绿脓杆菌（Pseudomonas aeruginosa）感染的发病机制可能包括细菌在上皮细胞内的存活。我们以前曾表明，这涉及到三型分泌系统（T3SS）及其效应分子ExoS所扮演的多重作用。这包括ExoS依赖性抑制宿主细胞裂解反应，从而使细菌能够在细胞内复制。在本研究中，我们研究了细菌内部的P. aeruginosa引起的细胞死亡反应，比较了能够引发细胞死亡并定位在不同细胞内区域的T3SS突变株与野生型菌株之间的差异。结果显示，缺乏T3SS的细菌引起的角膜上皮细胞死亡，伴随着核因子-κB活化（以p65重定位和肿瘤坏死因子α的转录和分泌作为衡量指标）。通过CRISPR-Cas9突变技术删除caspase-4可延迟由这些细菌内部的T3SS突变株引起的细胞死亡。caspase-4的缺失也能够抵抗T3SS效应因子缺失的突变株引起的更快细胞死亡，这些突变株仍然表达T3SS装置并可定位于宿主细胞的细胞质内，并且通过这种方式拯救了通常依赖ExoS的细菌内部复制。尽管HeLa细胞对T3SS突变株缺乏裂解性死亡反应，但干扰素γ的处理能够启动该反应。综上所述，这些结果表明上皮细胞可以通过非典型炎症小体通路来限制细菌内部的P. aeruginosa增殖，这并不完全依赖于细菌驱动的泡内逸出。由于ExoS抑制了裂解反应，我们的数据将caspase-4作为另一个内源性模式识别受体的靶点，推测其在绿脓杆菌细菌内部生存方式中的作用。 重要性：绿脓杆菌在体内和体外的上皮细胞内可以显示出细菌的内部生存方式。三型分泌系统（T3SS）的效应分子ExoS通过多种机制，包括延长宿主细胞的寿命，对细菌内部生存方式起到贡献。在本研究中，我们旨在了解P. aeruginosa细菌内部时ExoS抑制的潜在细胞死亡反应。结果表明，缺乏T3SS效应因子的细菌能够通过非典型炎症小体通路迅速引起细胞溶解。在这些细菌内部缺乏整个T3SS并因此无法逸出泡内的情况下，caspase-4也促使细胞溶解，表明这种情况在野生型感染中是一种自然存在的亚种。综上所述，我们的数据显示caspase-4炎症小体作为上皮细胞对细菌内的P. aeruginosa的防御机制，而且推测其是ExoS保护宿主细胞复制位点的另一种机制。

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.