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Microbiology Spectrum Sep 2021Bacteria have necessarily evolved a protective arsenal of proteins to contend with peroxides and other reactive oxygen species generated in aerobic environments....
Bacteria have necessarily evolved a protective arsenal of proteins to contend with peroxides and other reactive oxygen species generated in aerobic environments. Listeria monocytogenes encounters an onslaught of peroxide both in the environment and during infection of the mammalian host, where it is the causative agent of the foodborne illness listeriosis. Despite the importance of peroxide for the immune response to bacterial infection, the strategy by which L. monocytogenes protects against peroxide toxicity has yet to be illuminated. Here, we investigated the expression and essentiality of all the peroxidase-encoding genes during L. monocytogenes growth and during infection of murine cells in tissue culture. We found that and were required for aerobic growth , and and were each required for L. monocytogenes to survive acute peroxide stress. Despite increased expression of , and during infection of macrophages, only proved necessary for cytosolic growth. In contrast, the proteins encoded by , , , , and were dispensable for aerobic growth, acute peroxide detoxification, and infection. Together, our results provide insight into the multifaceted L. monocytogenes peroxide detoxification strategy and demonstrate that L. monocytogenes encodes a functionally diverse set of peroxidase enzymes. Listeria monocytogenes is a facultative intracellular pathogen and the causative agent of the foodborne illness listeriosis. L. monocytogenes must contend with reactive oxygen species generated extracellularly during aerobic growth and intracellularly by the host immune system. However, the mechanisms by which L. monocytogenes defends against peroxide toxicity have not yet been defined. Here, we investigated the roles of each of the peroxidase-encoding genes in L. monocytogenes growth, peroxide stress response, and virulence in mammalian cells.
Topics: Animals; Bacterial Proteins; Disease Models, Animal; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Listeriosis; Macrophages; Mice; Mice, Inbred C57BL; Oxidative Stress; Peroxidases; Virulence; Virulence Factors
PubMed: 34287055
DOI: 10.1128/Spectrum.00440-21 -
Cellular Microbiology Apr 2020
Topics: History, 21st Century; Host-Pathogen Interactions; Laboratory Personnel; Listeria monocytogenes; Listeriosis; Research
PubMed: 32185896
DOI: 10.1111/cmi.13165 -
Methods in Molecular Biology (Clifton,... 2021Proteomics has become an essential tool to answer biologists' questions. For bacteriologists, the proteome of bacteria is much less complex than that of eukaryotic...
Proteomics has become an essential tool to answer biologists' questions. For bacteriologists, the proteome of bacteria is much less complex than that of eukaryotic organisms. However, not all the different cell "compartments" are easily accessible, and the analysis of cell envelope proteins is particularly challenging. For the Gram-positive bacterium Listeria monocytogenes, one of the main foodborne pathogen microorganisms, the study of surface proteins is crucial to better understand the mechanisms of pathogenicity, as well as adaptation/resistance to and persistence in hostile environments. The evolution of proteomic techniques, and particularly the possibility of separating and analyzing complex protein samples by off-gel (LC-MS/MS) versus in-gel (two-dimensional electrophoresis) approach, has opened the doors to new extraction and preparation methods to target the different subproteomes. Here, we describe three procedures to prepare and analyze intracellular, exocellular, and cell surface proteins: (1) the cell fractionation, based on cell broken and separation of protein subfractions by differential centrifugation; (2) the biotinylation, based on the labeling of cell surface proteins and their selective extraction; and (3) the enzymatic shaving by the action of trypsin on intact cells. These complementary methods allow to encompass all L. monocytogenes subproteomes for general profiling or target studies and could be applicable to other Gram-positive bacteria.
Topics: Bacterial Proteins; Biotinylation; Cell Fractionation; Centrifugation; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Humans; Listeria monocytogenes; Listeriosis; Proteomics; Tandem Mass Spectrometry
PubMed: 32975772
DOI: 10.1007/978-1-0716-0982-8_11 -
Food Microbiology Jun 2022The behaviour of Listeria monocytogenes was investigated in soft pasteurized milk cheese elaborated with different salt concentrations (1.17 and 0.30% w/w) and in cured...
