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The International Journal of... Feb 2020The recombinases present in the all kingdoms in nature play a crucial role in DNA metabolism processes such as replication, repair, recombination and transcription....
The recombinases present in the all kingdoms in nature play a crucial role in DNA metabolism processes such as replication, repair, recombination and transcription. However, till date, the role of RecA in the deadly foodborne pathogen Listeria monocytogenes remains unknown. In this study, the authors show that L. monocytogenes expresses recA more than two-fold in vivo upon exposure to the DNA damaging agents, methyl methanesulfonate and ultraviolet radiation. The purified L. monocytogenes RecA protein show robust binding to single stranded DNA. The RecA is capable of forming displacement loop and hydrolyzes ATP, whereas the mutant LmRecA fails to hydrolyze ATP, showing conserved walker A and B motifs. Interestingly, L. monocytogenes RecA and LmRecA perform the DNA strand transfer activity, which is the hallmark feature of RecA protein with an oligonucleotide-based substrate. Notably, L. monocytogenes RecA readily cleaves L. monocytogenes LexA, the SOS regulon and protects the presynaptic filament from the exonuclease I activity. Altogether, this study provides the first detailed characterization of L. monocytogenes RecA and presents important insights into the process of homologous recombination in the gram-positive foodborne bacteria L. monocytogenes.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Bacterial Proteins; DNA, Bacterial; Homologous Recombination; Listeria monocytogenes; Rec A Recombinases; SOS Response, Genetics; Sequence Homology, Amino Acid; Serine Endopeptidases
PubMed: 31698090
DOI: 10.1016/j.biocel.2019.105642 -
International Journal of Food... Oct 2022Listeria monocytogenes is considered a major challenge for the food industry as it can persist for long periods in food processing plants by forming biofilms. The aims...
Listeria monocytogenes is considered a major challenge for the food industry as it can persist for long periods in food processing plants by forming biofilms. The aims of this study were: i) to assess the biofilm producing ability of 57 Listeria monocytogenes isolates previously subjected to whole-genome sequencing (WGS); ii) to compare the levels of biofilm formation with the presence or absence of biofilm associated genes. To determine the presence or absence of a known set of biofilm associated genes, a comparative genomic analysis was performed on each strain. Among Listeria monocytogenes isolates, 58 %, 38.5 % and 3.5 % exhibited weak, moderate or strong biofilm production, respectively. No difference in biofilm production was observed between food and environmental isolates. The percentage of Listeria monocytogenes strains isolated from meat products (57 %) classified as moderate or strong biofilm producers was higher than the percentage obtained for strains isolated from dairy products (28 %). The presence of the Stress Survival Islet 1, the arsD stress gene and the truncated inlA protein was significantly associated with increased levels of biofilm. Combining biofilm phenotype with molecular and genotyping data may provide the opportunity to better understand the relationship between genes linked to biofilm formation in Listeria monocytogenes.
Topics: Biofilms; Dairying; Food Microbiology; Genomics; Humans; Listeria monocytogenes; Listeriosis; Meat
PubMed: 35749910
DOI: 10.1016/j.ijfoodmicro.2022.109784 -
World Journal of Microbiology &... Jun 2021This research was carried out to investigate the differences in adhesion and growth during biofilm formation of L. monocytogenes from different sources and clonal...
This research was carried out to investigate the differences in adhesion and growth during biofilm formation of L. monocytogenes from different sources and clonal complexes. Biofilm by L. monocytogenes (isolates CLIST 441 and 7: both lineage I, serotype 1/2b, CC3; isolates 19 and 508: both lineage II, serotype 1/2c, CC9) was grown on stainless steel coupons under different stressing conditions (NaCl, curing salts and quaternary ammonium compounds-QAC), to determine the expression of different genes involved in biofilm formation and stress response. CLIST 441, which carries a premature stop codon (PMSC) in agrC, formed high-density biofilms in the presence of QAC (7.5% w/v) or curing salts (10% w/v). Reverse Transcriptase-qPCR results revealed that L. monocytogenes isolates presented differences in transcriptional profile of genes related to biofilm formation and adaptation to environmental conditions. Our results demonstrated how L. monocytogenes can survive, multiply and form biofilm under adverse conditions related to food processing environments. Differences in transcriptional expression were observed, highlighting the role of regulatory gene networks for particular serotypes under different stress responses.
