Genomic evidence for a complete sexual cycle in Candida albicans.

Candida albicans is a diploid fungus that has become a medically important opportunistic pathogen in immunocompromised individuals. We have sequenced the C. albicans genome to 10.4-fold coverage and performed a comparative genomic analysis between C. albicans and Saccharomyces cerevisiae with the objective of assessing whether Candida possesses a genetic repertoire that could support a complete sexual cycle. Analyzing over 500 genes important for sexual differentiation in S. cerevisiae, we find many homologues of genes that are implicated in the initiation of meiosis, chromosome recombination, and the formation of synaptonemal complexes. However, others are striking in their absence. C. albicans seems to have homologues of all of the elements of a functional pheromone response pathway involved in mating in S. cerevisiae but lacks many homologues of S. cerevisiae genes for meiosis. Other meiotic gene homologues in organisms ranging from filamentous fungi to Drosophila melanogaster and Caenorhabditis elegans were also found in the C. albicans genome, suggesting potential alternative mechanisms of genetic exchange.

In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis.

Secreted aspartyl proteinases are putative virulence factors in Candida infections. Candida albicans possesses at least nine members of a SAP gene family, all of which have been sequenced. Although the expression of the SAP genes has been extensively characterized under laboratory growth conditions, no studies have analyzed in detail the in vivo expression of these proteinases in human oral colonization and infection. We have developed a reliable and sensitive procedure to detect C. albicans mRNA from whole saliva of patients with oral C. albicans infection and those with asymptomatic Candida carriage. The reverse transcription-PCR protocol was used to determine which of the SAP1 to SAP7 genes are expressed by C. albicans during colonization and infection of the oral cavity. SAP2 and the SAP4 to SAP6 subfamily were the predominant proteinase genes expressed in the oral cavities of both Candida carriers and patients with oral candidiasis; SAP4, SAP5, or SAP6 mRNA was detected in all subjects. SAP1 and SAP3 transcripts were observed only in patients with oral candidiasis. SAP7 mRNA expression, which has never been demonstrated under laboratory conditions, was detected in several of the patient samples. All seven SAP genes were simultaneously expressed in some patients with oral candidiasis. This is the first detailed study showing that the SAP gene family is expressed by C. albicans during colonization and infection in humans and that C. albicans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of oral candidiasis.

Telomerase in kinetoplastid parasitic protozoa.

We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species: Trypanosoma brucei, Leishmania major, and Leishmania tarentolae. Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N., et al. (1994) Science 266, 2011-2015], although better results were subsequently achieved with the two-tube telomere repeat amplification protocol [Autexier, C., Pruzan, R., Funk, W. & Greider, C. (1996) EMBO J. 15, 5928-5935]. The activity in T. brucei extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of T. brucei. Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.

Cloning and regulated expression of the Candida albicans phospholipase B (PLB1) gene.

Degenerate oligonucleotides (derived from conserved regions of PLB1 genes from S. cerevisiae and other fungi) were used to amplify PLB1 homolog fragments from C. albicans and C. tropicalis by using the polymerase chain reaction. The C. albicans PLB1 fragment was then used as a probe to clone the full-length gene and to monitor PLB1 mRNA expression. The C. albicans PLB1 gene consists of a 1815-bp open reading frame encoding a putative protein of 605 amino acids. It contains the highly conserved Gly-X-Ser-X-Gly catalytic motif, found in all lipolytic enzymes, and exhibits significant homology with other fungal PLB1 gene products (approximately 63% similarity, approximately 45% identity). Blastospores and pseudohyphae expressed higher levels of PLB1 mRNA than germ-tube-forming cells. TUP1, a general transcriptional repressor, may regulate PLB1 expression in C. albicans, since PLB1 expression was the highest in tup1 delta mutants and did not vary in response to environmental stimuli. Together, these results suggest that expression of the C. albicans PLB1 gene is regulated as a function of morphogenic transition.

Single-step multiplex PCR assay for characterization of New World Leishmania complexes.

We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmania parasites, particularly where different Leishmania complexes are found in the same geographical area.

Three small nucleolar RNAs identified from the spliced leader-associated RNA locus in kinetoplastid protozoans.

