Candida glabrata: Adopting pathogenicity and resistant mechanisms for survival

Candida glabrata is a yeast of increasing medical relevance, particularly in critically ill patients. It is the second most isolated Candida species associated with invasive candidiasis (IC) behind C. albicans. The attributed higher incidence is primarily due to an increase in the acquired immunodeficiency syndrome (AIDS) population, cancer, and diabetes patients. The elderly population and the frequent use of indwelling medical devices are also predisposing factors. The work aimed to review various virulence factors that facilitate the survival of pathogenic C. glabrata in IC. The available published research articles related to the pathogenicity of C. glabrata were retrieved and reviewed from four credible databases, mainly Google Scholar, ScienceDirect, PubMed, and Scopus. The articles highlighted many virulence factors associated with pathogenicity in C. glabrata, including adherence to a susceptible host surface, evading host defences, and producing hydrolytic enzymes (e.g., phospholipases, proteases, and haemolysins). The factors facilitate infection initiation. Other virulent factors include iron regulation and genetic mutations. Accordingly, biofilm production, tolerance to high-stress environments, and development of resistance to the antifungal drug, notably to fluconazole and other azole derivatives, were reported. The review provided evident pathogenic mechanisms and antifungal resistance associated with C. glabrata in ensuring its sustenance and survival.


Introduction
Invasive candidiasis (IC) is a clinical condition that is not associated with a single Candida species. Each Candida species holds unique characteristics comparative to invasive potential, virulence, and antifungal susceptibility pattern [1]. Invasive candidiasis causes substantial morbidity and mortality of approximately 40% -60%, perhaps due to the inherent low susceptibility of C. glabrata to the most commonly used azoles [2]. It is an infection with many clinical manifestations that potentially affect any organ. Invasive candidiasis is associated with nosocomial bloodstream infections (BSIs) in tertiary health facilities worldwide [3]. Candida species also pose a significant threat to patients in the immune system. The treatment approach for C. glabrata infections is challenging due to the limited knowledge of its pathogenicity, the reduced antifungal drug susceptibility, and the limited choices of effective antifungal agents Yu et al. [28]. Other virulent factors include biofilm formation associated with adherence to host epithelial surfaces and hospital medical devices [8]. Despite the less destructive nature of C. glabrata in comparison to C. albicans, a high mortality rate associated with C. glabrata and rapidity of disease spread would argue otherwise [29]. Candida glabrata seems to have evolved a strategy based on secrecy, evasion, and persistence without causing severe damage in murine models [30]. Skrzypek et al. [31] also believed that C. glabrata exhibits a unique escape mechanism of the immune system and subsequently survives cellular engulfment and can resist antifungal treatment. This review summarises current information on pathogenic, virulence, and drug resistance mechanisms associated with C. glabrata ( Figure   1).

Enzyme secretion
Secretion of hydrolytic enzymes is a significant determinant of pathogenicity in C.
In addition to enzyme secretion, it is thought that host cell penetration occurs via endocytosis induction [15]. The study conducted by Nahas et al. [35] reported three gene families of phosphatases (CgPMU1-3) encoding phosphatase enzymes of different specificity. Accordingly, CgPMU2 was identified as analogous to the PHO5 gene found in S. cerevisiae. It serves as the phosphate-starvation inducible acid phosphatase gene. relevance of microbial biofilms in human diseases, with an estimated 65% of all human infections of biofilm aetiology [42]. Biofilm formation is another pathogenic mechanism observed in C. albicans with high biofilm mass, densely packed with pseudohyphae.
However, C. glabrata produces sparse biofilm (less weight) with yeast cells. It is an essential pathogenic mechanism for its survival [43] (Figure 2).

