Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein

To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogeues could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.     

Interactions between the environmental pathogen Listeria monocytogenes and a free-living protozoan (Acanthamoeba castellanii)

Listeria monocytogenes can cause severe disease in animal hosts, but it has no recognized animal host reservoir. We tested the hypothesis that L. monocytogenes retains virulence traits to survive predation by amoebae and that listeriolysin O plays a crucial role in this process. Co-culturing of L. monocytogenes and Acanthamoeba castellanii demonstrated that L. monocytogenes does not actively kill amoebae, but in the presence of amoebae, high bacterial population densities can be maintained over a period of at least 96 h. A gentamicin protection assay demonstrated that there is no significant difference in the ability to survive predation between serovars (4b versus 1/2a and 1/2c; P  = 0.08) and between five species of Listeria ( P  = 0.14). Three of these species do not harbour the hly gene responsible for listeriolysin O production. A hly knockout strain had poorer survival compared with the parental strain ( P  = 0.04 at 24 h; P  = 0.04 at 48 h; P  = 0.02 at 72 h) and electron microscopy was consistent with a wild-type strain being able to escape the phagosome whereas the hly knockout strain did not appear to have this ability. Thus, while there is weak evidence that listeriolysin O can contribute to improved survival after ingestion by amoebae, listeriolysin O does not appear to provide a significant selective advantage under the conditions of this study.     

携带新城疫病毒融合蛋白基因的重组减毒单核细胞增多性李斯特菌构建与鉴定

以鸡新城疫病毒F基因(NDV-F)为模式外源基因,通过基因切割-重叠延伸PCR法(SOE-PCR)将其插入到单核细胞增多性李斯特菌(Listeria monocytogenes)毒力基因hly的启动子和信号肽序列下游,并将该融合片段克隆入穿梭质粒pKSV7,随后将重组质粒电转李斯特菌进行同源重组。NDV-F基因的PCR扩增表明该重组菌构建成功,RT-PCR结果表明F基因在重组菌中得到了转录。比较了重组菌和野生型菌株的溶血性、黏附和侵袭力、对小鼠和鸡胚的毒力和生长特性以及重组菌的体内外稳定性,结果表明:hly基因中F片段的整合消除了单核细胞增多性李斯特菌溶血素基因的表达,其培养上清液没有溶血性,而野生型菌株的溶血价达24;细胞试验表明重组菌对细胞的黏附力和相对侵袭力均有不同程度的降低,而相对侵袭力与野生型菌株具有显著性差异(P<0.05);重组菌对小鼠及鸡胚的毒力(LD50)与野生型相比分别下降3.7和6.5个对数数量级;重组菌在BHI肉汤和小鼠体内连续5次后,仍然可以扩增出目的基因NDV-F,初步表明该重组菌较为稳定。     

Drosophila S2 cells: an alternative infection model for Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that infects humans and animals. Its pathogenic strategy involves the expression of virulence proteins that mediate intracytosolic growth and cell-to-cell spread. A key virulence protein is the cholesterol-dependent cytolysin, listeriolysin O (LLO), which is largely responsible for mediating escape from the phagosome into the host cytosol. To study further the host processes exploited during L. monocytogenes infection, we sought to develop Drosophila S2 cells as a model for infection. Here, we show that S2 cells share a number of properties with mammalian cell culture models of infection. As with mouse macrophages, LLO was required for phagosomal escape from S2 cells. Furthermore, vacuolar escape was dependent on their acidification via the ATPase proton pumps, as bafilomycin A1 treatment sharply decreased escape. However, unlike in mouse macrophages, LLO mutants replicated in the phagosome of S2 cells. Drosophila cells are cholesterol auxotrophs, and exogenous cholesterol increased the infection rate of L. monocytogenes (LLO independent) and also augmented the efficiency of vacuolar escape (LLO dependent). With available genetic tools such as RNA interference, S2 cells could become an important model in the study of host-pathogen interactions.     

Listeriolysin O-dependent activation of endothelial cells during infection with Listeria monocytogenes: activation of NF-kappa B and upregulation of adhesion molecules and chemokines

