Virulence Genes In Listeria Monocytogenes Research Articles

Investigating the Effect of Melittin Peptide in Preventing Biofilm Formation, Adhesion and Expression of Virulence Genes in Listeria monocytogenes.

Listeria monocytogenes is a notable food-borne pathogen that has the ability to create biofilms on different food processing surfaces, making it more resilient to disinfectants and posing a greater risk to human health. This study assessed melittin peptide's anti-biofilm and anti-pathogenicity effects on L. monocytogenes ATCC 19115. Melittin showed minimum inhibitory concenteration (MIC) of 100μg/mL against this strain and scanning electron microscopy images confirmed its antimicrobial efficacy. The OD measurement demonstrated that melittin exhibited a strong proficiency in inhibiting biofilms and disrupting pre-formed biofilms at concentrations ranging from 1/8MIC to 2MIC and this amount was 92.59 ± 1.01% to 7.17 ± 0.31% and 100% to 11.50 ± 0.53%, respectively. Peptide also reduced hydrophobicity and self-aggregation of L. monocytogenes by 35.25% and 14.38% at MIC. Melittin also significantly reduced adhesion to HT-29 and Caco-2 cells by 61.33% and 59%, and inhibited invasion of HT-29 and Caco-2 cells by 49.33% and 40.66% for L. monocytogenes at the MIC value. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed melittin's impact on gene expression, notably decreasing inlB (44%) and agrA (45%) gene expression in L. monocytogenes. flaA and hly genes also exhibited reduced expression. Also, significant changes were observed in sigB and prfA gene expression. These results underscore melittin's potential in combating bacterial infections and biofilm-related challenges in the food industry.

Evaluation of the constituent compounds, antioxidant, anticancer, and antimicrobial potential of Prangos ferulacea plant extract and its effect on Listeria monocytogenes virulence gene expression.

Prangos ferulacea plant is very popular in Iran due to its unique properties in treating diseases and its special flavor. To check the characteristics of this plant, first, its extract was extracted using the maceration method. Its chemical composition was investigated using high-performance liquid chromatography (HPLC) that p-coumaric was identified as its main compound, and Fourier-transform infrared spectroscopy (FTIR) showed the presence of functional groups related to phenolic, flavonoid, tannins, and carboxylic acids such as caffeic acid and coumaric acid composition. Total phenol content (TPC), total flavonoid content (TFC), and beta-carotene were equal to 202.04 ± 5.46 mg gallic acid equivalent (GAE)/g dry weight, 1,909.46 ± 13 μg quercetin (QE)/g of dry weight, and 2.91 mg/100 g. The antioxidant property of the extract was evaluated using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) free radical scavenging and ferric reducing antioxidant power assay (FRAP). According to the IC50 obtained for DDPH (274 ± 7.2 μg/mL) and ABTS (120.45 ± 9.6 μg/mL) and FRAP values [1.92 ± 0.05 μg ascorbic acid equivalent (AAE)/g of extract], this extract had high antioxidant properties. Cytotoxicity was evaluated against the survival of HT 29 cells that IC50 was 82.15 ± 0.02 μg/mL. The antimicrobial property of the extract was calculated using disk diffusion agar (DDA), well diffusion agar (WDA), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). Listeria monocytogenes has the highest sensitivity to this extract and inhibition zone based on DDA and WDA method and with an MIC and MBC equal to 16 and 128 mg/mL has the least resistance. The morphology change of L. monocytogenes strain was proved through scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM). The extract caused a significant reduction in the transcription of genes involved in the film formation ability of L. monocytogenes. The obtained results fully prove the very practical and pragmatic characteristics of P. ferulacea.

