Microbiology (1 996), 142, 173-1 80 Printed in Great Britain The inlA gene required for cell invasion is conserved and specific to Listeria monocytogenes Claire Poyart, Patrick Trieu-Cuot and Patrick Berche Author for correspondence: Patrick Berche. Tel: +33 1 40 61 53 79. Fax: +33 1 40 61 55 92. INSERM U.411, Facult6 de M6decine Necker-Enfants Malades, 156 rue de Vaugirard, 75724 Paris Cedex 15, France The Gram-positive bacterium Listeria monocytogenes can actively induce its own uptake by epithelial cells and fibroblasts through a surface-exposed 80 kDa protein, internalin (InlA), encoded by inIA. We studied the distribution and the DNA polymorphism of inIA sequences in a wide variety of wild strains of L. monocytogenes as compared to other Listeria species. This was done by PCR-amplifying inIA sequences encoding the fifteen repeats A and the three repeats B of InlA. inIA-repeatedsequences were only found in L. monocytogenes. The amplified fragment of inIA encoding the repeats A displayed an AIul DNA polymorphism which arises from point mutations. These results indicate that inIA required for cell invasion is specific to L. monocytogenes and that the intragenic repeats only exhibit a genetic heterogeneitydue to point mutations and not to recombinations. Keywords : Listeria monoqtogenes, inlA gene, internalin INTRODUCTION Listeria monoytogenes is a Gram-positive bacterium which is widely distributed in the environment and causes severe food-borne infections in humans and most warm-blooded animals (Gray & Killinger, 1966). Its pathogenicity is due to its capacity to survive and to multiply in a wide variety of cell types, including macrophages, fibroblasts, epi- thelial cells, hepatocytes and enterocytes (Mackaness, 1962 ; Racz et al., 1972 ; Havell, 1986 ; Gaillard et al., 1987 ; Woods et al., 1993). Like other intracellular bacteria, including the Gram-negative Yersinia psendotnbercnlosis (Isberg et al., 1987 ; Isberg & Leong, 1990), Sbigellaflexneri (Hale & Bonventre, 1979) and Salmonella tJvpbimurinm (Elsinghorst et al., 1989; Galan & Curtiss, 1989), L. monoytogenes can actively induce its own uptake by host cells through a specific interaction between a surface- exposed 80 kDa protein, termed internalin, and an un- identified receptor (Gaillard et al., 1987; Gaillard et al., 1991). The role of internalin has been established by isolating insertion mutants, which are non-adherent and non-invasive (Gaillard et al., 1991). Gene complemen- tation with inlA restores the invasive phenotype of these L. monoytogenes mutants and enables the non-invasive species Listeria innocna to penetrate enterocytes (Gaillard etal., 1991). However, inlA has only been characterized in two L. monoytogenes reference strains, E G D and LO28 (Gaillard et al., 1991), and almost nothing is known about the distribution of the gene in clinical isolates of this species. The gene inlA encodes a protein of 800 amino acids composed, from its amino- to its carboxy-terminus, of a signal peptide, 15 successive repeats of 22 amino acids (repeats A), three successive repeats of 70, 70 and 49 amino acids (repeats B), a proline/glycine-rich segment, a hexapeptide (LPTTGD) preceding a stretch of 20 hydro- phobic amino acids, and a short positively charged tail (Gaillard et al., 1991; Dramsi et al., 1993) (Fig. 1). Sequence analysis of the segments of inlA encoding regions A and B revealed that they are made up of DNA repeats with an identity of 3 3 4 9 % for repeats A, and 61-71 % for the repeats B (Table 1) (Gaillard et al., 1991 ; Dramsi et a/., 1993). Several pairs of segments encoding repeats A have in common a region of identity larger than 10 bp, and the two contiguous segments coding for repeats B2 and B3 share an identical 25 bp motif (Table 1). Moreover, Southern blot analysis indicates that inlA is also part of a gene family (Gaillard et al., 1991). One of them, designated inlB, is located immediately downstream of inlA and displays 56% identity with inlA (Gaillard et al., 1991). These structural data suggest that inlA might generate variants by intragenic recombination between repeated segments or by extragenic recombination with inlA-related sequences, as previously described for the emm genes of Streptococcnsp_yogenes encoding the M protein (Fischetti, 1989). 0002-0077 0 1996 SGM 173