Soluble Enolase 1 of Candida albicans and Aspergillus fumigatus Stimulates Human and Mouse B Cells and Monocytes.

Because of the growing numbers of immunocompromised patients, the incidence of life-threatening fungal infections caused by Candida albicans and Aspergillus fumigatus is increasing. We have recently identified enolase 1 (Eno1) from A. fumigatus as an immune evasion protein. Eno1 is a fungal moonlighting protein that mediates adhesion and invasion of human cells and also immune evasion through complement inactivation. We now show that soluble Eno1 has immunostimulatory activity. We observed that Eno1 from both C. albicans and A. fumigatus directly binds to the surface of lymphocytes, preferentially human and mouse B cells. Functionally, Eno1 upregulated CD86 expression on B cells and induced proliferation. Although the receptor for fungal Eno1 on B lymphocytes is still unknown, the comparison of B cells from wild-type and MyD88-deficient mice showed that B cell activation by Eno1 required MyD88 signaling. With respect to infection biology, we noted that mouse B cells stimulated by Eno1 secreted IgM and IgG2b. These Igs bound C. albicans hyphae in vitro, suggesting that Eno1-induced Ab secretion might contribute to protection from invasive fungal disease in vivo. Eno1 also triggered the release of proinflammatory cytokines from monocytes, particularly IL-6, which is a potent activator of B cells. Together, our data shed new light on the role of secreted Eno1 in infections with C. albicans and A. fumigatus. Eno1 secretion by these pathogenic microbes appears to be a double-edged sword by supporting fungal pathogenicity while triggering (antifungal) immunity.

Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans.

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20 µM with a Vmax of 0.079 nmol/(µg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

Production of autolysis-proof Kex2 protease from Candida albicans in Saccharomyces cerevisiae for in vitro processing of fusion proteins.

Protein expression with a fusion partner followed by the removal of the fusion partner via in vitro processing with a specific endoprotease is a favored method for the efficient production of intact recombinant proteins. Due to the high cost of commercial endoproteases, this process is restricted to laboratories. Kex2p is a membrane-bound serine protease that cleaves after dibasic residues of substrates in the late Golgi network. Although Kex2p is a very efficient endoprotease with exceptional specificity, it has not yet been used for the in vitro processing of fusion proteins due to its autolysis and high production cost. In this study, we developed an alternative endoprotease, autolysis-proof Kex2p, via site-directed mutagenesis of truncated KEX2 from Candida albicans (CaKEX2). Secretory production of manipulated CaKex2p was improved by employing target protein-specific translational fusion partner in Saccharomyces cerevisiae. The mass production of autolysis-proof Kex2p could facilitate the use of Kex2p for the large-scale production of recombinant proteins. KEY POINTS: • A soluble and active CaKex2p variant was produced by autocatalytic cleavage of the pro-peptide after truncation of C-terminus • Autolysis-proof CaKex2p was developed by site-directed mutagenesis • Secretion of autolysis-proof CaKex2p was improved by employing optimal translational fusion partner in Saccharomyces cerevisiae.

Ino80 is required for H2A.Z eviction from hypha-specific promoters and hyphal development of Candida albicans.

ATP-dependent chromatin remodeling complexes play important roles in many essential cellular processes, including transcription regulation, DNA replication, and repair. Evicting H2A.Z, a variant of histone H2A, from the promoter of hypha-specific genes is required for hyphal formation in Candida albicans. However, the mechanism that regulates H2A.Z removal during hyphal formation remains unknown. In this study, we demonstrated that Ino80, the core catalytic subunit of the INO80 complex, was recruited to hypha-specific promoters during hyphal induction in Arp8 dependent manner and facilitated the removal of H2A.Z. Deleting INO80 or mutating the ATPase site of Ino80 impairs the expression of hypha-specific genes (HSGs) and hyphal development. In addition, we showed that Ino80 was essential for the virulence of C. albicans during systemic infections in mice. Interestingly, Arp5, an INO80 complex-specific component, acts in concert with Ino80 during DNA damage responses but is dispensable for hyphal induction. Our findings clarified that Ino80 was critical for hyphal development, DNA damage response, and pathogenesis in C. albicans.

Candida albicans SR-Like Protein Kinases Regulate Different Cellular Processes: Sky1 Is Involved in Control of Ion Homeostasis, While Sky2 Is Important for Dipeptide Utilization.

