Zhuofei Xu

Genome biology of Actinobacillus pleuropneumoniae JL03, an isolate of serotype 3 prevalent in China.

Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a cause of considerable world wide economic losses in the swine industry. We sequenced the complete genome of A. pleuropneumoniae, JL03, an isolate of serotype 3 prevalent in China. Its genome is a single chromosome of 2,242,062 base pairs containing 2,097 predicted protein-coding sequences, six ribosomal rRNA operons, and 63 tRNA genes. Preliminary analysis of the genomic sequence and the functions of the encoded proteins not only confirmed the present physiological and pathological knowledge but also offered new insights into the metabolic and virulence characteristics of this important pathogen. We identified a full spectrum of genes related to its characteristic chemoheterotrophic catabolism of fermentation and respiration with an incomplete TCA system for anabolism. In addition to confirming the lack of ApxI toxin, identification of a nonsense mutation in apxIVA and a 5′-proximal truncation of the flp operon deleting both its promoter and the flp1flp2tadV genes have provided convincing scenarios for the low virulence property of JL03. Comparative genomic analysis using the available sequences of other serotypes, probable strain (serotype)-specific genomic islands related to capsular polysaccharides and lipopolysaccharide O-antigen biosyntheses were identified in JL03, which provides a foundation for future research into the mechanisms of serotypic diversity of A. pleuropneumoniae.

Genomic characterization of Haemophilus parasuis SH0165, a highly virulent strain of serovar 5 prevalent in China.

Haemophilus parasuis can be either a commensal bacterium of the porcine respiratory tract or an opportunistic pathogen causing Glässers disease, a severe systemic disease that has led to significant economical losses in the pig industry worldwide. We determined the complete genomic sequence of H. parasuis SH0165, a highly virulent strain of serovar 5, which was isolated from a hog pen in North China. The single circular chromosome was 2,269,156 base pairs in length and contained 2,031 protein-coding genes. Together with the full spectrum of genes detected by the analysis of metabolic pathways, we confirmed that H. parasuis generates ATP via both fermentation and respiration, and possesses an intact TCA cycle for anabolism. In addition to possessing the complete pathway essential for the biosynthesis of heme, this pathogen was also found to be well-equipped with different iron acquisition systems, such as the TonB system and ABC-type transport complexes, to overcome iron limitation during infection and persistence. We identified a number of genes encoding potential virulence factors, such as type IV fimbriae and surface polysaccharides. Analysis of the genome confirmed that H. parasuis is naturally competent, as genes related to DNA uptake are present. A nine-mer DNA uptake signal sequence (ACAAGCGGT), identical to that found in Actinobacillus pleuropneumoniae and Mannheimia haemolytica, followed by similar downstream motifs, was identified in the SH0165 genome. Genomic and phylogenetic comparisons with other Pasteurellaceae species further indicated that H. parasuis was closely related to another swine pathogenic bacteria A. pleuropneumoniae. The comprehensive genetic analysis presented here provides a foundation for future research on the metabolism, natural competence and virulence of H. parasuis.

Enhanced biofilm formation and reduced virulence of Actinobacillus pleuropneumoniae luxS mutant.

Actinobacillus pleuropneumoniae is the pathogen of porcine contagious pleuropneumonia causing great economic losses globally. LuxS is known to be involved in the interspecies quorum sensing by mediating the production of AI-2 signal molecule and play important roles in regulating various behaviors of bacteria. In this study, A. pleuropneumoniae was tested to be able to produce AI-2 like molecule. A luxS deletion mutant strain was constructed by homologous replacement, and its growth features, biofilm formation and virulence were characterized. The luxS mutant showed obvious growth deficiency when cultured in the serum-free medium. Biofilm formation of the mutant strain was significantly enhanced. In a mouse infection model, the 50% lethal dose of the mutant strain was increased up to 96-folds, and the ability to colonize in different mouse tissues was significantly decreased. Down-regulation of apxIIA, a major virulence factor of A. pleuropneumoniae, may contribute to the virulence attenuation of the mutant strain. Our data demonstrated that LuxS is functional in the AI-2-mediated quorum sensing in A. pleuropneumoniae and LuxS may regulate different behaviors including biofilm formation and virulence of this important pathogen.

