Mitogen-activated protein kinases (MAPK) or Extracellular signal-regulated protein kinase (EPK) is involved in the activation of transcription factors which play important role in cell cycle regulation, hyphal development, stress responses and changes in glycosylation, etc (11). They are serine/threonine or tyrosine specific kinases which get phosphorylated upon activation. It is regulated by a coordinated sequence of the phosphorylation events (12). They function in signaling cascade which is activated by ligand binding site lying on the cell surface receptor which in turn activates many MAPK/EPK kinases that phospholyrates the substrates (13). Generally, it comprises of three modules involving the MAPKKK (MAP kinase kinase kinase) which becomes phosphorylated on the advent of upstream signaling, which in turn phosphorylates the MAPKK (MAP kinase kinase) and, passes on the signal to the MAPK. A final stimulus is again perceived by the transcription factors which gives a necessary response. The most well-studied MAP kinase mediated pathways are HOG pathway, CEK mediated pathway and cell wall integrity pathway (Table 1). All these pathways have well- defined roles in C. albicans commonly serving in the morphogenesis but at the same time are required under distinct conditions also to be activated by different stimuli (14, 15).

cAMP-dependent protein kinase or protein kinase A (PKA) pathway regulates vital processes in cells. This pathway involves cyclic AMP which acts as a key player produced by ATP. There is role of adenylate cyclase and phosphodiesterase enzymes. The dual regulation of this pathway occurs by adenylate cyclase membrane-bound Ras protein and by glucose via a G-protein-coupled receptor system (29). This protein consists of two catalytic units and two regulatory units. The pathway starts with the binding of cAMP to the regulatory subunit of PKA, in turn liberating the catalytic subunit, which in turn phosphorylates the target proteins involved in stress resistance, cell growth and intermediary metabolism (30). Intracellular cAMP is produced by enzyme CaCdc35p in C. albicans. The catalytic unit of PKA are Tpk1 and Tpk2 and transcription factors Efg1 and Flo8 are found in C. albicans (31, 32).

Cyclin dependent kinase pathway is involved in the regulation of cell cycle. Cyclin- dependent kinases (CDK) are key players in this pathway and systematically coordinate the cell cycle through intracellular and extracellular signals. CDK modulate the activity by associating with regulatory subunits like inhibitors, cyclins and assembly factors by various mechanisms like reversible protein phosphorylation, transcriptional regulation, subcellular localization and by selective proteolysis (33, 34). Cyclins are important for the cyclin kinase activity as they bind to cyclin kinases and leads to phosphorylation. CDKsare regulated at the transcriptional level and synthesized or degraded periodically during the cell cycle and degraded by ubiquitin-mediated proteolysis. In S. cerevisiae, only single Cdc28 unit is found to regulate the cell cycle cascade (35). The regulation of Cdc28 requires the action catalyzed by the CDK-activating kinase gene CAK1 involved in all phases of the cell cycle to degrade the cyclins and inhibitors (36). Cak1p belongs to the serine/threonine protein kinases family. Far1p and Sic1p are two well known Cdc28p inhibitors. Far1p is required for the arrest of pheromone-induced cell cycle and inhibits the kinase activities of Cln1p-Cdc28p. Sic1p acts as a specific inhibitor of cyclin-Cdc28p complexes. It also inhibits premature S-phase entry in G1 phase before bud initiation and duplication of spindle pole body (SPD). Along with that it also inhibits the Clb-Cdc28p activity which promotes mitotic exit (37).