Topics: Bacterial Adhesion; Bacterial Proteins; Bacteriological Techniques; Biofilms; Culture Media; Food Microbiology; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Quaternary Ammonium Compounds; Reverse Transcriptase Polymerase Chain Reaction; Sodium Chloride; Stainless Steel; Stress, Physiological
PubMed: 34131813
DOI: 10.1007/s11274-021-03092-5 -
BMC Microbiology May 2020Listeria monocytogenes (L. monocytogenes) is a global opportunistic intracellular pathogen that can cause many infections, including meningitis and abortion in humans...
BACKGROUND
Listeria monocytogenes (L. monocytogenes) is a global opportunistic intracellular pathogen that can cause many infections, including meningitis and abortion in humans and animals; thus, L. monocytogenes poses a great threat to public safety and the development of the aquaculture industry. The isolation rate of Listeria monocytogenes in fishery products has always been high. And the pore-forming toxin listeriolysin O (LLO) is one of the most important virulence factors of L. monocytogenes. LLO can promote cytosolic bacterial proliferation and help the pathogen evade attacks from the host immune system. In addition, L. monocytogenes infection can trigger a series of severe inflammatory reactions.
RESULTS
Here, we further confirmed that morin lacking anti-Listeria activity could inhibit LLO oligomerization. We also found that morin can effectively alleviate the inflammation induced by Listeria in vivo and in vitro and exerted an obvious protective effect on infected cells and mice.
CONCLUSIONS
Morin does not possess anti-Listeria activity, neither does it interfere with secretion of LLO. However, morin inhibits oligomerisation of LLO and morin does reduce the inflammation caused during Listeria infection.
Topics: Animals; Bacterial Toxins; Cell Line; Disease Models, Animal; Flavonoids; Gene Expression Regulation, Bacterial; Heat-Shock Proteins; Hemolysin Proteins; Humans; Listeria monocytogenes; Listeriosis; Mice; Protein Multimerization; Virulence
PubMed: 32398085
DOI: 10.1186/s12866-020-01807-6 -
Methods in Molecular Biology (Clifton,... 2021Listeria monocytogenes is a major food-borne pathogen and causative agent of a fatal disease, listeriosis. Stringent regulatory guidelines and zero tolerance policy...
Listeria monocytogenes is a major food-borne pathogen and causative agent of a fatal disease, listeriosis. Stringent regulatory guidelines and zero tolerance policy toward this bacterium necessitate rapid, accurate, and reliable methods of identification and subtyping. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) has recently become a method of choice for routine identification of pathogens in clinical settings and has largely replaced biochemical assays. Identification relies on well-curated databases such as SARAMIS. Extensive use of SARAMIS to generate consensus mass spectra, in conjunction with statistical analysis, such as partial least square-discriminant analysis and hierarchical cluster analysis, is useful in subtyping bacteria. While MALDI-ToF MS has been extensively used for pathogen detection, its application in bacterial subtyping has been limited. The protocol describes a MALDI-ToF MS workflow as a single tool for simultaneous identification and subtyping of L. monocytogenes directly from solid culture medium.
Topics: Bacterial Typing Techniques; Humans; Listeria monocytogenes; Listeriosis; Software; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors; Workflow
PubMed: 32975763
DOI: 10.1007/978-1-0716-0982-8_2 -
MBio Aug 2020The facultative intracellular pathogen , like many related , uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability,...
The facultative intracellular pathogen , like many related , uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in , the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for -dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.
Topics: Animals; Bacterial Proteins; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Homeostasis; Listeria monocytogenes; Mice; Protein Binding; Second Messenger Systems; Signal Transduction
PubMed: 32843560
DOI: 10.1128/mBio.01625-20 -
Journal of Food Science Sep 2020This study was aimed to investigate the presence of Listeria monocytogenes in raw water buffalo milk and milk products, besides determining its serotype and the extent...
