Invasion of human epithelial cells by Pseudomonas aeruginosa involves src-like tyrosine kinases p60Src and p59Fyn - PubMed (original) (raw)
Invasion of human epithelial cells by Pseudomonas aeruginosa involves src-like tyrosine kinases p60Src and p59Fyn
M Esen et al. Infect Immun. 2001 Jan.
Abstract
Pseudomonas aeruginosa plays a major role in respiratory tract infections or sepsis in patients with cystic fibrosis or upon suppression of the immune system. Several P. aeruginosa strains have been shown to be internalized by human epithelial cells; however, the molecular mechanisms of the invasion process are poorly characterized. Here, we show that the internalization of P. aeruginosa into human epithelial cells results in and requires activation of the Src-like tyrosine kinases p59Fyn and p60Src and the consequent tyrosine phosphorylation of several eukaryotic proteins. The significance of Src-like tyrosine kinase activation is shown by an almost complete blockade of P. aeruginosa internalization, but not adhesion, upon inhibition of Src-like tyrosine kinases. Likewise, inhibition of P. aeruginosa binding to CFTR, which has been shown to block P. aeruginosa internalization, prevents Src and Fyn activation, supporting a pivotal role of Src-like tyrosine kinases for invasion by P. aeruginosa.
Figures
FIG. 1
Internalization of P. aeruginosa into human epithelial cells induces cellular tyrosine phosphorylation. Chang conjunctiva epithelial cells were infected with the invasive P. aeruginosa strain ATCC 27853; then the cells were lysed, and proteins were separated by SDS–10% PAGE and analyzed for tyrosine phosphorylation by Western blotting using the monoclonal anti-phosphotyrosine antibody 4G10. Results reveal a rapid and marked tyrosine phosphorylation of several cellular proteins, in particular those with molecular sizes of 140, 95, 70, 58, and 42 kDa, as soon as 10 min after infection with P. aeruginosa ATCC 27853. Samples were normalized by in vivo labeling of mammalian cells prior to infection and counting aliquots of the lysates.
FIG. 2
Infection of Chang epithelial cells with P. aeruginosa activates p60Src and p59Fyn. Infection of Chang or WI-38 epithelial cells with P. aeruginosa 696 or ATCC 27853 for the indicated times (in minutes) results in rapid activation of p60Src or p59Fyn. Control immunoprecipitates (up) from infected cells with irrelevant goat sera did not show phosphorylation of the substrate enolase. Likewise, immunoprecipitates (Ipt.) obtained from P. aeruginosa 696 or ATCC 27853 only did not reveal any cross-reactivity. Chang epithelial cells (A) or WI-38 (B) cells were infected with P. aeruginosa 696 or ATCC 27853 for the indicated times in minutes, lysed, and subjected to immunoprecipitation with goat polyclonal anti-Src or anti-Fyn antibodies. Cells were labeled with [3H]choline chloride prior to infection, permitting us to use the supernatants of the agarose-immobilized immunoprecipitates for normalization of the samples prior to the kinase assay. The kinase reaction was initiated by resuspending the immunoprecipitates in kinase buffer supplemented with 10 μg of enolase/ml and 10 μCi of [32P]γ-ATP. The samples were separated by SDS–10% PAGE and analyzed by autoradiography. The activities of p60Src and p59Fyn were determined by phosphorylation of the substrate enolase (P-enolase). Aliquots of the immunoprecipitates were blotted with anti-Fyn or anti-Src, followed by HRP-coupled protein L and enhanced chemiluminescence development, demonstrating similar amounts of Src or Fyn in each experiment. Control experiments were performed by incubation of bacterial lysates from P. aeruginosa 696 or ATCC 27853 with anti-Src or anti-Fyn antisera and reveal that the bacteria do not contain a cross-reacting kinase. Further, lysates from infected cells were subjected to precipitation with an irrelevant antiserum, demonstrating the specificity of the kinase assay.
FIG. 3
Inhibition of P. aeruginosa LPS binding to CFTR prevents activation of p60Src and p59Fyn. Prevention of P. aeruginosa binding to CFTR by incubation with a CFTR peptide encompassing amino acids 103 to 117 blocks activation of Src-like tyrosine kinases (A) and invasion (B) in Chang cells upon infection with the indicated strains. (A) Chang epithelial cells were infected with P. aeruginosa 696 or ATCC 27583 for the indicated times (in minutes) and lysed; then p60Src or p59Fyn was immunoprecipitated and subjected to kinase assays. Chang cells were labeled as above with [3H]choline chloride, the supernatants of the immobilized immunoprecipitates were counted, and the samples were normalized accordingly. P. aeruginosa 696 or ATCC 27853 was incubated with the CFTR peptide 10 min prior to the infection assay. Activities of p60Src and p59Fyn were determined by phosphorylation of enolase as above. An aliquot of the immunoprecipitates was blotted with anti-Src or anti-Fyn, displaying similar amounts of protein in all lanes. Control experiments were performed as above. (B) Chang epithelial cells were infected for 30 min as above with CFTR peptide-treated or untreated P. aeruginosa 696 or ATCC 27583, and invasion was determined by crystal violet staining. Results shown are means ± SD from three independent experiments. Significance was determined by t test, and significant differences are indicated by asterisks.
FIG. 4
Blockade of Src-like tyrosine kinases by herbimycin A or PP1 prevents P. aeruginosa uptake into different human epithelial cells. Chang and WI-38 epithelial cells were either pretreated for 12 h with 1 μM herbimycin A (HA) or for 15 min with 50 nM PP1 or left untreated. Cells were infected for 30 min, and invasion was scored by polymyxin survival (A) or crystal violet assay (B). Shown is the invasion of untreated Chang epithelial cells by two P. aeruginosa strains, (control), compared with the uptake of the bacteria into the indicated cells pretreated with HA or PP1. Means ± SD from three independent experiments are shown. Significance was determined by post hoc ANOVA done by the Bonferroni and Dunn method. Asterisks indicate significant differences.
References
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