Characterisation of Listeria monocytogenes using targeted proteomic analysis.

Abstract

Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection that is increasing significantly in Europe and North America. A correlating factor contributing to the resurgence of listeriosis is the rise in consumption of cold-stored ready-to-eat (RTE) foods. The steady upsurge in disease requires more focused research to control the pathogen, L. monocytogenes. Currently, there is a plethora of diagnostic methods for the causative agent, however, each has limitations, one of which is the inability to correlate results across laboratories. This is a particular hindrance to an outbreak investigation in an age when food is transported widely across the globe. In this study, proteomic approaches were used to search for biomarkers that facilitate rapid characterisation of isolates against a background of differentially expressed proteins. A preamble to this investigation necessitated incorporation of an efficient lysis procedure to release maximum proteins. This was eventually achieved using a Listeria specific enzyme, endolysin, and a disruptive mechanical method. Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) data showed that bead beating and enzymatic lysis were the most efficient methods for analysis of the proteome. Dendrogram lineages, derived from MALD-TOF-MS spectra, strongly correlated with 16S rRNA analyses. Selective protein capture and analysis by MALD-TOF-MS (designated SELDI-TOF-MS) demonstrated considerable intraspecies diversity as revealed by dendrograms which were also visualised by „Heat Maps‟. One-dimensional polyacrylamide gel electrophoresis and LC-MS/MS analysis of seven L. monocytogenes isolates, led to the successful identification of two proteins; a hypothetical protein, designated lwe06778 and a phosphoribosyl-AMP cyclohydrolase which were uniquely present at 4°C. This finding suggests that L. monocytogenes depends on the histidine biosynthesis pathway in order to survive at cold temperatures. It is hypothesised that the addition of inhibitors, specific to both proteins in RTE cold foods may be a useful means for controlling outbreaks of listeriosis in the future.