM AUTHOR]

Streptococcus suis ( S. suis ) is an emerging zoonotic agent that is responsible for significant economic losses to the porcine industry worldwide. However, most research regarding the pathogenic mechanisms has used in vitro cultures of S. suis , which may not provide an accurate representation of the in vivo biological activities. In this study, 188 differential abundance S. suis proteins were identified in in vivo samples obtained from the blood of the infected pigs. These were compared with in vitro samples by a Tandem Mass Tags (TMT) experiment. Thus, a virulence associated network was established using the enriched differential abundance proteins (obtained via bioinformatics analysis in this study) and the previously reported putative virulence factors associated with in vivo infection. One of the most important up-regulated hubs in this network, adhE (an acetaldehyde-CoA/alcohol dehydrogenase) was found. Furthermore, knocking out adh E in S. suis serotype 2 strain ZY05719 decreased virulence. Cell culture experiments and far-western blot analysis showed that adhE is involved in adhesion to Caco-2 cells; Hsp60 could be one of the receptors for this protein. Significance This study is a systematical research to identify in vivo regulated virulence associated proteins of S. suis in pigs. It constructs a network consisting of in vivo infection related factors for the first time to get to know the coordinated actions of a multitude of factors that lead to host pathogenicity and filter the most important hubs. The individual factors that contribute to infection is also identified. A novel differential protein adhE which is one of the most important hubs of this network and is up-regulated in abundance in vivo is found to moonlight as an important adhesion by binding Hsp60 and finally contributes to virulence. [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]

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]

Fibronectin-/fibrinogen-binding protein (FBPS) of Streptococcus suis serotype 2 (SS2) is an atypical anchorless microbial surface components recognizing adhesive matrix molecules (MSCRAMM) for which the role in bacterial infection are not clearly established. To investigate the biological functions of FBPS, an fbps knockout mutant was constructed in SS2 strain ZY05719 to explore the phenotypic changes between the wild-type and mutant strains. Cell morphology analyses combined with the basic growth curves showed that deletion of fbps does not significantly influence neither the thickness of the capsule of SS2 nor the cell growth characteristic. In addition to three previously identified host components fibronectin (FN), factor H (FH) and fibrinogen (FG), we also found that both laminin (LN) and immunoglobulin G (IgG) could bind specifically to FBPS. Furthermore, we confirmed that FBPS play an important role in adherence of SS2 to host cells. The in vitro assays demonstrated that an inactivation of fbps does not inhibit the intracellular survival of SS2 in RAW246.7 macrophages, attenuate the ability of invasion of host cells or the growth ability in pig blood. Additionally, the fbps mutation failed to decrease the virulence of SS2 in both BALB/c mice and zebrafish. Finally, immunization with recombinant FBPS showed no significant difference from the control groups in terms of murine viability after SS2 challenge. Taken together, we concluded that FBPS is not a critical virulence factor for the SS2 strain ZY05719. [ABSTRACT FROM AUTHOR]