The recent outbreak of fungal meningitis caused by Exserohilum rostratum in patients receiving contaminated steroid injections resulted in 64 deaths, receiving a lot of press and briefly bringing into the public eye the difficulty of treating systemic fungal infections [1]. What is generally less well appreciated, however, is that there are several other, much more common fungal pathogens that pose a serious health threat. Indeed, currently more people die from these fungal diseases worldwide than from tuberculosis or malaria [2]. The fungal pathogens most frequently responsible for human mortality are: Aspergillus fumigatus, Candida spp. (predominantly C. albicans), Cryptococcus neoformans, Pneumocystis carinii, and dimorphic fungi that cause endemic mycoses (Coccidioides immitis, Histoplasma capsulatum, Blastomyces dermatitides, and Paracoccidioides brasiliensis). Fungal pathogens pose an especially high risk to individuals with compromised immunity, and this population of susceptible hosts is growing [3,4]. There has been a steady increase in the incidence of fungal infections over recent decades, primarily due to the AIDS pandemic, an increase in patients receiving cancer chemotherapy and allogeneic bone marrow transplants, a higher incidence of seriously ill patients in intensive care units, and the aging of the human population [3–8].

Despite the extensive list of fungal pathogens and the increasing frequency of their occurrence, we have at our disposal only a very limited number of antifungal drugs. The past two decades have seen the emergence of two classes of antifungals: those that target ergosterol synthesis (the azoles) and those that target cell wall β-1,3 glucan synthase (echinocandins). Of the azoles, the triazoles have gained importance as alternatives to the more toxic amphotericin B. Triazoles are fungistatic and their continued use has resulted in an increase in triazole resistance among formerly sensitive species and a rising number of disease cases caused by intrinsically azole-resistant non-albicans Candida species [4]. Echinocandins are fungicidal and are the drug of choice for treating most fungal infections, but these drugs are not effective in treating infections caused by C. neoformans, and echinocandin resistance is increasing in some Candida species [9]. Clearly, there is an urgent need to discover new drug targets to meet the challenges posed by fungal infections.

An ideal drug target is a fungal-specific protein that (1) functions as a virulence factor or is essential for fungal viability and (2) is absent from the host organism, such that its inhibition causes no toxicity in the host. The low number of drugs in our antifungal armamentarium is in part due to the relative evolutionary relatedness of fungi and mammals and the resulting paucity of fungal-specific proteins that meet these criteria. One class of molecules that do fit both of these criteria is comprised of proteins that function in the so-called two-component signal transduction pathways. These signaling pathways are based on the transfer of phosphoryl groups among their components (phosphorelays) and are one of the primary means by which bacteria and fungi sense and respond to environmental cues. Two-component signal transduction pathways present attractive targets for antifungal drug discovery because they exist in prokaryotes, plants, and lower eukaryotes but not in mammalian cells. Furthermore, while the genes encoding two-component system proteins are frequently not essential for viability, multiple studies have demonstrated the importance of two-component signal proteins for virulence in fungal pathogens, including A. fumigatus [10], C. albicans [11–13], C. neoformans [14], B. dermatitidis, and H. capsulatum [15] (Table 1).

The term “two-component” is derived from bacterial systems where the phosphorelay generally involves two proteins: a histidine kinase (HK) and a response regulator (RR) protein (Fig. 1). In response to an environmental signal, the HK, which is frequently localized in the bacterial outer membrane, is autophosphorylated on a conserved histidine residue, followed by a transfer of the phosphoryl group to a cognate response regulator protein (RR) on a conserved aspartate residue. The phosphorylated RR then usually acts directly as a transcription factor to activate genes associated with chemotaxis, stress response, quorum sensing, sporulation, virulence factor expression, and antibiotic resistance [16]. Fungal two-component phosphorelays are more intricate in two respects (Fig. 1). First, the signaling cascade involves three proteins: HK, RR, and a histidine phosphotransferase (HPt) whose function is to shuttle the phosphate moiety from HK to RR [17]. Second, in fungi, the phosphorelay typically comprises four phosphorylation events: (1) the HK is autophosphorylated on a histidine residue within its histidine kinase domain; (2) the phosphate is transferred intramolecularly to an aspartate (His→Asp) in the HK receiver domain; (3) a third, intermolecular phosphotransfer occurs to the histidine residue present in the HPt domain on the transferase (His→Asp→His); and (4) the phosphoryl group is relayed to an aspartate on the RR protein (His→Asp→His→Asp). Thus, two-component–like phosphorelay systems are unusual in terms of mechanism: the amino acids that accept phosphoryl groups are either aspartate or histidine residues. These unique features may be exploited in designing specific inhibitors that would not affect the activity of conventional Ser/Thr/Tyr kinases more prevalent in mammalian systems.

Depending on the system and the specific factors involved, the initial HK phosphorylation may occur either in response to stress or in response to removal of stress, with the ultimate outcome of either activating or down-regulating the transcription of stress response genes. A comprehensive list of all known fungal two-component signal transduction proteins and their roles in virulence-related processes can be found in Table 1. For a detailed discussion on the function of HKs and RRs in medically relevant fungi, the reader is directed to several excellent reviews [17–19].

The limited data available from studies of two-component proteins in fungal pathogens have revealed the critical functions of these proteins in adaptation to stress, regulation of virulence factors, and sensitivity to antifungal drugs, underscoring the importance of these signaling pathways in fungal pathogenesis. These features, together with the absence of two-component pathways in animals, make these proteins very attractive targets for antifungal drug discovery. Because two-component systems are found in all major fungal pathogens, drugs targeting these factors may have broad spectra. Recently developed tools for phosphohistidine analysis are likely to facilitate in vitro screening efforts, while complementary searches for pathway-based inhibitors may identify compounds that specifically inactivate two-component signal transduction in vivo.