Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa.

Yani Zhang,Gee W Lau,Haihua Liang,Liang Yang,Xiao Du,Chao Zhang,Gukui Chen,Tietao Wang,Weina Kong,Yun Zhou,Gurjeet Singh Kohli

doi:10.3389/fcimb.2019.00164

Abstract

Glutathione (GSH) is the most abundant antioxidant in all living organisms. Previously, we have shown that a deletion mutant in the glutathione synthetase gene (ΔgshB) decreases the expression of type III secretion system (T3SS) genes of Pseudomonas aeruginosa. However, the mechanism remains elusive. In this study, a comprehensive transcriptomic analysis of the GSH-deficient mutant ΔgshAΔgshB was used to elucidate the role of GSH in the pathogenesis of P. aeruginosa. The data show that the expression of genes in T3SS, type VI secretion system (T6SS) and some regulatory genes were impaired. ΔgshAΔgshB was attenuated in a mouse model of acute pneumonia, swimming and swarming motilities, and biofilm formation. Under T3SS inducing conditions, GSH enhanced the expression of T3SS in both wild-type PAO1 and ΔgshAΔgshB, but not in Δvfr. Genetic complementation of Δvfr restored the ability of GSH to induce the expression of T3SS genes. Site-directed mutagenesis based substitution of cysteine residues with alanine in Vfr protein abolished the induction of T3SS genes by GSH, confirming that GSH regulates T3SS genes through Vfr. Exposure to H2O2 decreased free thiol content on Vfr, indicating that the protein was sensitive to redox modification. Importantly, GSH restored the oxidized Vfr to reduced state. Collectively, these results suggest that GSH serves as an intracellular redox signal sensed by Vfr to upregulate T3SS expression in P. aeruginosa. Our work provides new insights into the role of GSH in P. aeruginosa pathogenesis.

Highlights

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that cause acute and chronic infections (Gellatly and Hancock, 2013)
When compared to PAO1, gshB and gshA gshB were more susceptible to H2O2, while gshAgshB mutant completely lacking GSH described greater susceptibility to H2O2 than gshB mutant (Figure S1B; Table S4), confirming the protective function of GSH against oxidative stress and injury
We have previously shown GSH is important for the expression of P. aeruginosa T3SS genes

Summary

Introduction

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that cause acute and chronic infections (Gellatly and Hancock, 2013). Large multicomponent secretion apparatus, including both type III secretion system (T3SS) and type VI secretion system (T6SS), facilitate P. aeruginosa infection by directly injecting effector proteins into the cytoplasm of host cells to subvert the host immune response. In response to varieties of environmental signals (e.g., low Ca2+) and direct contact with eukaryotic host cells, ExsA autoregulates its own transcription through direct activation of the exsC promoter (Brutinel et al, 2008). Under inducible or low Ca2+ conditions, ExsE is secreted, which in turn, free ExsC to form a complex with ExsD, freeing ExsA to activate the expression of T3SS genes (King et al, 2012). Prior genomic and proteomic studies demonstrate that more than 100 genes and 60 proteins in P. aeruginosa are positively or negatively regulated by Vfr (Suh et al, 2002; Wolfgang et al, 2003). A recent study showed that Vfr directly binds to the promoter of exsA and activates its transcription (Marsden et al, 2016)

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Results

Conclusion