The AraC/XylS protein MxiE and its co-regulator IpgC control a negative feedback loop in the transcriptional cascade that regulates type III secretion in Shigella flexneri

Abstract

Members of the AraC Family of Transcriptional Regulators (AFTRs) control the expression of many genes important to cellular processes, including virulence. In Shigella species, the type III secretion system (T3SS), a key determinant for host cell invasion, is regulated by the three-tiered VirF/VirB/MxiE transcription cascade. Both VirF and MxiE belong to the AFTRs and are characterized as positive transcriptional regulators. Here, we identify a novel regulatory activity for MxiE and its co-regulator IpgC, which manifests as a negative feedback loop in the VirF/VirB/MxiE transcription cascade. Our findings show that MxiE and IpgC down-regulate the virB promoter and hence VirB protein production, thus, decreasing VirB-dependent promoter activity at ospD1, one of the nearly 50 VirB-dependent genes. At the virB promoter, regions required for negative MxiE- and IpgC-dependent regulation were mapped and found to be coincident with regions required for positive VirF-dependent regulation. In tandem, negative MxiE- and IpgC-dependent regulation of the virB promoter only occurred in the presence of VirF suggesting that MxiE and IpgC can function to counter VirF activation of the virB promoter. Lastly, MxiE and IpgC do not down-regulate another VirF-activated promoter, icsA, demonstrating that this negative feedback loop targets the virB promoter. Our study provides insight into a mechanism that may reprogram Shigella virulence gene expression following type III secretion and provides the impetus to examine if MxiE and IpgC homologs in other important bacterial pathogens such as Burkholderia pseudomallei and Salmonella enterica serovars Typhimurium and Typhi coordinate similar negative feedback loops. IMPORTANCEThe large AraC Family of Transcriptional Regulators (AFTRs) control virulence gene expression in many bacterial pathogens. In Shigella species, the AraC/XylS protein MxiE and its co-regulator IpgC positively regulate the expression of type III secretion system genes within the three-tiered VirF/VirB/MxiE transcription cascade. Our findings suggest a negative feedback loop in the VirF/VirB/MxiE cascade in which MxiE and IpgC counter VirF-dependent activation of the virB promoter, thus, making this the first characterization of negative MxiE- and IpgC-dependent regulation. Our study provides insight into a mechanism that likely reprograms Shigella virulence gene expression following type III secretion, which has implications for other important bacterial pathogens with functional homologs of MxiE and IpgC.