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ABSTRACTProtozoan predation has been demonstrated to be a strong driving force for bacterial defence strategies in the environment. Our previous study demonstrated that Aeromonas hydrophilaNJ-35, which evolved small-colony variants (SCVs), displayed various adaptive traits in response to Tetrahymena thermophilapredation, such as enhanced phage resistance. However, the evolutionary mechanisms are largely unknown. In this study, we performed a genome- and transcriptome-wide analysis of the SCV1, representing one strain of the SCVs, for identification of the genes of mutation and altered expression underlying this phage resistance phenotype. Our study demonstrated that phage resistance caused by T. thermophilapredation was due to the downregulation of a flagellar biosynthesis regulator, flhF, in SCV1. Interestingly, we confirmed that phage resistance in SCV1 was not straightforwardly attributable to the absence of flagella but to FlhF-mediated secretion of extracellular protein that hinders phage adsorption. This finding improves our understanding of the mechanisms by which A. hydrophilalowers the susceptibility to phage infection under predation pressure, and highlights an important contribution of bacterium–protozoan interactions in driving the adaptive evolution of pathogens in complex environments.

ABSTRACTStreptococcus suisis a major porcine bacterial pathogen and emerging zoonotic agent. S. suis5ʹ-nucleotidase is able to convert adenosine monophosphate to adenosine, resulting in inhibiting neutrophil functions in vitroand it is an important virulence factor. Here, we show that S. suis5ʹ-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 vivoby 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 vivoinfection experiment showed that 2ʹ-deoxyadenosine synthesized by 5ʹ-nucleotidase caused monocytopenia in mouse blood. The in vivotranscriptome 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. suissynthesizes 2ʹ-deoxyadenosine and adenosine by 5ʹ-nucleotidase to dampen host immune responses, which represents a new mechanism of S. suispathogenesis.

ABSTRACTMuramidase-released protein (MRP) of Streptococcus suisserotype 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 Δmrp, Δmrp 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-d1mutation significantly inhibited bacteremia and brain invasion in a mouse infection model. The mrp-d1mutation 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.

ABSTRACTThe 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-et3and an orphan et4(et4O1) genes caused by a termination-codon 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.

ABSTRACTAvian pathogenic Escherichia coli(APEC) strains cause one of the three most significant infectious diseases in the poultry industry and are also potential food-borne pathogens threating human health. In this study, we showed that ArcA (aerobic respiratory control), a global regulator important for E. coli's adaptation from anaerobic to aerobic conditions and control of that bacterium's enzymatic defenses against reactive oxygen species (ROS), is involved in the virulence of APEC. Deletion of arcAsignificantly attenuates the virulence of APEC in the duck model. Transcriptome sequencing (RNA-Seq) analyses comparing the APEC wild type and the arcAmutant indicate that ArcA regulates the expression of 129 genes, including genes involved in citrate transport and metabolism, flagellum synthesis, and chemotaxis. Further investigations revealed that citCEFXGcontributed to APEC's microaerobic growth at the lag and log phases when cultured in duck serum and that ArcA played a dual role in the control of citrate metabolism and transportation. In addition, deletion of flagellar genes motAand motBand chemotaxis gene cheAsignificantly attenuated the virulence of APEC, and ArcA was shown to directly regulate the expression of motA, motB, and cheA. The combined results indicate that ArcA controls metabolism, chemotaxis, and motility contributing to the pathogenicity of APEC.

ABSTRACTPorcine epidemic diarrhea has become pandemic in the Asian pig-breeding industry, causing significant economic loss. In the present study, 11 complete genomes of porcine epidemic diarrhea virus (PEDV) field isolates from China were determined and analyzed. Frequently occurring mutations were observed, which suggested that full understanding of the genomic and epidemiological characteristics is critical in the fight against PEDV epidemics. Comparative analysis of 49 available genomes clustered the PEDV strains into pandemic (PX) and classical (CX) groups and identified four hypervariable regions (V1 to V4). Further study indicated key roles for the spike (S) gene and the V2 region in distinguishing between the PX and CX groups and for studying genetic evolution. Genotyping and phylogeny-based geographical dissection based on 219 S genes revealed the complexity and severity of PEDV epidemics in Asia. Many subgroups have formed, with a wide array of mutations in different countries, leading to the outbreak of PEDV in Asia. Spatiotemporal reconstruction based on the analysis suggested that the pandemic group strains originated from South Korea and then extended into Japan, Thailand, and China. However, the novel pandemic strains in South Korea that appeared after 2013 may have originated from a Chinese variant. Thus, the serious PED epidemics in China and South Korea in recent years were caused by the complex subgroups of PEDV. The data in this study have important implications for understanding the ongoing PEDV outbreaks in Asia and will guide future efforts to effectively prevent and control PEDV.

