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Infectious Diseases and Translational Medicine • Review • Candida Infections: An Update on Host Immune Defenses and Anti-Fungal Drugs Ning Gao, Changbin Chen ABSTRACT From Key Laboratory of Molecular Virology and Immunology, Unit of Pathogenic Fungal Infection and Host Immunity, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Correspondence to: Changbin Chen, Tel: (+86)02154923055, Fax: (+86)02154923056, Email: cbchen@ ips.ac.cn. Open access DOI: 10.11979/idtm.201601005 Citation: Gao N, Chen CB. Candida Infections: An Update on Host Immune Defenses and Anti-Fungal Drugs. Infect Dis Transl Med, 2016; 2(1): 30-40. Copyright © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Received: Dec 29, 2015 Accepted: Jan 20, 2016 Published: Apr 10, 2016 30 Infections by fungal pathogens such as Candida albicans and non-albicans Candida species are becoming increasing prevalent in the human population. Such pathogens cause lifethreatening diseases with high mortality, particularly in immunocompromised patients. Host defenses against fungal infections are provided by an exquisite interplay between innate and adaptive immune responses. However, effective anti-fungal agents for Candida infections are limited, and fungal drug resistance is a significant treatment challenge. In this review, we summarize the current understanding of host–fungal interactions, discuss the modes action of antifungal drugs, explore host defense mechanisms, and define the new challenges for treating Candida infections. Keywords: Candida spp.; Candida albicans; Drug resistance; Host immune defenses I nfections by pathogenic fungi have become an increasing problem in human health, although their effects are not widely recognized and deaths caused by such infections are often overlooked. Based on reports from the World Health Organization, most people have experienced superficial fungal infections in their lifetimes[1]. In most cases, these infections are easy to cure, but millions of individuals worldwide are suffering from life-threatening invasive infections that are hard to diagnose and treat, a situation possibly attributable to the immunosuppression caused by HIV-AIDS, cancer, metabolic disorders like diabetes, and long-term antibiotic use. Invasive fungal infections are coming under the spotlight because of their unacceptably high mortality rates and because more than 90% of all the fungal-related deaths reported result from species that belong to one of the following four genera: Candida, Cryptococcus, Aspergillus, and Pneumocystis (Table 1)[2]. These fungi are saprophytes in soil and the general environment (e.g., Cryptococcus neoformans, Aspergillus fumigatus) or can be present as commensals in healthy organisms (e.g., Candida species). Among them, candidemia, which is caused by infection with Candida species, is one of the leading causes of blood-stream infections and has a mortality rate of more than 30%. There is an increased incidence of infections caused by non-albicans Candida species such as Candida tropicalis, Candida parapsilosis, and Candida glabrata. Although these fungi are isolated much more frequently as the causative agents of invasive candidiasis worldwide, Candida albicans remains the most common isolate from hospitalized Table 1. Statistics of the 4 most significant invasive fungal infections. Disease (Most common species Location Estimated life-threatening infections/year at that location Mortality rates (% in infected populations) Candidiasis (Candida albicans) Worldwide > 400,000 46-75 Worldwide > 200,000 30-95 Worldwide > 1,000,000 20-70 Worldwide > 400,000 20-80 Aspergillosis (Aspergillus fumigatus) Cryptococcosis (Cryptococcus neoformans) Pneumocystis (Pneumocystis jirovecii) Infect Dis Transl Med 2016;2(1):30-40. Candida Infections: a Review Figure 1. An overview of fungal pathogenicity mechanisms. (A) Fungal cells adhere to the surfaces of host cells and invade the host cell surfaces through damage of cell integrity or secretion of proteinases. (B) Fungal proteins affecting the adherence to host cells. Epa, ALS family and Hwp1 proteins can promote the adherence and enhance host-pathogen interactions. (C) Network of transcriptional factors involved in regulation of fungal biofilm formation. Six key factors, such as Efg1, Bcr1, Tec1, Ndt80, Rob1, and Brg1, form the regulation network to control C. albicans biofilm development. In addition, Ume6 and Ywp1 are positive factors and Nrg1 is a negative factor. patients. In this review, we will focus on Candida species as pathogens by dividing our discussion into three parts: the first part concentrates on fungal infection mechanisms, the second part focusses on host–fungus interactions, and the final part looks at anti-fungal drugs. 1. MECHANISMS OF INFECTION BY CANDIDA To infect a diverse range of host niches, Candida requires a wide range of virulence factors and fitness attributes. In Fig. 1, we have summarized several major factors and fitness traits for Candida. 