TOR related protein kinases have been identified in S. cerevisiae as a target of the antifungal and immunosuppressive agent rapamycin. TOR is a part of the phosphoinositide 3-kinase-related protein kinase family, which was originally reported from yeast, S. cerevisiae (38). TOR signaling pathway, plays an important role in various crucial growth-related phenomenon of yeast. TOR complex 1 (TORC1) and TOR complex 2 (TORC2), are the two functionally distinct TOR complexes, which has been also reported in Schizosaccharomyces. pombe, C. albicans and Cryptococcus neoformans and mammals (39). Two Tor proteins known as Tor1 and Tor2 have been recognized that are involved in regulating cellular growth in response to nutritional changes. In S. cerevisiae, it is found that Tor signaling by FKBP12-rapamycin stimulates autophagy, induces expression of retrograde response (RTG), inhibits translation and represses ribosomal gene expression (40). Contrary, in C. albicans, only one homolog Tor1 protein is present responsible for responses to nutrient starvation.

Ras proteins are small G-binding proteins. Ras activation depends on the exchange of bound GDP for GTP and its deactivation through hydrolysis of GTP to GDP (41). Ras signaling enhances GPI-GnT activity. The activation of Ras signaling pathway is initiated by a conformational change that occurs when it binds to GTP. The inactivation occurs when it is GDP-bound conformation through the exchange of GDP into GTP via Ras-GTPase-activating factors (Ras-GAPs) and guanine nucleotide exchange factors (Ras-GEFs) (42). Of the two Ras proteins, in C. albicans only CaRas1 (a close homolog of S. cerevisiae Ras2) enhances GPI-GnT activity. It has been studied that it is the key player in activation of Ras signaling as well as hyphal morphogenesis (43).

They are two-component system based signaling pathway. It is composed of membrane-associated sensor protein (histidine kinase, HK) and a cytoplasmic, response regulator (RR) protein which typically acts as a transcription factor to adapt cells to the environmental signal (44). In C. albicans, there are three two-component histidine kinases called as CaSln1p, CaNik1p/Cos1p and Chk1p along with three response regulators know as SRR1 (Stress response regulator 1), SSK1 (Suppressor of sensor kinase) and SKN7 (Suppressor of Kre Null). They work as sensor proteins that can mediate signaling which in turn regulates hyphal development, morphogenesis, osmoadaptation, cell wall regulation and virulence (45).

The CCS pathway is the main calcium-signaling pathway that is involved in cell survival under various environmental variations. It mediates influx of calcium ions via Cch1-Mid1 (found in fungal cell membranes) channel for the activation of calcineurin and Crz1p (downstream transcription factor). Calcineurin or protein phosphatase 3 is a calcium and calmodulin dependent serine/threonine protein phosphatase. Calcineurin is a protein, highly associated with growth and hyphal elongation, cation homeostasis, cell wall synthesis and regulation of cell cycle (46). The study on CCS signaling has demonstrated that Crz1p is a major mediator of calcineurin-activated gene expression. (47). It has been also reported that phosphorylated form of Crz1p accumulates in the cytosol. When there is an increase in Ca²⁺ ions, the activated calcineurin dephosphorylate Crz1p, which leads to its nuclear localization (48). There is a presence of C2H2 zinc fingers motif in Crz1p that combines to a specific element present on the promoter in the genes called CDRE (calcineurin-dependent response element) that are required for Ca2+ and calcineurin-dependent gene expression (49). Calcineurin, Crz1p, along with Rim101 and Nrg1 (having a similar role to Crz1) are involved in the pathogenicity of Candida hence devising new drug targets (50).

It is a Phosphoinositide-dependent protein kinase-1 (PDK1) signaling pathway in which PDK1 is an important protein, which mediates PI3K-AKT pathway, making it a potential drug target (51). The structure of PDPK1 can be divided into two domains; the kinase or catalytic domain and the PH domain. The interaction of PDPK1 with phosphatidylinositol (3,4)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate is accomplished by PH domain. This interaction is important for localization and activation of some of membrane associated PDPK1's substrates including AKT (protein kinase B) (52). The activity of PDK1 is mainly regulated by allosteric and orthosteric modulators, by binding to the PIF (Interacting Fragment) pocket of the PDK1. Perhaps, there are number of advantages of binding of allosteric modulators to the less conserved PIF-pocket, like high specificity, minimum side effect and reduced toxicity (53). However, the binding of the allosteric inhibitors to the PIF-pocket is shown to cause the rearrangement of αB and αC helices in the inert conformation, which leads to the inhibition of PDK1 activity (54).

