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4 MDR inCandida: TheReal Storm
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C. albicans transcription factor Bcr1p. Curr Biol 15(12):1150–1155 Nobile CJ, Fox EP, Nett JE, Sorrells TR, Mitrovich QM, Hernday AD etal (2012) A recently
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P. Srivastava et al.
Surviving theStorm: How Candida Species Master Adaptation forPathogenesis
AishwaryaRana, NidhiGupta, ShumaizaAsif, andAnilThakur
Abstract
The complex interplay between hosts and microbes in fungal pathogenesis, espe-
cially within Candida infections, presents a signicant healthcare concern.
Candida species, highlighted as high-priority pathogens by the WHO, pose a
global threat exacerbated by escalating drug resistance. This comprehensive
chapter delves into Candida’s multifaceted strategies in negotiating the host
environment, emphasizing regulatory mechanisms, phenotypic switching, and
metabolic adaptations crucial for virulence.
Phenotypic switching, a pivotal transformation, enables Candida to adjust cellular forms in response to environmental cues, augmenting adhesion, tissue penetration, and virulence. Metabolic exibility emerges as a pivotal virulence factor, facilitating survival in diverse host niches marked by nutrient depletion and stress, extending the focus beyond C. albicans to encompass metabolic path­ways in other Candida species.
Adhesion, a critical infection step, relies on various adhesins—ALS family, HWP adhesin, HYR/IFF family, and EPA family—facilitating host cell attach­ment and establishing footholds within the host. Enzymes like hemolysins, can­didalysin, and lipases contribute to host surface interactions, inuencing colonization and invasion.
As Candida species evolve resistance to existing drugs, the imperative to comprehend molecular networks intensies to identify innovative therapeutic targets. Here, we explore these intricate mechanisms, providing insights into potential areas for disrupting fungal pathogenesis and addressing the growing
5
A. Rana · N. Gupta · S. Asif · A. Thakur (*) Laboratory of Protein Translation and Fungal Pathogenesis, Regional Centre for Biotechnology, Faridabad, Haryana, India e-mail: anil.thakur@rcb.res.in
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2024 S. Hameed, P. Vijayaraghavan (eds.), Recent Advances in Human Fungal Diseases, https://doi.org/10.1007/978-981-97-4909-6_5
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challenge of drug resistance. These ndings guide the development of inventive therapeutic strategies against Candida infections worldwide.
Keywords
Non-albicans Candida species · Pathogenesis · Drug resistance · Virulence fac­tors · Biolm · Secreted aspartyl protease · Candidalysin · Amino acid permease · Oligopeptide transporter

5.1 Introduction

Host-pathogen interaction describes the intriguing relationship between a host and its microbe, which is critical to fungal pathogenesis. Fungal infection by Candida species is emerged as a pressing concern within the healthcare sector, reecting the dynamic interplay between the human immune system and opportunistic pathogens. Candida species (spp.) are placed in critical priority and high groups by the WHO fungal pathogen priority list in 2022 (Fisher and Denning 2023). The emergence of drug resistance lately has imparted further credence to the potential threat posed by Candida globally. Candida infections pose a signicant threat to individuals with weakened immune systems, as this typical benign microorganism can transform into a formidable pathogen. According to the SENTRY Antifungal Surveillance program in 2016, reports suggest a decline in Candida albicans infections, whereas a sudden surge takes place in cases of non-albicans species such as Candida gla- brata and Candida auris (Pfaller etal. 2019). Fungal pathogens, in general, are armed with weapons ranging from adhesion and invasion to phenotype switching and metabolic adaptations to navigate the challenges the host environment presents. Survival in a dynamic host environment requires the fungi to alter their protein pool, efciently respond to stressors and administer morphogenetic changes. Central to these strategies are the regulatory mechanisms that control virulence (Staniszewska 2020).
