Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.     

Comparative assessment of acid, alkali and salt tolerance in Listeria monocytogenes virulent and avirulent strains

Listeria monocytogenes is an opportunistic bacterial pathogen of man and animals that has the capacity to survive under extreme environmental conditions. While our knowledge on L. monocytogenes and its ability to sustain within wide pH and temperature ranges and salt concentrations has been largely built on the virulent strains of this species, relatively little is known about avirulent strains in this regard. In this study, we extend our analysis on avirulent L. monocytogenes strains. By subjecting three virulent (EGD, 874 and ATCC 19196) and three avirulent (ATCC 19114, HCC23 and HCC25) strains to various pH and salt concentrations, it was found that L. monocytogenes recovered well after treatment with 100 mM Tris at pH 12.0, and to a lesser extent at pH 3.0. Interestingly, avirulent L. monocytogenes strains showed a somewhat higher tolerance to alkali than virulent strains. This unique feature of avirulent L. monocytogenes strains may potentially be exploited for the development of a rapid technique for differentiation between avirulent and virulent strains. Furthermore, all L. monocytogenes strains tested were resistant to saturated NaCl (about 7 M, or 40% w/v) for a long period of time (20 h and possibly longer). Together, these results highlight that acid, alkali, and/or salt treatments commonly used in food product processing may not be sufficient to eliminate L. monocytogenes, and therefore stringent quality control measures at the beginning and end of the food manufacturing process is essential to ensure that such food products are free of listerial contamination.     

Differential contribution of sodC1 and sodC2 to intracellular survival and pathogenicity of Salmonella enterica serovar Choleraesuis

Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host's phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain.     

Simultaneous deficiency of both MurA and p60 proteins generates a rough phenotype in Listeria monocytogenes

We examined eight spontaneously occurring rough mutants of Listeria monocytogenes for their ability to express two previously reported autolysins, p60 and MurA. All mutants lack MurA expression and show strongly reduced levels of extracellular p60. One rough strain harbors a variant of the p60 protein with a partially truncated catalytic domain. In seven cases there were shifts in the localization of p60 to the membrane fraction. Mutations within the secA2 gene, encoding an auxiliary protein secretion system paralog, were previously shown to be involved in the smooth-rough phenotypic variation seen with Listeria strains. An isogenic DeltasecA2 EGDe deletion strain displays a strong pleiotropic reduction of p60 and MurA, in addition to a large number of secreted and surface proteins. However, we observed no apparent SecA2 dysfunction in several of the investigated strains as determined by direct sequencing of the secA2 gene and complementation of the DeltasecA2 mutant with the respective allele cloned from the rough mutant. To determine the gene products required for the smooth-rough transition, we created mutants lacking the individual iap and murA genes as well as a Deltaiap DeltamurA double mutant. The double mutant displays a rough phenotype and exhibits many of the properties seen with the DeltasecA2 mutant. Our results implicate p60 and MurA as important determinants in controlling the cell shape of L. monocytogenes. We also identified homologous MurA and SecA2 proteins in other Listeria species. The muramidase in two species, L. innocua and L. welshimeri, shows activity similar to that of the MurA protein in L. monocytogenes.     

Tools for functional postgenomic analysis of listeria monocytogenes

We describe the development of genetic tools for regulated gene expression, the introduction of chromosomal mutations, and improved plasmid transfer by electroporation in the food-borne pathogen Listeria monocytogenes. pIMK, a kanamycin-resistant, site-specific, integrative listeriophage vector was constructed and then modified for overexpression (pIMK2) or for isopropyl-beta-d-thiogalactopyranoside (IPTG)-regulated expression (pIMK3 and pIMK4). The dynamic range of promoters was assessed by determining luciferase activity, P60 secretion, and internalin A-mediated invasion. These analyses demonstrated that pIMK4 and pIMK3 have a stringently controlled dynamic range of 540-fold. Stable gene overexpression was achieved with pIMK2, giving a range of expression for the three vectors of 1,350-fold. The lactococcal pORI280 system was optimized for the generation of chromosomal mutations and used to create five new prfA star mutants. The combination of pIMK4 and pORI280 allowed streamlined creation of "IPTG-dependent" mutants. This was exemplified by creation of a clean deletion mutant with deletion of the universally essential secA gene, and this mutant exhibited a rapid loss of viability upon withdrawal of IPTG. We also improved plasmid transfer by electroporation into three commonly used laboratory strains of L. monocytogenes. A 125-fold increase in transformation efficiency for EGDe compared with the widely used protocol of Park and Stewart (S. F. Park and G. S. Stewart, Gene 94:129-132, 1990) was observed. Maximal transformation efficiencies of 5.7 x 10(6) and 6.7 x 10(6) CFU per mug were achieved for EGDe and 10403S, respectively, with a replicating plasmid. An efficiency of 2 x 10(7) CFU per mug is the highest efficiency reported thus far for L. monocytogenes F2365.     

