Characterization by molecular cloning and sequencing of the gene encoding an aminopeptidase from Listeria monocytogenes.

The pepC gene of Listeria monocytogenes encodes aminopeptidase C that is predicted to share 72% amino acid sequence similarity and 53% sequence identity with the cysteine aminopeptidase PepC from Lactococcus lactis. The gene product also shows strong similarity to aminopeptidase C from Streptococcus thermophilus and Lactobacillus helveticus, and to a cysteine proteinase/bleomycin hydrolase from Saccharomyces cerevisiae. The enzyme from L. monocytogenes displayed broad N-terminal hydrolytic activity, with a similar substrate specificity to its lactic acid bacterial counterpart. The inhibition spectrum shows a great deal of similarity with enzymes from the family of lactic acid bacteria. In addition, one of the clones studied contained DNA sequences that could encode a regulatory protein of the deoR helix-turn-helix DNA binding protein family. The organization of the locus, designated pep, is presented along with the characterization of the gene products of the pep locus.

Rapid detection of Listeria monocytogenes by a PCR assay specific for an aminopeptidase.

Specific and rapid detection of Listeria monocytogenes is very important with regard to food safety since all other species of Listeria appear to be non-pathogenic to humans. Conventional microbiological detection methods are very time consuming. The polymerase chain reaction (PCR) is one of the most promising techniques for rapid detection of micro-organisms in food products. We have developed a PCR assay, specific for L. monocytogenes, based on the gene encoding an aminopeptidase, which previously has not been described for this species. The L. monocytogenes aminopeptidase shares strong sequence similarity with aminopeptidase C from Streptococcus thermophilous, Lactobacillus lactis, Lactobacillus helveticus, and with a cysteine proteinase from Saccharomyces cerevisiae. Polymerase chain reaction primers were synthesized based on the DNA sequence of the aminopeptidase gene. A 90 bp product was apparent with all L. monocytogenes strains tested but not with other species of Listeria or other bacterial genera. The PCR assay, which is performed directly from whole bacterial cells, does not involve DNA purification and can be conducted in 4 h. It provided positive identification of L. monocytogenes in mixed culture.

A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources.

An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10(8) cfu/ml into a suspension of Ped-2E9 (10(6)/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test. Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 microns filter.