One key element of this transformation is phenotypic switching, where Candida
spp. adapt its cellular forms in response to environmental cues. This switching involves a shift from a yeast form to a hyphal form, which enhances adhesion, tissue penetration, and virulence. Environmental factors that trigger phenotypic switching include temperature, pH, serum, nitrogen availability, glucose, and osmotic stress. By modulating their cellular form based on these cues, Candida spp. can dynami­cally adapt to their surroundings and ensure their persistence in the host. The most crucial step in Candida infections is adhesion to the host surface before pathogenic­ity ensues. Adhesins are the proteins involved in host cell attachment; several adhe­sion proteins include the ALS family, HWP adhesin, HYR/IFF family, and EPA family. These adhesins facilitate the binding of Candida to host cells and tissues, allowing the fungi to establish a foothold within the host. This adhesion is a critical step, as it determines the success of subsequent colonization and invasion, which are central to the pathogenicity of Candida infections. The persistence of a pathogen
5 Surviving the Storm: How Candida Species Master Adaptation…
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and its ability to cause infection is further inuenced by a repertoire of enzymes such as hemolysins (which regulate iron acquisition), candidalysin, lipases, and many more, which interact with host surfaces and damage them for colonization and invasion.
For survival and subsequent virulence, pathogens rely on metabolic exibility,
allowing the pathogens to thrive and infect diverse host niches characterized by nutrient depletion and stress. This adaptability aids in assimilating host nutrients and establishing infections a phenomenon not exclusive to C. albicans but extend­ing to another Candida spp.
These metabolic pathways play a crucial role in surviving under nutrient deple-
tion. To efciently sense and utilize amino acids, Candida employs extracellular sensing pathways, including nitrogen catabolite repression (NCR), the target of rapamycin (TOR), and the SPS-mediated amino acid sensing pathway.
As we unravel the intricacies of these molecular networks, the quest to develop
novel therapeutic targets becomes crucial as all the Candida spp. have become resistant to the drugs in use. With better insights into molecular mechanisms and pathways, we can develop novel, innovative strategies to disrupt fungal pathogene­sis with much better efciency and better control of the prevailing drug resistance paradigm. This chapter meticulously explores how these mechanisms and pathways that underscore the pathogenicity of Candida infections, thus highlighting the sig­nicant potential target areas for therapeutic strategies to impede the onslaught of infections globally.
5.2 Phenotypic Switching inCandida Species
Phenotypic switching is a strategy employed by organisms to adapt to their environ­ment by altering their different cellular forms. In colonized or infected hosts, this switching profoundly impacts the host-pathogen relationship. This phenomenon has profound implications, as it plays a pivotal role in determining the virulence and pathogenicity of these organisms. Among the fungal pathogens, Candida spp., responsible for a range of human infections, have drawn signicant attention due to their ability to undergo phenotypic switching.
Different morphological forms of pathogens become indispensable at various
stages of infection, contributing signicantly to adhesion, tissue penetration, and immune evasion. However, it is noteworthy that not all Candida spp. undergo mor­phogenesis, and in most cases, the propensity for morphological switching is cor­related with their virulence.
Like other fungi, Candida spp. employ a range of regulatory mechanisms to cope
with environmental changes, such as pH, temperature, and exposure to host serum. This adaptability bolsters their resilience and enhances genetic diversity, ensuring their survival in the host environment. Candida spp., for instance, employ two dis­tinct switching systems, morphological transitions (bud to hyphae) and white opaque switching in response to changing environmental conditions (Jain etal. 2008).
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5.2.1 Morphogenetic Switching (Yeast-Hyphal Transition) inCandida Species
Due to its morphological alterations, Candida spp. can easily transmogrify from a commensal to a pathogenic form. The yeast form prevails under typical conditions, colonizing tissue surfaces. However, exposure to host-associated cues can trigger the transition to the hyphal state. Hyphal forms of Candida are elongated, branched laments with a larger surface area, enhancing their adhesion and tissue-penetration capabilities.
The switch to hyphal formation is orchestrated by the hyphal-specic gene regu-
latory network, activated in response to environmental conditions (Witchley etal.
