Xiangru Wang

Effect of the glycosyltransferases on the capsular polysaccharide synthesis of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (S. suis 2) is a serious zoonotic pathogen causing septicemia and meningitis in piglets and humans. The capsular polysaccharide (CPS) is an essential virulence factor for S. suis 2 to infect the host. The synthesis of CPS repeating units involves multiple glycosyltransferases. In this study, four genes (cps2E, cps2G, cps2J and cps2L) encoding different glycosyltransferases involved in CPS synthesis were researched in S. suis 2. Four deletion mutants (Δcps2E, Δcps2G, Δcps2J and Δcps2L) with their CPS incomplete and their sialic acid content significantly decreased were constructed in S. suis 2 SC19. All these four mutant strains showed enhanced adhesion to Hep-2 cells and increased sensitivity to phagocytosis. Flow cytometric analysis also revealed that these four mutants were more susceptible to the attack by the complement system. In a mouse model of infection, the mutant strains were rapidly cleared by the immune system, compared with the wild-type strain. In summary, this study characterized four genes (cps2E, cps2G, cps2J and cps2L) involved in CPS synthesis of S. suis 2 SC19 and it revealed that these genes were all crucial for SC19 to invade and survive in the host.

Induction of VEGFA and Snail-1 by meningitic Escherichia coli mediates disruption of the blood-brain barrier

Escherichia coli is the most common Gram-negative bacterium that possesses the ability to cause neonatal meningitis, which develops as circulating bacteria penetrate the blood-brain barrier (BBB). However, whether meningitic E. coli could induce disruption of the BBB and the underlying mechanisms are poorly understood. Our current work highlight for the first time the participation of VEGFA and Snail-1, as well as the potential mechanisms, in meningitic E. coli induced disruption of the BBB. Here, we characterized a meningitis-causing E. coli PCN033, and demonstrated that PCN033 invasion could increase the BBB permeability through downregulating and remodeling the tight junction proteins (TJ proteins). This process required the PCN033 infection-induced upregulation of VEGFA and Snail-1, which involves the activation of TLR2-MAPK-ERK1/2 signaling cascade. Moreover, production of proinflammatory cytokines and chemokines in response to infection also promoted the upregulation of VEGFA and Snail-1, therefore further mediating the BBB disruption. Our observations reported here directly support the involvement of VEGFA and Snail-1 in meningitic E. coli induced BBB disruption, and VEGFA and Snail-1 would therefore represent the essential host targets for future prevention of clinical E. coli meningitis.

Virulence determinants, antimicrobial susceptibility, and molecular profiles of Erysipelothrix rhusiopathiae strains isolated from China

The aim of this study was to understand the epidemiology, serotype, antibiotic sensitivity, and clonal structure of Erysipelothrix rhusiopathiae strains in China. Forty-eight strains were collected from seven provinces during the period from 2012 to 2013. Pulse-field electrophoresis identified 32 different patterns which were classified into clonal groups A–D. Most pulsed-field gel electrophoresis (PFGE) patterns were observed in clonal complex A and B, suggesting high diversity of genetic characterization in these two predominant clonal complexes. Antibiotic sensitivity test shows that all the stains were susceptible to ampicillin, erythromycin, and cefotaxime, and resistant to kanamycin, cefazolin, sulfadiazine, and amikacin. Erythromycin and ampicillin are recommended as first-line antibiotics for treatment of E. rhusiopathiae in China. The high variation in PFGE pattern among the main clonal groups shows that the E. rhusiopathiae in China may originate from different lineages and sources instead of from expansion of a single clonal lineage across different regions.

Plasmid-mediated multidrug resistance and virulence in an avian pathogenic Escherichia coli strain isolated in China

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colisepticaemia and has been previously reported as a potential zoonotic risk [1]. APEC strains often display a multidrug resistance pattern including resistance to aminoglycosides, blactams, chloramphenicol, fluoroquinolones, tetracyclines and trimethoprim/sulfamethoxazole [2]. In this study, an APEC strain (ACN001) isolated from the liver of a diseased chicken in China in 2010 was characterised. ACN001 was shown to belong to phylogenetic group B2 and was confirmed to be highly virulent in chicken and mouse models using previously described methods [3–5]. Antimicrobial susceptibility testing performed according to Clinical and Laboratory Standards Institute (CLSI) standards [6] revealed that the strain was multidrug-resistant, with high minimum inhibitory concentrations to amikacin (>16 mg/mL), ampicillin (256 mg/mL), chloramphenicol (256 mg/mL), ciprofloxacin (64 mg/mL), florfenicol (256 mg/mL), gentamicin (256 mg/ mL), norfloxacin (64 mg/mL), streptomycin (256 mg/mL) and tetracycline (256 mg/mL). To investigate further whether these resistance and virulence phenotypes were plasmid-mediated, the draft whole genome of ACN001 was sequenced using a Roche 454 GS-FLX Sequencer (Roche, Basel, Switzerland), and all contigs were analysed using Cytoscape software v.2.8.2 (http://www.cytoscape.org/). The predicted plasmid-related contigs were all BLASTed with the National Center for Biotechnology Information (NCBI) database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm their accuracy. The gaps between these contigs were filled by sequencing PCR products and were assembled using Newbler v.2.3 (454 Life Sciences, Branford, CT). A total of six plasmids were obtained (pACN001-A, 60 043 bp; pACN001-B, 168 543 bp; pACN001-C, 5784 bp; pACN001-D, 6747 bp; pACN001-E, 6822 bp; pACN001F, 92 447 bp). The sequences were analysed using Glimmer 3.0

Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli

Accumulating studies have indicated the influence of long non-coding RNAs (lncRNAs) on various biological processes as well as disease development and progression. However, the lncRNAs involved in bacterial meningitis and their regulatory effects are largely unknown. By RNA-sequencing, the transcriptional profiles of host lncRNAs in primary human brain microvascular endothelial cells (hBMECs) in response to meningitic Escherichia coli were demonstrated. Here, 25,257 lncRNAs were identified, including 24,645 annotated lncRNAs and 612 newly found ones. A total of 895 lncRNAs exhibited significant differences upon infection, among which 382 were upregulated and 513 were downregulated (≥2-fold, p < 0.05). Via bioinformatic analysis, the features of these lncRNAs, their possible functions, and the potential regulatory relationships between lncRNAs and mRNAs were predicted. Moreover, we compared the transcriptional specificity of these differential lncRNAs among hBMECs, human astrocyte cell U251, and human umbilical vein endothelial cells, and demonstrated the novel regulatory effects of proinflammatory cytokines on these differential lncRNAs. To our knowledge, this is the first time the transcriptional profiles of host lncRNAs involved in E. coli-induced meningitis have been reported, which shall provide novel insight into the regulatory mechanisms behind bacterial meningitis involving lncRNAs, and contribute to better prevention and therapy of CNS infection.

EGFR transactivation contributes to neuroinflammation in Streptococcus suis meningitis

BackgroundStreptococcus suis serotype 2 (SS2) is an important zoonotic bacterial pathogen in both humans and animals, which can cause high morbidity and mortality. Meningitis is one of the major clinical manifestations of SS2 infection. However, the specific process of SS2 meningitis and its molecular mechanisms remain unclear. Epidermal growth factor receptor (EGFR) has been reported to initiate transduction of intracellular signals and regulate host inflammatory responses. Whether and how EGFR contributes to the development of S. suis meningitis are currently unknown.MethodsThe tyrosine phosphorylation of cellular proteins, the transactivation of EGFR, as well as its dimerization, and the associated signal transduction pathways were investigated by immunoprecipitation and western blotting. Real-time quantitative PCR was used to investigate the transcriptional level of the ErbB family members, EGFR-related ligands, cytokines, and chemokines. The secretion of cytokines and chemokines in the serum and brain were detected by Q-Plex™ Chemiluminescent ELISA.ResultsWe found an important role of EGFR in SS2 strain SC19-induced meningitis. SC19 increasingly adhered to human brain microvascular endothelial cells (hBMEC) and caused inflammatory lesions in the brain tissues, with significant induction and secretion of proinflammatory cytokines and chemokines in the serum and brains. SC19 infection of hBMEC induced tyrosine phosphorylation of cellular EGFR in a ligand-dependent manner involving the EGF-like ligand HB-EGF, amphiregulin (AREG), and epiregulin (EREG) and led to heterodimerization of EGFR/ErbB3. The EGFR transactivation did not participate in SS2 strain SC19 adhesion of hBMEC, as well as in bacterial colonization in vivo. However, its transactivation contributed to the bacterial-induced neuroinflammation, via triggering the MAPK-ERK1/2 and NF-κB signaling pathways in hBMEC that promote the production of proinflammatory cytokines and chemokines.ConclusionsWe investigated for the first time the tyrosine phosphorylation of cellular proteins in response to SS2 strain SC19 infection of hBMEC and demonstrated the contribution of EGFR to SS2-induced neuroinflammation. These observations propose a novel mechanism involving EGFR in SS2-mediated inflammatory responses in the brain, and therefore, EGFR might be an important host target for further investigation and prevention of neuroinflammation caused by SS2 strains.

