Biology subjects

Munson, G. P.

Publications and source records attributed to Munson, G. P..

6 recordsLinked to original sources

bioRxiv2026

Regulation and Characterization of the Cex system of enteric pathogens: a pathway for the lipidation and secretion of noncanoncial bacterial lipoproteins

CexE is an outer membrane lipoprotein of enterotoxigenic Escherichia coli (ETEC); one of the most prevalent etiological agents of diarrheal disease. Homologs of CexE are present in enteroaggregative E. coli, Citrobacter rodentium, and other enteropathogens. We have previously shown that expression of CexE is dependent upon Rns, the master virulence regulator of ETEC and a member of the AraC/XylS superfamily of transcription factors. In this study we report that the expression of cexD and cexPABC, the genes required for lipidation and delivery of CexE to the outer leaflet of the outer membrane, are also Rns-dependent. Although cexE and cexPABC are arranged in the same orientation, they are separated by an intergenic region sufficiently large to accommodate a promoter and transcription factor binding sites. Nevertheless, expression of the four gene cluster does not originate within the intergenic region. Rather, cexPABC are expressed from the Rns-dependent cexE promoter. The remaining gene of the system, cexD, is monocistronic and expressed from its own Rns-dependent promoter. Mutagenesis of a predicted Rns binding site upstream of cexD abolished Rns binding to a cexD promoter fragment in vitro and activation of the promoter in vivo. CexD is a novel bacterial acyltransferase. Unlike canonical bacterial acyltransferases that lipidate amino-terminal cysteines, CexD attaches a lipid to the amino-terminal glycine of CexE. Lipidation must occur in the periplasm because topological mapping revealed that CexD has six transmembrane domains with both its amino-terminus and enzymatic domain in the periplasm. We further show that CexD is required for CexE localization to the outer leaflet of the outer membrane and that a cexD mutant of C. rodentium is attenuated in a murine model. These findings further define the Rns virulence regulon, the enzymology of a noncanonical acyltranferase and its contributions to pathogenicity.

microbiology↗

bioRxiv2025

Characterization of the ligand binding pocket of the virulence regulator Rns, a Member of the AraC/XylS family of transcription factors

Diarrheal disease caused by Gram-negative enteric pathogens such as enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella spp., and, Salmonella spp. are a leading cause of morbidity and mortality of children; especially, in low resource nations. While progress has been made in reducing this burden, there remains a need to develop efficacious therapies. Recently, we determined the structure of Rns, a member of the AraC/XylS family that regulates the expression of pili and other virulence factors in ETEC. The structure revealed decanoic acid bound between the N- and C-terminal domains. To test the hypothesis that bound decanoic acid directly inhibits Rns, we used its structure to identify residues that are necessary for ligand binding. Removal of the positive side chains of R75 and H20 rendered Rns insensitive to fatty acid inhibition. Additionally, mutations designed to occlude decanoic acid binding also produced a variant that was fatty acid insensitive. We also observed that this variant is structurally more flexible than wildtype Rns with decanoic acid; suggesting that bound fatty acid contributes to structural rigidity. These studies precisely demonstrate Rns binding pocket residues critical for binding fatty acids and inhibition of DNA binding. This supports our hypothesis that fatty acids must bind in the binding pocket to inhibit AraC regulators. Further work by us and others suggests inhibition of AraC virulence regulators by fatty acids is a common paradigm among many bacterial pathogens. Therefore, understanding the molecular basis of this inhibition lays the groundwork for the development of small molecule therapeutics targeting enteric disease.

microbiology↗

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↗

bioRxiv2020

The structure of Rns from enterotoxigenic Escherichia coli reveals a small molecule inhibitor

