Candida and Candidiasis, Second Edition

In recent years DNA sequence-based methods have helped to confirm taxonomic relationships within the genus and have been used to confirm that both sexual and nonsexual Candida species are ascomycetes. Molecular methods have shown that many of the medically important Candida species belong to a phylogenetic subgroup known as the CTG clade, a group of largely commensal yeast species that translate CTG as serine instead of leucine. Most women suffer from vulvovaginal candidiasis (VVC) at least once in life, with as many as 8% experiencing regular recurrent infections. VVC is primarily caused by Candida albicans, while C. glabrata is the second most common cause of this infection. Candida species are an important component of the normal flora of the human oral cavity, and if given the opportunity, these can overgrow and cause oropharyngeal candidiasis (OPC). The introduction of highly active antiretroviral therapy (HAART) in the mid-1990s has led to a marked decrease in the incidence of OPC in HIV-infected individuals. In general, the majority of C. albicans isolates are fully susceptible to all major classes of antifungal agents, including the azoles, echinocandins, and polyenes. Candida parapsilosis is frequently isolated from physical surfaces in the hospital environment, making it unique among Candida species. The remaining Candida species associated with human disease are only rarely detected, and therefore relatively little is known about the etiology or the epidemiology of the diseases they cause.

This chapter focuses on the Candida species within the CTG clade. Several of the species (Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis, and Lodderomyces elongisporus) have diploid genomes, so it is possible to compare the single nucleotide polymorphisms (SNPs) between the homologous chromosomes. Families of long terminal repeat (LTR) and non-LTR retrotransposons have been well characterized in C. albicans. The precise number of agglutinin-like sequence (ALS) genes in the other Candida species is likely to be overestimated, caused by difficulties in assembling the repeated regions. Species-specific amplifications have also occurred in the other Candida species. The large-scale analysis of Candida genomes confirmed that many of the multigene families described for C. albicans are also present in the genomes of other pathogenic Candida species. C. albicans and C. dubliniensis are the only two Candida species that generate true filaments, although C. dubliniensis is much less efficient. The change in the genetic code may have helped protect Candida species from infection by RNA-based viruses. Comparative genomic tools for Candida species are available from the Broad Institute, the Pasteur Institute, and University College Dublin. In conclusion, analysis of the vast array of genomic data available has already made, and is likely to continue to make, major contributions to our understanding of the biology of Candida species.

This chapter discusses the most recent findings on the reassignment mechanism of CUG codons from leucine to serine in various Candida and non-Candida species, the so-called CTG clade. It highlights how the Candida albicans model system improves one's understanding of the evolution of the genetic code, and explains how this genetic code alteration shaped the biology of the CTG clade species. Codon reassignments show that the genetic code evolves even in organisms with complex genomes and proteomes. The codon capture theory postulates that genetic code changes result from genome G+C biases on codon usage. There is significant flexibility in the genetic code to support codon reassignment. The phenotypic diversity induced by CUG ambiguity exposes some of these virulence traits, suggesting that CUG ambiguity may be relevant to pathogenesis and that C. albicans may have evolved unique mechanisms to take advantage of its genetic code alteration. The double identity of the CUG codon implies that each protein is represented by a mixture of molecules containing Leu or Ser at CUG positions. This creates a statistical proteome whose biological implications are still poorly understood. Nevertheless, the probability of finding identical cells in nature is extremely small.

This chapter describes DNA repair systems that have not been described for Candida species even though orthologues are found at least in the Candida albicans genome databases. A section of the chapter describes the genetic plasticity as it relates to drug resistance. In Saccharomyces cerevisiae haploid cells, the rate of spontaneous mutation in the nuclear genome is rather low under laboratory conditions. An additional marker of the genetic instability in C. albicans is represented by aneuploidies. Aneuploidies are common in laboratory strains of C. albicans but are especially abundant when those strains have been subjected to genetic manipulations, including several laboratory strains successively derived from CAI-4, or treated with mutagenic agents such as UV light. Genetic instability could be caused by an increase in the rate of mutations in the form of single base substitutions, microinsertions, and microdeletions. These alterations are known to arise from errors during normal DNA replication by polymerases δ and ε and are usually corrected before being fixed by methyl mismatch repair (MMR). It was suggested that C. albicans has evolved additional DNA repair systems to defend itself against killing by the oxygen radicals generated by macrophages. For an opportunistic pathogen, drug resistance represents an excellent and practical system to correlate phenotypic traits with genomic changes. Azoles are drugs commonly used in clinics. Hypermutable subpopulations are characterized by the presence of secondary mutations unrelated to that selected, which are distributed throughout the genome.

The chapter focuses on the switching and mating processes of Candida species. Hull and Johnson found orthologs of three Saccharomyces cerevisiae mating-type genes in the Candida albicans mating type-like (MTL) locus. Although the scheme for mating and the regulation of the response to the mating pheromones proved in subsequent studies to be in general similar to that of S. cerevisiae and hence that of the hemiascomycetes in general, the biggest surprise was the fundamental and unique role that white-opaque switching played in the mating process of C. albicans. The response of an opaque cell to pheromone produced by the opposite mating type was shown to be highly similar to that of S. cerevisiae, including the components of the major regulatory pathway that transduces the pheromone signal. The spontaneous transition between the white and opaque phenotypes by a selected group of strains had been shown to affect a number of phenotypic and virulence traits before the discovery by Miller and Johnson that it was an essential step in the mating process. The sensitivity of the opaque phenotype to high temperature caused a conundrum regarding the relationship of host and mating. The host signals that have been identified stimulate the rate of white-to-opaque switching, but switching occurs spontaneously and in both directions. Gradients of pheromone would be extremely prone to mechanical disruption and dissipation by diffusion if in a purely liquid environment. A biofilm would provide a protective environment against disruption, and a porous matrix would reduce diffusion.

Strain variability is a central topic in the discussion of Candida biology. Most of the Candida isolates that are studied are derived from clinical specimens. Therefore, a discussion of variability among Candida isolates within a particular species starts with decisions that are made in the diagnostic microbiology laboratory. Studies of Candida genetic variability may also be conducted from the perspective of assessing genomic rearrangements. The nature of genomic rearrangements in the diploid Candida albicans was understood more clearly from construction of a physical map of its eight pairs of chromosomes. DNA fingerprinting probe sequences useful in phylogenetic and epidemiological studies are derived from the major repeat sequence (MRS). The MRS is also present in the closely related Candida dubliniensis. Events such as mutation and mitotic recombination also can contribute to variability among C. albicans strains. One study examined whether the local wildlife population was responsible for maintaining a reservoir of C. albicans isolates specific to a defined geographic area in the midwestern United States. The work was expanded to include collection of C. albicans isolates from domestic animals. Results showed that there is a significant difference in the clade distribution of isolates from humans and wildlife, demonstrating population isolation between the groups. The work demonstrates the impressive display of genetic variability that C. albicans can develop when challenged with exposure to antifungal drugs. A given isolate of C. albicans can undergo an impressive range of genetic changes at the level of point mutation to alterations in whole chromosomes.

This chapter describes the major Candida albicans morphologies and the current understanding of the cell biological and cell cycle features that distinguish them. It highlights recent insights into how cell cycle regulators influence the formation of hypha-specific cellular features in particular. Since morphogenesis and cell cycle regulation have been studied most extensively in C. albicans, the chapter primarily focuses on work in C. albicans. The important distinction between yeast and pseudohyphae is that pseudohyphae spend more time in G2 phase of the cell cycle than yeast cells , and they continue to elongate during this time. There has long been a controversy as to how pseudohyphae are related to true hyphae. Initial models suggested that yeast cells, pseudohyphae, and true hyphae reside along a continuum. Later, based on differences in cell cycle dynamics and subcellular structures, it was proposed that pseudohyphae and hyphae represent two distinct morphological states, with pseudohyphae being more like yeast form growth with respect to cell cycle progression and cell biological markers. Recent work has shed light on cell biological features associated with cell cycle progression in chlamydospores and is discussed in theis chapter. In the C. albicans genome sequence, there are three G1 cyclins (Ccn1, Cln3, and Hgc1) and two G2 or B-type/mitotic cyclins (Clb2 and Clb4) that are predicted to associate with Cdc28.

Invasive candidiasis is a life-threatening opportunistic infection and has emerged as a major cause of morbidity and mortality in critically ill patients. Until recently, Candida albicans was by far the predominant species, causing up to two-thirds of all cases of invasive candidiasis. This chapter highlights how the past several years have seen remarkable advances in understanding the basic cellular and immunological mechanisms underlying resistance to the fungus but also organ dysfunction and failure of recovery relating to invasive candidiasis. In experimental candidiasis, both defense mechanisms are activated through the delicate equilibrium between Th/Th17 cells and regulatory T cells (Tregs) limiting the consequences of the associated inflammatory pathology. The inflammatory response is initially mediated by cells of the innate immune system, followed by a later adaptive immune response, which is triggered by the signals originated by the innate immune system. The inflammatory response, initiated by cells of the innate immune system, is followed by adaptive immunity, which responds to, and at the same time regulates, signals emanating from the innate system. Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and limiting step in the kynurenine pathway of tryptophan catabolism. Our increasing understanding of the basic mechanisms that dictate development and function of Th17 cells, as well as our better knowledge of how Th17/Tregs regulate each other as well as other immune and nonimmune cells, provides guidelines for rational design of novel immunomodulatory therapies that limit inflammation in order to stimulate an effective immune response.

Prior to the human immunodeficiency virus (HIV) epidemic, host defense against Candida albicans at mucosal sites was largely considered one-dimensional. This chapter is divided into in-depth reviews of host defense mechanisms against oral, vaginal, and Gastrointestinal (GI) candidiasis, with emphasis on the uniqueness of the responses at each site. Then it has a short discussion of the emerging role of mucosal Candida biofilms in pathogenesis and host defense. The chapter concludes with suggested future directions for the field of mucosal immunity and candidiasis. Oropharyngeal candidiasis (OPC) encompasses infections of the hard and soft palate, tongue, buccal mucosa, and floor of the mouth and can present as reddened patches (erythematous) or white curdlike lesions (pseudomembranous). Interestingly, highly active antiretroviral therapy (HAART) has reduced the incidence of OPC. Polymorphonuclear cells (PMNs) are considered to play a role in innate defenses against OPC because neutropenic cancer patients are susceptible to disease. Another form of oral candidiasis is Candida-associated denture stomatitis (DS). Candida albicans is the most common cause of DS and can readily form biofilms on denture material. The most recent data on host defense against GI candidiasis involve understanding the role of dendritic cells (DCs) in directing both local and systemic adaptive immunity. Vulvovaginal candidiasis (VVC) affects a significant number of women, predominantly in their reproductive years.

The tasks of recognition of the invading pathogen and host defense activation are accomplished by pattern recognition receptors (PRRs) that sense conserved chemical signatures of the microorganisms called pathogen-associated molecular patterns (PAMPs). The most important cell populations involved in the phagocytosis of fungal pathogens are neutrophils and macrophages, and together with the monocytes they have been shown to represent the major producers of proinflammatory cytokines. These innate immunity cell populations have subsequently been shown to be the most important cellular component of host defense against disseminated Candida infections. C-type lectin receptors (CLRs) are members of a large family of PRRs including dectin-1, the macrophage mannose receptor (MR), dendritic cells (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), dectin-2, and the circulating mannose-binding lectin (MBL). Recent progress in understanding host defense against fungal infections in general, and Candida infections in particular, has provided important novel targets for potential novel immunotherapeutic approaches. As vaccination is one of the most cost-effective treatment strategies and probably the most powerful tool to protect humans and animals against infectious disease, further vaccine development could significantly lessen the burden of Candida infections in patients at risk.

This chapter reviews the rationale for and latest advances in development of passive and active immunization against invasive candidal infections. The major form of immunosuppression that predisposes to development of disseminated candidiasis is a defect in innate phagocytic activity. Treatment with antibiotics is the most promising risk factor to identify a high attack rate of vulvovaginal candidiasis (VVC) as a basis for enrollment into clinical trials of an active vaccine. Development of a vaccine for oropharyngeal candidiasis (OPC) in patients with human immunodeficiency virus (HIV)/AIDS is complicated by the host immunosuppression and by the fact that effective immune reconstitution therapy is now available for most patients. The majority of patients at risk for disseminated candidiasis are those undergoing emergent or elective gastrointestinal or cardiac surgery, those with central venous catheters, those receiving broad-spectrum antibiotics, or in general those patients receiving their care in intensive care units, including both civilian and military trauma patients receiving abdominal, chest, or burn wounds. Passive immunization strategy focusing on β- glucan has been based upon raising protective antibodies by actively vaccinating with β-glucan conjugated to a carrier protein. To determine the potential for an Als3-based vaccine, the recombinant N terminus of Als3p (rAls3p-N) was expressed in Saccharomyces cerevisiae and purified by Ni-agarose affinity purification, in the same manner as described for rAls1p-N. The concept of niche vaccination of acutely at-risk patients or patients in restricted geographical areas is a new idea in vaccinology.

Immunohistochemical studies identified serous cells of the glandular acini as the cells responsible for production of salivary histatins. Histatins exhibit fungicidal activity against several Candida species, Aspergillus fumigatus, some strains of Saccharomyces cerevisiae, and Cryptococcus neoformans. Studies of levels of salivary histatins in vivo show large intersubject variation in both the concentrations of histatins and their rates of degradation. The total concentration of histatins in whole saliva is balanced between secretion of new proteins and removal of “older” proteins by degradation. Endocytosis was initially suggested as a means of histatin cellular entry based upon the observation that bafilomycin, an inhibitor of endosomal acidification, significantly decreased antifungal activity. Confocal imaging of C. albicans cells showed that some histatin 5 was localized to the vacuole but that cells containing only vacuolar histatin were viable. The cell wall of C. albicans is a thick multilayered structure of glucans, chitin, and mannoproteins that protects cells from osmotic stress and maintains structural integrity. Animal and human clinical studies to evaluate histatins as topical agents in prevention of gingivitis reported therapeutic efficacy without adverse side effects. The major requirements for effective use of salt-insensitive fungicidal peptides are selective and specific binding and uptake by candidal cells, efficacy at low concentrations that allow rapid eradication of yeast pathogens within the ionic strength of saliva, and minimal fungal resistance.

This chapter provides an update on cell wall structure and, in particular, cell wall glycoproteins (CWPs), cell wall remodeling, and cell wall regulation. Proteins associated with glycolysis, like enolase and alcohol dehydrogenase, are well-known cytoplasmic proteins but have also been identified at the cell surface; this dual location has led them to be termed “moonlighting” proteins. Glycosylphosphatidylinositol (GPI) anchoring is encountered in every eukaryotic cell, including unicellular yeast cells, several parasites, and highly specialized mammalian cells. Analyses of the Candida albicans and C. dubliniensis genomes to identify intragenic tandem repeats found a significant enrichment of putative CWPs. Mutants of GAS genes in C. albicans, Saccharomyces cerevisiae, C. glabrata, and Aspergillus fumigatus had defects in cell wall organization and morphogenesis. Single and double disruption of the A. fumigatus Gas orthologues GEL1 and GEL2 showed that the enzymatic activity was required for morphogenesis and virulence. The poor resolution of fungal glycoproteins through sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis has prompted alternative nongel technologies to study the complement of proteins in the cell wall. Transcript profiling of C. albicans cells from in vivo models has identified a number of cell wall-associated genes whose expression is altered compared to those in in vitro-grown cells. As well as being exposed to different stimuli that are independently known to alter expression of cell wall-related genes, the invading fungus will also be under attack by the host’s enzymes and immune cells.

This chapter summarizes the current understanding of stress responses in Candida albicans. Of all the pathogenic Candida species, the stress responses of C. albicans have been investigated in the greatest depth. Cellular responses to stresses in C. albicans include heat shock response, osmotic stress response and oxidative stress response. Stress-signaling pathways include mitogen-activated protein kinase (MAPK) pathway, Hog1 pathway and Mkc1 pathway. The chapter describes one's understanding of the molecular mechanisms that regulate stress responses in C. albicans and, where information is available, C. glabrata, contrasting these mechanisms mainly with those in Saccharomyces cerevisiae and S. pombe. Redox-sensitive antioxidant proteins, with roles in the detoxification of reactive oxygen species, can also act as sensors and regulators of reactive oxygen species-induced signal transduction pathways. Transcription factors that drive stress responses includes Cap1, Skn7 and Msn4. The structures of Hog1 signaling networks differ between C. glabrata and C. albicans. As these differences must contribute to the behavior of these pathogens in their hosts, it is important that these differences are addressed at a molecular level. Quantitative mathematical modeling of these responses will provide an invaluable foil to our more classical molecular and genomic approaches.

This chapter deals with cell surface proteins in two Candida species, C. albicans and C. glabrata, and in their nonpathogenic relative Saccharomyces cerevisiae, specifically focusing on the diversity and function of proteins implicated in adherence of the yeasts to each other or to other surfaces, including host cells. The most extensively studied family of adhesins in any Candida species is the one corresponding to the C. albicans ALS gene family, which includes eight family members. Two adhesins in family IV have similarity to the protein corresponding to DAN1 in S. cerevisiae, another subtelomeric adhesin whose expression is regulated by oxygen availability in the environment. Mass spectrometry was identify covalently linked cell wall proteins (CWPs), including adhesins, on the surface of C. glabrata. In total, five adhesins were detected: Epa6 and four uncharacterized proteins that were subsequently named Awp1 to -4 (adhesin-like wall protein). The FLO genes play essential roles during flocculation, a process by which yeast cells bind to the mannose residues on the surface of neighboring cells, forming clusters of cells called flocs, and sediment. CWPs are key to the interaction of fungi with the environment, and not surprisingly, changes within the CWP repertoire accompany the evolutionary adaptation to a particular niche. The relatively limited repertoire of FLO genes in S. cerevisiae primarily serves to mediate yeast-yeast interactions that adapt it to growth in communities/biofilms as well as to respond to nutritional cues.

This chapter reviews the changes in cellular physiology that follow contact with host cells and the impact of these changes on the host-pathogen interaction. The focus is on Candida albicans, for the simple reason that the vast majority of the published research is on this species. The chapter focuses on the interaction between Candida cells and the most important and relevant mammalian cell types. Reflecting the scope of available literature, the chapter concentrates on Candida interactions with neutrophils and macrophages. Stress responses include reactive oxygen species (ROS), reactive nitrogen species (RNS) and superoxide dismutases (SODs). The chapter focuses on the effectors and their roles in mediating the interaction with host cells. Invasion and endocytosis are terms primarily used to describe the internalization of Candida into normally nonphagocytic cells, mostly endothelial and epithelial cells. A surprising finding from the analysis of macrophagephagocytosed cells was the role of carbon starvation, which represented nearly two-thirds of the genes whose expression changed. The adhesion between Candida cells and phagocytes is primarily driven by receptors on the mammalian cell that recognize carbohydrate moieties on the cell wall, though there are some fungal proteins that mediate adhesion as well.

This chapter summarizes some of the known and proposed infection strategies and underlying transcriptional responses which govern them. Although similar strategies may be used by other pathogenic Candida species which are commonly found as commensals, the focus is on Candida albicans as the most common and best-investigated species. The ability of C. albicans to colonize surfaces therefore constitutes a vital first step in many forms of candidiasis. In nature, the gastrointestinal (GI) tracts of neither rats nor mice are colonized by C. albicans, and in order to establish colonization, some form of treatment such as the use of antibiotics is usually necessary. Certain predisposing conditions permit C. albicans to switch from a harmless commensal of the oral mucosa to an aggressive pathogen able to cause superficial infections of the oral cavity and oropharyngeal regions. The commensally colonized tissue could then be monitored to detect the natural fluctuations in C. albicans gene expression in the oral cavity. Simultaneously, the transcript profile of C. albicans in the disease state could be determined over time. To study the virulence properties of C. albicans in a mouse model, two infection routes are commonly used. Intravenous infection results in direct hematogenous dissemination via the bloodstream. Another method for inducing invasive candidiasis is via intraperitoneal infection.

This chapter focuses on biofilm formation by the pathogenic fungus Candida albicans. Biofilms are surface-associated microbial communities surrounded by an extracellular matrix. The chapter examines the steps of biofilm formation, from the genes known to function in C. albicans biofilm development, the cell-cell communication within the biofilm, the environmental responses that contribute to biofilm formation, the drug resistance of biofilms, and experimental techniques used to study biofilms. Cell wall genes and adhesins provide mechanisms that promote biofilm initiation, and the transcription factors that regulate their expression couple biofilm initiation with internal and external signals. The study of transcription factors has laid the framework for gene regulatory networks. While much is now known, detailed testing of other known adherence factors, cell wall proteins, transcription factors, kinases, and others is required to identify more genes involved in biofilm formation, and the elucidation of upstream regulation of these factors will allow for greater insights into the signaling events important for biofilm development. Quorum sensing governs functions as diverse as bioluminescence and virulence. It has a vital role in bacterial biofilm dynamics, and its role in C. albicans biofilms is now beginning to be understood. While major challenges lie ahead to further refine the gene regulatory networks and correlate them to in vivo results, it is nonetheless an exciting time for the field of C. albicans biofilm formation.

This chapter emphasizes the underlying mechanisms that govern the physical and chemical interactions between organisms and their potential relevance to disease. Many biofilms within the host are not simply single species assemblages, but rather, dynamic polymicrobial communities. Candida spp. in biofilms on medical devices are most often derived from the host’s own endogenous flora, and other microflora organisms, such as staphylococci, are also often associated with catheters in both the presence and absence of Candida albicans. Direct consequences of mixed infections could include C. albicans enhancement of Pseudomonas aeruginosa biofilm formation, antibiotic resistance or virulence factor production, or changes in immune responses or host tissues that result from simultaneous infection by bacteria and fungi. While C. albicans frequently encounters large numbers of gram-positive bacteria in the oral, intestinal, and skin microfloras, a relatively small number of studies describe the molecular interactions that occur between C. albicans and gram-positive microbes. A series of clinical trials have investigated the use of probiotics for the management of recurrent vulvovaginal candidiasis, and while the data are in conclusive, further in vitro and animal studies are warranted. Unicellular organisms often produce small, diffusible chemical signals, referred to as quorum-sensing molecules (QSMs), that coordinate group behavior within single-species populations.

This chapter reviews the role of mitochondria in human fungal pathogens, focusing upon Candida albicans, Candida glabrata, and Candida parapsilosis. The respiratory pathways of Candida species are highlighted along with the roles of mitochondria in morphogenesis, adaptive responses to oxidant stress and carbon depletion, and antifungal drug resistance and sensitivity. The focus therefore is on the role of mitochondria in the pathogenesis of candidiasis and developing the concept that there are differences in mitochondria of mammalian cells versus pathogens. The study of mitochondria in Candida species first followed those that were of industrial importance, examples being Candida utilis and Candida lipolytica. The morphogenesis of C. albicans has long been featured as critical to the establishment of candidiasis. Mitochondria play an active role in detoxifying reactive oxidant species (ROS) produced during cell metabolism. This has been convincingly shown through the use of inhibitors of respiratory complexes. 7-Chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) increases the activity of several antimycotics, including flucytosine as well as azoles. The target of histatin 5, a human basic salivary peptide that has demonstrated antifungal activity, was initially shown to be the energized mitochondria. There is ample proof that mitochondrial functions as well as regulation of these functions vary among pathogenic and model fungi, and that mammalian mitochondria have less complexity with regard to respiratory pathways.

Antifungal therapeutic outcomes have been historically suboptimal, in part, due to a relatively small number of safe and effective antifungal drugs. There are many important characteristics of antifungal drugs to consider in treatment of invasive fungal infection. Among these traits, spectrum of activity, pharmacokinetics, pharmacodynamics, potential drug-drug interactions, and toxicities are the most critical. This chapter focuses on these antifungal traits for available systemic agents for treatment of Candida infection. The main toxicities include renal damage, electrolyte abnormalities from renal toxicity, hepato-toxicity, and infusion-related reactions. The majority of the drug-drug interactions are related to potentiation of the electrolyte disturbances and renal dysfunction typical of amphotericin B. For example, the risk of renal toxicity is increased if amphotericin B is used concomitantly with the organ transplant immuno-suppressants cyclosporine and tacrolimus. In addition, lipid-based amphotericin B formulations are preferred for treatment of pregnant patients due to gestational toxicity with the triazole drug class. Studies have found the primary toxicity of flucytosine, bone marrow toxicity, to be associated with high peak concentrations. The fact that the pharmacodynamic drivers of success and toxicity are different provides an opportunity to design dosing strategies to both optimize treatment efficacy and reduce toxicity. The major toxicities of flucytosine include bone marrow suppression and hepato-toxicity. The major route of elimination for echinocandins is non-enzymatic degradation to inactive molecules which are excreted primarily in the bile.

This chapter critically evaluates some common assumptions regarding antifungal resistance and highlights key clinical problems that arise when managing patients with invasive infections caused by antifungal-resistant Candida species. Although this paper gives perhaps the first example of in vivo-acquired mutations in a fungal gene with a positive impact on in vivo fitness, all of the clinical isolates used in the study were acquired from semi-invasive infections of the oropharynx rather than the bloodstream of patients. The majority of patients with invasive mycoses probably fail therapy because of underlying host factors, rather than acquired resistance to the drug. One of the most underappreciated causes of treatment failures in Candida species is biofilm-mediated resistance. Problems associated with timely diagnosis and early detection of antifungal resistance in Candida species have not improved over the last two decades, as current testing approaches still rely primarily on blood cultures, which may be negative for up to 50% of patients. It is critical that the diagnostics and treatment tools to manage antifungal resistance in Candida species be developed now, along with clinical stewardship programs to effectively integrate their use in clinic, so that we are better equipped to deal with the challenges on the horizon that will be encountered in an increasingly complex and aged population of patients in the health care system.

This chapter is divided into two sections. The first section focuses on clinical perspectives, especially of global candidiasis. The second section discusses antifungal drug discovery by offering two different but interacting approaches: traditional, or classical, and genomic. A large study was recently published on a 10.5-year surveillance of susceptibility of Candida species to fluconazole and voriconazole. This study reported a slight trend toward an increasing resistance, in some regions, of NAC species like Candida tropicalis and Candida parapsilosis. In human immunodeficiency virus (HIV)/AIDS patients especially, mucosal candidiasis, or oropharyngeal candidiasis (OPC), remains one of the most common types of infection throughout the world, but especially in both adult and pediatric age groups in developing countries. Information on the incidence of vulvovaginal candidiasis (VVC) is incomplete as it is not reportable. However, the estimate is that VVC caused by Candida species affects about 70 to 75% of young women of childbearing age (most frequent); 40 to 50% of these individuals will have a recurrence, and 5 to 8% will develop recurrent VVC, defined as four or more episodes per year. Systemic candidiasis and bloodstream infections (BSI) also qualify as global infectious diseases. An increased incidence of invasive candidiasis (IC), aspergillosis, and zygomycosis has been reported in tertiary care facilities in India. The currently used echinocandins (caspofungin, micafungin, and anidulafungin) are still relatively new, and studies relating to their in vivo efficacy are ongoing.

Candida species are an important cause of both mucosal and invasive opportunistic infections among immuno-compromised patient populations, such as cancer patients receiving cytotoxic chemotherapy, solid-organ and bone marrow transplant patients receiving immunosuppressant therapy, and patients infected with human immunodeficiency virus (HIV) and suffering from AIDS. Moreover, recent reports have highlighted a paradoxical OPC infection rate of 30% in HIV-infected individuals who have shown improvements in CD4+ counts and have been classified as ''immune reconstituted''. This chapter focuses on the transcriptional regulation of azole antifungal resistance as well as transcriptional regulators that influence azole susceptibility in Candida species. Much of our understanding of azole antifungal resistance in Candida species has been greatly facilitated by the study of the pleiotropic drug resistance phenotype in Saccharomyces cerevisiae, which is largely driven by ATP-binding cassette (ABC) transporters such as Pdr5p. In a study to generate a more global assessment of Ndt80p, it was found that Ndt80p was bound to the promoters of many genes previously identified as being associated with azole resistance, including the ABC transporter genes CDR1, CDR2, and CDR4; the MFS transporter genes MDR1, FLU1, NAG3, and NAG4; the flippase genes RTA2 and RTA3; and other azole resistance-associated genes, PDR16, ERG3, and ERG11.

Mucosal candidiasis is extremely common and vastly more common than invasive, systemic candidiasis, although the latter receives far more attention due to the accompanying mortality. Although considerable progress has been made in the understanding of pathogenesis of mucosal candidiasis, considerable deficits in our knowledge persist, accompanied by dynamic changes in microbiology of the responsible Candida species and antifungal drug susceptibility, including drug resistance. Mucosal candidiasis involving the oral, gastrointestinal, and vaginal mucosae represents the commonest forms of superficial candidiasis and contrasts significantly with systemic and visceral candidiasis and disease. Although involving widely different anatomical sites with profoundly divergent physiological influences, the forms of mucosal candidiasis at the various sites have much in common with regard to pathophysiology, diagnostic principles, clinical manifestations, and therapy. Oral candidiasis (OC) is a commonly encountered condition in clinical practice, almost invariably associated with underlying disease or risk factors. Esophageal candidiasis (EC) is seen less commonly than OC, though usually in more immuno-compromised patients. The hormonal dependence of vulvovaginal candidiasis (VVC) is illustrated by the facts that Candida is seldom isolated from premenarchial girls and the prevalence of Candida vaginitis is lower after menopause, except in women taking hormone replacement therapy. In spite of progress in understanding the pathogenesis of mucosal candidiasis, there continues to be significant defects in one's knowledge of normal mucosal host defense mechanisms, both innate and acquired, effective in preventing symptomatic mucosal candidiasis.

This chapter reviews the clinical characteristics and specific treatment recommendations for the various types of invasive candidiasis, beginning with candidemia. Systemic candidiasis refers to end-organ disease stemming from hematogenous seeding, since any blood-borne Candidainfection may infect the organs through which it circulates. The risk factors for candidemia are numerous and well described in the chapter, including neutropenia, receipt of immunosuppressive drugs and other conditions associated with suppressed immune function, receipt of broad-spectrum antibiotics, use of central venous catheters and total parenteral nutrition, gastrointestinal surgery, burn injuries, and hemo-dialysis and other forms of renal replacement therapy. The chapter also reviews empirical therapy and treatment of proven candidemia in both nonneutropenic and neutropenic hosts. Unlike the case with non-neutropenic hosts, the use of empirical antifungal therapy among febrile neutropenic hosts is well established and validated by outcome data in clinical trials. In considering the treatment of proven candidemia it is useful to divide management decisions into those made prior to the identification of Candida species and those made afterwards. Candidemia due to Candida parapsilosis is generally catheter related, favoring routine catheter removal. All patients with candidemia require an ophthalmologic examination to exclude Candida endophthalmitis. This examination is best undertaken after the initiation of therapy and sterilization of the bloodstream, in order to minimize the possibility of subsequently seeding the eye. Similarly, any findings suggestive of an infectious process in other end organs should be investigated to exclude invasive candidiasis.

Invasive candidiasis is an infection of increasing prevalence which is associated with high morbidity and mortality. The rising incidence of invasive candidiasis is likely related to increasing numbers of immuno-compromised patients and lifesaving medical/surgical interventions that prolong life but break down both immunological and physical host defenses. The available tests include serologies focusing on the fungal cell wall components mannan, galactomannan, and (1,3)-ß-d-glucan (BG) or antibodies against these antigens, as well as genetic material detection tests. This chapter focuses on these new markers and their use for advanced prevention and treatment strategies. Research has shown that BG levels are not affected by Candida colonization. Aside from BG, which is currently commercially available throughout the world, there is intense research for new biomarkers. This is driven by the fact that current diagnostic methods are less than optimal, by the interest of pharmaceutical companies in finding new surrogate markers for diagnosis and outcomes assessment for drug development trials, and by the recent interest of commercial diagnostic laboratories in these methods. Antifungal prophylaxis is an attractive strategy, as the best way to deal with a fungal infection is to prevent it from ever occurring.

This chapter discusses the epidemiological profile of candidemia and invasive candidiasis (IC) worldwide as well as risk factors for infection with selected species. The susceptibility of the various species of Candida to antifungal agents is also discussed in this chapter. Important new findings in U.S. survey include an apparent increase in fluconazole resistance among C. glabrata bloodstream infections (BSI) isolates from pediatric and adolescent patients (7% in the period from 1992 to 2001 and 27% in the period from 2001 to 2007) as well as a very low rate of fluconazole resistance (6.8%) among BSI isolates from older (greater than equal to 70 years) patients. The current international guidelines for the management of sepsis recommend (in addition to numerous supportive interventions) the initiation of empirical antimicrobial therapy within 1 h of presentation with severe sepsis. Among the 16 Candida species discussed in this chapter, C. albicans, C. parapsilosis, C. tropicalis, C. lusitaniae, C. dubliniensis, C. metapsilosis, and C. orthopsilosis remain reliably susceptible to fluconazole and voriconazole. IC is an important and persistent public health problem. The incidence and mortality rates associated with this infectious disease have remained unchanged for more than two decades despite major advances in the field of antifungal therapy. Knowledge of the local and regional epidemiology as to the prevalent species and their susceptibility to the available antifungal agents is now more important than ever.

Interactions between proteins lie at the core of many crucial processes in the cell, including signal transduction, translation, transcription, DNA replication, and metabolic pathways. As a genetic approach, the yeast two-hybrid system has been highly successful in confirming and discovering protein-protein interactions. The principle of this method is based on the creation of two hybrid proteins, one fused to a DNA-binding domain (DBD; the "bait") and the other one fused to a transcription activation domain (AD; "prey"). Recently, an alternative Candida albicans two-hybrid system was developed, called the vesicle capture assay. This method is based upon the construction of two hybrid proteins, one protein that is fused to the vesicle targeted protein Vps32 and one protein fused to green fluorescent protein (GFP). This chapter describes the C. albicans two-hybrid system based upon the classic approach of transcription factor complementation. To develop a two-hybrid system suitable for C. albicans, all components of a yeast two-hybrid system needed to be included, with the requirement that each component was functional in C. albicans. In conclusion, this C. albicans two-hybrid system can be used to enhance one's knowledge of protein-protein interactions in C. albicans. Although only one-to-one interactions were tested until now, the method should be compatible for screening experiments due to the selective step involved.

The cell surface of Candida albicans is necessary for colonization of the human host and is also the target of the immune system when C. albicans enters the bloodstream as an opportunistic pathogen. In a study to develop C. albicans cell surface protein microarray, cell surface antigens that are specific to different phases (i.e., acute phase and early and midconvalescence) of candidemia, were identified. The study identified a set of 13 cell surface antigens capable of distinguishing acute candidemia from healthy individuals and uninfected hospital patients with commensal colonization. Studies need to address whether the serodiagnostic antigens identified in this study could provide protection from hematogenously disseminated candidiasis and whether sero-logical differences exist between superficial (i.e., thrush and vaginal candidiasis) and systemic infections. The increasing number of candidal infections necessitates the assay development of protein microarrays that include the whole proteome of not just C. albicans but also other pathogenic Candida species, as these studies may elucidate additional sero-diagnostic antigens and/or vaccine candidates. A high-throughput in vivo transformation system using a pXT7 linear vector was employed for cloning PCR products of genes encoding cell surface proteins. For PCR amplification, all forward and reverse primers had common 33-nucleotide-long sequences at the 5' end, followed by a gene-specific sequence (20 to 26 nucleotides). The protein microarray was produced by printing the in vitro-expressed proteins in duplicate onto nitrocellulose-coated FAST glass slides at a density of 960 spots per slide using an automatic GMS417 robot and the OmniGrid 100.

Although the genetic study of Candida albicans is generally less convenient than that of Saccharomyces cerevisiae, recent advances in the molecular genetics of C. albicans have greatly facilitated the study of this important human fungal pathogen. This chapter discusses the development of a novel strategy for large-scale synthetic genetic analysis in C. albicans based on complex haploin-sufficiency (CHI). An actin null mutant (act1?) containing ACT1 on a plasmid was mated to the set of single-gene-deletion strains and then cured of the plasmid to give a set of complex heterozygotes (act1Δ/ACT1 yfgΔ/YFG). Many of the genes identified using this genetic approach demonstrated defects in the actin cytoskeleton, confirming the ability of CHI to identify interacting pairs of genes and genes with similar cellular functions. The development of a large-scale synthetic genetic screen using CHI to understand the role of the RAM network in C. albicans morphological transition is described. As new mutagenesis techniques are developed and as large collections of mutants are created, it is likely that the creative application of these principles to genetic interaction analysis will lead to a deeper understanding of the complex regulatory networks that orchestrate C. albicans biology and pathogenesis.

Studies investigating the interaction of Candida with a single host cell type such as phagocytes or epithelial cells are typically performed in vitro. The advantages of in vitro studies include the ability to compare multiple conditions in one experiment, the ability to perform thorough biochemical and immunologic analyses, the relatively short duration of experiments, lower experimental costs, and the ability to reduce the use of laboratory animals. The major disadvantage of in vitro experiments is that it is not possible to fully replicate the myriad of cellular and extracellular signals provided by the host environment. Several animal models of candidiasis are regularly used, including models of oropharyngeal, vaginal, and disseminated candidiasis. Information about the interaction of specific host cell types with Candida organisms in vivo can sometimes be obtained through conventional histology or immunohistochemistry, but these analyses require sacrifice of the animal and thus are often limited to a single time point during the course of infection. Several fluorescent proteins have recently been codon optimized for expression in yeast. The authors suspect that any strain of Candida that has relatively bright fluorescence could be used in this model. For imaging, an inverted confocal microscope with an enclosed stage incubator chamber and generic stage insert is used. Recently, a stage insert was constructed allowing the use of an Olympus FV1000 laser scanning confocal microscope at the confocal microscopy core facility. Images were successfully obtained using both 10x, 0.4to 0.45-numerical-aperture and 20x, 0.75-NA dry objective lenses.

This chapter presents the strategy developed to generate a Candida albicans ORFeome collection in a versatile Gateway vector, which allows the transfer of the cloned genes into a variety of C. albicans Gateway-compatible expression vectors. The development of collections of over-expression strains is facilitated by the pre-establishment of a complete set of cloned open reading frames (ORFs), or ORFeome. Importantly, ORFeomes represent useful resources for the implementation of other approaches used to elucidate gene function apart from gene deletion or overexpression. Individual characterization of mutants on the genome-wide scale can be slow and laborious. In this respect, signature-tagged mutagenesis (STM) provides an attractive alternative. In STM, each mutant is tagged with a different DNA sequence, allowing all tags to be amplified from the DNA of mixed populations of mutants in a single PCR. In the C. albicans ORFeome, the start codon of the 6,205 ORFs has been included, whereas the stop codon has been excluded to allow insertion of C-terminal tags. The authors have favored C-terminal tagging since tags at the amino termini may possibly interfere with targeting of proteins to the secretory pathway. A panel of libraries of strains expressing tagged proteins should facilitate the systematic execution of high-throughput biochemical and microscopic assays of the C. albicans proteome. The construction of the ORFeome could pave the way to the generation of the C. albicans promoterome (library of gene promoters) in order to better characterize expression profiles.