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BMC Microbiology Nov 2019High temperature requirement A (HtrA) is a widely expressed chaperone and serine protease in bacteria. HtrA proteases assemble and hydrolyze misfolded proteins to...
BACKGROUND
High temperature requirement A (HtrA) is a widely expressed chaperone and serine protease in bacteria. HtrA proteases assemble and hydrolyze misfolded proteins to enhance bacterial survival under stress conditions. Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that induces listeriosis in humans. In previous studies, it was shown that deletion of htrA in the genome of L. monocytogenes increased the susceptibility to cellular stress and attenuated virulence. However, expression and protease activity of listerial HtrA (LmHtrA) were never analyzed in detail.
RESULTS
In this study, we cloned LmHtrA wildtype (LmHtrA) and generated a proteolytic inactive LmHtrA mutant. Recombinant LmHtrA and LmHtrA were purified and the proteolytic activity was analyzed in casein zymography and in vitro cleavage assays. LmHtrA activity could be efficiently blocked by a small molecule inhibitor targeting bacterial HtrA proteases. The expression of LmHtrA was enhanced in the stationary growth phase of L. monocytogenes and significantly contributed to bacterial survival at high temperatures.
CONCLUSIONS
Our data show that LmHtrA is a highly active caseinolytic protease and provide a deeper insight into the function and mechanism, which could lead to medical and biotechnological applications in the future.
Topics: Bacterial Proteins; Caseins; Food Microbiology; Gene Expression Regulation, Bacterial; Heat-Shock Proteins; Heat-Shock Response; Listeria monocytogenes; Microbial Viability; Protein Folding; Protein Multimerization; Proteolysis; Up-Regulation
PubMed: 31726993
DOI: 10.1186/s12866-019-1633-1 -
FEMS Microbiology Letters Jan 2023The growing number of Listeria monocytogenes strains displaying increased tolerance to sanitizers widely applied in the food industry is becoming a problem. The aims of...
The growing number of Listeria monocytogenes strains displaying increased tolerance to sanitizers widely applied in the food industry is becoming a problem. The aims of this study were to evaluate the susceptibility of L. monocytogenes isolates from food and food industry environments to sanitizers (benzalkonium chloride, sodium hypochlorite, peracetic acid, and chlorhexidine) and heavy metals (cadmium chloride), as well as to investigate the presence of the main genes related to efflux pumps. All 82 isolates showed reduced susceptibility to benzalkonium chloride (MIC from 16 to 128 µg mL-1), sodium hypochlorite (MIC of ≥ 2048 µg mL-1), and peracetic acid (MIC from 512 to ≥ 2048 µg mL-1), while 22 isolates showed reduced susceptibility to cadmium chloride (MIC > 70 µg mL-1). Susceptibility to chlorhexidine was found (MIC from 2 to 16 µg mL-1). PCR-based analysis revealed that mdrl and lde genes were harbored by 14.6% (12/82) and 40.2% (33/82) of the isolates, respectively. This study demonstrates the presence of L. monocytogenes from food and food industry environments with reduced susceptibility to sanitizers commonly used in food processing environments, highlighting the importance of continuous monitoring of the tolerance profile of this microorganism to sanitizers, as well as the need for strict control of sanitation conditions in food industries.
Topics: Benzalkonium Compounds; Listeria monocytogenes; Peracetic Acid; Sodium Hypochlorite; Cadmium Chloride; Chlorhexidine; Food Handling
PubMed: 36931897
DOI: 10.1093/femsle/fnad019 -
International Journal of Food... May 2021The foodborne pathogen L. monocytogenes can be present in food processing environments where it is exposed to various stressors. These antimicrobial factors, which aim...
The foodborne pathogen L. monocytogenes can be present in food processing environments where it is exposed to various stressors. These antimicrobial factors, which aim to eliminate the pathogen, can induce sub-lethal injury to the bacterial cells. In the present study, we investigated the efficacy of different treatments (stresses) relevant to food processing and preservation as well as sanitation methods, in generating sub-lethal injury at 4 °C and 20 °C to two L. monocytogenes strains, ScottA and EGDe. Additionally, we evaluated the survival and extent of L. monocytogenes injury after exposure to commonly used disinfectants (peracetic acid and benzalkonium chloride), following habituation in nutrient-deprived, high-salinity medium. Each stress had a different impact on the survival and injury kinetics of L. monocytogenes. The highest injury levels were caused by peracetic acid which, at 4 °C, generated high populations of injured cells without loss of viability. Other injury-inducing stresses were lactic acid and heating. Long-term habituation in nutrient-limited and high salinity medium (4 °C) and subsequent exposure to disinfectants resulted in higher survival and injury in benzalkonium chloride and increased survival, yet with lower injury levels, in peracetic acid at 20 °C. Taken together, these results highlight the potential food safety risk emerging from the occurrence of injured cells by commonly used food processing methods. Consequently, in order to accurately assess the impact of an antimicrobial method, its potential of inducing sublethal injury needs to be considered along with lethality.
