Abstract
The enteropathogen Yersinia pseudotuberculosis and the related plague agent Y. pestis require the Ysc type III secretion system (T3SS) to subvert phagocyte defense mechanisms and cause disease. Yet type III secretion (T3S) in Yersinia induces growth arrest and innate immune recognition, necessitating tight regulation of the T3SS. Here we show that Y. pseudotuberculosis T3SS expression is kept low under anaerobic, iron-rich conditions, such as those found in the intestinal lumen where the Yersinia T3SS is not required for growth. In contrast, the Yersinia T3SS is expressed under aerobic or anaerobic, iron-poor conditions, such as those encountered by Yersinia once they cross the epithelial barrier and encounter phagocytic cells. We further show that the [2Fe-2S] containing transcription factor, IscR, mediates this oxygen and iron regulation of the T3SS by controlling transcription of the T3SS master regulator LcrF. IscR binds directly to the lcrF promoter and, importantly, a mutation that prevents this binding leads to decreased disseminated infection of Y. pseudotuberculosis but does not perturb intestinal colonization. Similar to E. coli, Y. pseudotuberculosis uses the Fe-S cluster occupancy of IscR as a readout of oxygen and iron conditions that impact cellular Fe-S cluster homeostasis. We propose that Y. pseudotuberculosis has coopted this system to sense entry into deeper tissues and induce T3S where it is required for virulence. The IscR binding site in the lcrF promoter is completely conserved between Y. pseudotuberculosis and Y. pestis. Deletion of iscR in Y. pestis leads to drastic disruption of T3S, suggesting that IscR control of the T3SS evolved before Y. pestis split from Y. pseudotuberculosis.
Highlights
Iron availability and oxygen concentration play important roles in bacterial growth and gene regulation
As the mechanism by which iron and oxygen control the T3SS is completely conserved between Y. pseudotuberculosis and Y. pestis, yet Y. pestis is not transmitted through the intestinal route, we propose that Y. pestis has retained this T3SS regulatory mechanism to suit its new infection cycle
As IscR is a global regulator in Yersinia [24], the lcrFpNull mutant should only be defective in T3SS control, while a ΔiscR mutant displays additional T3SS-independent phenotypes
Summary
Iron availability and oxygen concentration play important roles in bacterial growth and gene regulation. Iron and oxygen regulate the Yersinia T3SS to enable disseminated infection iron-replete overnight cultures (M9+6.58 μM FeSO4) grown at 26 ̊C aerobically were diluted to an OD600 of 0.1 into Chelex-treated M9/high glucose media and grown for 8 hrs at 26 ̊C aerobically with agitation. Cultures were diluted a third time to OD600 0.2 in M9/high glucose supplemented with 6.58 μM FeSO4 (iron-replete) or with 0.0658 μM FeSO4 (iron limitation), grown for 2 hrs at 26 ̊C with agitation, and shifted to 37 ̊C for 4 hrs with agitation to induce type III secretion. The cultures were instead diluted a second time to OD600 0.1 in M9/high glucose supplemented with 6.58 μM FeSO4 (iron-replete,) or with 0.0658 μM FeSO4 (iron limitation), and transferred to a vinyl anaerobic chamber where they were grown at 26 ̊C for 12 hrs (most experiments) or 4 hrs (experiment shown in S5 Fig). Cultures were shifted to 37 ̊C for another 4 hrs to induce type III secretion
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