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Abstract: Bacterial ghosts may be generated by the controlled expression of the phiX174 lysis gene E in Gram-negative bacteria and they are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. The Edwardsiella tarda ghost (ETG) vaccine was prepared using this technology and tested in vaccination trials. Control groups included mice immunized with formalin-killed E. tarda (FKC) or mice treated with phosphate-buffered saline (PBS), respectively. The results showed that serum IgA and IgG antibody titers were significantly higher in the ETG-vaccinated group compared to the other groups. In addition, CD8+ T cell counts in peripheral blood were elevated in the ETG groups. Most important, ETG-immunized mice were significantly protected against E. tarda challenge (86.7% survival) compared to 73.3 and 33.3% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG oral vaccine could confer protection against infection in a mouse model of disease. [Copyright &y& Elsevier]

Highlights: [•] This study is the first MLST analysis of Pasteurella multocida strains from China. [•] For the first time, a common genotype (MLST sequence type ST129) was identified in China. [•] Our study suggests that P. multocida ST129 circulates in Jiangsu province, China. [•] Our study provides important information regarding the global population structure of P. multocida. [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]

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]

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]

Streptococcus suis (SS), an important pathogen for pigs, is not only considered as a zoonotic agent for humans, but is also recognized as a major reservoir of antimicrobial resistance contributing to the spread of resistance genes to other pathogenic Streptococcus species. In addition to serotype 2 (SS2), serotype 9 (SS9) is another prevalent serotype isolated from diseased pigs. Although many SS strains have been sequenced, the complete genome of a non-SS2 virulent strain has been unavailable to date. Here, we report the complete genome of GZ0565, a virulent strain of SS9, isolated from a pig with meningitis. Comparative genomic analysis revealed five new putative virulence or antimicrobial resistance-associated genes in strain GZ0565 but not in SS2 virulent strains. These five genes encode a putative triacylglycerol lipase, a TipAS antibiotic-recognition domain protein, a putative TetR family transcriptional repressor, a protein containing a LPXTG domain and a G5 domain, and a type VII secretion system (T7SS) putative substrate (EsxA), respectively. Western blot analysis showed that strain GZ0565 can secrete EsxA. We generated an esxA deletion mutant and showed that EsxA contributes to SS virulence in a mouse infection model. Additionally, the antibiotic resistance gene vanZ SS was identified and expression of vanZ SS conferred resistance to teicoplanin and dalbavancin in Streptococcus agalactiae . We believe this is the first experimental demonstration of the existence of the T7SS putative substrate EsxA and its contribution to bacterial virulence in SS. Together, our results contribute to further understanding of the virulence and antimicrobial resistance characteristics of SS. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV), is the etiological agent of porcine epidemic diarrhea virus (PED), causes high mortality with severe vomiting, diarrhea, and dehydration in swine farms. In this study, the PEDV strain 85-7 could be proliferated effectively in MARC-145 cells, and caused a distinct inhibition of the expression of interferon-β (IFN-β, encoded by IFNB1 ), which suggested that a full understanding how this virus manipulates the host immune responses is critical in the fight against the spread of PEDV. We found that, the infection of PEDV strain 85-7 significantly downregulated HSP27 production in MARC-145 cells, and overexpression of HSP27 (encoded by HSPB1 ) decreased the virus titer by about 28-fold. Further study revealed that HSP27 could significantly activate the NF-κB phosphorylation, and thus increase the mRNA level of IFNB1 and downstream interferon-stimulated genes (ISGs) in MARC-145 cells. Indeed, treatment with IFN-β and the ISGs, including murine myxovirus resistance 1 and 2 (Mx1 and Mx2), showed direct anti-PEDV activity. Notably, the antiviral activity and transcription of the antiviral effectors induced by overexpression of HSP27 could be counteracted by the knockdown of IFNB1 via RNA interference, indicating that HSP27 was an upstream regulator of the intracellular antiviral effect against PEDV infection. This study is the first to link HSP27 to PEDV replication via the innate immunity response, which contributed to further clarify the mechanism of PEDV infection and the development of novel antiviral therapies. [ABSTRACT FROM AUTHOR]

Factor H (FH), a regulatory protein of the complement system, can bind specifically to factor H-binding proteins (FHBPs) of Streptococcus suis serotype 2 (SS2), which contribute to evasion of host innate immune defenses. In the present study, we aimed to identify novel FHBPs and characterize the biological functions of FH in SS2 pathogenesis. Here, a method that combined proteomics and Far-western blotting was developed to identify the surface FHBPs of SS2. With this method, fourteen potential novel FHBPs were identified among SS2 surface proteins. We selected eight newly identified proteins and further confirmed their binding activity to FH. The binding of SS2 to immobilized FH decreased dramatically after pre-incubation with anti-FHBPs polyclonal antibodies. We showed for the first time that SS2 also interact specifically with mouse FH. Furthermore, we found that FH play an important role in adherence and invasion of SS2 to HEp-2 cells. Additionally, using a mouse model of intraperitoneal challenge, we confirmed that SS2 pre-incubated with FH enhanced bacteremia and brain invasion, compared with SS2 not pretreated with FH. Taken together, this study provides a useful method to characterize the host-bacteria interactions. These results first indicated that binding of FH to the cell surface improved the adherence and invasion of SS2 to HEp-2 cells, promoting SS2 to resist killing and leading to enhance virulence. [ABSTRACT FROM AUTHOR]

The TonB system is required for the active transport of iron compounds across the outer membrane in Gram-negative bacteria. Our previous data indicated that three TonB systems act coordinately to contribute to the motility of Aeromonas hydrophila NJ-35. In this study, we found that flagellum biogenesis was defective in the Δ tonB123 mutant. Subcellular localization indicated that the flagellin subunits FlaA and FlaB were trapped in the cytoplasm of Δ tonB123 mutant with reduced molecular mass. Overexpression of FlaA or FlaB in the Δ tonB123 mutant was unable to restore the secretion of flagellin subunits. Further investigation demonstrated that flagellins in the Δ tonB123 mutant showed a weak affinity for the flagellin-specific chaperone FliS, which is necessary for the export of flagellins. Deglycosylation analysis indicated that flagellins in the cytoplasm of the Δ tonB123 mutant were almost nonglycosylated. Our data suggested that disruption of tonB123 impairs the formation of flagella by inhibiting flagellin glycosylation and decreasing the binding affinity of flagellin for the chaperone FliS. Taken together, our findings indicate a new role of the TonB system in flagellar biogenesis in A. hydrophila. [ABSTRACT FROM AUTHOR]

Avian pathogenic Escherichia coli (APEC) causes colibacillosis, which results in significant economic losses to the poultry industry worldwide. Due to the drug residues and increased antibiotic resistance caused by antibiotic use, bacteriophages and other alternative therapeutic agents are expected to control APEC infection in poultry. Two APEC phages, named P483 and P694, were isolated from the feces from the farmers market in China. We then studied their biological properties, and carried out high-throughput genome sequencing and homology analyses of these phages. Assembly results of high-throughput sequencing showed that the structures of both P483 and P694 genomes consist of linear and double-stranded DNA. Results of the electron microscopy and homology analysis revealed that both P483 and P694 belong to T7-like virus which is a member of the Podoviridae family of the Caudovirales order. Comparative genomic analysis showed that most of the predicted proteins of these two phages showed strongest sequence similarity to the Enterobacteria phages BA14 and 285P, Erwinia phage FE44, and Kluyvera phage Kvp1; however, some proteins such as gp0.6a, gp1.7 and gp17 showed lower similarity (< 85%) with the homologs of other phages in the T7 subgroup. We also found some unique characteristics of P483 and P694, such as the two types of the genes of P694 and no lytic activity of P694 against its host bacteria in liquid medium. Our results serve to further our understanding of phage evolution of T7-like coliphages and provide the potential application of the phages as therapeutic agents for the treatment of diseases. [ABSTRACT FROM AUTHOR]

Streptococcus suis is one of the most important swine pathogens worldwide. In this study, a total of 22 virulence-related genes in 101 strains of S. suis serotype 2 (SS2) were detected by PCR, namely, mrp , epf , sly , fbps , rgg , ofs , srtA , pgdA , gapdh , iga , endoD , ciaRH , salKR , manN , purD , rgg , DppIV , neuB , dltA , SMU_61-like, SpyM3_0908 (Permease) and the SspA gene. The distribution of virulence-related genes among isolates was visualized using BioNumerics software to study similarities among the isolates. Two clusters of SS2 were apparent on the phylogenetic tree, namely, Clusters A and B. Both mouse and zebrafish infection models revealed that strains in Cluster B were more virulent than those in Cluster A. Statistical comparison between the two clusters was performed, and structure analysis demonstrated that epf , sly , rgg , endoD , SMU_61-like and SpyM3_0908 were possible predictive markers for SS2 virulence. The transcription levels of highly prevalent genes in both clusters were detected by qRT-PCR in representative strains. For Cluster A isolates, the transcription levels of neuB , dltA , fbps and pgdA were significantly lower, but the transcription level of iga was significantly higher than that in Cluster B isolates. Although encoded in the genomes of the selected Cluster A isolates, DppIV and mrp genes were not expressed. These results revealed the genetic differences between virulent and low-virulence SS2 isolates from China and provide a better understanding of the SS2 pathogenic mechanism. [ABSTRACT FROM AUTHOR]