Figure 2. Biofilm formation in a blood vessel and dissemination into multiple organs
Candidiasis associated with biofilm production has clinical implications. The formation of biofilm on medical devices can cause the device's failure. In addition, it can serve as a point source for further infections [44]. Fungal biofilms show properties different from planktonic (free-living) populations, including a higher antifungal resistance level [42]. The resistance development due to biofilm is complex and multifactorial, among the assumed mechanisms include (i) the elevated cellular density within the biofilm, (ii) the exopolymeric protective effect of the biofilm, (iii) differential up-regulation of genes linked to resistance and those encoding efflux pumps, and (iv) the presence of a subpopulation within the biofilm community.
The emergence of echinocandins and liposomal formulations of amphotericin B drugs show increasing efficacy against fungal biofilms [13,42]. Recent evidence indicates that most IC caused by C. glabrata is associated with biofilm growth [45]. Candida glabrata biofilms show antifungal resistance characterised by a compact, dense structure of yeast cells. The cells become nested in an extracellular matrix composed of high proteins and carbohydrates β-1,3 glucan contents [10]. Several genes are associated with biofilm formation in C. glabrata. For example, the EPA6 gene encodes adhesin regulated by multiple factors, including the CgYak1p kinase, subtelomeric silencing, chromatin remodelling Swi/Snf complex components, and the transcription factor CgCst6, which plays an important role.  [46]. According to da Silva Dantas et al. [47], low-level colonisation of epithelial surfaces may create a mature surface biofilm. Nevertheless, it is unclear how such a Candida biofilm structure may affect mucosal surface infection and host immunity. However, such mature biofilms formed with dense biomass would severely challenge the cellular immune system to contain and clear from the host system.
According to Jeffery-Smith et al. [48], C. auris biofilms demonstrated higher biomass than C. glabrata and reduced biomass compared with C. albicans. Resistance to drug sequestration in the biofilm matrix also reduces drug efficacy. It lowers the exposure of C.
glabrata to the drug, facilitating the selection of acquired resistance [46]. Al-Dhaheri and Douglas [49] found that the presence of persister cells in biofilms is mainly responsible for biofilm resistance. Accordingly, C. krusei and C. parapsilosis appear to possess persister cells that may become tolerant to drugs. In contrast, biofilms of C. glabrata and C. tropicalis do not possess such persister cells [15,46].

Presence of a stable cell wall
The fungal cell wall is the primary contact site for host-pathogen interaction [50]. The fungal cell wall consists of complex biomolecule structures made up of polysaccharides, proteins, and lipids. The composition is dynamic, responding to changes in the local environment [28,51]. Candida cell wall consists of an inner layer of polysaccharides (chitin, 1,3-β-glucans, and 1,6-β-glucans). An outer layer of proteins glycosylated with mannan constitutes the pathogen-associated molecular patterns (PAMPs). The PAMPs are recognised by specific innate immune receptors known as pathogen recognition receptors (PRRs) [23]. The cell wall is dynamic and necessary to maintain the osmotic pressure exertion and morphology during vegetative growth. Other environmentally induced developmental changes such as sporulation, sexual reproduction, or pseudohyphal growth are often necessary for survival and growth. The cell wall is vital as a target for antifungal agents because of species specificity [52]. The fungal cell wall comprises three significant polysaccharides: glucans, mannoproteins, and chitin [53]. Moreover, the findings of Srivastava et al. [54] showed that the cysteine-rich common in fungal extracellular membranes (CFEM) domain-harbouring cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3. They are vital for the maintenance of intracellular iron content, adherence to epithelial cells, and virulence.
During fungal growth, the cell wall expansion causes permanent remodelling of the polysaccharide network, consisting of mannans, β-glucans, and chitin. Chitin is a homopolymer of β1,4-N-acetylglucosamine (GlcNAc). Chitin is essential for fungal biological functions, including cell division, septa formation, hyphal growth, and virulence [50]. The chitin synthases enzyme carries out chitin synthesis in C. glabrata.
Deregulation of chitin biosynthesis is a potential mechanism of virulence and resistance to antifungal therapy-the presence of drugs, such as echinocandin, results in the corresponding increase in chitin synthesis. The chitin maintains the cell wall's structural integrity, as chitin replaces β-1,3 glucan. High chitin content restricts the penetration of the drug through the cell wall [55]. Candida glabrata presents strange features related to cell wall organisation, such as overexpression of genes encoding adhesion-like GPIanchored proteins or the implication of GPI-anchored aspartyl proteases (yapsins) in the infection process. These features indicate key virulence factors, with multiple roles in the high tolerance to azole drugs, adhesion to susceptible host cells, or survival inside macrophages [56].
Genetic mutations confer susceptibility to patients against Candida species [23].
Candida glabrata has well-characterised genes, including ACE2 (CgACE2), a transcription factor that serves as a negative regulator of virulence. It was studied in an invasive infection of an immunocompromised mice model. The evolved (Evo) strain is another hyper-virulent C. glabrata strain with a single nucleotide mutation in the chitin synthase gene CHS2. Both mutants have enhanced virulence. Moreover, they stimulate inflammatory response factors, such as tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Thus, the ace2 mutant and Evo strain exhibit a clumpy pseudohyphalike structure [28]. Other strains with enhanced virulence characters include a strain with the PDR1 gain-of-function mutation, a strain with mitochondrial dysfunction, and the anp1 and mnn2 glycosylation mutants [28].

