Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence.

The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence.     

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.     

Investigation of the role of ZurR in the physiology and pathogenesis of Listeria monocytogenes

Listeria monocytogenes is a Gram positive pathogen that is ubiquitous in the environment. It is a facultative anaerobic rod that causes listeriosis, a disease with potentially lethal consequences for susceptible individuals. During infection, the pathogen is capable of sequestering metal ions to act as vital biocatalysts in cellular processes. The zinc uptake regulator (ZurR) is predicted to coordinate uptake of zinc from the external environment. An in-frame deletion of the zurR gene resulted in a mutant exhibiting a small colony phenotype and a smaller cell size. The zurR mutant was unaffected under conditions of zinc limitation but demonstrated increased sensitivity to toxic levels of zinc. The mutant also demonstrated a significant (1-log) reduction in virulence potential in the murine model of infection. Using a bioinformatic approach, we identified a number of potentially Zur-regulated genes in the genome of L. monocytogenes. Quantitative RT-PCR demonstrated significant de-repression of zurA, lmo0153, and lmo1671 in the zurR mutant background indicating that these putative transporters are ZurR regulated.     

Listeria monocytogenes CtaP is a multifunctional cysteine transport-associated protein required for bacterial pathogenesis

The bacterial pathogen Listeria monocytogenes survives under a myriad of conditions in the outside environment and within the human host where infections can result in severe disease. Bacterial life within the host requires the expression of genes with roles in nutrient acquisition as well as the biosynthesis of bacterial products required to support intracellular growth. A gene product identified as the substrate-binding component of a novel oligopeptide transport system (encoded by lmo0135 ) was recently shown to be required for L. monocytogenes virulence. Here we demonstrate that lmo0135 encodes a multifunctional protein that is associated with cysteine transport, acid resistance, bacterial membrane integrity and adherence to host cells. The lmo0135 gene product (designated CtaP, for c ysteine t ransport a ssociated p rotein) was required for bacterial growth in the presence of low concentrations of cysteine in vitro , but was not required for bacterial replication within the host cytosol. Loss of CtaP increased membrane permeability and acid sensitivity, and reduced bacterial adherence to host cells. ctaP deletion mutants were severely attenuated following intragastric and intravenous inoculation of mice. Taken together, the data presented indicate that CtaP contributes to multiple facets of L. monocytogenes physiology, growth and survival both inside and outside of animal cells.     

Defensins enable macrophages to inhibit the intracellular proliferation of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that infects a large diversity of host cells, including macrophages. To avoid the phagosome microbicidal environment, L. monocytogenes secretes a pore-forming toxin (listeriolysin O, LLO) that releases the bacterium into the cytoplasm. We hypothesized that the α-defensins (HNPs) and/or humanized θ-defensin (RC-1) peptides produced by human and non-human primate neutrophils, respectively, cooperate with macrophages to control L. monocytogenes infection. Our results establish that HNP-1 and RC-1 enable macrophages to control L. monocytogenes intracellular growth by inhibiting phagosomal escape, as a consequence, bacteria remain trapped in a LAMP-1-positive phagosome. Importantly, HNP-1 interaction with macrophages and RC-1 interaction with bacteria are required to prevent macrophage infection. In accordance with these results, RC-1 is a more potent anti-listerial peptide than HNP-1 and HNP-1 is acquired by macrophages and trafficked to the phagocytosed bacteria. Finally, HNP-1 and RC-1 antimicrobial activity is complemented by their ability to prevent LLO function through two mechanisms, blocking LLO-dependent perforation of macrophage membranes and the release of LLO from the bacteria. In conclusion, at the site of infection the cooperation between antimicrobial peptides, such as HNP-1, and macrophages likely plays a critical role in the innate immune defence against L. monocytogenes.     

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.     

Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria and often consists a polyglycerolphosphate backbone chain that is linked to the membrane by a glycolipid. In Listeria monocytogenes this glycolipid is Gal-Glc-DAG or Gal-Ptd-6Glc-DAG. Using a bioinformatics approach, we have identified L. monocytogenes genes predicted to be involved in glycolipid ( lmo2555 and lmo2554 ) and LTA backbone ( lmo0644 and lmo0927 ) synthesis. LTA and glycolipid analysis of wild-type and mutant strains confirmed the function of Lmo2555 and Lmo2554 as glycosyltransferases required for the formation of Glc-DAG and Gal-Glc-DAG. Deletion of a third gene, lmo2553 , located in the same operon resulted in the production of LTA with an altered structure. lmo0927 and lmo0644 encode proteins with high similarity to the staphylococcal LTA synthase LtaS, which is responsible for polyglycerolphosphate backbone synthesis. We show that both proteins are involved in LTA synthesis. Our data support a model whereby Lmo0644 acts as an LTA primase LtaP and transfers the initial glycerolphosphate onto the glycolipid anchor, and Lmo0927 functions as LTA synthase LtaS, which extends the glycerolphosphate backbone chain. Inactivation of LtaS leads to severe growth and cell division defects, underscoring the pivotal role of LTA in this Gram-positive pathogen.     

