Fungi are the most common human pathogen that causes severe cutaneous infections to life-threatening systemic infections. In the United States, hospital born Candida is a major pathogen and cause frequent systemic infections in the patients. It also causes frequent oral infection in immunocompromised individuals. There are several virulent factors like an expression of adhesion molecules, secretion of hydrolytic enzymes, biofilm formation and micro-environment conditions like pH, nutrients which are responsible to convert non-virulent yeast form to virulent hyphal form (Polymorphism).

In major surgeries, severe cases of superficial and systemic infections of C. albicans are reported in immunocompromised patients¹. Several groups of antifungal drugs attack or disrupt cell membrane of C. albicans e.g. echinocandins, polyenes, and azoles etc.², which act as stress to the pathogen. The severity of stress condition depends upon dose and type of drug. Various receptors on the membrane of pathogenic fungi are involved to perceive this stress by eliciting various conserved signaling pathways like MAP kinase (MAPK) and calcineurin signal transduction pathways etc.^3,4

In drug resistant pathogenic fungi, several counter mechanisms exist to minimize or nullify the stress produced due to exposure of antifungal agents. Fungi static drugs that inhibit growth at the minimum growth-inhibitory concentration (MIC) did not kill fungal pathogen because of the drug tolerance phenomenon⁵, whereas fungicidal drugs that are been used at a higher concentration than MIC did not kill drug resistant pathogens. Prolonged exposure to different fungistatic drugs can cause genetic mutation in fungal cells that leads to various mechanisms for drug resistance like overexpression of membrane efflux pump proteins, mutations in drug target enzymes, and upregulation of stress-response genes etc.^6-8.

Extensive use of these anti-fungal drugs in the clinics has increased resistance and ineffectiveness against C. albicans infections^9,10. Therefore it is an emergency to come up with new therapeutic targets that can be explored for developing novel antifungal agents against C. albicans^10,11. Occurrences of Heat shock proteins (HSPs) are integral to almost every organism and are also highly diverse and widely distributed among fungal groups. It is been shown that under thermal stress, these proteins are getting upregulated. Expression of the heat shock proteins under heat stress is regulated by the heat shock transcription factor 1 (Hsf1)¹². In C. albicans, Hsps are important not only for the growth but also for developing infection and virulence^13-15. Stress conditions are inducive to promote the expression of heat shock proteins. Hsps interact with various cellular signaling pathways like MAPK, calcium-calcineurin, cell cycle control signaling, etc. regulating homeostasis and virulence in C. albicans^14,16,17. It is been reported that inhibiting or disrupting Hsps causes growth inhibition of C. albicans, which leads to reverse tolerance to available antifungal drugs. Studies have shown their involvement to confer resistance to antifungal agents by modulating HSPs associated signaling pathways in C. albicans and other pathogenic fungi.

A group of heat shock proteins known as small heat shock proteins (sHSPs) with a molecular weight ranging from 12 to 43 kDa gets upregulated upon non-thermal cues like oxidative and heavy metal stress^18,19. sHSPs, not only act as chaperones but are involved in the many biological vital functions like development, cytoskeletal organization, apoptosis, membrane lipid polymorphism, differentiation, autophagy, and infection recognition^20,21. These proteins can be used as therapeutic targets in the direction of the development of antifungal agents.

Heat shock proteins (HSPs) in Fungi
Hsps are integral and conserved among all living species, they respond to heat and non-thermal processes like oxidative stress and starvation that leads to stress to the organism²². These stressful conditions cause the loss of three dimensional structures of proteins and their aggregations leading to cell death. Hsps are molecular chaperones and expression of Hsps is a protective mechanism of the cell to combat these changes to ensure cell survival under the stress. There are two groups of Hsps, First is ATP-dependent high molecular weight protein Hsps, having four different families (Hsp104, Hsp90, Hsp70, and Hsp60) and a second ATP-independent family of proteins – Hsp12 and Hsp21 having low molecular weight proteins ranging between 12-42 kDa called Small Heat Shock Proteins (sHSPs) (Table 1)²³. Hsps are expressed in stress and non-stress conditions while sHsps are mostly expressed under stress conditions²⁴. It has been shown that most of the antifungal drugs are perceived as stress by the fungal cell²⁵. Thus sHSPs can be explored as the putative targets to combat drug resistance and the development of effective therapeutics to treat fungal infections.

