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.     

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。     

Identification and characterization of Di- and tripeptide transporter DtpT of Listeria monocytogenes EGD-e

Listeria monocytogenes is a gram-positive intracellular pathogen responsible for opportunistic infections in humans and animals. Here we identified and characterized the dtpT gene (lmo0555) of L. monocytogenes EGD-e, encoding the di- and tripeptide transporter, and assessed its role in growth under various environmental conditions as well as in the virulence of L. monocytogenes. Uptake of the dipeptide Pro-[14C]Ala was mediated by the DtpT transporter and was abrogated in a DeltadtpT isogenic deletion mutant. The DtpT transporter was shown to be required for growth when the essential amino acids leucine and valine were supplied as peptides. The protective effect of glycine- and proline-containing peptides during growth in defined medium containing 3% NaCl was noted only in L. monocytogenes EGD-e, not in the DeltadtpT mutant strain, indicating that the DtpT transporter is involved in salt stress protection. Infection studies showed that DtpT contributes to pathogenesis in a mouse infection model but has no role in bacterial growth following infection of J774 macrophages. These studies reveal that DptT may contribute to the virulence of L. monocytogenes.     

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.     

Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes

The dlt operon of Gram-positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D-alanine residues into the cell wall-associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D-Ala-deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA- mutant did not show any morphological alterations and its growth rate was similar to that of the wild-type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA- mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow-derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA- mutant to non-phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D-alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.     

The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages

Under stress conditions, the facultative intracellular pathogen Listeria monocytogenes produces a ClpC ATPase, which is a general stress protein encoded by clpC and belonging to the HSP-100/Clp family. A ClpC-deficient mutant was obtained by gene disruption in strain LO28, which became highly susceptible to stress conditions in vitro . Intracellular growth of this mutant was restricted within macrophages, one of the major target cells of L . monocytogenes , during the infectious process. A quantitative electron microscope study showed that, contrary to wild-type bacteria that rapidly gain access to the cytoplasm of macrophages, mutant bacteria remained confined to membrane-bound phagosomes. Only a few mutant bacteria disrupted the phagosome membrane after 4 h of incubation, then polymerized actin filaments and multiplied within the cytoplasm. The ClpC ATPase, therefore, promotes early bacterial escape from the phagosome of macrophages, thus enhancing intracellular survival. The ClpC ATPase was produced in vivo during experimental infection by wild-type bacteria. The virulence of the ClpC-deficient mutant was severely attenuated in mice, with a three-log decrease in its 50% lethal dose compared with wild-type bacteria. Bacterial growth of mutant bacteria was strongly restricted in organs, presumably because of an impairment of intracellular survival in host tissues. Our results provide evidence that a general stress protein is required for the virulence of L . monocytogenes , which behaves as a virulence factor promoting intracellular survival of this pathogen.     

The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes

Listeria monocytogenes is a versatile bacterial pathogen that is able to accommodate to diverse environmental and host conditions. Presently, we have identified a L. monocytogenes two-component response regulator, ResD that is required for the repression of virulence gene expression known to occur in the presence of easily fermentable carbohydrates not found inside host organisms. Structurally and functionally, ResD resembles the respiration regulator ResD in Bacillus subtilis as deletion of the L. monocytogenes resD reduces respiration and expression of cydA, encoding a subunit of cytochrome bd. The resD mutation also reduces expression of mptA encoding the EIIABman component of a mannose/glucose-specific PTS system, indicating that ResD controls sugar uptake. This notion was supported by the poor growth of resD mutant cells that was alleviated by excess of selected carbohydrates. Despite the growth deficient phenotype of the mutant in vitro the mutation did not affect intracellular multiplication in epithelial or macrophage cell lines. When examining virulence gene expression we observed traditional induction by charcoal in both mutant and wild-type cells whereas the repression observed in wild-type cells by fermentable carbohydrates did not occur in resD mutant cells. Thus, ResD is a central regulator of L. monocytogenes when present in the external environment.     

