Dataset Information

A Novel RAYM_RS09735/RAYM_RS09740 Two-Component Signaling System Regulates Gene Expression and Virulence in Riemerella anatipestifer.

ABSTRACT: The Gram-negative bacterium Riemerella anatipestifer is an important waterfowl pathogen, causing major economic losses to the duck-producing industry. However, little is known of the virulence factors that mediate pathogenesis during R. anatipestifer infection. In this study, RAYM_RS09735 and RAYM_RS09740 were predicted to form a two-component signaling system (TCS) through bioinformatics analysis. This TCS was highly conserved across the Flavobacteriaceae. A mutant YM?RS09735/RS09740 strain was constructed to investigate the role of the RAYM_RS09735/RAYM_RS09740 TCS in R. anatipestifer virulence and gene regulation. The median lethal dose (LD50) of YM?RS09735/RS09740 was found to be >1011 CFU, equivalent to that of avirulent bacterial strains. The bacterial abundances of the YM?RS09735/RS09740 strain in the heart, brain, liver, blood, and spleen were significantly lower than that of the wild-type R. anatipestifer YM strain. Pathological analysis using hematoxylin and eosin staining showed that, compared to the wild-type, the mutant YM?RS09735/RS09740 strain caused significantly less virulence in infected ducklings. RNAseq and real-time PCR analysis indicated that the RAYM_RS09735/RAYM_RS09740 TCS is a PhoP/PhoR system. This is a novel type of TCS for Gram-negative bacteria. The TCS was also found to be a global regulator of expression in R. anatipestifer, with 112 genes up-regulated and 693 genes down-regulated in the YM?RS09735/RS09740 strain (~33% genes demonstrated differential expression). In summary, we have reported the first PhoP/PhoR TCS identified in a Gram-negative bacterium and demonstrated that it is involved in virulence and gene regulation in R. anatipestifer.

SUBMITTER: Wang Y

PROVIDER: S-EPMC5399024 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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A Novel RAYM_RS09735/RAYM_RS09740 Two-Component Signaling System Regulates Gene Expression and Virulence in Riemerella anatipestifer.

Wang Ying Y Lu Ti T Yin Xuehuan X Zhou Zutao Z Li Shaowen S Liu Mei M Hu Sishun S Bi Dingren D Li Zili Z

Frontiers in microbiology 20170421

The Gram-negative bacterium Riemerella anatipestifer is an important waterfowl pathogen, causing major economic losses to the duck-producing industry. However, little is known of the virulence factors that mediate pathogenesis during R. anatipestifer infection. In this study, RAYM_RS09735 and RAYM_RS09740 were predicted to form a two-component signaling system (TCS) through bioinformatics analysis. This TCS was highly conserved across the Flavobacteriaceae. A mutant YMΔRS097 ...[more]

PMID: 28484437

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Project description:UnlabelledRiemerella anatipestifer is a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of the AS87_01735 gene significantly decreased the bacterial virulence of R. anatipestifer strain Yb2 (mutant RA625). The AS87_01735 gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, the AS87_01735 gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated that R. anatipestifer PncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncA in this study) showed a similar growth rate but decreased NAD(+) quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that the R. anatipestifer AS87_01735 gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate.ImportanceRiemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. The pncA gene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, we identified and characterized the pncA-homologous gene AS87_01735 in R. anatipestifer strain Yb2. R. anatipestifer PncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of the pncA mutant Yb2ΔpncA led to a decrease in the NAD(+) content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence of R. anatipestifer and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate.

Project description:Duck infectious serositis, also known as Riemerella anatipestifer disease, infects domestic ducks, geese, and turkeys and wild birds. However, the regulatory mechanism of its pathogenicity remains unclear. The PhoPR two-component system (TCS) was first reported in Gram-negative bacteria in our previous research and was demonstrated to be involved in virulence and gene expression. Here, DNA affinity purification sequencing (DAP-seq) was applied to further explore the regulation of PhoPR in relation to pathogenicity in R. anatipestifer. A conserved motif was identified upstream of 583 candidate target genes which were directly regulated by PhoP. To further confirm the genes which are regulated by PhoR and PhoP, single-gene-deletion strains were constructed. The results of transcriptome analysis using next-generation RNA sequencing showed 136 differentially expressed genes (DEGs) between the ΔphoP strain and the wild type (WT) and 183 DEGs between the ΔphoR strain and the WT. The candidate target genes of PhoP were further identified by combining transcriptome analysis and DAP-seq, which revealed that the main direct regulons of PhoP are located on the membrane and PhoP is involved in regulating aerotolerance. Using the in vivo duck model, the pathogenicity of ΔphoP and ΔphoR mutants was found to be significantly lower than that of the WT. Together, our findings provide insight into the direct regulation of PhoP and suggest that phoPR is essential for the pathogenicity of R. anatipestifer. The gene deletion strains are expected to be candidate live vaccine strains of R. anatipestifer which can be used as ideal genetic engineering vector strains for the expression of foreign antigens. IMPORTANCE Riemerella anatipestifer is a significant pathogen with high mortality in the poultry industry that causes acute septicemia and infectious polyserositis in ducks, chickens, geese, and other avian species. Previously, we characterized the two-component system encoded by phoPR and found that R. anatipestifer almost completely lost its pathogenicity for ducklings when phoPR was deleted. However, the mechanism of PhoPR regulation of virulence in R. anatipestifer had not been deeply explored. In this study, we utilized DAP-seq to explore the DNA-binding sites of PhoP as a response regulator in the global genome. Furthermore, phoP and phoR were deleted separately, and transcriptomics analysis of the corresponding gene deletion strains was performed. We identified a series of directly regulated genes of the PhoPR two-component system. The duckling model showed that both PhoP and PhoR are essential virulence-related factors in R. anatipestifer.

Dataset Information

A Novel RAYM_RS09735/RAYM_RS09740 Two-Component Signaling System Regulates Gene Expression and Virulence in Riemerella anatipestifer.

Publications

A Novel RAYM_RS09735/RAYM_RS09740 Two-Component Signaling System Regulates Gene Expression and Virulence in <i>Riemerella anatipestifer</i>.

Similar Datasets

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