Abstract
It is generally thought that mucosal fluids protect underlying epithelial surfaces against opportunistic infection via their antimicrobial activity. However, our published data show that human tear fluid can protect against the major opportunistic pathogen Pseudomonas aeruginosa independently of bacteriostatic activity. Here, we explored the mechanisms for tear protection, focusing on impacts of tear fluid on bacterial virulence factor expression. Results showed that tear fluid suppressed twitching motility, a type of surface-associated movement conferred by pili. Previously, we showed that twitching is critical for P. aeruginosa traversal of corneal epithelia, exit from epithelial cells after internalization, and corneal virulence. Inhibition of twitching by tear fluid was dose-dependent with dilutions to 6.25% retaining activity. Purified lactoferrin, lysozyme, and contrived tears containing these, and many other, tear components lacked the activity. Systematic protein fractionation, mass spectrometry, and immunoprecipitation identified the glycoprotein DMBT1 (Deleted in Malignant Brain Tumors 1) in tear fluid as required. DMBT1 purified from human saliva also inhibited twitching, as well as P. aeruginosa traversal of human corneal epithelial cells in vitro, and reduced disease pathology in a murine model of corneal infection. DMBT1 did not affect PilA expression, nor bacterial intracellular cyclicAMP levels, and suppressed twitching motility of P. aeruginosa chemotaxis mutants (chpB, pilK), and an adenylate cyclase mutant (cyaB). However, dot-immunoblot assays showed purified DMBT1 binding of pili extracted from PAO1 suggesting that twitching inhibition may involve a direct interaction with pili. The latter could affect extension or retraction of pili, their interactions with biotic or abiotic surfaces, or cause their aggregation. Together, the data suggest that DMBT1 inhibition of twitching motility contributes to the mechanisms by which mucosal fluids protect against P. aeruginosa infection. This study also advances our understanding of how mucosal fluids protect against infection, and suggests directions for novel biocompatible strategies to protect our surface epithelia against a major opportunistic pathogen.
Highlights
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen ubiquitous in our environment
P. aeruginosa disease is increasing in prevalence while bacteria continue to evolve antibiotic resistance
It is not clear how mucosal fluids usually protect against opportunistic pathogens
Summary
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen ubiquitous in our environment. It is a leading cause of life-threatening infections in debilitated individuals in the hospital setting [1], and of sight-threatening corneal disease in healthy people who wear contact lenses [2, 3]. The surface of the eye is normally bathed in tear fluid, which like other mucosal fluids contains many proteins-peptides, lipids, small molecule metabolites, and electrolytes. In addition to playing antimicrobial roles, mucosal fluids function to provide lubrication, remove foreign debris, provide homeostatic factors, and repair epithelial damage [7], Our previous studies have confirmed that tear fluid collected from healthy people can protect corneas (of mice) against P. aeruginosa infection in vivo [8]
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