Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes

Abstract

To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin 0. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin 0, by immunoblotting with a specific antiserum against listeriolysin 0. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin 0 would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin 0 (58,000). The protein sequence is highly homologous to those of streptolysin 0 and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin 0 gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.

A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes

“…HindIII digestion of amplified DNA generated two products of 162 bp and 255 bp ( Fig. 2b) in agreement with previously published sequence data (Mengaud et al 1988).…”

Listeriolysin O: a key protein ofListeria monocytogeneswith multiple functions

“…The LLO protein is synthesized as a precursor composed of 529 residues and possesses at its N-terminus a typical signal sequence (SS) (Mengaud, 1988). After cleavage of the SS, the mature protein is secreted into the culture supernatant as a monomer (Geoffroy et al, 1989).…”

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

“…A specific RTi-PCR assay for the quantitative detection of L. monocytogenes has already been developed [25]. This assay targets the hly gene [26] (GenBank Accession No. M24199, positions 1602-1665), which codes for the virulence factor listeriolysin O.…”