J. Med. Microbiol. - Vol. 48 (1999), 117-124 0 1999 The Pathological Society of Great Britain and Ireland B ACTE R 1 AL PATH 0 G E N I C I TY Surface protein p104 is involved in adhesion of Listeria monocytogenes to h u man intestinal cel I line, Caco-2 V. K. PANDIRIPALLY, D. G. WESTBROOK, G. R. SUNK1 and A. K. BHUNIA Food Microbiology and lmmunochemistry Laboratory, Department of Life Sciences, Alabama A & M University, PO Box 264, Normal, AL 35762, USA Adhesion of Listeria monocytogenes to intestinal endothelial cells is an important initial event in the pathogenesis of infection which is not well understood. The suggestion has been made that some proteins, including internalin and actin polymerisation protein (ActA), and carbohydrate molecules mediate, at least in part, the adhesion of listeria to certain cultured mammalian cells. This study investigated the role of a L. monocytogenes cell-surface protein of 104 kDa (p104) in adhesion to human intestinal enterocyte-like Caco-2 cell lines by transposon (Tn916) mutagenesis and a pl04-specific monoclonal antibody (MAb-H7). Genotypic and phenotypic characteristics of Tn916-transformed L. monocytogenes strains, AAMU530 and AAMU572, revealed that these strains did not express p104, and the transposon had been inserted at a single locus in the structural gene. Strains AAMU530 and AAMU572 yielded only 10 and 6.3% adhesion to Caco-2 cells. Coating of L. monocytogenes and L. innocua wild-type strains with MAb-H7 reduced adhesion to Caco-2 cells from 100% to 50 and 45%, respectively, whereas on isotype control MAb EM-7G1 had no effect. Western blot analysis with MAb-H7 indicated that p104 is present in all Listeria spp. except in L. grayi. Furthermore, p104 is also present in internalin (BUGS) and ActA (LUT12) deficient strains, suggesting that p104 is indeed different from internalin or ActA proteins. Cytotoxicity analysis of strains AAMU530 and AAMU572 demonstrated that these strains, although haemolytic and phospholipase-positive, were avirulent when tested with a hybridoma B-lymphocyte cell line. Loss of virulence could be attributed to the interruption of adhesion of mutant strains to the hybridoma cell line. These results strongly suggest that p104 is an adhesion factor in L. monocytogenes and involved in adhesion to intestinal cells. Introduction Listeria monocytogenes, a food-borne human pathogen, is responsible for infections such as septicaemia and meningitis in immunocompromised individuals and is one cause of abortion in pregnant women [l]. A recent report indicated that L. monocytogenes also causes gastro-enteritis in healthy individuals [2]. Transmission in man is frequently linked to food contaminated by listeria and entry into the host normally occurs in the gut. In animal studies (following intragastric inocula- tion) bacteria pass the gastrointestinal barrier and possibly penetrate the intestinal epithelial cells over- Received 24 Feb. 1998; accepted 4 May 1998. Corresponding author: Dr A. IS. Bhunia, Purdue University, Department of Food Science, West Lafayette, IN 47907- 1 160, USA. E-mail: bhuniaa@foodsci.purdue.edu possibly in other Listeria species and is laying Peyer’s patches [3,4]. The organism then disseminates to the brain and to the spleen, liver and other lymphatic systems. A key aspect of the pathogenicity of L. monocyto- genes is its ability to invade and multiply in phagocytic and non-phagocytic cells. Adhesion of L. rnonocytogenes to host cells is an essential event for invasion leading to infection. Adhesion is presumed to be mediated by L. monocytogenes cell-surface mole- cules with complementary receptors present on the eukaryotic cells [5]. Internalin (80 kDa) [6, 71, actin polymerisation protein (ActA, 90 kDa) [S], and other surface molecules [5,9, 101 of L. monocytogenes have been shown to be partially responsible for adhesion to mammalian cells. Internalin A (InlA) is essential for entry into human enterocyte-like Caco-2 cells, whereas Internalin B (InlB) is required for entry into cultured