Abstract
A companion manuscript revealed that deletion of the Pseudomonas aeruginosa (Pae) PA1006 gene caused pleiotropic defects in metabolism including a loss of all nitrate reductase activities, biofilm maturation, and virulence. Herein, several complementary approaches indicate that PA1006 protein serves as a persulfide-modified protein that is critical for molybdenum homeostasis in Pae. Mutation of a highly conserved Cys22 to Ala or Ser resulted in a loss of PA1006 activity. Yeast-two-hybrid and a green-fluorescent protein fragment complementation assay (GFP-PFCA) in Pae itself revealed that PA1006 interacts with Pae PA3667/CsdA and PA3814/IscS Cys desulfurase enzymes. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) “top-down” analysis of PA1006 purified from Pae revealed that conserved Cys22 is post-translationally modified in vivo in the form a persulfide. Inductively-coupled-plasma (ICP)-MS analysis of ΔPA1006 mutant extracts revealed that the mutant cells contain significantly reduced levels of molybdenum compared to wild-type. GFP-PFCA also revealed that PA1006 interacts with several molybdenum cofactor (MoCo) biosynthesis proteins as well as nitrate reductase maturation factor NarJ and component NarH. These data indicate that a loss of PA1006 protein’s persulfide sulfur and a reduced availability of molybdenum contribute to the phenotype of a ΔPA1006 mutant.
Highlights
Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen capable of causing many different kinds of infections including localized acute infections of the eye, chronic localized lung infections prevalent in Cystic Fibrosis (CF) patients, as well as disseminated infections such as occurs after severe burns [1,2]
The goal of this study was to determine a function of the PA1006 protein that could account for the biological data reported in the companion manuscript [9]
Based upon data in the accompanying manuscript and the homology of PA1006 with the E. coli YhhP/TusA protein, we originally hypothesized that: 1) PA1006 Cys22 may be modified as a persulfide in vivo, and 2) PA1006 may be connected to molybdenum pathways since nitrate reductases which contain molybdenum cofactor (MoCo) are inactive and several MoCo biosynthesis genes display altered expression in the DPA1006 mutant [9]
Summary
Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen capable of causing many different kinds of infections including localized acute infections of the eye, chronic localized lung infections prevalent in Cystic Fibrosis (CF) patients, as well as disseminated infections such as occurs after severe burns (thermal injury) [1,2]. Drug resistance is increasing among Pae strains in the clinic, and options to combat it are limited [4]. One approach to avoiding drug resistance may be to target virulence-specific pathways [5]. While an in-depth understanding of Pae metabolism during localized eyes infections or in disseminated infections is lacking, transcriptome and metabolome studies of Pae clinical isolates obtained from CF patients have begun to emerge. In these cases, long-term infection appears to involve complex physiological adaptations in Pae that distinguish them from cells cultured in the laboratory (in vitro) in rich media [7,8]. We set out to provide a mechanistic basis for PA1006 function that will facilitate future drug design efforts
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