Protease inhibitors (PIs) are a diverse group of proteins divided into 82 families and 39 clans according to MEROPS [21]. Overall, 6700 plant protease inhibitors (PPIs) were identified that include proteins (> 15 kDa) [6], which act to prevent degradation of storage proteins in plants and defend against phytopathogens and are considered promising tools to combat human microbial infections [5]. However, PPIs are rarely explored in the development of therapeutic peptides, even in view of their broad spectrum of action and interesting characteristics for the development of drugs, such as stability and specificity, and are considered safe because they are metabolized into nontoxic amino acids [6].
Recently, a thermostable trypsin inhibitor, named ShTI (11.558 kDa), from chia seeds was isolated by our research group. ShTI was remarkably stable under high temperatures (100°C, 120 min) and a wide range of pH values (2–10). Furthermore, ShTI demonstrated antibacterial activity against Staphylococcus aureus alone and in combination with oxacillin via cell membrane pore formation and increased production of reactive oxygen species (ROS) [8]. Despite differences in bacterial and fungal cells, such as cell wall composition and the presence of more specific cytoplasmic organelles in yeasts, numerous studies have found that protease inhibitors act on both types of cells [22, 23]. This inspired us to examine the effects of ShTI on fungal species of interest to human health.
Among fungi, Candida species are recognized as the most pathogenic organisms. C. albicans is the most commonly isolated species in bloodstream infections (candidemia), and it is characterized as highly adaptable and capable of developing resistance to clinical conventional antifungals [3]. Moreover, nonalbicans species, including C. parapsilosis, C. glabrata, C. tropicalis, and C. krusei, represent 35–65% of cases of candidemia. Notably, infection by C. krusei exhibits high mortality (40–58%) compared to several nonalbicans species and displays a low response to conventional antifungal therapies [24]. Thus, emphasizing the importance of looking for new strategies to solve Candida infections.
In this work, it was reported for the first time that a trypsin inhibitor from Chia exhibited antifungal effects against resistant clinical and susceptible strains of Candida spp. (MIC50 ranging from 2.05 µM to 4.01 µM and MIC100: 8.2 µM). The literature reports others to plant serine protease inhibitors with anticandidal effects. When compared to ShTI, a similar concentration of anticandidal effect was reported in ClTI (trypsin inhibitor from Cassia leiandra) seeds (MIC100: 8.39 µM) [25], while a lower concentration than those reported in IETI (trypsin inhibitor purified from Inga edulis) with MIC50 10.2 µM [26] and a trypsin inhibitor from Enterolobium timbouva named EtTI, capable of inhibiting Candida spp. species with MIC50 values varying from 11.66 to 21.63 µM [27].
The fungal species evaluated include C. albicans, C. parapsilosis, C. krusei, and C. tropicalis, the most frequent species found in bloodstream infections associated with high human mortality [28]. In this work, C. parapsilosis (ATCC® 22019) was the most susceptible yeast tested in comparison to the other analyzed species. This result confirmed the epidemiological studies that indicate differential susceptibility modes among Candida species to drug treatments. According to scientific evidence, C. parapsilosis is more resistant to echinocandins (drugs that act on lipids in the cell membrane) and more susceptible to azoles, whereas C. krusei responds in the opposite direction and is more resistant to azole drugs [29]. This difference in fungal susceptibility to ShTI may vary due to its different modes of action and the response of the yeast to the treatment, a common characteristic also evidenced in conventional antifungals.
Considering the anticandidal activity of ShTI, its mode of action was characterized by the intrinsically resistant strains of C. krusei (ATCC® 6258™) and the fluconazole-resistant clinical strains of C. albicans (strain 1) through fluorescence microscopy and scanning electron microscopy (SEM). The results indicated that ShTI disrupted the cell wall and caused damage to the membrane, as confirmed by the uptake of propidium iodide, a marker of cellular damage that only permeates the membrane-compromised cells, culminating in the loss of functional integrity of the membrane and promoting changes in its organization [30].
A serine peptidase N-ρ-tosyl-l-phenylalanine chloromethyl ketone (TPCK) inhibitor was also able to alter the integrity of the cell membrane of clinical isolates from Candida spp. by inhibiting the biosynthesis of ergosterol, the main lipid of the composition of the fungal cell membrane [31]. A hypothesis can be raised that the action of drugs based on protease inhibitors, such as TPCK and possibly ShTI, on ergosterol biosynthesis occurs through the inhibition of serine proteases involved in the proteolytic cleavage of the Steroid Regulatory Element Binding Proteins (SREBPs), essential in the regulation of ergosterol biosynthesis genes [32]. Thus, the anticandidal effect of ShTI could be the result of a combination of factors that alter cell homeostasis and cause energetic stress in the mitochondria, resulting in the production of ROS [33]. Therefore, our results confirmed that ShTI treatment induced ROS overproduction in C. krusei (ATCC® 6258™) and C. albicans (strain 1) cells, as confirmed by the oxidation of DCFH-DA to DCF by intracellular ROS, leading to cell death pathways by apoptosis.