Topics: Benzalkonium Compounds; Disinfectants; Food Handling; Kinetics; Listeria monocytogenes; Microbial Viability; Peracetic Acid; Stress, Physiological
PubMed: 33773356
DOI: 10.1016/j.ijfoodmicro.2021.109159 -
Methods in Molecular Biology (Clifton,... 2021The pathogen Listeria monocytogenes is a facultative intracellular bacterium, which targets a large range of cell types. Following entry, bacteria disrupt the invasion...
The pathogen Listeria monocytogenes is a facultative intracellular bacterium, which targets a large range of cell types. Following entry, bacteria disrupt the invasion vacuole and reach the cytoplasm where they replicate and use the actin cytoskeleton to propel themselves from cell to cell. Mammalian epithelial cells grown in vitro can be used to study the different steps of the intracellular life of Listeria. However, rapid multiplication and dissemination of bacteria can induce important cell death and detachment, resulting in the formation of lytic plaques. Thus, in vitro infections with L. monocytogenes are usually restricted to short time courses, from a few minutes to one day. Here, we present a method to study long-term L. monocytogenes infections in epithelial cells using epifluorescence microscopy. This protocol enables the observation of actin-based motility, intercellular dissemination foci, and entrapment of L. monocytogenes within vacuoles of persistence termed "Listeria-Containing Vacuoles" (LisCVs). We also describe a protocol to study the recruitment of cytoskeletal proteins at Listeria actin comet tails, as well as a method to assess the membrane integrity of intracellular bacteria using a LIVE/DEAD viability assay.
Topics: Cell Line; Cytoskeletal Proteins; Epithelial Cells; Fluorescent Antibody Technique; Host-Pathogen Interactions; Humans; Listeria monocytogenes; Listeriosis; Microscopy, Fluorescence
PubMed: 32975777
DOI: 10.1007/978-1-0716-0982-8_16 -
Methods in Molecular Biology (Clifton,... 2021The behavior of Listeria monocytogenes communities in the food chain is closely associated with their spatial organization. Whether as biofilms on industrial surfaces or...
The behavior of Listeria monocytogenes communities in the food chain is closely associated with their spatial organization. Whether as biofilms on industrial surfaces or as microcolonies in food matrices, the resulting physiological diversification combined with the presence of extracellular polymeric substances (EPS) triggers emergent community functions involved in the pathogen survival and persistence (e.g., tolerance to dehydration, biocides, or preservatives). In this contribution, we present a noninvasive confocal laser microscopy (CLM) protocol allowing exploration of the spatial organization of L. monocytogenes communities on various inert or nutritive materials relevant for the food industry.
Topics: Biofilms; Food Microbiology; Humans; Listeria monocytogenes; Listeriosis; Microscopy, Confocal
PubMed: 32975771
DOI: 10.1007/978-1-0716-0982-8_10 -
Food Microbiology Sep 2019The susceptibility of four L. monocytogenes isolates from pork to sodium hypochlorite (SHY) and benzalkonium chloride (BZK) was tested. Minimum inhibitory concentration...
The susceptibility of four L. monocytogenes isolates from pork to sodium hypochlorite (SHY) and benzalkonium chloride (BZK) was tested. Minimum inhibitory concentration (MIC) values of 3500 ppm (SHY), or between 3 ppm and 13 ppm (BZK), were found. Minimum bactericidal concentration (MBC) values ranged from 3500 ppm to 4500 ppm (SHY), and from 3 ppm to 14 ppm (BZK). The effect of SHY and BZK on the architecture and cellular viability of 24-h-old biofilms formed by such strains on polystyrene was determined through confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9) and dead cells (propidium iodide). Strains were able to form biofilm (biovolume values in the observation field of 14,161 μm ranged between 103,928.3 ± 6730.2 μm and 276,030.9 ± 42,291.9 μm). Treatment of biofilms for 10 min with SHY (1MIC or 1.5MIC) or BZK (0.5MIC, 1MIC or 1.5MIC) decreased the biovolume of live (potentially dangerous) cells. SHY reduced the cellular viability of biofilms by more than 90%. On the other hand, BZK was able to remove most biofilm mass (live and dead cells), but decreased cellular viability only to a lesser extent, this suggesting strong biofilm detachment and dissemination of live cells.