C. albicans being diploid is capable of undergoing morphogenetic transition from yeast to filamentous form under nutrient starvation conditions. There are three interchangeable morphological forms critically important for pathogenesis, known as yeast form, pseudohyphae and true hypha (55). The hyphal form is induced under certain conditions like nitrogen starvation, high temperature, neutral pH etc. (56). The hyphal formation is a multifactorial phenomenon governed by more than one signaling pathway comprising protein kinases (57). Dimorphism is crucial in serving many functions like adhesion to host cell, escape from phagocytic cells and invasion into another host cell (58). The hyphal development is activated by MAPK kinase pathway or cAMP protein kinase A pathway. Transcription factor, Cphlp which is homologue of Stel2p in S. cerevisiae is involved in the hyphal formation along with other transcription factors like Cst2Op, Hst7p (59). Under starving conditions, there is an inactivation of CPP1 which induces hyphal development. Cpp1p protein suppresses the hyphal development at ambient temperature. Another transcription factor, Efg1p is activated in cAMP protein kinase A cascade (60). The CAP1 gene mediates induction of yeast to hyphal transition and favors filamentous growth to give a response to serum or other environmental conditions. Ssk1p and Chk1p are essential for the yeast to hyphal transition (13).

HOG pathway represses the serum-induced yeast-to-hypha transition in C. albicans suggesting its role as another determinant of virulence. In C. albicans, TUP1 gene is responsible for hyphal development (61). It has been reported that repression of hyphal gene was dependent on Ssn6-Tup1 complex which in turn depends on HOG1-dependent expression or activation of a DNA binding protein (62). Mds3 which is a negative regulator of Tor signaling pathway involved in the yeast-hyphal transition. RAS2 gene acts upstream of signaling pathways which can lead to hyphal differentiation whereas Candida RAS1 gene is essential for hyphal differentiation in response to serum (63).

Cyclin dependent kinases are also involved in cytoskeleton polarization for morphogenesis in candidal cells. Cdk cdc28 binds temporarily to cyclins namely Ccn1 and Hgc1. The Cdc28-Ccn1 pair binds to septin complexes which in turn results in phosphorylation of Cdc11 which is a septin cytoskeleton protein required for hyphal development (36). Hsl1p-Swe1p-Cdc28p is essential for cell elongation in morphogenesis and imparts virulence to candidal cells. Cdc28 exerts inhibitory phosphorylation on Tyr-19 via Swe1p. It is also involved in the delay of the cell cycle in G2 phase and it is a ‘morphogenesis checkpoint pathway’ (64). Swe1p is also regulated negatively by Hsl1p protein kinase. Under low Cdc28p activity, Swe1p activates and leads to G2 delay which results in elongated bud formation. Relatively, Hsl1p activates when colocalized with the cytoskeletal proteins septins, around the neck. Cyclin namely Cdc11p or Cdc12p, a component of the septin complex, interacts directly with Hsl1p. It acts as a reliever of the autoinhibition which is imposed by the kinase inhibitory domain. Hsl1p localization results in inhibition of Swe1p which allows the cells to undergo mitosis (65). Similarly, the calcium signaling of a cell is required for various physiological activities in yeast, which includes morphogenesis, and virulence (66,67).

