Evidence for Rho-dependent control of a virulence switch in Acinetobacter baumannii

Abstract

ABSTRACTAcinetobacter baumannii strain AB5075 is able to interconvert at high frequency between virulent cells that form opaque colonies (VIR-O) and avirulent cells that form translucent colonies (AV-T). Cells switch from the VIR-O to the AV-T state by the combinatorial activation of at least three TetR-type transcriptional regulators (TTTRs). A genetic screen identified the transcription termination factor Rho as a major contributor that controls expression differences of these TTTRs between VIR-O and AV-T cells. Each TTTR has a long mRNA leader region where transcripts are terminated in VIR-O cells. However, in AV-T cells, the degree of termination in each TTTR leader was greatly reduced, allowing for higher levels of TTTR expression. In a strain with decreased Rho expression, or in wild-type VIR-O cells treated with the Rho inhibitor bicyclomycin, the degree of termination in each TTTR mRNA leader region was reduced. Mutations in the leader region of one TTTR, ABUW_1645, were identified that reduced the degree of termination. Purified Rho protein bound the leader region of the most frequently activated TTTR ABUW_1645 and the least frequently activated TTTR ABUW_1959 with similar affinities. This together with the observation that the levels of Rho protein were unchanged between VIR-O and AV-T cells, suggested that additional factors differentially modulate Rho activity between each variant. Finally, we demonstrate in AV-T cells that nutrient depletion is a condition that increases the levels of Rho-dependent transcription termination in the TTTR leader regions to regulate this phenotypic switch.IMPORTANCEAcinetobacter baumannii is a significant cause of infections in the healthcare setting. More recently, A. baumannii has been a leading cause of secondary bacterial pneumonia in patients infected with SARS-CoV-2 and the overall frequency of A. baumannii infection increased 78% during the COVID-19 pandemic. A. baumannii can exist in virulent or avirulent subpopulations and this interconversion is mediated by the expression of a family of TetR-type transcriptional regulators. In this study, we demonstrate that Rho is a key regulatory component in the expression of these TetR regulators. Overall, this study is the first to address a role for Rho in A. baumannii and provides additional evidence for the role of Rho in regulating diversity in bacterial subpopulations.

Keywords

• Acinetobacter baumannii
• phenotypic heterogeneity
• TetR regulator transcriptional regulator
• Rho-dependent termination