The OpdQ porin of Pseudomonas aeruginosa is regulated by environmental signals associated with cystic fibrosis including nitrate‐induced regulation involving the NarXL two‐component system

Abstract

Pseudomonas aeruginosa is a versatile opportunistic pathogen that causes chronic infections in immunocompromised hosts. Multiple porins modulate outer membrane permeability under various environmental conditions. The lung environment of cystic fibrosis (CF) patients is unique with changes occurring in nutrient availability, osmolarity, and oxygen content. Although P. aeruginosa gene expression is modified under these conditions, little is known about how they influence porin regulation. In this study, we evaluated the regulation of the outer membrane porin OpdQ, a member of the OprD family of porins, with regard to oxygen, nitrate, and/or NaCl levels. We demonstrated using promoter::fusion clones of P. aeruginosa PAO1 and clinical strains collected from CF patients that OpdQ was transcriptionally repressed under low oxygen but increased in the presence of nitrate. The nitrate‐induced regulation of OpdQ was found to be dependent on the transcription factor NarL via the NarXL two‐component system. In addition, NaCl‐induced osmotic stress increased OpdQ production among most of the clinical strains evaluated. In conclusion, these data identify for the first time that specific environmental cues associated with the CF microenvironment influence porin regulation, and that the nitrate‐induced regulation of OpdQ is associated with nitrate metabolism via the NarXL two‐component system of P. aeruginosa.

The absence of SigX results in impaired carbon metabolism and membrane fluidity in Pseudomonas aeruginosa

“…Similar data were previously reported when PAOSX was grown in M9G 8 , 13 . Conversely, genes encoding OpdQ (a putative nitrate transporter 19 ) and OpdP (also called OpdD, a glycine-glutamate dipeptide transporter) were increased by 14.8 and 3.2-fold, respectively in the sigX mutant strain compared to H103 WT. Expression of many carboxylic acids, carbohydrates and amino acids inner membrane (IM) transporters was also dysregulated.…”

Evaluation of CTX-M steady-state mRNA, mRNA half-life and protein production in various STs ofEscherichia coli

“…The linear response range of the Stain-Free fluorescence and the anti-CTX-M antibody for bacterial lysates was determined by performing a western blot on a dilution series of total protein ranging from 40 to 0.625 μg as previously described. 27 The protein linearity ranged from 5 to 40 μg ( Figure S1 ) and the linear range of the anti-CTX-M antibody was from 2.5 to 40 μg ( Figure S2 ). Therefore, 10 μg of total protein and an antibody dilution factor of 1: 45 000 were used for immunoblot analyses.…”

“… 28 Stain-Free SDS-PAGE, imaging and total protein normalization were carried out as previously described. 27 A custom polyclonal antibody specific for CTX-M-14 and CTX-M-15, directed toward the peptide sequence CAIPGDPRDTT was generated by GenScript (Piscataway, NJ, USA) and the secondary antibody (horseradish peroxidase-goat anti-rabbit IgG) was used with a dilution factor of 1: 50 000. The chemiluminescent signal intensity of CTX-M was normalized to the Stain-Free fluorescent signal intensity of total protein for each isolate.…”

Integrated Genome-Wide Analysis of an Isogenic Pair of Pseudomonas aeruginosa Clinical Isolates with Differential Antimicrobial Resistance to Ceftolozane/Tazobactam, Ceftazidime/Avibactam, and Piperacillin/Tazobactam

“…Previously, OprD porin has been shown to serve as a channel for the β-lactam antibiotic imipenem, whereby mutation or loss of OprD can lead to imipenem resistance [21][22][23]. In addition, OpdQ porin has been shown to be regulated by specific environmental cues associated with cystic fibrosis microenvironment, such as oxygen, nitrate, and NaCl levels [25]. Considering these together, we proposed a model for a methylation-regulated antimicrobial resistance ( Figure 3B).…”