Genome-wide analysis reveals PhoP regulates pathogenicity in Riemerella anatipestifer

Abstract

AbstractDuck infectious serositis, also known as the Riemerella anatipestifer disease infects domestic ducks, geese, turkeys, and wild birds. However, the regulatory mechanism of its pathogenicity remains unclear. The phoP/phoR two-component system was first reported in gram-negative bacteria in our previous research and was demonstrated to be involved in virulence and gene expression. Here, the DNA-affinity-purified sequencing (DAP-seq) was applied to further explore the regulation of phoP/phoR to pathogenicity in R. anatipestifer. A conserved motif was identified in the upstream of 583 candidate target genes which were directly regulated by phoP. To further confirm the genes which are regulated by phoP/phoR, phoR and phoP, the single-gene deletion strains were constructed. The results of transcriptome analysis using next-generation RNA sequencing showed 136 differential expression genes (DEGs) between ΔphoP and RA-YM, and 183 DEGs between ΔphoR and WT. The candidate target genes of PhoP were further identified by combining transcriptome analysis and DAP-seq. The results of DAP-seq and RNA-seq of ΔphoP in combination 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 or ΔphoR was significantly lower than that of the WT. Together, our findings provided a perception about the direct regulation of PhoP and suggested that phoP/phoR is essential for the pathogenicity of R. anatipestifer, and phoP/phoR is related to the aerotolerance of R. anatipestifer. The gene deletion strains are expected to be the candidate live vaccine strains of R. anatipestifer which can be used as ideal genetic engineering vector strains for the expression of foreign antigens.Author summaryRiemerella anatipestifer is a severe pathogen in the poultry industry with high mortality in ducks and geese mainly due to acute septicemia and infectious polyserositis. A two-component system phoP/phoR was previously characterized in R. anatipestifer, and the phoP/phoR TCS was reported for the first time in Gram-negative bacteria. A deleted phoP/phoR in R. anatipestifer has been reported to almost lose its pathogenicity in ducklings. However, the mechanism of phoP/phoR regulating the virulence of R. anatipestifer had not been explored in detail. This study has utilized DAP-seq to explore the DNA-binding sites of PhoP as a response regulator in the global genome. Furthermore, the phoP and phoR were deleted separately and the transcriptomics of the corresponding gene-deleted strains was analyzed. A series of directly regulated genes of phoP/phoR TCS were determined in combination. The duckling model revealed both PhoP and PhoR as essential virulence-related factors of R. anatipestifer.