Flagellar regulation mediated by the Rcs pathway is required for virulence in the fish pathogen Yersinia ruckeri

The flagellum is a complex surface structure necessary for a number of activities including motility, chemotaxis, biofllm formation and host attachment. Flagellin, the primary structural protein making up the flagellum, is an abundant and potent activator of innate and adaptive immunity and therefore expression of flagellin during infection could be deleterious to the infection process due to flagellin-mediated host recognition. Here, we use quantitative RT-PCR to demonstrate that expression of the flagellin locus fiiC is repressed during the course of infection and subsequently up-regulated upon host mortality in a motile strain of Yersinia ruckeri. The kinetics of fliC repression during the infection process is relatively slow as full repression occurs 7-days after the initiation of infection and after approximately 3-logs of bacterial growth in vivo. These results suggests that Y. ruckeri possesses a regulatory system capable of sensing host and modulating the expression of motility in response. Examination of the master flagellar operon (flhDC) promoter region for evidence of transcriptional regulation and regulatory binding sites revealed potential interaction with the Rcs pathway through an Rcs(A)B Box. Deletion of rcsB (Delta rcsB) by marker-exchange mutagenesis resulted in overproduction of flagellin and unregulated motility, showing that the Rcs pathway negatively regulates biosynthesis of the flagellar apparatus. Experimental challenge with Delta rcsB and Delta rcsB Delta fliC1 Delta fliC2 mutants revealed that mutation of the Rcs pathway results in virulence attenuation which is dependent on presence of the flagellin gene. These results suggest that the inappropriate expression of flagellin during infection triggers host recognition and thus immune stimulation resulting in attenuation of virulence. In addition, RNAseq analyses of the Delta rcsB mutant strain verified the role of this gene as a negative regulator of the flagellar motility system and identified several additional genes regulated by the Rcs pathway.