Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

The presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogen Vibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in a lonA mutant compared to the wild type. We further demonstrated that a lonA mutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence of V. cholerae. IMPORTANCEBacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogen V. cholerae is in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding of V. cholerae physiology.

Type III secretion systems (T3SSs) are used by Gram-negative pathogens to form pores in host membranes and deliver virulence-associated effector proteins inside host cells. In pathogenic Yersinia, the T3SS pore-forming proteins are YopB and YopD. Mammalian cells recognize the Yersinia T3SS, leading to a host response that includes secretion of the inflammatory cytokine interleukin-1␤ (IL-1␤), Toll-like receptor (TLR)-independent expression of the stress-associated transcription factor Egr1 and the inflammatory cytokine tumor necrosis factor alpha (TNF-␣), and host cell death. The known Yersinia T3SS effector proteins are dispensable for eliciting these responses, but YopB is essential. Three models describe how the Yersinia T3SS might trigger inflammation: (i) mammalian cells sense YopBD-mediated pore formation, (ii) innate immune stimuli gain access to the host cytoplasm through the YopBD pore, and/or (iii) the YopB-YopD translocon itself or its membrane insertion is proinflammatory. To test these models, we constructed a Yersinia pseudotuberculosis mutant expressing YopD devoid of its predicted transmembrane domain (YopD ⌬TM ) and lacking the T3SS cargo proteins YopHE-MOJTN. This mutant formed pores in macrophages, but it could not mediate translocation of effector proteins inside host cells. Importantly, this mutant did not elicit rapid host cell death, IL-1␤ secretion, or TLR-independent Egr1 and TNF-␣ expression. These data suggest that YopBD-mediated translocation of unknown T3SS cargo leads to activation of host pathways influencing inflammation, cell death, and response to stress. As the YopD ⌬TM Y. pseudotuberculosis mutant formed somewhat smaller pores with delayed kinetics, an alternative model is that the wild-type YopB-YopD translocon is specifically sensed by host cells.

SummaryThe Yersinia type III secretion system (T3SS) translocates Yop effector proteins into host cells to manipulate immune defenses such as phagocytosis and reactive oxygen species (ROS) production. The T3SS translocator proteins YopB and YopD form pores in host membranes, facilitating Yop translocation. While the YopD amino and carboxy termini participate in pore formation, the role of the YopD central region between amino acids 150-227 remains unknown. We assessed the contribution of this region by generating Y. pseudotuberculosis yopD Δ150-170 and yopDΔ207-227 mutants and analyzing their T3SS functions. These strains exhibited wild-type levels of Yop secretion in vitro and enabled robust pore formation in macrophages. However, the yopDΔ150-170 and yopDΔ207-227 mutants were defective in Yop translocation into CHO cells and splenocyte-derived neutrophils and macrophages. These data suggest that YopD-mediated host membrane disruption and effector Yop translocation are genetically separable activities requiring distinct protein domains. Importantly, the yopDΔ150-170 and yopDΔ207-227 mutants were defective in Yopmediated inhibition of macrophage cell death and ROS production in neutrophil-like cells, and were attenuated in disseminated Yersinia infection. Therefore, the ability of the YopD central region to facilitate optimal effector protein delivery into phagocytes, and therefore robust effector Yop function, is important for Yersinia virulence.

Human pathogenic Yersinia express a type III secretion system (T3SS) critical for virulence. The T3SS proteins YopD and YopB form pores in host membranes and mediate injection of effector proteins inside host cells to disrupt innate immune defenses such as production of reactive oxygen species (ROS). Discrete functional domains within YopD have been identified, yet the YopD central region (amino acids 150-227) remains largely uncharacterized. We assessed the importance of the YopD central region by generating Y. pseudotuberculosis YopDΔ150-170 and YopDΔ207-227 mutants and analyzing their virulence in mouse infection models. Y. pseudotuberculosis YopDΔ150-170 and YopDΔ207-227 grew normally in mesenteric lymph nodes and Peyer’s patches following oral infection, but displayed a 20-29,000-fold defect in the spleen and liver. These mutants were also highly attenuated following intraperitoneal infection. These data indicate that the YopD150-227 central region is critical for Y. pseudotuberculosis disseminated infection. To identify the underlying cause of this virulence defect, we examined the ability of the YopDΔ150-170 and YopDΔ207-227 mutants to translocate T3SS cargo and inhibit ROS production in macrophages. While only the YopDΔ150-170 mutant exhibited translocation defects, both mutants failed to fully inhibit ROS production. We hypothesize that the YopD central region facilitates optimal T3SS effector protein delivery to effectively disarm innate immune defenses.