The Occurrence of Serotypes and Virulence Genes of Listeria monocytogenes in Various Food Products

Background: Given that controlling Listeria contamination is very important in food chain system, the knowledge of their prevalence in food is very important. Therefore, this study aims to examine the prevalence of important Listeria species in various food types and evaluate serotype distribution, as well as the study of the virulence factors of L. monocytogenes.
 Methods: During July 2018 and January 2020, 900 food samples were collected in the North of Iran, including beef, chicken, fish, shrimp, milk and yogurt, green vegetables, mixed vegetable salad, Olivier salad, and cottage cheese. After isolation and identification steps, each bacterial DNA was extracted. Then, using specific primers, species, serotypes, and virulence genes of Listeria isolates were evaluated in the samples by Polymerase Chain Reaction (PCR) method.
 Results: The test results of 136 samples (15.1%) were positive for Listeria spp. and the most contaminated food was beef (35%) followed by chicken (29%) and green vegetables (23%). The most isolated Listeria spp. was L. monocytogenes and L. ivanovii. Among L. monocytogenes isolates, the dominant serogroups were 1/2a and 4b; furthermore, all of the isolates of this species harbored four virulence genes, including hlyA, plc, iap, and actA.
 Conclusion: These reports highlighted the importance of food safety in various food products, particularly raw meats and vegetables. Moreover, contamination of healthy foods such as fish and vegetables with Listeria is an indicator of public health.

A Highly Substituted Ring-Fused 2-Pyridone Compound Targeting PrfA and the Efflux Regulator BrtA in Listeria monocytogenes.

Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents. IMPORTANCE Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor-2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

Evaluation of the microbiological and chemical aspects of autochthonous wild snails in Sardinia.

This study was conducted to acquire knowledge on the epidemiology and ecology of some zoonotic agents in snails. Chemical and microbiological analysis was carried out on 46 samples of snails belonging to the species of Helix aspersa and Helix (Eobania) vermiculata. The association between heavy metals and wild snails, a native consumer product in the Region of Sardinia, was determined.The molecular characterisation of Listeria monocytogenes virulence genes has shown a genetic profile that deserves more attention for the improvement of surveillance and risk prevention. Specimens of H. vermiculata showed higher concentrations of cadmium (M=0.80±0.56 mg/kg) than H. aspersa (M=0.61±0.17 mg /kg).A further objective was to determine whether the samples showed significant differences from the point of view of secretion characterisation, in terms of protein content, and to identify species-specific correlations and possible relationships with the environment. The presence of Salmonella enterica sub.sp houtenae (6,14: z4, z23) and Salmonella enterica subsp diarizonae (47: k: e, n, z15) (1 sample), Listeria monocytogenes (2 samples) with Molecular characterization of virulence genes together with the measurement of heavy metals in samples of wild snails has shown a health and hygiene profile that would deserve greater attention for the improvement of the surveillance and prevention of microbiological and chemical risk in such products which currently show a tendency towards increase in consumption.

Multiple Anti-microbial Resistance Profile and Molecular Detection of Some Virulence ‎Genes of Listeria Monocytogenes Isolated from Fresh Raw Meat Retailed in Zaria, ‎Northwestern Nigeria ‎

Multiple Anti-microbial Resistance Profile and Molecular Detection of Some Virulence ‎Genes of Listeria Monocytogenes Isolated from Fresh Raw Meat Retailed in Zaria, ‎Northwestern Nigeria ‎

Growth and Expression of Virulence Genes of Listeria monocytogenes during the Processing of Dry-Cured Fermented "Salchichón" Manufactured with a Selected Lactilactobacillus sakei.

