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Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes.

ABSTRACT: Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Delta lgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Delta lgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Delta lgt Delta prfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.

SUBMITTER: Baumgartner M

PROVIDER: S-EPMC1797373 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes.

Baumgärtner Maja M Kärst Uwe U Gerstel Birgit B Loessner Martin M Wehland Jürgen J Jänsch Lothar L

Journal of bacteriology 20061013 2

Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular prote ...[more]

PMID: 17041050

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Project description:This project was undertaken to determine the kinetic parameters of thermal inactivation of Listeria monocytogenes on pecans, macadamia nuts, and sunflower seeds subjected to heat treatments simulating industry processes. Five strains were grown in nonselective medium, mixed, and resuspended before inoculating macadamia nuts, pecans, and sunflower seeds (6 to 9 Log CFU/g). Redried inoculated pecans and macadamia nuts were heated in an oven at a temperature range of 90 to 140°C. Unshelled sunflower seeds were heated in sunflower seed oil. The thermal inactivation was determined by measuring viable cell counts using standard microbiological methods. Average count data were fit to the log-linear model, and thermal-death kinetics were calculated. On pecans, the viable Listeria counts were reduced by 3 and 3.5 Log CFU/g after 40 min at 110°C and 8 min at 140°C, respectively. On macadamia nuts, the L. monocytogenes population was reduced by 5 Log CFU/g after 20 min at 120°C. Unshelled sunflower seeds were subjected to heat treatment via a hot-oil bath. On sunflower seeds, >7 Log CFU/g reductions were observed after 15 min at 120°C. The thermal resistance (D value) for inactivation on pecans at 140°C was 3.1 min and on macadamia nuts at 120°C was 4.4 min. The inactivation of L. monocytogenes was influenced by the kind of nut or seed. These results suggest that L. monocytogenes has a relatively high thermal tolerance. The findings from this study will contribute to the assessment of the effectiveness of heat treatment for control of this pathogen on nuts and seeds. IMPORTANCE Listeria monocytogenes is a major concern for the food industry in ready-to-eat (RTE) foods. In recent years, large-scale recalls have occurred with contaminated sunflower seeds and macadamia nuts that triggered product withdrawals. These events stress the importance of understanding Listeria's ability to survive heat treatments in these low-water activity foods. Nuts and seeds are subjected to a variety of thermal treatments typically referred as roasting. To date, no listeriosis outbreak has been linked to nuts and seeds, but the recent recognition that this pathogen can be detected in commercial products stresses the need for research on thermal treatments. The characterization of heat inactivation kinetics at temperatures typically used during roasting processes will be very beneficial for validation studies. This manuscript reports inactivation rates of L. monocytogenes strains inoculated onto macadamia nuts, sunflower seeds, and pecan halves subjected to temperatures between 90 and 140°C.

Project description:BackgroundListeria monocytogenes is a common cause of bacterial meningitis. We developed an animal model of listerial meningitis.MethodsIn survival studies, C57BL/6 mice received intracisternal injections with different L. monocytogenes sequence type 1 (ST1) colony forming units per milliliter (CFU; n = 48, 105, 106, 107, 108, and 109 CFU/ml). Second, mice were inoculated with 108 CFU/ml ST1 and sacrificed at 6 h and 24 h (n = 12/group). Outcome parameters were clinical score, CFUs, cyto- and chemokine levels, and brain histopathology. Third, 84 mice were inoculated (109 CFU/ml ST1) to determine optimal antibiotic treatment with different doses of amoxicillin and gentamicin. Fourth, mice were inoculated with 109 CFU/ml ST1, treated with amoxicillin, and sacrificed at 16 h and 24 h (n = 12/group) for outcome assessment. Finally, time point experiments were repeated with ST6 (n = 24/group).ResultsMedian survival time for inoculation with 108 and 109 CFU/ml ST1 was 46 h and 40 h; lower doses of bacteria led to minimal clinical signs of disease. Brain levels of IL-6, IL-17A, and IFN-γ were elevated at 24 h, and IL-1β, IL-6, IL-10, IFN-γ, and TNF-α were elevated in blood at 6 h and 24 h. Histopathology showed increased meningeal infiltration, vascular inflammation of meningeal vessels, hemorrhages, and ventriculitis. In the treatment model, brain levels of IL-6 and IL-17A and blood levels of IL-6 and IFN-γ were elevated. Compared to ST6, infection with ST1 led initially to higher levels of IL-1β and TNF-α in blood and more profound neuropathological damage. At 16 h post inoculation, IL-1β, IL-10, and TNF-α in blood and IL-6, IL17A, TNF-α, and IFN-γ levels in brain were higher in ST1 compared to ST6 without differences in CFUs between STs. At 24 h, neuropathology score was higher in ST1 compared to ST6 (p = 0.002) infected mice.ConclusionsWe developed and validated a murine model of listerial meningitis. ST1-infected mice had a more severe inflammatory response and brain damage as compared to ST6-infected mice.

Project description:The application of cold atmospheric pressure plasma (CAP) for decontamination of sliced ready-to-eat (RTE) meat products (in this case, rolled fillets of ham), inoculated with Salmonella (S.) Typhimurium and Listeria (L.) monocytogenes was investigated. Cold atmospheric plasma (CAP) is an ionised gas that includes highly reactive species and ozone, interacting with cell membranes and DNA of bacteria. The mode of action of CAPs includes penetration and disruption of the outer cell membrane or intracellular destruction of DNA located in the cytoplasm. Inoculated ham was treated for 10 and 20 min with CAP generated by a surface-micro-discharge-plasma source using cost-effective ambient air as working gas with different humidity levels of 45-50 and 90%. The chosen plasma modes had a peak-to-peak voltage of 6.4 or 10 kV and a frequency of 2 and 10 kHz. Under the tested conditions, the direct effectiveness of CAP on microbial inactivation was limited. Although all treated samples showed significant reductions in the microbial load subsequent to plasma treatment, the maximum inactivation of S. Typhimurium was 1.14 lg steps after 20 min of CAP-treatment (p<0.05), and L. monocytogenes was reduced by 1.02 lg steps (p<0.05) using high peak-to-peak voltage of 10 kV and a frequency of 2 kHz regardless of moisture content. However, effective inactivation was achieved by a combination of CAP-treatment and cold storage at 8°C ± 0.5°C for 7 and 14 days after packaging under sealed high nitrogen gas flush (70% N2, 30% CO2). Synergistic effects of CAP and cold storage for 14 days led to a clearer decrease in the microbial load of 1.84 lg steps for S. Typhimurium (p<0.05) and 2.55 lg steps for L. monocytogenes (p<0.05). In the case of L. monocytogenes, subsequent to CAP-treatment (10 kV, 2 kHz) and cold storage, microbial counts were predominantly below the detection limit. Measurement showed that after CAP-treatment, surface temperature of ham did not exceed the room temperature of 22°C ± 2°C. With the application of humidity levels of 45-50%, the colour distance ΔE increased in CAP treated samples due to a decrease in L* values. In conclusion, effectiveness of CAP-treatment was limited. However, the combination of CAP-treatment and cold storage of samples under modified-atmospheric-conditions up to 14 days could significantly reduce microorganisms on RTE ham. Further investigations are required to improve effectiveness of CAP-treatment.

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Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes.

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Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes.

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