Molecular mechanisms of host-pathogen interactions in pseudomonas aeruginosa microbial keratitis

Purpose: To examine the molecular mechanisms of interactions between P. aeruginosa bacteria and primary human corneal fibroblasts (hCF) in an invitro model of microbial keratitis. Methods: Human CF were extracted from clinical samples, cultured to confluence in vitro and incubated with live PAO1 wild type bacteria as well as mutant strains deficient in type IV pilus (∆pilA), flagella (∆fliM) or double mutants (∆pilA∆fliM) for 3h. Bacterial association was quantified and compared across the strains. Confluent hCF were also pre-treated with SRC kinase inhibitors genstein (GST) and PP2 and actin microfilament inhibitor cytochalasin D (CD), and bacterial internalization was compared across pre-treated cells at 3h using a gentamicin protection assay. Wild type (WT) PA14 as well as mutant strains deficient in type III secretion system (T3SS) needle apparatus (∆popB) and flagella (∆flgK) were incubated with confluent hCF for 9h, and bacterial cytotoxicity was assessed by a lactate dehydrogenase (LDH) assay. Results: Mutant PAO1strains ∆pilA, ∆fliM and ∆pilA∆fliM adhered significantly less than WT to hCF (P<0.05). Bacterial internalization in hCF pre-treated with CD, GST and PP2 was significantly less than untreated cells (P<0.05). Mutant PA14 strains ∆popB and ∆flgK caused significantly less cytotoxicity to hCF than WT PA14 (P<0.05). Conclusions: PAO1 bacteria use type IV pilus and flagella to adhere to hCF. Bacterial internalization to hCF is dependent on the actin microfilament and SRC kinase systems. Bacteria use flagella to adhere to hCF and T3SS to induce cytotoxicity