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

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 ΔsecA2prlA1 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 ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.     

Fibronectin‐binding protein,FbpA, is the adhesin responsible for pathogenesis of Listeria monocytogenes infection

The role of fibronectin binding protein A (FbpA) in Listeria monocytogenes infection and its pathogenesis were studied in vivo and in vitro by constructing a fbpA‐deficient mutant of L. monocytogenes (ΔfbpA). In vivo, ΔfbpA was less pathogenic in mutant mice than was wild‐type L. monocytogenes. FbpA did not affect the amounts of various virulence‐determining factors, including internalin B and listeriolysin O. However, adherence to, and invasion of, mouse hepatocytes by the ΔfbpA mutant were reduced. In contrast, adherence to, but not invasion of, the ΔfbpA mutant to macrophages was attenuated. Fibronectin contributed to the efficient adherence and invasion of wild‐type L. monocytogenes, but not to those of the ΔfbpA mutant. Attenuation of adhesion and uptake of the ΔfbpA mutant were reversed by overexpression of FbpA in it. FbpA was not involved in intracellular growth, autophagy induction or actin tail formation. Thus, the present findings clearly show that FbpA acts as an important adhesion molecule of L. monocytogenes, especially regarding hepatocytes, without modulating the expression of other virulence factors that have been implicated in the pathogenesis of L. monocytogenes infection.     

Influence of Temperature and Growth Phase on Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117–124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42°C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42°C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25°C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37°C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.     

Investigating the therapeutic potential of herbal leads against drug resistant Listeria monocytogenes by computational virtual screening and in vitro assays

Listeria monocytogenes, a Gram-positive opportunistic food-borne pathogen, naturally resistant to many antibiotics and acquired resistance may be a concern in the nearer future. Hence, there is a scope for screening of novel therapeutic agents and drug targets, toward the treatment of fatal listeria infections. The SecA homologs, SecA1 and SecA2 are the essential components of the general secretion (Sec) pathway, a specialised protein export system, present in L. monocytogenes. This study evaluates the use of botanicals against L. monocytogenes MTCC 1143 by considering SecA proteins as probable drug targets by high-throughput screening approaches. The 3D structure of SecA proteins with good stereochemical validity was generated by comparative modelling. The druglikeness and pharmacokinetic properties of 97 phytoligands identified through the extensive literature survey were predicted for druglikeness and ADMET properties. The inhibitory properties of best candidates were studied by molecular docking. The effect of the selected candidate molecules were further analysed in vitro well diffusion and cell aggregation assays. The antibiotic sensitivity profiling applied to L. monocytogenes MTCC 1143 using clinically relevant antibiotics showed that the bacteria became drug resistant to many tested antibiotics. The virtual screening suggested that .05 M cinnamic aldehyde from Cinnamomum camphora and 1, 2-Epoxycyclododecane from Cassia auriculata were identified as potential SecA inhibitors. The well diffusion assays suggested that the selected herbal substances have antibacterial activities. Further, preliminary validation suggested that incorporation of cinnamic aldehyde and methanolic or ethyl acetate extract of C. auriculata in broth medium shows growth reduction, misassembly and cell aggregation. This indicates the inhibition of SecA targets.     

Recombinant probiotic expressing Listeria adhesion protein attenuates Listeria monocytogenes virulence in vitro

Background

Listeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.

Methodology/Principal Findings

The objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp^LAP) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp^LAP for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp^LAP pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp^LAP also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp^LAP cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.

Conclusions/Significance

Wild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp^LAP blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.     

Biotechnological applications of Listeria's sophisticated infection strategies

Listeria monocytogenes is a Gram‐positive bacterium that is able to survive both in the environment and to invade and multiply within eukaryotic cells. Currently L. monocytogenes represents one of the most well‐studied and characterized microorganisms in bacterial pathogenesis. A hallmark of L. monocytogenes virulence is its ability to breach bodily barriers such as the intestinal epithelium, the blood–brain barrier as well as the placental barrier to cause severe systemic disease. Curiously, this theme is repeated at the level of the interaction between the individual cell and the bacterium where its virulence factors contribute to the ability of the bacteria to breach cellular barriers. L. monocytogenes is a model to study metabolic requirements of bacteria growing in an intracellular environment, modulation of signalling pathways in the infected cell and interactions with cellular defences involving innate and adaptive immunity. Technical advances such as the creation of LISTERIA‐susceptible mouse strains, had added interest in the study of the natural pathogenesis of the disease via oral infection. The use of attenuated strains of L. monocytogenes as vaccines has gained considerable interest because they can be used to express heterologous antigens as well as to somatically deliver recombinant DNA to eukaryotic cells. A novel vaccine concept, the use of non‐viable but metabolically active bacteria to induced immunoprotective responses, has been developed with L. monocytogenes. In this mini‐review, we review the strategies used by L. monocytogenes to subvert the cellular functions at different stages of the infection cycle in the host and examine how these properties are being exploited in biotechnological and clinical applications.     

