BackgroundCandida auris is a multidrug resistant, emerging agent of fungemia in humans. Its actual global distribution remains obscure as the current commercial methods of clinical diagnosis misidentify it as C. haemulonii. Here we report the first draft genome of C. auris to explore the genomic basis of virulence and unique differences that could be employed for differential diagnosis.ResultsMore than 99.5 % of the C. auris genomic reads did not align to the current whole (or draft) genome sequences of Candida albicans, Candida lusitaniae, Candida glabrata and Saccharomyces cerevisiae; thereby indicating its divergence from the active Candida clade. The genome spans around 12.49 Mb with 8527 predicted genes. Functional annotation revealed that among the sequenced Candida species, it is closest to the hemiascomycete species Clavispora lusitaniae. Comparison with the well-studied species Candida albicans showed that it shares significant virulence attributes with other pathogenic Candida species such as oligopeptide transporters, mannosyl transfersases, secreted proteases and genes involved in biofilm formation. We also identified a plethora of transporters belonging to the ABC and major facilitator superfamily along with known MDR transcription factors which explained its high tolerance to antifungal drugs.ConclusionsOur study emphasizes an urgent need for accurate fungal screening methods such as PCR and electrophoretic karyotyping to ensure proper management of fungemia. Our work highlights the potential genetic mechanisms involved in virulence and pathogenicity of an important emerging human pathogen namely C. auris. Owing to its diversity at the genomic scale; we expect the genome sequence to be a useful resource to map species specific differences that will help develop accurate diagnostic markers and better drug targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1863-z) contains supplementary material, which is available to authorized users.

Hsp90 is important for normal growth and development in eukaryotes. Together with Hsp70 and other accessory proteins, Hsp90 not only helps newly synthesized proteins to fold but also regulates activities of transcription factors and protein kinases. Although the gene coding for heat shock protein 90 from Plasmodium falciparum (PfHsp90) has been characterized previously, there is very little known regarding its function in the parasite. We have analyzed PfHsp90 complexes and addressed its role in parasite life cycle using Geldanamycin (GA), a drug known to interfere with Hsp90 function. Sedimentation analysis and size exclusion chromatography showed PfHsp90 to be in 11 s 20, w complexes of ϳ300 kDa in size. Similar to the hetero-oligomeric complexes of Hsp90 in mammals, PfHsp70 was found to be present in PfHsp90 complexes. Homology modeling revealed a putative GA-binding pocket at the amino terminus of PfHsp90. The addition of GA inhibited parasite growth with LD 50 of 0.2 M. GA inhibited parasite growth by arresting transition from Ring to trophozoite. Transition from trophozoite to schizonts and reinvasion of new erythrocytes were less significantly affected. While inducing the synthesis of PfHsp70 and PfHsp90, GA did not significantly alter the pattern of newly synthesized proteins. Pre-exposure to heat shock attenuated GA-mediated growth inhibition, suggesting the involvement of heat shock proteins. Specificity of GA action on PfHsp90 was evident from selective inhibition of PfHsp90 phosphorylation in GA-treated cultures. In addition to suggesting an essential role for PfHsp90 during parasite growth, our results highlight PfHsp90 as a potential drug target to control malaria.Plasmodium falciparum is responsible for the most severe form of malaria in humans, causing approximately 2 million deaths every year. During its asexual life cycle in human erythrocytes, the parasite progresses through three growth phases (1). The early form following invasion called the Ring stage is the phase of establishment in the erythrocyte. Trophozoite stage is the metabolically most active biosynthetic phase, whereas the schizont stage represents the phase of nuclear division before release of merozoites from the erythrocyte. Heat shock proteins of the class Hsp60, Hsp70, and Hsp90 are known to be expressed by the parasite during the intraerythrocytic stages in the vertebrate host (2-4). Although these heat shock proteins share significant homologies with their mammalian counterparts, there is very limited information available regarding their functional roles in parasite development. We have focused our study on the role of parasite-heat shock protein 90 expressed during erythrocytic cycle in humans.Previous reports have shown that the gene coding for Hsp90 from P. falciparum is present on chromosome 7, has a single intron of 800 bp, and encodes protein with 745 amino acids, giving a molecular mass of 86 kDa (5, 6). Sequence comparison shows 59% identity and 69% similarity to human Hsp90. There is significant similarity at the N...

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

Calnexin is a membrane-bound lectin and a molecular chaperone that binds newly synthesized glycoproteins in the endoplasmic reticulum (ER). To analyze the oligomeric properties of calnexin and calnexin-substrate complexes, sucrose velocity gradient centrifugation and chemical cross-linking were used. After CHAPS solubilization of Chinese Hamster Ovary cells, the unoccupied calnexin behaved as a monomer sedimenting at 3.5 S20,W. For calnexin-substrate complexes the S-values ranged between 3.5–8 S20,W, the size increasing with the molecular weight of the substrate. Influenza hemagglutinin, a well-characterized substrate associated with calnexin in complexes that sedimented at 5–5.5 S20,W. The majority of stable complexes extracted from cells, appeared to contain a single calnexin and a single substrate molecule, with about one third of the calnexin in the cell being unoccupied or present in weak associations. However, when chemical cross-linking was performed in intact cells, the calnexin-substrate complexes and calnexin itself was found to be part of a much larger heterogeneous protein network that included other ER proteins. Pulse-chase analysis of influenza-infected cells combined with chemical cross-linking showed that HA was part of large, heterogeneous, cross-linked entities during the early phases of folding, but no longer after homotrimer assembly. The network of weakly associated resident ER chaperones which included BiP, GRP94, calreticulin, calnexin, and other proteins, may serve as a matrix that binds early folding and assembly intermediates and restricts their exit from the ER.

Using influenza hemagglutinin (HA0) and vesicular stomatitis virus G protein as model proteins, we have analyzed the effects of dithiothreitol (DTT) on conformational maturation and transport of glycoproteins in the secretory pathway of living cells. While DTT caused reduction of folding intermediates and misfolded proteins in the endoplasmic reticulum (ER), it did not affect molecules that had already acquired a mature trimeric conformation, whether present in the ER or elsewhere. The conversion to DTT resistance was therefore a pre‐Golgi event. Reduction of folding intermediates was dependent on the intactness of the ER and on metabolic energy, suggesting cooperativity between DTT and ER folding factors. DTT did not inhibit most cellular functions, including ATP synthesis and protein transport within the secretory pathway. The results established DTT as an effective tool for analyzing the folding and compartmental distribution of proteins with disulfide bonds.