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Pseudomonas aeruginosa host-pathogen interactions in human corneal infection models

Pseudomonas aeruginosa host-pathogen interactions in human corneal infection models
Pseudomonas aeruginosa host-pathogen interactions in human corneal infection models
Purpose: to examine over time, the electron microscopic changes associated with Pseudomonas aeruginosa (PA) and human corneal tissue interactions in the context of microbial keratitis.

Methods: corneal stromal fibroblast monolayer and whole tissue models were made from human eye bank eyes and from residual tissue after corneal transplantation.

In the whole tissue model (WTM), donor buttons were infected with PAO1 by scoring and intrastromal injection. Tissue was examined after 3, 9 and 24 hours post challenge by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

In the cell culture model (CCM), corneal fibroblasts (CF) were infected in vitro with PAO1. Bacterial adherence and internalization were assayed at 3, 6 and 9h by SEM and TEM. Adherent and internalized bacteria were measured by the gentamicin protection invasion assay. A subset of infected fibroblasts was treated with gentamicin to study intracellular bacterial survival and cell viability using a lactose dehydrogenase assay (LDH).

Results: in the WTM, bacteria were seen to penetrate the epithelium at the scored sites only. At 3h bacteria were seen in the stroma and by 9h distinct intrastromal bacterial colonies were observed. Clusters of intracellular bacteria were observed in keratocytes in the intrastromal injection model.

In CCM, SEM demonstrated bacteria adherent to the surface of CF and the saponin lysis assay demonstrated adherence and internalization in a dose- and time-dependent manner. Bacterial internalization was detected as early as 3h. Intracellular bacteria survived and replicated without affecting cell viability.

Conclusion: PAO1 bacterial can infect stromal keratocytes only when the epithelium and basement membrane are breached, or bypassed by direct injection. PA interaction with CF occurs very early leading to internalization of bacteria where they are protected and can multiply intracellularly without affecting CF viability for 24 hrs. This may have relevance to ideal timing of medical intervention.
cornea, Pseudomonas aeruginosa, keratitis, fibroblasts, keratocyte, infection
2452-4034
Elsahn, Ahmad
db5d3c4e-61a6-4069-8a20-64b077127ca6
Cendra gascon, Maria del mar DM
150d34a4-4598-4138-96c0-06f3f338cca2
Humbert, Maria
82134d25-24b8-4fdd-bd1c-461683b5322e
Christodoulides, Myron
eba99148-620c-452a-a334-c1a52ba94078
Dua, Harminder S
dd8109b7-81da-4c4c-9cb1-9f205896392a
Hossain, Parwez
563de5fc-84ad-4539-9228-bde0237eaf51
Elsahn, Ahmad
db5d3c4e-61a6-4069-8a20-64b077127ca6
Cendra gascon, Maria del mar DM
150d34a4-4598-4138-96c0-06f3f338cca2
Humbert, Maria
82134d25-24b8-4fdd-bd1c-461683b5322e
Christodoulides, Myron
eba99148-620c-452a-a334-c1a52ba94078
Dua, Harminder S
dd8109b7-81da-4c4c-9cb1-9f205896392a
Hossain, Parwez
563de5fc-84ad-4539-9228-bde0237eaf51

Elsahn, Ahmad, Cendra gascon, Maria del mar DM, Humbert, Maria, Christodoulides, Myron, Dua, Harminder S and Hossain, Parwez (2020) Pseudomonas aeruginosa host-pathogen interactions in human corneal infection models. Journal of EuCornea. (doi:10.1016/j.xjec.2020.02.002). (In Press)

Record type: Article

Abstract

Purpose: to examine over time, the electron microscopic changes associated with Pseudomonas aeruginosa (PA) and human corneal tissue interactions in the context of microbial keratitis.

Methods: corneal stromal fibroblast monolayer and whole tissue models were made from human eye bank eyes and from residual tissue after corneal transplantation.

In the whole tissue model (WTM), donor buttons were infected with PAO1 by scoring and intrastromal injection. Tissue was examined after 3, 9 and 24 hours post challenge by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

In the cell culture model (CCM), corneal fibroblasts (CF) were infected in vitro with PAO1. Bacterial adherence and internalization were assayed at 3, 6 and 9h by SEM and TEM. Adherent and internalized bacteria were measured by the gentamicin protection invasion assay. A subset of infected fibroblasts was treated with gentamicin to study intracellular bacterial survival and cell viability using a lactose dehydrogenase assay (LDH).

Results: in the WTM, bacteria were seen to penetrate the epithelium at the scored sites only. At 3h bacteria were seen in the stroma and by 9h distinct intrastromal bacterial colonies were observed. Clusters of intracellular bacteria were observed in keratocytes in the intrastromal injection model.

In CCM, SEM demonstrated bacteria adherent to the surface of CF and the saponin lysis assay demonstrated adherence and internalization in a dose- and time-dependent manner. Bacterial internalization was detected as early as 3h. Intracellular bacteria survived and replicated without affecting cell viability.

Conclusion: PAO1 bacterial can infect stromal keratocytes only when the epithelium and basement membrane are breached, or bypassed by direct injection. PA interaction with CF occurs very early leading to internalization of bacteria where they are protected and can multiply intracellularly without affecting CF viability for 24 hrs. This may have relevance to ideal timing of medical intervention.

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Accepted/In Press date: 29 February 2020
Keywords: cornea, Pseudomonas aeruginosa, keratitis, fibroblasts, keratocyte, infection

Identifiers

Local EPrints ID: 438594
URI: http://eprints.soton.ac.uk/id/eprint/438594
ISSN: 2452-4034
PURE UUID: 8d470d29-f48e-4fb3-81f7-7a310e0717e8
ORCID for Maria Humbert: ORCID iD orcid.org/0000-0002-5728-6981
ORCID for Myron Christodoulides: ORCID iD orcid.org/0000-0002-9663-4731
ORCID for Parwez Hossain: ORCID iD orcid.org/0000-0002-3131-2395

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Date deposited: 18 Mar 2020 17:30
Last modified: 07 Oct 2021 01:46

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Contributors

Author: Ahmad Elsahn
Author: Maria del mar DM Cendra gascon
Author: Maria Humbert ORCID iD
Author: Harminder S Dua
Author: Parwez Hossain ORCID iD

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