Expression of listeriolysin O by intracellular Listeria monocytogenes following infection of lipopolysaccharide-treated or untreated J774.1 macrophage-like cells

Abstract Listeria monocytogenes replicates in a phagocytic cell following escape into the host cytoplasm. Listeriolysin O, secreted by L. monocytogenes , which belongs to the thiol-activated hemolysin family, is known to play an important role in the escape of the bacterium into the host cytoplasm. In this study, we demonstrated that expression of listeriolysin O by infecting L. monocytogenes was lightly induced in J774.1 macrophage-like cells pretreated with lipopolysaccharide, although the growth of the bacteria was suppressed. The number of viable L. monocytogenes decreased until 4 h post-infection and then increased between 4 and 8 h post-infection in untreated J774.1 host cells, but it decreased until 8 h post-infection in lipopolysaccharide-treated host cells. However, expression of listeriolysin O by L. monocytogenes was not induced in the untreated host cells, while it increased between 0 and 4 h post-infection in the lipopolysaccharide-treated host cells. Expression of listeriolysin O mRNA in the lipopolysaccharide-treated host cells was also induced at 2 h post-infection, suggesting that listeriolysin O was newly synthesized in the macrophage-like cells. These results suggest that macrophage activation induced with lipopolysaccharide could lead to the expression of the listeriolysin O gene and the synthesis of listeriolysin O protein under suppression of the intracellular growth of L. monocytogenes .     

Factors affecting survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Listeria innocua</Emphasis> in soil samples

We investigated the ability of several strains of L. monocytogenes and Listeria innocua strains to survive in local soil samples in vitro. Survival of three L. monocytogenes strains, EGDe, CD83, and CD1038, and three L. innocua strains, CLIP, FH2117, FH2152, was monitored in soil samples by direct enumeration of colony-forming units on selective agar. The study did not demonstrate any species-specific difference in soil survival, and all Listeria strains exhibited a marked decline in numbers over time. Bioluminescence imaging approaches to detect lux-tagged strains in soil proved largely ineffective, most likely due to the reduced metabolic activity of strains in this environment. We investigated the influence of specific factors including the presence of a background microbiota, growth temperature, moisture and strain motility upon persistence in this environment. A sequenced L. monocytogenes strain, EGDe, was capable of active growth in sterile soil yet exhibited a decline in the presence of the normal soil microbiota. Furthermore, greater survival was seen at lower incubation temperatures in normal soil. Finally, we demonstrated a direct correlation between motility and survival of L. monocytogenes in soil with highly motile L. monocytogenes strains exhibiting greater soil survival than non-motile mutants.     

Survival of nontypeable Haemophilus influenzae in macrophages

In this study we have investigated the ability of nonencapsulated, nontypeable Haemophilus influenzae, NT477 to survive in the J774 mouse macrophage-like cell line. Viable, intracellular nontypeable H. influenzae could still be recovered from macrophages 72 h after phagocytosis. In contrast, H. influenzae strain Rd, an avirulent, nonencapsulated variant of a serotype d strain, was killed within 24 h. These differences suggest that NT477, in comparison to Rd, possesses unique attributes that enable it to survive in macrophages for prolonged periods. To determine whether this trait is ubiquitous amongst nontypeable H. influenzae, 33 primary clinical isolates obtained from children with otitis media were screened for their ability to survive in macrophages. Of these isolates, 82% were able to persist in an intracellular environment for periods of at least 24 h. The number of viable organisms recovered at this time ranged from 2x10(4) to 50 colony-forming units per strain indicating that the extent to which nontypeable H. influenzae can resist macrophage-mediated killing varies between strains.     

