Biology subjects

Spratt, M.

Publications and source records attributed to Spratt, M..

3 recordsLinked to original sources

bioRxiv2026

Regulator-derived growth and fitness costs of Salmonella SPI-2 expression are environment specific and T3SS independent

Bacterial pathogens must evolve regulatory mechanisms that balance the necessity of virulence genes with the costs associated with their expression. Salmonella Pathogenicity Island-2 (SPI-2) is a genomic locus that encodes a Type 3 Secretion System (T3SS) and secreted effectors necessary for intracellular survival and replication. Recent work has shown that SPI-2 is expressed heterogeneously, suggesting uniform or aberrant expression of the system carries fitness costs. Here, we report a negative correlation between microcolony growth and SPI-2 expression, suggesting a growth cost to expression of SPI-2. To quantify and mechanistically evaluate this cost, we employ knockout and synthetic expression of the SPI-2 master regulator ssrB to eliminate or force the expression of SPI-2 genes. We find that ssrB expression causes deficits throughout the growth curve and puts cells at a competitive disadvantage in acidic, nutrient limited media. We further show that these deficits are environment specific, and not observed until stationary phase in neutral, rich media. We also observe unexpected cell morphology effects of ssrB expression, suggesting its effects on the cell extend beyond its regulation of SPI-2 genes. Using known ssrB mutations we find that DNA-binding activity, but not phosphorylation, of SsrB is necessary for growth costs. Lastly, we show that this growth cost is incurred even in the absence of the SPI-2 genetic locus, indicating that it is inherent to expression of ssrB, rather than its primary downstream virulence gene targets. These results demonstrate that SPI-2 expression is coupled to context specific cell growth and fitness deficits induced by its master regulator, offering a potential benefit to heterogeneous expression. SIGNIFICANCEVirulence factors are often heterogeneously expressed in bacteria, offsetting the resource and energetic burdens they impose. Characterizing such burdens reveals the evolutionary pressures that act on virulence genes and uncovers the logic behind regulatory mechanisms that drive heterogeneity in their expression. This study evaluates the growth and fitness costs associated with expression of Salmonella Pathogenicity Island-2 (SPI-2), which encodes a Type 3 Secretion System and secreted effectors required for intracellular survival and replication. The expression of the SPI-2 master regulator ssrB is shown to impose environment-specific growth and fitness deficits. These deficits persist in the absence of T3SS genes, suggesting that costs originate from SsrB acting on other target genes. These results provide critical context to recent observations of heterogeneous SPI-2 expression, elucidating the fitness cost this heterogeneity has evolved to offset.

microbiology↗

bioRxiv2026

Perturbing H-NS function reveals roles in restricting virulence heterogeneity and pathogen adaptation

Bacterial pathogens must balance rapid expression of virulence genes in host niches with tight repression when not needed to avoid fitness costs and ensure survival. Integration of virulence gene regulatory networks with the conserved global repressor H-NS was critical to achieve this balance. H-NS-mediated repression of virulence genes in non-inducing environments is essential for maintaining virulence genes and has shaped pathogen evolution. However, the role of H-NS-mediated repression in virulence gene activation and pathogen evolution in virulence-inducing conditions is less clear. For instance, although virulence gene expression is often heterogeneous, whether relief of H-NS repression contributes to this heterogeneity remains unknown. Furthermore, whether H-NS repression shapes pathogen evolution in virulence-inducing environments is unclear. Here, we use a Salmonella strain with reduced H-NS DNA-binding affinity to investigate the role of H-NS in virulence gene expression in individual bacteria and pathogen adaptation. We find that reduced H-NS repression increases the fraction of virulence gene expressing cells without eliminating bimodality, resulting in enhanced epithelial cell infection in vitro. Using experimental evolution, we demonstrate that the hns genotype constrains adaptive mutations and that disabling virulence gene expression is a common path to improved fitness in intracellular-like environments. Our results expand the role of H-NS-mediated repression from silencing virulence genes in non-inducing conditions to regulating heterogeneity in inducing conditions and demonstrate that evolutionary conservation of H-NS constrains adaptive strategies in intracellular-like environments.

systems biology↗

bioRxiv2025

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

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 Typhimurium (STm), to evaluate whether it has evolved the capacity to tune transcriptional responses to intracellular-like environments. Using live-cell reporters and smFISH in in vitro inducing environments, we identify bimodal expression of SPI-2 in clonal populations. We show cells progressively transition into a SPI-2 expressing state, with multi-generational variation in the timing at which this transition occurs. The rate of these single-cell transitions are modulated by the strength of intracellular-like signals and environmental manipulations of growth rate. We determine that heterogeneity originates at the level of sensor kinase SsrA signaling and is dependent on response regulator SsrB concentration. We identify switch-like dynamics in SPI-2 activation and link single-cell responses to features of cell growth and lineage. Combined, our results show that the fraction of cells expressing SPI-2 in a given timeframe is probabilistically tuned to the likelihood they are experiencing an intracellular environment, a strategy that resolves uncertainty in ambiguous environments.

systems biology↗