Biotin is essential for the growth and pathogenicity of microorganisms. Damage to biotin biosynthesis results in impaired bacterial growth and decreased virulence in vivo. However, the mechanisms of biotin biosynthesis in Riemerella anatipestifer remain unclear. In this study, two R. anatipestifer genes associated with biotin biosynthesis were identified. AS87_RS05840 encoded a BirA protein lacking the N-terminal winged helix-turn-helix DNA binding domain, identifying it as a group I biotin protein ligase, and AS87_RS09325 encoded a BioX protein, which was in the helix-turn-helix xenobiotic response element family of transcription factors. Electrophoretic mobility shift assays demonstrated that BioX bound to the promoter region of bioF. In addition, the R. anatipestifer genes bioF (encoding 7-keto-8-aminopelargonic acid synthase), bioD (encoding dethiobiotin synthase), and bioA (encoding 7,8-diaminopelargonic acid synthase) were in an operon and were regulated by BioX. Quantitative reverse transcription-PCR showed that transcription of the bioFDA operon increased in the mutant Yb2ΔbioX in the presence of excessive biotin, compared with that in the wild-type strain Yb2, suggesting that BioX acted as a repressor of biotin biosynthesis. Streptavidin blot analysis showed that BirA caused biotinylation of BioX, indicating that biotinylated BioX was involved in metabolic pathways. Moreover, as determined by the median lethal dose, the virulence of Yb2ΔbioX was attenuated 500-fold compared with that of Yb2. To summarize, the genes birA and bioX were identified in R. anatipestifer, and BioX was found to act as a repressor of the bioFDA operon involved in the biotin biosynthesis pathway and identified as a bacterial virulence factor. IMPORTANCE Riemerella anatipestifer is a causative agent of diseases in ducks, geese, turkeys, and various other domestic and wild birds. Our study reveals that biotin synthesis of R. anatipestifer is regulated by the BioX through binding to the promoter region of the bioF gene to inhibit transcription of the bioFDA operon. Moreover, bioX is required for R. anatipestifer pathogenicity, suggesting that BioX is a potential target for treatment of the pathogen. R. anatipestifer BioX has thus been identified as a novel negative regulator involved in biotin metabolism and associated with bacterial virulence in this study.
Newcastle disease virus (NDV) causes a highly devastating and contagious disease in poultry, which is mainly attributed to extensive tissue damages in the digestive, respiratory and nervous systems. However, nature and dynamics of NDV-induced oxidative stresses in the intestine of chickens remain elusive.In this study, we examined the magnitude of intestinal oxidative stress and histopathological changes caused by the virulent NDV infection, and explored the protective roles of vitamin E (vit. E) in ameliorating these pathological changes. For these purposes, chickens were divided into four groups namely i) non supplemented and non-challenged (negative control, CON); ii) no supplementation of vit. E but challenged with ZJ1 (positive control, NS+CHA); iii) vit. E supplementation at the dose of 50 IU/day/Kg body weight and ZJ1 challenge (VE50+CHA); and 4) vit. E supplementation at the dose of 100 IU/day/Kg body weight and ZJ1 challenge (VE100+CHA). In all groups, we analyzed concentrations of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T-AOC), and activity of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) using biochemical methods. The virus loads were determined by quantitative RT-PCR and antibody titers by hemagglutination inhibition assays. We also examined the histopathological changes in the duodenal and jejunal mucosa at 3 and 5-day post infection (dpi) with NDV.A significant elevation in the NO level was observed in NDV challenged chickens compared to the CON chickens at 2 dpi. The MDA contents were significantly increased whereas GSH was significantly decreased in NDV-challenged chickens compared to control. Furthermore, activities of GST, CAT, SOD, as well as the TOAC were markedly decreased in challenged chickens in comparison with control. Virus copy numbers were higher in NDV infected NS+CHA group compared to other groups. Severe histopathological changes including inflammation, degeneration and broken villi were observed in the intestine of NDV challenged chickens. However, all these malfunctions of antioxidant system and pathological changes in the intestine were partially or completely reversed by the vit. E supplementation.Our results suggest that NDV infection causes oxidative stress and histopathological changes in the duodenum and jejunum of chickens, which can be partially or fully ameliorated by supplementation of vit. E. Additionally, these findings suggest that oxidative stress contributes to the intestinal damages in NDV infected chickens. These findings will help to understand the pathogenesis of NDV and further investigation of therapeutic agents for control of Newcastle disease.