ABSTRACTType VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. The VgrG protein, a core component and effector of T6SS, has been demonstrated to perform diverse functions. The N-terminal domain of VgrG protein is a homologue of tail fiber protein gp27 of phage T4, which performs a receptor binding function and determines the host specificity. Based on sequence analysis, we found that two putative T6SS loci exist in the genome of the avian pathogenic Escherichia coli(APEC) strain TW-XM. To assess the contribution of these two T6SSs to TW-XM pathogenesis, the crucial clpVclusters of these two T6SS loci and their vgrGgenes were deleted to generate a series of mutants. Consequently, T6SS1-associated mutants presented diminished adherence to and invasion of several host cell lines cultured in vitro, decreased pathogenicity in duck and mouse infection models in vivo, and decreased biofilm formation and bacterial competitive advantage. In contrast, T6SS2-associated mutants presented a significant decrease only in the adherence to and invasion of mouse brain microvascular endothelial cell (BMEC) line bEnd.3 and brain tissue of the duck infection model. These results suggested that T6SS1 was involved in the proliferation of APEC in systemic infection, whereas VgrG-T6SS2 was responsible only for cerebral infection. Further study demonstrated that VgrG-T6SS2 was able to bind to the surface of bEnd.3 cells, whereas it did not bind to DF-1 (chicken embryo fibroblast) cells, which further proved the interaction of VgrG-T6SS2 with the surface of BMECs.

ABSTRACTStreptococcus agalactiaeis the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiaeHyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl+isolate. The isogenic hylbmutant (?hylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-a), than the wild type in macrophages as well as in mice. Furthermore, only Hyl+strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylbmutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ?hylBmutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiaeand proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiaepathogenicity.

In this differential RNA-seq study, Wu et al. identified 29 small RNAs expressed by the pig pathogen Streptococcus suis. The authors then went on to show that S. suisstrains lacking some of these small RNAs were attenuated in a zebrafish infection model or were more sensitive to killing by pig blood. The study thus provides further evidence that small, regulatory RNAs play roles in bacterial virulence.

ABSTRACTAutotransporter (AT) proteins constitute a large family of extracellular proteins that contribute to bacterial virulence. A novel AT adhesin gene, aatB, was identified in avian pathogenic Escherichia coli(APEC) DE205B via genomic analyses. The open reading frame of aatBwas 1,017 bp, encoding a putative 36.3-kDa protein which contained structural motifs characteristic for AT proteins: a signal peptide, a passenger domain, and a translocator domain. The predicted three-dimensional structure of AatB consisted of two distinct domains, the C-terminal ß-barrel translocator domain and an N-terminal passenger domain. The prevalence analyses of aatBin APEC indicated that aatBwas detected in 26.4% (72/273) of APEC strains and was strongly associated with phylogenetic groups D and B2. Quantitative real-time reverse transcription-PCR analyses revealed that AatB expression was increased during infection in vitroand in vivo. Moreover, AatB could elicit antibodies in infected ducks, suggesting that AatB is involved in APEC pathogenicity. Thus, APEC DE205B strains with a mutated aatBgene and mutated strains complemented with the aatBgene were constructed. Inactivation of aatBresulted in a reduced capacity to adhere to DF-1 cells, defective virulence capacity in vivo, and decreased colonization capacity in lung during systemic infection compared with the capacities of the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatB makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivoand in vitro. In addition, biofilm formation assays with strain AAEC189 expressing AatB indicated that AatB mediates biofilm formation.

ABSTRACTStreptococcus suisis an emerging zoonotic pathogen causing severe infections in pigs and humans. Thirty-three serotypes of S. suishave been identified using serum agglutination. The capsular polysaccharides synthesis (cps) locus is usually conserved among different strains of the same serotype. The cpsloci of 15 serotypes have been sequenced, while the loci of the other serotypes remain unknown. In the present study, two to six serotype-specific genes of each of eight serotypes, i.e., serotypes 3, 4, 5, 8, 10, 19, 23, and 25, were identified using cross-hybridization with 93 nucleic acid probes specific to genes in the cpslocus, and serotype-specific PCR assays for rapid and sensitive detection of the eight serotypes were then developed. The PCR typing results of the 148 serologically typeable isolates were completely consistent with agglutination results. Furthermore, some autoagglutinating, acapsular, and multiagglutinating strains which could not be differentiated by traditional serum agglutination assays were positive in the PCR assays. Use of the PCR assays with clinical tonsillar specimens showed that the assays are sensitive and able to identify samples with autoagglutinating isolates. To our knowledge, this is the first study to identify the serotype-specific genes of the eight Streptococcus suisserotypes and develop rapid and sensitive PCR assays for the eight serotypes which can be identified only by serum agglutination.

