Effects of vancomycin and ceftazidime on biofilm formation in Elizabethkingia

Elizabethkingia infections are associated with high mortality rates which is due in part to the antibiotic resistance expressed by all Gram-negative Elizabethkingia species. Biofilm formation by Elizabethkingia provides the bacteria with further defense against the action of antibiotics. Vancomycin is an unusual drug used to treat Elizabethkingia infections, but its use is controversial, since it is normally only used to treat Gram-positive infections. Our study tests the effect of vancomycin and ceftazidime on biofilm formation for all known species of Elizabethkingia. Biofilm formation was measured at 24, 48, and 72-hour timepoints using a crystal violet biofilm assay. Biofilm formation occurred in all positive control wells for all three timepoints for all species. The biofilms of Elizabethkingia meningoseptica in the presence of subinhibitory vancomycin concentrations, and Elizabethkingia ursingii in the presence of inhibitory ceftazidime concentrations, were reduced compared to the controls. This suggests that both drugs can inhibit biofilm formation in a species-specific manner.Howard Hughes Medical Institute Science Education ProgramBiochemistry and Molecular Biolog

G2019s leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization

The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinson's disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain. The presence of G2019S LRRK2 mutation did not influence LRRK2 expression in fibroblasts. However, the expression of the G2019S LRRK2 mutation in both fibroblast and neuroblastoma cells was associated with mitochondrial uncoupling. This was characterized by decreased mitochondrial membrane potential and increased oxygen utilization under basal and oligomycin-inhibited conditions. This resulted in a decrease in cellular ATP levels consistent with compromised cellular function. This uncoupling of mitochondrial oxidative phosphorylation was associated with a cell-specific increase in uncoupling protein (UCP) 2 and 4 expression. Restoration of mitochondrial membrane potential by the UCP inhibitor genipin confirmed the role of UCPs in this mechanism. The G2019S LRRK2-induced mitochondrial uncoupling and UCP4 mRNA up-regulation were LRRK2 kinase-dependent, whereas endogenous LRRK2 levels were required for constitutive UCP expression. We propose that normal mitochondrial function was deregulated by the expression of G2019S LRRK2 in a kinase-dependent mechanism that is a modification of the normal LRRK2 function, and this leads to the vulnerability of selected neuronal populations in PD