Topics: Benzalkonium Compounds; Biofilms; Disinfectants; Listeria monocytogenes; Microbial Sensitivity Tests; Microbial Viability; Microscopy, Confocal; Sodium Hypochlorite
PubMed: 31027816
DOI: 10.1016/j.fm.2019.03.020 -
International Journal of Food... Feb 2021The food processing environments of a newly opened meat processing facility were sampled in ten visits carried out during its first 1.5 years of activity and analyzed...
The food processing environments of a newly opened meat processing facility were sampled in ten visits carried out during its first 1.5 years of activity and analyzed for the presence of Listeria monocytogenes. A total of 18 L. monocytogenes isolates were obtained from 229 samples, and their genomes were sequenced to perform comparative genomic analyses. An increase in the frequency of isolation of L. monocytogenes and in the diversity of sequence types (STs) detected was observed along time. Although the strains isolated belonged to six different STs (ST8, ST9, ST14, ST37, ST121 and ST155), ST9 was the most abundant (8 out of 18 strains). Low (0 and 2) single nucleotide polymorphism (SNP) distances were found between two pairs of ST9 strains isolated in both cases 3 months apart from the same processing room (Lm-1267 and Lm-1705, with a 2 SNPs distance in the core genome; Lm-1265 and Lm-1706, with a 0 SNPs distance), which suggests that these strains may be persistent L. monocytogenes strains in the food processing environment. Most strains showed an in silico attenuated virulence potential either through the truncation of InlA (in 67% of the isolates) or the absence of other virulence factors involved in cell adhesion or invasion. Twelve of the eighteen L. monocytogenes isolates contained a plasmid, which ranged in size from 4 to 87 Kb and harbored stress survival, in addition to heavy metals and biocides resistance determinants. Identical or highly similar plasmids were identified for various sets of L. monocytogenes ST9 isolates, which suggests the clonal expansion and persistence of plasmid-containing ST9 strains in the processing environments of the meat facility. Finally, the analysis of the L. monocytogenes genomes available in the NCBI database, and their associated metadata, evidenced that strains from ST9 are more frequently reported in Europe, linked to foods, particularly to meat and pork products, and less represented among clinical isolates than other L. monocytogenes STs. It also showed that the ST9 strains here isolated were more closely related to the European isolates, which clustered together and separated from ST9 North American isolates.
Topics: Animals; Disinfectants; Equipment Contamination; Europe; Floors and Floorcoverings; Food Handling; Food Microbiology; Genes, Bacterial; Genetic Variation; Listeria monocytogenes; Manufacturing and Industrial Facilities; Meat; Plasmids; Swine; Virulence; Virulence Factors; Whole Genome Sequencing
PubMed: 33454520
DOI: 10.1016/j.ijfoodmicro.2021.109043 -
Chemistry (Weinheim An Der Bergstrasse,... Jan 2023The glmS ribozyme regulates the expression of the essential GlmS enzyme being involved in cell wall biosynthesis. While >450 variants of the glmS ribozyme were...
The glmS ribozyme regulates the expression of the essential GlmS enzyme being involved in cell wall biosynthesis. While >450 variants of the glmS ribozyme were identified by in silico approaches and homology searches, only a few have yet been experimentally investigated. Herein, we validate and characterize the glmS ribozymes of the human pathogens Clostridium difficile and Listeria monocytogenes. Both ribozymes, as their previous characterized homologs rely on glucosamine-6-phosphate as co-factor and the presence of divalent cations for exerting the cleavage reaction. The observed EC values in turn were found to be in the submicromolar range, at least an order of magnitude lower than observed for glmS ribozymes from other bacteria. The glmS ribozyme of L. monocytogenes was further shown to bear unique properties. It discriminates between co-factors very stringently and other than the glmS ribozyme of C. difficile retains activity at low temperatures. This finding illustrates that albeit being highly conserved, glmS ribozymes have unique characteristics.
