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Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential.

ABSTRACT: A total of 133 Listeria monocytogenes isolates were characterized by ribotyping and allelic analysis of the virulence genes hly, actA, and inlA to uncover linkages between independent phylogenetic and specific virulence markers. PCR-restriction fragment length polymorphisms revealed 8 hly, 11 inl4, and 2 actA alleles. The combination of these virulence gene alleles and ribotype patterns separated L. monocytogenes into three distinct lineages. While distinct hly and inlA alleles were generally found to cluster into these three lineages, actA alleles segregated independently. These three phylogenetic lineages were confirmed when 22 partial actA DNA sequences were analyzed. The clinical history of the L. monocytogenes strains showed evidence for differences in pathogenic potential among the three lineages. Lineage I contains all strains isolated during epidemic outbreaks of listeriosis, while no human isolates were found in lineage III. Animal isolates were found in all three lineages. We found evidence that isolates from lineages I and III have a higher plaquing efficiency than lineage II strains in a cell culture assay. Strains from lineage III also seem to form larger plaques than strains from lineage II. A distinctive ribotype fragment and unique 16S rRNA gene sequences furthermore suggest that lineage III might represent a L. monocytogenes subspecies. None of the 20 human isolates available but 11% of our animal isolates were grouped in this lineage, indicating that strains in this lineage might have reduced virulence for humans.

SUBMITTER: Wiedmann M

PROVIDER: S-EPMC175382 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

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Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential.

Wiedmann M M Bruce J L JL Keating C C Johnson A E AE McDonough P L PL Batt C A CA

Infection and immunity 19970701 7

A total of 133 Listeria monocytogenes isolates were characterized by ribotyping and allelic analysis of the virulence genes hly, actA, and inlA to uncover linkages between independent phylogenetic and specific virulence markers. PCR-restriction fragment length polymorphisms revealed 8 hly, 11 inl4, and 2 actA alleles. The combination of these virulence gene alleles and ribotype patterns separated L. monocytogenes into three distinct lineages. While distinct hly and inlA alleles were generally fo ...[more]

PMID: 9199440

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Project description:Listeriamonocytogenes lineages III and IV represent two uncommon lineages of the human and animal pathogen L. monocytogenes, characterized by occurrence of unusual phenotypic and genetic characteristics that differentiate them from the common lineages I and II. To gain further insights into the evolution of lineages III and IV, we amplified and sequenced housekeeping genes (i.e., gap, prs, purM, ribC, and sigB), internalin genes (i.e., inlA, inlB, inlC, inlG, inlC2, inlD, inlE, inlF, and inlH) and the virulence gene cluster containing prfA, plcA, hly, mpl, actA, and plcB for lineages III (n = 7) and IV (n = 4) isolates. Phylogenetic analyses of the sequences obtained along with previously reported sequence data for 40 isolates representing lineages I (n = 18), II (n = 21), and III (n = 1), showed that lineages III and IV represent divergent and monophyletic lineages. The virulence gene cluster as well as the inlAB operon were present in all isolates, with inlF absent from all lineages III and IV isolates. While all lineage IV isolates contained only inlC (in addition to inlAB), lineage III isolates showed considerable diversity with regard to internalin gene presence, including presence of (i) only inlC (n = 2), (ii) inlC and inlGC2DE (n = 3), (iii) only inlGC2DE (n = 2), and (iv) inlC and inlC2DE (n = 1). In addition to evidence for horizontal gene transfer events, among lineages III and IV isolates, in prs, actA, plcB, mpl, inlA, inlB, inlG, inlD, and inlE, we also found significant evidence for positive selection in the hly promoter region and, along the lineages III and IV branches, for actA (including in sites recognized for interactions with proteins involved in actin tail polymerization). In conclusion, lineages III and IV represent two distinct monophyletic groups with contributions of intragenic recombination to the evolution of their internalin genes as well as contributions of positive selection to evolution of the virulence genes island.

