Shengqing Yu

OmpA is a virulence factor of Riemerella anatipestifer

Riemerella anatipestifer infection is probably the most economically important disease of farm ducks worldwide. The pathogen R. anatipestifer causes septicemia anserum exsudativa in ducks, but little is known about the molecular basis of its pathogenesis and the virulence factors involved. In this study, by deleting ompA gene from R. anatipestifer serotype 2 strain Th4, we constructed a mutant strain Th4ΔompA to investigate whether R. anatipestifer OmpA is an important virulence factor. Results showed that although the growth curve, bacterial and colony morphology of Th4ΔompA in tryptic soybean broth (TSB) or on TSB agar were similar to its parent strain Th4, the adhesion and invasion capacities of mutant strain to Vero cells were decreased significantly. Furthermore, the median lethal dose (LD(50)) of both strains was determined to measure the virulence with 10-day-old Cherry Valley ducklings. The results showed that LD(50) of Th4ΔompA mutant was >10(10) colony forming units (CFU), it was attenuated significantly in comparison with that of Th4 which LD(50) was 4.41 × 10(8) CFU. Additional analysis indicated that blood bacterial loading of ducklings infected with the Th4ΔompA mutant were much lower than those of Th4-infected ducklings. The results demonstrate that OmpA is a virulence factor of R. anatipestifer, and that it may act as an adhesin.

Characterization of biofilm formation by Riemerella anatipestifer.

Riemerella anatipestifer (RA) causes epizootics of infectious disease in poultry and results in serious economic losses, especially for the duck industry. The present study focuses on understanding the biofilm-producing ability of RA strains in attempt to explain the intriguing persistence of RA post-infection on duck farms. Four RA serotype reference strains and 39 field RA isolates were measured for the biofilm formation by crystal violet staining. Eighteen out of the 43 RA strains produced biofilms. Furthermore, RA isolate CH3 was treated with carbohydrates (sucrose; glucose), disodium EDTA (EDTA), antibiotics (ampicillin; chloramphenicol) or detergent (Triton X-100) to determine the effect of the treatments on biofilm formation. Biofilm formation by RA isolate CH3 was independent of sucrose but significantly inhibited by 5% glucose and 0.1 mmol/L EDTA. Biofilmed CH3 culture (CH3 grown with a biofilm) was 5-31 times more resistant to the treatments of ampicillin, chloramphenicol or Triton X-100 than planktonic CH3 culture on the basis of minimal inhibitory concentration and minimal bactericidal concentration. The development and architecture of the biofilm formed by CH3 were also assessed using confocal laser scanning microscopy, scanning electron microscopy and fluorescence microscopy. In addition, animal experiment was performed to determine the median lethal doses (LD(50)) of three RA isolates with different biofilm formation abilities. Despite the result that virulence is strain-dependent as a result of various factors other than biofilm-producing ability, the fact that biofilmed isolate is more resistant to antibiotic and detergent treatments than planktonic isolate suggest that biofilm formation by RA may contribute to the persistent infections on duck farms.

Newcastle disease virus triggers autophagy in U251 glioma cells to enhance virus replication

Newcastle disease virus (NDV) can replicate in tumor cells and induce apoptosis in late stages of infection. However, the interaction between NDV and cells in early stages of infection is not well understood. Here, we report that, shortly after infection, NDV triggers the formation of autophagosomes in U251 glioma cells, as demonstrated by an increased number of double-membrane vesicles, GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) a dot formations, and elevated production of LC3II. Moreover, modulation of NDV-induced autophagy by rapamycin, chloroquine or small interfering RNAs targeting the genes critical for autophagosome formation (Atg5 and Beclin-1) affects virus production, indicating that autophagy may be utilized by NDV to facilitate its own production. Furthermore, the class III phosphatidylinositol 3-kinase (PI3K)/Beclin-1 pathway plays a role in NDV-induced autophagy and virus production. Collectively, our data provide a unique example of a paramyxovirus that uses autophagy to enhance its production.

