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

Molecular mechanisms of host-pathogen interactions in pseudomonas aeruginosa microbial keratitis
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.
0146-0404
Elsahn, Ahmad
db5d3c4e-61a6-4069-8a20-64b077127ca6
Cendra gascon, Maria del mar DM
150d34a4-4598-4138-96c0-06f3f338cca2
Hossain, Parwez
563de5fc-84ad-4539-9228-bde0237eaf51
Christodoulides, Myron
eba99148-620c-452a-a334-c1a52ba94078
Elsahn, Ahmad
db5d3c4e-61a6-4069-8a20-64b077127ca6
Cendra gascon, Maria del mar DM
150d34a4-4598-4138-96c0-06f3f338cca2
Hossain, Parwez
563de5fc-84ad-4539-9228-bde0237eaf51
Christodoulides, Myron
eba99148-620c-452a-a334-c1a52ba94078

Elsahn, Ahmad, Cendra gascon, Maria del mar DM, Hossain, Parwez and Christodoulides, Myron (2015) Molecular mechanisms of host-pathogen interactions in pseudomonas aeruginosa microbial keratitis. Investigative Ophthalmology & Visual Science, 56 (7).

Record type: Meeting abstract

Abstract

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.

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Published date: June 2015

Identifiers

Local EPrints ID: 448088
URI: http://eprints.soton.ac.uk/id/eprint/448088
ISSN: 0146-0404
PURE UUID: bd15225c-5a44-4a26-abf0-29012f335dab
ORCID for Parwez Hossain: ORCID iD orcid.org/0000-0002-3131-2395
ORCID for Myron Christodoulides: ORCID iD orcid.org/0000-0002-9663-4731

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Date deposited: 01 Apr 2021 15:58
Last modified: 13 Apr 2021 01:41

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