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

Abstract

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. Author SummarySlow-growing bacterial cells have reduced antibiotic susceptibility, rendering them very difficult to eliminate during antibiotic treatment. However, for many key virulence factors (bacterial factors required to promote infection), it remains unclear whether expression is sufficient to slow bacterial growth and impact antibiotic susceptibility. Using Yersinia pseudotuberculosis, we found ribosomal protein expression fluctuated based on growth rate, and we generated a fluorescent reporter construct to detect altered ribosomal protein expression within individual bacterial cells. We then asked if expression of a key virulence factor in Yersinia, the type-III secretion system (T3SS), is sufficient to lower ribosomal protein expression, since it has been well established that T3SS induction results in growth arrest. We found high levels of T3SS expression promotes slowed growth and antibiotic tolerance, and bacterial cells that survive treatment with a ribosome-targeting antibiotic, gentamicin, have heightened levels of T3SS and lower levels of S10 expression.