This study was aimed to investigate the presence of Listeria monocytogenes in raw water buffalo milk and milk products, besides determining its serotype and the extent of its resistance against various antibiotics. A total of 188 samples of raw water buffalo milk and milk products were collected from Samsun Province, Turkey between November 2012 and May 2013. The classical culture technique was used to isolate and identify L. monocytogenes, as described in EN ISO 11290-1. The isolates were confirmed as L. monocytogenes by using PCR with (hylA) primers specific for the hemolysin gene. The antimicrobial susceptibility test was achieved by using the VITEK 2 compact system and VITEK 2 AST-P640 card. L. monocytogenes was found in 7 (3.7%) of the 188 samples. Four of them were obtained from cheese and three from milk samples. Whereas, L. monocytogenes was not detected in any of the clotted cream samples. A total of 13 isolates were confirmed by PCR as L. monocytogenes. Among these isolates, one was 1/2c (or 3c) (7.6%), three were 4b (or 4d, 4e) (23%), four were 1/2b (or 3b) (30.7%), and the other five isolates were serotype 1/2a (or 3a) (38.4%). The highest antimicrobial resistance was recorded against fosfomycine (100%) followed by oxacillin (92%), penicillin (84%), and erythromycin (69%). However, no resistance was determined against ciprofloxacin, gentamicin, and tigecycline. PRACTICAL APPLICATION: This study showed that some samples of raw buffalo milk and the milk products were contaminated with Listeria monocytogenes. The serotype with the highest prevalence was determined as L. monocytogenes 1/2a. This study also demonstrated that most of the L. monocytogenes isolates had developed multiresistance to many frequently used medical antimicrobial agents.
Topics: Animals; Anti-Bacterial Agents; Buffaloes; Cheese; DNA Primers; Drug Resistance, Bacterial; Food Contamination; Food Microbiology; Listeria monocytogenes; Milk; Polymerase Chain Reaction; Serotyping; Turkey
PubMed: 32794185
DOI: 10.1111/1750-3841.15376 -
Molecular Omics Jun 2021In this work, the effect of antimicrobial lipopeptide P34 on Listeria monocytogenes was evaluated for the first time through a proteomics approach. Bacteria were treated... (Comparative Study)
Comparative Study
In this work, the effect of antimicrobial lipopeptide P34 on Listeria monocytogenes was evaluated for the first time through a proteomics approach. Bacteria were treated with sub-lethal doses of peptide P34 (F-P34) and P34 encapsulated into nanoliposomes (N-P34), while empty nanoliposomes (NE) and fresh buffer were used as controls. The proteomic analysis allowed the detection of one group of proteins commonly differentially represented in response to free and encapsulated P34 exposure. A second group of proteins was found to be exclusively differentially represented after exposure with encapsulated P34 only. The antimicrobial peptide P34 caused a significant downregulation of proteins associated with the transport of manganese and the over-representation of proteins related with iron transport in L. monocytogenes. In addition, reduction of stress tolerance proteins related to the σB and VirR regulons, together with the modulation of phosphoenolpyruvate phosphotransferase systems (PTS) for sugar transport were observed. The sugar and oligopeptide transporters regulated by antimicrobial action may influence the key virulence factor PrfA, reducing the pathogenicity of this microorganism.
Topics: Antimicrobial Peptides; Bacterial Proteins; Biological Transport; Drug Compounding; Gene Expression Regulation, Bacterial; Iron; Lipopeptides; Listeria monocytogenes; Manganese; Nanostructures; Proteomics
PubMed: 33735358
DOI: 10.1039/d0mo00178c -
Molecular Microbiology Mar 2020The universe of Molecular Microbial Pathogenesis is filled with many female and male stars. But there are two particularly bright shining supernovae-like stars: the late...
The universe of Molecular Microbial Pathogenesis is filled with many female and male stars. But there are two particularly bright shining supernovae-like stars: the late Stanley Falkow and the very lively and creative Pascale Cossart. These two outstanding luminaries, surrounded by numerous planets, do not only belong to different scientific generations but their splendor also comes from very different scientific concepts. Stanley Falkow, often referred to as the 'Father of Molecular Microbial Pathogenesis', made many groundbreaking contributions to this field by addressing almost all important bacterial pathogens. Pascale Cossart, who could be called in analogy the 'Queen of Modern Molecular Microbial Pathogenesis' by combining the Microbiology and Cell Biology, concentrates in her similarly impressive scientific work essentially on a single bacterial species which she studied and still studies in great depth: the facultative intracellular bacterial pathogen Listeria monocytogenes-and the vast majority of her most prominent publications deals with this pathogen in almost all facets. It is certainly not an exaggeration to say that she together with her co-workers and collaborators developed this model bacterium into a paradigm among the intracellular bacterial pathogens.
Topics: Female; History, 20th Century; History, 21st Century; Host-Pathogen Interactions; Humans; Listeria monocytogenes; Listeriosis; Virulence; Virulence Factors
PubMed: 32185837
DOI: 10.1111/mmi.14450 -
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