Biology subjects

Auerbuch, V.

Publications and source records attributed to Auerbuch, V..

3 recordsLinked to original sources

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↗

bioRxiv2024

The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria

Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37{degrees}C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence. Author SummaryMany pathogens carry extrachromosomal DNA elements known as plasmids, which encode genes that confer bacterial virulence or antimicrobial resistance (AMR). Acquisition of these plasmids by bacteria can lead to the emergence of new pathogenic traits and the spread of AMR, yet the mechanisms by which plasmids are retained in bacterial populations particularly in the absence of selective pressure remain incompletely understood. Here we show that the major bacterial polyadenylase enzyme PAP I, encoded by the pcnB gene, is critical for the human pathogen Yersinia pseudotuberculosis to maintain its native virulence plasmid as well as AMR plasmids. Very little is known about the process of polyadenylation in bacteria, or the post-transcriptional addition of adenosine residues to the 3 end of transcripts. This study represents the first demonstration that PAP I-mediated polyadenylation contributes to bacterial pathogenesis.

microbiology↗

bioRxiv2021

The transcription factor IscR promotes Yersinia type III secretion system activity by antagonizing the repressive H-NS-YmoA histone-like protein complex

The type III secretion system (T3SS) is a 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 YmoA/H-NS histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. Although IscR has been shown to bind the lcrF promoter and is required for in vivo expression of lcrF, in this study we show IscR alone fails to enhance lcrF transcription in vitro. Rather, we find that in a ymoA mutant, IscR is no longer required for LcrF expression or T3SS activity. Additionally, a mutation in YmoA that prevents H-NS binding (ymoAD43N) rescues the T3SS defect of a {Delta}iscR mutant, suggesting that a YmoA/H-NS complex is needed for this repressive activity. Furthermore, chromatin immunoprecipitation analysis revealed that H-NS is enriched at the lcrF promoter at environmental temperatures, while IscR is enriched at this promoter at mammalian body temperature under aerobic conditions. Importantly, CRISPRi knockdown of H-NS leads to increased lcrF transcription. Collectively, our data suggest that as IscR levels rise with iron limitation and oxidative stress, conditions Yersinia experiences during extraintestinal infection, IscR antagonizes YmoA/H-NS-mediated repression of lcrF transcription to drive T3SS activity and manipulate host defense mechanisms. Author SummaryFacultative pathogens must silence virulence gene expression during growth in the environment, while retaining the ability to upregulate these genes upon infection of a host. H-NS is an architectural DNA binding protein proposed to silence horizontally acquired genes, regulating virulence genes in a number of pathogens. Indeed, H-NS was predicted to regulate plasmid-encoded virulence genes in pathogenic Yersinia. However, Yersinia H-NS is reported to be essential, complicating testing of this model. We used chromatin immunoprecipitation and inducible CRISPRi knockdown to show that H-NS binds to the promoter of a critical plasmid-encoded virulence gene, silencing its expression. Importantly, under conditions that mimic Yersinia infection of a mammalian host, the transcriptional regulator IscR displaces H-NS to drive virulence factor expression.

microbiology↗