Yersinia pseudotuberculosis growth arrest during type-III secretion system expression is associated with altered ribosomal protein expression and decreased gentamicin susceptibility.

It has been long appreciated that expression of the Yersinia type-III secretion system (T3SS) in culture is associated with growth arrest. Here we sought to understand whether this impacts expression of ribosomal protein genes, which were among the most highly abundant transcripts in exponential phase Yersinia pseudotuberculosis based on RNA-seq analysis. To visualize changes in ribosomal protein expression, we generated a fluorescent transcriptional reporter with the promoter upstream of rpsJ/S10 fused to a destabilized gfp variant. We confirmed reporter expression significantly increases in exponential phase and decreases as cells transition to stationary phase. We then utilized a mouse model of systemic Y. pseudotuberculosis infection to compare T3SS and S10 reporter expression during clustered bacterial growth in the spleen, and found that cells expressing high levels of the T3SS had decreased S10 levels, while cells with lower T3SS expression retained higher S10 expression. In bacteriological media, growth inhibition with T3SS induction and a reduction in S10 expression were observed in subsets of cells, while cells with high expression of both T3SS and S10 were also observed. Loss of T3SS genes resulted in rescued growth and heightened S10 expression. To understand if clustered growth impacted bacterial gene expression, we utilized droplet-based microfluidics to encapsulate bacteria in spherical agarose droplets, and also observed growth inhibition with high expression of T3SS and reduced S10 levels that better mirrored phenotypes observed in the mouse spleen. Finally, we show that T3SS expression is sufficient to promote tolerance to the ribosome-targeting antibiotic, gentamicin. Collectively, these data indicate that the growth arrest associated with T3SS induction leads to decreased expression of ribosomal protein genes, and this results in reduced antibiotic susceptibility.

Protocol for detecting Yersinia pseudotuberculosis nitric oxide exposure during in vitro growth.

Yersinia pseudotuberculosis (Yptb) is a bacterial pathogen that causes foodborne illness. Defense against the host antimicrobial gas, nitric oxide (NO), by the bacterial NO-detoxifying gene, hmp, promotes Yptb replication in mouse models of infection. Here, we detail the use of fluorescent signals as readouts for NO exposure within individual cells and subsequent detection of heterogeneity within a population, using single-cell imaging and analysis. This protocol quantifies NO exposure in culture, without capturing the full complexity of the host environment. For complete details on the use and execution of this protocol, please refer to Patel et al. (2021).

NO-Stressed Y. pseudotuberculosis Has Decreased Cell Division Rates in the Mouse Spleen.

Fluorescence dilution approaches can detect bacterial cell division events and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein and then tracking partitioning of fluorescence into daughter cells. However, fluorescence can be diluted very quickly within a rapidly replicating population, such as pathogenic bacterial populations replicating within host tissues. To overcome this limitation, we have generated two revTetR reporter constructs, where either mCherry or yellow fluorescent protein (YFP) is constitutively expressed and repressed by addition of tetracyclines, resulting in fluorescence dilution within defined time frames. We show that fluorescent signals are diluted in replicating populations and that signal accumulates in growth-inhibited populations, including during nitric oxide (NO) exposure. Furthermore, we show that tetracyclines can be delivered to the mouse spleen during Yersinia pseudotuberculosis infection and defined a drug concentration that results in even exposure of cells to tetracyclines. We then used this system to visualize bacterial cell division within defined time frames postinfection. revTetR-mCherry allowed us to detect slow-growing cells in response to NO in culture; however, this strain had a growth defect within mouse tissues, which complicated results. To address this issue, we constructed revTetR-YFP using the less toxic YFP and showed that heightened NO exposure correlated with heightened YFP signal, indicating decreased cell division rates within this subpopulation in vivo. This revTetR reporter will provide a critical tool for future studies to identify and isolate slowly replicating bacterial subpopulations from host tissues.