Protein kinases play a crucial role in regulating cellular processes such as growth, proliferation, environmental adaptation and stress responses. Serine-arginine (SR) protein kinases are highly conserved in eukaryotes and regulate fundamental processes such as constitutive and alternative splicing, mRNA processing and ion homeostasis. The Candida albicans genome encodes two (Sky1, Sky2) and the Candida glabrata genome has one homolog (Sky1) of the human SR protein kinase 1, but their functions have not yet been investigated. We used deletion strains of the corresponding genes in both fungi to study their cellular functions. C. glabrata and C. albicans strains lacking SKY1 exhibited higher resistance to osmotic stress and toxic polyamine concentrations, similar to Saccharomyces cerevisiae sky1Δ mutants. Deletion of SKY2 in C. albicans resulted in impaired utilization of various dipeptides as the sole nitrogen source. Subsequent phosphoproteomic analysis identified the di- and tripeptide transporter Ptr22 as a potential Sky2 substrate. Sky2 seems to be involved in Ptr22 regulation since overexpression of PTR22 in the sky2Δ mutant restored the ability to grow on dipeptides and made the cells more susceptible to the dipeptide antifungals Polyoxin D and Nikkomycin Z. Altogether, our results demonstrate that C. albicans and C. glabrata Sky1 protein kinases are functionally similar to Sky1 in S. cerevisiae, whereas C. albicans Sky2, a unique kinase of the CTG clade, likely regulates dipeptide uptake via Ptr22.

Sth1, the ATPase subunit of the RSC chromatin remodeler has important roles in stress response and DNA damage repair in the pathogenic fungi Candida albicans.

Candida albicans, the most prevalent fungal pathogen, exists as a commensal in the human host. It is subjected to myriad physiological stress conditions in different host niches, which jeopardizes its fitness to survive and propagate as an established commensal. C. albicans has highly labile chromatin which gets remodeled in response to the stress conditions to facilitate the expression of several stress-responsive genes. Several epigenetic factors including histone variants, histone modifiers and chromatin remodelers that define the chromatin architecture play crucial roles in the regulation of the stress-responsive genes in this organism. Here we investigated the roles of the ATP-dependent chromatin remodeler RSC (Remodel the Structure of Chromatin) in several stress responses in C. albicans, by targeting the key ATPase component, Sth1, given its profound and similar roles exist in Saccharomyces cerevisiae. We have unraveled the crucial roles of the RSC complex (Sth1) in maintaining cell wall integrity and fighting against osmotic and oxidative stresses. We found that the mutant conditionally depleted of Sth1 was sensitive to the cell wall disrupting agents, and the mutant without exposure to any stressor accumulated higher chitin content in the cell wall as a defense mechanism to restore the cell wall integrity. Further, this was supported by the phosphorylation of MAPK1 protein Mkc1, which happens due to activation of the cell wall integrity pathway PKC1. We also observed the Sth1 mutant to be sensitive to oxidative and osmotic stresses in vitro, which are very important and imparted by the host defense mechanism. This suggests that the mutant could get attenuated and hence become less virulent than the wild-type when loss of function of Sth1 happens. We also found that Sth1 has a crucial role in maintaining genomic integrity as sth1 mutant cells accumulate extensive DNA damages and show the loss in cell viability. Overall this work suggests that Sth1 has an important role in fighting against some of the clinically relevant and physiologically important stresses. It also has a crucial role in fighting against stress to the genomic integrity and hence functions in DNA damage repair.

Inhibitory Effects of Nitrogenous Metabolites from a Marine-Derived Streptomyces bacillaris on Isocitrate Lyase of Candida albicans.

Two nitrogenous metabolites, bacillimide (1) and bacillapyrrole (2), were isolated from the culture broth of the marine-derived actinomycete Streptomyces bacillaris. Based on the results of combined spectroscopic and chemical analyses, the structure of bacillimide (1) was determined to be a new cyclopenta[c]pyrrole-1,3-dione bearing a methylsulfide group, while the previously reported bacillapyrrole (2) was fully characterized for the first time as a pyrrole-carboxamide bearing an alkyl sulfoxide side chain. Bacillimide (1) and bacillapyrrole (2) exerted moderate (IC50 = 44.24 µM) and weak (IC50 = 190.45 µM) inhibitory effects on Candida albicans isocitrate lyase, respectively. Based on the growth phenotype using icl-deletion mutants and icl expression analyses, we determined that bacillimide (1) inhibits the transcriptional level of icl in C. albicans under C2-carbon-utilizing conditions.

ABC typing and extracellular enzyme production of Candida albicans isolated from Candida vulvovaginitis.

