Luiz Roberto Basso

Cryptococcus gattii from the North American Northwest (NW) have higher azole MICs than do non-NW C. gattii or Cryptococcus neoformans. Since mechanisms of azole resistance in C. gattii are not known, we identified C. gattii and C. neoformans plasma membrane azole efflux pumps and characterized their properties. The C. gattii R265 genome was searched for orthologues of known fungal azole efflux genes, expression of candidate genes was assessed by RT–PCR and the expressed genes' cDNAs were cloned and expressed in Saccharomyces cerevisiae. Azole MICs and intracellular [³H]fluconazole were measured in C. gattii and C. neoformans and in S. cerevisiae expressing each cDNA of interest, as was [³H]fluconazole uptake by post-Golgi vesicles (PGVs) isolated from S. cerevisiae sec6-4 mutants expressing each cDNA of interest. Intracellular [³H]fluconazole concentrations were inversely correlated with fluconazole MICs only in 25 NW C. gattii strains. S. cerevisiae expressing three C. gattii cDNAs (encoded by orthologues of C. neoformans AFR1 and MDR1 and the previously unstudied gene AFR2) and their C. neoformans counterparts had higher azole MICs and lower intracellular [³H]fluconazole concentrations than did empty-vector controls. PGVs from S. cerevisiae expressing all six Cryptococcus cDNAs also accumulated more [³H]fluconazole than did controls, and [³H]fluconazole transport by all six transporters of interest was ATP dependent and was inhibited by excess unlabelled fluconazole, voriconazole, itraconazole and posaconazole. We conclude that C. gattii and C. neoformans AFR1, MDR1 and AFR2 encode ABC transporters that pump multiple azoles out of S. cerevisiae cells, thereby causing azole resistance.

Cryptococcus gattii is responsible for an expanding epidemic of serious infections in Western Canada and the Northwestern United States (Pacific Northwest). Some patients with these infections respond poorly to azole antifungals, and high azole MICs have been reported in Pacific Northwest C. gattii . In this study, multiple azoles (but not amphotericin B) had higher MICs for 25 Pacific Northwest C. gattii than for 34 non-Pacific Northwest C. gattii or 20 Cryptococcus neoformans strains. We therefore examined the roles in azole resistance of overexpression of or mutations in the gene ( ERG11 ) encoding the azole target enzyme. ERG11 / ACT1 mRNA ratios were higher in C. gattii than in C. neoformans , but these ratios did not differ in Pacific Northwest and non-Pacific Northwest C. gattii strains, nor did they correlate with fluconazole MICs within any group. Three Pacific Northwest C. gattii strains with low azole MICs and 2 with high azole MICs had deduced Erg11p sequences that differed at one or more positions from that of the fully sequenced Pacific Northwest C. gattii strain R265. However, the azole MICs for conditional Saccharomyces cerevisiae erg11 mutants expressing the 5 variant ERG11 s were within 2-fold of the azole MICs for S. cerevisiae expressing the ERG11 gene from C. gattii R265, non-Pacific Northwest C. gattii strain WM276, or C. neoformans strains H99 or JEC21. We conclude that neither ERG11 overexpression nor variations in ERG11 coding sequences was responsible for the high azole MICs observed for the Pacific Northwest C. gattii strains we studied.

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.

Septins are evolutionarily conserved proteins that contain a GTPase domain and are capable of forming filaments at the cell periphery. Septins are involved in many essential cellular processes, such as cytokinesis and cell polarization, and are used as markers of morphogenesis in several fungi. Dimorphism in fungi enables cells to switch between morphologies (yeast or filament forms), due to changes in the temperature of the environment. We analysed the localization of septin proteins in yeast and filamentous cells of the dimorphic fungus Paracoccidioides brasiliensis, a common cause of granulomatous mycosis. In order to determine septin localization, we first cloned Cdc12p, a septin homolog from P. brasiliensis, and expressed it in Escherichia coli. Following PbCdc12p purification, specific serum against PbCdc12p were raised for use in immunofluorescence assays. We observed the hourglass and ring forms of septin filaments during cell division in yeast. Septin filaments were also simultaneously localized in the necks of multiple budding cells. A distinctive pattern of punctuate and/or diffuse localization was also seen in the periphery of multinucleate yeast cells and at the tips and septa of filamentous cells. A more diffuse and punctuate pattern of localization observed in P. brasiliensis cells seems to be unique to filamentous and dimorphic fungi and may be related to their specialization in cell wall deposition, morphogenesis and cell cycle control. Copyright © 2011 John Wiley & Sons, Ltd.

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2‐containing plasmids or single‐copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT‐1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild‐type C. albicans. We used PCR to fuse CaHygB or SAT‐1 to approximately 1 kb of 5′ and 3′ noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild‐type C. albicans strains. Homologous targeting frequencies were approximately 50–70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates. Copyright © 2010 John Wiley & Sons, Ltd.

A major cause of azole resistance in Candida albicans is overexpression of CDR1 , CDR2 , and/or MDR1 , which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1 - and GAL1 -regulated expression plasmids to overexpress CDR1 , CDR2 , or MDR1 in a C. albicans cdr1 cdr2 mdr1 -null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [ ³ H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1 -regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1- , CDR2 -, or MDR1- overexpressing cells accumulated much more [ ³ H]fluconazole than did PGVs from empty-vector controls. The K _m s (expressed in micromolar concentrations) and V _max s (expressed in picomoles per milligram of protein per minute), respectively, for [ ³ H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [ ³ H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [ ³ H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [ ³ H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC ₅₀ s; expressed as proportions of the [ ³ H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi.

The DNA puff BhC4‐1 gene of the sciarid Bradysia hygida is induced in salivary glands prior to the pupal molt as a secondary response to the increase in ecdysone titers. Previous studies demonstrated that the BhC4‐1 promoter is activated in transgenic Drosophila melanogaster salivary glands as a late response to the ecdysone peak that triggers metamorphosis, revealing that this aspect of BhC4‐1 transcriptional regulation is conserved in the Drosophila background. To identify regulators of BhC4‐1 expression, we utilized a candidate gene approach and tested the roles of the ecdysone‐induced genes BR‐C, E74, and E75. Our results reveal that the BR‐C Z3 isoform is essential for BhC4‐1‐lacZ induction in prepupal salivary glands and constitute the first demonstration of the participation of early genes products on DNA puff genes regulation. genesis 44:505–514, 2006.

The mechanisms that control DNA puff BhC4‐1 expression in the salivary gland of sciarid late larvae have been shown to be conserved in Drosophila . By analysing Drosophila transformed with constructs carrying progressive deletions of the BhC4‐1 promoter fragment (−3314/+40) fused to the lacZ reporter gene we show that the elements required for the correct BhC4‐1‐lacZ developmental regulation in prepupal salivary glands are contained in a 226 bp fragment (−186/+40). Also, interestingly, this study identified a 67 bp fragment (−253/−187) that activates BhC4‐1‐lacZ expression specifically in the ring gland.