Simple SummaryDuring the ripening process of the dry-cured fermented sausage “salchichón”, Listeria monocytogenes could fail to be eliminated. In addition, the food safety criterion for L. monocytogenes in the European Union sets up a maximum level of 100 units of this microorganism per gram in ready-to-eat products throughout their shelf-life. Thus, since L. monocytogenes could be present in this product, it is necessary to evaluate the impact of the dry-cured fermented processing in the potential virulence of this pathogen, even considering the possible effect of the usual microbiota (lactic-acid bacteria) of “salchichón”. In this work, the effect of the processing of “salchichón”, inoculated with a selected strain of Lactilactobacillus sakei, on the growth of L. monocytogenes and on the expression of its virulence genes, was evaluated. The processing of “salchichón” provoked a relevant reduction in L. monocytogenes, but this pathogen was not completely eliminated. However, a downregulation in the expression of the tested virulence genes was found, which could suppose a reduction in the pathogenic effect of this microorganism. These findings could be of great interest to consider the dry-cured ripening of “salchichón” as a safe process to control the pathogen L. monocytogenes.The effect of the dry-cured fermented processing of “salchichón” inoculated with a selected strain of Lactilactobacillus sakei (205) on the growth and transcriptional response of three virulence genes (plcA, hly, and iap) of Listeria monocytogenes was evaluated. For this, three different batches of “salchichón” were analyzed: batch B (inoculated only with L. sakei), batch L (inoculated only with L. monocytogenes), and batch L + B (inoculated with both microorganisms). Sausages were ripened for 90 days according to a traditional industrial process. The processing of “salchichón” provoked a reduction in L. monocytogenes counts of around 2 log CFU/g. The downregulation of the expression of the three genes was found at the end of ripening when the water activity (aw) of “salchichón” was <0.85 aw. The combined effect on the reduction in L. monocytogenes counts together with the downregulation in the expression of the virulence genes throughout the “salchichón” processing could be of great interest to control the hazard caused by the presence of this pathogenic bacterium.

Occurrence, antimicrobial resistance, serotyping and virulence genes of Listeria monocytogenes isolated from foods

Listeria monocytogenes is a pathogen contaminated food, it is the cause of listeriosis worldwide. The aims of this study were to investigate the occurrence, antimicrobial resistance, serotyping and virulence genes of L. monocytogenes isolated from foods in Meknes city of Morocco. From June 2017 to May 2018, 520 food samples were randomly collected from a traditional market and two overcrowded popular neighborhoods (Lahdim and Hamria) and subjected to the detection of L. monocytogenes. Then, the antimicrobial susceptibility of the isolated strains were evaluated using the standard disk diffusion method and the determination of serotypes and virulence genes was performed by PCR. The results showed the detection of L. monocytogenes in fifteen (2.9%) of 520 samples, including three (5.7%) isolates in traditional whey, raw minced meat and raw sausage, two (3.8%) in raw milk and one (1.9%) in smen (traditional butter), raw bovine meat, raw poultry meat and raw fish, while salads and rayeb (traditional coagulated milk) were not contaminated. Among the fifteen isolated L. monocytogenes, nine (60%) belonged to the serogroup (1/2a, 1/2c, 3a and 3c), two (13.3%) belonged to the serogroup (1/2b, 3b, 4b and 4d) and four (26.6%) do not belong to any studied serogroup. Furthermore, fifteen (100%) isolates showed the presence of actA gene, fourteen (93.3%) harbored hlyA, prfA and plcB genes, thirteen (86.7%) carried inlA and inlC genes and twelve (80%) showed inlJ gene. The antimicrobial susceptibility analysis showed that the isolated strains were more resistant to amoxicillin/clavulanic acid (67.0%), erythromycin (60.0%), sulphamethoxazole (40.0%), ampicillin and sulphamethoxazole/trimethoprim (33.0%) and tetracycline (20.0%). Furthermore, 66.7% (10/15) were multidrug-resistant. From this study, we can conclude that foods marketed in Meknes city were contaminated by multidrug-resistant strains of L. monocytogenes harboring virulence genes, which may cause a serious risk to public health.

Effect of Zataria multiflora Boiss. Essential oil, time, and temperature on the expression of Listeria monocytogenes virulence genes in broth and minced rainbow trout

The virulence potential of Listeria monocytogenes is not a stable property and can be greatly affected by diverse environmental conditions and stresses. For example, the addition of essential oils (EOs) into the formulation of foods, as well as the time and temperature of the treatment process, can affect the virulence gene expression of L. monocytogenes. The aim of the present study was to assess the transcriptomic response of L. monocytogenes isolate on the exposure to Zataria multiflora Boiss. essential oil (ZEO) in fish storage resembling conditions. The expression of four different genes of L. monocytogenes (hly, prfA, inlA, and inlB) was assessed by the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) during growth in two different growth mediums (broth and minced rainbow trout fish), treated by sub inhibitory concentrations (subMIC) of ZEO at select times (0 h, 24 h and 48 h) and temperatures (12 °C, 22 °C and 37 °C). The mixed responses of gene regulation for L. monocytogenes were obtained in response to the differences in the type of growth medium, dose of ZEO, time, and temperature of treatment. Our results show that the application of EO at acceptable sensory amounts (<0.7%) can significantly increase the expression of virulence genes. Therefore, EOs can be important in virulence potential and pathogenicity of L. monocytogenes. The level and trend of transcription of hly gene was affected differently compared to the other genes.

RNA- and protein-mediated control of Listeria monocytogenes virulence gene expression

ABSTRACTThe model opportunistic pathogen Listeria monocytogenes has been the object of extensive research, aiming at understanding its ability to colonize diverse environmental niches and animal hosts. Bacterial transcriptomes in various conditions reflect this efficient adaptability. We review here our current knowledge of the mechanisms allowing L. monocytogenes to respond to environmental changes and trigger pathogenicity, with a special focus on RNA-mediated control of gene expression. We highlight how these studies have brought novel concepts in prokaryotic gene regulation, such as the ‘excludon’ where the 5′-UTR of a messenger also acts as an antisense regulator of an operon transcribed in opposite orientation, or the notion that riboswitches can regulate non-coding RNAs to integrate complex metabolic stimuli into regulatory networks. Overall, the Listeria model exemplifies that fine RNA tuners act together with master regulatory proteins to orchestrate appropriate transcriptional programmes.

Tannin-Rich Pomegranate Rind Extracts Reduce Adhesion to and Invasion of Caco-2 Cells by Listeria monocytogenes and Decrease Its Expression of Virulence Genes

Pomegranate rind is rich in tannins that have remarkable antimicrobial activities. This study aimed to evaluate the effects of a tannin-rich fraction from pomegranate rind (TFPR) on Listeria monocytogenes virulence gene expression and on the pathogen's interaction with human epithelial cells. Growth curves were monitored to determine the effect of TFPR on L. monocytogenes growth. The 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide and fluorescence staining assays were used to examine the cytotoxicity of TFPR. The effects of TFPR on L. monocytogenes adhesion to and invasion of epithelial cells were investigated using Caco-2 cells. Real-time quantitative PCR analysis was conducted to quantify mRNA levels of three virulence genes in L. monocytogenes. Results showed that a MIC of TFPR against L. monocytogenes was 5 mg/ml in this study. TFPR exhibited cytotoxicity against Caco-2 cells when the concentration was 2.5 mg/ml. Subinhibitory concentrations of TFPR significantly reduced, in a dose-dependent manner, adhesion to and invasion of Caco-2 cells by L. monocytogenes. When L. monocytogenes was grown in the presence of 2.5 mg/ml TFPR, the transcriptional levels of prfA, inlA, and hly decreased by 17-, 34-, and 28-fold, respectively.

Seafood a potential source of some zoonotic bacteria in Zagazig, Egypt, with the molecular detection of Listeria monocytogenes virulence genes.

