Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice

Roche, Sylvie; Velge, Philippe; Bottreau, Elisabeth; Durier, Christine; Mee, N. Marquet-Van Der; Pardon, Pierre

doi:10.1016/s0168-1605(01)00460-3

Cited by 96 publications

(115 citation statements)

References 28 publications

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“…A critical aspect of L. monocytogenes pathogenesis is the capacity to spread from cell to cell and form plaques in monolayers of tissue culture cells, which correlates well with mouse virulence (48). The secA2 mutant forms plaque sizes that are approximately 30% of those of the WT (Fig.…”

Section: Resultsmentioning

confidence: 86%

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

2015

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The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ⌬secA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ⌬secA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.T he essential general secretory (Sec) pathway is responsible for exporting the majority of secreted proteins across the bacterial cell membrane (1-3). Much of what we know about this pathway was discovered in Escherichia coli by using genetic screens to identify loss-of-function mutations in components of the Sec system. This led to the identification of components of the SecYEG complex that forms the translocation channel and the SecA ATPase that binds to signal sequences to drive the export of precursor proteins across the channel (3-9). In contrast to loss-of-function sec mutations, prl alleles are gain-of-function mutations, which expand the repertoire of substrates being exported across the SecYEG channel (6-8, 10, 11). The most dominant prl variants are in secY, known as prlA mutations, which allow for the secretion of proteins with altered signal sequence (7, 9, 11) without significantly altering the secretion of other proteins (6,12).In addition to the canonical SecA, an accessory cytosolic ATPase, SecA2, has been identified in Mycobacterium and a subset of other Gram-positive bacteria. Unlike SecA, SecA2 is not essential for cell viability but is required for virulence in some pathogenic bacteria (13-15). SecA1 and SecA2 are homologous but are not interchangeable (16)...

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Section: Resultsmentioning

confidence: 86%

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

2015

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“…Briefly, the Listeria monocytogenes strains (culturable, VBNC and recovered cells) were tested with HT-29 cell monolayers in a plaqueforming assay [48,49] and the strains were also tested for their potential to colonise mouse spleens (Swiss female OF1) after intravenous inoculation [1,8] according to the methods described by Roche et al [42]. The level of virulence determined by in vitro (PFA assay) and in vitro (inoculation in mice) techniques, for each strain used, in the culturable state and in the VBNC state were previously described in Cappelier et al [12] (Tab.…”

Section: Virulence Testsmentioning

confidence: 99%

Avirulent Viable But Non Culturable cells ofListeria monocytogenesneed the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

et al. 2007

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-The aim of this study was to assess the efficiency of the embryonated egg model to recover Viable But Non Culturable (VBNC) cells of Listeria monocytogenes. L. monocytogenes cells were incubated in filtered sterilised distilled water. The VBNC state was obtained after a 25 to 47 days incubation period (concentration of culturable cells less than 1 cfu/mL). Fifteen days after the VBNC state was reached, non culturability was checked in various media. One milliliter of each VBNC suspension that contained 10 4 metabolically active cells (i.e. Direct Viable Count + cells) was inoculated into the vitellus fluid of embryonated and non-embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs (18/32), but not in the non-embryonated eggs (1/32). The recovery rate was higher after culture of the vitellus fluid plus embryo (18/32) than after culture of the vitellus fluid alone (6/32). The results indicate that the embryo likely plays a prominent part in the recovery process. The virulence of recovered cells was assessed by the ability to form plaques in HT-29 cell monolayers and by the ability to colonise mouse spleens. Although the cells were classified as avirulent when in the VBNC state, the virulence was recovered after resuscitation.VBNC / virulence / embryonated eggs / Listeria monocytogenes

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“…The mouse virulence assay provides an in vivo measurement of virulence and often serves as a reference standard for other methods (Pine et al, 1991;Nishibori et al, 1995;Erdenlig et al, 2000;Roche et al, 2001). In vitro culture techniques measure the ability of L. monocytogenes to cause cytopathogenic effects in the enterocytelike cell line Caco-2 (Pine et al, 1991), to form plaques in the human adenocarcinoma cell line HT-29 (Roche et al, 2001) or to cause death in chicken embryos (Olier et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes

Liu

Ainsworth

Austin

et al. 2003

Journal of Medical Microbiology

103

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Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide. However, L. monocytogenes strains demonstrate considerable variation in pathogenic potential. In this report, virulent and avirulent L. monocytogenes isolates were compared by using a comparative screening strategy. Two clones were identified that contained DNA that was only present in virulent L. monocytogenes strains. PCR primers were designed for three genes from these clones and for five other selected L. monocytogenes genes. All eight primer sets predominantly detected virulent L. monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent. Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L. monocytogenes isolates, rather than environmental isolates. The findings from this study suggest that virulent L. monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L. monocytogenes virulence.

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Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice

Cited by 96 publications

References 28 publications

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

Avirulent Viable But Non Culturable cells ofListeria monocytogenesneed the presence of an embryo to be recovered in egg yolk and regain virulence after recovery

Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes

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