Invasion assay of Listeria monocytogenes using Vero and Caco-2 cells

The invasion ability of Listeria monocytogenes into cultured cells has been used to evaluate its pathogenicity. In this study, invasive ability was investigated using Vero and Caco-2 cell lines. The form of invasion showed no morphological differences between both cell lines inoculated with L. monocytogenes L89-H2 or L96-23C1 strains when double fluorescence stained with rhodamine and FITC or with Giemsa staining. Recovery count and recovery rate of L. monocytogenes from Vero cells was related to the number of inoculated bacteria (2 x 10(5) to 2 x 10(7)/ml) in a bell-shape pattern, though the relationship was unclear in Caco-2 cells. Recovery rate of L. monocytogenes was higher in Vero cells than Caco-2 cells at a multiplicity of infection (MOI) 10, though the rates in both cells showed different stable stages over a considerably wide range of MOI. The recovery rate of all five L. monocytogenes strains from listeriosis patients was 15% at MOI 10 from infected Vero cells, while meat-derived strains showed variable rates regardless of the serovar. These results suggest that the Vero cell line is suitable for an invasion assay and that a recovery rate of 15% may be the critical limit for the expression of pathogenicity in the host.     

单核细胞增生李斯特菌中CcpA缺失株的构建及对毒力的影响

CcpA是革兰氏阳性菌中由ccpA基因编码的介导碳分解代谢物阻遏的全局调控因子。近年来的研究表明,CcpA不仅参与CCR效应,还直接或间接参与病原细菌毒力基因的表达调控。为探讨CcpA对单核细胞增生李斯特菌（Lm）毒力的影响,应用同源重组方法构建CcpA缺失菌株。以BLAB/c小鼠为实验动物模型,检测野生株EGDe和缺失株EGDeΔccpA侵染小鼠后的半数致死剂量 LD₅₀和肝脾细菌载菌量,观察小鼠肝脏和脾脏的病理形态变化。结果显示：缺失CcpA后,Lm的LD₅₀降低了10倍,虽然肝脾细菌载菌量没有显著变化,但EGDe△ccpA对小鼠肝和脾的损害更为严重,表明CcpA缺失增强了细菌的毒力,CcpA 对Lm 毒力基因的表达可能具有间接或者直接的调控作用。     

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.     

The role of the sigB gene in the general stress response of Listeria monocytogenes varies between a strain of serotype 1/2a and a strain of serotype 4c

The ability of Listeria monocytogenes to resist many adverse environmental conditions has been attributed in part to activation of the alternative sigma factor sigma(B), encoded by the sigB gene. The ability of this pathogen to survive and grow under stress conditions varies between strains within the species. The current study was undertaken to determine whether the role played by the sigB gene in the stress response varies among strains of different serotypes. Null mutations were generated in the sigB genes of L. monocytogenes L61 (serotype 1/2a) and L99 (serotype 4c), and the survival of the resulting mutants was compared with that of the wild-type strains under osmotic, oxidative, and carbon starvation stress conditions and on exposure to bacteriocins, ethanol, acid, and heat. Except in a few cases, strain L61 displayed greater dependence on the sigB products for survival of adverse conditions than did strain L99. The results of this study indicated that the relative importance of the sigB gene in the stress response is not the same in all strains of L. monocytogenes, and this difference may be specific to serotype groupings within the species.     

Comparative Proteomic Analysis of Listeria monocytogenes Strains F2365 and EGD

Listeria monocytogenes is a gram-positive, food-borne pathogen that causes disease in both humans and animals. There are three major genetic lineages of L. monocytogenes and 13 serovars. To further our understanding of the differences that exist between different genetic lineages/serovars of L. monocytogenes, we analyzed the global protein expression of the serotype 1/2a strain EGD and the serotype 4b strain F2365 during early-stationary-phase growth at 37°C. Using multidimensional protein identification technology with electrospray ionization tandem mass spectrometry, we identified 1,754 proteins from EGD and 1,427 proteins from F2365, of which 1,077 were common to both. Analysis of proteins that had significantly altered expression between strains revealed potential biological differences between these two L. monocytogenes strains. In particular, the strains differed in expression of proteins involved in cell wall physiology and flagellar biosynthesis, as well as DNA repair proteins and stress response proteins.     

