Binding of Azole Antibiotics to Staphylococcus aureus Flavohemoglobin Increases Intracellular Oxidative Stress

Abstract

In this work, we report that flavohemoglobin contributes to the azole susceptibility of Staphylococcus aureus. We first observed that deletion of the flavohemoglobin gene leads to an increase in the viability of imidazoletreated S. aureus cells and that reversion to the wild-type phenotype occurs upon expression of flavohemoglobin from a multicopy plasmid. Further spectroscopic analyses showed that miconazole, the most efficient azole antibiotic against S. aureus, ligates to heme of both oxidized and reduced flavohemoglobin. The binding of miconazole to oxidized flavohemoglobin, with an association constant of 1.7 ؋ 10 6 M ؊1 , typical of a tight, specific binding equilibrium, results in augmentation of the superoxide production by the enzyme. These results are corroborated by in vivo studies showing that imidazole-treated S. aureus cells expressing flavohemoglobin contain a larger amount of reactive oxygen species. Moreover, it was observed that the survival of miconazole-treated S. aureus internalized by murine macrophages is higher for cells lacking flavohemoglobin. Altogether, the present data revealed that in S. aureus, flavohemoglobin enhances the antimicrobial activity of imidazoles via an increase of intracellular oxidative stress.Staphylococcus aureus is an opportunistic pathogen responsible for a large number of human infections that cause systemic diseases from a mild to life-threatening character. The increasing incidence of methicillin-resistant S. aureus (MRSA) strains observed in the past few years makes S. aureus infections a leading threat to public health, causing more deaths in the United States and Europe than human immunodeficiency virus (AIDS) (11). Like other Gram-positive bacteria, staphylococci are sensitive to imidazoles (27). Imidazoles (such as clotrimazole, miconazole, ketoconazole, and sulconazole) ( Fig. 1) represent one of the major classes of azole antifungal that are useful in the treatment of infections, including cutaneous and vaginal candidiasis (8). The activity of these antifungal drugs derives primarily from inhibition of the biosynthesis of ergosterol, an essential component of the fungal plasma membrane, at the level of lanosterol 14-alpha demethylase. Furthermore, in fungi and yeast, azole treatment leads to an increase in the endogenous production of reactive oxygen species (ROS) (12, 25). For example, in Candida albicans and Saccharomyces cerevisiae, the miconazole inhibition of cytochrome c oxidase, peroxidase, and catalase has been reported to be responsible for a high level of ROS production (3, 4). It has also been reported that clotrimazole inhibition of Plasmodium falciparum hemoperoxidase leads to ROS accumulation in this protozoan pathogen (26). For S. cerevisiae, C. albicans, and Escherichia coli, the action of imidazoles was also correlated with the inhibition of the nitric oxide (NO) scavenger activity of flavohemoglobin (7).Flavohemoglobins (Hmp) are widespread among bacteria and yeast and contain three domains: C-terminal NAD-and flavin adenine dinucleot...

The antibacterial effect of nitric oxide against ESBL-producing uropathogenic E. coli is improved by combination with miconazole and polymyxin B nonapeptide

“…Furthermore, the fact that miconazole did not increase the DETA/NO-induced growth inhibition in an hmp -mutant strain, support that inhibition of flavohemoglobin [14] contributes to the antibacterial effect of miconazole in our experiments. Thus, the prolonged growth inhibition evoked by DETA/NO and miconazole in combination may be a result of interactions of miconazole with flavohemoglobin, causing both inhibition of NO dioxygenase activity and oxidative stress following high levels of cytotoxic superoxide production [14,15]. In agreement with our results, intracellular survival studies in activated NO-producing macrophages demonstrated decreased survival of miconazole-treated S. aureus compared to untreated bacteria [15].…”

Biophysical Screening of a Focused Library for the Discovery of CYP121 Inhibitors as Novel Antimycobacterials

“…An explanation for the antibacterial activity of econazole against E. coli and S. aureus was already provided before. In these studies econazole was described as an inhibitor of flavohemoglobin . Further possible target systems of azole antifungals within Mtb metabolism have also been described .…”

In vitro miconazole susceptibility of meticillin‐resistant Staphylococcus pseudintermedius and Staphylococcus aureus

“…This does not apply directly to bacteria; however, Mycobacterium smegmatis, another bacterium that is susceptible to miconazole, has been shown to contain a cytochrome P450 monooxygenase that is orthologous to 14α‐sterol demethylase and strongly binds miconazole 12 . Direct membrane damage has also been suggested as a mechanism of action in staphylococci, 6 and it is has recently been reported that miconazole induces intracellular reactive oxygen species in S. aureus 13 . As a clear mechanism is not understood (and multiple mechanisms may be present), it is difficult to hypothesize possible explanations for the impact of species or meticillin resistance on miconazole susceptibility.…”