Insights into the intracellular mechanisms of citronellal in Candida albicans: implications for reactive oxygen species-mediated necrosis, mitochondrial dysfunction, and DNA damage

Abstract INTRODUCTION Citronellal (Cit) possesses antifungal activity and has possible implications for reactive oxygen species (ROS) generation in Candida albicans. In this study, the effects of Cit on ROS generation and the mechanisms by which Cit exerts anti-Candida effects were examined. METHODS A 2′,7′-dichlorodihydrofluorescein diacetate assay was used to assess oxidative damage. Cell necrosis was determined by flow cytometry after FITC-Annexin V staining. Mitochondrial function was studied based on mitochondrial potential, metabolic activity (MTT assay), and phenotypic susceptibility on a non-fermentable carbon source. Membrane intactness and DNA damage were estimated by a propidium iodide (PI) uptake assay and 4',6-diamidino-2-phenylindole (DAPI) staining. RESULTS ROS generation was enhanced in response to Cit, leading to necrosis (2%). Additional hallmarks of cell death in response to Cit, such as mitochondrial membrane depolarization and DNA damage, were also observed. Cit treatment resulted in dysfunctional mitochondria, as evidenced by poor labeling with the mitochondrial membrane potential-sensitive probe rhodamine B, reduced metabolic activity (61.5%), and inhibited growth on a non-fermentable carbon source. Furthermore, Cit induced DNA damage based on DAPI staining. These phenotypes were reinforced by RT-PCR showing differences in gene expression (30-60%) between control and Cit-treated cells. Finally, PI uptake in the presence of sodium azide confirmed non-intact membranes and suggested that Cit activity is independent of the energy status of the cell. CONCLUSIONS Cit possesses dual anticandidal mechanisms, including membrane-disruptive and oxidative damage. Taken together, our data demonstrated that cit could be used as a prominent antifungal drug.

Influence of iron deprivation on virulence traits of mycobacteria

ABSTRACT Novel strategies to combat the ever increasing burden of drug resistance in Mycobacterium tuberculosis (MTB) causing tuberculosis (TB) remains a global concern. The ability of MTB to sense and adapt to restricted iron conditions in the hostile environment is essential for their survival and confers the basis of their success as dreadful pathogen. The striking and clinically relevant virulence trait of MTB is its ability to form biofilms and adhere to the host cells. The present study elucidated the effect of iron deprivation on biofilm formation and cell adherence of Mycobacterium smegmatis, a non-pathogenic surrogate of MTB. Firstly, we showed that iron deprivation leads to enhanced cell sedimentation rate and altered colony morphology depicting alterations in cell surface envelope properties. We explored that biofilm formation and cell adherence to polystyrene surface as well as human oral epithelial cells were considerably reduced under iron deprivation both in presence of 2,2 BP (iron chelator) and siderophore mutant Δ011-14 strain. We further investigated that the potency of three first line anti-TB drugs (Isoniazid, Ethambutol, Rifampicin) to inhibit both biofilm formation and cell adhesion were enhanced under iron deprivation in contrast to the drugs when tested alone. Taken together, by virtue of the indispensability of iron for functional virulence traits in mycobacteria, iron deprivation strategies could be further exploited against this notorious human pathogen to explore novel drug targets.

Citronellal-induced disruption of membrane homeostasis in candida albicans and attenuation of its virulence attributes

Abstract: INTRODUCTION There is an increasing burden of multidrug resistance. As a result, deciphering the mechanisms of action of natural compounds with antifungal activity has gained considerable prominence. We aimed to elucidate the probable mechanism of action of citronellal, a monoterpenoid found in the essential oil extracted from Cymbopogon plants, against Candida albicans. METHODS Drug susceptibility was measured by broth microdilution and spot assays. Ergosterol levels were estimated using the alcoholic potassium hydroxide method and H+ extrusion was assessed by monitoring the glucose-induced acidification of the external medium. Virulence traits were studied by hyphal morphogenesis and biofilm formation, along with fungal cell adherence to polystyrene surface and human oral epithelial cells. RESULTS Citronellal showed anticandidal activity against C. albicans and non-albicans species of Candida at a minimum inhibitory concentration of 1 mg/ml. Citronellal interfered with membrane homeostasis, which is the major target of known antifungal drugs, by increasing the hypersensitivity of the fungi to membrane-perturbing agents, reducing ergosterol levels, and diminishing glucose-induced H+ extrusion. In addition, oxidative and genotoxic stresses were induced via an increased production of reactive oxygen species. Furthermore, citronellal inhibited the virulent attributes of yeast-to-hypha transition and biofilm formation. It also reduced cell adherence to polystyrene surface and the human oral epithelial cells. CONCLUSIONS This is the first study to propose the cell membrane, morphogenetic switching, biofilm formation, and cell adherence of Candida albicans as potential targets for the anticandidal activity of citronellal. However, clinical investigations on the therapeutic applications of citronellal are required.