Biofilms contribute to the resistance of Edwardsiella tarda to antibiotics and host immunity. AroC in the shikimate pathway produces chorismate to synthesize crucial intermediates such as indole. In this study, the differences between biofilms produced by aroC mutants (△aroC), wild-type (WT) strains, and △aroC complementary strains (C△aroC) were detected both in vitro with 96-well plates, tubes, or coverslips and in vivo using a mouse model of subcutaneous implants. When examining potential mechanisms, we found that the diameters of the movement rings in soft agar plates and the flagellar sizes and numbers determined by silver staining were all lower for △aroC than for WT and C△aroC. Moreover, qRT-PCR showed that the transcription levels of flagellar synthesis genes, fliA and fliC , were reduced in △aroC. AroC, FliC, or FliA may accompany the motility of △aroC strains. In addition, compared with the WT and C△aroC, the amounts of indole in △aroC were significantly decreased. Notably, the formation of biofilms by these strains could be promoted by exogenous indole. Therefore, the aroC gene could affect the biofilm formation of E. tarda concerning its impact on flagella and indole. [ABSTRACT FROM AUTHOR]

In early 2013, a Bengal tiger ( Panthera tigris ) in a zoo died of respiratory distress. All specimens from the tiger were positive for HPAI H5N1, which were detected by real-time PCR, including nose swab, throat swab, tracheal swab, heart, liver, spleen, lung, kidney, aquae pericardii and cerebrospinal fluid. One stain of virus, A/Tiger/JS/1/2013 , was isolated from the lung sample. Pathogenicity experiments showed that the isolate was able to replicate and cause death in mice. Phylogenetic analysis indicated that HA and NA of A/Tiger/JS/1/2013 clustered with A/duck/Vietnam/OIE-2202/2012 (H5N1) , which belongs to clade 2.3.2.1. Interestingly, the gene segment PB2 shared 98% homology with A/wild duck/Korea/CSM-28/20/2010 (H4N6) , which suggested that A/Tiger/JS/1/2013 is a novel reassortant H5N1 subtype virus. Immunohistochemical analysis also confirmed that the tiger was infected by this new reassortant HPAI H5N1 virus. Overall, our results showed that this Bengal tiger was infected by a novel reassortant H5N1, suggesting that the H5N1 virus can successfully cross species barriers from avian to mammal through reassortment. [ABSTRACT FROM AUTHOR]

Abstract: Streptococcus suis (SS) is an important swine pathogen worldwide that occasionally causes serious infections in humans. SS infection may result in meningitis in pigs and humans. The pathogenic mechanisms of SS are poorly understood. Here, we provide the complete genome sequence of S. suis serotype 2 (SS2) strain SC070731 isolated from a pig with meningitis. The chromosome is 2,138,568bp in length. There are 1933 predicted protein coding sequences and 96.7% (57/59) of the known virulence-associated genes are present in the genome. Strain SC070731 showed similar virulence with SS2 virulent strains HA9801 and ZY05719, but was more virulent than SS2 virulent strain P1/7 in the zebrafish infection model. Comparative genomic analysis revealed a unique 105K genomic island in strain SC070731 that is absent in seven other sequenced SS2 strains. Further analysis of the 105K genomic island indicated that it contained a complete nisin locus similar to the nisin U locus in S. uberis strain 42, a prophage similar to S. oralis phage PH10 and several antibiotic resistance genes. Several proteins in the 105K genomic island, including nisin and RelBE toxin–antitoxin system, contribute to the bacterial fitness and virulence in other pathogenic bacteria. Further investigation of newly identified gene products, including four putative new virulence-associated surface proteins, will improve our understanding of SS pathogenesis. [Copyright &y& Elsevier]

