The Pseudomonas aeruginosa T3SS can contribute to traversal of an in situ epithelial multilayer independently of the T3SS needle

Abstract

Multilayered epithelia lining our tissue surfaces normally resist traversal by opportunistic bacteria. Previously, we developed a strategy to experimentally perturbate this resistance in situ in the corneas of mouse eyes and used it to show that traversal of a multilayered epithelium by Pseudomonas aeruginosa requires ExsA, the transcriptional activator of its type 3 secretion system (T3SS). Here, we developed a novel strategy for quantitively localizing individual traversing bacteria within the in situ multilayered corneal epithelium and explored contributions of T3SS components. The results showed that T3SS translocon and T3SS effector mutants had reduced epithelial traversal efficiency. Surprisingly, a {Delta}pscC mutant unable to assemble the T3SS needle traversed as efficiently as wild-type P. aeruginosa, while a {Delta}exsD mutant constitutively on for T3SS expression was traversal defective. Dispensability of the T3SS needle for effector-mediated traversal was confirmed using a mutant lacking the T3SS operon except the effector genes ({Delta}pscU-L mutant). That mutant reacquired the ability to traverse if complemented with rhamnose-inducible exsA, but not if the effector genes were also deleted ({Delta}pscU-L{Delta}exoSTY). Western immunoblot confirmed ExoS in culture supernatants of rhamnose-induced exsA-complemented {Delta}pscU-L mutants lacking all T3SS needle protein genes. Together, these results show that epithelial traversal by P. aeruginosa can involve T3SS effectors and translocon proteins independently of the T3SS needle previously thought essential for T3SS function. This advances our understanding of P. aeruginosa pathogenesis and has relevance to development of therapeutics targeting the T3SS system. IO_SCPLOWMPORTANCEC_SCPLOWWhile the capacity to cross an epithelial barrier can be a critical step in bacterial pathogenesis, our understanding of mechanisms involved is derived largely from cell culture experimentation. The latter is due to practical limitations of in vivo/in situ models and challenge of visualizing individual bacteria in the context of host tissue. Here, factors used by P. aeruginosa to traverse an epithelial multilayer in situ were studied by: 1) leveraging the transparent properties and superficial location of the cornea, 2) using our established method for enabling bacterial traversal susceptibility, and 3) developing a novel strategy for accurate and quantitative localization of individual traversing bacteria in situ. Outcomes showed that T3SS translocon and T3SS effector proteins synergistically contribute to epithelial traversal efficiency independently of the T3SS needle. These findings challenge the assumption that the T3SS needle is essential for T3SS effectors or translocon proteins to contribute to bacterial pathogenesis.