BACKGROUND: Candida albicans is the most common and virulent genus Candida. Detection of virulence factors in this species plays an important role in the better understanding of pathogenesis and antifungal treatment. Molecular typing investigations are important in the epidemiological interpretation of infection. This study aimed to evaluate extracellular enzyme activity and genotyping of C. albicans species isolated from vulvovaginal samples. METHODS: One hundred and three vaginal C. albicans isolates were tested for esterase, phospholipase, proteinase, and hemolysin activities by specific media. Besides, the DNA of C. albicans isolates was extracted and amplified for ABC genotyping. RESULTS: The highest enzyme production of C. albicans isolates was for proteinase (97.1%) and esterase (95.2%), whereas 59.2% of C. albicans isolates were negative for hemolysin secretion. Genotype C (83.5%) was the most frequent genotype followed by genotype B (12.6%) and genotype A (3.9%). CONCLUSION: It is concluded that genotype C was the predominant genotype in all examined vulvovaginal C. albicans isolates. Also, there was a significant difference between enzyme production in each genotype (except for proteinase).

A Suppressor Mutation in the ß-Subunit Kis1 Restores Functionality of the SNF1 Complex in Candida albicans snf4Δ Mutants.

The heterotrimeric protein kinase SNF1 is a key regulator of metabolic adaptation in the pathogenic yeast Candida albicans, and mutants with a defective SNF1 complex cannot grow on carbon sources other than glucose. We identified a novel type of suppressor mutation in the ß-subunit Kis1 that rescued the growth defects of cells lacking the regulatory γ-subunit Snf4 of the SNF1 complex. Unlike wild-type Kis1, the mutated Kis1A396T could bind to the catalytic α-subunit Snf1 in the absence of Snf4. Binding of Kis1A396T did not enhance phosphorylation of Snf1 by the upstream activating kinase Sak1, which is impaired in snf4Δ mutants. Nevertheless, the mutated Kis1A396T reestablished SNF1-dependent gene expression, confirming that SNF1 functionality was restored. The repressor proteins Mig1 and Mig2 were phosphorylated even in the absence of Snf1, but their phosphorylation patterns were altered, indicating that SNF1 regulates Mig1 and Mig2 activity indirectly. In contrast to wild-type cells, mutants lacking Snf4 were unable to reduce the amounts of Mig1 and Mig2 when grown on alternative carbon sources, and this deficiency was also remediated by the mutated Kis1A396T. These results provide novel insights into the regulation of SNF1 and the repressors Mig1 and Mig2 in the metabolic adaptation of C. albicans. IMPORTANCE The highly conserved protein kinase SNF1 plays a key role in the metabolic adaptation of the pathogenic yeast Candida albicans, but it is not clear how it regulates its downstream targets in this fungus. We show that the repressor proteins Mig1 and Mig2 are phosphorylated also in cells lacking the catalytic α-subunit Snf1 of the SNF1 complex, but the amounts of both proteins were reduced in wild-type cells when glucose was replaced by alternative carbon sources, pointing to an indirect mechanism of regulation. Mutants lacking the regulatory γ-subunit Snf4 of the SNF1 complex, which cannot grow on alternative carbon sources, were unable to downregulate Mig1 and Mig2 levels. We identified a novel type of suppressor mutation, an amino acid substitution in the ß-subunit Kis1, which enabled Kis1 to bind to Snf1 in the absence of Snf4, thereby restoring Mig1 and Mig2 downregulation, SNF1-dependent gene expression, and growth on alternative carbon sources. These results provide new insights into the SNF1 signaling pathway in C. albicans.

Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads.

Aspergillus fumigatus is a fungal pathogen whose effects can be debilitating and potentially fatal in immunocompromised patients. Current drug treatment options for this infectious disease are limited to just a few choices (e.g. voriconazole and amphotericin B) and these themselves have limitations due to potentially adverse side effects. Furthermore, the likelihood of the development of resistance to these current drugs is ever present. Thus, new treatment options are needed for this infection. A new potential antifungal drug target is acetohydroxyacid synthase (AHAS; EC 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway, and a target for many commercial herbicides. In this study, we have expressed, purified and characterised the catalytic subunit of AHAS from A. fumigatus and determined the inhibition constants for several known herbicides. The most potent of these, penoxsulam and metosulam, have Ki values of 1.8 ± 0.9 nM and 1.4 ± 0.2 nM, respectively. Molecular modelling shows that these compounds are likely to bind into the herbicide binding pocket in a mode similar to Candida albicans AHAS. We have also shown that these two compounds inhibit A. fumigatus growth at a concentration of 25 µg/mL. Thus, AHAS inhibitors are promising leads for the development of new anti-aspergillosis therapeutics.

Crystal structure of glutamate dehydrogenase 3 from Candida albicans.

