The Listeria monocytogenes DnaK chaperone is required for stress toleranceand efficient phagocytosis with macrophages

Listeria monocytogenes is a facultative intracellular pathogen which can es cape bactericidal mechanisms and grow within macrophages. The intracellular environment of macrophages is one of the most stressful environments encou ntered by an invading bacterium during the course of infection. To study th e role of the major stress protein, DnaK, of L. monocytogenes in survival u nder intracellular stress induced by macrophage-phagocytosis as well as und er extracellular environmental stresses, we cloned, sequenced, and analyzed the dnaK locus from L. monocytogenes. Then we constructed an insertional m utation in the dnaK gene by homologous recombination and characterized it. Sequencing has revealed that the dnaK locus consists of four open reading f rames in the order hrcA-grpE-dnaK-dnaJ. The mutant grows neither at tempera tures above 39 degrees C nor under acidic conditions e.g. pH 3.0. Using the macrophage cell line JA-4, the ability of the dnaK mutant to grow intracel lularly was examined. Immediately after phagocytosis, the number of viable dnaK mutant bacteria found within macrophages was significantly lower compa red to that of intracellular wild type bacteria. However, following a 1-3 h latency period, the mutant multiplied in a similar fashion to the wild typ e within macrophage cells. A quantitative analysis of intracellular bacteri a in macrophage cells by microscope and a binding assay of bacteria to the surface of macrophages by ELISA revealed that the lower number of viable dn aK mutant in macrophages after phagocytosis is due to the low efficiency of phagocytosis resulting from the reduced binding capacity of the dnaK mutan t. These results demonstrate that DnaK of L. monocytogenes is essentially r equired for survival under high temperatures and acidic conditions. Though it does not largely contribute to the survival of L. monocytogenes in macro phage cells, it is essential for efficient phagocytosis, This is the first evidence that DnaK is required for the efficient phagocytosis of a facultat ive intracellular pathogen with macrophages.