Progress 07/01/11 to 06/30/13
Outputs
Target Audience: Life Science Community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Targeting and dissemination through brain microvascular endothelium is considered as one of the routes for L. monocytogenes to cause CNS infection. To date, the molecular mechanisms of how L. monocytogenes interact with brain endothelium and cross the blood brain barrier are not completely understood. Due to the low invasiveness of L. monocytogenes on human brain microvascular endothelial cells (HBMEC), we hypothesized that overcome of HBMECs layer by L. monocytogenes is mediated by other mechanism such as the cytotoxic effect of listerial toxins rather than direct invasion. In this study, secreted proteins were collected from bacterial culture supernatant using ethanol precipitation and their cytotoxicity effect on HBEMCs was evaluated using live/dead staining method. Interestingly, cytotoxicity was induced by supernatant from the EGD wild type strain but the supernatant from the isogenic ?prfA strain did not induce cytotoxicity. Therefore, we further investigated the cytotoxicity effects of supernatants from the isogenic mutant strains (?plcA, ?mpl, and ?hly) that are incapable of producing the bacterial products (PlcA, Mpl, and LLO, respectively) that are under regulation of prfA. Using fluorescent microscopy and flow cytometry, supernatants from mutant strain ?hly did not induce the cytotoxicity on HBMEC but ?plcA and ?mpl did. The results suggest that LLO mediated the cytotoxicity on brain endothelial cells may contribute to the invasion of CNS by L. monocytogenes.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Abstract/Presentation PARTICIPANTS: Chinling Wang (PI), DVM, MS, Ph.D. Department of Basic Science, College of Veterinary Medicine, Mississippi State University Ting Zhang (Ph.D. student) is working on this project. TARGET AUDIENCES: Life Science Community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Listerial surface protein and host receptor interactions make paramount contributions to adherent and entry of Listeria to non-phagocytic cells. Interestingly, L. monocytogenes possesses a large set of leucine rich repeats (LRRs) molecules; however, those functions largely remain unknown. To determine the pathogenicity, 11 clinical isolates and an EGD strain of L. monocytogenes were tested in various non-phagocytic cells using adhesion and invasion assays. Among these isolates, L. monocytogenes 4b serotype H7858 strain demonstrated high invasiveness to both Caco-2 cell and HepG2 cells. We generated seven deletion mutants targeting LRRs motif of L. monocytogenes H7858 strain and tested its virulence. Among these seven mutants, H7858∆0369 strain, H7858∆2546 strain, and H7858∆InlB strain demonstrated attenuated phenotypes in vitro with significant impaired invasiveness in HepG-2 cells. We further tested the virulence of H7858∆0369 strain and H7858∆2546 strain in BALB/c mice via intravenous injection. Interestingly, H7858∆0369 strain showed significant defects in host organ colonization, bacteremia formation, as well as CNS infection. This study suggests that the 0369 gene is a novel virulence factor.
Publications
- Zhang, T., D. Bae, K. Seo and C. Wang. Characterization of surface proteins of Listeria monocytogenes that involve in adhesion and invasion of non-phagocytic cells. Missississippi State University Research Day. August 16, 2012. Mississippi State, Mississippi. (Abstract/oral presentation)
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Abstract/Presentation PARTICIPANTS: Chinling Wang (PI), DVM, MS, Ph.D. Department of Basic Science, College of Veterinary Medicine, Mississippi State University Ting Zhang (Ph.D. student) is working on this project. TARGET AUDIENCES: Life Science Community PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Listerial surface protein and host receptor interactions make paramount contributions to adherent and entry of Listeria to non-phagocytic cells. Interestingly, L. monocytogenes possesses a large set of leucine rich repeats (LRRs) molecules; however, those functions largely remain unknown. To determine the pathogenicity, 11 clinical isolates and an EGD strain of L. monocytogenes were tested in various non-phagocytic cells using adhesion and invasion assays. Among these isolates, L. monocytogenes 4b serotype H7858 strain demonstrated high invasiveness to both Caco-2 cell and HepG2 cells. All tested strains and isolates were less invasive to human brain microvascular endothelial cells. To further characterize the function of LRRs molecules, 8 deletion mutants were generated in H7858 strain using in-frame deletion mutagenesis. The entry of L. monocytogenes to HepG2 cells was significantly impaired when the function of three LRRs molecules Hinl1, Hinl2, and Hinl3 were inactivated. This is the first report that elucidates the role of these three LRRs molecules in L. monocytogenes's invasion.
Publications
- Zhang, T., D. Bae, K. Seo and C. Wang. Characterization of surface proteins of Listeria monocytogenes that involve in adhesion and invasion of non-phagocytic cells. Missississippi State University Research Day. August 16, 2012. Mississippi State, Mississippi. (Abstract/oral presentation)
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