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

Protective immunity induced by a LLO‐deficient Listeria monocytogenes

Listeria monocytogenes is a food‐borne pathogen able to cause serious disease in human and animals. Listeriolysin O (LLO), a major virulence factor secreted by this bacterium, is a vacuole‐specific lysin that facilitates bacterial entrance into the host cytosol. Thus, LLO plays a key role in the translocation and intracellular spread of L. monocytogenes. To study the effect of LLO on virulence and immunopotency, a LLO‐deficient L. monocytogenes mutant was constructed using a shuttle vector followed by homologous recombination. The mutant strain had lost hemolytic activity, which resulted in an extremely reduced virulence, 5 logs lower than that of the parent strain, yzuLM4, in BALB/c mice. The number of bacteria detected in the spleens and livers of mice infected with the mutant was greatly reduced, and the bacteria were rapidly eliminated by the host. Kinetics studies in this murine model of infection showed that the invasion ability of the mutant strain was much lower than that of the parent strain. Moreover, immunization with the mutant strain conferred protective immunity against listerial infection. In particular, stimulation with Ag85B_240‐259, strong specific Th1 type cellular immunity was elicited by vaccination C57BL/6 mice with hly deficient strain delivering Mycobacterium tuberculosis fusion antigen Ag85B‐ESAT‐6 via intravenous inoculation. These results clearly show that highly attenuated LLO‐deficient L. monocytogenes is an attractive vaccine carrier for delivering heterologous antigens.     

The p60 and NamA autolysins from Listeria monocytogenes contribute to host colonization and induction of protective memory

Inducing long‐term protective memory CD8⁺ T‐cells is a desirable goal for vaccines against intracellular pathogens. However, the mechanisms of differentiation of CD8⁺ T‐cells into long‐lived memory cells capable of mediating protection of immunized hosts remain incompletely understood. We have developed an experimental system using mice immunized with wild type (WT) or mutants of the intracellular bacterium Listeria monocytogenes (Lm) that either do or do not develop protective memory CD8⁺ T‐cells. We previously reported that mice immunized with Lm lacking functional SecA2, an auxiliary secretion system of gram‐positive bacteria, did not differentiate functional memory CD8⁺ T‐cells that protected against a challenge infection with WT Lm. Herein we hypothesized that the p60 and NamA autolysins of Lm, which are major substrates of the SecA2 pathway, account for this phenotype. We generated Lm genetically deficient for genes encoding for the p60 and NamA proteins, ΔiapΔmurA Lm, and further characterized this mutant. Δp60ΔNamA Lm exhibited a strong filamentous phenotype, inefficiently colonized host tissues, and grew mostly outside cells. When Δp60ΔNamA Lm was made single unit, cell invasion was restored to WT levels during vaccination, yet induced memory T‐cells still did not protect immunized hosts against recall infection. Recruitment of blood phagocytes and antigen‐presenting cell activation was close to that of mice immunized with ΔActA Lm, which develop protective memory. However, key inflammatory factors involved in optimal T‐cell programming such as IL‐12 and type I IFN (IFN‐I) were lacking, suggesting that cytokine signals may largely account for the observed phenotype. Thus, altogether, these results establish that p60 and NamA secreted by Lm promote primary host cell invasion, the inflammatory response and the differentiation of functional memory CD8⁺ T‐cells, by preventing Lm filamentation during growth and subsequent triggering of innate sensing mechanisms.     

单增李斯特菌胎盘感染的体内外模型研究进展

单核细胞增生李斯特氏菌（Listeria monocytogenes）是重要的食源性致病菌,能引发人类的李斯特菌病,是全球公共卫生问题之一。该菌易感染孕妇,引起胎儿和新生儿的侵袭性李斯特菌病,严重威胁母婴健康。因此,建立有效的单增李斯特菌感染胎盘体内外模型,解析和探究单增李斯特菌经胎盘感染机制,是预防和控制单增李斯特菌感染母婴的关键所在。本文综述了可用于研究单增李斯特菌母婴感染的体内外胎盘模型,总结和讨论了各类模型的优势和局限性;并着重分析了体外三维胎盘屏障模型在单增李斯特菌感染方面的研究进展和未来研究方向。以期为深入解析该菌经胎盘感染的途径、发病机制提供支持,并为预防和控制母婴李斯特菌病提供科学参考。     

Glucose and Nutrient Concentrations Affect the Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K₂HPO₄ reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.     