Novel hybrid iron regulation and acquisition strategies
Candida glabrata requires iron as an essential micronutrient for its growth during infection. Thus, it is necessary to strategise the mechanism for its acquisition for disease establishment [57]. Among the known iron uptake mechanisms in fungi include siderophore-interceded uptake of Fe 3+ , reductive iron procurement, and haemoglobin/haem uptake. All these frameworks are operational in C. glabrata except for the receptor-interceded haem uptake [58]. The underscore tight regulation of all processes involving iron in the organism, including uptake, distribution, utilisation, and storage.
Candida glabrata has high-affinity iron uptake mechanisms as critical virulence determinants.
Hosts' fundamental approach uses "nutritional immunity" to limit the iron required by invading pathogenic microorganisms, like in humans, available iron seized by various carriers and storage proteins, including haemoglobin, transferrin, and ferritin. They virtually deprive the available iron system, leaving no option for invading organisms. It thus exploits other iron source mechanisms (reductive, non-reductive and haemoglobinbound iron acquisition and degradation) [54]. Iron is usually incorporated into haeme or bound iron-sulfur, acting as a cofactor in many vital processes. These processes include the tricarboxylic acid cycle (TCA), DNA replication, mitochondrial respiration, and detoxification of reactive oxygen species (ROS) [59]. Iron effectively works due to its redox potentiality to switch between the two states as ferric iron (Fe 3+ ) and ferrous iron (Fe 2+ ).
Both ionic states have different effects on pathogenic microorganisms. For instance, Fe 3+ is poorly soluble in alkaline conditions, and Fe 2+ becomes toxic by promoting ROS production via the Fenton reaction [60]. According to the findings of Srivastava et al. [54] that the high-affinity reductive iron uptake system is necessary for metabolism in the presence of alternate carbon sources and for growth under both in vitro and in vivo ironlimiting conditions. The phenotypic, biochemical, and molecular analyses of 13 C. glabrata strains deleted for proteins [Cth1, Cth2 and common in fungal extracellular membranes (CFEM) domain-containing structural proteins CgCcw14, CgMam3 and putative haemolysin] confirmed that these proteins are potentially implicated in iron metabolism.
While Saccharomyces cerevisiae is a non-pathogenic yeast belonging to whole-genome duplication clade (WGD), having significant similarities with pathogenic C. glabrata [2]. It is poorly understood whether the different pathogenic clades, including CTG, may use common infection strategies or use lineage-specific mechanisms, or both combinations for pathogenicity [2,57]. C. glabrata combines the iron regulation network properties of both pathogenic and non-pathogenic fungi (S. cerevisiae). Candida glabrata, like S. cerevisiae, uses the Aft1 gene as the primary positive regulator during the sub-optimal iron condition. At the same time, Cth2 degrades mRNAs encoding iron-requiring enzymes. However, it contrasts with S. cerevisiae in that it requires Sef1 ortholog for total growth under ironlimited conditions. The iron homeostasis mechanisms in C. glabrata is still unknown.
Candida glabrata showed host-specific iron acquisition mechanisms by utilising siderophores and haemoglobin as a source of iron and haemolysin. It also uses cell wall structural protein to maintain iron homoeostasis [54].

