Enhancement of β-Lactam-Mediated Killing of Gram-Negative Bacteria by Lysine Hydrochloride

ABSTRACT Widespread bacterial resistance among Gram-negative bacteria is rapidly depleting our antimicrobial arsenal. Adjuvants that enhance the bactericidal activity of existing antibiotics provide a way to alleviate the resistance crisis, as new antimicrobials are becoming increasingly difficult to develop. The present work with Escherichia coli revealed that neutralized lysine (lysine hydrochloride) enhances the bactericidal activity of β-lactams in addition to increasing bacteriostatic activity. When combined, lysine hydrochloride and β-lactam increased expression of genes involved in the tricarboxylic acid (TCA) cycle and raised reactive oxygen species (ROS) levels; as expected, agents known to mitigate bactericidal effects of ROS reduced lethality from the combination treatment. Lysine hydrochloride had no enhancing effect on the lethal action of fluoroquinolones or aminoglycosides. Characterization of a tolerant mutant indicated involvement of the FtsH/HflkC membrane-embedded protease complex in lethality enhancement. The tolerant mutant, which carried a V86F substitution in FtsH, exhibited decreased lipopolysaccharide levels, reduced expression of TCA cycle genes, and reduced levels of ROS. Lethality enhancement by lysine hydrochloride was abolished by treating cultures with Ca2+ or Mg2+, cations known to stabilize the outer membrane. These data, plus damage observed by scanning electron microscopy, indicate that lysine stimulates β-lactam lethality by disrupting the outer membrane. Lethality enhancement of β-lactams by lysine hydrochloride was also observed with Acinetobacter baumannii and Pseudomonas aeruginosa, thereby suggesting that the phenomenon is common among Gram-negative bacteria. Arginine hydrochloride behaved in a similar way. Overall, the combination of lysine or arginine hydrochloride and β-lactam offers a new way to increase β-lactam lethality with Gram-negative pathogens. IMPORTANCE Antibiotic resistance among Gram-negative pathogens is a serious medical problem. The present work describes a new study in which a nontoxic nutrient increases the lethal action of clinically important β-lactams. Elevated lethality is expected to reduce the emergence of resistant mutants. The effects were observed with significant pathogens (Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa), indicating widespread applicability. Examination of tolerant mutants and biochemical measurements revealed involvement of endogenous reactive oxygen species in response to outer membrane perturbation. These lysine hydrochloride–β-lactam data support the hypothesis that lethal stressors can stimulate the accumulation of ROS. Genetic and biochemical work also revealed how an alteration in a membrane protease, FtsH, abolishes lysine stimulation of β-lactam lethality. Overall, the work presents a method for antimicrobial enhancement that should be safe, easy to administer, and likely to apply to other nutrients, such as arginine

Carbonylation Induces Heterogeneity in Cardiac Ryanodine Receptor Function in Diabetes Mellitus

Heart failure and arrhythmias occur at 3 to 5 times higher rates among individuals with diabetes mellitus, compared with age-matched, healthy individuals. Studies attribute these defects in part to alterations in the function of cardiac type 2 ryanodine receptors (RyR2s), the principal Ca2+-release channels on the internal sarcoplasmic reticulum (SR). To date, mechanisms underlying RyR2 dysregulation in diabetes remain poorly defined. A rat model of type 1 diabetes, in combination with echocardiography, in vivo and ex vivo hemodynamic studies, confocal microscopy, Western blotting, mass spectrometry, site-directed mutagenesis, and [3H]ryanodine binding, lipid bilayer, and transfection assays, was used to determine whether post-translational modification by reactive carbonyl species (RCS) represented a contributing cause. After 8 weeks of diabetes, spontaneous Ca2+ release in ventricular myocytes increased ∼5-fold. Evoked Ca2+ release from the SR was nonuniform (dyssynchronous). Total RyR2 protein levels remained unchanged, but the ability to bind the Ca2+-dependent ligand [3H]ryanodine was significantly reduced. Western blotting and mass spectrometry revealed RCS adducts on select basic residues. Mutation of residues to delineate the physiochemical impact of carbonylation yielded channels with enhanced or reduced cytoplasmic Ca2+ responsiveness. The prototype RCS methylglyoxal increased and then decreased the RyR2 open probability. Methylglyoxal also increased spontaneous Ca2+ release and induced Ca2+ waves in healthy myocytes. Treatment of diabetic rats with RCS scavengers normalized spontaneous and evoked Ca2+ release from the SR, reduced carbonylation of RyR2s, and increased binding of [3H]ryanodine to RyR2s. From these data, we conclude that post-translational modification by RCS contributes to the heterogeneity in RyR2 activity that is seen in experimental diabetes