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

ABSTRACT Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.

Identification of the Genes Involved in Riemerella anatipestifer Biofilm Formation by Random Transposon Mutagenesis

Riemerella anatipestifer causes epizootics of infectious disease in poultry that result in serious economic losses to the duck industry. Our previous studies have shown that some strains of R. anatipestifer can form a biofilm, and this may explain the intriguing persistence of R. anatipestifer on duck farms post infection. In this study we used strain CH3, a strong producer of biofilm, to construct a library of random Tn4351 transposon mutants in order to investigate the genetic basis of biofilm formation by R. anatipestifer on abiotic surfaces. A total of 2,520 mutants were obtained and 39 of them showed a reduction in biofilm formation of 47%–98% using crystal violet staining. Genetic characterization of the mutants led to the identification of 33 genes. Of these, 29 genes are associated with information storage and processing, as well as basic cellular processes and metabolism; the function of the other four genes is currently unknown. In addition, a mutant strain BF19, in which biofilm formation was reduced by 98% following insertion of the Tn4351 transposon at the dihydrodipicolinate synthase (dhdps) gene, was complemented with a shuttle plasmid pCP-dhdps. The complemented mutant strain was restored to give 92.6% of the biofilm formation of the wild-type strain CH3, which indicates that the dhdp gene is associated with biofilm formation. It is inferred that such complementation applies also to other mutant strains. Furthermore, some biological characteristics of biofilm-defective mutants were investigated, indicating that the genes deleted in the mutant strains function in the biofilm formation of R. anatipestifer. Deletion of either gene will stall the biofilm formation at a specific stage thus preventing further biofilm development. In addition, the tested biofilm-defective mutants had different adherence capacity to Vero cells. This study will help us to understand the molecular mechanisms of biofilm development by R. anatipestifer and to study the pathogenesis of R. anatipestifer further.

Activation of the PKR/eIF2α signaling cascade inhibits replication of Newcastle disease virus

BackgroundNewcastle Disease virus (NDV) causes severe and economically significant disease in almost all birds. However, factors that affect NDV replication in host cells are poorly understood. NDV generates long double-stranded RNA (dsRNA) molecules during transcription of single-stranded genomic RNA. Protein kinase R (PKR) is activated by dsRNA. The aim of this study was to elucidate the role of PKR in NDV infection.ResultsNDV infection led to the activation of dsRNA-dependent PKR and phosphorylation of its substrate, translation initiation factor eIF2α, in a dose-dependent manner by either the lentogenic strain LaSota or a velogenic strain Herts/33. PKR activation coincided with the accumulation of dsRNA induced by NDV infection. PKR knockdown remarkably decreased eIF2α phosphorylation as well as IFN-β mRNA levels, leading to the augmentation of extracellular virus titer. Furthermore, siRNA knockdown or phosphorylation of eIF2α or okadaic acid treatment significantly impaired NDV replication, indicating the critical role of the PKR/eIF2α signaling cascade in NDV infection.ConclusionPKR is activated by dsRNA generated by NDV infection and inhibits NDV replication by eIF2α phosphorylation. This study provides insight into NDV-host interactions for the development of candidate antiviral strategies.

Immunoproteomics analysis of whole cell bacterial proteins of Riemerella anatipestifer

