Reading the host terrain: how pathogen sensing of host environmental cues controls the outcome of bacterial infection - To successfully colonize a host, bacterial pathogens must precisely regulate virulence gene expression in response to the host 9ssue microenvironment. Key among these cues are iron availability and oxida9ve stress, which fluctuate across 9ssues and vary between individuals. Chronic condi9ons such as anemia or iron overload disrupt iron homeostasis and oxida9ve stress, altering the host terrain sensed by bacterial pathogens. Notably, iron overload increases suscep9bility to severe infec9on with Yersinia and several other pathogens. Yersinia, Salmonella enterica, and Vibrio vulnificus all use the iron-sulfur (Fe-S) cluster coordina9ng transcrip9on factor IscR to regulate virulence gene expression in response to cellu- lar Fe-S cluster demand, which is shaped by iron availability and oxida9ve stress. Although previous studies have shown that iron and oxida9ve stress affect IscR DNA-binding specificity, how IscR integrates these signals to regulate bacterial virulence and promote infec9on remains poorly understood. Preliminary studies indicate that IscR is essen9al for bacte- ria to express horizontally-acquired virulence genes that are silenced by the global repressor of foreign DNA, H-NS. More- over, our preliminary data suggest that IscR enables bacteria to overcome boPlenecks during 9ssue coloniza9on. We hy- pothesize that IscR strategically overrides H-NS–mediated silencing of key virulence factors in response to spaCotemporal fluctuaCons in host iron and oxidaCve stress during criCcal stages of Cssue colonizaCon, shaping the course of infecCon. To test this hypothesis, we will u9lize Yersinia pseudotuberculosis as an ideal model pathogen to carry out two independ- ent, complementary aims. In Aim 1, we will determine how IscR orchestrates regula9on of the Yersinia type III secre9on system (T3SS) virulence factor in response to cellular Fe-S cluster demand. To do this, we will determine how purified apo-IscR, [2Fe-2S]-IscR, H-NS, the H-NS binding partner YmoA, and RNA polymerase interact with the promoter of the Yersinia T3SS master regulator LcrF, as well as how IscR and YmoA/H-NS affect RNAP ac9vity. These experiments will guide the design of Yersinia mutants that will be used to test our in vitro findings in bacterial culture. In Aim 2, we will determine how bacterial sensing of Fe-S cluster demand controls the outcome of infec9on in heathy hosts as well as those suffering from anemia or hereditary hemochromatosis, a common iron overload disorder. To do this, we will assess infec9on kine9cs by monitoring luminescent bacteria in living mice over 9me, determine the spa9al distribu9on of bac- terial T3SS expression within 9ssues using fluorescence microscopy, assess Yersinia iron bioavailability and exposure to oxida9ve stress in infected 9ssues, and quan9fy coloniza9on boPlenecks and pathogen dissemina9on paPerns through lineage tracing of barcoded bacterial popula9ons. The conceptual framework for IscR virulence factor regula9on estab- lished in Aim 1 will inform interpreta9on of bacterial infec9on dynamics in Aim 2. Together, this work will provide mecha- nis9c understanding of how bacteria sense the host 9ssue microenvironment to express key virulence programs. In addi- 9on, this study will shed light on how infec9on dynamics are influenced by underlying host condi9ons.