Table (1):
Different types of heat shock proteins (HSPs).

Roles of Heat Shock Proteins (HSPs) in pathogenic fungi
High molecular weight HSPs are the major proteins that are involved in protecting cellular proteins by acting as chaperons. HSPs in pathogenic fungi are assisting in the process of making and maintaining three dimensional conformations of proteins during various stress and unfavorable conditions to counter the changes in the fungal cells. Expression and functions of different Hsps are variable with the kind of stress to the fungal pathogen.

Hsp104
Hsp104 was firstly reported in Saccharomyces cerevisiae (S. cerevisiae) and induced in elevated temperature^23,26,27. It also acts as a pro-survival protein under high temperature, suggesting its role as thermal tolerance²⁶. Mutant of hsp104Δ/Δ have shown morphological defects in hyphae²⁸. In C. albicans, Hsp104 has a greater role in biofilm formation and virulence²⁸. Cytosolic Hsp104 of C. albicans is not equivalent to human Hsp104, thus can be used as a promising antifungal target against C. albicans.

Hsp90
Hsp90 determines antifungal drug resistance in several diseases causing fungi like Candida albicans, A. fumigatus, and C. neoformans^29-31. Hsp90 is involved in several cellular functions like development, regulation, homeostasis, and drug resistance in C. albicans^16,17. A recent report showed that mutations in the region of Hsp90, responsible for post-translational modifications affect colony morphology³². Inhibitors of Hsp90 function have shown additive effects with fluconazole (FLC) against Fluconazole resistant Candida albicans^33,34. In yeast, the Hsp90 function is mediated by its C-terminal phosphorylation, S-nitrosylation, and acetylation^35-37. Several findings showed that Hsp90 contributes a vital function in confirming resistance to antimycotic drugs. Thus inhibiting Hsp90 function or development of pathogen specific histone deacetylase inhibitor can be the effective therapeutics to treat candidiasis.

Hsp70
Hsp70 is uniformly present from prokaryotes like bacteria to higher eukaryotes like mammals. Ssa1 and Ssa2 are two important members of the Hsp70 family on the cell surface of C. albicans^38,39. C. albicans Ssa1/1 mutant showed altered virulence in vitro as well as in vivo. Ssa1 and many antimicrobial peptides have Ssa2 as receptors that also have antifungal effects.Hsp70 alone or in combination with Hsp90 plays a major role in morphogenesis and dimorphism. A report have shown that the Hsp70 works with the heat shock transcription factor 1 (Hsf1) to regulate heat shock response in the yeast⁴⁰.

Hsp60
Hsp60 encodes a predictive mitochondrial heat shock protein, whose function is not known. A heterozygous mutant of Hsp60 (hsp60Δ/HSP60) has been shown to increase sensitivity with increasing temperatures. This is an indication of Hsp60 can be essential to overcome thermal stress⁴¹. Four fold increased expression of hsp60 in wild type yeast can resist oxidative stress in comparison to the mutant of hsp60. It was because iron/sulfur containing enzymes were protected from oxidative inactivation⁴². Another study showed an increase in the levels of Hsp60 under thermal stress and is important for differentiation, infection, and colonization⁴³. Expression of hsp60 mRNA level increased by 5.9-6.9-foldat 40°C in A. fumigatus and A. terreus⁴⁴. Hsp60 functions as an immunological trigger and play a role in fungal diseases in humans⁴⁵.

Role of Small Heat shock proteins (sHSPs) in C. albicans
Reports have shown that sHSPs of different species function as molecular chaperones and have conserved the a-crystallin domain (ACD). sHsps are cellular chaperones, involve in the proper folding of proteins during normal and extreme conditions. Under stress conditions, sHSPs contribute in refolding of partially unfolded proteins. They bind to the irreversible aggregation of denaturing proteins and are prevented in an ATP-independent fashion^46-49. Exposure to a variety of stresses like elevated temperature or oxidative stress causes unfolding of proteins and form early intermediates that can aggregates. These partially unfolding protein molecules are stabilized by the sHSPs^48,50-52. Early expression of sHSPs allows rescuing proteins that are getting unfolded under stressed conditions. Completely unfolded proteins and pre-aggregated proteins are not refolded by sHSPs. Thus, sHSPs protect unfolding proteins that otherwise become irreversible aggregates under stressful conditions to maintain homeostasis within the cell (Fig. 1).