The behaviour of Listeria monocytogenes was investigated in soft pasteurized milk cheese elaborated with different salt concentrations (1.17 and 0.30% w/w) and in cured raw sheep milk cheese over storage up to 189 days at different isothermal conditions. Commercial 25-g cheese samples were inoculated with a 4-strain cocktail of L. monocytogenes (serovars 4b, 1/2a, 1/2b and 1/2c) at approximately 10 CFU/g. The inoculated samples were stored at 4 and 22 °C and withdrawn at proper intervals for L. monocytogenes enumeration. The prevalence of the different serovar strains of L. monocytogenes was characterized on soft cheese samples over storage at 4 °C using multiplex PCR. Salt reduction did not affect the survival of L. monocytogenes in soft cheeses and a maximum of 1-log reduction was observed in both regular and low-salt cheeses after 189 days of storage at 4 °C. The pathogen showed greater survival capacity in both soft and cured cheeses during storage at 4 °C compared to the storage at 22 °C, where more than 2.5 log reductions were computed. The fate of L. monocytogenes was described through a Weibull model fitted to survival data. The time required for a first tenfold reduction of the L. monocytogenes population (δ) at 4 °C is around 150 days in soft and 72 days in cured cheeses. At 22 °C, the estimated δ values are at least 60% lower in both cheese types. Among the four L. monocytogenes serovars present in the inoculated cocktail, the serovar 4b strain was the most sensitive to refrigerated storage, while the prevalence of serovar 1/2c strain increased over time in soft cheeses. Overall, the data obtained in this study help to deepen knowledge into factors affecting L. monocytogenes behaviour on cheeses and evidenced the variability between serovars in terms of survival capacity, which may be considered when performing microbial risk assessments.
Topics: Animals; Cheese; Food Microbiology; Food Storage; Listeria monocytogenes; Sheep; Temperature; Time Factors
PubMed: 35287808
DOI: 10.1016/j.fm.2022.103979 -
International Journal of Food... Jul 2021The ability of Listeria monocytogenes isolates to survive within the food production environment (FPE), as well as virulence, varies greatly between strains. There are...
The ability of Listeria monocytogenes isolates to survive within the food production environment (FPE), as well as virulence, varies greatly between strains. There are specific genetic determinants that have been identified which can strongly influence a strains ability to survive in the FPE and/or within human hosts. In this study, we assessed the FPE fitness and virulence potential, including efficacy of selected hygiene or treatment intervention, against 52 L. monocytogenes strains isolated from various food and food environment sources. Phenotypic tests were performed to determine the minimum inhibitory concentration of cadmium chloride and benzalkonium chloride and the sensitivities to five clinically relevant antibiotics. A genomic analysis was also performed to identify resistance genes correlating to the observed phenotypic resistance profiles, along with genetic determinants of interest which may elude to the FPE fitness and virulence potential. A transposon element containing a novel cadmium resistance gene, cadA7, a Tn916 variant insert in the hypervariable Listeria genomic island 1 region and an LGI2 variant were identified. Resistance to cadmium and disinfectants was prevalent among isolates in this study, although no resistance to clinically important antimicrobials was observed. Potential hypervirulent strains containing full length inlA, LIPI-1 and LIPI-3 were also identified in this study. Cumulatively, the results of this study show a vast array of FPE survival and pathogenicity potential among food production-associated isolates, which may be of concern for food processing operators and clinicians regarding L. monocytogenes strains colonising and persisting within the FPE, and subsequently contaminating food products then causing disease in at risk population groups.
