β-amyrin-induced apoptosis in Candida albicans triggered by calcium

Highlights

• Generation of ROS is triggered by β-amyrin in C. albicans.

• β-amyrin induces intracellular calcium increment in C. albicans.

• β-amyrin-induced calcium increase cause mitochondrial dysfunction and oxidative damage in C. albicans.

• β-amyrin treatment led to occurrence of apoptosis in Candida albicans.

Abstract

The emergence of drug-resistant pathogens has urged researchers to discover alternatives for traditional antibiotics. β-amyrin, which is included in the category of triterpenoids extracted from plants, is known for its antimicrobial activity, although the underlying mechanism has not yet been revealed. This study was conducted to elucidate the antifungal mode of action of β-amyrin against Candida albicans. Based on the relevance between triterpenoids and oxidative molecules, reactive oxygen species (ROS) concentrations were detected, which showed a noticeable increment. Disruption of Ca²⁺ homeostasis in the cytosol was additionally analyzed, which was supported by interactions between two. Subsequently, decrease in mitochondrial membrane potential, increment of mitochondrial Ca²⁺, and ROS concentration were monitored, which suggested mitochondrial dysfunction modulated by Ca²⁺. Further investigation confirmed oxidative damage through glutathione reduction and DNA fragmentation. Accumulation of lethal damages resulted in the activation of caspases and externalization of phosphatidylserine, indicating the induction of yeast apoptosis by β-amyrin in C. albicans.

Introduction

Triterpenoids, often known as triterpenes based on their functionalized structure, are extracts primarily derived from various plants. Their structural form includes hydrocarbons made up of isoprene units as their building blocks. Due to their diverse possible structures, triterpenoids are considered as the largest class of all known plant metabolites (Haque et al., 2016). Research shows that triterpenoids exhibit diverse biological activities, including anticancer, anti-inflammatory, immunological, antiviral, antibacterial, and antifungal effects (Johann et al., 2007). In a study, triterpenoids have been also discovered to induce apoptotic cell death through production of mitochondrial reactive oxygen species (ROS) generation (Shin et al., 2018). Due to these various properties, triterpenoids are frequently investigated as substances to enhance human health. There is a wide variety of members of pentacyclic triterpenoids, including lupeol, α-amyrin, and β-amyrin (JC Furtado et al., 2017). Among these triterpenoids, β-amyrin is known to exhibit potential regulatory biological activities related to pathogens by demonstrating its antibacterial and antifungal effects.

Included among the pentacyclic triterpenoids, β-amyrin possesses an oleanane skeleton and is extracted from several medical plants such as Protium heptaphyllum, Catharanthus roseus, and Camellia tenuifolia (Huang et al., 2012). β-amyrin is a well-known material used in medical treatment and also plays the role of a plant metabolite, an Aspergillus metabolite, inhibiting collagen synthesis (Ching et al., 2010). Recent studies have additionally reported the pharmacological activities of β-amyrin, suggesting the potential of its use in medicine. Some of these studies have reported its antiproliferative, antitumor, anti-parkinsonian, anti-inflammatory, and antimicrobial activities (Johann et al., 2007). Despite these diverse discoveries concerning β-amyrin, the exact mechanisms underlying its several activities are still unknown. However, its apoptotic activity, along with cell cycle arrest, has been monitored in a recent study, which disclosed the mode of action of β-amyrin in terms of cellular death (Wen et al., 2018). As an advancement, the antimicrobial activities of β-amyrin are likely to be investigated in an objective to clarify its influence on cell survival, ranging from necrosis to apoptosis (Abdel-Raouf et al., 2015).

In general, apoptosis in microorganisms, especially in yeasts, occurs through their eukaryotic structure. Although bacterial apoptosis-like death processes have been discovered recently, most of the reported antimicrobial apoptotic properties were antifungal, which were widely demonstrated using Candida albicans as a model organism (Ramsdale, 2008). Due to the presence of various pathogenic yeasts, investigation of antifungal activity is considered as an important strategy for the treatment of diseases (Kim et al., 2019). Regarding the treatment for lethal diseases, including aspergillosis, zygomycosis, dermatophytosis, and candidiasis, several methods have been discovered to regulate the cellular processes of these fungi. Of these processes, yeast apoptosis has been widely investigated in several studies, displaying several interesting characteristics. Apoptosis is a form of programmed cell death that is important for the regulation of cellular homeostasis and is different from simple cell death in a manner that it takes advantage by being free from costly complications such as wound healing (Fink and Cookson, 2005). Yeast apoptosis occurs under various conditions and environments. Several characteristic markers derived from such circumstances include DNA cleavage, externalization of phosphatidylserine (PS), ROS accumulation, and cytochrome c release (Madeo et al., 2004). These hallmarks directly lead to apoptosis because of the critical damage induced during the process. Meanwhile, there are also preceding events found in the initial stage, which are generally the primary cause of the entire process. Events such as ion influx and generation of nitric oxide are some of the examples discovered till date. Among them, Ca²⁺ is one of the most frequently referred ions due to its role as a ubiquitous intracellular messenger in regulating biological processes (Lee and Lee, 2018b). In this study, along with the antifungal mechanism of β-amyrin, the influence of Ca²⁺ during the process was investigated to identify the possible role of Ca²⁺ in yeast apoptosis.