Phytochemicals and Their Antifungal Potential against Pathogenic Yeasts

1. Introduction

Fungal infections are considered a serious health problem, especially in people with weakened immune systems, and are a main cause of morbidity and mortality worldwide [1].

However, the impact of these “opportunistic” diseases on human health is not widely highlighted [2]. Due to this, research related to fungi occurs slowly compared to those caused by other pathogens.

Among the different mycotic infections, those caused by Candidaand Cryptococcusare the most threatening due to severity of the disease and higher worldwide occurrence [3]. The pathogenicity of fungal infections proceeds in well-organized steps. For example, Candidacell surface adhesion factors first promote its adherence to host surface, followed by releasing of various hydrolytic enzymes and other virulence factors for invasion and damage of the host tissues [4].

Candidaspecies can cause a variety of infections from the mildest to the most severe being candidemia the most frequent hospital infection accounting for up to 15% of bloodstream infections. Candidaspecies are the main causative agents in 50–70% of systemic fungal infections [5].

Cryptococcusspecies are other yeasts of medical importance, with more than 39 species, among which Cryptococcus gattiiand Cryptococcus neoformansare the most clinically relevant [6, 7, 8]. However, other species such as Cryptococcus albidusand Cryptococcus laurentiiare emerging pathogens involved in several types of infections [6, 9, 10, 11].

These yeasts are present in several environmental niches, such as woody sites (decomposing tree trunks, mainly eucalyptus, and soil), vegetable remains, domestic dust, and bird excrement, more precisely in Columba livia[12, 13, 14]. The source of the infection is exogenous and occurs primarily by inhalation or by direct inoculation into the tissue after trauma of desiccated spores or yeasts. It is believed that the only source of infection is environmental, since there are no reports of transmission between animals and humans or between humans [15].

The main virulence factors of Cryptococcusspecies are growth capacity at 37°C, polysaccharide capsule, melanin synthesis, and production of urease and antioxidant enzymes, causing primary or opportunistic cryptococcosis, such as pulmonary, cutaneous, and meningitis diseases [6, 8, 13, 16, 17, 18, 19]. Cryptococcosis is the third opportunistic infection associated with AIDS [20].

In addition to delays in yeast diagnosis, there is currently a limited antifungal armamentarium in use against yeast diseases including only four chemical classes: polyenes, triazoles, echinocandins, and flucytosine. Antifungals act by binding specific components of fungal plasma membrane or its biosynthetic pathways or even cell wall components [21]. However, most of the antifungal agents used in the clinic is fungistatic and often led to the development of resistance by fungal species. Modern early antifungal treatment strategies, such as prophylaxis and empirical and preemptive therapy, result in long-term exposure to antifungal agents, which is a major driving force for the development of resistance.

Among the available antifungal agents, azoles are the preferred and most frequently used drugs for treatment of Candidaand Cryptococcusinfections. Fluconazole (FLZ), a type of azole, is often preferred in treatments of Candidainfections because of its low cost and toxicity, in addition to availability in varied formulations [22]. However, there are many reports that described resistance development among Candidaspecies, especially in relation to azoles.

Infectious Diseases Society of America recommends the treatment of cryptococcosis through FLZ and amphotericin B (AMB) with or without combination with 5-flucytosine (5-FC), followed by prolonged maintenance with fluconazole. Other azole compounds such as itraconazole (ITC), voriconazole, and posaconazole may be used as an alternative to FLZ in cases of contraindication or inefficacy of the latter [23, 24]. However, there has been a progressive increase in isolates of Cryptococcusspp. resistant to FLZ, which complicates the management of cryptococcal meningitis [25]. On the other hand, AMB and 5-FC are not available in all countries and are, respectively, nephrotoxic and hepatotoxic, limiting the anti-cryptococcal therapeutic [24].

Considering the limited availability of antifungals in use and the emergence of resistance, the control of Candidaand Cryptococcusinfections is a challenge in the modern clinic. In this way there is a continuous need for the search for new substances with new mechanisms of action with the aim of developing novel broad spectrum antifungal drugs with better efficacy.

