Proteomics for the Elucidation of Cold Adaptation Mechanisms in Listeria monocytogenes

Listeria monocytogenes is a gram-positive rod-shaped bacterium found in food of different origin (fish meat, milk, soft cheese, etc) and frequently involved in foodborne disease outbreaks. It causes listeriosis, a potentially life-threatening illness. Notably, it is able to survive hostile environments and stress conditions such as those encountered in food-processing technologies (high salt concentration, wide range of pH and temperature, low water availability) and also to grow in biofilm mode. In particular, this psychrotolerant organism has a minimum growth temperature estimated to be just below 2°C. Cold adaptation mechanisms are important in L. monocytogenes, enabling this food pathogen to survive and proliferate, thus reaching minimal infectious levels also on refrigerated foods (1). In this light, L. monocytogenes represents an interesting and well studied model system; the knowledge of the genome of this pathogen, which has been completely sequenced (2), opened up new prospects to proteomic studies (3). Proteomics could in fact provide a suitable tool also to gain an improved understanding of survival and cold adaptation mechanisms implemented by L. monocytogenes. With this aim we carried out a comparative study on L. monocytogenes grown at three different temperatures (4°C, 25°C and 37°C), by integrating two dimensional electrophoretic separation, image analysis of the 2D-maps and identification of proteins differentially expressed by Peptide MALDI Fingerprint strategy. The 70 differentially expressed proteins were grouped based on their cellular functions and metabolic pathways, using the KEGG (Kyoto Encyclopedia of Genes and Gemomes) resource (http://www.genome.jp/kegg/) (4). This classification helped to highlight a stringent correlation between proteins with similar general function and their regulation in the expression pattern in relationship with the different growth conditions. Primary results highlighted, as major difference, a lower amount of enzymes involved in carbohydrate metabolism expressed by L monocytogenes grown both at 25°C and 4°C. Moreover, other down-regulated proteins included enzymes associated with amino acid metabolism, mainly of small hydrophobic residues, and nucleotide (purine and pyrimidine) biosynthesis, thus indicating that a shift in growth temperature basically induces a reduction in the general growth rate. Interestingly, in L. monocytogenes grown at 4°C, proteins involved in membrane transport resulted to be down regulated, while proteins involved in cell motility, folding, sorting and degradation were up-regulated. A study of the functional role of the identified proteins clearly suggests that cold stress at 4°C more significantly affects several, seemingly unrelated, cellular processes and a rationalization of results is still in progress to gain a deeper insight in the mechanisms of cold acclimatation. These findings may have an impact in the development of better ways of controlling this pathogen in food and related environments.