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Probing the role of protein surface charge in the activation of PrfA, the central regulator of Listeria monocytogenes pathogenesis.

ABSTRACT: Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor.

SUBMITTER: Xayarath B

PROVIDER: S-EPMC3155570 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Probing the role of protein surface charge in the activation of PrfA, the central regulator of Listeria monocytogenes pathogenesis.

Xayarath Bobbi B Volz Karl W KW Smart Jennifer I JI Freitag Nancy E NE

PloS one 20110812 8

Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur ...[more]

PMID: 21858145

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Project description:Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes serious disease in immunocompromised individuals, pregnant women, and neonates. Bacterial virulence is mediated by the expression of specific gene products that facilitate entry into host cells and enable bacterial replication; the majority of these gene products are regulated by a transcriptional activator known as PrfA. L. monocytogenes strains containing prfA E77K or prfA G155S mutations exhibit increased expression of virulence genes in broth culture and are hypervirulent in mice. To define the scope of the influences of the prfA E77K and prfA G155S mutations on L. monocytogenes pathogenesis, multiple aspects of bacterial invasion and intracellular growth were examined. Enhanced bacterial invasion of host epithelial cells was dependent on the expression of a number of surface proteins previously associated with invasion, including InlA, InlB, and ActA. In addition to these surface proteins, increased production of the hly-encoded secreted hemolysin listeriolysin O (LLO) was also found to significantly enhance bacterial invasion into epithelial cell lines for both prfA mutant strains. Although prfA E77K and prfA G155S strains were similar in their invasive phenotypes, the infection of epithelial cells with prfA E77K strains resulted in host cell plasma membrane damage, whereas prfA G155S strains did not alter plasma membrane integrity. Bacterial infection of human epithelial cells, in which the production of LLO is not required for bacterial entry into the cytosol, indicated that prfA E77K cytotoxic effects were mediated via LLO. Both prfA E77K and prfA G155S strains were more efficient than wild-type bacteria in gaining access to the host cell cytosol and in initiating the polymerization of host cell actin, and both were capable of mediating LLO-independent lysis of host cell vacuoles in cell lines for which L. monocytogenes vacuole disruption normally requires LLO activity. These experiments illuminate the diverse facets of L. monocytogenes pathogenesis that are significantly enhanced by the constitutive activation of PrfA via prfA mutations and underscore the critical role of this protein in promoting L. monocytogenes virulence.

Project description:Listeria monocytogenes is able to efficiently utilize glycerol as a carbon source. In a defined minimal medium, the growth rate (during balanced growth) in the presence of glycerol is similar to that in the presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed high-level transcriptional upregulation of the genes known to be involved in glycerol uptake and metabolism (glpFK and glpD) in the presence of glycerol (compared to that in the presence of glucose and/or cellobiose). Levels of expression of the genes encoding a second putative glycerol uptake facilitator (GlpF(2)) and a second putative glycerol kinase (GlpK(2)) were less enhanced under these conditions. GlpK(1) but not GlpK(2) was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while the loss of GlpK(1) affected replication in Caco-2 cells less than did the loss of GlpK(2) and GlpD. Additional genes whose transcription levels were higher in the presence of glycerol than in the presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression control. Transcriptional downregulation in the presence of glycerol (compared to those in the presence glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N metabolism, and the biosynthesis of branched-chain amino acids. The highest level of transcriptional upregulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions, a low level of HPr-Ser-P and a high level of HPr-His-P were present in the cells, suggesting that all enzyme IIA (EIIA) (or EIIB) components of the phosphotransferase system (PTS) permeases expressed will be phosphorylated. These and other data suggest that the phosphorylation state of PTS permeases correlates with PrfA activity.

Project description:Naturally occurring free fatty acids (FFAs) are recognized as potent antimicrobial agents that also affect the production of virulence factors in bacterial pathogens. In the foodborne pathogen Listeria monocytogenes, some medium- and long-chain FFAs act as antimicrobial agents as well as signaling compounds, causing a repression of transcription of virulence genes. We previously observed that the master virulence regulator PrfA is involved in both the antimicrobial and virulence-inhibitory response of L. monocytogenes to selected FFAs, but the underlying mechanisms are presently unknown. Here, we present a systematic analysis of the antimicrobial and PrfA-inhibitory activities of medium- and long-chain FFAs of various carbon chain lengths and degrees of saturation. We observed that exposure to specific antimicrobial and nonantimicrobial FFAs prevented PrfA-dependent activation of virulence gene transcription and reduced the levels of PrfA-regulated virulence factors. Thus, an antimicrobial activity was not compulsory for the PrfA-inhibitory ability of an FFA. In vitro binding experiments revealed that PrfA-inhibitory FFAs were also able to prevent the constitutively active variant PrfA* from binding to the PrfA box in the promoter region of the virulence gene hly, whereas noninhibitory FFAs did not affect its ability to bind DNA. Notably, the unsaturated FFAs inhibited the DNA binding activity of PrfA* most efficiently. Altogether, our findings support a model in which specific FFAs orchestrate a generalized reduction of the virulence potential of L. monocytogenes by directly targeting the key virulence regulator PrfA.IMPORTANCE Listeria monocytogenes is a Gram-positive pathogen able to cause foodborne infections in humans and animals. Key virulence genes in L. monocytogenes are activated by the transcription regulator PrfA, a DNA binding protein belonging to the CRP/FNR family. Various signals from the environment are known to affect the activity of PrfA, either positively or negatively. Recently, we found that specific medium- and long-chain free fatty acids act as antimicrobial agents as well as signaling compounds in L. monocytogenes Here, we show that both antimicrobial and nonantimicrobial free fatty acids inhibit PrfA-dependent activation of virulence gene transcription by interfering with the DNA binding activity of PrfA. Our findings suggest that free fatty acids could be candidates for alternative therapies against L. monocytogenes.

Dataset Information

Probing the role of protein surface charge in the activation of PrfA, the central regulator of Listeria monocytogenes pathogenesis.

Publications

Probing the role of protein surface charge in the activation of PrfA, the central regulator of Listeria monocytogenes pathogenesis.

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