Yeast infections in human have increased significantly in recent years. Among the pathogens, Candida albicans is the most dominant one. Currently, the available antifungal drugs have undesirable issues such as side effects, ineffective against new or reemerging fungi, and leading to the rapid development of resistance (White et al., 1998; Yang and Lo, 2001). Candida albicans is an opportunistic fungal pathogen, commensally colonizes various anatomical sites in humans. In the
immunocompromise individuals, such as the ones with HIV infection, diabetes, organ transplantation and cancer chemotherapy, C. albicans can become virulent and
invasive (Edwards, 1990). Candida albicans not only causes mucosal diseases (such as oropharyngeal/esophageal and vulvovaginal candidiasis), but can also invade the bloodstream (candidaemia). It has emerged as the fourth most common cause of nosocomial infection (Beck-Sague and Jarvis, 1993). The estimated cost for treating this Candida nosocomial infection approaches 1 billion US Dollars per year in the United States (Miller et al., 2001). Understanding the regulatory mechanisms and gene functions of pathogenesis-related pathways may provide us the knowledge and drug targets for anti-fungal application. Although several factors associated with pathogenesis, such as environmental cues, nutrition availability, drug resistance, and virulence factors (such as morphogenesis, extracellular hydrolytic activities, and phenotype switch) have been identified in C. albicans, the picture of the global
networking and coordination of those regulations and signaling pathways contributing to pathogenesis in Candida albicans is still lacking. Noteworthy, those pathways are intertwined and only handful controlling points are known to modulate the global activities. There are the well-known major controlling point EFG1 and the two lesser candidates, CPH1, and TUP1. In this project, we will focus on the
relationship between environmental cues, stress, and morphogenesis, with emphasis on the connection to one of the major pathogenesis control point, Efg1 pathway.
Complexity of pathogenesis in C. albicans and Efg1 as a major controlling point
Signaling pathways commonly sense and transfer environmental signals to downstream regulators that lead to the expression of subsets of genes potentially related to Candida pathogenesis. Those subsets of genes are known as virulence factors. Several virulence factors of C. albicans have been proposed, including hyphal morphogenesis, extracellular hydrolytic activities (e.g. secreted aspartyl proteinases and lipases) (Calderone and Fonzi, 2001; Gow et al., 2002; Naglik et al., 2004; Stehr et al., 2004; Sundstrom, 2002; Yang, 2003), and phenotypic switching.
In hyphal morphogenesis, cells transit from an ovoid yeast shape to filament forms (pseudohyphae and hyphae); both filamentous forms are able to again produce yeast forms (Lo et al., 1997). Hyphal morphogenesis can be induced by a number of environmental cues, including the presence of serum, N-acetylglucosamine (GlcNAc), proline, neutral pH and elevated temperature (Ernst, 2000). The secreted aspartyl proteinases are encoded by a large SAP gene family of ten members, each
differentially regulated under a variety of conditions (e.g. pH, temperature and cell morphology) (Naglik et al., 2004). Finally, C. albicans reversibly switches phenotypes with a high frequency (Soll, 2002) known as the phenotypic switching.
It occurs spontaneously and is also induced by temperature and low doses of UV irradiation (Soll, 1997). These cells that undergo the switch of phenotypes vary in morphology, physiology, metabolism and pathogenicity (Lan et al., 2002; Soll, 1997).
But then how do these "factors" connect to the pathogenesis? In C. albicans yeast-hyphae morphogenesis, the presence of serum activates adenylate cyclase (Cdc35), and thus promotes cyclic adenosine monophosphate (cAMP) production.
Cyclic AMP acts as an intracellular regulator and in turn activates protein kinase A (PKA) that mediates its downstream signal via Efg1. Expression of the C. albicans HMX1 gene, which encodes a heme oxygenase required for utilization of exogenous heme and hemoglobin, is shown to be strongly de-regulated in an EFG1-null mutant (Santos et al., 2003). In C. albicans, the pH response is governed in part by the transcription factor Rim101. Rim101 promotes alkaline responses by repressing expression of Nrg1, itself a transcriptional repressor (Bensen et al., 2004). Rim101 and Nrg1 are also shown to act in parallel pathways to control hyphae morphogenesis.
Interestingly, an alkaline pH condition induces expression of subsets of genes, including several iron acquisition genes also mediated by Rim101 (Bensen et al., 2004). Moreover, in the yeast model system, Saccharomyces cerevisiae, cAMP controls the activity of ferrireductases, components of a high-affinity iron uptake system. Another example is the Tpk2, a catalytic subunit of protein kinase A, whose expression negatively regulates iron uptake genes (Lesuisse et al., 1991; Robertson et al., 2000). And the complication does not stop here. Recently, we have reported that in addition to being a virulence factor, Efg1 is also involved in drug resistance (Lo et al., 2005). Together, these studies implicate that the decision-making presides over the onset of pathogenesis is made of a complex signaling and regulatory network, which includes multiple components and each of them may controls subsets of gene expression. However, those studies also point out the fact that situating among this complex network, there is the key regulator Efg1, which through coordination of different components/pathways, regulates various cell functions (ie. drug resistance, morphogenesis, and gene expression) in response to different environmental cues (e.g.
iron availability, serum, and temperature). Hence, we are interested in what is the networking centering at Efg1.