Comparative Genomic Characterization of Actinobacillus pleuropneumoniae

The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13 were sequenced by using rapid high-throughput approach. Based on 12 genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b, and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes, and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O antigen relative to serovar diversity were compared, and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.

Contribution of glutamine synthetase to the virulence of Streptococcus suis serotype 2

Streptococcus suis serotype 2 (S. suis 2) is an important pathogen, responsible for diverse diseases in swine and human. In this study, we investigated the role of the glutamine synthetase (GlnA) in the pathogenesis of S. suis 2 in mice. To assess the contribution of glutamine synthetase (GlnA) to the virulence of S. suis 2, an knockout mutant (DeltaglnA) unable to produce GlnA was constructed, and the virulence level of wild-type (WT) SC19 and the DeltaglnA mutant strain were compared in an in vitro adherence assay and murine infection models. The data showed that DeltaglnA mutant exhibited a significant decrease in adherence to the epithelial cells HEp-2. The DeltaglnA mutant strain was attenuated and could reduce mortality and morbidity in murine infection models. Furthermore, organ cultures showed that GlnA plays a role in the colonization of the specific organs involved in S. suis infection. Functional complementation of the glnA gene into the knockout mutant DeltaglnA or incubated with extracellular glutamine restored its ability to adhere to the epithelial cells HEp-2. These findings suggested that glnA is required for the full virulence in S. suis 2. Therefore, the DeltaglnA mutant was considered as an attenuated mutant.

Analysis on Actinobacillus pleuropneumoniae LuxS regulated genes reveals pleiotropic roles of LuxS/AI-2 on biofilm formation, adhesion ability and iron metabolism.

LuxS is an enzyme involved in the activated methyl cycle and the by-product autoinducer-2 (AI-2) was a quorum sensing signal in some species. In our previous study, the functional LuxS in AI-2 production was verified in the porcine respiratory pathogen Actinobacillus pleuropneumoniae. Enhanced biofilm formation and reduced virulence were observed in the luxS mutant. To comprehensively understand the luxS function, in this study, the transcriptional profiles were compared between the A. pleuropneumoniae luxS mutant and its parental strain in four different growth phases using microarray. Many genes associated with infection were differentially expressed. The biofilm formation genes pgaABC in the luxS mutant were up-regulated in early exponential phase, while 9 genes associated with adhesion were down-regulated in late exponential phase. A group of genes involved in iron acquisition and metabolism were regulated in four growth phases. Phenotypic investigations using luxS mutant and both genetic and chemical (AI-2) complementation on these virulence traits were performed. The results demonstrated that the luxS mutant showed enhanced biofilm formation and reduced adhesion ability and these effects were not due to lack of AI-2. But AI-2 could increase biofilm formation and adhesion of A. pleuropneumoniae independent of LuxS. Growth under iron restricted condition could be controlled by LuxS through AI-2 production. These results revealed pleiotropic roles of LuxS and AI-2 on A. pleuropneumoniae virulence traits.

Global Effects of Catecholamines on Actinobacillus pleuropneumoniae Gene Expression