The facultative intracellular bacterium Listeria monocytogenes is an invasive pathogen that crosses the vascular endothelium and disseminates to the placenta and the central nervous system. Its interaction with endothelial cells is crucial for the pathogenesis of listeriosis. By infecting in vitro human umbilical vein endothelial cells (HUVEC) with L. monocytogenes, we found that wild-type bacteria induced the expression of the adhesion molecules (ICAM-1 and E-selectin), chemokine secretion (IL-8 and monocyte chemotactic protein-1) and NF-kappa B nuclear translocation. The activation of HUVEC required viable bacteria and was abolished in prfA-deficient mutants of L. monocytogenes, suggesting that virulence genes are associated with endothelial cell activation. Using a genetic approach with mutants of virulence genes, we found that listeriolysin O (LLO)-deficient mutants inactivated in the hly gene did not induce HUVEC activation, as opposed to mutants inactivated in the other virulence genes. Adhesion molecule expression, chemokine secretion and NF-kappa B activation were fully restored by a strain of Listeria innocua transformed with the hly gene encoding LLO. The relevance in vivo of endothelial cell activation for listerial pathogenesis was investigated in transgenic mice carrying an NF-kappa B-responsive lacZ reporter gene. NF-kappa B activation was visualized by a strong lacZ expression in endothelial cells of capillaries of mice infected with a virulent haemolytic strain, but was not seen in those infected with a non-haemolytic isogenic mutant. Direct evidence that LLO is involved in NF-kappa B activation in transgenic mice was provided by injecting intravenously purified LLO, thus inducing stimulation of NF-kappa B in endothelial cells of blood capillaries. Our results demonstrate that functional listeriolysin O secreted by bacteria contributes as a potent inflammatory stimulus to inducing endothelial cell activation during the infectious process.     

Identification of the Listeria monocytogenes virulence factors involved in the CAMP reaction

There is a need to identify the virulence factors involved in the synergistic lysis of erythrocytes (CAMP reaction) by Listeria monocytogenes and either Staphylococcus aureus or Corynebacterium equi , in order to assess the relationship between the CAMP reaction and virulence of L. monocytogenes . The ability of various L. monocytogenes mutants to secrete listeriolysin O and phospholipases, and to produce lysis of sheep blood agar was determined. The results suggest that the CAMP reaction with Coryne. equi involves listeriolysin O and Coryne. equi cholesterol oxidase, and that the reaction with Staph. aureus involves either of the phospholipases C produced by L. monocytogenes . A modified CAMP test, which incorporates cholesterol oxidase into sheep blood agar, is proposed for the rapid (4–6 h) identification of L. monocytogenes.     

Sec16p potentiates the action of COPII proteins to bud transport vesicles

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.     

T cell recognition of listeriolysin O is induced during infection with Listeria monocytogenes

During bacterial multiplication, Listeria monocytogenes (strain EGD) secretes sulfhydryl-dependent cytotoxin, termed listeriolysin O, a virulence factor presumable promoting intracellular growth of this ubiquitous pathogen. The role of this exotoxin in the process of T cell activation was studied in vivo during the course of an experimental infection in the mouse. By using highly purified listeriolysin O, it was found that infection with viable, replicative bacteria induced in vivo the emergence of T cells specifically reacting against this exotoxin, as demonstrated by eliciting the expression of delayed-type hypersensitivity to listeriolysin O in Listeria-immune mice. The kinetics of this inflammatory reaction followed the same pattern as that observed with crude Listeria antigenic preparation classically used for the detection of delayed-type hypersensitivity, with a peak of expression by day 6 and a slow decline over the next 3 wk to a residual level, indicating the presence of memory T cells reacting with the exotoxin. This result, therefore, allowed us to identify for the first time that a pure immunogenic molecule secreted by L. monocytogenes is specifically recognized by sensitized T cells induced during the course of infection by L. monocytogenes. The expression of T cell-mediated immunity to listeriolysin O was generated by very low amounts of replicative bacteria, indicating that the exotoxin released in host tissues during the process of intracellular growth is highly immunogenic. Our data favor the view that the binding of listeriolysin O to the membrane cholesterol might be a critical event potentiating the in vivo expression of delayed sensitivity against this exotoxin. Indeed, the insertion of listeriolysin O into the cell membrane induced resistance to enzymatic proteolysis and membrane-bound listeriolysin O was significantly more effective in inducing delayed inflammatory reaction in Listeria-immune mice.     

Host-induced, stage-specific virulence gene activation in Candida albicans during infection

An understanding of the complex interactions between pathogenic microbes and their host must include the identification of gene expression patterns during infection. To detect the activation of virulence genes in the opportunistic fungal pathogen Candida albicans in vivo by host signals, we devised a reporter system that is based on FLP-mediated genetic recombination. The FLP gene, encoding the site-specific recombinase FLP, was genetically modified for expression in C. albicans and fused to the promoter of the SAP2 gene that codes for one of the secreted aspartic proteinases, which are putative virulence factors of C. albicans. The SAP2P-FLP fusion was integrated into one of the SAP2 alleles in a strain that contained a deletable marker that conferred resistance to mycophenolic acid and was flanked by direct repeats of the FLP recognition target (FRT). Using this reporter system, a transient gene induction could be monitored at the level of single cells by the mycophenolic acid-sensitive phenotype of the colonies generated from such cells after FLP-mediated marker excision. In two mouse models of disseminated candidiasis, SAP2 expression was not observed in the initial phase of infection, but the SAP2 gene was strongly induced after dissemination into deep organs. In contrast, in a mouse model of oesophageal candidiasis in which dissemination into internal organs did not occur, no SAP2 expression was detected at any time. Our results support a role of the SAP2 gene in the late stages of an infection, after fungal spread into deep tissue. This new in vivo expression technology (IVET) for a human fungal pathogen allows the detection of virulence gene induction at different stages of an infection, and therefore provides clues about the role of these genes in the disease process.     