Glutamate dehydrogenase 3 from Candida albicans (CaGdh3) catalyzes the reversible oxidative deamination of l-glutamate, playing an important role in the yeast-to-hyphal transition of C. albicans. Here we report the crystal structures of CaGdh3 and its complex with α-ketoglutarate and NADPH. CaGdh3 exists as a hexamer, with each subunit containing two domains. The substrate and coenzyme bind in the cleft between the two domains and their binding induces a conformational change in CaGdh3. Our results will help to understand the catalytic mechanism of CaGdh3 and will provide a structural basis for the design of antifungal drugs targeting the CaGdh3 pathway.

Pervasive transcription of the mitochondrial genome in Candida albicans is revealed in mutants lacking the mtEXO RNase complex.

The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3'-5' exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wild-type strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense ('mirror') transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments.

Candida albicans and Candida glabrata triosephosphate isomerase - a moonlighting protein that can be exposed on the candidal cell surface and bind to human extracellular matrix proteins.

BACKGROUND: Triosephosphate isomerase (Tpi1) is a glycolytic enzyme that has recently been reported also to be an atypical proteinaceous component of the Candida yeast cell wall. Similar to other known candidal "moonlighting proteins", surface-exposed Tpi1 is likely to contribute to fungal adhesion during the colonization and infection of a human host. The aim of our present study was to directly prove the presence of Tpi1 on C. albicans and C. glabrata cells under various growth conditions and characterize the interactions of native Tpi1, isolated and purified from the candidal cell wall, with human extracellular matrix proteins. RESULTS: Surface plasmon resonance measurements were used to determine the dissociation constants for the complexes of Tpi1 with host proteins and these values were found to fall within a relatively narrow range of 10- 8-10- 7 M. Using a chemical cross-linking method, two motifs of the Tpi1 molecule (aa 4-17 and aa 224-247) were identified to be directly involved in the interaction with vitronectin. A proposed structural model for Tpi1 confirmed that these interaction sites were at a considerable distance from the catalytic active site. Synthetic peptides with these sequences significantly inhibited Tpi1 binding to several extracellular matrix proteins suggesting that a common region on the surface of Tpi1 molecule is involved in the interactions with the host proteins. CONCLUSIONS: The current study provided structural insights into the interactions of human extracellular matrix proteins with Tpi1 that can occur at the cell surface of Candida yeasts and contribute to the host infection by these fungal pathogens.

Kinetic characterisation and inhibitor sensitivity of Candida albicans and Candida auris recombinant AOX expressed in a self-assembled proteoliposome system.

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (Km and Vmax) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose-response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia.

CaBir1 functions as an inhibitor-of-apoptosis and affects caspase-like activitiy in Candida albicans.

CaMca1 is the only metacaspase in Candida albicans, which shows structural homology to the mammalian caspases. CaMca1 consists of the caspase domain, the P20 and P10 regions, and the conserved catalytic histidine-cysteine dyad that is required for executing apoptosis in C. albicans. However, little is known about the proteolytic processing of CaMca1 or its activation under apoptosis-inducing conditions. To understand the regulation of this process, we characterized CaBir1 which is the single IAP (inhibitor-of-apoptosis protein) in C. albicans. IAPs are a family of proteins whose members all harbor a BIR (baculovirus IAP repeat) domain and negatively regulate apoptosis by inhibiting caspases. We found that the Cabir1/Cabir1 deletion mutant exhibited increased apoptotic phenotypes, such as ROS accumulation, nuclear segmentation, and cell survival, under apoptosis-inducing conditions. Examination of CaMca1 cleavage patterns in response to various apoptotic stresses revealed that these cleavages were stress-specific and dependent on the catalytic histidine-cysteine residues of CaMca1. The Cabir1/Cabir1 mutation was not associated with altered CaMca1 processing with or without apoptotic stimuli, but the Cabir1/Cabir1 mutant exhibited significantly increased caspase-like activities. These results suggest that CaBir1 acts as an apoptosis inhibitor by regulating caspase-like activity, but not CaMca1 processing.

Genotyping, antifungal susceptibility, enzymatic activity, and phenotypic variation in Candida albicans from esophageal candidiasis.