Topics: Anti-Bacterial Agents; Benzalkonium Compounds; Cadmium Chloride; DNA Transposable Elements; Disinfectants; Drug Resistance, Bacterial; Food Handling; Food Microbiology; Genomic Islands; Humans; Listeria monocytogenes; Microbial Sensitivity Tests; Virulence; Virulence Factors
PubMed: 34023680
DOI: 10.1016/j.ijfoodmicro.2021.109247 -
Applied and Environmental Microbiology Nov 2021In this study, we addressed different aspects regarding the implementation of quasimetagenomic sequencing as a hybrid surveillance method in combination with enrichment...
In this study, we addressed different aspects regarding the implementation of quasimetagenomic sequencing as a hybrid surveillance method in combination with enrichment for early detection of Listeria monocytogenes in the food industry. Different experimental enrichment cultures were used, comprising seven L. monocytogenes strains of different sequence types (STs), with and without a background microbiota community. To assess whether the proportions of the different STs changed over time during enrichment, the growth and population dynamics were assessed using colony sequencing and and 16S rRNA amplicon sequencing. There was a tendency of some STs to have a higher relative abundance during the late stage of enrichment when L. monocytogenes was enriched without background microbiota. When coenriched with background microbiota, the population dynamics of the different STs was more consistent over time. To evaluate the earliest possible time point during enrichment that allows the detection of L. monocytogenes and at the same time the generation of genetic information that enables an estimation regarding the strain diversity in a sample, quasimetagenomic sequencing was performed early during enrichment in the presence of the background microbiota using Oxford Nanopore Technologies Flongle and Illumina MiSeq sequencing. The application of multiple displacement amplification (MDA) enabled detection of L. monocytogenes (and the background microbiota) after only 4 h of enrichment using both applied sequencing approaches. The MiSeq sequencing data additionally enabled the prediction of cooccurring L. monocytogenes strains in the samples. We showed that a combination of a short primary enrichment combined with MDA and Nanopore sequencing can accelerate the traditional process of cultivation and identification of L. monocytogenes. The use of Illumina MiSeq sequencing additionally allowed us to predict the presence of cooccurring L. monocytogenes strains. Our results suggest quasimetagenomic sequencing is a valuable and promising hybrid surveillance tool for the food industry that enables faster identification of L. monocytogenes during early enrichment. Routine application of this approach could lead to more efficient and proactive actions in the food industry that prevent contamination and subsequent product recalls and food destruction, economic and reputational losses, and human listeriosis cases.
Topics: Food Microbiology; Genes, Bacterial; Listeria monocytogenes; Metagenomics; Microbiota; Population Dynamics; RNA, Ribosomal, 16S
PubMed: 34613762
DOI: 10.1128/AEM.01774-21 -
Microbiology and Molecular Biology... Nov 2019The foodborne pathogen can modulate its transcriptome and proteome to ensure its survival during transmission through vastly differing environmental conditions. While... (Review)
Review
The foodborne pathogen can modulate its transcriptome and proteome to ensure its survival during transmission through vastly differing environmental conditions. While utilizes a large array of regulators to achieve survival and growth in different intra- and extrahost environments, the alternative sigma factor σ and the transcriptional activator of virulence genes protein PrfA are two key transcriptional regulators essential for responding to environmental stress conditions and for host infection. Importantly, emerging evidence suggests that the shift from extrahost environments to the host gastrointestinal tract and, subsequently, to intracellular environments requires regulatory interplay between σ and PrfA at transcriptional, posttranscriptional, and protein activity levels. Here, we review the current evidence for cross talk and interplay between σ and PrfA and their respective regulons and highlight the plasticity of σ and PrfA cross talk and the role of this cross talk in facilitating successful transition of from diverse extrahost to diverse extra- and intracellular host environments.