This article describes the results of a study conducted on 71 fresh seafood samples (fish and shellfish) marketed in Zagazig city, Sharkia province, Egypt, as well as on 50 human stool samples collected at the Zagazig University Hospital. The samples were examined for the presence of Listeria monocytogenes and Escherichia coli. The investigation of L. monocytogenes virulence genes was performed using Polymerase Chain Reaction (PCR), while the microbiological quality of the seafood samples was evaluated using the coliform count and aerobic plate count (APC) as indicators. Out of the examined 71 seafood samples, 20 (28.2%) were identified as L. monocytogenes, 15 (75%) of which were confirmed as virulent strains. Also, out of 50 human stool samples, only 1 (2%) was identified as virulent L. monocytogenes. E. coli serotypes were isolated from only 11.3% of seafood and 30% of human stool samples. In shellfish, the APC and most probable number of coliforms (MPC) were higher than those obtained from other fish samples. Multiplex PCR targeting internalin genes allowed simultaneous identification of L. monocytogenes and differentiation of virulent strains, thus enabling more timely detection of cases and sources of food borne listeriosis. The article concludes by stressing that the isolation of potentially virulent L. monocytogenes and E. coli from both seafood samples and humans emphasises the potential public health hazard caused by eating raw or undercooked shellfish.

On-chip parallel detection of foodborne pathogens using loop-mediated isothermal amplification

According to estimates issued by the Center for Disease Control and Prevention, one out of six Americans will get sick during this year due to consumption of contaminated products and there will be 50,000 related hospitalizations. To control and treat the responsible foodborne diseases, rapid and accurate detection of pathogens is extremely important. A portable device capable of performing nucleic acid amplification will enable the effective detection of infectious agents in multiple settings, leading to better enforcement of food safety regulations. This work demonstrates the multiplexed detection of food pathogens through loop-mediated isothermal amplification on a silicon chip. Silane passivation is used to prevent the adsorption of the polymerase on silicon oxide, which can severely inhibit nucleic acid amplification. We demonstrate the multiplexed screening of virulence genes of Listeria monocytogenes, Escherichia coli, and Salmonella by dehydrating the corresponding primers in oxidized silicon wells. Droplets of 30nL with reagents for nucleic acid amplification and lysate of suspected pathogens are arrayed on micro-machined wells with an automated microinjection system. We show that dehydrated primers re-suspend when other reagents are microinjected, and the resulting mix can be used to specifically amplify the targeted gene. Results of characterization experiments demonstrate sensitivity down to a few templates per reaction, specificity that enables multiplexed screening, and robustness that allows amplification without DNA extraction.

The transcriptional response of virulence genes in Listeria monocytogenes during inactivation by nisin

The influence of preservatives on the expression of the virulence genes in Listeria monocytogenes (L. monocytogenes) is significant in assessing their functions and effectiveness against pathogens. In this study, the transcriptional response of the virulence genes (inlA, prfA, plcA, and hlyA) in L. monocytogenes during inactivation by nisin was investigated at 37 °C and 4 °C. The expression of these virulence genes decreased rapidly and remained at a constant level when nisin was added both at 37 °C and 4 °C. The decrease in the expression of the virulence genes caused by nisin at 37 °C was higher than that at 4 °C; thus, the ability of nisin to inhibit virulence genes expression at 37 °C is more effective than that at 4 °C. Regarding the effect of the nisin concentration on transcriptional inhibition, 500 IU/mL nisin caused significantly lower expression of the virulence genes than 100 IU/mL nisin at both temperatures. With the cell count increasing after the early bactericidal activity of nisin, the expression of the virulence genes also began to increase. Interestingly, the expression of these virulence genes all responded uniformly to nisin of different concentrations at different temperatures. A comprehensive understanding of the transcriptional response of the virulence genes in L. monocytogenes during inactivation by nisin could contribute to the effective application of nisin in the food industry.