In this way, plants stand out as the major producers of promising substances, the phytochemicals. Identification of new molecules with antifungal potential for the manufacture of new drugs, more effective and less toxic, is essential to facing the challenge. The use of phytochemicals alone or in combination with traditional drugs represents an important alternative to conventional therapy. The combination of drugs usually requires lower doses of antimicrobials. This reduction might lead to a toxicity decrease, which results in a higher tolerance to the antimicrobial by the patient.

2. Pathogenic yeast infections: a serious health problem

In the last two decades, fungal infections have shown a significant increment. In addition to the increase in the number of patients with compromised immune system, factors such as increasing number of patients using catheters, the use of broad-spectrum antibiotics, the rising number of patients requiring organ transplantations, as well as those with hematological malignancies and diabetes also contribute to this phenomenon [26, 27].

Even though fungal infections cause significant amount of human morbidity and mortality, the impact of these “opportunistic” diseases on human health is not widely highlighted [2]. Due to this, the research into the pathophysiology of human fungal infections is slow in comparison to other disease-causing pathogens. Recently, an editorial published in the journal Nature Microbiology[28] ratified the importance of not neglecting fungi. The call proposed a reflection on fungi and how these microorganisms have been neglected, even with studies already consolidated showing their medical relevance.

The most frequent fungal diseases affecting populations in the world are candidiasis [29, 30, 31, 32, 33, 34] and cryptococcosis [8, 20, 25]. There are several types of candidiasis as mucosal candidiasis, cutaneous candidiasis, onychomycosis, systemic candidiasis [35, 36], and pulmonary candidiasis. An important fact is that candidiasis is an infection that can affect both immunocompromised and healthy people [37, 38]. Candidemia is the most relevant and prevalent nosocomial fungal infection associated with a high mortality rate (up to 49%) in patients with a compromised immune system [39, 40]. The association of Candidawith bloodstream infections depends on patient’s condition, age, and geographic region. Candidemia is such an important infection that in 10–40% of cases, it is associated with sepsis or septic shock [41].

Candida albicanscontinues to be the most prevalent species isolated from fungal infections [27, 42, 43, 44]. However, the prevalence of other Candidaspecies has increase substantially. These species are C. parapsilosis, C. tropicalis, C. krusei, C. glabrata, C. guilliermondii, C. orthopsilosis, C. metapsilosis, C. famata, and C. lusitaniae[44, 45, 46].

Candidaspecies presents high degree of flexibility, being able to grow in extremely different environments regarding to the availability of nutrients, temperature variation, pH, osmolarity, and amount of available oxygen [47]. This fact associated with the high resistance capacity of species to antifungals, their virulent features, and capability of forming biofilms with other species [48, 49] makes the genus Candidaa serious risk to human health [50]. Thus, Candidaspecies are highly adaptable and possess numerous strategies to survive in conditions that can affect their overgrowth and alter their susceptibility profiles.

Cryptococcusspp. may remain latent in the lungs, leading to asymptomatic infection, or may cause multifocal lung disease. The latency period of Cryptococcuscan range from 6 weeks to more than 1 year after inhalation [51]. The fungus presents neurotrophism and can migrate to the central nervous system (CNS) through hematogenous dissemination and, when crossing the blood-brain barrier, can cause meningoencephalitis [13, 18]. Episodes of mental confusion in patients with cryptococcosis have been described [52, 53]. Neurocryptococcosis is the most severe form of the disease with high mortality rates in the absence of adequate treatment [18, 23]. The mortality due to cryptococcosis is higher than the mortality caused by tuberculosis and similar to that caused by malaria [54].

Another clinical manifestation is cutaneous cryptococcosis, which is rare and usually secondary to hematogenous dissemination. Cutaneous lesions are characterized by an infiltrative plaque of a solid tumor mass that can present ulcerative and necrotic lesion [17]. Pulmonary and cutaneous lesions due to nodular features may be misdiagnosed as tumor lesions [55]. In addition to the respiratory tract, CNS and skin, other sites may be affected: prostate, eyes, adrenal glands, lymph nodes, bone marrow, and liver [51].

