Genetic Markers and Pathogenesis Features of Listeria Monocytogenes Serotype 4b

NON-TECHNICAL SUMMARY: Listeria monocytogenes of serotype 4b are responsible for a substantial fraction of food-borne listeriosis, and are involved in the vast majority of common-source, food-borne outbreaks of the disease. This project examines the suitability of serotype-specific sequences for develioment of acucrate "molecular serotypeing" schemes for the pathogen. The project also aims at the investigation fo the possible role of serotype-specific sequences in virulence. Virulence will be evaluated in a human endothelial cell model.

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APPROACH: <BR> Objective 1. To achieve Objective 1, we will identify candidate serotype-specific sequences that are unique to major serotypes of special epidemiologic relevance. Of key interest will be sequences specific to serotypes 4b, 1/2a and 1/2b, which are associated with the majority of human cases of food borne listeriosis. Oligonucleotide primers will be designed on the basis of these sequences and will be used to amplify the corresponding DNA fragments by Polymerase Chain Reaction (PCR). The amplified fragments will be used as probes against genomic DNAs from a panel of strains of diverse serotypes, of both human and food origin, to determine the serotype-specificity of the sequences. The results will be combined with DNA subtyping tools derived from the sequence of the flagellin genes to enhance the resolution of the PCR-based "molecular serotyping" scheme. In addition, the amplified fragments of the serotype-specific genes will be used as digoxigenin-labeled probes in Southern blots to more accurately determine the distribution of the corresponding sequences in the genome of different strains of Listeria monocytogenes.<BR><BR> Objective 2. To achieve Objective 2, we will investigate the ability of Listeria monocytogenes of serotype 4b, and isogenic derivatives deficient in the expression of selected serotype-specific genes, to evaluate the possible involvement of serotype-specific antigens in activiation of human endothelial cells in culture. We will concentrate on the serotype-specific genes gtcA, gltA and gltB, present in serotype 4b strains, and the gene mtrA, present in strains of serotype 1/2a and 1/2b. The ability of the mutants to bind to the endothelial cells and invade these cells will be determined. The stimulation of the endothelial cells by the bacteria will be quantitatively determined by whole cell ELISA-based measurements of E-selectin, ICAM-1 and VCAM-1 expression on the endothelial cells, using commercially available reagents and established procedures. At this time, we will investigate the possible involvement of four distinct serotype-specific genes in endothelial cell binding, invasion and activation.

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PROGRESS: 2002/05 TO 2005/05<BR>
Listeria monocytogenes of serotype 4b is responsible for a large fraction of sporadic cases of foodborne listeriosis, and for mot of the outbreaks of illness. This project has focused on characterization of two serotype -specific genomic regions of L. monocytogenes, the region that harbors the gene gtcA and the region that harbors the gltAgltB genes. We have confirmed that gtcA is specific to serogroup 4 (serotypes 4a, 4b, 4c, 4e and 4e) whereas gltA and gltB are unique to the serotype 4b complex 9serotype 4b and the closely related serotypes 4d and 4e). In terms of potential of these genes as genetic markers for rapid detection of serotype 4b isolates, we have found that the gene gltA, which is required for serotype 4-specific decoration of the teichoic acid of the cell wall, offers reliable detection, both by Polymerase Chain Reaction and by Southern blots or other hybridization formats. Serotype 4d and 4e isolates remain associated with serotype 4b in terms of their reactivity with gltA probes, and all other tests that we have devised, suggesting that the use of the term serotype 4b complex, that encompasses serotypes 4b, 4d and 4e, may be appropriate. Screening of a population of Listeria monocytogenes isolates from foods, food processing environments, and recent clinical cases with the gltA probes readily identified those belonging to the serotype 4b complex. Rare genotypes were identified among sporadic clinical isolates, that had the typical serotype 4b markers but harbored other, rarely encountered genes and had unique pulsed field gel electrophoresis patterns. Substantial effort has been invested in determining how the genomic content of sporadic serotype 4b strains, which constitute the majority of clinical listeriosis strains, differs from the more extensively characterized epidemic clonal groups.
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IMPACT: 2002/05 TO 2005/05<BR>
Listeria monocytogenes strains of serotype 4b are of special public health relevance, as they are involved in the majority of outbreaks of human listeriosis and in a large fraction of sporadic listeriosis cases. However, the genetic features unique to this serotype, and their possible involvement in virulence, remain poorly understood. The research findings from this project have provided DNA reagents useful for the detection and monitoring of strains of this important serotype, and have also increased our understanding of the unique genetic determinants of epidemiolgically relevant sub-populations of L. monocytogenes.