Inhibition of calpain blocks the phagosomal escape of Listeria monocytogenes

Listeria monocytogenes is a gram-positive facultative intracellular bacterium responsible for the food borne infection listeriosis, affecting principally the immunocompromised, the old, neonates and pregnant women. Following invasion L. monocytogenes escapes the phagosome and replicates in the cytoplasm. Phagosome escape is central to L. monocytogenes virulence and is required for initiating innate host-defence responses such as the secretion of the cytokine interleukin-1. Phagosome escape of L. monocytogenes is reported to depend upon host proteins such as γ-interferon-inducible lysosomal thiol reductase and the cystic fibrosis transmembrane conductance regulator. The host cytosolic cysteine protease calpain is required in the life cycle of numerous pathogens, and previous research reports an activation of calpain by L. monocytogenes infection. Thus we sought to determine whether host calpain was required for the virulence of L. monocytogenes. Treatment of macrophages with calpain inhibitors blocked escape of L. monocytogenes from the phagosome and consequently its proliferation within the cytosol. This was independent of any direct effect on the production of bacterial virulence factors or of a bactericidal effect. Furthermore, the secretion of interleukin-1β, a host cytokine whose secretion induced by L. monocytogenes depends upon phagosome escape, was also blocked by calpain inhibition. These data indicate that L. monocytogenes co-opts host calpain to facilitate its escape from the phagosome, and more generally, that calpain may represent a cellular Achilles heel exploited by pathogens.     

The molecular mechanisms of listeriolysin O-induced lipid membrane damage

Listeria monocytogenes is an intracellular food-borne pathogen that causes listeriosis, a severe and potentially life-threatening disease. Listeria uses a number of virulence factors to proliferate and spread to various cells and tissues. In this process, three bacterial virulence factors, the pore-forming protein listeriolysin O and phospholipases PlcA and PlcB, play a crucial role. Listeriolysin O belongs to a family of cholesterol-dependent cytolysins that are mostly expressed by gram-positive bacteria. Its unique structural features in an otherwise conserved three-dimensional fold, such as the acidic triad and proline-glutamate-serine-threonine-like sequence, enable the regulation of its intracellular activity as well as distinct extracellular functions. The stability of listeriolysin O is pH- and temperature-dependent, and this provides another layer of control of its activity in cells. Moreover, many recent studies have demonstrated a unique mechanism of pore formation by listeriolysin O, i.e., the formation of arc-shaped oligomers that can subsequently fuse to form membrane defects of various shapes and sizes. During listerial invasion of host cells, these membrane defects can disrupt phagosome membranes, allowing bacteria to escape into the cytosol and rapidly multiply. The activity of listeriolysin O is profoundly dependent on the amount and accessibility of cholesterol in the lipid membrane, which can be modulated by the phospholipase PlcB. All these prominent features of listeriolysin O play a role during different stages of the L. monocytogenes life cycle by promoting the proliferation of the pathogen while mitigating excessive damage to its replicative niche in the cytosol of the host cell.     

Use of PCR methods for identification of Listeria monocytogenes in milk

The aim of this work was to estimate the limit of Listeria monocytogenes cfu in polymerase chain reaction (PCR) for a DNA fragment of listeriolysine O (hly A) gene. The PCR method, with used primers selected in areas of the listeriolysin O gene, allows to differentiate L. monocytogenes strains from other Listeria species. The amplified fragment (456 bp) of hly A gene was obtained for all strains L. monocytogenes and no other Listeria species. The PCR method with the selected primers allowed to detect 50-500 cfu L. monocytogenes/ml suspended in water or milk. Among 20 samples of raw milk from cows, 10 samples contained > 50 cfu L. monocytogenes/ml. Obtained results indicate that the PCR assay of L. monocytogenes identification is technically simple and may be conduct with minimal time. So, it could be recommended as quick diagnostic method in identification L. monocytogenes in milk.     

Isolation of Listeria monocytogenes mutants with high-level in vitro expression of host cytosol-induced gene products

The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA, the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfA-dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.