Until now, there are three proposals to explain fungal neurotropism. The first is that neuronal substrates present in the basal ganglia promote cryptococcal growth and survival, and, thus, perivascular spaces may serve as a niche for Cryptococcus, as described by [56] in a healthy female patient who had evidence of Cryptococcusinfection within the perivascular spaces of the parenchyma. The second proposal describes that it is possible that there are specific neuronal receptors that can attract Cryptococcusto the CNS [57]. The third hypothesis, one of the most widespread, is that the fungus uses neurotransmitters such as dopamine that aids in the synthesis of melanin [19, 57, 58].

Besides the clinical importance of fungal infections caused by theses pathogenic yeasts, interestingly, climatic abnormalities due to phenomena such as La Niña and El Niño have recently been described as important in the distribution and occurrence of mycoses in countries influenced by them [59].

3. Traditional antifungal agents against yeasts

In the last two decades, there has been an increasing, but limited, discovery of antifungal agents [47]. These include azoles, such as fluconazole, itraconazole, ketoconazole (KTC), miconazole, and clotrimazole, polyenes (amphotericin B [AMB] and nystatin), allylamines, thiocarbamates, morpholines, 5-fluorocytosine, and echinocandins (for instance, caspofungins) [21]. However, fungal cells and human cells are eukaryotic, so antifungal compounds target both cell types, resulting in considerable side effects in patients and fewer available targets for drug action. Antifungals target three cellular components of fungi (Figure 1). Azoles inhibit ergosterol biosynthesis by interfering with the enzyme lanosterol 14-α-demethylase in endoplasmic reticulum of the fungal cell. This enzyme is involved in the transformation of lanosterol into ergosterol, a component that is part of the plasma membrane structure of the fungus (Figures 1 and 2). Thus, as the concentration of ergosterol is reduced, the cell membrane structure is altered, thereby inhibiting fungal growth [60].

Azoles comprise a five-member azole ring containing two (imidazole) or three nitrogen atoms (triazole) attached to a complex side chain [61, 62]. Imidazoles include KTC, miconazole, econazole, and clotrimazole, and triazoles include FLZ, ITC, voriconazole (synthetic triazole derivative of FLZ of second generation), and posaconazole (hydroxylated analog of itraconazole) [63].

AMB and nystatin bind to ergosterol causing the disruption of the membrane structure and promoting extravasation of intracellular constituents such as ions and sugars and, consequently, cell death [21] (Figure 1).

Pyrimidine analogs include 5-fluorocytosine and 5-fluorouracil (5FU). The first has fungistatic properties and enters the fungal cell through cytosine permease, inhibiting the thymidylate-synthetase enzyme and interfering with DNA. 5-fluorouracil, which in turn can be phosphorylated to 5-fluorodeoxyuridine monophosphate, can be incorporated into RNA molecules [63]. Due to toxicity [64]; stronger side effects, such as hepatic impairment; interference with bone marrow function; and rapid occurrence of resistance especially among Candidaspecies, the clinical use of 5-FC is preferred in association with AMB [65, 66]. In addition, the nephrotoxicity and hepatotoxicity of AMB and 5-FC, respectively, and the unavailability of these antifungals in many countries have limited their use in cryptococcal therapeutic [24].

Host’s immunity, type of infection, site of origin of the samples, toxicity, bioavailability of the drug, and the sensitivity/resistance profile of the isolates interfere in the choice of the type of agent to be used [22]. AMB is considered the gold standard drug for most mycoses that affect patients at risk [67], although it has high toxicity. Azoles have fungistatic properties that affect cell growth and proliferation [65]. Among azoles, KTC was one of the firsts to emerge and was the first alternative to AMB [68]. Currently, FLZ is the drug of choice for most Candidaand Cryptococcusinfections [64] and is the most recommended antifungal agent for use in invasive candidiasis [47, 49].

For cryptococcosis, the choice of treatment depends on the patient’s immunological status and mainly on the clinical of the disease, if it is just a pulmonary manifestation or if the infection is systemic. Fluconazole is recommended in cases of lung disease with mild to moderate symptoms. Amphotericin B with or without combination with 5-flucytosine is the recommended therapy for more serious infections such as meningoencephalitis, followed by prolonged maintenance with fluconazole [23, 24].

