Effects of Depleting the Essential Central Metabolic Enzyme Fructose-1,6-Bisphosphate Aldolase on the Growth and Viability of Candida albicans : Implications for Antifungal Drug Target Discovery

Abstract

The central metabolic enzyme fructose-1,6-bisphosphate aldolase (Fba1p) catalyzes a reversible reaction required for both glycolysis and gluconeogenesis. Fba1p is a potential antifungal target because it is essential in yeast and because fungal and human aldolases differ significantly. To test the validity of Fba1p as an antifungal target, we have examined the effects of depleting this enzyme in the major fungal pathogen Candida albicans. Using a methionine/cysteine-conditional mutant (MET3-FBA1/fba1), we have shown that Fba1p is required for the growth of C. albicans. However, Fba1p must be depleted to below 5% of wild-type levels before growth is blocked. Furthermore, Fba1p depletion exerts static rather than cidal effects upon C. albicans. Fba1p is a relatively abundant and stable protein in C. albicans, and hence, Fba1p levels decay relatively slowly following MET3-FBA1 shutoff. Taken together, our observations can account for our observation that the virulence of MET3-FBA1/fba1 cells is only partially attenuated in the mouse model of systemic candidiasis. We conclude that an antifungal drug directed against Fba1p would have to be potent to be effective.Candida albicans is the major systemic fungal pathogen of humans (5, 35). This fungus is carried as a commensal in the oral and gastrointestinal tracts of many individuals but can cause oral and vaginal infections if normal fungus-host interactions are disturbed. In severely immunocompromised patients, C. albicans can cause life-threatening systemic infections (35). A range of virulence attributes, including adhesion, morphogenesis, phenotypic switching, and the secretion of hydrolytic enzymes, contributes to the pathogenicity of this fungus (5,6,35).A limited range of antifungal drugs is available to combat Candida infections. Those drugs in routine clinical use include the polyenes, azoles, and echinocandins (36). Polyenes, such as amphotericin B, are thought to bind ergosterol in the fungal plasma membrane, the azoles inhibit ergosterol biosynthesis, and the echinocandins inhibit glucan synthesis (36). The search for novel, broad-spectrum drugs that are fungicidal (rather than fungistatic) and yet exert no significant side effects upon the patient continues. Not surprisingly, this search has focused mainly upon fungus-specific processes, such as cell wall or ergosterol biosynthesis. However, metabolic enzymes have been targeted as potential antibiotic targets in other microbial pathogens (9, 34, 37).The ability of C. albicans to thrive in its mammalian host is due not only to its virulence factors but also to its metabolic flexibility. This fungus can assimilate fermentative or nonfermentative carbon sources, depending upon the host niche it occupies (1,4,16,27). For example, C. albicans activates the glyoxylate cycle and gluconeogenesis following phagocytosis by white blood cells, whereas the glycolytic pathway is activated in most fungal cells infecting the kidney (1, 17). These pathways are critical for the virulence of C. albicans (1,27). Therefore, an...

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

“…Attempts to inactivate fba-tb in M. tuberculosis H37Ra and M. tuberculosis H37Rv in the absence of a rescue copy of this gene yielded no candidate mutant at the last selection step of the procedure, suggesting that, unlike the situation in Neisseria meningitidis (40) but similar to those in E. coli, Streptomyces, and Candida albicans (41)(42)(43), the class II FBA gene of M. tuberculosis is required for growth even under optimal laboratory growth conditions where both glucose and oleic acid are present in the culture medium. As proposed in the case of E. coli (44), it is likely that this requirement is related to the role of FBA-tb in preventing the toxic effect of FBP accumulation in the cells.…”

Role of Acetyl Coenzyme A Synthesis and Breakdown in Alternative Carbon Source Utilization in Candida albicans

“…lele using a tetracycline-regulated promoter (23), as the MET3 promoter is not appropriate for in vivo studies. However, the growth defects of ACS2-depleted strains are so severe that it is extremely unlikely that they would retain virulence; it has been shown that other mutants that cannot grow on glucose are avirulent in vivo (2,31).…”

Candida albicans Uses Multiple Mechanisms To Acquire the Essential Metabolite Inositol during Infection

“…Previous work had indicated that the presence of the P MET3 promoter on the essential gene CaFBA1 can compromise virulence (37). The methionine in the mouse bloodstream is presumably able to prevent the expression of the CaFBA1 gene sufficiently to compromise growth and virulence in the mouse.…”