Topics: Clostridioides difficile; Listeria monocytogenes; RNA, Catalytic; Bacterial Proteins
PubMed: 36194523
DOI: 10.1002/chem.202202376 -
Molecular Microbiology Dec 2021Listeria monocytogenes is a food-borne bacterium that causes gastroenteritis, meningitis, or abortion. L. monocytogenes induces its internalization (entry) into human... (Review)
Review
Listeria monocytogenes is a food-borne bacterium that causes gastroenteritis, meningitis, or abortion. L. monocytogenes induces its internalization (entry) into human cells and either spreads laterally in tissues or transcytoses to traverse anatomical barriers. In this review, we discuss mechanisms by which five structurally related proteins of the "internalin" family of L. monocytogenes (InlA, InlB, InlC, InlF, and InlP) interact with distinct host receptors to promote infection of human cells and/or crossing of the intestinal, blood-brain, or placental barriers. We focus on recent results demonstrating that the internalin proteins InlA, InlB, and InlC exploit exocytic pathways to stimulate transcytosis, entry, or cell-to-cell spread, respectively. We also discuss evidence that InlA-mediated transcytosis contributes to traversal of the intestinal barrier, whereas InlF promotes entry into endothelial cells to breach the blood-brain barrier. InlB also facilitates the crossing of the blood-brain barrier, but does so by extending the longevity of infected monocytes that may subsequently act as a "Trojan horse" to transfer bacteria to the brain. InlA, InlB, and InlP each contribute to fetoplacental infection by targeting syncytiotrophoblast or cytotrophoblast layers of the placenta. This work highlights the diverse functions of internalins and the complex mechanisms by which these structurally related proteins contribute to disease.
Topics: Animals; Bacterial Proteins; Humans; Listeria monocytogenes; Listeriosis; Membrane Proteins; Transcytosis
PubMed: 34704304
DOI: 10.1111/mmi.14836 -
Microbiology Spectrum Feb 2022Listeria monocytogenes is a major pathogen contributing to foodborne outbreaks with high mortality. Nisin, a natural antimicrobial, has been widely used as a food...
Listeria monocytogenes is a major pathogen contributing to foodborne outbreaks with high mortality. Nisin, a natural antimicrobial, has been widely used as a food preservative. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. A transposon library was constructed in L. monocytogenes, leading to the identification of 99 genes associated with the innate resistance to nisin via Transposon sequencing (Tn-seq) analysis. To validate the accuracy of the Tn-seq results, we constructed five mutants (Δ, Δ, Δ, Δ, and Δ) in L. monocytogenes. The results revealed that and , the lipoteichoic acid-related genes, were essential for resistance to nisin, while the and mutants showed substantially enhanced nisin resistance. Densely wrinkled, collapsed surface and membrane breakdown were shown on Δ and Δ mutants under nisin treatment. Deletion of and altered the surface charge, and decreased the resistance to general stress conditions and cell envelope-acting antimicrobials. Furthermore, YqgS and LafA are required for biofilm formation and cell invasion of L. monocytogenes. Collectively, these results reveal novel mechanisms of nisin resistance in L. monocytogenes and may provide unique targets for the development of food-grade inhibitors for nisin-resistant foodborne pathogens. Listeria monocytogenes is an opportunistic Gram-positive pathogen responsible for listeriosis, and is widely present in a variety of foods including ready-to-eat foods, meat, and dairy products. Nisin is the only licensed lantibiotic by the FDA for use as a food-grade inhibitor in over 50 countries. A prior study suggests that L. monocytogenes are more resistant than other Gram-positive pathogens in nisin-mediated bactericidal effects. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. Here, we used a mariner transposon library to identify nisin-resistance-related genes on a genome-wide scale via transposon sequencing. We found, for the first time, that YqgS and LafA (Lipoteichoic acid-related proteins) are required for resistance to nisin. Subsequently, we investigated the roles of YqgS and LafA in L. monocytogenes stress resistance, antimicrobial resistance, biofilm formation, and virulence in mammalian cells.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Foodborne Diseases; Gene Expression Regulation, Bacterial; Lipopolysaccharides; Listeria monocytogenes; Listeriosis; Nisin; Teichoic Acids; Virulence
PubMed: 35196823
DOI: 10.1128/spectrum.02095-21