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 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 based on RNA-seq analysis. To visualize changes in ribosomal protein expression, we generated a fluorescent transcriptional reporter with the promoter upstream of /S10 fused to a destabilized 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 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.