Mucosal fluid glycoprotein DMBT1 suppresses twitching motility and virulence of the opportunistic pathogen Pseudomonas aeruginosa

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.Author summary: Pseudomonas aeruginosa is an opportunistic pathogen that causes life-threatening infections. 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. Knowing the key ingredients would help us understand susceptibility and develop novel biocompatible therapeutics. Mucosal fluid factors suppressing bacterial virulence may induce less bacterial resistance than traditional antimicrobials. Here we show that DMBT1, an abundant mucosal fluid glycoprotein, enabled tear fluid to inhibit P. aeruginosa twitching motility. We also show DMBT1 directly binds pili, which mediate twitching motility, suggesting a potential mechanism for twitching inhibition. Reflecting the known importance of twitching motility in virulence, purified DMBT1 reduced P. aeruginosa traversal of human cornea epithelial cell layers in vitro, and protected against P. aeruginosa induced disease in vivo, as does whole human tear fluid. These findings contribute to our understanding of mucosal fluid protection against infection, and suggest that DMBT1, or its derivatives, have potential as novel anti-virulence agents that protect against infection.