Evolutionary history of the genus Listeria and its virulence genes

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9 kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence of the virulence gene cluster locus and its flanking regions was either determined in this study or assembled from public databases. Furthermore, some virulence-associated internalin loci were compared among the six species. Phylogenetic analyses were performed on a data set containing the sequences of prs, ldh, vclA, and vclB (all directly flanking the virulence gene cluster), as well as the iap gene and the 16S and 23S-rRNA coding genes which are located at different sites in the listerial chromosomes. L. grayi represents the deepest branch within the genus. The remaining five species form two groupings which have a high bootstrap support and which are consistently found by using different treeing methods. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch. Based on this perception, we tried to reconstruct the evolution of the virulence gene cluster. Since no traces of lateral gene transfer events could be detected the most parsimonious scenario is that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri and that the pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. This hypothesis is also supported by the location of the putative deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri.     

A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins

Listeria ivanovii differs from the human pathogen Listeria monocytogenes in that it specifically affects ruminants, causing septicaemia and abortion but not meningo-encephalitis. The genetic characterization of spontaneous L. ivanovii mutants lacking the virulence factor SmcL (sphingomyelinase) led us to identify LIPI-2, the first species-specific pathogenicity island from Listeria. Besides SmcL, this 22 kb chromosomal locus encodes 10 internalin (Inl) proteins: i-InlB1 and -B2 are large/surface-associated Inls similar to L. monocytogenes InlB; i-InlE to -L are small/excreted (SE)-Inls, i-InlG being a tandem fusion of two SE-Inls. Except i-inlB1, all LIPI-2 inl genes are controlled by the virulence regulator, PrfA. LIPI-2 is inserted into a tRNA locus and is unstable - half of it deleting at approximately 10(-4) frequency with a portion of contiguous DNA. The spontaneous mutants were attenuated in vivo in mice and lambs and showed impaired intracellular growth and apoptosis induction in bovine MDBK cells. Targeted knock-out mutations associated the virulence defect with LIPI-2 genes. The region between the core genome loci ysnB-tRNA(arg) and ydeI flanking LIPI-2 contained different gene complements in the different Listeria spp. and even serovars of L. monocytogenes, including remnants of the PSA bacteriophage int gene in serovar 4b, indicating it is a hot spot for horizontal genome diversification. LIPI-2 is conserved in L. ivanovii ssp. ivanovii and londoniensis, suggesting an early acquisition during the species' evolution. LIPI-2 is likely to play an important role in the pathogenic and host tropism of L. ivanovii.     

Effect of growth temperature on virulence of strains of Listeria monocytogenes in the mouse: evidence for a dose dependence

Growth of Listeria monocytogenes at 4 degrees C significantly increased its virulence for mice by the intravenous route and the effect was dose-dependent. Virulence was apparent only at a dose of about or above 10(4) viable listerias. At slightly lower doses of about 10(3), no such effect was observed. Growth at 4 degrees C did not increase the virulence of the strains for mice by oral-gastric challenge when given at doses of approximately 10(10).     

Biodiversity of the species Listeria monocytogenes and the genus Listeria

This review describes the Listeria monocytogenes genome sequences available today and their comparison with that of Listeria innocua and Listeria welshimeri by highlighting their characteristic features and common traits. The diversity present among them is analysed with emphasis on putative virulence and host-pathogen interaction related functions. Then large-scale studies comparing gene content of Listeria and how these studies contributed to typing applications will be discussed. Finally, evolutionary conclusions and future perspectives in Listeria genomics are presented.     

Effect of growth temperature on virulence of strains of Listeria monocytogenes in the mouse: evidence for a dose dependence

Growth of Listeria monocytogenes at 4°C significantly increased its virulence for mice by the intravenous route and the effect was dose-dependent. Virulence was apparent only at a dose of about or above 10⁴ viable listerias. At slightly lower doses of about 10³, no such effect was observed. Growth at 4°C did not increase the virulence of the strains for mice by oral-gastric challenge when given at doses of approximately 10¹⁰.     