Characterization and distinction of two flagellar systems in extraintestinal pathogenic Escherichia coli PCN033

Extraintestinal pathogenic Escherichia coli (ExPEC) can invade and colonize multiple extraintestinal tissues and can cause a wide range of infections; however the mechanisms of its pathogenicity are not well understood. Flagella contribute to the infection of E. coli strains by mediating adhesion and invasion. Our previous bioinformatic analysis revealed two flagella gene clusters in the genome of an ExPEC isolate, PCN033. One encodes the conventional flagellum system (Flag-1) and the other encodes the Flag-2 system, whose function is uncharacterized. Here we aimed to characterize these two flagellum systems and determine their contributions to the flagellum formation and certain pathogenicity-associated phenotypes. Our observations support the involvement of Flag-1 system, but not Flag-2 system, in the synthesis and maturation of the flagellum structure, and in mediating bacterial swimming and swarming. Moreover, flgD, which encodes a flagellar-hook scaffolding protein in the Flag-1 system, is required for flagellum assembly by influencing the production of FliC (flagellin). Deletion of flgD attenuated ExPEC strain PCN033 invasion and colonization in vivo, probably by affecting bacterial adhesion and invasion, and by reducing resistance to phagocytosis by circulating monocytes. In contrast, these phenotypes were not observed in the strain with deletion of lfgD, encoding the FlgD-like protein in the Flag-2 system. Taken together, these findings indicate that Flag-1 flagellum system is the determinative component of bacterial flagella that contributes to the infection.

Roles of Hcp family proteins in the pathogenesis of the porcine extraintestinal pathogenic Escherichia coli type VI secretion system.

Hcp (hemolysin-coregulated protein) is considered a vital component of the functional T6SS (Type VI Secretion System), which is a newly discovered secretion system. Our laboratory has previously sequenced the whole genome of porcine extraintestinal pathogenic E. coli (ExPEC) strain PCN033, and identified an integrated T6SS encoding three different hcp family genes. In this study, we first identified a functional T6SS in porcine ExPEC strain PCN033, and demonstrated that the Hcp family proteins were involved in bacterial competition and the interactions with other cells. Interestingly, the three Hcp proteins had different functions. Hcp2 functioned predominantly in bacterial competition; all three proteins were involved in the colonization of mice; and Hcp1 and Hcp3 were predominantly contributed to bacterial-eukaryotic cell interactions. We showed an active T6SS in porcine ExPEC strain PCN033, and the Hcp family proteins had different functions in their interaction with other bacteria or host cells.

Haemophilus parasuis CpxRA two-component system confers bacterial tolerance to environmental stresses and macrolide resistance

Haemophilus parasuis is an opportunistic pathogen localized in the upper respiratory tracts of pigs, its infection begins from bacterial survival under complex conditions, like hyperosmosis, oxidative stress, phagocytosis, and sometimes antibiotics as well. The two-component signal transduction (TCST) system serves as a common stimulus-response mechanism that allows microbes to sense and respond to diverse environmental conditions via a series of phosphorylation reactions. In this study, we investigated the role of TCST system CpxRA in H. parasuis in response to different environmental stimuli by constructing the ΔcpxA and ΔcpxR single deletion mutants as well as the ΔcpxRA double deletion mutant from H. parasuis serotype 4 isolate JS0135. We demonstrated that H. parasuis TCST system CpxRA confers bacterial tolerance to stresses and bactericidal antibiotics. The CpxR was found to play essential roles in mediating oxidative stress, osmotic stresses and alkaline pH stress tolerance, as well as macrolide resistance (i.e. erythromycin), but the CpxA deletion did not decrease bacterial resistance to abovementioned stresses. Moreover, we found via RT-qPCR approach that HAPS_RS00160 and HAPS_RS09425, both encoding multidrug efflux pumps, were significantly decreased in erythromycin challenged ΔcpxR and ΔcpxRA mutants compared with wild-type strain JS0135. These findings characterize the role of the TCST system CpxRA in H. parasuis conferring stress response tolerance and bactericidal resistance, which will deepen our understanding of the pathogenic mechanism in H. parasuis.

Binding of Fibronectin to SsPepO Facilitates the Development of Streptococcus suis Meningitis

Background SsPepO is an important virulence in Streptococcus suis. Methods In this study, we showed that SsPepO contributes to the human fibronectin-mediated adherence ability of S. suis to human brain microvascular endothelial cells. Results The addition of an antifibronectin antibody or an arginine-glycine-aspartic acid peptide that blocks fibronectin binding to integrins significantly reduced adherence of the wild-type but not the SspepO mutant strain, indicating the importance of the SsPepO-fibronectin-integrin interaction for S. suis cellular adherence. Conclusions By analyzing Evans blue extravasation in vivo, we showed that the interaction between SsPepO and human fibronectin significantly increased permeability of the blood-brain barrier. Furthermore, the SspepO mutant caused lower bacterial loads in the brain than wild-type S. suis in models of meningitis. These data demonstrate that SsPepO is a fibronectin-binding protein, which plays a contributing role in the development of S. suis meningitis.