BACKGROUND: Esophageal candidiasis is the most frequent form of esophagitis. The pathogenicity of Candida spp. is related to a combination of microbial factors, hydrolytic enzyme secretion and phenotypic switching. This study was designed to investigate esophageal candidiasis, antifungal activity, enzymatic activity patterns, phenotyping, and genotyping profiles of Candida albicans species. METHODS: Nine hundred thirty-three visited patients were evaluated, and esophageal biopsies from patients were included in this study during 2019-2020. Direct smear, Gram staining, and culture on CHROMagar were performed for each sample. Isolated species were identified with conventional procedures and PCR-RFLP. Susceptibility to antifungals was determined according to CLSI guidelines. ABC typing, phenotype switching, hemolysin, proteinase, phospholipase, and esterase activity were also determined with the appropriate protocols. RESULTS: Twenty-three (2.5%) patients (mean age 55.2 years) were diagnosed with esophageal candidiasis. The species isolated were 19(82.6%) C. albicans, 3(13.1%) C. glabrata, and 1(4.3%) C. tropicalis. Genotype A (57.9%) was the predominant type in C. albicans isolates. 50% of C. albicans isolates exhibited a white phenotype. A high level of phospholipase (47.4%), hemolysin (68.4%), and proteinase activity (36.8%) was observed in the C. albicans isolates. Only three C. glabrata isolates displayed non-wild type susceptibility to voriconazole and itraconazole. CONCLUSION: This study shows that C. albicans are still the most frequent isolates from patients with esophageal candidiasis. The predominance of genotype A, the white phenotype, and strong hemolysin activity may indicate a high prevalence of pathogenicity in these isolates. Sensitivity to antifungal drugs was greatest for amphotericin and fluconazole.

Genetic Analysis of Sirtuin Deacetylases in Hyphal Growth of Candida albicans.

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell's metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here, we studied the roles of three sirtuin deacetylases-Sir2, Hst1, and Hst2-in the hyphal growth of C. albicans We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hypha formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2 Moreover, the expression of hypha-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to the wild type. This regulation of hypha formation was likely dependent on the deacetylase activity of Sir2, as a similar defect in hypha formation was observed when an asparagine known to be required for deacetylation was mutated. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and nonredundant role in hyphal growth of C. albicansIMPORTANCECandida albicans is one of the most common causes of hospital-acquired systemic fungal infections in the United States. It can switch between ovoid yeast and elongated hyphal growth forms in response to environmental cues. This morphological transition is essential for its survival in the host. Thus, identifying regulators involved in this process can lead to new therapies. In this study, we examined the contribution of three regulators called sirtuins (Sir2, Hst1, and Hst2) to the yeast-to-hypha transition of C. albicans We found that loss of Sir2 but not Hst1 or Hst2 hampered hypha formation. Moreover, the defect was caused by the loss of the catalytic activity of Sir2. Our study may lay the groundwork for discovering novel targets for antifungal therapies.

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

Analysis of GTP addition in the reverse (3'-5') direction by human tRNAHis guanylyltransferase.

Human tRNAHis guanylyltransferase (HsThg1) catalyzes the 3'-5' addition of guanosine triphosphate (GTP) to the 5'-end (-1 position) of tRNAHis, producing mature tRNAHis In human cells, cytoplasmic and mitochondrial tRNAHis have adenine (A) or cytidine (C), respectively, opposite to G-1 Little attention has been paid to the structural requirements of incoming GTP in 3'-5' nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson-Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5'-end of tRNAHis with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNAHis Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5'-terminal modification of RNAs.

Kinome analyses of Candida albicans, C. parapsilosis and C. tropicalis enable novel kinases as therapeutic drug targets in candidiasis.

Candida spp. have attracted considerable attention as they cause serious human diseases in immunocompromised individuals. The genomes of the pathogenic Candida spp. have been sequenced, but systemic characterizations of their kinomes are yet to be reported. As in various eukaryotes, the protein kinases play crucial regulatory roles in pathogenicity of Candida. Increased frequency of antifungal resistance in Candida spp. requires significant attention to explore novel therapeutic molecules for their control. The present in-silico study involves novel bioinformatics strategies to identify the kinase proteins and their potential drug targets with the purpose to combat fungal infections. The study reports 103, 107 and 106 kinase proteins from 3 Candida spp., C. albicans, C. parapsilosis and C. tropicalis, respectively. Moreover, 79 common kinase proteins were identified, of which 54 proteins play essential roles in Candida spp. and 42 proteins were human non-homologues. Among the essential and human non-homologous protein kinases, 9 were found to be common essential human non-homologues, of which 6 are uniquely present in Candida. These 6 protein kinases namely, Hsl1, Npr1, Ptk2, Kin2, Ksp1 and orf19.3854 (CAALFM_CR06040WA) are involved in various molecular and cellular processes regulating virulence or pathogenicity. Further, these 6 kinases are prioritized as potential drug targets and explored for discovering new lead compounds against candidiasis. The drug repurposing approach for these 6 kinases show 13 approved drugs and investigational compounds that might play substantial inhibitory roles during combating candidiasis.