MATERIALS AND METHODS: We identified differentially expressed mitochondrial proteins in 50 infertile men with varicocele and in 10 fertile controls by secondary liquid chromatography-tandem mass spectroscopy data driven in silico analysis. Identified proteins were validated by Western blot and immunofluorescence. Seminal oxidation-reduction potential was measured.
RESULTS: We identified 22 differentially expressed proteins related to mitochondrial structure (LETM1, EFHC, MIC60, PGAM5, ISOC2 and import TOM22) and function (NDFSU1, UQCRC2 and COX5B, and the core enzymes of carbohydrate and lipid metabolism). Cluster analysis and 3-dimensional principal component analysis revealed a significant difference between the groups. All proteins studied were under expressed in infertile men with varicocele. Liquid chromatography-tandem mass spectroscopy data were corroborated by Western blot and immunofluorescence. Impaired mitochondrial function was associated with decreased expression of the proteins (ATPase1A4, HSPA2, SPA17 and APOA1) responsible for proper sperm function, concomitant with elevated seminal oxidation-reduction potential in the semen of infertile patients with varicocele.
CONCLUSIONS: Impaired mitochondrial structure and function in varicocele may lead to oxidative stress, reduced ATP synthesis and sperm dysfunction. Mitochondrial differentially expressed proteins should be explored for the development of biomarkers as a predictor of infertility in patients with varicocele. Antioxidant therapy targeting sperm mitochondria may help improve the fertility status of these patients.
METHODS: Regulators were overproduced using a pBAD expression vector. Antibiotic susceptibility was measured using disc testing. Envelope permeability was estimated using a fluorescent dye accumulation assay. Porin and efflux pump production was quantified using proteomics and validated using real-time quantitative RT-PCR.
RESULTS: Envelope permeability and antibiotic disc inhibition zone diameters both reduced during overproduction of RamA and to a lesser extent RarA or SoxS, but did not change following overproduction of MarA. These effects were associated with overproduction of the efflux pumps AcrAB (for RamA and SoxS) and OqxAB (for RamA and RarA) and the outer membrane protein TolC (for all regulators). Effects on porin production were strain specific.
CONCLUSIONS: RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates.
Objectives: To define, in an otherwise isogenic background, the relative effects of OqxR and RamR loss-of-function mutations on envelope protein production, envelope permeability and antimicrobial susceptibility. We also investigated the clinical relevance of an OqxR loss-of-function mutation, particularly in the context of β-lactam susceptibility.
Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. Antimicrobial susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and quantitative RT-PCR was used to measure transcript levels.
Results: Loss of RamR or OqxR reduced envelope permeability in K. pneumoniae by 45%-55% relative to the WT. RamR loss activated AcrAB efflux pump production ∼5-fold and this reduced β-lactam susceptibility, conferring ertapenem non-susceptibility even in the absence of a carbapenemase. In contrast, OqxR loss specifically activated OqxAB efflux pump production >10 000-fold. This reduced fluoroquinolone susceptibility but had little impact on β-lactam susceptibility even in the presence of a β-lactamase.
Conclusions: Whilst OqxR loss and RamR loss are both seen in K. pneumoniae clinical isolates, only RamR loss significantly stimulates AcrAB efflux pump production. This means that only RamR mutants have significantly reduced β-lactamase-mediated β-lactam susceptibility and therefore represent a greater clinical threat.