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Structural basis of VHH-mediated neutralization of the food-borne pathogen Listeria monocytogenes.

ABSTRACT: Listeria monocytogenes causes listeriosis, a potentially fatal food-borne disease. The condition is especially harmful to pregnant women. Listeria outbreaks can originate from diverse foods, highlighting the need for novel strategies to improve food safety. The first step in Listeria invasion is internalization of the bacteria, which is mediated by the interaction of the internalin family of virulence factors with host cell receptors. A crucial interaction for Listeria invasion of the placenta, and thus a target for therapeutic intervention, is between internalin B (InlB) and the receptor c-Met. Single-domain antibodies (VHH, also called nanobodies, or sdAbs) from camel heavy-chain antibodies are a novel solution for preventing Listeria infections. The VHH R303, R330, and R326 all bind InlB with high affinity; however, the molecular mechanism behind their mode of action was unknown. We demonstrate that despite a high degree of sequence and structural diversity, the VHH bind a single epitope on InlB. A combination of gentamicin protection assays and florescent microscopy establish that InlB-specific VHH inhibit Listeria invasion of HeLa cells. A high-resolution X-ray structure of VHH R303 in complex with InlB showed that the VHH binds at the c-Met interaction site on InlB, thereby acting as a competitive inhibitor preventing bacterial invasion. These results point to the potential of VHH as a novel class of therapeutics for the prevention of listeriosis.

SUBMITTER: King MT

PROVIDER: S-EPMC6120195 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Structural basis of VHH-mediated neutralization of the food-borne pathogen Listeria monocytogenes.

King Moeko Toride MT Huh Ian I Shenai Akhilesh A Brooks Teresa M TM Brooks Cory L CL

The Journal of biological chemistry 20180705 35

Listeria monocytogenes causes listeriosis, a potentially fatal food-borne disease. The condition is especially harmful to pregnant women. Listeria outbreaks can originate from diverse foods, highlighting the need for novel strategies to improve food safety. The first step in Listeria invasion is internalization of the bacteria, which is mediated by the interaction of the internalin family of virulence factors with host cell receptors. A crucial interaction for Listeria</i ...[more]

PMID: 29976754

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Project description:Listeria monocytogenes is one of the most heat-resistant non-spore-forming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmid-mediated heat resistance of L. monocytogenes for the first time. This resistance is mediated by the ATP-dependent protease ClpL. We tested the survival of two wild-type L. monocytogenes strains-both of serotype 1/2c, sequence type ST9, and high sequence identity-at high temperatures and compared their genome composition in order to identify genetic mechanisms involved in their heat survival phenotype. L. monocytogenes AT3E was more heat resistant (0.0 CFU/ml log10 reduction) than strain AL4E (1.4 CFU/ml log10 reduction) after heating at 55°C for 40 min. A prominent difference in the genome compositions of the two strains was a 58-kb plasmid (pLM58) harbored by the heat-resistant AT3E strain, suggesting plasmid-mediated heat resistance. Indeed, plasmid curing resulted in significantly decreased heat resistance (1.1 CFU/ml log10 reduction) at 55°C. pLM58 harbored a 2,115-bp open reading frame annotated as an ATP-dependent protease (ClpL)-encoding clpL gene. Introducing the clpL gene into a natively heat-sensitive L. monocytogenes strain (1.2 CFU/ml log10 reduction) significantly increased the heat resistance of the recipient strain (0.4 CFU/ml log10 reduction) at 55°C. Plasmid-borne ClpL is thus a potential predictor of elevated heat resistance in L. monocytogenes. IMPORTANCEListeria monocytogenes is a dangerous food pathogen causing the severe illness listeriosis that has a high mortality rate in immunocompromised individuals. Although destroyed by pasteurization, L. monocytogenes is among the most heat-resistant non-spore-forming bacteria. This poses a risk to food safety, as listeriosis is commonly associated with ready-to-eat foods that are consumed without thorough heating. However, L. monocytogenes strains differ in their ability to survive high temperatures, and comprehensive understanding of the genetic mechanisms underlying these differences is still limited. Whole-genome-sequence analysis and phenotypic characterization allowed us to identify a novel plasmid, designated pLM58, and a plasmid-borne ATP-dependent protease (ClpL), which mediated heat resistance in L. monocytogenes. As the first report on plasmid-mediated heat resistance in L. monocytogenes, our study sheds light on the accessory genetic mechanisms rendering certain L. monocytogenes strains particularly capable of surviving high temperatures-with plasmid-borne ClpL being a potential predictor of elevated heat resistance.

Dataset Information

Structural basis of VHH-mediated neutralization of the food-borne pathogen Listeria monocytogenes.

Publications

Structural basis of V<sub>H</sub>H-mediated neutralization of the food-borne pathogen <i>Listeria monocytogenes</i>.

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