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Type III secretion (TTS) mediated translocation of exoenzymes is a key virulence strategy utilised by the opportunistic pathogen Pseudomonas aeruginosa to deliver exoenzyme effectors into the eukaryotic cell. We have previously shown that type III mediated translocation is a contact dependent process, which requires the secreted translocator proteins PerV, PopB and PopD. To further analyse this mechanism, HeLa cells were infected with the wild-type strain PAK as well as isogenic pcrV, popB, popD, pcrG and popN mutants. In the presence of eukaryotic cells, expression of exoenzyme S (ExoS) increased. When cells were infected with the wild-type strain PAK no ExoS was detected in the tissue culture medium. This confirms that ExoS translocation by P. aeruginosa occurs by a polarised mechanism. In contrast, high levels of ExoS were recovered in the tissue Culture medium when cells were infected with pcrG, pcrV and popN mutants. Additionally, ExoS expression levels were higher for these mutants regardless of inducing conditions. This suggests that PcrG, PcrV and PopN are involved in negative regulation of ExoS expression and secretion, and are required to ensure polarised delivery of effectors into target cells.