Infection-associated gene regulation of L-tartrate metabolism in Salmonella enterica serovar Typhimurium.

Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU, genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for the sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, the regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella-induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, i.e., substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes the utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection. IMPORTANCE: Bacterial pathogens must adapt their gene expression profiles to cope with diverse environments encountered during infection. This coordinated process is carried out by the integration of cues that the pathogen senses to fine-tune gene expression in a spatiotemporal manner. Many studies have elucidated the regulatory mechanisms of how Salmonella sense metabolites in the gut to activate or repress its virulence program; however, our understanding of how Salmonella coordinates its gene expression to maximize the utilization of carbon and energy sources found in transitional nutrient niches is not well understood. In this study, we discovered how Salmonella integrates two infection-relevant cues, substrate availability and exogenous electron acceptors, to control L-tartrate metabolism. From our experiments, we propose a model for how L-tartrate metabolism is regulated in response to different metabolic cues in addition to characterizing two previously unknown transcriptional regulators. This study expands our understanding of how microbes combine metabolic cues to enhance fitness during infection.

Gene regulation of infection-associated L-tartrate metabolism in Salmonella enterica serovar Typhimurium.

Enteric pathogens such as Salmonella enterica serovar Typhimurium experience spatial and temporal changes to the metabolic landscape throughout infection. Host reactive oxygen and nitrogen species non-enzymatically convert monosaccharides to alpha hydroxy acids, including L-tartrate. Salmonella utilizes L-tartrate early during infection to support fumarate respiration, while L-tartrate utilization ceases at later time points due to the increased availability of exogenous electron acceptors such as tetrathionate, nitrate, and oxygen. It remains unknown how Salmonella regulates its gene expression to metabolically adapt to changing nutritional environments. Here, we investigated how the transcriptional regulation for L-tartrate metabolism in Salmonella is influenced by infection-relevant cues. L-tartrate induces the transcription of ttdBAU, genes involved in L-tartrate utilization. L-tartrate metabolism is negatively regulated by two previously uncharacterized transcriptional regulators TtdV (STM3357) and TtdW (STM3358), and both TtdV and TtdW are required for sensing of L-tartrate. The electron acceptors nitrate, tetrathionate, and oxygen repress ttdBAU transcription via the two-component system ArcAB. Furthermore, regulation of L-tartrate metabolism is required for optimal fitness in a mouse model of Salmonella-induced colitis. TtdV, TtdW, and ArcAB allow for the integration of two cues, substrate availability and availability of exogenous electron acceptors, to control L-tartrate metabolism. Our findings provide novel insights into how Salmonella prioritizes utilization of different electron acceptors for respiration as it experiences transitional nutrient availability throughout infection.

Repression by the H-NS/YmoA histone-like protein complex enables IscR dependent regulation of the Yersinia T3SS.

The type III secretion system (T3SS) is an appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive H-NS/YmoA histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. While deletion of iscR or ymoA has been shown to decrease and increase LcrF expression and type III secretion, respectively, the role of H-NS in this system has not been definitively established because hns is an essential gene in Yersinia. Using CRISPRi knockdown of hns, we show that hns depletion causes derepression of lcrF. Furthermore, we find that while YmoA is dispensable for H-NS binding to the lcrF promoter, YmoA binding to H-NS is important for H-NS repressive activity. We bioinformatically identified three H-NS binding regions within the lcrF promoter and demonstrate binding of H-NS to these sites in vivo using chromatin immunoprecipitation. Using promoter truncation and binding site mutation analysis, we show that two of these H-NS binding regions are important for H-NS/YmoA-mediated repression of the lcrF promoter. Surprisingly, we find that IscR is dispensable for lcrF transcription in the absence of H-NS/YmoA. Indeed, IscR-dependent regulation of LcrF and type III secretion in response to changes in oxygen, such as those Yersinia is predicted to experience during host infection, only occurs in the presence of an H-NS/YmoA complex. These data suggest that, in the presence of host tissue cues that drive sufficient IscR expression, IscR can act as a roadblock to H-NS/YmoA-dependent repression of RNA polymerase at the lcrF promoter to turn on T3SS expression.

Genome Scale Analysis Reveals IscR Directly and Indirectly Regulates Virulence Factor Genes in Pathogenic Yersinia.

The iron-sulfur cluster coordinating transcription factor IscR is important for the virulence of Yersinia pseudotuberculosis and a number of other bacterial pathogens. However, the IscR regulon has not yet been defined in any organism. To determine the Yersinia IscR regulon and identify IscR-dependent functions important for virulence, we employed chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of Y. pseudotuberculosis expressing or lacking iscR following iron starvation conditions, such as those encountered during infection. We found that IscR binds to the promoters of genes involved in iron homeostasis, reactive oxygen species metabolism, and cell envelope remodeling and regulates expression of these genes in response to iron depletion. Consistent with our previous work, we also found that IscR binds in vivo to the promoter of the Ysc type III secretion system (T3SS) master regulator LcrF, leading to regulation of T3SS genes. Interestingly, comparative genomic analysis suggested over 93% of IscR binding sites were conserved between Y. pseudotuberculosis and the related plague agent Yersinia pestis. Surprisingly, we found that the IscR positively regulated sufABCDSE Fe-S cluster biogenesis pathway was required for T3SS activity. These data suggest that IscR regulates the T3SS in Yersinia through maturation of an Fe-S cluster protein critical for type III secretion, in addition to its known role in activating T3SS genes through LcrF. Altogether, our study shows that iron starvation triggers IscR to coregulate multiple, distinct pathways relevant to promoting bacterial survival during infection. IMPORTANCE How bacteria adapt to the changing environment within the host is critical for their ability to survive and cause disease. For example, the mammalian host severely restricts iron availability to limit bacterial growth, referred to as nutritional immunity. Here, we show that pathogenic Yersinia use the iron-sulfur (Fe-S) cluster regulator IscR, a factor critical for pathogenesis, to sense iron availability and regulate multiple pathways known or predicted to contribute to virulence. Under low iron conditions that mimic those Yersinia encounter during infection, IscR levels increase, leading to modulation of genes involved in iron metabolism, stress resistance, cell envelope remodeling, and subversion of host defenses. These data suggest that IscR senses nutritional immunity to coordinate processes important for bacterial survival within the mammalian host.