2019). Several hyphal-specic factors, including adhesins (Als), secreted proteases
(Sap), hyphal wall proteins (HWPs), and antioxidant proteins (Sod5), play a crucial role in this transition (Noble etal. 2017). Yue and colleagues demonstrated that la­mentation in C. auris can be induced by passing the fungal cells through a mam­malian host’s body. The cells recovered from the liver and kidney showed lamentous transition, whereas cells from the spleen, brain, and lung did not show any yeast-to­lament growth. In Candida spp., morphogenesis is signicantly inuenced by a multitude of environmental factors. Among these, several key determinants are out­lined as follows:
5.2.2 Role ofTemperature inMorphogenetic Switching inCandida Species
Temperature is a crucial element mainly involved in the yeast to hyphal transition. C. albicans assumes a singular, rounded yeast-like form at standard ambient tem­perature. However, an increase in temperature triggers a notable morphogenetic transition, resulting in the adoption of a lamentous structure. This transforma­tion is meticulously regulated by the heat shock protein Hsp90, operating through the cAMP/PKA pathway. Hsp90, in conjunction with the cochaperone Sgt1, mod­ulates the pathway by interacting with Cyr1, subsequently impacting the cAMP/ PKA signalling pathway (Fig.5.1). In instances of thermal stress, the pivotal role of this chaperone magnies signicantly, managing widespread protein misfolding.
Consequently, it relinquishes its repressive effect on Cyr1, thereby depressing the signalling pathway (Robbins and Cowen 2023). In C. auris, a nonheritable switch from a lament-competent (FC) to a lamentous form is temperature depen­dent. FC cells are single-celled yeast forms originating from lamentous cells, and their transition is favored under low temperatures (Fan etal. 2021). This transition is regulated by Hsp90, with its inhibition or depletion leading to upregulated cell surface genes similar to those found in C. albicans, emphasizing the role of Hsp90in morphogenesis (Kim etal. 2019). Similar to C. auris, wherein lament formation is favored at lower temperatures, it is also observed in Candida haemulonii (Deng etal. 2021).
5 Surviving the Storm: How Candida Species Master Adaptation…
Fig. 5.1 Factors inuencing yeast hyphae morphogenetic transition: (a) environmental factors such as temperature, CO2, N-acetyl glucosamine, nitrogen starvation, serum, carbon source, and peptidoglycan induce lamentation, whereas farnesol inhibits the yeast hyphal transition. (b) Transcription factors, namely, Efg1, Czf1, Cph1, and Cph2 promote lament growth and Tpk1, Tpk2, and Nrg1 prevent lamentation
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5.2.3 Other Environmental Factors Influencing Morphogenesis
inCandida Species
Apart from temperature, several other environmental factors signicantly inuence the yeast-hyphal transition in Candida spp.:
5.2.3.1 Serum
C. albicans typically coexist as a commensal in the gut environment, frequently encountering bacterial peptidoglycans (PGN) within the serum. Candida recog­nizes PGNs and muramyl dipeptides through the pathogen recognition receptor Cyr1, which possesses 14 leucine-rich repeats (LRR). These LRR domains enable the detection of PGNs and muramyl dipeptides, subsequently initiating morpho­genic responses by activating Ras1 within the cAMP/PKA signalling pathway (Xu etal. 2008) (Fig.5.1).
5.2.3.2 Low Nitrogen
When the nitrogen content in the surrounding medium is signicantly depleted, Candida spp. like C. albicans detect the presence of ammonium sulphate through the ammonium permease Mep2. This detection event initiates the activation of MAPK and cAMP signalling pathways. Consequently, these pathways trigger the expression of hyphal-specic genes through the action of Cph1 and Efg1 (Biswas and Morschhäuser 2005) (Fig.5.1). Similarly, in Candida glabrata, Ste12 protein is
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required for lamentation under nitrogen starved conditions. ste12 resulted in very few pseudohyphae (Calcagno etal. 2003).
5.2.3.3 CO
2
Carbon dioxide (CO2), present in higher concentrations in the body, is another fac­tor contributing to morphogenesis. In C. albicans, CO2 is detected by carbonic anhydrase Nce3, which directly activates the adenylyl cyclase Cyr1 and cAMP/ PKA signalling (Klengel etal. 2005).
5.2.3.4 Osmotic Stress andCell Wall Damage
In response to osmotic stress or cell wall damage, the MAPK signalling pathway is activated. The disturbance is detected by a protein complex consisting of three pro­teins: Sho1, Opy2, and Msb2. This complex interacts with Cdc42 and Cst20, ulti­mately triggering the phosphorylation of Cek1 which activates Cph1 inducing lamentous growth. Mutants lacking these proteins exhibit sensitivity to cell wall­damaging agents like Congo Red, zymolyase, and tunicamycin, underscoring their involvement in cell wall formation. Interestingly, only sho1∆/∆ displayed sensitiv­ity to osmotic stress among these mutants, emphasizing its specic role in osmotic stress tolerance (Herrero de Dios etal. 2013).