A comparison of selected methods for measuring the virulence properties of Listeria spp

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.     

Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes

Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways. We investigated whether L. monocytogenes modulates T-cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells. In vitro culture of murine splenocytes with L. monocytogenes resulted in a specific and dose-dependent upregulation of Fas ligand (FasL). Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non-pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s). Examination of L. monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine-preferring phospholipase C (PC-PLC). Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC-PLC synergized with LLO for the induction of FasL expression. FasL-expressing cells induced by L. monocytogenes were capable of killing Fas-expressing target cells. Furthermore, L. monocytogenes infection results in upregulation of FasL on T cells in mice. These results describe a novel function for LLO and PC-PLC and suggest that L. monocytogenes may use these virulence factors to modulate the host immune response.     

Taxonomy of the genus Listeria by using multilocus enzyme electrophoresis

Seventy-three strains of the seven recognized Listeria species were studied by performing a multilocus enzyme electrophoresis analysis of 18 enzyme loci. The mean number of alleles per locus was 9.5 and all of the loci were polymorphic. A total of 56 electrophoretic types were distinguished. Cluster analysis of a matrix of the genetic distances between paired electrophoretic types revealed that there were six principal clusters at the species level (genetic distances between clusters greater than 0.8). Listeria monocytogenes, Listeria innocua, Listeria welshimeri, Listeria seeligeri, and Listeria ivanovii each corresponded to one of these clusters with no overlap. Our results are in agreement with those of previous DNA hybridization experiments (Rocourt et al., Curr. Microbiol. 7:383-388, 1982). Listeria grayi and Listeria murrayi electrophoretic types formed a unique cluster, thus reinforcing the suggestion of Wilkinson and Jones (J. Gen. Microbiol. 98:399-421, 1977) that these two species should be considered two biovars of a single species.     

Phosphatidylinositol-specific phospholipase C of Bacillus anthracis down-modulates the immune response

Phosphatidylinositol-specific phospholipases (PI-PLCs) are virulence factors produced by many pathogenic bacteria, including Bacillus anthracis and Listeria monocytogenes. Bacillus PI-PLC differs from Listeria PI-PLC in that it has strong activity for cleaving GPI-anchored proteins. Treatment of murine DCs with Bacillus, but not Listeria, PI-PLC inhibited dendritic cell (DC) activation by TLR ligands. Infection of mice with Listeria expressing B. anthracis PI-PLC resulted in a reduced Ag-specific CD4 T cell response. These data indicate that B. anthracis PI-PLC down-modulates DC function and T cell responses, possibly by cleaving GPI-anchored proteins important for TLR-mediated DC activation.     

A putative P-type ATPase required for virulence and resistance to haem toxicity in Listeria monocytogenes

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem.     

Sigma B在单核细胞增生李斯特菌耐受环境压力胁迫中的作用

摘要：Sigma B（σB）因子在单核细胞增生李斯特菌（Listeria monocytogenes）的压力应答和毒力调控中起着重要的作用。研究发现单核细胞增生李斯特菌的致病力与耐受环境条件的胁迫息息相关。在压力存在的情况下能启动一些基因的表达,以增强细菌对环境的耐受性,这些基因包括与耐受渗透压、酸碱性环境、氧化、极端温度和宿主体内胆碱等环境压力相关的基因。本文综述了σB因子在上述几种环境压力胁迫中的作用,为深入了解该菌的生理特征、探讨食品的最佳生产和保藏条件、防止细菌的感染等方面提供新的理论依据。     

The use of listeriolysin to identify in vivo induced genes in the gram-positive intracellular pathogen Listeria monocytogenes