Strain dependent expression of stress response and virulence genes of Listeria monocytogenes in meat juices as determined by microarray

A subgenomic array, encompassing 54 probes targeting genes responsible for virulence, adhesion and stress response in Listeria monocytogenes, was used in order to study their expression in food systems. RNA extracted from L. monocytogenes inoculated in BHI and in situ (i.e. in minced meat and fermented sausage juices) and incubated at 4°C, was hybridized on the array and the results obtained were compared in order to understand the effect that the food juice has on the expression. Three different strains of L. monocytogenes were tested, in order to determine the effect of the strain provenience. As determined by cluster analysis, each strain behaved in a different way when inoculated in food juices. The goal was to respond to acidic and osmotic stresses encountered in the food, particularly in the fermented sausage juice. No differences in the expression profile between the three strains were observed, when they were inoculated in BHI. On the other hand, in the meat and sausage juices, the iap, gadC and gadE genes, together with different internalin encoding genes, were significantly differentially expressed in the three strains.

Modulation of PrfA activity in Listeria monocytogenes upon growth in different culture media

PrfA is the major transcriptional activator of most virulence genes of Listeria monocytogenes. Its activity is modulated by a variety of culture conditions. Here, we studied the PrfA activity in the L. monocytogenes wild-type strain EGD and an isogenic prfA deletion mutant (EGDDeltaprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDDeltaprfApPrfA and EGDDeltaprfApPrfA*) in response to growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented with one of the three phosphotransferase system (PTS) carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type strain in BHI and LB with all of these carbon sources, while PrfA activity was high in minimal medium in the presence of glycerol. EGDDeltaprfApPrfA*, expressing a large amount of PrfA* protein, showed high PrfA activity under all growth conditions. In contrast, strain EGDDeltaprfApPrfA, expressing an equally high amount of PrfA protein, showed high PrfA activity only when cultured in BHI, and not in LB or MM (in the presence of any of the carbon sources). A ptsH mutant (lacking a functional HPr) was able to grow in BHI but not in LB or MM, regardless of which of the four carbon sources was added, suggesting that in LB and MM the uptake of the used PTS carbohydrates and the catabolism of glycerol are fully dependent on the functional common PTS pathway. The BHI culture medium, in contrast, apparently contains carbon sources (supporting listerial growth) which are taken up and metabolized by L. monocytogenes independently of the common PTS pathway. The growth rates of L. monocytogenes were strongly reduced in the presence of large amounts of PrfA (or PrfA*) protein when growing in MM, but were less reduced in LB and only slightly reduced in BHI. The expression of the genes encoding the PTS permeases of L. monocytogenes was determined in the listerial strains under the applied growth conditions. The data obtained further support the hypothesis that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases.

Lineage specific recombination and positive selection in coding and intragenic regions contributed to evolution of the main Listeria monocytogenes virulence gene cluster

The major virulence cluster of Listeria monocytogenes harbors six virulence genes that encode proteins critical for the intracellular life cycle of this human and animal pathogen. In this study, we determined the sequence (8709 nt) of the virulence gene cluster (including the six main virulence genes) in 40 L. monocytogenes isolates from different source populations (human clinical cases, animal clinical cases, foods, and natural environments). An alignment of the full length cluster as well as individual gene alignments and alignments of intragenic regions were used for phylogenetic, recombination, and positive selection analyses. Initial phylogenetic analyses showed that the sequences represented two main clusters, consistent with previously defined L. monocytogenes phylogenetic lineages. The 40 sequences represented 25 distinct allelic types and the overall alignment included 592 polymorphic sites. Overall, our data show that (i) virulence genes in the main L. monocytogenes virulence gene cluster include highly conserved genes (i.e., hly and prfA) as well as diverse genes that appear to have evolved by positive selection ( mpl, actA, and plcA), (ii) recombination has played an important role in the evolution of the virulence gene cluster, but is limited to lineage II isolates, and (iii) the promoter region driving the transcription of virulence genes transcribed early in intracellular infection (i.e., hly and plcA) has evolved by positive selection. The genes and intragenic regions in the L. monocytogenes virulence gene cluster thus have evolved independently, despite their close physical linkage, likely reflecting distinct selective pressures associated with expression and function of the proteins encoded in this region.