Tor protein kinase is involved in the cell to cell adhesion in C. albicans. It regulates transcriptional regulation of cell surface adhesins and results in cell adherence. The inhibition of Tor1 facilitates the increase in expression of genes encoding the adhesins Als1p, Als3p, Hwp1p and Ece1p by genes ALS1, ALS3, HWP1, and ECE1 respectively which in turn promotes biofilm formation (68, 69). Thus, in other words, it can be said that Tor1 inhibits cell adhesion by repression of adhesin genes expression. However, Tor1 regulates and governs cell adhesion by multiple mechanisms. It controls the expression of NRG1 and TUP1 by downregulating Efg1 and Bcr1 (69). Mds3 is involved in a variety of morphogenetic processes, including the yeast-hyphal transition, chlamydospore formation and biofilm formation (70). A study by Flanagan et al (71) has shown that TOR-Activating GTPases Gtr1 and Rhb1 regulate the biofilm formation and nutrient starvation. Ras1-Cyr1-PKA signaling is found to be important as it regulates the expression of adhesins, such as Als1p, and also control the activity of the key transcription factors EFG1, TEC1 and BCR (72). The calcium signaling is also required for adhesion, drug tolerance in C. albicans (66, 67). The PDK1 orthologs (Pkh kinases) of PDK pathway is also involved in the biofilm formation (73).

Under high oxidative potential, there is a formation of reactive oxygen species (ROS) such as superoxide anion (O₂^-), hydrogen peroxide (H₂O₂), hydroxyl radical (OH˙) which damages cellular components like DNA, lipids, and proteins (74). All these factors have toxic effect on yeast cell which leads to cell death. To cope up with oxidative stress, yeast cells have developed several antioxidant mechanisms (75). The activation of detoxifying enzymes such as superoxide dismutases and catalases, to prevent the ROS action, nonenzymatic scavenging substances are formed (76). In S. cerevisiae, Yap1 transcription factor is involved in the oxidative stress response. , A Yap1 homolog called as Cap1 in C. albicans is involved in mediating the oxidative stress resistance. HOG pathway plays an important role in an acute and adaptive response to oxidants (77). A study by Alonso-Monge et al (21)demonstrates that Hog 1 MAP kinase gene plays a crucial role in the oxidative stress response via different mechanisms from CAP1. The activation of Hog1 occurs due to phosphorylation in the presence of H₂O₂ or by increase in external osmolarity which depicts the specificity of the Hog1-mediated response to oxidative stress. This study broadens the role of MAP kinase in morphogenesis and osmoadaptation (21).

Chlamydospores are thick-walled cells which are formed under certain environmental conditions like defined nutritional media, low temperature and oxidative stress. Its formation is the distinctive morphological feature of C. albicans (78). They usually arise as elongated suspensor cells which are situated on pseudohyphae or hyphae. It has been reported that six genes namely ISW2, MDS3, RIM13, RIM101, SCH9, and SUV3 are required for efficient chlamydospore formation. The mutations in ISW2, SCH9 and SUV3 completely remove chlamydospore formation whereas mutations in RIM13, RIM101, and MDS3 delays normal chlamydospore formation (79). Efg1p is the protein which belongs to a group of bHLH proteins termed as APSES-proteins. Efg1p as a key regulator of C. albicans morphogenesiswhich influences yeast hyphae interconversion as well as chlamydospore formation (80). A study by Alonso-Monge et al (21) stated that HOG1 gene also takes part in chlamydospore formation.

The candidal cell wall is composed of a dynamic structure comprising of β-1,3-glucans which is covalently linked to β-1,6-glucans and chitin, along with matrix composed mainly of mannose-glycosylated proteins (81). It was reported that Cek1 plays a role in the maintenance and structure of candial cell wall by regulating the β-glucan exposure. It also maintains the cell wall glycoproteins by regulating the mannosylation status. Cek1 pathway was also found to be responsive against the antifungal agents like caspofungin and tunicamycin by masking the cell surface damage (25). Similarly, Cek1 pathway is also responsive to the glycosylation defects in the cell wall (82). Two component histidine related gene CHK1 is involved in the regulation of cell wall mannan and glucan biosynthesis (45). The activation of HOG1 pathway also triggers cell wall modification. A study by Alonso-Monge et al (21) suggested the role of PBS2 gene in maintaining cytoskeleton by β- (1,6)-glucan assembly. The activation of glycosylphosphatidylinositol anchor biosynthesis through the GPI-N-acetylglucosaminyl transferase (GPI-GnT) is accomplished by Ras signaling. (44).

