Biology subjects

McKenna, J. A.

Publications and source records attributed to McKenna, J. A..

3 recordsLinked to original sources

bioRxiv2023

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp., at [≤] 30 {degrees}C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid. Upon a switch to 37 {degrees}C, VirB, a DNA binding protein and key transcriptional regulator of Shigella virulence, is produced. VirB functions to counter H-NS-mediated silencing in a process called transcriptional anti-silencing. Here, we show that VirB mediates a loss of negative DNA supercoils from our plasmid-borne, VirB-regulated PicsP-lacZ reporter, in vivo. The changes are not caused by a VirB-dependent increase in transcription, nor do they require the presence of H-NS. Instead, the VirB-dependent change in DNA supercoiling requires the interaction of VirB with its DNA binding site, a critical first step in VirB-dependent gene regulation. Using two complementary approaches, we show that VirB:DNA interactions in vitro introduce positive supercoils in plasmid DNA. Subsequently, by exploiting transcription-coupled DNA supercoiling, we reveal that a localized loss of negative supercoils is sufficient to alleviate H-NS-mediated transcriptional silencing, independently of VirB. Together, our findings provide novel insight into VirB, a central regulator of Shigella virulence and more broadly, a molecular mechanism that offsets H-NS-dependent silencing of transcription in bacteria.

molecular biology↗

bioRxiv2022

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

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.

microbiology↗

bioRxiv2021

Gut regulatory T cells mediate immunological tolerance in Salmonella Typhimurium-infected superspreader hosts by suppressing cytotoxic activity of T cells.

Superspreader hosts carry out most pathogen transmission events and are often disease tolerant since they remain asymptomatic despite high pathogen burdens. Here we describe the superspreader immune state that allows for disease tolerance. In a model of Salmonella infection, superspreader mice develop colitis with robust CD4+ and CD8+ T-cell responses, however, they remain asymptomatic. We found that superspreaders have significantly more regulatory T cells (Tregs) in the distal gut compared to non-superspreader infected hosts. Surprisingly, the depletion of Tregs did not induce pathogen clearance but rather exacerbated weight loss, increased gut inflammation, and compromised epithelial intestinal barrier. This loss of tolerance correlated with dramatic increases in cytotoxic CD4+ and CD8+ T cells. Interestingly, CD4 neutralization in Tregs-depleted superspreaders was sufficient to rescue tolerance. Our results indicate that Tregs play a crucial role in maintaining immunologic tolerance in the guts of superspreader mice by suppressing cytotoxic CD4+ and CD8+ T-cell activities. AUTHOR SUMMARYSuperspreader hosts are the main cause of disease transmission and a very important public health concern. Here, we evaluated the immunological tolerance of the Salmonella infected superspreaders in a mouse model. By manipulating Tregs, we demonstrated the immunological mechanism from the host to maintain health status and high pathogen burden. Tregs depletion in the superspreaders led to severe disease, with damage of the intestinal epithelia, and high morbidity without having any effect on shedding and systemic Salmonella burden. Furthermore, we demonstrated that the damage of the intestinal epithelia was related to cytotoxic activity of T cells. When Tregs were depleted, CD8+ T cells produced high levels of granzyme B and perforin. CD8+ T cells neutralization in Tregs depleted mice led to increased cytotoxic CD4+ T cells. Interestingly, neutralization of CD4+ T cells in the Tregs depleted mice led to a reduction in the CD8+ T cells producing granzyme B and it was sufficient to rescue host tolerance in this model. We demonstrate for the first time that cytotoxic CD4+ T cells damage the epithelial intestinal barrier and contribute to loss of tolerance in the context of a superspreader host. These findings open new perspectives to understand mechanisms of tolerance in the intestine of a superspreader host.

immunology↗