Pseudomonas aeruginosa host-pathogen interactions in human corneal infection models

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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Identifiers

ORCID for Maria Humbert: orcid.org/0000-0002-5728-6981

ORCID for Myron Christodoulides: orcid.org/0000-0002-9663-4731

ORCID for Parwez Hossain: orcid.org/0000-0002-3131-2395

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