Bacteria can use mammalian hormones to modulate pathogenic processes that play essential roles in disease development. Actinobacillus pleuropneumoniae is an important porcine respiratory pathogen causing great economic losses in the pig industry globally. Stress is known to contribute to the outcome of A. pleuropneumoniae infection. To test whether A. pleuropneumoniae could respond to stress hormone catecholamines, gene expression profiles after epinephrine (Epi) and norepinephrine (NE) treatment were compared with those from untreated bacteria. The microarray results showed that 158 and 105 genes were differentially expressed in the presence of Epi and NE, respectively. These genes were assigned to various functional categories including many virulence factors. Only 18 genes were regulated by both hormones. These genes included apxIA (the ApxI toxin structural gene), pgaB (involved in biofilm formation), APL_0443 (an autotransporter adhesin) and genes encoding potential hormone receptors such as tyrP2, the ygiY-ygiX (qseC-qseB) operon and narQ-narP (involved in nitrate metabolism). Further investigations demonstrated that cytotoxic activity was enhanced by Epi but repressed by NE in accordance with apxIA gene expression changes. Biofilm formation was not affected by either of the two hormones despite pgaB expression being affected. Adhesion to host cells was induced by NE but not by Epi, suggesting that the hormones affect other putative adhesins in addition to APL_0443. This study revealed that A. pleuropneumoniae gene expression, including those encoding virulence factors, was altered in response to both catecholamines. The differential regulation of A. pleuropneumoniae gene expression by the two hormones suggests that this pathogen may have multiple responsive systems for the two catecholamines.

Complete Genome Sequence of Pasteurella multocida HN06, a Toxigenic Strain of Serogroup D

Pasteurella multocida is an important etiological agent that can cause many economically important diseases in a wide range of mammals and birds. Here, we report the complete genome sequence of P. multocida HN06, a toxigenic serogroup D strain isolated from a diseased pig in China.

Immunogenic characterization of outer membrane porins OmpC and OmpF of porcine extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen that can cause systemic infections in a broad spectrum of mammals and birds. To date, commercial vaccines against ExPEC infections in pigs are rare and antibiotic resistance has become a serious clinical problem. Identification of protective antigens is helpful for developing potentially effective vaccines. In this study, two outer membrane porins, OmpC and OmpF, of porcine ExPEC were cloned and expressed to investigate their immunogenicity. Intraperitoneal immunization of mice with the purified recombinant proteins OmpC and OmpF stimulated strong immunoglobulin G (IgG) antibody responses. Both IgG1 and IgG2a subclasses were induced, with a predominance of IgG1 production. After challenge with 2.5 × 10(7) CFU (5 × LD50 ) of the highly virulent ExPEC strain PCN033, 62.5% and 87.5% protection was observed in mice immunized with OmpC and OmpF, respectively. In addition, both anti-OmpC and anti-OmpF sera can mediate complement-dependent opsonophagocytosis. Phylogenetic analysis showed that the ompC gene was ubiquitously present in all E. coli strains, whereas the ompF gene was mutated in certain strains. Furthermore, the selection analysis indicated that gene ompC may be subject to strong immune pressure. Our results demonstrated that OmpC is a promising vaccine target against ExPEC infections in swine.

Genome-wide evidence for positive selection and recombination in Actinobacillus pleuropneumoniae

BackgroundActinobacillus pleuropneumoniae is an economically important animal pathogen that causes contagious pleuropneumonia in pigs. Currently, the molecular evolutionary trajectories for this pathogenic bacterium remain to require a better elucidation under the help of comparative genomics data. For this reason, we employed a comparative phylogenomic approach to obtain a comprehensive understanding of roles of natural selective pressure and homologous recombination during adaptation of this pathogen to its swine host.ResultsIn this study, 12 A. pleuropneumoniae genomes were used to carry out a phylogenomic analyses. We identified 1,587 orthologous core genes as an initial data set for the estimation of genetic recombination and positive selection. Based on the analyses of four recombination tests, 23% of the core genome of A. pleuropneumoniae showed strong signals for intragenic homologous recombination. Furthermore, the selection analyses indicated that 57 genes were undergoing significant positive selection. Extensive function properties underlying these positively selected genes demonstrated that genes coding for products relevant to bacterial surface structures and pathogenesis are prone to natural selective pressure, presumably due to their potential roles in the avoidance of the porcine immune system.ConclusionsOverall, substantial genetic evidence was shown to indicate that recombination and positive selection indeed play a crucial role in the adaptive evolution of A. pleuropneumoniae. The genome-wide profile of positively selected genes and/or amino acid residues will provide valuable targets for further research into the mechanisms of immune evasion and host-pathogen interactions for this serious swine pathogen.