Cell wall surface plays a crucial interface between host and pathogen. When the microbial cells are phagocytosed by host cells, they have highly conserved pathogen-associated molecular patterns, which are recognized by different pathogen recognition receptors (PRRs) which include C-type lectins and Toll-like receptors (TLRs) (83). The recognition of these PRRs further mediates microbial killing as well as antigen presentation which finally results in the production of proinflammatory cytokines. In C. albicans, the β- (1,3)-glucan on cell wall is recognized by C-type lectin receptor called as Dectin 1 which is expressed by myeloid cells (84). The coating of mannoprotein on cell surface helps the candidal cells to escape from the host cell recognition. The Cek1 MAPK kinase pathway favors the β- (1,3)-glucan masking and alters the binding of Dectin1 to the β-glucan which helps in evasion of immune response and recognition by host cell . It has been reported that Cek1p MAPK kinase cascade involves the Ste11-Hst7-Cek1 which aids in controlling the β (1,3)-glucan masking in C. albicans (85). Another study also demonstrated that exposure of α-1,2 and β-1,2-mannosides in the cell wall was mediated by Cek1-mediated MAP kinase pathway which modulates immune recognition. Additionally, even the calcium signaling is also required for maintaining cell wall integrity (66, 67).

Mating in yeast cells is simple unlike complex eukaryotes and sexes are designated as a and α (86). They communicate by signaling molecules called pheromones. The pheromones are detected by receptors on cell surfaces and communication is further disseminated and preceded by heterotrimeric G protein complex that activates mitogen-activated protein (MAP) kinase cascade module (87). In S. cerevisiae, two mating MAP kinases are involved called as Fus3 and Kss1. Contrary, in C. albicans, Cek1 and Cek2 MAP kinases are involved in the mating process. Mating in C. albicans relies on a reversible phenotypic switching between two states termed as ‘white’ and ‘opaque’. Wor1 transcription factor is the key player in regulating the white-opaque switch. Ras1-Cyr1-PKA pathway is known to have a role in the white-opaque switch (88).

Regulation of cell cycle is critical for the precise and appropriate manner as to ensure the sequential coordinated events to bring equal distribution of genetic material to daughter cells. In higher eukaryotes, the cell cycle is controlled by activation or deactivation of cyclin dependent kinases (CDKs) (89)

HOG pathway is considered to be a potential intracellular drug target with reduced toxicity to the host, due to the fact that, TCS is conserved in a broad number of living organisms, from microorganisms to plants, except mammals. The compounds, 4- and 5-substituted 1,2,3-triazoles, and pyridinylimidazole-based inhibitors, potentially have been demonstrated to specifically inhibit Hog1 in S. cerevisiae (90). Simultaneously, HOG pathway overactivation can also be lethal to a pathogen, as observed by the activity of fludioxonil (Figure 1). The study suggests that development of drugs which targets the negative regulators of the HOG pathway could provide better results in killing a pathogen (91).

Figure 1

Various inhibitors designed to target protein kinase pathways governing several functions in C. albicans. The color codes depict common functions shared by one or more pathways derived from previous literature.

The inhibition of CPH1 could prove as a beneficial drug target for Candida therapy, because of the abundance of functions contributing to its virulence. The interaction of ligand and receptor has a leading role in all cellular metabolic activities (92). The in silico work conducted by Jha et al (93), demonstrated that several ligands, like, VT1161, T-2307, 2-O-prenyl coumaric acid, 2-nitro-4 methyl-cinnamaldehyde, and 3,5-diprenyl-4 coumaric acid, profoundly blocks Cst20, Hst7, and Cek1. Such ligands can disrupt the cell wall organization, block biofilm formation and topography, due to the ability of the lead molecule to interact with the kinase by binding to the amino acid residues (93). Pepstatin A which is the known inhibitor of aspartic proteases inhibits the Msb2 shedding from the cell surface and phosphorylation of Cek1 (25).

