NrtR Regulates the Type III Secretion System Through cAMP/Vfr Pathway in Pseudomonas aeruginosa.

Yongxin Jin,Feng Zhu,Shouguang Jin,Fang Bai,Chang Liu,Qianqian Peng,Yuding Weng,Qiang Zhao,Mengjing Zhang,Weihui Wu,Zhihui Cheng

doi:10.3389/fmicb.2019.00085

Yongxin Jin, Feng Zhu + Show 9 more

Open Access

https://doi.org/10.3389/fmicb.2019.00085

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Abstract

The type III secretion system (T3SS) plays an important role in the pathogenesis of Pseudomonas aeruginosa. Expression of the T3SS is controlled under a complicate regulatory network. In this study, we demonstrate that NrtR (PA4916) is involved in the T3SS expression and pathogenesis of P. aeruginosa in a mouse acute pneumonia model. Overexpression of the T3SS central activator ExsA or exogenous supplementation of cAMP restored the expression of T3SS in the ΔnrtR mutant, suggesting that NrtR might regulate T3SS through the cAMP-Vfr signaling pathway. Further experiments demonstrated that the decrease of cAMP content is not due to the expression change of adenylate cyclases or phosphodiesterase in the ΔnrtR mutant. As it has been shown that nadD2 is upregulated in the ΔnrtR mutant, we overexpressed nadD2 in wild type PAK, which reduced the intracellular cAMP level and the expression of the T3SS genes. Meanwhile, deletion of nadD2 in the ΔnrtR mutant restored the expression and secretion of the T3SS. Co-immunoprecipitation assay revealed an interaction between NadD2 and the catalytic domain of the adenylate cyclase CyaB. Further in vitro assay indicated that NadD2 repressed the enzymatic activity of CyaB. Therefore, we have identified a novel regulatory mechanism of T3SS in P. aeruginosa.

Highlights

We identified that NrtR (PA4916) is required for the expression of T3SS in P. aeruginosa
We identified that NrtR is required for the T3SS and involved in pathogenesis of P. aeruginosa in a murine acute pneumonia model
Further experimental results demonstrated that NrtR regulates expression of T3SS through the cAMP/Vfr signaling system

Summary

Introduction

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can cause both acute and chronic infections in individuals with compromised immunity such as cancer patients and those with cystic fibrosis (Crowe et al, 1982; Sherertz and Sarubbi, 1983; Lyczak et al, 2002; Sousa and Pereira, 2014).The type III secretion system (T3SS) is an important virulence factor of P. aeruginosa, through which effector proteins are directly injected into the cytosols of eukaryotic host cells, inhibiting host defense by inducing cell death in polymorphonuclear phagocytes, macrophages, and epithelial cells (Dacheux et al, 1999; Hauser and Engel, 1999; Kaufman et al, 2000). Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that can cause both acute and chronic infections in individuals with compromised immunity such as cancer patients and those with cystic fibrosis (Crowe et al, 1982; Sherertz and Sarubbi, 1983; Lyczak et al, 2002; Sousa and Pereira, 2014). Expression of the T3SS confers an increased virulence in P. aeruginosa and is associated with poor clinical outcomes (ElSolh et al, 2012), whereas strains with defective T3SS display attenuated virulence in mouse acute infection models (Smith et al, 2004). Four effector proteins have been identified and well characterized in P. aeruginosa, i.e., ExoS, ExoT, ExoU, and ExoY (Hauser, 2009). Strain PAK expresses ExoS, ExoT, and ExoY, while strain PA14 expresses ExoU, ExoT, and ExoY

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