Various studies have shown a link between HSPs and virulence potential of pathogenic microorganisms^18,53-58 including Hsp90 and Hsp70 in Candida albicans^33,59. Biofilm formation is an important virulence phenomenon in C. albicans infection and HSPs are required for its progression¹⁴. In C. albicans have reported three sHSPs i.e. Hsp10, Hsp12, and Hsp30/Hsp31.

Hsp12
Small heat shock protein (Hsp12) contributes to heat-shock resistance and hybridization of hsp12 mRNA analysis demonstrated its co-regulation with environmental pH and CO₂  in C. albicans^60-63. Intracellular amount of trehalose (Protectant against cell freezing) was minimized in C. albicans by TPS1 deletion. TPS1 deletion showed overexpression of Hsp12 in C. albicans. C. albicans have shown increased cell adhesion and decrease susceptibility to the quorum sensing molecule, farnesol^64,65. SSK1 mutant of C. albicans was susceptible to several oxidative agents like H₂O₂ and has also shown a high level of HSP12⁶⁶. This shows Hsp12p has a crucial role in combating various kinds of stresses. Another study showed that increased expression of Hsp12 leads to sensitivity to itraconazole, ketoconazole, and FLC in C. albicans⁶⁵.

Hsp21
Small heat shock protein 21 (Hsp21p) is another protein which plays an important role under various environmental stress for C. albicans^61,67-72. It was found that Hsp21 promotes virulence of C. albicans. Tolerance to heat and oxidative stress requires Hsp21 in C. albicans⁷³. Activation of the mitogen activated protein (MAP) kinase and normal filamentation require Hsp21. Hsp21 mutant and chemical inhibitors have shown a correlation in inhibiting germ tube formation and filamentation at the initial time point⁷³. In-vitro, analysis of hsp21 Δ/Δ mutant strain showed an inability to damage endothelial and oral epithelial cells⁷³. Growth of Hsp21 mutant was significantly reduced when treated with membrane perturbing agents that target ergosterol biosynthetic pathway i.e. terbinafine, caspofungin⁷³. At high temperatures, it is involved in maintaining homeostasis of glycerol, glycogen, and trehalose⁷³. Susceptibility to various antifungal drugs was seen in the deletion mutant of hsp21of C. albicans⁷⁴. Hsp21 can be an effective target to develop a treatment strategy for C. albicans infection.

Hsp10
Hsp10p is present in association with Hsp60p and assisting Hsp60 function⁷⁵. In-vivo Hsp60p and Hsp10p did not always act as a single functional unit in-vitro. Hsp10p is crucial for cell survival and acting as a co-chaperon to assist the folding of the proteins in the mitochondrial matrix⁷⁶. Functionally defective protein aggregation was seen in the mutant of Hsp10. It imparts in the sorting of the RieskeFeS protein during the transport from the matrix to the intermembrane space⁷⁷.

Hsp30/31
In C. albicans, oxidative stress upregulates Hsp30p besides other heat shock proteins⁶¹. A gene of A. nidulans, Hsp30 is homologous to the Hsp26 gene of Saccharomyces cerevisiae and found upregulated in numerous stress conditions including low pH^18,78. In another study, Iron deprivation is sensed as the nutritional deficiency in Candida cells, leading to the upregulation of Hsp30p⁷⁹.

Targeting small heat shock proteins (sHSPs) can be an effective combat strategy to overcome drug resistance in pathogenic fungi. Sequence alignment analysis of sHSPs showed no similarity in the nucleotides or protein sequences with that of humans (data not shown). Understanding sHSPs and its interactive partners not only allow us to study drug resistance but also can be the promising lead therapeutic molecules. It will be an interesting study to see their expression pattern during the progression of drug resistance in pathogenic fungi.