Adaptation to various environmental conditions
Candida species can quickly adapt to host environmental changes as commensal pathogens even under nutrients bioavailability restriction [61]. Candida species use different nutrients available in the vast host niche. The Candida pathogens possess a high degree of metabolic flexibility because of the adaptive metabolic mechanisms necessary for significant nutrient acquisition [62]. Fungal pathogens require the adaptation to different host immune defence mechanisms and environmental stresses. Transcription factors Msn2p and Msn4p are crucial for resistance to various stresses in C. glabrata [63].
Stress conditions include disturbed cellular integrity, osmostress, elevated temperature, and the presence of antifungal drugs [25].
Temperature is a critical environmental variable that influences an organism's physiology for a certain period within a particular space [64]. Temperature and ambient pH are significant elements required for the optimal growth of Candida species. The ability to withstand a wide range of temperature and pH are regarded as virulence factors used by Candida species [65]. Candida glabrata grows optimally at 37°C and therefore thrives glabrata virulence are switched on only at 37°C [67].
Flexibility in carbon metabolism is critical for the survival, propagation, and pathogenicity of many human fungal pathogens [62]. (CgASG1, CAGL0G08844g) deletion resulted in increased tolerance to salt stress [63].
Active pH modulation is one likely fungal approach to change the pH of the phagosome.
Candida glabrata makes its extracellular environment alkaline when grown on amino acids as the sole carbon source in vitro. Mutant C. glabrata that lacks fungal mannosyltransferases resulted in strictly reduced alkalinisation in vitro. The condition subjects C. glabrata to acidified phagosomal activity [24]. Proteomic analysis of the pH response showed that C. glabrata observes low pH as less stressful than high pH [63]. The low acidic environment of the vaginal tract (pH ~4-4.5) contributes to the increased resilience to azoles against C. glabrata and C. albicans. Thus, this demonstrates the decreased efficacy of azole drugs in vitro at acidic pHs [70].
During phagocytosis, the internalised microbes become lysed in lysosomes -a specialised compartment in which oxidative and non-oxidative mechanisms kill and degrade the internalised microbes [24]. Candida glabrata lacks hyphal formation and phagosomal extrusions to escape the phagocytic cells attack contrary to C. albicans and Cryptococcus neoformans. The less aggressive mechanism helps in an autophagy process by mobilising its intracellular resources for metabolism and survival during prolonged starvation [71,72].. Evidence suggests that growth in the presence of alternative carbon sources affects the phagocytosis of Candida species. C. glabrata has high-stress resistance. Perhaps its enhanced sustenance during starvation allows it to survive and replicate inside the immune system cells (macrophages). The C. glabrata are engulfed during bloodstream circulation [15,20]. The findings of Chew et al. [73] revealed that the ICL1 gene helps to promote the growth and prolonged survival of C. glabrata during macrophage engulfment. Thus, C. glabrata shows a unique immune system evasion mechanism and survives after cellular engulfment despite the antifungal presence.
Perhaps through concealment within intracellular niches [24,31]. Lactate-grown C. glabrata cells, for example, resist killing by macrophages and have developed distinct tactics for intracellular survival killing and escaping phagocytosis [44]. Following extended division, the macrophages rupture, and yeast cells escape and disseminate into the blood system for further spread [15] (Figure 3).

Figure 3. Candida glabrata replication inside the macrophage cells before organ dissemination
Successful clearance of pathogens depends on phagocytes' rapid actions of the innate immune system, such as macrophages, dendritic cells, and neutrophils [24]. The primary factor aiding the persistence of C. glabrata is its less aggressive nature to stimulate the strong reaction of the host immune system [27]. Because of the low host cell damage, C.
glabrata cells elicit a cytokine profile significantly different from that of C. albicans.
Consequently, C. glabrata is associated with mononuclear cell proliferation (macrophages).
In contrast, neutrophil emergence becomes typical of C. albicans [9]. Despite the medical importance of C. glabrata, it is less lethal because it provokes a low inflammatory immune response. The systemic mouse infection models indicated that even at high inocula doses of intravenous infection [24]. Furthermore, the upregulation of Trx1p as a stress-response protein exerts defences to C. glabrata against oxidative stress [74]. Considering the role of dimorphism as a factor for pathogenicity in some Candida species, C. glabrata is exceptional; it does not germinate into hyphae yet is virulent [75].

Drug resistance mechanisms of Candida glabrata
The emergence of antifungal resistance becomes a problem in clinical medicine, significantly associated with Candida species. Knowledge of C. glabrata infection symptoms is essential because Candida species commonly share indices of suspicion of the disease. C. glabrata among the non-albicans Candida species can acquire drug resistance.
Moreover, it can develop secondary resistance to other available antifungal classes, resulting in poor treatment outcomes. It is a well-known fact that both C. krusei and some C. glabrata have intrinsic resistance to fluconazole. In such a situation, proper diagnosis is essential to justify appropriate treatment [76].
The incidence of candidemia caused by fluconazole-resistant strains and derivatives is high [61]. Azole drugs are among the four classes of antifungals commonly used in clinical practice to treat cancer, AIDS, patients on chemotherapy, and bone marrow transplant patients with fungal infections [77]. The most prevalent Candida species, C.
albicans, and C. glabrata differ significantly in response to antifungal therapy (Irinyi et al., 2016). Fluconazole is extensively prescribed and administered because of its availability for oral administration, has low toxicity, and less expensive. However, the extensive use of fluconazole has led to the increasing emergence of resistant isolates [79,80].
Candida glabrata infections are complicated to treat due to their inherent resistance to antifungals, especially against azoles [44]. Sardi et al. [45] viewed that C. glabrata has intrinsic antifungal resistance, especially to fluconazole. Arendrup and Patterson [46] argued that C. glabrata developed acquired resistance to antifungal drugs through Moreover, multi-drug resistance is a rare phenomenon in Candida species. Mutations in the MSH2 gene, encoding a DNA mismatch repair protein, occur in C. glabrata. Its effects have been found in clinical isolates to facilitate the selection of resistance to azoles, echinocandins, and polyenes in vitro [1]. On a general note, the published in vitro data have shown that deoxycholate amphotericin B (dAmB) and echinocandins such as caspofungin or micafungin demonstrated high activity against C. albicans and C. glabrata growing in biofilms settings [82].