Although azoles are generally well-tolerated, they have limitations such as hepatotoxicity and the emergence of resistance among fungal isolates [69] which provide motivation for improving this class of antifungal agents [68]. For instance, alterations in triazole molecule gave rise to voriconazole (structurally related to FLZ) and posaconazole (related to ITC), both available for systemic therapy [66].

Echinocandins, which include caspofungin, micafungin, and anidulafungin, are a new class of antifungals and have fungicidal effects in all Candidaspecies [66]. They inhibit (1,3) β-d-glucan synthase, thereby preventing glucan synthesis, which is present in the cell membrane of fungi (Figure 1). As this drug acts on the wall structure of the fungus, it has the advantage of a lower side effect in animal cells [47].

Allylamines (terbinafine and naftifine) and thiocarbamates inhibit the enzyme squalene epoxidase, which participates in the synthesis of ergosterol and is encoded by the ERG1gene (Figure 2). This activity leads to membrane rupture and accumulation of squalene. Allylamine effects can also prevent the production of other sterol derivatives.

To minimize toxicity and resistance, some pharmacological strategies were developed. The preparation and use of new antifungal formulas (liposomal AMB (Ambisome®), AMB lipid complexes (Abelcet®), AMB colloidal dispersions (Amphocil®/Amphotech®), and AMB lipid nanosphere formulations and β-cyclodextrin itraconazole) are one strategy [68]. Others include combination therapies of antifungal compounds (e.g., AMB + 5-FC, FLZ + 5-FC, AMB + FLZ, caspofungin + liposomal AMB, and caspofungin + FLZ) and nanostructuring of conventional antifungal agents [70, 71, 72, 73].

However, all traditional antimycotic drugs have at least one restriction related to their use. Some do not have a broad spectrum of action or are fungistatic. Others have high toxicity and low bioavailability with significant side effects [74]. Therefore, limitations of treatment and drug resistance associated with pathogenicity of the clinical isolates support the urgent need to identify substances that are more effective, with new mechanisms of action in the fight against Candidaand Cryptococcusinfections.

4. Resistance in pathogenic yeasts: a significant problem

Most antifungals target sterols or the enzymes that synthesize them. However, the fungistatic nature of many of these antifungals and emergence of clinical drug resistance limits their success. Increased drug resistance in fungi is a problem that cannot be avoided, particularly for FLZ, which is the preferred antifungal for treating yeast infections [75].

The number of people at risk for fungal infections has been increasing, resulting in an increased use of antifungal agents, even as prophylaxis. Thus, besides the existence of some non-albicans Candida(NAC) species presenting inherent resistance to azoles, higher minimum inhibitory concentrations (MICs) for antifungals against C. albicansstrains have been observed [76]. The World Health Organization (2014) categorizes antimicrobial resistance as that developed by the microorganism to an antimicrobial drug, which was initially effective in treatment of such infections. Low-dose prophylactic administration of azole derivatives, such as FLZ, for prolonged periods to prevent the occurrence of opportunistic infections in immunosuppressed patients also results in resistant phenotypes [27, 75]. Therapeutic failures and empiric treatment are facts which are likely to collaborate to the increased incidence of fungal infections.

In the last decade, a number of new clinical problems have arisen, requiring new guidelines regarding the treatment of cryptococcosis, mainly because clinical data have suggested that cryptococcal strains have become more resistant to drugs [23, 25]. Some relates say that clinical Cryptococcusisolates are frequently less susceptible to fluconazole than environmental isolates. However, Chowdhary et al. [77] evaluated the susceptibility profile of environmental and clinical strains of C. gattiiand observed that environmental samples were less susceptible to fluconazole, itraconazole, and voriconazole in comparison to clinical isolates.

Heteroresistance is also a worrying phenomenon. It consists of the ability of a subpopulation of microorganism to adapt to high concentrations of the drug, resulting in resistant homogenous populations. However, heteroresistant strains return to the initial phenotype when the stimulus with the drug is withdrawn [78].

