Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

SignificanceThe “centromere paradox” refers to rapidly evolving and highly diverse centromere DNA sequences even in closely related eukaryotes. However, factors contributing to this rapid divergence are largely unknown. Here, we identified large regional, LTR retrotransposon-rich centromeres in a group of human fungal pathogens belonging to the Cryptococcus species complex. We provide evidence that loss-of-functional RNAi machinery and possibly cytosine DNA methylation trigger instability of the genome by activation of centromeric retrotransposons presumably suppressed by RNAi. We propose that RNAi, together with cytosine DNA methylation, serves as a critical determinant that maintains repetitive transposon-rich centromere structures. This study explores the direct link between RNAi and centromere structure evolution.

Because pure cultures and a stable transformation system are not available for arbuscular mycorrhizal fungi, the role of their phosphate transporters for the symbiotic interaction with the plant up till now could not be studied. Here we report the cloning and the functional analysis of a gene encoding a phosphate transporter (PiPT) from the root endophytic fungus Piriformospora indica, which can be grown axenically. The PiPT polypeptide belongs to the major facilitator superfamily. Homology modeling reveals that PiPT exhibits twelve transmembrane helices divided into two halves connected by a large hydrophilic loop in the middle. The function of the protein encoded by PiPT was confirmed by complementation of a yeast phosphate transporter mutant. The kinetic analysis of PiPT (K m 25 M) reveals that it belongs to the high affinity phosphate transporter family (Pht1). Expression of PiPT was localized to the external hyphae of P. indica colonized with maize plant root, which suggests that external hyphae are the initial site of phosphate uptake from the soil. To understand the physiological role of PiPT, knockdown transformants of the gene were prepared using electroporation and RNA interference. Knockdown transformants transported a significantly lower amount of phosphate to the host plant than wild-type P. indica. Higher amounts of phosphate were found in plants colonized with wild-type P. indica than that of non-colonized and plants colonized with knockdown PiPT P. indica. These observations suggest that PiPT is actively involved in the phosphate transportation and, in turn, P. indica helps improve the nutritional status of the host plant.

The bioprotection performance of Piriformospora indica against the root parasite Fusarium verticillioides was studied. We found that maize plants first grown with F. verticillioides and at day 10 inoculated with P. indica showed improvements in biomass, and root length and number as compared with plants grown with F. verticillioides alone. To validate our finding that inoculation with P. indica suppresses colonization by F. verticillioides, we performed PCR analyses using P. indica-and F. verticillioides-specific primers. Our results showed that inoculation with P. indica suppresses further colonization by F. verticillioides. We hypothesized that as the colonization by P. indica increases, the presence of/colonization by F. verticillioides decreases. In roots, catalase (CAT), glutathione reductase (GR), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were found to be higher in F. verticillioides-colonized plants than in noncolonized plants. Increased activity of antioxidant enzymes minimizes the chances of oxidative burst (excessive production of reactive oxygen species), and therefore F. verticillioides might be protected from the oxidative defence system during colonization. We also observed decreased antioxidant enzyme activities in plants first inoculated with F. verticillioides and at day 10 inoculated with P. indica as compared with plants inoculated with F. verticillioides alone. These decreased antioxidant enzyme activities due to the presence of P. indica help the plant to overcome the disease load of F. verticillioides. We propose that P. indica can be used as a bioprotection agent against the root parasite F. verticillioides. INTRODUCTIONMycorrhizal associations are among the most widespread, intimate and important symbioses in terrestrial ecosystems. Among these, arbuscular mycorrhiza (AM) represent the most ancient and widespread mycorrhizal symbiosis, and are found in a wide range of land plant species (Harrison, 1997). The successful establishment of this mutualistic association constitutes a strategy to fulfil the nutritional demands of both partners . This requires a balance between the defence responses of the host plant and the nutrient demands of the endophyte, resulting in an altered defence-related gene expression, which has been extensively studied during host colonization by obligate biotrophic arbuscular mycorrhizal fungi (AMF). Induction of defence-related genes is most prominent during the early stages of colonization (García-Garrido & Ocampo, 2002), but can also be detected during arbuscule development (Grunwald et al., 2004).The active resistance of plants to colonization by fungi is often expressed by the hypersensitive reaction (HR) of challenged plant cells, and is characterized by reactive oxygen species (ROS), as well as by induced rapid and localized death of plant tissue at the site of infection (Ingram, 1978;Tenhaken et al., 1995). At the biochemical level, the rapid generation of ROS, such as superoxide (O { 2 ) and hydrogen peroxide (H 2 O 2 ), kno...

Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing approximately 1 million annual infections globally. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large mating type (MAT) locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with 2 MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species, as well as its possible link with the origin and evolution of pathogenesis. In this study, we sequenced, assembled, and annotated the genomes of 2 C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the 2 C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Bioinformatic and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that, similar to those of the pathogenic Cryptococcus species, C. amylolentus has regional centromeres (CENs) that are enriched with species-specific transposable and repetitive DNA elements. Additionally, we found that while neither the P/R nor the HD locus is physically closely linked to its centromere in C. amylolentus, and the regions between the MAT loci and their respective centromeres show overall synteny between the 2 genomes, both MAT loci exhibit genetic linkage to their respective centromere during meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the MAT-CEN intervening regions. Furthermore, genomic comparisons between C. amylolentus and related pathogenic Cryptococcus species provide evidence that multiple chromosomal rearrangements mediated by intercentromeric recombination have occurred during descent of the 2 lineages from their common ancestor. Taken together, our findings support a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by similar repetitive centromeric DNA elements shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and further chromosomal rearrangements then resulted in the 2 MAT loci becoming physically linked and eventually fusing to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species.

Microbial attachment and subsequent colonization onto surfaces lead to the spread of deadly community-acquired and hospital-acquired (nosocomial) infections. Noncovalent immobilization of water insoluble and organo-soluble cationic polymers onto a surface is a facile approach to prevent microbial contamination. In the present study, we described the synthesis of water insoluble and organo-soluble polymeric materials and demonstrated their structure-activity relationship against various human pathogenic bacteria including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and beta lactam-resistant Klebsiella pneumoniae as well as pathogenic fungi such as Candida spp. and Cryptococcus spp. The polymer coated surfaces completely inactivated both bacteria and fungi upon contact (5 log reduction with respect to control). Linear polymers were more active and found to have a higher killing rate than the branched polymers. The polymer coated surfaces also exhibited significant activity in various complex mammalian fluids such as serum, plasma, and blood and showed negligible hemolysis at an amount much higher than minimum inhibitory amounts (MIAs). These polymers were found to have excellent compatibility with other medically relevant polymers (polylactic acid, PLA) and commercial paint. The cationic hydrophobic polymer coatings disrupted the lipid membrane of both bacteria and fungi and thus showed a membrane-active mode of action. Further, bacteria did not develop resistance against these membrane-active polymers in sharp contrast to conventional antibiotics and lipopeptides, thus the polymers hold great promise to be used as coating materials for developing permanent antimicrobial paint.

Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes.

Toxoplasma gondii is the most common cause of secondary CNS infections in immunocompromised persons such as AIDS patients. The major route of adenosine metabolism in T. gondii is direct phosphorylation to adenosine 5'-monophosphate (AMP) catalyzed by the enzyme adenosine kinase (EC 2.7.1.20). Adenosine kinase in T. gondii is significantly more active than any other purine salvage enzyme in this parasite and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Subversive substrates of T. gondii,but not the human, adenosine kinase are preferentially metabolized to their monophosphorylated forms and become selectively toxic to the parasites but not their host. 6-Benzylthioinosine (BTI) was identified as an excellent subversive substrate of T. gondii adenosine kinase. Herein, we report the synthesis of new analogues of BTI as subversive substrates for T. gondii adenosine kinase. These new subversive substrates were synthesized starting from tribenzoyl protected d-ribose. To accomplish the lead optimization process, a divergent and focused combinatorial library was synthesized using a polymer-supported trityl group at the 5'-position. The combinatorial library of 20 compounds gave several compounds more active than BTI. Structure-activity relationship studies showed that substitution at the para position plays a crucial role. To investigate the reasons for this discrimination, substrates with different substituents at the para position were studied by molecular modeling using Monte Carlo Conformational Search followed by energy minimization of the enzyme-ligand complex.