To investigate the prevalence and genetic characterization of Pigeon circovirus(PiCV) circulating in Chinese flocks, the genomic DNA of 144 samples collected from pigeons in 6 different geographic regions of eastern China between 2009 and 2010 were amplified using previously published PiCV primers. The PiCV sequence was detected in 83 of 104 unhealthy pigeons (79.8%) and 25 of 40 healthy pigeons (62.5%). The overall positive rate was 75% for all samples. An inverse primer polymerase chain reaction (IP-PCR) assay was performed to amplify the full-length sequence from a random sample of each region, and 6 specific DNA fragments were gel-purified and sequenced. The 6 full-length sequences were designated as SHWH-AB4 (2,031 bp), NJPK-21 (2,035 bp), HBLF-E2 (2,031 bp), JSJN (2,039 bp), SDDZ (2,037 bp), and AHBZ (2,035 bp) after BLAST analyses. The phylogenic tree and amino acid comparison indicated that all the strains examined were derived from a common strain, but had undergone genetic mutations through time. Pairwise comparisons revealed 93.4%–100% amino acid identity for the putative replication-associated proteins and 67.5%–100% for the putative capsid proteins.

ABSTRACTThe ibeAgene is located on a genomic island, GimA, which is involved in the pathogenesis of neonatal meningitis Escherichia coli(NMEC) and avian pathogenic E. coli(APEC). The prevalence of ibeAin the APEC collection in China was investigated, and 20 of 467 strains (4.3%) were positive. In addition, analysis of the association of the E. colireference (ECOR) groups with positive strains revealed that ibeAwas linked to group B2. The ibeAgene in DE205B was analyzed and compared to those of APEC and NMEC, which indicated that the specificity of ibeAwas not consistent along pathotypes. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. The inactivation of ibeAin APEC DE205B led to the reduced capacity to invade DF-1 cells, defective virulence in vivo, and decreased biofilm formation compared to the wild-type strain. In addition, strain AAEC189 expressing ibeAexhibited enhanced invasion capacity and biofilm formation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the loss of ibeAdecreased the colonization and proliferation capacities of APEC in the brain during system infection.

The ibeA gene is located on a genomic island, GimA, which is involved in the pathogenesis of neonatal meningitis Escherichia coli (NMEC) and avian pathogenic E. coli (APEC). The prevalence of ibeA in the APEC collection in China was investigated, and 20 of 467 strains (4.3%) were positive. In addition, analysis of the association of the E. coli reference (ECOR) groups with positive strains revealed that ibeA was linked to group B2. The ibeA gene in DE205B was analyzed and compared to those of APEC and NMEC, which indicated that the specificity of ibeA was not consistent along pathotypes. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. The inactivation of ibeA in APEC DE205B led to the reduced capacity to invade DF-1 cells, defective virulence in vivo, and decreased biofilm formation compared to the wild-type strain. In addition, strain AAEC189 expressing ibeA exhibited enhanced invasion capacity and biofilm formation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the loss of ibeA decreased the colonization and proliferation capacities of APEC in the brain during system infection.

Cysticercosis caused by Taenia solium metacestodes is a worldwide public health problem. Important progress in the development of effective and practical vaccines against this disease has been made. In this study, the promising T. solium oncospheral vaccine candidate named TSOL18 antigen was produced in a 5-liter fermentor. During the process of fermentation, the pH of the culture was always kept below 5.0, and in order to prevent foaming, an antifoam agent was added. In addition, the oxygen content of the culture was constantly kept at >50% in our experiment. A high level of the glycosylated protein (2.5 g/liter) was obtained, and the protein was easily purified by gel chromatography. Vaccination trials showed that the recombinant TSOL18 antigen induced 94 and 100% reductions in metacestode burdens in vaccinated pigs, obviously higher than the 89% reduction in pigs immunized with cysticercus crude extracts in trial 1. These are very promising results in the development of an efficient tool to control cysticercosis in Asia.

ABSTRACTCysticercosis caused by Taenia soliummetacestodes is a worldwide public health problem. Important progress in the development of effective and practical vaccines against this disease has been made. In this study, the promising T. soliumoncospheral vaccine candidate named TSOL18 antigen was produced in a 5-liter fermentor. During the process of fermentation, the pH of the culture was always kept below 5.0, and in order to prevent foaming, an antifoam agent was added. In addition, the oxygen content of the culture was constantly kept at >50% in our experiment. A high level of the glycosylated protein (2.5 g/liter) was obtained, and the protein was easily purified by gel chromatography. Vaccination trials showed that the recombinant TSOL18 antigen induced 94 and 100% reductions in metacestode burdens in vaccinated pigs, obviously higher than the 89% reduction in pigs immunized with cysticercus crude extracts in trial 1. These are very promising results in the development of an efficient tool to control cysticercosis in Asia.

