Pneumocystis carinii appears to scavenge exogenous sterols, including cholesterol, from its mammalian host. As a result, it has long been held that their ability to scavenge cholesterol from their hosts, and their inability to undergo sterol biosynthesis, makes them resistant to antifungal drugs that target ergosterol or ergosterol biosynthesis. However, genome scans and in vitro assays indicate the presence of sterol biosynthetic genes within the P. Heterologous expression of P. Pneumocystis carinii , fungal sterol biosynthesis , Pneumocystis sterol uptake Introduction Members of the genus Pneumocystis are opportunistic fungi capable of causing a lethal pneumonia in mammalian hosts.
Pneumocystis colonization of immunocompetent hosts appears to have minimal clinical consequences, but colonization in hosts with debilitated or compromised immune systems may result in the development of Pneumocystis pneumonia PCP. Before the AIDS epidemic in the early s, PCP was a rare occurrence seen only in malnourished children, transplant recipients, cancer patients and those with immune deficiencies Gajdusek, Despite the fungal nature of Pneumocystis, drugs used for the treatment of PCP include pentamidine, atovaquone and combinations of either trimethoprim and sulfamethoxazole TMP-SMX or clindamycin and primaquine Hughes et al.
Pneumocystis are resistant to many standard antifungal drugs that target either enzymes involved in sterol biosynthesis or ergosterol, the end product of the sterol biosynthesis in fungal cells.
Resistance to these drugs has been attributed in part to the lack of detectable ergosterol within the membranes of Pneumocystis. Affinity binding analysis reveals azoles directly bind with high affinity to Cyp51 class of proteins in various organisms including A.
It is worth noting that azoles are effective in controlling growth of Aspergillus, but they are mostly fungistatic against the majority of A. Thus, a better understanding of the molecular mechanisms associated with sterol biosynthesis is needed to develop new therapeutic strategies particularly in the face of emerging triazole resistance. Sterol Biosynthesis in Aspergillus fumigatus Sterols are isoprenoid derived molecules and are a major component of eukaryotic cell membranes; necessary for fluidity, permeability and protein function.
Fungi are among the oldest eukaryotes known to synthesize sterols reviewed in Parks and Casey, The membrane was hybridized with the bp CgAUS1 probe left panel or 1. Lanes from the left : 1. Cg, clinical isolate; 2. CgTnS, Cgerg1 mutant; 3. DNA cloning and hybridization analyses were done according to the standard protocol.
GD mutation, regardless of fluconazole treatment, showed a full constitutive activation of Upc2, which is 1. This observation is consistent with the previous report that the CTD is essential for the transcription activation of Upc2 ref. The MR showed no increase of transcriptional activity on sterol depletion suggesting that dimerization is essential for the regulatory function. These data suggest that the C-terminal activation loop is essential not only for the regulation of protein localization but for the full transcription activity of Upc2.
Disruption of sterol binding by LF or LW showed constitutive nuclear localization. It seems that introduction of bulky residues to the sterol-binding pocket not only disrupt sterol binding but also perturb the proper conformation of Upc2 for full transcriptional activity.
These observations together with ligand binding and cell localization experiments suggest that sterol binding not only regulates protein localization but also regulates transcriptional activity of Upc2. To confirm that Upc2 is involved in the regulation of ergosterol level in the cells, we quantified total ergosterol contents of yeast cells containing wild-type or mutant UPC2 alleles Fig.
Since Upc2 activates the expression of ergosterol biosynthetic genes, it is conceivable that the sterol level of the yeast cells indirectly correlates with the transcriptional activity of Upc2. Wild-type Upc2 displayed a similar ergosterol level in the cell with those of LF and LW in the absence of fluconazole.
Upc2 lacking LBD showed some basal level of sterol synthesis in the absence of fluconazole. The cells expressing GD mutant have three times higher ergosterol levels compared with the cells expressing wild-type Upc2. Ergosterol levels were higher in fluconazole-treated GD mutant cells compared with those found in wild-type cells grown without fluconazole.
These data suggest that the GD mutation constitutively activates Upc2 for the biosynthesis of ergosterol, which leads to an increased resistance to azole antifungals. Figure 7: Ergosterol level and antibiotic resistance. The assay values represent the average of three independent experiments. All error bars indicate s. Each data point is the average of three measurements. The strains were initially grown without fluconazole and the dilution series were spotted on the agar plates containing fluconazole and incubated for 36 h.