1.1 Adherence. For most fungal infections, the ability of the host to resist the physical clearing of the infectious agent is important. During interactions with the host, Candida spp. rely on their cell wall proteins for tissue adhesion and invasion, biofilm formation, and evasion of the host immune system[3, 4]. For successful infection, the first step Candida has to achieve is adherence to host cell surfaces[5]. Thus, adhesion is an extremely important step in the infection process, and the extent of adhesion is dependent on the properties of the microbe, the host, and the abiotic surface, with two such properties being cell-surface hydrophobicity and cell wall composition[6, 7]. Dysfunction in cell wall regulation may cause inappropriate exposure or aberrant surface localization of adhesins, which can reduce the adhesion properties of C. albicans[8]. Molecules are present in the most external layers of Candida cells that are essential for its successful adherence to host surfaces and these molecules must play a pivotal role in the pathophysiology of candidiasis[9]. Below, we summarize some key factors involved in Candida adherence. 1.1.1 Lectin-like proteins: C. glabrata, a species of yeast that is normally a commensal member of the human microbiome, is localized predominantly in the mucosa of different organs. However, like other Candida species, this fungus can cause localized infections in patients whose immune systems have been compromised[10, 11]. Adherence of C. glabrata to human epithelial cells relies mainly on Epa (epithelial adhesins) proteins, which are glycosylphosphatidylinositol-anchored cell proteins with well-defined modular structures[12]. The C. glabrata cell wall contains 67 adhesin-like proteins; these are classifiable into seven groups with the largest being the Epa family, which contains 17–23 members depending on the strain[13]. Several Epa protein members mediate adhesion to epithelial and endothelial cells, and contribute to the establishment of infection. Genome analysis indicates that the EPA1 cluster harbors three gene loci (EPA1, EPA2 and EPA3), and the transcriptional profiles of these genes have revealed that EPA1 is induced in the first cell division, EPA2 is induced by oxidative stress, and EPA3 is induced by biofilm formation or osmotic/nutritional stressors[6, 13, 14]. Epa1p is a Ca2+-dependent lectin, and its deletion significantly reduces adherence in vitro, supporting the importance of this protein in adherence[6, 15]. The structures and functions of Epa proteins have been thoroughly studied at both the cellular and molecular levels, and further information is available from recent reviews[3, 4, 16, 17]. 1.1.2 Agglutinin-like sequence (ALS) family: The C. albicans ALS family includes eight genes (ALS1–7 and ALS9) that encode a family of cell-surface glycoproteins[5]. Functional studies of ALS family proteins have focused on the notion that Als proteins act as the adhesins involved primarily in host–pathogen interactions in C. albicans [18]. To date, two different approaches have been used by researchers to test this hypothesis. One approach evaluated the effect on adherence of knocking out individual Als proteins, while the other used heterogenous overexpression of Candida ALS genes in S. cerevisiae. The latter approach has a disadvantage in that it may not accurately reflect the protein function in C. albicans because of differences in codon usage and glycosylation between Candida spp. and S. cerevisiae. Although the three-domain structures of the ALS gene family are similar, differences may exist in terms of their expression profiles and functions. Some ALS genes appear to be regulated by obvious increases and decreases at the mRNA level (ALS1, ALS2, and ALS3), whereas others (ALS6 and ALS7) are consistently transcribed at lower levels[19-21], suggesting that evaluated transcript levels alone may not be enough to explain the importance of some ALS proteins in superficial adherence to the host. A 31 Infectious Diseases and Translational Medicine substantial number of studies have supported the key role played by Als proteins in host–fungus interactions. Als3 is a major component of the hyphal cell wall, but not for the yeast phase[22]. Deletion of C. albicans ALS3 results in striking adhesion and biofilm formation defects. ALS3 is also required for C. albicans cell binding to E-cadherin on epithelial cells, and for N-cadherin on endothelial cells and extracellular matrix proteins[23, 24]. ALS3 expression, which is regulated by a number of transcription factors, is strongly induced by Efg1, Tec1 and Bcr1 and inhibited by Nrg1 and Tup1. Additionally, recent studies suggest that the Cph1 transcription factor plays an essential role in the ALS3 activation process[25, 26]. Knocking out ALS4 significantly slowed down germ tube formation and decreased fungal adhesion to endothelial cells. Interestingly, similar to that of Als2p, ALS4 expression has no impact on epithelial cell binding or biofilm formation in a catheter model[20]. Deletion of ALS5, ALS6 or ALS7, which normally exhibit lower expression levels than other ALS family genes, was found to significantly increase adhesion of C. albicans to human vascular endothelial cell monolayers and buccal epithelial cells, indicating low transcript levels are sufficient to translate enough protein for the required function[21]. ALS9 contains two alleles (ALS9-1 and ALS9-2), and based on sequence variations within the 5 and 3′ domains of the ALS9 coding region, this protein exhibits the greatest allelic variability in the ALS gene family. ALS9-2, rather than ALS9-1, plays a role in vascular endothelium adhesion, suggesting allelic functional differences between strains[27]. Allelic diversity in ALS genes is an important area of study because information about a protein encoded by a single allele provides a context for studying allelic variation within the larger population of C. albicans strains. Southern blot hybridization and genomic DNA amplification using degenerate PCR primers designed against the C. albicans ALS sequences indicate that ALS genes probably exist in C. dubliniensis and C. tropicalis, and potentially in C. parapsilosis[28]. In addition to the comprehensive studies that have outlined the structures and functions of the Als proteins, and the role played by this gene family in C. albicans biology and pathogenesis, ALS genes and proteins are attracting more attention largely because they are potential targets for inclusion in an anti-Candida vaccine[29-32]. 1.1.3 Hyphal wall protein 1 (HWP1): Hwp1, a GPI anchor-dependent cell wall protein family member responsible for adhesion processes and biofilm formation, is involved in the pathogenesis of C. albicans[33-35]. HWP1 expression is strongly induced during germ tube formation but is absent during yeast growth, indicating that its expression appears to be coordinately controlled by the transcriptional activators and repressors that control morphology[36]. The N-terminal domain of Hwp1, which mimics host cell transglutaminase substrates, can form tight attachments to host epithelial cells through a coiled disulfide bond, whereas the C-terminal domain, which can be modified by glycosylphosphatidylinositol, is required for the N-terminal functioning in host 32 cell wall binding[37, 38]. As a hypha-specific gene, HWP1 expression during hyphae development and biofilm formation is specifically regulated by a number of key developmental regulators such as EFG1, TUP1, and RBF1, but is not affected by CPH1. HWP1 expression is dependent upon Efg1 and is repressed by Tup1, while Rbf1 appears to act as an inducer of expression[39]. In addition, the Hwp1 adhesin protein may play a pivotal role in C. albicans virulence and drug resistance, as shown by the observation that its expression is regulated by the Kex2 endoproteinase, which also regulates the activity of C. albicans proteinases[40, 41]. Interestingly, Hwp1 and Als3 (along with the closely related Als1) seem to have distinct and complementary roles in cell-cell adhesion and biofilm formation, because the Hwp1 function in promoting cell adherence was attenuated in an als1−/− als3−/− double mutant strain[42]. 1.2 Biofilm formation. Biofilms display an organized three-dimensional structure comprising a dense network of yeast and filamentous cells embedded in an exopolymeric matrix of carbohydrates, proteins and nucleic acids[43]. Biofilm formation is a step-wise process in the host–fungal interaction, and occurs in surfaceassociated microbial communities that are surrounded by an extracellular matrix, such as teeth and the foreign surfaces of implanted medical devices as well as mucosal surfaces[7, 9]. Biofilm formation can be partitioned into four temporal stages: adherence, initiation, maturation and dispersal[44, 45]. At the beginning, in the adherence step, yeast-form cells adhere to the substrate and then propagate to form a basal layer and germ tubes form to yield hyphae (the initiation stage). Next, the biofilm forms the dense bilayers of yeast, hyphae and pseudohyphae (the maturation stage). Finally, the yeast-form cells are released from the biofilm to attach and colonize new sites in the surrounding environment (the dispersal stage)[46]. Although these steps might occur concurrently rather than sequentially during natural biofilm development in vivo, they provide a useful framework for analyzing the mechanisms involved in biofilm development. A comprehensive study, based on high-throughput deepsequencing technologies, has unraveled a transcriptional circuit governing biofilm formation[47, 48]. This complex, but typical network, consists of six master transcriptional regulators (i.e., Efg1, Tec1, Bcr1, Ndt80, Brg1, and Rob1) that control each other’s expression and bind directly to the promoters of 44 additional biofilm regulators. The adherence step, for example, requires participation of Bcr1, Efg1 and some of their downstream targets[34, 49-52]; these targets include the Ywp1 regulator (which plays an inhibitory role in adherence)[53], the Tec1 and Bcr1 regulators, while Ece1 and Als9 (cell wall proteins) also affect the initiation of biofilm development[54]. Ume6 affects biofilm formation by inhibiting dispersal, while the Nrg1 positive regulator promotes dispersal development[55-59]. 1.3 Yeast and hyphal morphologies. The ability to switch between yeast and hyphal forms represents one of the most discussed and extensively investigated Candida Infections: a Review virulence attributes of the human pathogenic fungus C. albicans[16, 60]. Numerous studies have shown that the yeast-to-hyphal transition is important for virulence and contributes to various distinct functions during the different stages of disease development, including adhesion to epithelial and endothelial cells, invasion through induced endocytosis and active penetration, iron acquisition and utilization, escape from phagocytes and immune evasion, and mucosal immune activation and triggering of specific sepsis-like immune responses during systemic infection[17, 61, 62]. Therefore, fungal dimorphism is vital for virulence at both superficial and systemic levels. The ability to express hypha-specific genes accounts for the pathogenic potential of hyphal formation and a number of environmental signals trigger its development. These signals include the presence of serum, elevated temperature, neutral pH, the presence of certain nutrients, starvation signals, matrix embedding, CO2 and O2 levels, cell density, and contact with physical surfaces[16]. Interestingly, these signals resemble unfavorable growth conditions or indicate the possibility of a hostile environment. Such environmental signals stimulate activation of a range of signaling pathways that include the following: inhibition of heat shock protein 90 (Hsp90) by elevated temperatures and subsequent activation of Ras1[63], the cAMP/PKA-signaling pathway via direct or indirect activation of the Cyr1 adenylyl cyclase [64], mitogen activated protein kinase (MAPK) signaling via Ras1/ Hst7 and Cek1[65], activation of the Rim101 pathway by neutral to alkaline pH[66], Czf1 activation under nonembedded conditions via Rac1[67], hypoxia-induced Efg1/ Efh1 activation[68], and reactive oxygen species (ROS) signaling induced by genotoxic stress[69]. Consequently, activation of these signaling pathways triggers activation or inhibition of the key transcriptional regulators (e.g., Efg1, Czf1, Cph1, Tec1, Flo8, and Nrg1) that control expression of the genes necessary for hyphae formation and hypha-associated genes[70, 71]. The immune system is able to discriminate between yeast and hyphal growth. Morphological recognition of C. albicans, together with detection of fungal burden and the damage caused by invading hyphae, may hold the key to host discrimination between colonization and infection, and may also explain the presence of different lifestyles of C. albicans, either as a commensal or a pathogen. 1.4 pH adaptation. The pH of the human host differs dramatically according to the anatomical site. Normally, the pH in the blood and tissues is somewhat neutral (pH 7.4 ±e0.1), whereas the human vaginal cavity is acidic (pH ~4) and the pH along the digestive tract differs significantly, ranging from pH 2 to 8[72]. C. albicans is able to survive in both the acidic and basic pH of the host environment, and this ability enables this fungus to colonize different host niches, such as the acidic vagina or the neutral oropharyngeal tract. Accumulating evidence has demonstrated that tolerance of different pH levels is important for C. albicans pathogenicity. PHR1 and PHR2 are two pH-regulated C. albicans genes that are differentially expressed according to the pH conditions in the host. Transcriptional profiling has shown that PHR2 is expressed under acidic pH, while PHR1 expression is enriched at neutral and basic pH[73, 74]. Deletion of PHR2 leads to growth inhibition at acidic pH conditions, whereas deletion of PHR2 results in an inability to grow at neutral and basic pH conditions. Importantly, the contribution of each gene to virulence depends on the host niche, as noted by the observation that a PHR2 deletion mutant was avirulent in the vagina, whereas the PHR1 deletion mutant showed reduced virulence in a systemic infection model. Furthermore, the RIM101 gene, which encodes a pH-regulated transcription factor and is well characterized in other fungi such as Cryptococcus neoformans, when deleted also reduced fungal virulence in a systemic mouse model and in an endothelial-celldamage-model[66]. More recently, researchers found that C. albicans is not only able to sense and adapt to a wide range of pH conditions, but can also actively modulate environmental pH. A good example of such modulation is the research that showed that C. albicans is able to utilize amino acids to promote neutralization of phagosomal pH and that increasing pH levels resulted in hyphae development and escape from macrophages[75]. 2. HOST DEFENSE MECHANISMS Of the approximately 200 Candida species that have been described to date, only five species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei) are attributed to the vast majority of Candida infections, suggesting that these species in particular are well adjusted for survival within the human host. For example, C. glabrata and C. tropicalis are more commonly seen in patients with hematological or solid organ malignancies and neutropenia, while C. krusei infections occur predominately in patients with hematopoietic stem cell transplantations[76]. C. parapsilosis, which causes infections mainly in neonates rather than in adults, is a common pathogen of catheter-related infections[76]. To fight fungal infections, the human host has evolved several different effective strategies, including mechanical (e.g., epithelial) barriers, as well as biochemical, chemical and physical antagonists (e.g., bile, mucus, pH, and antimicrobial peptides), microbial competition (normal human microbiota), and the innate and adaptive immune systems. The increased prevalence of fungal infections has prompted the development of one branch of fungi-related human disease: research that involves investigations into the two major host anti-fungal protective mechanisms. One of these mechanisms was active early in the evolution of multicellular organisms (innate immunity), while the other involves the sophisticated adaptive mechanisms that are induced specifically during infection and disease (adaptive immunity). Fig.2 shows most of the major events involved in host immune recognition upon fungal infection. Defense against fungal infection is supported by an exquisite interplay between the innate and adaptive arms of the host immune system, which act together to 33 Infectious Diseases and Translational Medicine Figure 2. Innate and adaptive immunity involved in host antifungal defense activities. (A) Innate immunity. Recognition of Candida species is mediated by TLRs, CLRs and NOD receptors. Both TLR4/TLR2 can induce production of proinflammatory signals and cytokines through the MyD88-dependent NF-κB pathway. However, TLR3-mediated recognition is through activation of the IRF3 transcription factor. The CLR receptors, such as Dectin-1, Dectin-2, Dectin-3 and Mincle, stimulate release of inflammatory cytokines by activating T cell lineage-specific tyrosine kinase (Syk) and downstream complex (CARD9-Bcl10-MALT1) to initiate the canonical and non-canonical NF-κB pathways. Dectin-1 promotes maturation of inflammasome to induce production of cytokines. DC-specific intracellular adhesion molecule-grabbing non-integrin (DC-SIGN) receptor can induce the TH cell responses through Raf1 activation. In addition, NLRP3 stimulates ROS production through an Erk dependent pathway, and NOD2 receptor activates inflammatsome by affecting maturation of the pro-IL-1β via caspase-1. (B) Adaptive immunity. T cells form an integral part of adaptive immunity, which classified into two distinct lineages (CD4+ and CD8+ cells). Based on the effector functions, CD4+ cells are further subdivided into Th1, Th2, Th17, and Treg cells. eliminate pathogens from the human body. 2.1 Innate immunity. Traditionally recognized as the first line of defense against pathogens, innate immunity utilizes the body surfaces and the mucosal epithelial surfaces of the respiratory, gastrointestinal and genito-urinary tracts as barriers against the continuous interactions between fungi and host[77, 78]. The cell wall of Candida spp. has two layers: the inner layer, which contains β-1, 3-glucan and β-1, 6-glucan, and the outer layer, which is composed mainly of O- and N-linked glycoproteins[3]. The polysaccharide structures in the C. albicans cell wall are recognized by two classes of membrane-bound pat34 tern-recognition receptors (PRRs): the Toll-like receptors (TLRs) and the C-type lectin receptors (CLRs) [79]. The several TLRs that have been extensively studied for their roles in fungal recognition include TLR1, TLR2, TLR4, TLR6, TLR7 and TLR9[80]. Remarkably, different TLRs can recognize several structurally divergent pathogenassociated molecular pattern (PAMP) ligands in fungi. For example, TLR4 can recognize mannans and mannoproteins from C. albicans [81-83], while TLR2 responds to phospholipomannan from C. albicans[84]. However, other TLRs, such as TLR1 and TLR6, may not be important for host innate recognition and they do not seem to be essential for antifungal defense in candidiasis[85]. CLRs are another type of PRR family that can recognize both endogenous and exogenous ligands via the characteristics of the conserved domain from CRD, which allows them to recognize a range of molecules such as carbohydrates, lipids and proteins. Dectin-1 specifically recognizes β-glucans [86], and the macrophage mannose receptor recognizes N-linked mannan and mediates recognition and phagocytosis of different yeast species by macrophages[82, 87], while α-mannan was reported to be recognized by both yeast and hyphae[88-90]. DC-SIGN is another important dendritic cell (DC)specific receptor that is able to internalize C. albicans by recognizing the mannan structure exposed on Candida cells[91]. Also, galectin-3, which was found to be essential for recognizing the β-1,2 mannosides of C. albicans [92], associates with TLR-2 when macrophages interact with yeasts [93]. A soluble CLR MBL (mannose-binding lectin) was found to facilitate opsonophagocytosis by binding to Candida mannan and to the surface of the C1q receptor on the phagocyte[94]. Although the nature of the macrophage-inducible C-type lectin (Mincle) ligand has not yet been characterized, this C-lectin receptor also binds to yeast cell wall components and participates in the cell response with TLR-2[95]. In addition to the membrane-bound receptors described above, several PRRs are able to recognize Candida intracellularly. TLR9 has been shown to recognize C. albicans DNA and induce cytokine production in dendritic cells [96]. The nucleotide-binding domain, leucinerich, repeat-containing receptors (NOD-like receptors, NLRs) are PRRs that recognize intracellular PAMPs, and one of their main functions is to activate the inflammasome via caspase 1, thereby leading to processing and activation of interleukin 1 (IL-1) family cytokines[97]. These different PRRs have been implicated in the activation of highly characterized innate host responses against fungal pathogens. Most TLRs (with the exception of TLR3) are able to signal through adaptor protein MyD88, which is essential for responses against a broad range of microbial components and crucial for the clearance of infections [79]. In contrast, TLR3 mediates the activation of interferon (IFN)-regulatory factor 3 and this activation is operated in a MyD88-independent manner. Spleen tyrosine kinase (Syk), a primary signal transduction factor in the immune regulation of CLRs, operates to either activate the classic CARD9-Bcl10-MALT1 com- Candida Infections: a Review plex to induce the canonical NF-κB pathway or activate NIK to induce non-canonical NF-κB activation [98-100]. Although most CLRs like Dectin-1, Dectin-2 and Mincle signal mainly through Syk activation, their major roles in fungal infections are quite distinct. Dectin-1 was found to play a major role in activating the inflammasome via the induction of phagocytosis and other antimicrobial mechanisms, such as NLRP3 activation via ERK-induced ROS production[101]. With human DCs, host recognition by Dectin-2 activates the NF-κB subunit c-Rel via Malt1 activation, thereby triggering a Th17 response. Th17 cells produce IL-17A/F cytokines that bind to the IL-17 receptor and induce secretion of a number of proinflammatory factors including IL-6, IL-8 and GM-CSF as well as CXCL1 and CCL20 chemokines, which are involved in macrophage and neutrophil recruitment[102]. Additionally, interplay between TLRs and CLR-mediated signaling is required for inducing optimal immunity and antifungal responses. Co-stimulation with both Dectin-1 and TLR2 or TLR4 ligands, for example, leads to an increase in TNF, IL-10 and IL-23 and a decrease in IL-12 production[103]. NLRs act as scaffold proteins that assist with assembling the signal platforms required for triggering NF-κB and MAPK pathways that control the activation of inflammatory caspases and proinflammatory cytokine release[104]. 2.2 Adaptive immunity. Generally, acquired immune responses are slower to act than innate immune responses. Acquired immunity is mediated mainly by B cells that produce specific antibodies, CD8+ T cells that kill pathogen-infected cells, and CD4+ T cells that produce effector cytokines, and these cells are able to respond to a wide range of potential antigens[105, 106]. As specialized antigen presenting cells, DCs play a central role in immune defense against pathogens. Immature DCs are recruited to the site of infection in response to chemokines and antimicrobial peptides. It has been reported that DCs are vital for the initiation of adaptive T-cellmediated immune protection against C. albicans and an important conclusion is that mixed populations of DCs have non-redundant functionalities and can drive CD4+ and CD8+ T-cell responses against C. albicans. DCs recognize C. albicans through interactions between PRRs expressed on the surface of the DCs and PAMPs exposed on the fungal cell wall[107]. Upon recognition, activated DCs induce different antigen-specific immune responses. For example, induction of Th17 responses requires the presentation of C. albicans antigens by Langerhans cells. However, this interaction has no impact on the development of CD8+ cytotoxic T-lymphocyte (CTL) responses. Surprisingly, evidence has shown that specific Langerin+ dermal DCs are able to trigger both Th1 and CTL responses, and activation of CTL responses suggests that fungal antigens are cross-presented via the MHC I pathway, thereby promoting antigen display to CD8+ Tlymphocytes[108]. Several immune and non-immune cells are also known to contribute to antifungal responses and these include epithelial cells, neutrophils, natural killer cells, DCs, monocytes, macrophages and T cells. Epithelial cells play an important role as barriers against tissue invasion by fungi. After sensing the cell wall component signals of Candida yeast or hyphae, epithelial cells produce cytokines via MAPK1- and FOS-dependent pathways, leading to the recruitment of phagocytic immune cells[109]. Neutrophil activation, which is essential for fungal clearance, occurs through the mechanisms of both oxidative and non-oxidative effectors that are capable of killing fungi[110]. Macrophages produce the inflammatory cytokines and chemokines that recruit and activate other immune cells, such as blood monocytes, at the site of infection. Additionally, natural killer cells are in most cases sufficient contributors to the rapid innate immune response that prevents surface colonization and tissue invasion of the host. 3. ANTI-FUNGAL AGENTS Fungal infections pose a continuous and serious threat to human health and longevity. Although C. albicans is recognized as the most common Candida pathogen, other non-albicans Candida species are being isolated with increasing frequency from patients with non-fungal clinical diseases. The dramatic increase in fungal infections has intensified the search for new, safer, and more efficacious agents to combat serious fungal infections. In general, the anti-fungal drugs currently available can be classified into the following five categories: azoles, ployenes, allylamines, flucytosine and echinocandins. Unravelling the mechanisms of action of the different anti-fungal agents listed above will significantly improve our understanding of the mechanisms of fugal resistance to them. Indeed, determining resistance mechanisms has enabled or enhanced our understanding of the specific mechanisms of action of many drugs. 3.1 Azoles. Azoles (e.g., fluconazole, voriconazole, itraconazole and posaconazole) and allylamines (e.g., terbinafine) inhibit ergosterol biosynthesis by binding to the 14-α-lanosterol demethylase (14α-DM) in yeasts or 14-α-sterol demethylase (CYP51p) in molds (cytochrome P450 enzymes encoded by the EFG11 gene or CYP51A and CYP51B genes, respectively)[111-113]. Fluconazole, an inhibitor of cytochrome CYP3A4, is recommended as the primary treatment for candidemia and remains the most frequently used anti-fungal agent because it is safe, cheap and tolerable[114]. Voriconazole, an inhibitor of the cytochrome enzymes CYP2C19 and CYP2C9, is an azole with a broader spectrum than fluconazole. Intraconazole is out-of-date, but a parenteral form has become available recently. Fungal resistance to azoles has been attributed to a number of mechanisms, such as activation of efflux pumps, genetic alterations and genomic instability, transient gene overexpression, impaired drug penetration, modification and up-regulation of the target enzyme concentration, and ergosterol replacement[111, 115, 116]. 3.2 Polyenes. Polyenes like amphotericin B bind to ergosterol, which is the major component of the fungal plasma membrane, by creating large pores that allow 35 Infectious Diseases and Translational Medicine leakage of cell contents and disrupt cell function. For a long time, amphotericin B was considered to be the ‘gold standard’ in the treatment of invasive fungal infections[117]. The molecular structure of amphotericin B shows it has poor water solubility but excellent lipid solubility [118]. The two-domain structure of this polyene comprises a polyene hydrocarbon chain and a ployhydroxyl chain, both of which are important for its antifungal effect. These domains in amphotericin B mediate the interaction between eight amphotericin B and eight ergosterol molecules by forming a pore, and this results in the rapid efflux of K+, inhibition of fungal glycolysis and subsequent Mg2+ efflux[117, 119]. Amphotericin B stimulates immune cells via Toll-like receptor TLR2 and CD14 protein, the latter of which is a transmembrane signaling protein on the surface of mononuclear cells. By triggering the intracellular signaling pathways involving the adapter protein MyD88 and NF-κB, amphotericin B induces the expression and release of inflammatory cytokines, such as pro-inflammatory cytokines (e.g., interleukin (IL)-1β, TNF-α, IL-6 and IL-1Rα) and chemokines (e.g., IL-8, MCP-1 and MIP-1β)[120]. 3.3 Allylamines. The allylamines are a class of synthetic anti-fungal agent used in clinical applications. Allylamines, which are represented mainly by naftifine and terbinafine, possess activity against a wide range of filamentous, dimorphic and yeast-like fungi. The primary mode of action of these anti-fungals is the inhibition of fungal ergosterol biosynthesis at the point of squalene epoxidation. Squalene epoxidase is another enzyme required for ergosterol synthesis[121]. 3.4 Flucytosine. Flucytosine (e.g., 5-fluorocytosine) is a base pyrimidine analogue that functions to inhibit cellular pyrimidine metabolism, transcription, DNA replication and protein synthesis[122]. This agent is active mainly against Candida spp., C. neoformans and some molds. Normally, 5-flucytosine use is combined with other antifungal agents such as amphotericin B or fluconazole to attenuate secondary resistance to it[123, 124]. Two kinds of resistance have been found to be mediated by 5-flucytosine in clinically relevant Candida species: primary resistance is related to decreased drug uptake by cytosine permease, which is encoded by the FCY2 gene, while secondary resistance is limited by alterations in the enzyme activities of cytosine deaminase or uracil phosphoribosyltransferase, which are encoded by FCY1 and FUR1 genes, respectively[125]. In a study that sought to decipher the resistance mechanism of flucytosine, a chemogenomics analysis of C. albicans identified 183 genes that may contribute to flucytosine resistance[126]. Among these genes, 5 are involved in DNA repair, 8 are involved in chromatin remodeling, and 17 appear to participate in RNA metabolism. Moreover, another study revealed that a family of two genes, CgFPS1 and CgFPS2, which both encode aquaglyceroporin-regulated glycerol transport, were found to mediate 5-flucytosine resistance by decreasing the accumulation of 5-flucytosine in C. glabrata cells[127]. 3.5 Echinocandins. Echinocandins (i.e., caspofungin, 36 anidulafungin and micafungin), a group of unique antifungal agents, target the fungal cell wall through noncompetitive inhibition of the biosynthesis of β-1, 3-Dglucan, a major structural component of the fungal cell wall and a fungal-specific target [128, 129]. Caspofungin received FDA approval in 2002, micafungin in 2005, and anidulafungin in 2006. These three agents are approved for treatment of serious fungal infections, such as esophageal candidiasis, candidemia and other Candida infections, but not infections by Aspergillus spp. In Candida spp., the β-1, 3-D-glucan synthase is a multi-subunit enzyme complex and the UDP-glycosyltransferases are major ones. The β-glucan synthase complex has a minimum of two subunits: Rho (regulatory subunit, which is a GTP-binding protein in the Rho/Rac subfamily of Raslike GTPases) and Fksp (catalytic subunit), and these two subunits are the major targets of echinocandins[128]. In C. albicans, echinocandin resistance was found to correlate with point mutations in FKS1[128]. However, this type of resistance in C. glabrata is often caused by mutations in both FKS1 and FKS2 genes[130]. Interestingly, studies have shown that two ‘hot spot’ regions in Fksp can explain the mechanism of drug resistance against echinocandin in C. albicans clinical isolates. These ‘hot spots’ span amino acids Phe641 to Pro649 and Asp1357 to Leu1364, suggesting that FKS gene mutations in hot spots account for most of the universally observed resistance to echinocandin [131]. Resistance to anti-fungal agents is the logical and inevitable consequence of using these agents to treat human infections. The availability of molecular genetic tools will undoubtedly greatly improve our understanding of the mechanisms by which anti-fungal drug resistance emerges and spreads and promises to greatly inform efforts to develop novel and effective compounds for future use. In addition, the introduction of new antifungal agents (e.g., echinocandins, second-generation triazoles) in the past decade has greatly increased the number of treatments for invasive fungal infections, and drug toxicity is no longer the major limiting factor in treatment regimes. Although many of these newer antifungal agents have limitations in their activities, pharmacokinetics, unique predispositions for pharmacokinetic drug-drug interactions and unusual toxicities associated with their long-term use, we still believe that their benefits, in terms of delivering safer and more effective antifungal therapies, vastly outweigh these drawbacks. 4. CONCLUDING REMARKS The rapidly increasing numbers of immune-compromised patients and the increase in fungal resistance to fungicides mean that invasive fungal infections, which have become an important problem in both the hospital and the community and are costly to treat, are an everincreasing cause of morbidity and mortality. In the battle against fungal infections, a concerted effort to raise public awareness of fungal pathogens by clinicians, researchers, the pharmaceutical industry and public health Candida Infections: a Review officials is required. As this review has sought to show, gaining better knowledge of fungal pathogen biology and host immunity against these pathogens seems an equally important goal, and the complexity of the interactions between them is a fascinating area of research. Work on one gene or one property, no matter how detailed and careful, will REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 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