Modification of a virulence-associated phenotype after growth of Listeria monocytogenes on food

AIM: To assess the effect of different foods, which have been implicated or not in cases of listeriosis, on the in vitro virulence-associated phenotype level of different Listeria monocytogenes strains. METHODS AND RESULTS: The virulence-associated phenotype level of L. monocytogenes was studied with the in vitro cell test based on a plaque-forming assay with a human adenocarcinoma cell line (HT-29) monolayer. Three strains of L. monocytogenes were grown in preparations (homogenate, 1-mum filtrate or 0.2-mum filtrate) of different food extracts ['rillettes' (potted minced pork), milk, raw salmon and cold-smoked salmon] or in a control medium, brain heart infusion (BHI). The bacterial suspensions grown in food extracts or in BHI at 37 degrees C were diluted with their growth medium (food extract or BHI) or with minimum essential medium before seeding on confluent HT-29 cell monolayers. Filtration of food extracts had no significant effect on the plaque numbers formed by the bacteria. A significant decrease in the plaque numbers was noted for the three strains when they grew in the rillettes extracts, compared with the other food extracts and BHI. The levels of in vitro virulence-associated phenotype of the strains after growth in the rillettes extract were similar to or lower than that of the hypovirulent internal reference strain L. monocytogenes 442. After growth in milk and cold-smoked salmon, the impact on virulence-associated phenotype depended on the strain. In contrast, plaque-forming assay indicated increased virulence-associated phenotype when the strains were switched from a nutrient-rich medium (food extract or BHI) to a minimum essential medium. CONCLUSIONS: In vitro virulence-associated phenotype level of the studied strains grown in BHI or cold-smoked salmon was the same as the control virulent strain EGD. In contrast, the nutrients present in rillettes may therefore substantially reduce the number of plaques but not the growth of L. monocytogenes. The utilization of minimum essential medium as diluent attenuates changes the effect of the food extract on virulence-associated phenotype in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: In the experimental design of this study, we showed that the nature of the food could affect the in vitro virulence-associated phenotype level of L. monocytogenes.     

Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes

We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of the opuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of L-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting L-carnitine and which plays an important role in osmoregulation in this pathogen.     

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.     

Characteristics of induction of virulence factor expression by activated charcoal in Listeria monocytogenes

Activated charcoal has been previously shown to induce in vitro expression of virulence factors by Listeria monocytogenes. In trying to elucidate the nature of the charcoal action, we found that the treatment of brain heart infusion medium with activated charcoal followed by charcoal removal does not result in an increase of virulence factor expression. At the same time, the addition of fresh charcoal to the charcoal-treated medium induces expression, suggesting that the effect of activated charcoal cannot be explained only by changes in medium composition. In addition, we observed that activated charcoal induced expression of virulence factors even when L. monocytogenes was physically separated from charcoal particles by either a nitrocellulose membrane or a thin layer of agar. We propose that the interaction of charcoal with some listerial product(s) might be responsible for the effect observed.     

Listeria monocytogenes ferritin protects against multiple stresses and is required for virulence

In this study, the role of Listeria monocytogenes ferritin was investigated. The fri gene encoding the ferritin was deleted and the phenotype of the mutant was analyzed demonstrating that ferritin is necessary for optimal growth in minimal medium in both presence and absence of iron, as well as after cold- and heat-shock. We also showed that ferritin provides protection against reactive oxygen species and is essential for full virulence of L. monocytogenes. A comparative proteomic analysis revealed an effect of the fri deletion on the levels of listeriolysin O and several stress proteins. Together, our study demonstrates that fri has multiple roles that contribute to Listeria virulence.     

Cloning of rel from Listeria monocytogenes as an osmotolerance involvement gene

Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.     

The yeast-phase virulence requirement for α-glucan synthase differs among Histoplasma capsulatum chemotypes

Histoplasma capsulatum strains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype II Histoplasma virulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucan in vitro, yet they remain fully virulent in vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishes AGS1 expression. Nonetheless, AGS1 mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced during in vivo growth despite its absence in vitro. To directly test whether AGS1 contributes to chemotype I strain virulence, we prevented AGS1 function by RNA interference and by insertional mutation. Loss of AGS1 function in chemotype I does not impair the cytotoxicity of ags1(-) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, the ags1(-) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate that AGS1 expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a unique Histoplasma chemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.