Some mechanisms for cellular and molecular resistance to FLZ in yeasts are described. In Candidaand Cryptococcus, the first is related to the induction of multidrug pumps, which decrease the concentration of drug available in the intracellular compartment of yeast cells. Various genes belonging to the ATP-binding cassette superfamily or to the major facilitator superfamily encode efflux pumps were identified in C. albicans. Overexpression of some transporter genes or of their regulated genes can confer cross-resistance to various azoles [21]. In C. gattiiand C. neoformans, AFR1, MDR1, and AFR2 genes encode ABC transporters that expel the azole out of the fungal cell, thereby causing resistance to these drugs [79].

A second mechanism of resistance involves modification of the target enzyme encoded by the ERG11gene, also known as cytochrome P₄₅₀ lanosterol 14-α-demethylase (Cyp51). Mutations in this gene prevent azoles from binding to enzyme sites. Another mechanism of resistance is related to mutations in the ERG3gene which does not convert 14-α-methylfecosterol into 14-α-methyl-3,6-diol in the ergosterol synthesis pathway. This substitution causes azoles to have no fungistatic effects on the fungal cell membrane [21].

Transcriptional regulation is also important for the development of resistance mechanisms. YAP1, a protein, is important for the mechanism of C. neoformansheteroresistance to fluconazole and oxidative stress. Mutant strains of C. neoformansthat lost protein YAP1 became hypersensitive to a variety of oxidizing agents and mainly to fluconazole [80].

Resistance to polyenes (AMB) in fungus is less common and in C. albicansis associated with the substitution of ergosterol with a precursor molecule or a general reduction of sterols in the plasma membrane [81]. Reduction of membrane ergosterol renders Cryptococcus neoformansand Aspergillusspp. less susceptible to amphotericin B [82]. Enzymes encoded by ERG3and ERG2genes participate in ergosterol biosynthesis and have the main alterations related to AMB resistance because mutations in their genes modify ergosterol content required for the action of polyenes [83].

The main resistance mechanism to echinocandins is related with point mutations in gene that encodes the major subunit of the glucan synthase enzyme (Fks subunit) (Figure 1) and can provide resistance to all echinocandin [84]. Other Candidaspecies also present this resistance mechanism such as C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, and C. dubliniensis[85, 86].

Resistance to 5-FC can be of two types: primary, occurring via cytosine permease (encoded by the FCY2 gene) whose mutation decreases drug uptake [87], and secondary, related to alterations in cytosine deaminase (encoded by FCY1) or uracil phosphoribosyltransferase (encoded by FUR1) activities. Cytosine permease is responsible by conversion of 5-FC to 5-fluorouridine or to 5-fluorouridine monophosphate (5-FUMP) [88]. Resistance is easily developed in fungal isolates from patients who are receiving the drug. However, other molecular mechanisms related to resistance to 5-FC must exist because most of them have not been observed in C. albicans[89].

The increase in the drug-resistant Candidaand Cryptococcusstrains to commercial antifungals has caught the attention of clinicians and researchers to medicinal plant products (commonly referred as phytochemicals). The use of phytochemicals with greater antifungal potential and different mechanisms of action may be useful in reducing the phenomenon of resistance. Lately, they have become a significant alternative for discovery of commercially viable, economically cheaper, and safe phytomedicines.

5. Medicinal plants as a source of antifungal agents

Global Action Fund for Fungal Infections (GAFFI), an international organization working to reduce infections and deaths associated with fungi, has reported that approximately 300 million people in the world suffer from a serious fungal infection every year and that among them over 1.35 million deaths are registered [90].

Despite the introduction of new and novel antifungal drugs, their production and impact are slow, and the development of antifungal resistance has forced the attention of researchers toward herbal products, mainly phytochemicals, in search of development of safe and economically viable antifungals.

Populations around the world have used folk medicine as an alternative therapy for various disorders. Currently, many species have been extensively studied in an attempt to discover new biologically active compounds with novel structures and mechanism of action for the development of new drugs.

