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Identification of novel Listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator, PrfA.

ABSTRACT: Upon bacterial entry into the cytosol of infected mammalian host cells, the central virulence regulator PrfA of Listeria monocytogenes becomes activated and induces the expression of numerous factors which contribute to bacterial pathogenesis. The mechanism or signal by which PrfA becomes activated during the course of infection has not yet been determined; however, several amino acid substitutions within PrfA (known as PrfA* mutations) that appear to lock the protein into a constitutively activated state have been identified. In this study, the PrfA activation statuses of several L. monocytogenes mutant strains were subjected to direct isogenic comparison and the mutant with the highest activity, the prfA(L140F) mutant, was identified. The prfA(L140F) strain was subsequently used as a tool to identify gene products secreted as a result of PrfA activation. By use of two-dimensional gel electrophoresis followed by liquid chromatography-electrospray ionization-tandem mass spectroscopy analyses, 15 proteins were identified as up-regulated in the prfA(L140F) secretome, while the secretion of two proteins was found to be reduced. Although some of the proteins identified were known to be subject to direct regulation by PrfA, the majority have not previously been associated with PrfA regulation and their expression or secretion may be influenced indirectly by a PrfA-dependent regulatory pathway. Plasmid insertion inactivation of the genes encoding four novel secreted products indicated that three of the four have significant roles in L. monocytogenes virulence. The use of mutationally activated prfA alleles therefore provides a useful approach towards identifying gene products that contribute to L. monocytogenes pathogenesis.

SUBMITTER: Port GC

PROVIDER: S-EPMC2168340 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Identification of novel Listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator, PrfA.

Port Gary C GC Freitag Nancy E NE

Infection and immunity 20071015 12

Upon bacterial entry into the cytosol of infected mammalian host cells, the central virulence regulator PrfA of Listeria monocytogenes becomes activated and induces the expression of numerous factors which contribute to bacterial pathogenesis. The mechanism or signal by which PrfA becomes activated during the course of infection has not yet been determined; however, several amino acid substitutions within PrfA (known as PrfA* mutations) that appear to lock the protein into a constitutively activ ...[more]

PMID: 17938228

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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

Identification of novel Listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator, PrfA.

Publications

Identification of novel Listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator, PrfA.

Similar Datasets

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