Project description:As for many opportunistic pathogens, the virulence potential of Listeria monocytogenes is highly heterogeneous between isolates and correlated, to some extent, with phylogeny and gene repertoires. In sharp contrast with copious data on intraspecies genome diversity, little is known about transcriptome diversity despite the role of complex genetic regulation in pathogenicity. The current study implemented RNA sequencing to characterize the transcriptome profiles of 33 isolates under optimal in vitro growth conditions. Transcript levels of conserved single-copy genes were comprehensively explored from several perspectives, including phylogeny, in silico-predicted virulence category based on epidemiological multilocus sequence typing (MLST) data, and in vivo virulence phenotype assessed in Galleria mellonella Comparing baseline transcriptomes between isolates was intrinsically more complex than standard genome comparison because of the inherent plasticity of gene expression in response to environmental conditions. We show that the relevance of correlation analyses and their statistical power can be enhanced by using principal-component analysis to remove the first level of irrelevant, highly coordinated changes linked to growth phase. Our results highlight the major contribution of transcription factors with key roles in virulence to the diversity of transcriptomes. Divergence in the basal transcript levels of a substantial fraction of the transcriptome was observed between lineages I and II, echoing previously reported epidemiological differences. Correlation analysis with in vivo virulence identified numerous sugar metabolism-related genes, suggesting that specific pathways might play roles in the onset of infection in G. mellonellaIMPORTANCEListeria monocytogenes is a multifaceted bacterium able to proliferate in a wide range of environments from soil to mammalian host cells. The accumulated genomic data underscore the contribution of intraspecies variations in gene repertoire to differential adaptation strategies between strains, including infection and stress resistance. It seems very likely that the fine-tuning of the transcriptional regulatory network is also a key component of the phenotypic diversity, albeit more difficult to investigate than genome content. Some studies reported incongruity in the basal transcriptome between isolates, suggesting a putative relationship with phenotypes, but small isolate numbers hampered proper correlation analyses with respect to their characteristics. The present study is the embodiment of the promising approach that consists of analyzing correlations between transcriptomes and various isolate characteristics. Statistically significant correlations were found with phylogenetic groups, epidemiological evidence of virulence potential, and virulence in Galleria mellonella larvae used as an in vivo model.

Project description:Background: L. monocytogenes meningoencephalitis has a mortality rate of up to 50% and neurofunctional sequelae are common. Type I restriction-modification systems (RMS) are capable of adding methyl groups to the host genome. Some contain multiple sequence recognition (hsdS) genes that recombine, resulting in distinct DNA methylation patterns and patterns of gene expression. These phenotypic switches have been linked to virulence and have recently been discovered in multiple clonal complexes of L. monocytogenes. In the present study, we investigated the significant of RMS on L. monocytogenes virulence during the acute phase of experimental meningitis. Methods: L. monocytogenes strains containing RMS systems were identified, and purified clones enriched for single hsdS alleles were isolated. In vivo, 11-day old Wistar rats were infected with an inoculum containing (a) one of 4 single RMS allele variants (A, B, C, D) treated with amoxicillin (AMX 50 mg/kg/dosis, q8h), (b) a mixture of all 4 variants with or without AMX treatment, or (c) different mixtures of 2 RMS allele variants. At selected time points after infection, clinical and inflammatory parameters, bacterial titers and brain damage were determined. Changes in the relative frequency of the occurring RMS alleles in the inoculum and in CSF or cerebellum of infected animals were analyzed by capillary electrophoresis. Results: We have identified a phase variable RMS locus within L. monocytogenes CC4 and generated stocks that stably expressed each of the possible hsdS genes within that loci. Generation of these allele variants (A, B, C, D) allowed us to determine the methylation pattern associated with each hsdS through SMRT sequencing. In vivo infections with these single allele variants revealed differences in disease severity in that C induced the worst clinical outcome and more pronounced hippocampal apoptosis; D showed the most pronounced weight loss and the highest bacterial titer in the cerebellum. A caused the least severe disease. Conclusion: We identified that L. monocytogenes expressing hsdS (A) causes less damage than when other hsdS genes are expressed. While expression of hsdSC and D worsened the outcome in L. monocytogenes meningitis. We also demonstrate a competitive advantage of variants C and B over variant A in this model. Phenotypical switching may therefore represent a mechanism of virulence regulation during the acute phase of CNS infections with L. monocytogenes.

Project description:BackgroundListeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.Methodology/principal findingsThe objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp(LAP)) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp(LAP) for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp(LAP) pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp(LAP) also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp(LAP) cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.Conclusions/significanceWild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp(LAP) blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.

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Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential.

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Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential.

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