The two-component system CpxAR controls biofilm formation by directly regulating the T3SS needle tip protein EseB in Edwardsiella piscicida.

The type III secretion system (T3SS) translocon protein EseB (needle tip protein) forms filamentous appendages on the surface of Edwardsiella piscicida to facilitate autoaggregation and biofilm formation. By contrast, another T3SS translocon protein EseC inhibits biofilm formation by sequestering EseC's chaperone EseE, which also functions as a positive regulator of the escC-eseE operon, in which EseB is encoded. The two-component system (TCS) EsrAB and the regulator EsrC tightly and positively regulate the T3SS in E. piscicida. The TCS CpxAR provides an adaptive response to external environmental changes. In this study, we have shown that disruption of the histidine kinase CpxA (sensor) instead of CpxR (response regulator) significantly reduces biofilm formation in E. piscicida. CpxR is negatively regulated by CpxA, and significant amounts of CpxR accumulate in E. piscicida in the absence of CpxA. CpxR, together with EsrB and EsrC, directly binds the promoter of the cpxR-cpxA operon to promote CpxR transcription and expression. The elevated phosphorylated CpxR (CpxR-P) binds to the promoter of the escC-eseE operon to repress eseB transcription and expression, while EseE, EsrB, and EsrC bind directly to the same promoter to promote EseB transcription and expression. E. piscicida is an enteric pathogen that senses microbiota-derived indole in the gut lumen. EseB filament-mediated biofilm formation in E. piscicida is inversely proportional to exogenous indole. Together, CpxR inhibits while EsrB, EsrC, and EseE stimulate transcription and expression of the escC-eseE operon, thereby coordinately controlling EseB filament-mediated biofilm formation in E. piscicida in response to environmental stimuli.IMPORTANCEEdwardsiella piscicida is primarily an enteric pathogen of fish and can form a biofilm to resist the lethal effects of host or antimicrobial agents. The assembly of filamentous appendages on the bacterial surface, mediated by the type III secretion system (T3SS) needle tip protein EseB, promotes bacterial-bacterial interactions and biofilm formation when E. piscicida is cultured in Dulbecco's modified Eagle's medium (DMEM). In this study, we have shown that the histidine kinase CpxA regulates biofilm formation in E. piscicida by negatively regulating its response regulator CpxR. Binding to the promoter of the escC-eseE operon, CpxR negatively regulates, whereas EsrB, EsrC, and EseE positively regulate the escC-eseE operon, of which EseB is encoded, coordinately regulating biofilm formation in E. piscicida.