Dr. Lula Smith, Associate Professor

Dr. Lula Smith, Associate Professor of Biology, has been awarded $10,000 in support of her project titled “ Role of Myosin light chain kinase isoforms in bacteria pathogenesis”.  These funds were also obtained by the National Institute of Health system.

Project Narrative: Bacterial pathogenesis depends on the ability of bacteria to enter, invade and spread throughout the cells of the target host. Several bacterial species employ the actin-based motility system as a means of  intracellular  spread.  In this system,  the bacteria polymerize the host’s actin to form motile actin comets or rockets.  These actin filaments serve as cables upon which the bacteria move through the initially infected cell(s) on to adjacent cells and ultimately, throughout the cytoplasm of eucaryotic cells. While the role of actin polymerization in bacterial pathogenesis is well established, not much is known about the precise mechanism of regulated myosin-dependent cytoskeletal contractile processes in pathogenesis. Myosin light chain kinase (MLCK) is a Ca²⁺/calmodulin-dependent protein kinase. Once activated, MLCK phosphorylates a serine residue in the N-terminus of the regulatory light chain (RLC) of myosin II.  Myosin regulatory light chain is the only known physiological substrate for MLCK. Thus, it is a dedicated protein kinase. Phosphorylated myosin through its association with actin, has been shown to  regulate smooth muscle contraction and a number of nonmuscle cellular  processes  which include cell division, endothelial cell retraction, fibroblast contraction, platelet aggregation, neurite cone advancement, cytoskeletal clustering of integrins at focal adhesions, and cell spreading and migration. As these processes all involve reorganization of the actin cytoskeleton, we hypothesize that MLCK is significant in the formation and/or stabilization of the actin tails used by pathogenic bacteria to spread from cell-to-cell within human cells. The vertebrate smooth muscle MLCK gene locus encodes two isoforms: (1) Long MLCK, a high molecular weight form (210-220 kDa), also referred to as nonmuscle (nmMLCK) and (2) short MLCK (130-150 kDa). A number of studies have demonstrated variation in expression of the long and short form MLCKs during development, and in smooth muscle and nonmuscle tissues.  In addition, a small number of  studies show that  the two isoforms are expressed simultaneously in several cell lines.  Therefore, the  objective of the proposed research is to determine the role of MLCK in the cell-to-cell spread of several species of pathogenic bacteria, with a particular focus on Shigella flexineri. To test this hypothesis the following specific aims will be investigated  (1) What MLCK isoform is present in various cells and tissues invaded by pathogenic bacteria? (2) What is the relationship between the phosphorylation state of MLCK and RLC? (3) Is the cell-to-cell spread method employed by Shigella similar in other species of pathogenic bacteria? (4) Can the MLCK-mediated cell-to-cell spread be assigned to a specific region of MLCK.? To address these issues, we will use western blotting and immunocytochemistry to analyze a panel of cultured smooth muscle and nonmuscle cell lines for endogenous MLCK expression, and cultured cells that have been transfected with cDNA from both isoforms.  These experiments will be accompanied by infection studies in which the cultured cells will be infected with Shigella species and other pathogenic bacteria to analyze the affect on MLCK on cell-to-cell spread. We expect that the level of MLCK expression and isoform type will vary with respect to tissue and cell type. Importantly, the degree of bacterial-induced infections in cells may be MLCK isoform-dependent. Studies will then focus on determining the role and mechanisms of the specific isoforms involved in MLCK-dependent bacterial pathogenesis. The  findings from the proposed study will lead to greater understanding of the role and mechanism of myosin in  actin-based motility, and indicate for the first time, whether  a specific isoform  of myosin light chain kinase is important in bacterial pathogenesis. As myosin has also been implicated in viral infections, vascular dysfunctions, and cancer, this study will also provide insight into the mechanisms of these health-related  issues as well.