The Importance of the Pseudomonas aeruginosa Type III Secretion System in Epithelium Traversal Depends upon Conditions of Host Susceptibility

Abstract

Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an ϳ16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88؊/؊ epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88 ؊/؊ mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30-to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain. P seudomonas aeruginosa remains a leading cause of respiratory infections, septicemia, and urinary tract and burn wound infections (1-4). It is also the most common cause of corneal infection associated with contact lens wear (5, 6). Our research has shown that clinical and laboratory isolates of P. aeruginosa can be divided into invasive or cytotoxic strains based upon how they interact with host cells (7). Invasive strains can survive and replicate inside epithelial cells dependent on the type III secretion system (T3SS) effector ExoS (8-10), while cytotoxic strains instead encode ExoU, which can induce rapid death of intoxicated host cells (11,12). While details of how invasive and cytotoxic strains impact host cells vary, both types can cause human disease, and they can each be virulent in animal models of infection (13-15).Several in vivo models exist for studying P. aeruginosa pathogenicity. They include Caenorhabditis elegans (16-18) and Drosophila melanogaster (19), in addition to mouse models of pneumonia with sepsis (20-22), burn wound infection with sepsis (23-25), and gastrointestinal colonization and sepsis (26,27) and corneal scarification models (28)(29)(30)...

Human Tear Fluid Reduces Culturability of Contact Lens-Associated Pseudomonas aeruginosa Biofilms but Induces Expression of the Virulence-Associated Type III Secretion System

“…The data presented here show that biofilm growth on a lens and exposure to human tear fluid can also upregulate expression of the T3SS, which can enable P. aeruginosa to traverse susceptible corneal epithelium, 25,43 an essential event in the pathogenesis of corneal infection. In the absence of lens wear, P. aeruginosa (and other microbes) cannot infect a healthy corneal surface, which has anti-adhesive properties.…”

“…Our published data show that the T3SS is involved in P. aeruginosa traversal of superificially-injured corneal epithelium in mouse models. 25,43 The net sum result on virulence of increasing T3SS expression while also reducing the number of culturable bacteria via tear fluid exposure will require further investigation. How virulence factors other than the T3SS are impacted by tear fluid also remains to be determined.…”

“…We focused on the type III secretion system ( T3SS ), because it is considered among the most significant virulence factors of this pathogen, 20–24 and we have shown that it can promote P. aeruginosa traversal across a susceptible corneal epithelium. 24, 25 …”

Pseudomonas aeruginosa: breaking down barriers

“…In addition to its pathogenic effects on cells grown in culture, ExoS causes decreased survival and greater bacterial dissemination from the lung in a mouse model of acute pneumonia (Shaver and Hauser 2004). Likewise, a functional type III secretion system was necessary for transversal of P. aeruginosa across a corneal epithelium (Sullivan et al 2015). However, until recently the mechanism behind enhanced bacterial dissemination and the cell types targeted by ExoS during pneumonia still remained unclear.…”

Corneal surface glycosylation is modulated by IL‐1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding

“…MyD88, which is a critical adaptor protein for TLR‐ and IL‐1 receptor (IL‐1R)‐mediated signaling, can modulate expression of various factors including mucins, cytokines, and antimicrobial peptides. Indeed, we have demonstrated that corneal lysates from MyD88 − / − mice exhibited reduced antimicrobial activity (18). The targets modulated by MyD88 to inhibit P. aeruginosa binding to the mouse cornea, as well as how those targets relate to enhanced P. aeruginosa adhesion after tissue paper blotting, remain unknown.…”