Balazs, G. I. | Search bioRxiv

bioRxiv2025

IscR-mediated sensing of iron-sulfur cluster demand coordinates virulence gene expression in Yersinia pseudotuberculosis

The type III secretion system (T3SS) is a needle-like appendage that translocates effector proteins into host cells to disrupt host defenses. Strict control of T3SS expression is critical for facultative pathogens such as Yersinia pseudotuberculosis, as the T3SS is indispensable for virulence but is metabolically costly. We previously showed that the iron-sulfur (Fe-S) cluster-coordinating transcription factor IscR controls expression of LcrF, the master regulator of the Yersinia T3SS and the YadA adhesin. Clusterless apo-IscR, the predominant IscR form during high cellular Fe-S cluster demand (aerobic, low iron conditions), promotes LcrF, T3SS, and YadA expression. Importantly, binding of apo-IscR to the lcrF promoter at a type II IscR binding site facilitates Yersinia disseminated infection. Here, we show that mutating the lcrF promoter to allow only [2Fe-2S]-IscR binding (lcrFpTypeI) results in hyperexpression of LcrF, the T3SS, and YadA during low Fe-S cluster demand (anaerobic, iron replete conditions). These data suggest that switching the form of IscR that can bind to the lcrF promoter reversed how iron and oxygen regulate Yersinia virulence factors. We used barcoded Y. pseudotuberculosis to probe how control of the lcrF promoter in response to iron and oxygen modifies infection dynamics. We found that the lcrFpTypeI mutant experiences a tighter bottleneck in the cecum, where Yersinia is expected to experience a low oxygen, iron replete environment. Taken together, these findings suggest that by tying T3SS and YadA expression to cellular Fe-S cluster demand, Yersinia can fine-tune its virulence repertoire to the host tissue microenvironment. ImportanceIron and oxygen availability fluctuate spatially across mammalian tissues as well as temporally during the course of bacterial infection. The [2Fe-2S] cluster coordinating transcription factor IscR senses changes in iron and oxygen levels, and plays a pivotal role in enabling pathogens like Yersinia, Salmonella, and Vibrio to express critical virulence genes. While prior research has established that iron availability and oxygen tension influence IscR abundance and DNA-binding specificity, it is unclear how these changes control the timing and location of virulence factor expression during infection. In this study, we engineered a bacterial strain to reverse the way in which iron and oxygen drive expression of two critical virulence factors through IscR. This mutant displayed altered host infection dynamics, revealing that uncoupling virulence gene expression from host tissue cues decreases bacterial fitness.

microbiology↗

bioRxiv2025

TNF signaling maintains local restriction of bacterial founder populations in intestinal and systemic sites during oral Yersinia infection

Enteroinvasive bacterial pathogens are responsible for an enormous worldwide disease burden that critically affects the young and immunocompromised. Yersinia pseudotuberculosis is a Gram-negative enteric pathogen, closely related to the plague agent Y. pestis, that colonizes intestinal tissues, induces the formation of pyogranulomas along the intestinal tract, and disseminates to systemic organs following oral infection of experimental rodents. Prior studies proposed that systemic tissues were colonized by a pool of intestinal replicating bacteria distinct from populations within Peyers patches and mesenteric lymph nodes. Whether bacteria within intestinal pyogranulomas serve as the source for systemic dissemination, and the relationship between bacterial populations within different tissue sites is poorly defined. Moreover, the factors that regulate Yersinia colonization and dissemination are not well understood. Here, we demonstrate, using Sequence Tag-based Analysis of Microbial Populations in R (STAMPR), that remarkably small founder populations independently colonize intestinal and systemic tissues. Notably, intestinal pyogranulomas contain clonal populations of bacteria that are restricted and do not spread to other tissues. However, populations of Yersinia are shared among systemic organs and the blood, suggesting that systemic dissemination occurs via hematogenous spread. Finally, we demonstrate that TNF signaling is a key contributor to the bottlenecks limiting both tissue colonization and lymphatic dissemination of intestinal bacterial populations. Altogether, this study reveals previously undescribed aspects of infection dynamics of enteric bacterial pathogens. ImportanceBacterial escape from the intestine can lead to severe disease, including sepsis, organ damage, and death. However, the intestinal bacterial population dynamics governing the colonization of mucosal and systemic tissues and the intestinal sites that seed systemic spread are not clear. Yersinia pseudotuberculosis is a rodent and human intestinal pathogen closely related to the plague agent and provides a natural rodent-adapted model to study systemic bacterial dissemination. Our findings define the infection dynamics of enteric Yersinia and the impact of the innate immune system on Yersinia colonization of the intestine and systemic organs.

microbiology↗