Two-Component Signal Transduction Proteins as Potential Drug Targets in Medically Important Fungi

Treatment of diseases such as invasive candidiasis and invasive aspergillosis (IA) remains problematic for the clinician. Costs of patient care management are staggering and are most often associated with an increased length of stay in the hospital associated with a delayed therapeutic intervention and other problems (3, 45, 54, 70, 71). But IA and invasive candidiasis are only two of several clinically relevant fungal diseases. A significant number of infections are common among healthy populations, including vulvovaginal candidiasis (61). Also, cryptococcosis occurs in human immunodeficiency virus/AIDS patients, especially in developing countries, but also has been reported in an outbreak that likely included mostly healthy individuals (33). The dimorphic, endemic fungi are also major pathogens of otherwise healthy individuals. For example, the incidence of coccidioidomycosis alone is about 100,000 cases per year (24). Immunocompromised patients are at risk for these diseases also, and in fact, a 5 to 7% crude mortality rate has been observed in hospitalized patients (20). Further, the endemic mycoses like histoplasmosis can present as common-source or focal epidemics, which can result in disease in a significant number of patients (59). The extensive and expanding list of fungal pathogens and the frequency of their occurrence demand the availability of drugs to counter disease. New antifungal drugs are sought because the former “gold standard,” amphotericin B (binds to membrane ergosterol causing changes in permeability), invariably causes toxicity in the patient, negating the importance of its fungicidal activity. Triazoles (target ergosterol synthesis) are now more often used in treatment of fungal disease given their reduced toxicity and in many cases ease of treatment. However, the emergence of new species (Candida species other than Candida albicans) among clinical isolates is due to their lack of susceptibility to the triazoles. The β-1,3-glucan inhibitors (caspofungin and micafungin) are fungicidal but ineffective against Cryptococcus neoformans and of questionable value in IA patients (67). Terbinafine (an allylamine that targets ergosterol synthesis) offers promise although it currently is recommended only for superficial fungal infections. Drug discovery is currently based upon the paradigm that a target must be a growth-essential gene product. This review is intended to suggest that compounds that inhibit virulence factors of fungal pathogens need consideration for new antifungal drug discovery. This hypothesis was recently discussed in regard to antibacterial drug discovery (15). Species-specific virulence factors of human fungal pathogens such as the capsule of Cryptococcus neoformans are known. But we will develop the theme that a conserved signal transduction pathway that regulates the expression of virulence factors across fungal pathogens but that is not found in humans could represent a target for drug discovery. We distinguish “virulence-essential” from “growth-essential” gene products since most in the former category are not required for growth in vitro. Specifically, this review will focus upon two-component proteins that are critical to a number of processes fungi pathogenic to humans use to adapt to the host environment. First described for both pathogenic and environmental, nonpathogenic bacteria, the term “two component” reflects a requirement for two proteins, one a histidine kinase (HK), usually a transmembrane protein that autophosphorylates using ATP upon perception of an environmental cue (47). Phosphorelay is accomplished on a response regulator (RR) protein, which usually acts as a transcription factor to adapt cells to the environmental signal. A major difference between bacteria and lower eukaryotes is that the latter usually (but not always) require an intermediate protein, a histidine phosphotransfer protein (Hpt), which shuttles phosphate from the HKs to RR proteins. The classic pathway which has been studied extensively in fungi is the HOG1 (hyperosmotic glycerol) mitogen-activated protein kinase (MAPK) pathway (30). Regulation of the HOG1 MAPK pathway requires three upstream proteins that participate in a phosphotransfer relay, including Sln1p (a transmembrane HK), Ypd1p (a cytoplasmic Hpt), and Ssk1p (an RR protein). In addition, other HKs and at least one other RR are found in a variety of fungi, and those fungi pathogenic to humans are depicted in Fig. 1A and B. Most domain functions indicated for each protein are inferred from studies of model fungi. FIG. 1. Two-component signal proteins of selected fungal pathogens. (A) Domains of HKs from fungi pathogenic to humans. Black lines represent the relative sizes of the proteins. GAF, cyclic GMP phosphodiesterase/adenylcyclase/FhlA; HAMP, hepcidin antimicrobial . Curiously, in the absence of stress, phosphotransfer among Sln1p-Ypd1-Ssk1p occurs but activation of the HOG1 MAPK does not since the phosphorylated RR protein Ssk1p is unable to activate the Ssk2p MAPK kinase kinase of the HOG1 MAPK pathway, at least in C. albicans and Saccharomyces cerevisiae (Fig. ​(Fig.2A). 2A). There are sound reasons for this, including the fact that, in the absence of stress, cellular machinery is minimally used so energy is conserved. When stress signals are detected by cells (oxidants, high salt, etc.), the RR protein is not phosphorylated and is now able to activate the HOG1 MAPK pathway to adapt cells to stress (Fig. ​(Fig.2B). 2B). In the case of human pathogens, functions of this pathway compared to those of model fungi are expanded to regulate a number of other attributes such as virulence, host cell recognition, morphogenesis/dimorphism, survival in neutrophils, mating, and quorum sensing (see below). It should be mentioned that mutants lacking genes in the HOG1 MAPK pathway (ssk2, pbs2, hog1) are also oxidant sensitive and, at least in the case of the hog1 mutant, avirulent (18). FIG. 2. Signal pathways that include each of the HKs and RRs of C. albicans. (A) The HOG1 MAPK kinase pathway and the upstream two-component phosphotransfer proteins, Sln1p, Ypd1p, and Ssk1p. In the absence of stress, phosphotransfer reactions occur on these .