Topics: Bacterial Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Humans; Listeria monocytogenes; Peptide Termination Factors; Sigma Factor; Signal Transduction; Stress, Physiological; Virulence
PubMed: 31484692
DOI: 10.1128/MMBR.00034-19 -
Microbiology Spectrum Jul 2019Bacterial metabolism represents the biochemical space that bacteria can manipulate to produce energy, reducing equivalents and building blocks for replication.... (Review)
Review
Bacterial metabolism represents the biochemical space that bacteria can manipulate to produce energy, reducing equivalents and building blocks for replication. Gram-positive pathogens, such as , show remarkable flexibility, which allows for exploitation of diverse biological niches from the soil to the intracytosolic space. Although the human host represents a potentially rich source for nutrient acquisition, competition for nutrients with the host and hostile host defenses can constrain bacterial metabolism by various mechanisms, including nutrient sequestration. Here, we review metabolism in the model Gram-positive bacterium, , and highlight pathways that enable the replication, survival, and virulence of this bacterial pathogen.
Topics: Animals; Bacterial Proteins; Gene Expression Regulation, Bacterial; Humans; Listeria monocytogenes; Listeriosis; Virulence
PubMed: 31418407
DOI: 10.1128/microbiolspec.GPP3-0066-2019 -
Frontiers in Immunology 2021The intestine harbors a complex community of bacterial species collectively known as commensal microbiota. Specific species of resident bacteria, as known as pathobiont,...
The intestine harbors a complex community of bacterial species collectively known as commensal microbiota. Specific species of resident bacteria, as known as pathobiont, have pathogenic potential and can induce apparent damage to the host and intestinal inflammation in a certain condition. However, the host immune factors that permit its commensalism under steady state conditions are not clearly understood. Here, we studied the gut fitness of by using germ-free (GF) mice orally infected with this food-borne pathogen. persistently exists in the gut of GF mice without inducing chronic immunopathology. at the late phase of infection is not capable of infiltrating through the intestinal barrier. established the commensalism through the reversible down regulation of virulence gene expression. CD8 T cells were found to be sufficient for the commensalism of . CD8 T cells responding to contributed to the down-regulation of virulence gene expression. Our data provide important insights into the host-microbe interaction and have implications for developing therapeutics against immune disorders induced by intestinal pathogens or pathobionts.
Topics: Animals; CD8-Positive T-Lymphocytes; Gastrointestinal Tract; Gene Expression Regulation, Bacterial; Germ-Free Life; Host Microbial Interactions; Intestinal Mucosa; Listeria monocytogenes; Mice; Pore Forming Cytotoxic Proteins; Symbiosis; Virulence
PubMed: 34012449
DOI: 10.3389/fimmu.2021.666088 -
Scientific Reports Jan 2022Listeria monocytogenes is a food-borne pathogen that can grow at very low temperatures close to the freezing point of food and other matrices. Maintaining cytoplasmic...
Listeria monocytogenes is a food-borne pathogen that can grow at very low temperatures close to the freezing point of food and other matrices. Maintaining cytoplasmic membrane fluidity by changing its lipid composition is indispensable for growth at low temperatures. Its dominant adaptation is to shorten the fatty acid chain length and, in some strains, increase in addition the menaquinone content. To date, incorporation of exogenous fatty acid was not reported for Listeria monocytogenes. In this study, the membrane fluidity grown under low-temperature conditions was affected by exogenous fatty acids incorporated into the membrane phospholipids of the bacterium. Listeria monocytogenes incorporated exogenous fatty acids due to their availability irrespective of their melting points. Incorporation was demonstrated by supplementation of the growth medium with polysorbate 60, polysorbate 80, and food lipid extracts, resulting in a corresponding modification of the membrane fatty acid profile. Incorporated exogenous fatty acids had a clear impact on the fitness of the Listeria monocytogenes strains, which was demonstrated by analyses of the membrane fluidity, resistance to freeze-thaw stress, and growth rates. The fatty acid content of the growth medium or the food matrix affects the membrane fluidity and thus proliferation and persistence of Listeria monocytogenes in food under low-temperature conditions.
Topics: Listeria monocytogenes
PubMed: 35087150
DOI: 10.1038/s41598-022-05548-6