Early intracellular events during internalization of Listeria monocytogenes by J774 cells.

The gram-positive bacillus Listeria monocytogenes gains entry into host cells through a phagosome membrane that forms around entering bacteria. During the early stages of internalization the invading bacteria appear to modify the protein composition of the forming phagosome membrane in J774 cells. MHC class II molecules on the cell surface and exposed surface molecules available for biotinylation are excluded from the bacteria-host cell membrane interface and from the forming phagosome. This exclusion of MHC class II molecules from the early phagosome may partially help to explain previous reports suggesting that L. monocytogenes is able to interfere with antigen presentation. Inside the host cell, MHC class II molecules are delivered to the phagosome membrane. This is followed by delivery of LAMP 1, a marker of late endocytic compartments, and fusion with low-pH compartments. The bacteria then escape into the cell cytoplasm, possibly assisted by rapid delivery of this low-pH environment.     

Rapid, transient, and proportional activation of σ(B) in response to osmotic stress in Listeria monocytogenes

The osmotic activation of sigma B (σ(B)) in Listeria monocytogenes was studied by monitoring expression of four known σ(B)-dependent genes, opuCA, lmo2230, lmo2085, and sigB. Activation was found to be rapid, transient, and proportional to the magnitude of the osmotic stress applied, features that underpin the adaptability of this pathogen.     

Thiamine plays a critical role in the acid tolerance of Listeria monocytogenes

Understanding the molecular basis of acid tolerance in the food-borne pathogen Listeria monocytogenes is important as this property contributes to survival in the food-chain and enhances survival within infected hosts. The aim of this study was to identify genes contributing to acid tolerance in L.?monocytogenes using transposon mutagenesis and subsequently to elucidate the physiological role of these genes in acid tolerance. One mutant harboring a Tn917 insertion in the thiT gene (formerly lmo1429), which encodes a thiamine (vitamin B1) uptake system, was found to be highly sensitive to acid. The acid-sensitive phenotype associated with loss of this gene was confirmed with an independently isolated mutant, from which the thiT gene was deleted (?thiT). Cells of both wild-type and ?thiT mutant that were thiamine depleted were found to be significantly more acid sensitive than control cultures. Thiamine-depleted cultures failed to produce significant concentrations of acetoin, consistent with the known thiamine dependence of acetolactate synthase, an enzyme required for acetoin synthesis from pyruvate. As acetoin synthesis is a proton-consuming process, we suggest that the acid sensitivity observed in thiamine-depleted cultures may be owing to an inability to produce acetoin.     

Inducible control of virulence gene expression in Listeria monocytogenes: temporal requirement of listeriolysin O during intracellular infection

We have constructed a lac repressor/operator-based system to tightly regulate expression of bacterial genes during intracellular infection by Listeria monocytogenes. An L. monocytogenes strain was constructed in which expression of listeriolysin O was placed under the inducible control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-dependent promoter. Listeriolysin O (LLO) is a pore-forming cytolysin that mediates lysis of L. monocytogenes-containing phagosomes. Using hemolytic-activity assays and Western blot analysis, we demonstrated dose-dependent IPTG induction of LLO during growth in broth culture. Moreover, intracellular growth of the inducible-LLO (iLLO) strain in the macrophage-like cell line J774 was strictly dependent upon IPTG. We have further shown that iLLO bacteria trapped within primary phagocytic vacuoles can be induced to escape into the cytosol following addition of IPTG to the cell culture medium, thus yielding the ability to control bacterial escape from the phagosome and the initiation of intracellular growth. Using the iLLO strain in plaque-forming assays, we demonstrated an additional requirement for LLO in facilitating cell-to-cell spread in L2 fibroblasts, a nonprofessional phagocytic cell line. Furthermore, the efficiency of cell-to-cell spread of iLLO bacteria in L2 cells was IPTG dose dependent. The potential use of this system for determining the temporal requirements of additional virulence determinants of intracellular pathogenesis is discussed.     

Presence of GadD1 glutamate decarboxylase in selected Listeria monocytogenes strains is associated with an ability to grow at low pH

The glutamate decarboxylase (GAD) system is critical to the survival of Listeria monocytogenes LO28 at low-pH stress (相似文献