Listeria adhesion protein and heat shock protein 60: Application in pathogenic Listeria detection and implication in Listeriosis prevention

Ok Kyung Koo, Purdue University

Abstract

Listeria adhesion protein (LAP, lmo1634), also known as a housekeeping enzyme alcohol acetaldehyde dehydrogenase, is a cell surface protein involved in adhesion and translocation of L. monocytogenes to human intestinal cells. The pathogen crosses the intestinal barrier following interaction with the receptor present on intestinal epithelial cells. Heat shock protein 60 (Hsp60), a eukaryotic mitochondrial chaperon protein is the receptor for LAP. LAP is present in all Listeria species except L. grayi, and the purified LAP from all Listeria species had similar molecular characteristics such as high sequence similarity, strong binding to Hsp60 and enzyme activity. Interestingly, secretion of LAP was only observed in pathogenic L. monocytogenes and one of our previous study suggested that LAP secretion is SecA2-dependent.Because of the specific interaction between surface exposed LAP and Hsp60, strategies could be employed to potentially protect the host from the infection by blocking the attachment. The overall goals of this project were to enhance our understanding of the adhesion mechanism and to apply this knowledge in developing a recombinant probiotic bacterial strain to prevent infection in a cell culture model and to develop method(s) to improve L. monocytogenes detection from food matrices. Antibodies have been widely used for detection of specific antigens; however, earlier studies have shown that non-specific interactions may reduce specificity of antibody detection. As an alternate method, Hsp60 was used on microfluidic biochip and paramagnetic beads to capture and concentrate L. monocytogenes from contaminated foods. The efficiency of Hsp60 to capture L. monocytogenes was 83 times greater than monoclonal antibody MAb-C11E9, on the biochip. Hsp60 also showed specific interaction with L. monocytogenes compared to other Listeria spp. and other food-borne bacteria on both detection platforms. On paramagnetic beads, Hsp60 was able to capture 2.1- to 6.2-fold more of L. monocytogenes than L. innocua in hotdog samples under a coculture environment. We also examined if LAP can be expressed in a probiotic bacterium to prevent Listeria attachment and colonization in an in vitro Caco-2 cell culture model. Wild type probiotic Lactobacillus species were unable to reduce the adhesion of L. monocytogenes; however, Lb. paracasei expressing LAP caused significant reduction in L. monocytogenes adhesion (25-31%) and transepithelial translocation (40%) in Caco-2 cells. Furthermore, re-association of secreted LAP to the surface of Listeria strain and recombinant Lactobacillus was found to be essential to promote interaction with host cells. Non-pathogenic Listeria lacks surface molecule(s) that aid in LAP re-association, hence it unables to interact with host cells. Collectively, our data show that Hsp60 interacts strongly with only pathogenic Listeria, including L. monocytogenes, and can be used to capture and detect the pathogen on microfluidic biochip or on magnetic beads. Furthermore, LAP expressing probiotic Lactobacillus has the potential to protect host against infection by specific blocking of Listeria interaction with the host receptor.

Degree

Ph.D.

Advisors

Bhunia, Purdue University.

Subject Area

Food Science|Microbiology

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