Azole resistance
Azole drugs play a critical role in clinical practice, especially fluconazole, clotrimazole, and imidazoles [77]. Fluconazole is the frontline drug used for prophylaxis and treatment of many fungal infections [83]. The disease candidiasis has predisposing factors including organ and bone marrow transplant, prolonged chemotherapy, and AIDS [77]. The reported ability of C. glabrata to show resistance to fluconazole in clinical isolates indicates the need to improve the diagnostic approach. In addition, it promotes new antifungal therapy for easy management of such cases [39].
Candida glabrata possesses numerous resistance mechanisms to fluconazole, including fluctuation of gene regulation, genetic mutations, and cross-resistance among azole derivatives [12]. Yoo et al. [80] described the primary tools of azole resistance associated with Candida species, including mutations in the ERG11 gene and the proliferation of copy number of azole targets. Other mechanisms include blockage of the ergosterol biosynthesis pathway. Over-expression of genes coding some adenosine triphosphate (ATP)-binding cassette is fluconazole resistance mechanism of C. glabrata as observed in Iranian isolates. Resistance mechanisms are also associated with significant facilitator superfamily efflux pumps, leading to the increasing efflux of azole drugs.
Although numerous possible tools have been reported previously, the exact resistance mechanism is not entirely clear on azole resistance. Approximately more than 140 alterations in the ERG11 target gene have been described. Some alterations are exclusively found in azole-resistant isolates, whereas some are obtained in susceptible isolates [46].
The mechanism of action of azole is to target the cytochrome P450 enzyme sterol 14αdemethylase. The enzyme converts lanosterol to ergosterol as an essential structural component of the fungal cell membrane [84]. According to the findings of Gohar et al. [12] and Farahyar et al. oxide (CgSNQ2) and Candida glabrata Candida drug resistance 1 and 2 (CgCDR1 and CgCDR2) genes. More specifically, the free nitrogen atom of the azole ring binds an iron atom within the enzyme haem group. Thus, it prevents oxygen activation and causes demethylation of lanosterol that inhibits the ergosterol biosynthesis process. The inhibition is toxic methylated sterols accumulated in the fungal cellular membrane, and cell growth is arrested [86].
For instance, Song et al. [87] reported from South Korea that two of the five C. glabrata isolates tested were resistant to fluconazole. The five isolates were resistant to itraconazole.
Similarly, all the isolates were resistant to itraconazole. In a similar resistance pattern, Premamalini et al. [88] study conducted in Chennai, India, indicated that 2 (66.7%) C.

Echinocandins resistance
Echinocandins are recommended as first-line therapy for non-neutropenic patients associated with C. albicans and C. glabrata in suspected severe IC conditions [91].
Echinocandins were launched in the early 2000s and became the first-line treatment following the emergence of C. glabrata with reduced susceptibility to fluconazole Colombo et al. [92]. The emergence of resistant C. glabrata usually correlates with high azole and frequent echinocandin usage in hospitals or specific hospital wards [1]. Candida species strains resistant to first-line antifungals (such as fluconazole and echinocandins) are increasingly documented. Echinocandins exist according to the international guidelines in three available types (caspofungin, anidulafungin, and micafungin). They differ based on the route of metabolism, half-life, and safety [93]. among Candida strains remains low, with around 4% observed in C. glabrata and less than 1% in C. albicans [94] (Sasso et al., 2017). Intrinsic resistance to echinocandins is rare and primarily only observed in Candida parapsilosis [94]. However, Echinocandins resistance is an emerging scourge in Candida species, particularly C. glabrata [1]. In infections associated with C. glabrata or C. krusei, echinocandins offered preference over azoles. One major setback of echinocandins oral administration is the inadequate bioavailability, and therefore it is often given intravenously [95]. The frequent prescription and usage of echinocandins result in resistance development by decreasing the targeted Candida species
The mechanism of action of AmB is to bind to ergosterol in the plasma membrane resulting in leakage of cytoplasmic materials and cellular destruction [55,90]. The resistance to AmB is not commonly observed in Candida species [14]. showed better activity than micafungin in the treatment of both C. glabrata and C.
parapsilosis blood stream infections in 80-year old woman.

Conclusion and way forward
Alteration of host interaction factors facilitates opportunistic harmless commensal glabrata. Moreover, acquired and intrinsic resistance to fluconazole and rapid development of resistance to a few available antifungal drugs are of significant concern.
These issues prompt an urgent and development of a better diagnostic approach to detect and identify C. glabrata for effective and timely treatment and management.