Listeria monocytogenes is capable of growth within the cytoplasm of infected host cells. Escape from the host cell phagosome is mediated primarily through secretion of listeriolysin, a haemolytic factor which functions to actively lyse the phagosomal membrane. Listeriolysin negative mutants of L. monocytogenes are non-haemolytic on blood agar plates and demonstrate a significant reduction of virulence in the mouse model of infection. We have developed a system for the identification of in vivo induced genes in L. monocytogenes which utilizes the listeriolysin gene, hly, as both a reporter of gene expression and as a means of selection of promoter elements expressed in vivo. The system is analogous to in vivo expression technology (IVET) first reported for Salmonella, however, as listeriolysin functions in the environment of the host phagosome the loci identified in this study are most likely expressed during residence in the phagosome. The system was successfully tested using the promoter of the inducible virulence gene plcA. A bank was created by fusing a promoterless copy of hly to random promoter elements in a listeriolysin negative IVET host. Sequential inoculations of mice with this bank resulted in the isolation of clones with increased survival potential in the mouse model relative to a negative control, but which remained haemolysin negative on blood agar plates. Nine in vivo induced loci were identified including genes encoding a DNA topoisomerase III, a cellobiose transporter and a fumarase. Two isolates represented fusions to proteins of unknown function and three isolates contained no significant homologues in the database. A mutant in the fumarase gene demonstrated reduced virulence for mice and an inability to grow in cultured mouse phagocytes.     

单核细胞增生李斯特菌PrfA蛋白转录调控毒力基因表达的分子机制

单核细胞增生李斯特菌 (Listeria monocytogenes LM) 属于典型的细胞内寄生革兰氏阳性菌, 是WHO公布的四大食源性致病菌之一。LM不仅是人畜共患传染病李斯特菌病 (listeriosis) 的主要病原菌, 也是研究胞内感染和细胞介导的免疫应答的模式细菌。绝大多数LM毒力基因的转录表达受到PrfA蛋白的调控。本文简单介绍了LM侵染宿主细胞必需的毒力基因及其产物; 重点对毒力基因调节蛋白PrfA的结构和功能, PrfA调节毒力基因表达的主要方式最新进展进行了综述和讨论。     

Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor?

A phospholipase C which cleaves phosphatidylinositol and glycosylphosphatidylinositol (GPI) anchors was identified in Listeria monocytogenes. This 36 kDa protein is encoded by the gene plcA, and is homologous to the Bacillus cereus, Bacillus thuringiensis and eukaryotic phosphatidylinositol-specific phospholipases C (PI-PLC). Expression of the plcA gene in Escherichia coli correlates with the appearance of PI-PLC activity in the cells. In Listeria monocytogenes, the activity is secreted to the culture medium. PI-PLC activity was only found in the two pathogenic species of the genus Listeria, namely L. monocytogenes and L. ivanovii. PI-PLC activity was lost and virulence decreased when the plcA gene was disrupted in the chromosome. This suggests that the PI-PLC of L. monocytogenes might be involved in virulence.     

单核细胞增生李斯特菌PrfA蛋白转录调控毒力基因表达的分子机制

单核细胞增生李斯特菌(Listeria monocytogenes LM)属于典型的细胞内寄生革兰氏阳性菌,是WHO公布的四大食源性致病菌之一.LM不仅是人畜共患传染病李斯特菌病(listeriosis)的主要病原菌,也是研究胞内感染和细胞介导的免疫应答的模式细菌.绝大多数LM毒力基因的转录表达受到PrfA蛋白的调控.本文简单介绍了LM侵染宿主细胞必需的毒力基因及其产物;重点对毒力基因调节蛋白PrfA的结构和功能,PrfA调节毒力基因表达的主要方式最新进展进行了综述和讨论.     

Comparison of virulence and activity of some enzymes ofListeria monocytogenes

The virulence of microorganisms and the activity of some enzymes were compared on 17 strains ofListeria monocytogenes, including 5 Patterson's strains, 4 strains maintained for longer periods on nutrient media and 8 strains freshly or recently isolated from animals in our field material. The LD₅₀ and invasivity index were determined as indicators of virulence. The activity of catalase, glucose and lactic acid dehydrogenases and glutamic-oxalo-acetic transaminase were determined quantitatively. Close agreement was found between the values of both indicators of virulence and the activity of catalase. The quantitative assay of catalase and to some extent of some dehydrogenases, as well as glutamic-oxaloacetic transaminase thus represents a simple and satisfactory method of assessing the virulence ofListeria monocytogenes.