Repeated isolation of an antibiotic-dependent and temperature-sensitive mutant of Pseudomonas aeruginosa from a cystic fibrosis patient.

BACKGROUND: Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy. OBJECTIVES: We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present. METHODS: Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing. RESULTS: P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content. CONCLUSIONS: A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections.

Normalization of NAD+ Redox Balance as a Therapy for Heart Failure.

BACKGROUND: Impairments of mitochondrial function in the heart are linked intricately to the development of heart failure, but there is no therapy for mitochondrial dysfunction. METHODS: We assessed the reduced/oxidized ratio of nicotinamide adenine dinucleotide (NADH/NAD(+) ratio) and protein acetylation in the failing heart. Proteome and acetylome analyses were followed by docking calculation, mutagenesis, and mitochondrial calcium uptake assays to determine the functional role of specific acetylation sites. The therapeutic effects of normalizing mitochondrial protein acetylation by expanding the NAD(+) pool also were tested. RESULTS: Increased NADH/NAD(+) and protein hyperacetylation, previously observed in genetic models of defective mitochondrial function, also are present in human failing hearts as well as in mouse hearts with pathologic hypertrophy. Elevation of NAD(+) levels by stimulating the NAD(+) salvage pathway suppressed mitochondrial protein hyperacetylation and cardiac hypertrophy, and improved cardiac function in responses to stresses. Acetylome analysis identified a subpopulation of mitochondrial proteins that was sensitive to changes in the NADH/NAD(+) ratio. Hyperacetylation of mitochondrial malate-aspartate shuttle proteins impaired the transport and oxidation of cytosolic NADH in the mitochondria, resulting in altered cytosolic redox state and energy deficiency. Furthermore, acetylation of oligomycin-sensitive conferring protein at lysine-70 in adenosine triphosphate synthase complex promoted its interaction with cyclophilin D, and sensitized the opening of mitochondrial permeability transition pore. Both could be alleviated by normalizing the NAD(+) redox balance either genetically or pharmacologically. CONCLUSIONS: We show that mitochondrial protein hyperacetylation due to NAD(+) redox imbalance contributes to the pathologic remodeling of the heart via 2 distinct mechanisms. Our preclinical data demonstrate a clear benefit of normalizing NADH/NAD(+) imbalance in the failing hearts. These findings have a high translational potential as the pharmacologic strategy of increasing NAD(+) precursors are feasible in humans.

Modifications of IL-6 by Hypochlorous Acids: Effects on Receptor Binding.

Interleukin-6 (IL-6) has been implicated in the pathogenesis of inflammatory events including those seen with COVID-19 patients. Positive clinical responses to monoclonal antibodies directed against IL-6 receptors (IL-6Rs) suggest that interference with IL-6-dependent activation of pro-inflammatory pathways offers a useful approach to therapy. We exposed IL-6 to hypochlorous acid (HOCl) in vitro at concentrations reported to develop in vivo. After HOCl treatment, binding of IL-6 to IL-6R was reduced in a dose-dependent manner using a bioassay with human cells engineered to provide a luminescence response to signal transduction upon receptor activation. Similar results followed the exposure of IL-6 to N-chlorotaurine (NCT) and hypobromous acid (HOBr), two other reactive species produced in vivo. SDS-PAGE analysis of HOCl-treated IL-6 showed little to no fragmentation or aggregation up to 1.75 mM HOCl, suggesting that the modifications induced at concentrations below 1.75 mM took place on the intact protein. Mass spectrometry of trypsin-digested fragments identified oxidative changes to two amino acid residues, methionine 161 and tryptophan 157, both of which have been implicated in receptor binding of the cytokine. Our findings suggest that exogenous HOCl and NCT might bring about beneficial effects in the treatment of COVID-19. Further studies on how HOCl and HOBr and their halogenated amine derivatives interact with IL-6 and related cytokines in vivo may open up alternative therapeutic interventions with these compounds in COVID-19 and other hyperinflammatory diseases.