Label‐Free Quantitative Proteomics Distinguishes General and Site‐Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface

Abstract

Mass spectrometry-based proteomics enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying dual biological systems in a single experiment, supporting investigation of infection from both the host and pathogen perspectives. At the ocular surface, Pseudomonas aeruginosa is commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa is profiled and neutrophil marker proteins are detected, indicating neutrophil recruitment to the site of infection. The first potential diagnostic markers of P. aeruginosa-associated keratitis are also identified. In addition, the deepest murine corneal proteome to date is defined and proteins, categories, and networks critical to the host response are detected. Moreover, the first identification of bacterial proteins attached to the ocular surface is reported. The findings are validated through in silico comparisons and enzymatic profiling. Overall, the work provides comprehensive profiling of the host-pathogen interface and uncovers differences between general and site-specific host responses to infection.

Exoproteomics for Better Understanding Pseudomonas aeruginosa Virulence

“…P. aeruginosa is involved in eye infection. With a quantitative approach, Yeung et al investigated for the first time the exoproteome of eye wash in mice after biofilm formation of the invasive P. aeruginosa clinical isolate 6294 [ 110 ]. They succeeded to detect bacterial proteins at the corneal surface that are involved in both bacterial virulence and survival.…”

Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis

“…This has allowed researchers to begin to characterise the process of biofilm formation at the ocular surface ( Table 2 ). In vivo infection models have also played an integral role in other areas of bacterial keratitis research, including: biofilm formation on contact lenses in rabbit [ 172 ] and mice [ 175 ], host–pathogen interactions on ocular samples using proteomics [ 184 , 185 ], activation of immune signalling pathways [ 186 ], the role of virulence factors in keratitis [ 142 , 173 , 178 , 187 ] and drug testing of new ophthalmic antimicrobials [ 174 , 176 , 188 ]. Drug testing has included synthetic analogues of host antimicrobial peptides, with one study reporting reduced corneal bioburden and improved ocular scores following treatment with their lead peptide [ 188 ].…”

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment

“…To explore the relationship between the host and pathogen during ocular keratitis and define the presence of bacterial biofilms at the ocular surface, we use bottom-up mass spectrometry-based proteomics (Yeung et al, 2020). Using a clinical isolate of P. aeruginosa (strain 6294) in a murine model of ocular infection, we detect neutrophil marker proteins within the extracellular environment (eye wash), indicating neutrophil recruitment to the site of infection.…”

“…In addition, proteomic profiling can provide valuable insight into the interplay between host and pathogen during infection and generate large datasets outlining changes from these dual perspectives in a single experiment (Ball, Bermas, Carruthers-Lay, & Geddes-McAlister, 2019;Jean Beltran, Federspiel, Sheng, & Cristea, 2017;Sukumaran et al, 2019). For example, proteomic profiling of the ocular surface during infection with Pseudomonas aeruginosa, a Gramnegative bacterial pathogen and the primary causative agent of ocular keratitis, defines global and site-specific host responses to infection and uncovers potential biomarkers for prognostic and diagnostic purposes (Yeung, Gadjeva, & Geddes-McAlister, 2020). To evaluate the impact of bacterial infection of the ocular surface on the host, response both at the site of infection (i.e., cornea) and the adjacent uninfected eye, as well as to define changes to the surface by analyzing eye wash samples (representing a non-invasive strategy for examining the relationship between host and pathogen), we devised a stepby-step protocol (Fig.…”