SEM images revealed morphological changes, pseudohyphae formation, perforations in their surface and lysis in Candida cells. The change in the surface of planktonic cells caused by ShTI compromises the cellular adhesion of Candida cells to substrate, an essential element for the establishment of infection [34] and the formation of microbial biofilms [35]. Therefore, we can infer that ShTI may act in this way to prevent fungal growth. In addition, Candida filamentous growth may be an indication that ShTI acts on the expression of yeast shape regulation genes. This effect was observed with another plant protein, soybean toxin (SbTX), capable of inducing the filamentous growth of C. albicans cells, in which the transcriptional corepressor (TUP1) gene was involved [36]. Furthermore, the presence of large pores in the membrane was demonstrated with internal content loss with consequent homeostasis imbalance, negatively impacting cell survival and compromising yeast pathogenicity [37].
These results corroborate those of other plant protease inhibitors, such as a trypsin inhibitor purified from C. leiandra (MM: 19.48 kDa) that induced oxidative stress in C. albicans cells [25], the cysteine protease from Cassia leiandra (MM: 16.63 kDa) induced disruption of the cell surface from C. tropicalis [38], the synthetic inhibitor bioinspired by the trypsin inhibitor from Jatropha curcas (MM: 600–1200 Da) showed an increase in membrane permeabilization in C. krusei cells [39], and a Kunitz trypsin inhibitor from Enterolobium timbouva (MM: 20 kDa) altered the integrity of the plasma membrane and induced morphological changes in C. albicans, C. buinensis, and C. tropicalis cells [40]. Although the scientific literature reports other studies of plant protease inhibitors against Candida species, our study differentially reports this effect against resistant strains of C. krusei and resistant clinical isolates of C. albicans, with emphasis on the evidenced synergistic effect with fluconazole.
Currently, the most commonly prescribed antifungal drug for Candida infections is fluconazole, a triazole antifungal with a high spectrum of action, large bioavailability, and few side effects [41, 42]. Nonetheless, widespread use and prolonged exposure have increased the number of Candida-resistant strains [43]. Our results suggest that the combination of ShTI-FLC significantly reduces the required dose of FLC, as confirmed in SEM images, where the Candida cells assumed a gross morphological appearance with an irregular and contorted surface. Such characteristics compromise the maintenance of cellular metabolism and consequently its survival and virulence [37] and encourage us to speculate that ShTI is a potential sensitizer of the effect of fluconazole against resistant strains of C. albicans (strain 1).
Therefore, we suggest that the drug combination had a species-dependent effect because the ShTI-FLC combination did not exhibit a synergistic effect against C. parapsilosis (ATCC® 22019) and C. krusei (ATCC® 6258™) strains. This response difference was probably due to the genotypic and phenotypic changes characteristic of the organisms [44]. The literature reported that, based on genotypic analysis, high genetic variability among Candida species was confirmed, mainly among infectious species that frequently inhabit humans, such as C. albicans [45]. Such genotypic differences may explain why the combination ShTI-FLC effectively inhibited the growth of C. albicans cells but was not effective against the nonalbicans cells tested.
Pathogenic fungi such as Candida secrete various serine proteases, such as trypsin, in the process of pathogenicity [46]. Therefore, molecules that act by inhibiting the action of these proteases are interesting compounds as new antifungal drugs. A secretory leucocyte proteinase inhibitor (SLPI) (MM: 11.7 kDa) exhibited an antifungal effect against a clinical isolate of C. albicans (MIC 18 µM) and was able to inhibit the proteolytic activity of secreted proteases [47]. Although studies show the action of PIs on fungal proteins, none of the antifungal compounds currently used act by inhibiting serine proteases, which is considered a clinical challenge since proteases are present in human metabolism. However, alternative application routes, such as topical applications, may be considered [46]. In addition, when evaluating the specificity of PI, plant protease inhibitors have a variety of characteristics, such as stability at high temperatures and pH variations, substrate specificity, and high solubility in aqueous solutions, which also allow their oral administration [48].
Based on the promising antimicrobial effect of ShTI, studies of its toxic effects were evaluated to obtain preliminary data on the safety of its application against mammalian cells. The results indicated that ShTI did not significantly alter cell viability in the MTT test and did not exhibit signs of apoptosis, necrosis, or DNA damage in mouse fibroblast cells. The literature has reported that plant protease inhibitors were found to be safe in in vitro toxicity assays [25], and there are several preclinical studies on the pharmacological effects of plant protease inhibitors on human health [49]. In addition, chia seeds are widely used as a food ingredient and have not reported cases of toxicity; on the other hand, beneficial effects such as antioxidant [50], antidiabetic and anticholesterol properties [51] associated with the consumption of seed proteins have been described. Thus, the European Commission, the EFSA Panel on Nutrition, Novel Foods, and Food Allergens (NDA), issued a technical opinion proving the safety of chia as a new food ingredient [52] currently considered one of the most popular seeds in the world [53]. Therefore, it has been suggested that the components present in Chia seeds, such as proteins, are considered safe for human use.
In this work, we started from a preliminary study of the in vitro anti-Candida effect of a trypsin inhibitor from Chia seeds, but new assays will be necessary as a test in vivo model of candidiasis and preclinical toxicity assays to enable the advancement of the application future use of ShTI as a new antimicrobial.