Genetic characterization of Listeria monocytogenes food isolates and pathogenic potential within serovars 1/2a and 1/2b

A total of 39 Listeria monocytogenes strains isolated from raw milk, smoked meat, chicken carcass and reference strains, belonging to serovars 1/2a, 4a, 1/2b, 3b and 4b, were analysed by RAPD and by polymorphisms of the virulent genes inlAB and iap. Ten isolates, belonging to serovars 1/2a and 1/2b and, collected from raw milk and smoked meat, were further tested for pathogenicity by IP injection into mice. The clustering of the 39 L. monocytogenes strains in 3 groups at 0.45 similarity level, based on molecular typing, was observed. Distribution of serovars in these clusters was in agreement with the proposed three Listeria monocytogenes lineages. Within serovar 1/2b, the 50% lethal dose (LD50) ranged from 8.4 x 10(4) to 1.7 x 10(6) cfu.ml(-1). One of the serovar 1/2b strains, isolated from smoked meat, exhibited the lowest virulence potential evaluated by LD50 and by mean time to death (MTD) and, from this point of view, was completely different from the other strains. Our results suggest the existence of heterogeneity in virulence levels within serovars 1/2a and 1/2b. However, when comparing the isolates based on genotyping, virulence indicators and food origin, no relation could be assessed.     

Murine pulmonary infection with Listeria monocytogenes: differential susceptibility of BALB/c, C57BL/6 and DBA/2 mice

Murine listeriosis is a paradigm to understand host pathogen interactions. Airway infections with Listeria monocytogenes, although representing a serious problem in early onset neonatal listeriosis, has not been investigated in detail in animal models so far. Here, the susceptibility of BALB/c, DBA/2 and C57BL/6 mice towards an intratracheal (i.t.) infection with virulent L. monocytogenes EGDe and the attenuated variant L. monocytogenes EGD hlyW491A(pERL3-CMVGFP) is reported. The course of infection was characterized by determination of bacterial numbers in the organs and assessment of the health condition of the mice. The distribution and cellular localization of Listeria in the airways was assessed by immunocytochemistry and confocal and electron microscopy. The differential susceptibility of inbred mouse strains to airway infections with L. monocytogenes could be assigned to the major virulence factor listeriolysin O. Resistant C57BL/6 mice were not affected by the two listerial strains. In contrast, BALB/c and DBA/2 mice showed differential susceptibility towards L. monocytogenes EGDe and attenuated bacteria, with all the mice being killed by the wild-type bacteria but rarely by the variant that secretes a listeriolysin of only 10% activity of that of the wild-type toxin. Thus, listeriolysin is a decisive factor for differential susceptibility against Listeria. After i.t. application, bacteria were predominantly localized in the peribronchiolar space and invaded alveolar macrophages but rarely lung epithelial cells. Dissemination from the lung into the deep organs started almost immediately after application, although a pulmonary bacterial reservoir remained during the first 4 days.     

Factors controlling acid tolerance of Listeria monocytogenes: effects of nisin and other ionophores.

The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.     

Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity

Listeria monocytogenes, an invasive opportunistic, food-borne pathogen, remains one of the leading causes of mortality from food-borne infections. The recently determined complete genome sequences of L. monocytogenes strain EGDe and of that of the closely related non-pathogenic species Listeria innocua strain CLIP11262 enhance our knowledge of the genetic basis of the virulence of L. monocytogenes and advance our understanding of the evolution of these Listeria species. Both genomes encode a high number of surface, transport and regulatory proteins. Comparison of the genome organisation revealed a perfect synteny between the two Listeria genomes. Comparison with other closely related bacteria also showed a high conservation in genome organisation among the Listeria, Staphylococcus and Bacillus group of low G+C content bacteria. Distinct G+C content of a number of strain-specific genes suggests intensive lateral gene transfer. The identification of a 55-kb locus encoding proteins with high homology to Salmonella enterica serovar Typhimurium vitamin B(12) synthesis proteins as well as those necessary for degradation of ethanolamine and propanediol further indicates acquisition of a complete metabolic pathway by horizontal gene transfer and a probable role of this locus in anaerobic growth in the host.     

The survival of Listeria monocytogenes in cottage cheese

Because of the difficulty of ensuring that cottage cheese is produced in conditions that prevent contamination with Listeria monocytogenes, the ability of this bacterium to survive in cottage cheese from three sources was investigated (a) during shelf-life at chill temperature and (b) in conditions of temperature abuse. Three batches of creamed cottage cheese, from three sources, received within 24 h of production, were inoculated with L. monocytogenes strain F6861 and stored at 4, 8 or 12 degrees C for 14 d. The three batches differed in their initial pH, titratable acidity and content of lactic acid and of lactic acid bacteria. No increase in numbers of L. monocytogenes occurred in the cottage cheeses during storage in these conditions. The numbers of listeria decreased; the rate of decrease differed in products from the three sources and was least in the product with the highest pH and lowest content of lactic acid. Acid formation by lactic acid bacteria during storage of the products probably contributed to the inhibition of listeria.