Role of anaerobiosis in virulence of Salmonella typhimuirium

Intestinal pathogens are exposed to various stress conditions during their infectious cycle. Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells is priority for the invading pathogen. In the present study, a virulent strain of S. typhimurium (1402/84) was grown under anaerobic conditions and its virulence characteristics such as host cell binding, penetration and intracellular survival were compared with aerobic S. typhimurium. Anaerobically grown S. typhimurium showed significantly higher binding to immobilized mice enterocytes and intestinal mucus as compared to bacteria grown aerobically. Anaerobic bacteria also showed an early penetration of mucus and subsequent binding to underlying immobilized enterocytes, in vitro. Anaerobic S. typhimurium exhibited increased intracellular survival within spleen macrophages of mice and caused significantly higher fluid accumulation in ligated rabbit ileal loops as compared to aerobic bacteria. LD₅₀ of anaerobic S. typhimurium was also observed to be 2 fold lower when compared to aerobic bacteria. Cell surface hydrophobicity of anaerobic S. typhimurium was also found to be significantly higher than aerobic bacteria. Thus, it appears that exposure of S. typhimurium to anaerobiosis results in its enhanced virulence, adhesion and penetration of host cells.     

<Emphasis Type="Italic">Listeria monocytogenes</Emphasis> virulence factor Listeriolysin O favors bacterial growth in co-culture with the ciliate <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>, causes protozoan encystment and promotes bacterial survival inside cysts

Background  

The gram-positive pathogenic bacterium Listeria monocytogenes is widely spread in the nature. L. monocytogenes was reported to be isolated from soil, water, sewage and sludge. Listeriolysin O (LLO) is a L. monocytogenes major virulence factor. In the course of infection in mammals, LLO is required for intracellular survival and apoptosis induction in lymphocytes. In this study, we explored the potential of LLO to promote interactions between L. monocytogenes and the ubiquitous inhabitant of natural ecosystems bacteriovorous free-living ciliate Tetrahymena pyriformis.     

I型干扰素在李斯特菌感染中免疫抑制作用机制的研究进展

I型干扰素在李斯特菌感染中的作用成为近年的研究热点。大量研究证实I型干扰素在李斯特菌感染中发挥免疫抑制作用,但其产生及作用机制仍不十分明确,本文就I型干扰素在单核细胞增多性李斯特菌感染中的产生及免疫抑制机制的研究进展进行综述。     

The RAB5‐GEF Function of RIN1 Regulates Multiple Steps During Listeria monocytogenes Infection

Listeria monocytogenes is a food‐borne pathogenic bacterium that invades intestinal epithelial cells through a phagocytic pathway that relies on the activation of host cell RAB5 GTPases. Listeria monocytogenes must subsequently inhibit RAB5, however, in order to escape lysosome‐mediated destruction. Relatively little is known about upstream RAB5 regulators during L. monocytogenes entry and phagosome escape processes in epithelial cells. Here we identify RIN1, a RAS effector and RAB5‐directed guanine nucleotide exchange factor (GEF), as a host cell factor in L. monocytogenes infection. RIN1 is rapidly engaged following L. monocytogenes infection and is required for efficient invasion of intestinal epithelial cells. RIN1‐mediated RAB5 activation later facilitates the fusion of phagosomes with lysosomes, promoting clearance of bacteria from the host cell. These results suggest that RIN1 is a host cell regulator that performs counterbalancing functions during early and late stages of L. monocytogenes infection, ultimately favoring pathogen clearance.      

Analysis of SecA2‐dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector

The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2‐dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinum secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2‐dependent substrates. Immunoblotting procedures confirmed SecA2‐dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG‐overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2‐dependent membrane localization of PknG is an important determinant for M. marinum virulence.     

单增李斯特菌耐药外排泵研究进展

单增李斯特菌是一种重要的人兽共患食源性胞内致病菌,广泛存在于自然环境中且易污染动物性食品,人及动物感染后可引起严重的李斯特菌病,死亡率高达30%。单增李斯特菌通常对多种药物敏感,然而,因不合理使用抗菌药或消毒剂形成的选择压力导致李斯特菌多重耐药情况的报道日渐增多。外排泵蛋白是细菌中一类重要的蛋白,可参与机体多种生物学过程,包括影响细菌对抗生素敏感性、促进有毒化合物泵出、影响细菌毒力等。本文综述了近年来关于单增李斯特菌耐药外排泵的功能及调控机制的研究进展,为深入理解李斯特菌耐药等环境适应机制及有效控制该病原污染传播和筛选抗感染药物新靶点提供理论基础。     

A study on the effects of some laboratory-derived genetic mutations on biofilm formation by Listeria monocytogenes

Biofilms formed by the human pathogen Listeria monocytogenes in food-processing environments can be a potential source of contamination. In this study, we investigated the ability of L. monocytogenes wild type and its laboratory-derived isogenic mutants in cwhA, prfA, agrA, flaA, degU, ami and sigB to adhere to and form biofilms on abiotic surfaces. The results suggest that inactivation of the two component regulatory system degU completely abolished biofilm formation, while inactivation of the flagellar gene flaA, two component response regulator agrA and the autolysin-adhesin gene ami lead to severe impairment of initial attachment and the subsequent development of a mature biofilm by L. monocytogenes. Mutants in the global regulator of virulence prfA and the alternative sigma factor sigB were unaffected and formed biofilms similar to wild type L. monocytogenes.