5.2.3.5 Carbon Source
The yeast-to-hypha transition of C. albicans can be triggered by specic nutrient sources. The glucose-sensing G-protein-coupled receptor Gpr1 is crucial in detect­ing glucose in the medium and inducing lamentation (Fig.5.1).
This glucose sensing via Gpr1 happens in the presence of methionine as wild­type cells induces lamentation as compared to gpr1/gpr1. However, the cells cease to transition into hyphae as the glucose concentration increases to 2%. This G protein-coupled receptor (Gpcr) also senses other sugars such as fructose, maltose, sucrose, and galactose, contributing to its regulatory role in morphogenesis (Maidan et al. 2005) (Fig.5.1). Similarly, in Candida haemulonii, when cells from YPD phloxine B plates were replated onto YPG (yeast peptone glycerol) agar, lamenta­tion was induced due to using a different carbon source (Deng etal. 2021).
5.2.3.6 pH
The pH of the environment is a crucial factor inuencing the yeast hyphae morpho­genetic transition in C. albicans. It notably contributes to the yeast-hyphae morpho­genetic transition. C. albicans, functioning as a commensal, colonizes various sites with varying pH levels, ranging from the stomach’s acidic environment to the gut’s alkaline pH.This transition is governed by the Rim101 pathway, activated by its upstream elements Rim8 and Rim21. Rim21 senses pH shifts and activates Rim8, which, in turn, triggers the Rim101 pathway, initiating pH-dependent responses (Bensen etal. 2004). However, the transcriptional regulator NRG1 opposes Rim101, serving as a negative regulator of the yeast-hyphae transition (Murad etal. 2001). Interestingly, contrast to above, the transition from yeast to hyphae occurs indepen­dently of the Rim101 pathway within macrophages, suggesting that pH changes do not inuence morphogenesis in this particular context (Wilson and Lorenz 2023). Mechanism of action is discussed in Sect. 5.8.
5 Surviving the Storm: How Candida Species Master Adaptation…
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5.2.3.7 N-acetylglucosamine (GlcNAc)
N-acetylglucosamine (GlcNAc) is a major structural component of the mucosa of the GI tract, PGN, fungal chitin, and extracellular matrix (ECM) of human cells. GlcNAc is intracellularly present and can also be uptaken externally from the envi­ronment via the Ngt1 receptor. In Candida albicans, GlcNAc can induce lamenta­tion by a catabolic pathway or cAMP/PKA signalling (Konopka 2012). During the catabolic pathway of GlcNAc, Nag1 deaminase converts glucosamine-6-PO4 to fructose-6-PO4, which releases NH3, which raises the extracellular pH and promotes hyphal development (Min etal. 2019). In the cAMP/PKA pathway, RAS activates Cyr1, leading to increased cAMP levels. Elevated cAMP binds to PKA, releasing Bcy1 from PKA catalytic subunits Tpk1 and Tpk2. Tpk1 and Tpk2 then phosphory­late target proteins, like Efg1, promoting lamentation (Cloutier et al. 2003) (Fig.5.1). Interestingly, in Candida haemulonii, TEC1, a transcription factor, works independently of the cAMP/PKA pathway and induces lamentation even in the
cyr/cyr and ras1/ras1 mutant. However, GlcNAc inhibits hyphal development in Candida tropicalis (Xie et al. 2012), and interestingly, the catabolic genes for GlcNAc are missing from S. cerevisiae and Candida glabrata.
5.2.3.8 Quorum Sensing Molecule
Farnesol, a quorum sensing molecule, uniquely regulates hyphal development in C. albicans, a key virulence factor. It operates through two mechanisms: cAMP/ PKA dependent and cAMP/PKA independent. Endogenously produced in response to cell density, farnesol acts as a negative regulator of morphogenesis by alleviating Nrg1 repression, inhibiting yeast to hyphae transition. In the cAMP/PKA pathway, farnesol inhibits Cyr1, adenylyl cyclase, downregulating Nrg1 expression, and impeding cAMP/PKA signalling (Fig. 5.1). Additionally, farnesol triggers Cup9 degradation through Ubr1 regulation, releasing the inhibition of Sok1 and dere­pressing Nrg1 independently of the cAMP/PKA pathway, preventing yeast to hyphae formation (Lu etal. 2014; Cao etal. 2006). Antifungal drugs, such as azoles, echinocandins, and polyenes, have been found to impact the yeast-hyphal transition in C. albicans. Azoles inhibit yeast hyphal transition in C. albicans as treatment with azoles led to increased production of farnesol, an inhibitor of morphogenetic transition. Echinocandins and polyenes change the cell wall structure or cause dis­ruption, possibly preventing the transition (Hawser et al. 1996; Hornby and Nickerson 2004).