Riemerella antipestifer is one of the most important duck pathogens. It has worldwide distribution, and the lack of the information on bacteria-host interactions and an effective vaccine are limitations on the control of this infection. In this study, an immunoproteomic assay was used to identify immunogenic proteins among the whole cell bacterial proteins of R. anatipestifer virulent strain Th4. Duck antiserum against R. anatipestifer Th4 recognized 64 protein spots which were transferred from two-dimensional electrophoresis (2-DE) gel of the whole cell bacterial proteins onto polyvinylidene fluoride (PVDF) membrane. Immunogenic proteins on a duplicate gel were excised and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprinting (PMF), a total of 34 immunogenic proteins were found. With the exception of OmpA and GroEL, the other 32 proteins were newly recognized immunogenic antigens of R. anatipestifer. In addition, TonB-dependent outer membrane receptor was found to be a cross immunogenic antigen among serotypes 1, 2 and 10 of R. anatipestifer. Bioinformatics analysis showed that most of the immunogenic proteins were located in the outer membrane and cytoplasm, and were involved in cellular processes and metabolism. The newly identified immunogenic proteins of R. anatipestifer may help us to uncover the pathogenesis of the bacteria, develop novel vaccine candidates and serological diagnosis marker.

Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks

Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10(3) colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.

Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein.

BackgroundMycoplasma synoviae is an avian pathogen that can lead to respiratory tract infections and arthritis in chickens and turkeys, resulting in serious economic losses to the poultry industry. Enolase reportedly plays important roles in several bacterial pathogens, but its role in M. synoviae has not been established. Therefore, in this study, the enolase encoding gene (eno) of M. synoviae was amplified from strain WVU1853 and expressed in E. coli BL21 cells. Then the enzymatic activity, immunogenicity and binding activity with chicken plasminogen (Plg) and human fibronectin (Fn) was evaluated.ResultsWe demonstrated that the recombinant M. synoviae enolase protein (rMsEno) can catalyze the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), the Km and Vmax values of rMsEno were 1.1 × 10-3 M and 0.739 μmol/L/min, respectively. Western blot and immuno-electron microscopy analyses confirmed that enolase was distributed on the surface and within the cytoplasm of M. synoviae cells. The binding assays demonstrated that rMsEno was able to bind to chicken Plg and human Fn proteins. A complement-dependent mycoplasmacidal assay demonstrated that rabbit anti-rMsEno serum had distinct mycoplasmacidal efficacy in the presence of complement, which also confirmed that enolase was distributed on the surface of M. synoviae. An inhibition assay showed that the adherence of M. synoviae to DF-1 cells pre-treated with Plg could be effectively inhibited by treatment with rabbit anti-rMsEno serum.ConclusionThese results reveal that M. synoviae enolase has good catalytic activity for conversion of 2-PGA to PEP, and binding activity with chicken Plg and human Fn. Rabbit anti-rMsEno serum displayed an obvious complement-dependent mycoplasmacidal effect and adherent inhibition effect. These results suggested that the M. synoviae enolase plays an important role in M. synoviae metabolism, and could potentially impact M. synoviae infection and immunity.

Inactivation of the ABC transporter ATPase gene in Brucella abortus strain 2308 attenuated the virulence of the bacteria

Brucella abortus is a Gram-negative, facultative intracellular bacterial pathogen of human and other animals. Brucella lipopolysaccharide has been identified as an important virulence factor. In this study, the ABC transporter ATPase gene (BAB1_0542) of B. abortus strain S2308 was inactivated by deleting a 446-bp fragment from the gene, thereby generating the mutant strain, S2308ΔATP. Real time PCR analysis confirmed the inactivation of this gene with no polar effect on the transcription of adjacent genes on the chromosome. The mutant was identified as a rough phenotype strain using heat agglutination test and crystal violet staining. The mutant strain had a different growth rate in Tryptic Soy Broth (TSB), compared to the wild type S2308 strain. Moreover, the mutant strain showed attenuated virulence in vitro and in vivo in RAW264.7 macrophages and Balb/c mice, respectively. Complementation of the mutant strain recovered the smooth phenotype of the bacteria and the complemented strain C2308ΔATP survived for more than four weeks in Balb/c mice, comparable to wild type strain S2308. Furthermore, immunization with the mutant strain protected mice from virulent strain challenge, which suggests the potential for the mutant strain S2308ΔATP as a future vaccine candidate. MHC I, MHC II and co-stimulatory molecule expression levels in mice following infection of S2308ΔATP and S2308 were also investigated.