title: Mutation of csk, encoding the C-terminal Src kinase, reduces Pseudomonas aeruginosa internalization by mammalian cells and enhances bacterial cytotoxicity.
abstract: Clinical isolates of Pseudomonas aeruginosa are either invasive or cytotoxic towards mammalian epithelial cells, endothelial cells, and macrophages. Invasion requires host cell actin cytoskeleton function, and ExsA-regulated proteins of P. aeruginosa that inhibit invasion (ExoS and ExoT) can disrupt the cytoskeleton. Another ExsA regulated protein, ExoU, is involved in the cytotoxic activity of cytotoxic strains. Src-family kinases are thought to participate in the regulation of cytoskeleton function. Recent studies have suggested that Src-family tyrosine kinases, p60-Src and p59-Fyn, are activated during P. aeruginosa invasion. Using fibroblasts homozygous for mutation of csk (-/-), we tested the hypothesis that mutation of csk, encoding a negative regulator of Src-family tyrosine kinases, would be important in P. aeruginosa invasion and cytotoxicity. Mutation of csk was found to reduce invasion by approximately 8-fold, without reducing bacterial adherence to cells (P=0.0001). Conversely, csk (-/-) cells were approximately 5-fold more susceptible to ExoU-dependent cytotoxicity (P=0.024), which was accompanied by a small increase in ExsA-regulated adherence. ExoT-dependent invasion inhibitory activity of cytotoxic P. aeruginosa was attenuated in csk (-/-) cells as compared to normal fibroblasts. These data show that fibroblasts, like epithelial cells, are susceptible to P. aeruginosa invasion and cytotoxicity. They also show a role for Csk in P. aeruginosa invasion, while providing further evidence that actin cytoskeleton disruption contributes to ExsA-regulated P. aeruginosa cytotoxicity and invasion inhibition.
authors.affiliation: Morton D. Sarver Laboratory for Cornea and Contact Lens Research, School of Optometry, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Evans
authors.forename: Tracy C
authors.initials: TC
authors.lastname: Kuo
authors.forename: Mary
authors.initials: M
authors.lastname: Kwong
authors.forename: Rajana
authors.initials: R
authors.lastname: Van
authors.forename: Suzanne M J
authors.initials: SM
authors.lastname: Fleiszig
mesh_terms: D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology*; D008322:Mammals; D051379:Mice; D016296:Mutagenesis; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000502:physiology*; D011550:Pseudomonas aeruginosa / Q000378:metabolism / Q000472:pathogenicity*; D014774:Virulence; D019061:src-Family Kinases
chemical_list: D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases