Greene Justin, Cotten Katherine L, Snyder Rhett A, Huiszoon Ryan C, Chu Sangwook, Braza Rezia Era D, Chapin Ashley A, Stine Justin M, Bentley William E, Ghodssi Reza, Davis Kimberly M
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
Institute for Systems Research, University of Maryland, College Park, Maryland, United States of America.
PLoS Pathog. 2025 Jul 7;21(7):e1012548. doi: 10.1371/journal.ppat.1012548. eCollection 2025 Jul.
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 T3SS expression is sufficient to trigger loss of exponential phase markers, and utilized a fluorescent reporter for ribosomal protein expression to detect changes in bacterial growth state. Using a fluorescent transcriptional reporter with the rpsJ/S10 promoter fused to a destabilized gfp variant, we confirmed reporter expression significantly increases in exponential phase and decreases as cells transition to stationary phase. In a mouse model of systemic Y. pseudotuberculosis infection, we found multiple subsets of bacterial cells in the mouse spleen, including cells with high T3SS and low S10 expression and cells with high expression of both markers. In bacterial media, growth inhibition with T3SS induction and a reduction in S10 expression were observed, but a significant proportion of cells retained high expression of both T3SS and S10. Paradoxically, while loss of T3SS expression rescued growth, lower S10 expression was detected, again indicating bacteria can express both markers simultaneously. In media, bacteria grow planktonically as individual cells, while in mouse tissues, bacteria form clustered extracellular communities. We utilized droplet-based microfluidics to encapsulate bacteria in spherical agarose droplets and model clustered growth, and observed high expression of T3SS without an impact on S10 levels. Finally, we show that T3SS expression is sufficient to promote antibiotic tolerance, but surviving bacteria in a gentamicin treatment mouse model specifically express low S10. Collectively, these data indicate that the growth arrest associated with T3SS induction can reduce antibiotic susceptibility, but cells surviving antibiotic treatment display lower levels of the exponential phase marker, S10.
长期以来，人们一直认识到耶尔森氏菌III型分泌系统（T3SS）在培养物中的表达与生长停滞有关。在这里，我们试图了解T3SS的表达是否足以引发指数期标志物的丧失，并利用核糖体蛋白表达的荧光报告基因来检测细菌生长状态的变化。使用与不稳定的gfp变体融合的rpsJ/S10启动子的荧光转录报告基因，我们证实报告基因的表达在指数期显著增加，并随着细胞过渡到稳定期而减少。在系统性假结核耶尔森氏菌感染的小鼠模型中，我们在小鼠脾脏中发现了多个细菌细胞亚群，包括T3SS高表达和S10低表达的细胞以及两种标志物都高表达的细胞。在细菌培养基中，观察到T3SS诱导导致生长抑制和S10表达降低，但相当一部分细胞仍同时高表达T3SS和S10。矛盾的是，虽然T3SS表达的丧失挽救了生长，但检测到较低的S10表达，这再次表明细菌可以同时表达这两种标志物。在培养基中，细菌以单个细胞的形式浮游生长，而在小鼠组织中，细菌形成聚集的细胞外群落。我们利用基于液滴的微流控技术将细菌封装在球形琼脂糖液滴中并模拟聚集生长，观察到T3SS的高表达而对S10水平没有影响。最后，我们表明T3SS的表达足以促进抗生素耐受性，但在庆大霉素治疗的小鼠模型中存活的细菌特异性地表达低水平的S10。总的来说，这些数据表明与T3SS诱导相关的生长停滞会降低抗生素敏感性，但抗生素治疗后存活的细胞显示出较低水平的指数期标志物S10。
