A Contingency Locus in prfA in a Listeria monocytogenes Subgroup Allows Reactivation of the PrfA Virulence Regulator during Infection in Mice

Abstract

A nonhemolytic Listeria monocytogenes strain isolated from a fish processing plant was avirulent in a plaque-forming assay and in a subcutaneous mouse virulence assay. However, it showed 60% lethality (9/15 mice) when 10 9 CFU were intraperitoneally injected into mice. Hemolytic L. monocytogenes bacteria were recovered from liver and spleen of the deceased mice, and the pulsed-field gel electrophoresis patterns were indistinguishable for the nonhemolytic and the hemolytic isolates. Sequencing of prfA from the nonhemolytic strain revealed a duplication of 7 bp in the helix-turn-helix region, resulting in a truncated PrfA protein. We propose that the direct repeat of 7 bp causes a reversible inactivation of prfA and that slipped-strand mispairing regulates the phase variation in hemolytic activity and virulence. Nonhemolytic L. monocytogenes strains with identical duplications in prfA were isolated from several sources in France, as well as in Norway, suggesting that the reversible inactivation described in this study is not an isolated event.Listeria monocytogenes is a Gram-positive, facultative intracellular bacterial pathogen causing the food-borne infection listeriosis, a rare but serious illness with a mortality rate of 30%. Malignancies, diabetes mellitus, old age, pregnancy, and immunosuppressive diseases and treatments are predisposing factors to listeriosis. In 2007, 0.3 cases per 100,000 population were reported in the EU states (2); however, the annual numbers of reported listeriosis cases are increasing in several European countries (1, 4, 6). More elderly people, an increase in diabetes mellitus in the population, and more individuals on cancer and immunosuppressive medications results in a higher risk imposed by the presence of L. monocytogenes in foods.PrfA, a pleiotropic transcriptional regulator, activates the transcription of L. monocytogenes virulence genes, such as hly, plcA, plcB, and inlA, by binding to a DNA sequence present in the promoter of its target genes. Low production of listeriolysin O results in low hemolytic activity and has been linked to low pathogenicity (3); thus, hemolysis on blood agar is an important criterion in the diagnosis of L. monocytogenes. Chromogenic agars, such as OCLA (Oxoid chromogenic listeria agar) and RapidЈL.mono, utilize the expression of phospholipases to distinguish between pathogenic and nonpathogenic Listeria species. Low-virulence L. monocytogenes strains vary in their expression of listeriolysin O and phospholipases (7) and may easily be overlooked when the identification of Listeria species is based upon hemolysis and phospholipase activity. The pathogenicity of low-virulence L. monocytogenes strains toward predisposed individuals is unknown.A survey of L. monocytogenes in smoked salmon was carried out in Norway by the Norwegian Food Safety Authority in 2003 (26) and indicated a low prevalence of L. monocytogenes in the tested products. However, one producer had a high prevalence of an atypical L. monocytogenes strain over a period of half a year. Fo...

The role of variable DNA tandem repeats in bacterial adaptation

“…In general, the effect of DNA replication on TR rearrangements provides evidence for the replication slippage mechanism, because a prerequisite of this mechanism is that DNA replication is stalled by the secondary loop structures formed by TRs. Notably, the SSM mechanism is not only widely accepted to account for the majority of SSR variations, it is also invoked to explain the genesis of TRs from unrepeated DNA (Levinson & Gutman, 1987;Waite et al, 2003;Lindb€ ack et al, 2011).…”

Low Potential Virulence Associated with Mutations in the inlA and prfA Genes in Listeria monocytogenes Isolated from Raw Retail Poultry Meat

“…Sequencing of the prfA gene of this R6 strain revealed that it had a sevennucleotide insertion at codon 171, which would give rise to a truncated PrfA protein (PrfAA 174-237) (23). This mutation can revert when using high infective doses in certain animal models, giving rise to a fully virulent phenotype (11).…”

“…According to Roche et al (24), if one includes all of the strains that are nonhemolytic, have either a truncated InlA (approximately 45% of the L. monocytogenes isolates from RTE foods (29)) or prfA mutations (about 25% of the low-virulence strains (23)), or present any other defects in potential virulence, avirulent or low virulence strains could easily represent 50% of all L. monocytogenes field strains. Thus, inlA PMSC appear the most common virulence attenuation mechanism at the population level (29), although prfA mutants could be common at specific food environments (11). One possibility for the underestimation of avirulent strains of L. monocy togenes is that most agar media currently used for the isolation of Listeria are based on the detection of virulenceassociated proteins (LLO, PI-PLC, and PC-PLC).…”

“…L. monocytogenes is subdivided into at least four evolu tionary lineages (I through IV), which have different levels of virulence (19). The virulence attenuation presented by a large number of lineage II isolates is because of mutations that give rise to premature stop codons (PMSC) in the inIA gene (which encodes intemalin A) (8,9), along with mutations in the prfA gene (which encodes the central vimlence regulator PrfA) (11,23). Nevertheless, multiple distinct genetic mechanisms could be responsible for natural virulence attenuation in L. monocytogenes.…”

Detection of Plasmid in Listeria monocytogenes Isolated from Fresh Raw Milk in Nigeria

“…Thus, it indicates that the prevalence of Listeria monocytogenes in foods poses a significant danger. The incidence of listeriosis in European Union in 2007 was reported to be 0.3 cases in every 100,000 population (8). In Nigeria, few sporadic cases of listeriosis have been reported (9), there is no data on outbreak of human listeriosis and the sources of contamination were unknown.…”