One of the kinases, reported to be essential for the survival of Candida is CaMps1p, i.e., C. albicans monopolar spindle 1, which also shares some sequence homology with hMps1p, a human ortholog. A study conducted by Tsuda (94), demonstrated the recombination of a catalytic domain of CaMps1p labeled as LY83583, which is 6-anilino-5,8-quinolinedione. LY83583, also termed as a guanylate cyclase inhibitor, inhibited the activity of CaMps1p kinase, hence reducing the growth of Candida cells. Additionally, LY83583 was found to have inhibition which is specific for CaMps1p without affecting hMps1p activity, suggesting that LY83583 could be an ideal inhibitor for anticandidal therapy (94).

One of the PDK1 inhibitors is KP-372-1, a potential molecule that inhibits the PDK1 orthologs (Pkh kinases) is involved in the biofilm (73). Two other PDK1 inhibitors, OSU-03012 and UCN-01, are also known for their activity against biofilms of C. albicans (95). Therefore, the search for inhibitors of PIF-pocket of PDK1, could provide an ideal target for drug development.

It has been reported that two fungicides called as Iprodione and fludioxonil targets the histidine kinases by inhibiting CaN1K1 and COS1 transduction system governing osmotic transduction system which disturbs glycerol synthesis and inhibits the hyphal formation (96).

Rapamycin, a microbial product, specifically inhibits TORC1, while TORC2 is indirectly sensitive to rapamycin, and inhibits, C. albicans and C. neoformans (97, 98). Rapamycin forms a complex with a cytoplasmic protein, i.e., FK506-binding protein (FKBP) receptor and inhibits the TORC1 activity (99). The Mds3, Sit4, Tor1, and Tco89, members of the TOR pathway have crucial roles in morphogenesis, which in turn demonstrates their role in virulence of C. albicans (98, 100,). Further studies on Mds3, Sit4, Tor1, and Tco89 could help in developing a structural modification of rapamycin to treat C. albicans infections.

A modulator, sphingosine 1-phosphate (S1P) receptor, known as fingolimod hydrochloride (FTY720), acts on calcium signaling in Candida. It has a potential to suppress the growth of Candida by inducing a dose-dependent calcium increase, due to an influx of the molecule across the cytoplasmic membrane (101). Beauvericin is reported to enhance cytoplasmic calcium which plays an important role in the apoptosis, particularly Ca²⁺ overload of mitochondria (102). Cyclosporine A (CsA), tacrolimus (FK506) and amlodipine in combination with fluconazole, shows excellent synergistic effect against resistant C. albicans. Cyclosporine is known inhibitor of calcineurin and down regulates expression of ALS3, HWP1, CDR1, MDR, ERG11 genes (100, 50). Even natural compounds belonging to class of phenolic compounds, terpenoids, alkaloids were found to inhibit the calcineurin signaling pathway. A phenolic compound like perilyll alcohol and terpenoid like geraniol have been reported to disrupt the calcineurin signaling (103, 104).Therefore, specific inhibitors for the calcium signaling pathway which is involved in cell survival could provide a novel platform for the development of antifungal agents.

Various compounds have been studied as inhibitors for targeting cAMP pathway namely 2-Alkyl-4-hydroxyquinolines which is derived from marine Streptomyces sp. inhibiting candidal hyphal development (105). Another compound staurosporine targets cyclic AMP (cAMP)-dependent protein kinase A and adenylyl cyclase Cyr1 resulting in abrogated fungal drug resistance and morphogenesis (106). Phorbasin H which has been isolated from marine sponge Phorbas sp inhibits the yeast-to-hyphal transition in C. albicans (107). Mammalian adenylate cyclase inhibitor MDL-12330A has been tested and found to inhibit the cyclic AMP signaling pathway in C. albicans (108).

The inhibition of cell cycle was demonstrated by delaying the expression of cyclins. The compounds named as atropine and trifluoperazine were found to prolong the cell cycle by delaying the expression of G1 cyclins (109).