Recently, we have reported that Efg1 and its downstream target CaEno1 have multiple functions in C. calbicans (Lo et al., 2005; Yang et al., 2006). CaENO1 encodes enolase and is under the control of Efg1 (Nantel et al., 2002). Enolase is a highly conserved protein throughout the phylogenetic tree (Van der et al., 1991). In addition to the known function in the glycolytic pathway, enolase is a major
glucan-associated protein found in the fungal cell wall. It also serves as a receptor for human plasmin/plasminogen (Jong et al., 2003) and the major immunogen of Candida infection. In different cellular locations, its function varies. Interestingly, the locality of enolase in cancer cells is associated with the ability for metastasis.
Therefore, it is our interest to known the function of CaEno1 in connection to pathogenesis and the locality-function relationship.
The adaptation of C. albicans to the environments and its relation to the pathogenesis factors.
The ability of C. albicans to sense and adapt to alterations in host environments is integrated in its survival and pathogenicity (Soll, 2002). As it has been mentioned, several environmental conditions are known to affect cellular growth and
morphogenesis. In fact, they can even have direct impact on pathogenictiy. Those factors include various forms of stress and the availability of nutrients, for example, iron. The iron-free forms of host lactoferrin and transferrin inhibit C. albicans growth and render it more susceptible to damage by neutrophils (Okutomi et al., 1998). Iron deprivation affects the adhesive properties and cell wall compositions of C. albicans (Paul et al., 1989; Sweet and Douglas, 1991) and studies on suspension cultures and biofilms indicate that drug resistance of C. albicans is affected by iron availability (Baillie and Douglas, 1998; Paul et al., 1991). The high-affinity iron permease (CaFtr1) is required for systemic infection in a mouse model whereas the siderophore transporter (CaArn1) is required for epithelial invasion. Besides the endothelial cell injury caused by C. albicans is iron-dependent (Fratti et al., 1998;
Heymann et al., 2002; Ramanan and Wang, 2000). Moreover, in the human host, iron is mostly bound to high-affinity ligands (e.g. transferrin, hemoglobin, lactoferrin and ferritin), and there is virtually no free iron available. This iron-withholding is an important defense mechanism for the host; the availability of iron has shown to be a common signal to induce the expression of virulence factors of pathogens (Paul et al., 1991). But how do the signals from those environmental cues link to the
pathogenesis pathway? Do they achieve this by sending signal to one of the virulence factors? To study the molecular mechanism of stimulus-responsive gene
regulation in C. albicans using iron availability as the model, we have identified iron-regulated genes and a potential DNA-binding protein, Sfu1, which negatively regulates gene expression under iron-repletion conditions (Lan et al., 2004).
Recently, the cellular levels of iron are shown to be crucial for the mode of action of a topical antifungal agent ciclopirox olamine, while the detail mechanism is most unknown (Sigle et al., 2005). Therefore, we are interested in how does the iron availability affect the pathogenesis of C. albicans, especially regarding to the Efg1 pathway.
Another system we are interested to study is the response of Candida albicans to the stimuli from the host. In addition to growth and proliferation on mucosal
surfaces, ingestion by human immune cells exposes C. albicans to novel
environments. The cells respond to phagocytosis of neutrophils by inducing genes related to arginine and methionine pathways, suggesting that the phagosome of the neutrophil is an amino acid-deficient environment; however, neither pathway is induced upon phagocytosis by monocytes. An analysis of its transcriptional response upon internalization by macrophages reveals that C. albicans activates alternate metabolic pathways, represses translation and induces genes related to the oxidative stress response, DNA damage repair, arginine biosynthesis and peptide utilization (Lorenz et al., 2004). These results suggest that the environment of the macrophage phagosome lacks usable nutrients (e.g. glucose) and contains reactive oxygen and nitrogen species, as part of its antimicrobial burst (Fang, 2004).
Moreover, C. albicans encounters many antimicrobial proteins/peptides and innate defense molecules that act synergistically to combat infections. For example, some host proteins secreted onto the oral surface can directly inhibit Candida growth, morphogenesis, and adhesion through the action of antimicrobial peptides, including calprotectin (Sweet and Douglas, 1991), lysozyme (Laibe et al., 2003), low molecular weight salivary mucins (Satyanarayana et al., 2000), secretory leukocyte protease inhibitor (Chattopadhyay et al., 2004), lactoferrin (Samaranayake et al., 2001), and histatins (Lupetti et al., 2004). Finally, in the treatment of candidiasis, C. albicans also encounter various therapeutically antifungal drugs. All those stresses or cues induce the response of C. albicans cells and eventually via drug resistance and virulence/morphogenesis pathways send the signal to the controlling points of pathogenesis. Do all those signals eventually go to the well-known Efg1? Or do some of them go to other controlling points? For example, there are two potential candidates, Cph1 and Tup1. Therefore, we are interested in knowing which of those pathways linked to Efg1 and if not, where do those responses send their signals.
In conclusion, this research is to continue our effort on the Efg1-CaEno1
pathway and to expand the study by using genome-wide analysis to explore the networking and cross-talk between multiple components/pathways.