The phylogenetic relationships between six isolates of Theileria spp. infective to small ruminants , and two isolates of Theileria spp. infective to yak, all transmitted by Haemaphysalis qinghaiensis, together with the Theileria orientalis/sergenti/buffeli group and T. sinensis, were analyzed using the 18S ssrRNA gene sequence. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was used either for direct sequencing or was ligated to the PCR II vector for sequencing. The length of the 18S ssrRNA gene of all Theileria spp. involved in this study was around 1,740 bp. Two phylogenetic trees were inferred based on the 18S ssrRNA gene sequence of the Chinese isolates only, and Chinese isolates and other species of Theileria available in GenBank. In the first tree, the Theileria sp. infective to yaks was found to be T. sinensis. The Theileria sp. infective to small ruminants was found to be composed of two separate species of Theileria. Theileria sp. from Qinghai, Madang, Ningxian and Lintan, which was identical to the unidentified Theileria sp. described previously, is designated Theileria sp (China 1). The Theileria sp. from Longde, Zhangjiachuan and Lintan, which has not been described previously, is designated Theileria sp. (China 2) in order to avoid confusion. In the second tree, Theileria sp. (China 1) was closely related to benign Theileria, such as T. buffeli and T. sergenti, while Theileria sp. (China 2) was separated from other Theileria spp. The results indicate that H. qinghaiensis transmit at least three species of Theileria, two which are infective to sheep and goats, but not yak and one which is infective to yaks and cattle, but not to sheep and goats.

Streptococcus agalactiae(group B streptococcus [GBS]) has received continuous attention for its involvement in invasive infections and its broad host range. Transcriptional regulators have an important impact on bacterial adaptation to various environments. Research on transcriptional regulators will shed new light on GBS pathogenesis. In this study, we identified a novel XRE-family transcriptional regulator encoded on the GBS genome, designated XtgS. Our data demonstrate that XtgS inactivation significantly increases bacterial survival in host blood and animal challenge test, suggesting that it is a negative regulator of GBS pathogenicity.

The transmission of a recently identified Theileriaspecies pathogenic for sheep and goats in northern China is described. Haemaphysalis qinghaiensisnymphs 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.

ABSTRACTStreptococcus suishas received increasing attention for its involvement in severe human infections worldwide as well as in multidrug resistance. Two-component signaling systems (TCSSs) play important roles in bacterial adaptation to various environmental stimuli. In this study, we identified a novel TCSS located in S. suisserotype 2 (SS2), designated VraSRSS, which is involved in bacterial pathogenicity and susceptibility to antimicrobials. Our data demonstrated that the yvqFSSgene, located upstream of vraSRSS, shared the same promoter with the TCSS genes, which was directly regulated by VraSRSS, as shown in electrophoretic mobility shift assays. Notably, YvqFSSand VraSRSSconstitute a novel multidrug resistance module of SS2 that participates in resistance to certain groups of antimicrobials. Further analyses showed that VraSRSSinactivation significantly attenuated bacterial virulence in animal models, which, coupled with the significant activation of VraSRSSexpression observed in host blood, strongly suggested that VraSRSSis an important regulator of SS2 pathogenicity. Indeed, RNA-sequencing analyses identified 106 genes that were differentially expressed between the wild-type and ΔvraSRSSstrains, including genes involved in capsular polysaccharide (CPS) biosynthesis. Subsequent studies confirmed that VraSRSSindirectly regulated the transcription of CPS gene clusters and, thus, controlled the CPS thickness shown by transmission electron microscopy. Decreased CPS biosynthesis caused by vraSRSSdeletion subsequently increased bacterial adhesion to epithelial cells and attenuated antiphagocytosis against macrophages, which partially clarified the pathogenic mechanism mediated by VraSRSS. Taken together, our data suggest that the novel TCSS, VraSRSS, plays critical roles for multidrug resistance and full virulence in SS2.

ABSTRACTClustered regularly interspaced palindromic repeats (CRISPR) and their associated casgenes have been demonstrated to regulate self-genes and virulence in many pathogens. In this study, we found that inactivation of cas9caused reduced adhesion and intracellular survival of the piscine Streptococcus agalactiaestrain GD201008-001 and significantly decreased the virulence of this strain in zebrafish and mice. Further investigation indicated that the regRtranscriptional regulator was upregulated in the Δcas9mutant. As regRmediates the repression of hyaluronidase, a critical factor involved in opening the blood-brain barrier (BBB) in mice, cas9-mediated repression of regRtranscription is important for S. agalactiaeto open the BBB and thereby cause meningitis in animals. This study expands our understanding of endogenous gene regulation mediated by CRISPR-Cas systems in bacteria.