Temporal tuning of switch-like virulence expression resolves environmental uncertainty through phenotypic...

Gene regulatory networks often evolve in the face of environmental uncertainty, as stimuli are rarely precise and uniform enough to make all-or-nothing responses advantageous. Virulence gene regulation in intracellular bacterial pathogens is shaped by unique selective pressures to resolve this uncertainty, as host environments are dynamic, hostile, and heterogeneous. Here, we investigate the regulation of Salmonella pathogenicity island 2 (SPI-2), a set of genes required for intracellular replication of the enteric pathogen Salmonella enterica serovar Typhimurium (STm), to evaluate whether it has evolved the capacity to tune transcriptional responses to intracellular-like environments. Using live-cell reporters and single-molecule fluorescence in situ hybridization in in vitro-inducing environments, we identify bimodal SPI-2 expression in clonal populations. We show cells progressively transition into an SPI-2-expressing state, with multi-generational variation in the timing at which this transition occurs. The rate of these single-cell transitions is modulated by the strength of intracellular-like signals. We determine that heterogeneity is dependent on sensor kinase SsrA and levels of the response regulator SsrB, with SsrA gating SPI-2 activation at low SsrB concentrations. We identify switch-like dynamics in SPI-2 activation and link single-cell responses to features of cell growth and lineage. Finally, we find that this heterogeneous activation also occurs during macrophage infection and demonstrate that inhibition of phagosome acidification alters the rate of SPI-2 activation. Combined, our results show that the fraction of cells expressing SPI-2 in a given time frame is probabilistically tuned to the likelihood they are experiencing an intracellular environment, a strategy that resolves uncertainty in ambiguous environments.