Highlights: [•] We provide the first evidence of three distinguishable T6SS loci in APEC genomes. [•] Results display multipurpose T6SS1, single-minded T6SS2 and unserviceable T6SS3. [•] The properties difference of T6SS may be supervised and managed by VgrG. [•] A possible receptor-banding protein, VgrG may responsible for cell recognition. [•] Similar to the phage, the host specificity may also present in different T6SS loci. [ABSTRACT FROM AUTHOR]

Highlights: [•] Immunoproteomics was applied to identify immunogenic proteins of APEC for the first time. [•] Two predominant immunogenic components, OmpA and Flic, were identified. [•] GroEL and other 8 proteins are reported as immunoreactive proteins in APEC for the first time. [•] GroEL stimulated antibody responses and supported protective efficacy against infections of heterogeneous APEC strains. [ABSTRACT FROM AUTHOR]

Experiments on the transmission of an unidentified Theileria sp. infective for small ruminants by Haemaphysalis qinghaiensis and Hyalomma anatolicum anatolicum were carried out. Three Theileria-free batches of adult, larvae, and nymphs of laboratory reared H. qinghaiensis and Hy. a. anatolicum ticks were infected by feeding them on sheep infected with Theileria sp. The Theileria sp. was originally isolated from adult ticks of H. qinghaiensis, by inoculation of blood stabilates or tick transmission. H. qinghaiensis has been shown to be capable of transmitting the Theileria sp. infective for small ruminants transstadially to sheep and goats. The nymphs developed from the larvae engorged on the sheep infected with the parasite transmitted the pathogen to splenectomized sheep with prepatent periods of 30, 31 days, respectively; but the subsequent adult ticks of H. qinghaiensis derived from the nymphs did not transmit the pathogen to sheep. However, adults developed from the nymphs engorged on the sheep infected with the parasite transmitted the pathogen to sheep with prepatent periods of 24–27 days. The larvae, nymphs and adult ticks derived from female H. qinghaiensis ticks engorged on infected sheep were not able to transmit the parasite transovarially. The same experiments were done with Hy. a. anatolicum, but examination for presence of piroplasma of Theileria sp. from all animals were negative, demonstrating that Hy. a. anatolicum could not transmit the organism to sheep or goats. [Copyright &y& Elsevier]

Eha is a virulence regulator in Edwardsiella tarda (E. tarda). The present study examined how Eha regulated its target genes to affect the bacterial survival within the cells. We constructed the reporter a pGEX-4T-ehaflag plasmid expressing Eha tagged at its C terminus with the flag epitope, and introduced the plasmid into an eha mutant ET13 strain, and obtained a Cehaflag strain. The expression and activity of an EhaFlag fusion protein restored the survival of the Cehaflag as the wild type in macrophages by Western blotting and intracellular survival experiments. We used a monoclonal anti-Flag antibody to precipitate EhaFlag-DNA complexes using chromatic immunoprecipitation (ChIP). We then designed primers based on the differentially-expressed genes identified from RNA-sequencing, and identified ten Eha-interacting genes by qPCR. We amplified the promoter regions of the ten genes and the eha gene from ET13 strain by PCR, constructed pBD-P target lacZ and pBD-P eha lacZ plasmids. The eha gene directly and positively regulated these target genes, and be negatively auto-regulated by Eha in E. tarda , as determined by comparing their β -Galactosidase activities. These target genes were distributed in the categories involved in the bacterial growth, movement and resistance to H 2 O 2 or acid. We further constructed a ETATCC_RS15225 mutant (△dcuA1), a ETATCC_ RS14855 mutant (△flgK) anda ETATCC_RS07650 mutant (ΔtnaA), and a partial complementary strains of △eha-tnaA and △eha-flgK and the complementary strains of CΔflgK and CΔtnaA. The ETATCC_RS15225 gene probably encoded a transporter protein DcuA1 at outer membrane with SDS-PAGE and RT-PCR. The ETATCC _RS14855 gene probably encoded FlgK protein and affected the bacterial motility. The ETATCC_RS07650 gene encoded Tryptophanase, which affected the bacterial survival within macrophages. With the assistance of these above strains, our results showed that the eha gene was able to regulate the ETATCC_RS15225 gene to express its outer membrane protein DcuA1, the ETATCC _RS14855 gene to control the flagellar motility and the ETATCC_RS07650 to affect the bacterial survival within macrophages. With the combination of other functions of above three genes, our results suggested that Eha directly regulates the target genes to affect E. tarda to survive within the cells. [ABSTRACT FROM AUTHOR]