Type III secretion system ofPseudomonas aeruginosaaffects mucin gene expression via NF-κB and AKT signaling in human carcinoma epithelial cells and a pneumonia mouse model

Abstract

25The type III secretion system (T3SS) in Pseudomonas aeruginosa has been linked to 26 severe disease and poor clinical outcomes in animal and human studies. Of the various T3SS 27 effector genes, ExoS and ExoT showed mutually exclusive distributions, and these two genes 28 showed varied virulence. We aimed to investigate whether the ExoS and ExoT effector proteins 29 of P. aeruginosa affect the expression of the proinflammatory mediators Muc7, Muc13, Muc15, 30 and Muc19 via the NF-κB and AKT signaling pathways. To understand the role of the T3SS, 31 we used ΔExoS, ΔExoT, and T3SS transcriptional activator ExsA mutants (ExsA::Ω), as well 32 as A549 cells stimulated with P. aeruginosa strain K (PAK). We investigated the effects of 33 ΔExoS, ΔExoT, and ExsA::Ω on the development of pneumonia in a mouse model and on 34 Muc7, Muc13, Muc15, and Muc19 production in A549 cells. ΔExoS and ΔExoT markedly 35 decreased the neutrophil count in the bronchoalveolar lavage fluid, with a reduction in Muc7, 36 Muc13, Muc15, and Muc19 expression. ΔExoS and ΔExoT reduced NF-κB and AKT 37 phosphorylation, together with Muc7, Muc13, Muc15, and Muc19 expression in PAK-infected 38 mice and A549 cells. In conclusion, P. aeruginosa infection induced the expression of Mucus, 39 and the P. aeruginosa T3SS appeared to be a key player in Muc7, Muc13, Muc15, and Muc19 40 expression, which is further controlled by NF-κB and AKT signaling. These findings might be 41 useful to devise a novel therapeutic approach for the treatment of chronic pulmonary infections 42 by targeting ExoS and ExoT.43 44 3 45 Author Summary 46Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium causing serious infections. 47Many clinical isolates of P. aeruginosa have a specialized apparatus for injecting toxins into 48 eukaryotic cells, namely, the type III secretion system (T3SS). The T3SS is a syringe-like 49 apparatus on the bacterial surface, with 4 effector toxins: ExoS, ExoT, ExoY, and ExoU. We 50 investigated the effect of ExoS and ExoT of the T3SS of P. aeruginosa K strain (PAK). Mucus 51 plays a vital role in protecting the lungs from environmental factors, but conversely, in muco-52 obstructive airway disease, mucus becomes pathologic. We showed that infection with ExoS 53 and ExoT induced Muc7, Muc13, Muc15, and Muc19 expression in host cells. PAK clinical 54 strains induce proinflammatory cytokine production through the T3SS, and this involves NF-55 κB and SP1/AKT activation in pneumonia mouse models. Mucus induction in response to 56 ExoS and ExoT infection relied on NF-κB and SP1/AKT activation. Our findings highlight the 57 roles of Muc7, Muc13, Muc15, and Muc19 in inducing proinflammatory cytokine expression 58 during ExoS and ExoT exposure in PAK infections, paving the way for a novel therapeutic 59 approach for the treatment of pulmonary infections.60 4 61 62The gram-negative bacterium Pseudomonas aeruginosa uses a complex type III 63 secretion system (T3SS) to inject effector proteins into host cells [1,2]. The T3SS is a major 64 v...