Homeostasis of glutathione is associated with polyamine-mediated β-lactam susceptibility in Acinetobacter baumannii ATCC 19606

Abstract

Glutathione is a tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) thiol compound existing in many bacteria and maintains a proper cellular redox state, thus protecting cells against toxic substances such as reactive oxygen species. Polyamines (spermine and spermidine) are low-molecular-weight aliphatic polycations ubiquitously presenting in all living cells and modulate many cellular functions. We previously reported that exogenous polyamines significantly enhanced β-lactam susceptibility of β-lactam-associated multidrug-resistant Acinetobacter baumannii. In this study, three genes differentially associated with the polyamine effects on β-lactam susceptibility were identified by transposon mutagenesis of A. baumannii ATCC 19606. All three genes encoded components of membrane transport systems. Inactivation of one of the genes encoding a putative glutathione transport ATP-binding protein increased the accumulation of intracellular glutathione (∼150 to ∼200%) and significantly decreased the polyamine effects on β-lactam susceptibility in A. baumannii ATCC 19606. When the cells were grown with polyamines, the levels of intracellular glutathione in A. baumannii ATCC 19606 significantly decreased from ∼0.5 to ∼0.2 nmol, while the levels of extracellular glutathione were correspondingly increased. However, the levels of total glutathione (intra- plus extracellular) were unchanged when the cells were grown with or without polyamines. Overall, these results suggest that exogenous polyamines induce glutathione export, resulting in decreased levels of intracellular glutathione, which may produce an improper cellular redox state that is associated with the polyamine-mediated β-lactam susceptibility of A. baumannii. This finding may provide a clue for development of new antimicrobial agents and/or novel strategies to treat multidrug-resistant A. baumannii.

Fig 1

Glutathione synthesis in A. baumannii ATCC 19606. Cell cultures collected at each optical density (OD₆₀₀) were used to measure the glutathione concentration as described in Materials and Methods. Open bars indicate extracellular levels of glutathione in cell supernatants; gray-filled bars indicate intracellular levels of glutathione in the cell pellet. Three independent measurements were used to determine standard deviations. GSH, glutathione.

Fig 2

Intracellular glutathione concentrations in A. baumannii ATCC 19606 and its mutant strain (a homolog of AB_AB1837::Km of A. baumannii AB0057). Cell pellets of the mutant strain and the parental strain collected at each optical density (OD₆₀₀) were used to measure the glutathione concentration as described in Materials and Methods. Open bars indicate A. baumannii ATCC 19606; gray-filled bars indicate the mutant strain. Three independent measurements were used to determine standard deviations. GSH, glutathione.

Fig 3

Polyamine effects on glutathione transport in A. baumannii ATCC 19606. Cells grown with or without polyamines (1 mM spermine or 10 mM spermidine) were collected at an OD₆₀₀ of 1.0. Cell supernatants and cell pellets were used to measure levels of extracellular and intracellular glutathione, respectively. Three independent measurements were used to determine standard deviations. GSH, glutathione.