Structural Analysis of the Bacterial HPr Kinase/Phosphorylase V267F Mutant Gives Insights into the Allosteric Regulation Mechanism of This Bifunctional Enzyme

The HPr kinase/phosphorylase (HPrK/P) is a bifunctional enzyme that controls the phosphorylation state of the phospho-carrier protein HPr, which regulates the utilization of carbon sources in Gram-positive bacteria. It uses ATP or pyrophosphate for the phosphorylation of serine 46 of HPr and inorganic phosphate for the dephosphorylation of Ser(P)-46-HPr via a phosphorolysis reaction. HPrK/P is a hexameric protein kinase of a new type with a catalytic core belonging to the family of nucleotide-binding protein with Walker A motif. It exhibits no structural similarity to eukaryotic protein kinases. So far, HPrK/P structures have shown the enzyme in its phosphorylase conformation. They permitted a detailed characterization of the phosphorolysis mechanism. In the absence of a structure with bound nucleotide, we used the V267F mutant enzyme to assess the kinase conformation. Indeed, the V267F replacement was found to cause an almost entire loss of the phosphorylase activity of Lactobacillus casei HPrK/P. In contrast, the kinase activity remained conserved. To elucidate the structural alterations leading to this drastic change of activity, the x-ray structure of the catalytic domain of L. casei HPrK/P-V267F was determined at 2.6A resolution. A comparison with the structure of the wild type enzyme showed that the mutation induces conformation changes compatible with the switch from phosphorylase to kinase function. Together with nucleotide binding fluorescence measurements, these results allowed us to decipher the cooperative behavior of the protein and to gain new insights into the allosteric regulation mechanism of HPrK/P.

Development of multilocus single strand conformation polymorphism (MLSSCP) analysis of virulence genes of Listeria monocytogenes and comparison with existing DNA typing methods

Development of rapid and simple typing methods is required for analyzing the distribution and contamination routes of food-borne pathogens. We established a simple typing method for Listeria monocytogenes using MLSSCP (Multilocus Single Strand Conformation Polymorphism) analysis. Four virulence genes, hlyA, iap, actA and inlB were amplified by PCR, digested with endonucleases and applied to gels for SSCP. As banding patterns have been shown to reflect even a single nucleotide difference, this method has a potential discriminatory power comparable to that of sequencing analysis. The 64 strains isolated from five meat processing plants were divided into 18 groups by this MLSSCP. Additionally, clustering obtained with this method showed strong correspondence with phylogenetic lineages I and II, and was achieved with much less expenditure in time and cost than is required for other methods, such as MLST. The validity of the MLSSCP lineage classification was confirmed by PFGE, AFLP and ribotyping results. This newly developed MLSSCP method is suitable when obtaining accurate results quickly and simply is crucial.

Characterization of Ers, a PrfA-like regulator ofEnterococcus faecalis

We have identified a transcriptional regulator, named Ers (for enterococcal regulator of survival), of Enterococcus faecalis, an important opportunistic bacterium commonly recovered from hospitalized patients. Ers is a member of the Crp/Fnr family and is 69% similar to Srv, a PrfA-like regulator of Streptococcus pyogenes implicated in virulence, and is the E. faecalis protein most closely related to PrfA, a positive regulator of virulence genes in Listeria monocytogenes. In an in vivo-in vitro macrophage infection model, the survival of an ers mutant was highly significantly decreased compared with that of the parental strain JH2-2. This mutant was more than 10-fold more sensitive to oxidative challenge by hydrogen peroxide. In order to identify genes whose expression was under Ers control, the RNA levels of 31 likely candidates were measured by real-time quantitative PCR. The results indicate that ers may be autoregulated and that the locus ef0082 appears to be positively regulated by Ers. Nevertheless, mutation of ef0082 did not result in any detectable changes in the survival of the bacterium within murine macrophages.