5.2.4 White Opaque Switching inCandida albicans:
APhenomenon ofPhenotypic Transformation
In the realm of Candida albicans, white opaque switching emerged in 1985, cour­tesy of Slutsky etal. showcasing its inheritability across seven distinct phenotypes (Slutsky etal. 1985).
Their pioneering work unveiled the inheritability of this intriguing phenomenon across seven distinct phenotypes. Two years later, in 1987, they documented a piv­otal event—the transition of the clinical strain WO-1 into two distinctive colonies: one large, at, and opaque, and the other bulged, white, and hemispherical. This
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event marked the onset of white opaque switching, a remarkable occurrence charac­terized by its random, stable, and high-frequency nature (Slutsky etal. 1987). The orchestration of white opaque switching is heavily inuenced by environmental fac­tors that signicantly regulate the transcriptional feedback loop (Morrow et al.
1989; Kolotila and Diamond 1990). Notably, these two phenotypic forms exhibit
diverse characteristics depending on their niches within the host. For instance, white cells exhibit higher virulence in intravenous infections, while opaque cells excel in skin colonization (Kvaal etal. 1997; 1999). The opaque cells showed attenuated virulence in oropharyngeal candidiasis due to the reduced expression of Als3, and they were unable to activate the epithelial cells EGFR (Phan etal. 2007). These forms signicantly differ in morphological appearance, virulence, and mating com­petence. The phenomenon of white opaque switching thus adds a captivating dimen­sion to the multifaceted world of Candida albicans, shedding light on the intricate mechanisms governing its phenotypic transformations.
5.2.5 MTL Locus Important forWhite Opaque Switching
In C. albicans, white opaque switching is regulated by the MTL (mating type like) locus. MTL locus exists in two allelic forms: MTLa and MTLα. MTL locus codes for four transcriptional regulators: a1, a2, α1, and α2. To facilitate mating, heterozy­gous a/α cells must undergo homozygosis (become a/a or α/α cells). Additionally, they need to transition from the mating incompetent white phenotype to the mating competent opaque phenotype, a switching process that adds complexity to the mat­ing mechanism (Miller and Johnson 2002). Candida glabrata is reported to have four distinct mating classes depending on the distribution of genes at three mating type loci, MTL1, MTL2, and MTL3. MTL1 codes for two genes, a and α, whereas
MTL2 and MTL3 encodes for a and α gene, respectively (Srikantha etal. 2003). MTL1 and MTL2 are transcriptionally active, and MTL3 undergoes telomeric silenc-
ing (Ramírez-Zavaleta etal. 2010).
The bistable switching mechanism in Candida albicans is orchestrated by the master regulator WOR1, inuenced by MTL a1-α2, which acts as a repressor and keeps cells in the white state. Deleting WOR1 impedes switching in homozygous a/a or α/α cells, although ectopic expression in a/α cells allows switching, albeit with reduced efciency (Huang etal. 2006). Transcriptional factors like Czf1 and WOR2 promote the opaque state, while Efg1 favors the white phenotype. Czf1 is crucial for switching, and WOR2 ensures stability in the opaque form. Notably, Efg1 deletion prompts a signicant shift from a white to an opaque state, especially in certain strains, underlining Efg1’s role as an upstream regulator of the a1-α2 repressor(Fig.5.2) (Zordan etal. 2007).
There are two switching systems in Candida glabrata: the core and irregular wrinkled switching system. The core switching system in Candida glabrata show­cases four colony variants, namely, dark brown (DB), light brown (LB), white (Wh), and very dark brown (vDB). These colors become apparent when the fungus grow on a CuSO4-containing agar medium. Additionally, Candida glabrata is known to spontaneously and reversibly switch to an irregular wrinkled phenotype, adding