Stochastic activation of a family of TetR type transcriptional regulators controls a virulence switch in Acinetobacter baumannii

Phenotypic heterogeneity is an important mechanism to regulate bacterial virulence, where a single regulatory switch is typically activated to generate virulent and avirulent subpopulations. The opportunistic pathogen Acinetobacter baumannii can transition at high-frequency between virulent (VIR-O) and avirulent (AV-T) subpopulations, distinguished by cells that form opaque or translucent colonies. We demonstrate that expression of twelve TetR-type transcriptional regulators (TTTRs) can drive cells from the VIR-O subpopulation to the AV-T state. Remarkably, in a subpopulation of VIR-O cells, four of these TTTRs were stochastically activated in different combinations to drive cells to the AV-T state, with each resulting AV-T subvariant exhibiting unique phenotypic differences. Due to their functional redundancy, a quadruple mutant with all four of these TTTRs inactivated was required to see a loss of VIR-O to AV-T switching. Further, we demonstrate a small RNA, SrvS, acts as a “rheostat”, where the levels of SrvS expression influences both the VIR-O to AV-T switching frequency and which TTTR is activated when VIR-O cells switch to AV-T. In summary, this work has revealed a new paradigm for phenotypic switching in bacteria, where an unprecedented number of related transcriptional regulators are activated in different combinations to control virulence and generate unique AV-T subvariants with distinct phenotypic properties. Overall design: Comparative gene expression profiling analysis of RNA-seq data for VIR-O pWH1266, VIR-O pWH1266_1959 and VIR-O pWH1266_2818. Gene expression was determined via RNA-sequencing. Three biological replicates per sample.