Newcastle disease (ND) is an acute, febrile, highly contagious disease caused by the virulent Newcastle disease virus (vNDV). The disease causes serious economic losses to the poultry industry. However, the metabolic changes caused by vNDV infection remain unclear. The objective of this study was to determine the metabolomic profiling after infection with vNDV. DF-1 cells infected with the vNDV strain Herts/33 and the lungs from Herts/33-infected specific pathogen-free (SPF) chickens were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 305 metabolites were found to have changed significantly after Herts/33 infection, and most of them belong to the amino acid and nucleotide metabolic pathway. It is suggested that the increased pools of amino acids and nucleotides may benefit viral protein synthesis and genome amplification to promote NDV infection. Similar results were also confirmed in vivo. Identification of these metabolites will provide information to further understand the mechanism of vNDV replication and pathogenesis.
Brucellosis is a zoonotic bacterial disease caused by Brucella spp. The virulence of these bacteria is dependent on their ability to invade and replicate within host cells. In a previous study, a putative gene bab_RS27735 encoding an amino acid ABC transporter substrate-binding protein homologous to AapJ protein was found to be involved in Brucella abortus virulence. In this study, we successfully constructed a bab_RS27735 deletion mutant, Δ27735. Compared with the wild-type strain, the lipopolysaccharide pattern of the mutant was not changed, but the growth ability was slightly defected in the exponential phase. In tolerance tests, sensitivity of the Δ27735 mutant to oxidative stress, bactericidal peptides or low pH was not different from that of the wild-type strain. Cell infection assay showed that the mutant was reduced survival within macrophages but could efficiently escape lysosome degradation. The results of a virulence test showed that the Δ27735 mutant was attenuated in a mouse model at the early stage of infection but recovered its virulence at the late stage of infection. Meanwhile, the development of splenomegaly and histopathological lesions was observed in mice infected with either the wild-type strain or the mutant. These results are in line with the release of IL-12p40 and TNF-α into the peripheral blood of infected mice. Besides, expression of diverse genes was up-regulated in the Δ27735 mutant, which may contribute to the reduced virulence of the mutant. These data elucidated that the bab_RS27735 gene is necessary for B. abortus virulence at the early stage of infection in a mouse model.
Pathogens could precisely alter their gene expression to facilitate their survival and successful infection. The LuxR family transcriptional regulator DctR (also known as YhiF) was shown to participate in the regulation of acid fitness and adhesion of enterohemorrhagic E. coli (EHEC) O157:H7. Avian pathogenic Escherichia coli (APEC) causes significant economic losses to the poultry industries and also potentially threatens human health. However, the effects of DctR on the fitness and virulence of APEC have not been investigated yet. To assess the function of DctR in APEC, the dctR gene mutant and complemented strains were constructed and biologically characterized. Our results show that inactivation of the dctR gene led to decreased biofilm formation, diminished serum resistance, reduced adherence capacity, attenuated colonization and virulence of APEC in ducks. The altered capacities of the mutant strain were restored by genetic complementation. In addition, we found that DctR positively regulates the expression of E. coli type III secretion system 2 (ETT2) core genes in APEC. The expression of the inflammatory cytokines interleukin (IL)-1β and IL-8 were decreased in HD-11 macrophages infected with the mutant strain compared with the wild-type strain. These observations indicate that regulator DctR contributes to the virulence of APEC through regulation of ETT2 expression.
The chicken is a model animal for the study of evolution, immunity and development. In addition to their use as a model organism, chickens also represent an important agricultural product. Pathogen invasion has already been shown to modulate the expression of hundreds of genes, but the role of alternative splicing in avian virus infection remains unclear. We used RNA-seq data to analyze virus-induced changes in the alternative splicing of Gallus gallus, and found that a large number of alternative splicing events were induced by virus infection both in vivo and in vitro. Virus-responsive alternative splicing events preferentially occurred in genes involved in metabolism and transport. Many of the alternatively spliced transcripts were also expressed from genes with a function relating to splicing or immune response, suggesting a potential impact of virus infection on pre-mRNA splicing and immune gene regulation. Moreover, exon skipping was the most frequent AS event in chickens during virus infection. This is the first report describing a genome-wide analysis of alternative splicing in chicken and contributes to the genomic resources available for studying host-virus interaction in this species. Our analysis fills an important knowledge gap in understanding the extent of genome-wide alternative splicing dynamics occurring during avian virus infection and provides the impetus for the further exploration of AS in chicken defense signaling and homeostasis.
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