Since fluconazole interferes with yeast cell growth by inhibiting ergosterol biosynthesis, the overall patterns of cell growths displayed a correlation with the sterol levels of the cells treated with fluconazole. However, GD mutant grew more weakly than wild type in the absence of fluconazole suggesting that full constitutive activation of Upc2 is disadvantageous under non-selective condition by imposing metabolic burden This is consistent with the previous observations that deletion of UPC2 results in ketoconazole sensitivity, whereas no effect was observed with ECM22 deletion suggesting that Ecm22 and Upc2 have specific targets with some essential overlapping functions 35 , Upc2 is a novel class of zinc finger TFs Recent studies proposed that several members of the fungal zinc cluster family of transcription regulators may represent functional analogues of metazoan NRs 13 , 18 , 19 , While fungal zinc cluster TFs have no apparent sequence or structural similarity to metazoan NRs, Upc2 displays conceptual similarities with steroid NRs in general domain architectures, sterol ligand binding, homodimerization, ligand-dependent transcriptional regulation and nuclear translocation.
Zinc fingers coordinating two zinc ions are composed of six cysteines for Upc2 and four cysteines in each of the two fingers for NRs Zinc finger TFs have a C-terminal negative regulatory domain, which for a number of family members mediates ligand binding or response to small molecules such as cellular metabolites and lipophilic ligands Transcriptional activity of Upc2 is regulated by ergosterol, which is functionally similar to Type I steroid NRs in terms of ligand specificities.
Upc2 and steroid receptors both form homodimers.CgTnS, Cgerg1 mutant; 3. In contrast, many mammalian NRs bind small molecules of extracellular origin. The dissociation of sterol ligand from Upc2 may lead to exposure of the NLS or release Upc2 from the capture by cytosolic proteins for nuclear transport.
As molecular oxygen is necessary for sterol production and sterols are only found in eukaryotes, Galea and Brown hypothesize a direct correlation between eukaryotic aerobic life-style and sterols Galea and Brown, These observations together with ligand binding and cell localization experiments suggest that sterol binding not only regulates protein localization but also regulates transcriptional activity of Upc2. The major drugs used to treat aspergillosis target ergosterol, the fungal cholesterol equivalent, and belong to the polyene and azole classes of antifungal drugs. These data suggest that the C-terminal activation loop is essential not only for the regulation of protein localization but for the full transcription activity of Upc2.
Though comprehensive epidemiology studies are currently lacking for aspergillosis, a recent estimate suggests more than 3 million people have invasive or chronic A. The cell wall is located outside the cell membrane and provides structural integrity and protection from external forces. The lack of sequence and structural similarities of fungal zinc cluster TFs and metazoan NRs suggests that the architectural and functional similarities are the product of convergent evolution with common mechanistic strategy. This mini-review summarizes our current understanding of sterol biosynthesis regulation with a focus on mechanisms of transcriptional regulation by the SREBP family of transcription factors.
Upc2 lacking LBD showed some basal level of sterol synthesis in the absence of fluconazole.
Pneumocystis do not appear to synthesize the major fungal sterol, ergosterol, and biochemical analyses have shown that they utilize cholesterol rather than ergosterol as the bulk sterol. DNA cloning and hybridization analyses were done according to the standard protocol. This is consistent with the previous observations that deletion of UPC2 results in ketoconazole sensitivity, whereas no effect was observed with ECM22 deletion suggesting that Ecm22 and Upc2 have specific targets with some essential overlapping functions 35 , The assay values represent the average of three independent experiments. Disruption of sterol binding by LF or LW showed constitutive nuclear localization. Upc2 specifically recognizes ergosterol, which is the final product of the sterol biosynthetic pathway in fungi.
Although Pneumocystis are fungi, this genus has characteristics that make it atypical among other fungi. However, genome scans and in vitro assays indicate the presence of sterol biosynthetic genes within the P. Upc2 and steroid receptors both form homodimers.
The MR mutation that disrupts dimerization of LBD and ligand binding shows mostly nuclear localization regardless of sterol level, suggesting that dimerization of Upc2 is essential for its regulatory function. Discussion The crystal structure of Upc2 LBD together with biochemical and yeast cell biology studies provides a detailed mechanistic picture of transcriptional regulation Fig. Lanes from the left : 1.