Medicinal plants are commonly preferred because of their wide level of functional chemical groups with comparatively poor toxic substances, low-cost extracts, fewer side effects, and easy accessibility to people. Various bioactive compounds have been abundantly found such as phytochemicals.

Leaves, as well as the seeds and fruits of plants, have higher levels of phenolic compounds. The concentration of these compounds also depends on the nature of the chemical used as solvent in the extraction process as well as on the growth and storage conditions [91].

The biological activity of plant products has been evaluated against fungi. The ethanol extract, Lonicera japonicaaerial parts, a medicinal plant of folk medicine of China that used to treat some diseases, showed a very strong antimicrobial activity against Candidaspecies and potent wound healing capacity [92]. Methanolic extract of Lannea welwitschiileaves was antimicrobial against clinical yeasts. A preliminary phytochemical screening of extracts revealed tannins, flavonoids, alkaloids, and glycosides as compounds [93]. Pyrostegia venustacrude flower extracts, fractions, and pure compounds showed an effective broad spectrum antifungal activity [94].

An extract of Piper betleleaves inhibited the growth of Candidaspecies [95], and four different extracts of Strychnos spinosashowed anti-Candidaactivity [96]. Hydro-methanolic extracts of leaves from Juglans regiaand Eucalyptus globulusand methanol extract of Cynomorium coccineumdemonstrated excellent antimycotic property against Candidastrains [91, 97]. Akroum [98] showed antifungal activity in an acetylic extract of Vicia fabaagainst C. albicansin vitro and reduced mortality rates in Candida-infected mice that were treated with the extract.

Berberine, a protoberberine-type isoquinoline alkaloid isolated from the roots, rhizomes, and stem bark of natural herbs, such as Berberis aquifolium, Berberis vulgaris, Berberis aristata, Hydrastis canadensis, Phellodendron amurense, Coptis chinensis,and Tinospora cordifolia, was described as powerful reducer of the viability of in vitro biofilms formed by fluconazole-resistant Candida tropicaliscells [99].

Ethanolic and aqueous extracts from different plants from Brazilian Cerrado commonly used in folk medicine such as Eugenia dysentericaand Pouteria ramiflorawere promising against C. tropicalis, C. famata, C. krusei, C. guilliermondii, and C. parapsilosis. A phytochemical screening of active extracts from these plants disclosed as main components flavonoids and catechins [100]. Crude extract and fractions (n-butanolic and ethyl acetate ones) from Terminalia catappaleaves showed antifungal properties against Candidaspp.; hydrolysable tannins (punicalin, punicalagin), gallic acid (GA), and flavonoid C-glycosides were the active components found in butanolic fraction [101].

Bottari et al. [102] determined the antimicrobial activity of the aqueous and ethanolic leaf extracts of Carya illinoensis. Both extracts had MIC values against seven Candidareference strains between 25 and 6.25 mg/mL. Phenolic acids (gallic acid and ellagic acid), flavonoids (rutin), and tannins (catechins and epicatechins) were likely responsible, in part, for the activity against Candidastrains. Further, the extracts inhibited the production of C. albicansgerm tubes.

6. Conclusions

The increase in Candidaand Cryptococcusinfections is alarming leading to high rates of morbidity and mortality worldwide. Concomitantly with the increase in fungal infections, species emerged, and the resistance phenomenon increased so that the available antifungal arsenal becomes irrelevant in the face of the problem. In addition, there are limitations manifested by some antifungal agents such as fungistatic character, severe toxicity, and renal dysfunction. Therefore, it is crucial to develop new drugs as alternative therapies that are potentially active against Cryptococcusand Candida. Plants are considered abundant and safe sources of phytochemicals endowed with many biological activities. Several polyphenols have been isolated and studied in relation to their anti-yeast and anti-virulence activities and may be useful in obtaining promising, efficient, and cost-effective drugs for the inhibition of Candidaand Cryptococcusinfections. Many phytosubstances are extremely effective in combination therapy with traditional or other phytochemicals, which can be further exploited to lead to novel drug therapies against recalcitrant infections.

Acknowledgments

Authors thank Ceuma University for their contribution to the work.

Conflict of interest

The authors declare no competing interests.