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

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