Exploring the Ocular Surface Microbiome and Tear Proteome in Glaucoma

Abstract

Although glaucoma is a leading cause of irreversible blindness worldwide, its pathogenesis is incompletely understood, and intraocular pressure (IOP) is the only modifiable risk factor to target the disease. Several associations between the gut microbiome and glaucoma, including the IOP, have been suggested. There is growing evidence that interactions between microbes on the ocular surface, termed the ocular surface microbiome (OSM), and tear proteins, collectively called the tear proteome, may also play a role in ocular diseases such as glaucoma. This study aimed to find characteristic features of the OSM and tear proteins in patients with glaucoma. The whole-metagenome shotgun sequencing of 32 conjunctival swabs identified Actinobacteria, Firmicutes, and Proteobacteria as the dominant phyla in the cohort. The species Corynebacterium mastitidis was only found in healthy controls, and their conjunctival microbiomes may be enriched in genes of the phospholipase pathway compared to glaucoma patients. Despite these minor differences in the OSM, patients showed an enrichment of many tear proteins associated with the immune system compared to controls. In contrast to the OSM, this emphasizes the role of the proteome, with a potential involvement of immunological processes in glaucoma. These findings may contribute to the design of new therapeutic approaches targeting glaucoma and other associated diseases.

Keywords: glaucoma; liquid chromatography–tandem mass spectrometry; ocular surface microbiome; tear proteome; whole-metagenome shotgun sequencing.

Figure 1

Taxonomic characterization of ocular surface microbiome. Box plots for relative abundances at phylum (a) and species level (b) are shown (red, glaucoma, n = 16; blue, healthy controls, n = 16).

Figure 2

Diversity in the ocular surface microbiome. There were no differences in Shannon index observed between glaucoma patients (n = 16) and controls (n = 16; p = 0.35, Welch’s t test).

Figure 3

Principal component analysis (PCA) of microbial abundances (a) and protein intensities (b). (a) PCA of microbial abundances did not separate glaucoma patients (red, n = 16) from healthy controls (blue, n = 16; p = 0.51, PERMANOVA, n repeat = 1000). (b) Log2−transformed protein group intensities before imputation were used for analysis. PCA separates glaucoma patients (n = 16) from controls (n = 16; p = 0.0060, PERMANOVA, n repeat = 1000). Orange, female glaucoma patients (n = 7); red, male glaucoma patients (n = 9); light blue, healthy control females (n = 10); blue, healthy control males (n = 6).

Figure 4

The ocular surface microbiome of POAG and PEXG patients. Box plots for relative abundances of all species are shown. PEXG, pseudoexfoliation glaucoma (n = 8); POAG, primary open-angle glaucoma (n = 8).

Figure 5

Quantification of the tear proteome. Volcano plot illustrating the 2250 identifed tear proteins. A total of 123 proteins with a maximum adjusted p value of 0.05 were either up-regulated (log2 fold change ≥ 1) or down-regulated (log2 fold change ≤ −1) in glaucoma patients (n = 16) versus healthy controls (n = 16).