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal disease worldwide and a frequent cause of travelers diarrhea. In addition to the production of enterotoxins, studies with human volunteers established ETEC virulence is dependent upon the production of proteinaceous adhesive pili for attaching to the intestinal wall. Although pilins are highly immunogenic, vaccines incorporating them have yet to be proven efficacious. An additional challenge for vaccines is the heterogeneity of ETEC pili, as 20 different pilus types have been identified. However, the expression of a significant number of pilus types is dependent upon Rns, an AraC family transcription factor. Furthermore, Rns also regulates the expression of the virulence factor CexE, an outer membrane coat protein. To determine how Rns functions and is regulated we solved its structure by X-ray crystallography to 3 [A] resolution. Rns forms a dimer via its N-terminal domain and its structure is consistent with the dimer binding looped DNA. Our analyses also revealed a fatty acid, decanoic acid, bound within the Rns structure. Although Rns was not known to specifically bind small molecule ligands, biochemical analysis showed decanoic acid specifically stabilized Rns in a dose dependent manner. Lac reporter assays further showed that decanoic acid inhibits Rns function at both activated and repressed promoters. In situ, exogenous decanoic acid inhibited the expression of Rns-dependent CFA/I pili and CexE in different ETEC strains. Thus, our study reveals for the first time a naturally occurring small molecule ligand specifically inhibits Rns activity and potently suppresses the expression of ETEC virulence factors. Our findings provide an alternative approach to vaccines for inhibiting ETEC pathogenesis by using the Rns structure as a framework for rational drug design.

biochemistry↗

bioRxiv2018

CexE Is A Virulence Factor of Citrobacter rodentium And Present In Enteric Pathogens Of Humans

CexE is a 12 kDa protein that was originally reported to be present in just three strains of enterotoxigenic Escherichia coli (ETEC); a frequent causes of diarrheal illnesses worldwide. However, an examination of recently sequenced genomes has revealed that CexE is actually present in a majority of ETEC strains. Homologs of CexE are also present in enteroaggregative E. coli (EAEC) and other enteric pathogens including Yersinia enterocolitica, Providencia alcalifaciens, and Citrobacter rodentium. CexE and its homologs are expressed within virulence regulons of ETEC, EAEC, and C. rodentium. This, along with its distribution across several species of enteric pathogens, suggest that CexE confers a selective advantage to these pathogens. However, this hypothesis has yet to be tested in vivo. Here we demonstrate that CexE is conditionally secreted to the external leaf of the outer membrane of ETEC. Although CexE does not appear to play a role in adherence in vitro, it does facilitate colonization of murine intestinal tissues by C. rodentium in vivo. In adult mice wild-type bacteria reached significantly higher loads and were shed in higher numbers than a cexE::kan mutant. A similar trend was observed in neonatal mice. In addition, all of the neonates infected with the wild-type strain succumbed to infection within 16 days of inoculation. In contrast, 45% of the neonates infected with the cexE::kan strain survived for the 30 day duration of the experiment. These finding indicate that CexE is a conditionally secreted virulence factor that increases the colonization of hosts by enteric pathogens.

microbiology↗

bioRxiv2018

Perforin-2 Permeabilizes the Envelope of Phagocytosed Bacteria

Perforin-2, the product of the MPEG1 gene, limits the spread and dissemination of bacterial pathogens in vivo. It is highly expressed in murine and human phagocytes, and macrophages lacking Perforin-2 are compromised in their ability to kill phagocytosed bacteria. In this study we used Salmonella typhimurium as a model intracellular pathogen to elucidate the mechanism of Perforin-2 s bactericidal activity. In vitro Perforin-2 was found to facilitate the degradation of antigens contained within the envelope of phagocytosed bacteria. In contrast, degradation of a representative surface antigen was found to be independent of Perforin-2. Consistent with our in vitro results a protease sensitive, periplasmic superoxide disumutase (SodCII) contributed to the virulence of S. typhimurium in Perforin-2 knockout but not wild-type mice. In aggregate our studies indicate that Perforin-2 breaches the envelope of phagocytosed bacteria facilitating the delivery of proteases and other antimicrobial effectors to sites within the bacterial envelope.

immunology↗