First characterized in Trypanosoma brucei, the spliced leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the T. brucei SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and approximately 450 or approximately 350 nt, respectively, each with significant sequence identity to transcripts previously described from the T. brucei SLA RNA locus. Cell fractionation studies localize the three additional RNAs to the nucleolus; the presence of box C/D-like elements in two of the transcripts suggests that they are members of a class of small nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs. Candidate rRNA-snoRNA interactions can be found for one domain in each of the C/D element-containing RNAs. The putative target site for the 75/76-nt RNA is a highly conserved portion of the small subunit rRNA that contains 2'-O-ribose methylation at a conserved position (Gm1830) in L. tarentolae and in vertebrates. The 92/94-nt RNA has the potential to form base pairs near a conserved methylation site in the large subunit rRNA, which corresponds to position Gm4141 of small rRNA 2 in T. brucei. These data suggest that trypanosomatids do not obey the general 5-bp rule for snoRNA-mediated methylation.

Entamoeba histolytica: computer-assisted modeling of phosphofructokinase for the prediction of broad-spectrum antiparasitic agents.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) is the rate-limiting glycolytic enzyme found in the pathogenic protists Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichomonas vaginalis, and Naegleria fowleri. The enzyme differs significantly from ATP-dependent phosphofructokinases found in humans and as such represents an important drug target. Current therapy for infections caused by these pathogens is inadequate, especially for children, pregnant women, and the immune compromised. The development of more selective, safer agents in imperative, as parasitic infections are currently a significant health threat worldwide and will likely become increasingly common agents of disease in the future. For the purpose of designing drugs to treat parasitic infections, we have constructed a model of PPi-PFK from E. histolytica based on the three-dimensional structure of the ATP-dependent PFK from Bacillus stearothermophilus. The model was used with the computer program Dock 3.5 (University of California, San Francisco) to predict the binding of pyrophosphate and selected bisphosphonates to the enzyme. The predicted drug-enzyme interactions suggested that two of these compounds would be competitive inhibitors of pyrophosphate. These drugs were tested against E. histolytica and inhibited the growth of amebae in vitro. This class of compounds may have broad-spectrum antiparasitic activity and, in the future, may facilitate the treatment of serious parasitic infections.

KEX2 influences Candida albicans proteinase secretion and hyphal formation.

Candida albicans possesses at least seven differentially expressed genes that encode virulence-related secretory aspartyl proteinases (Saps). Sap DNA sequences predict post-translational processing at lysine-arginine residues in the preproteins, reminiscent of the maturation of Saccharomyces cerevisiae alpha-factor, where a prepropolypeptide is converted into a biologically active pheromone by Kex2, a subtilisin-like proprotein convertase. To investigate involvement of a C. albicans KEX2 homologue in Sap activation, a genetic selection was performed based on KEX2 function. A kex2 strain of S. cerevisiae was transformed with a C. albicans genomic DNA library and screened for the production of active alpha-factor. Positive clones were assayed for killer toxin activity, another Kex2-dependent phenotype. Plasmids that rescued both defects contained a sequence encoding a protein homologous to S. cerevisiae Kex2. Both alleles of the C. albicans KEX2 were inactivated by successive mutations. Null mutants continued to secrete active Sap2; however, the enzyme was abnormally processed and secreted at reduced levels. Unexpectedly, null mutants were incapable of forming hyphae, instead differentiating into aberrantly shaped cells. The ability to normally process Sap2 and form hyphae was restored upon transformation of null mutants with a KEX2-containing plasmid.

Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid.

An IMP dehydrogenase gene was isolated from Candida albicans on a approximately 2.9-kb XbaI genomic DNA fragment. The putative Candida IMP dehydrogenase gene (IMH3) encodes a protein of 521 amino acids with extensive sequence similarity to the IMP dehydrogenases of Saccharomyces cerevisiae and various other organisms. Like the S. cerevisiae IMH3 sequence characterized in the genome sequencing project, the open reading frame of the C. albicans IMH3 gene is interrupted by a small intron (248 bp) with typical exon-intron boundaries and a consensus S. cerevisiae branchpoint sequence. IMP dehydrogenase mRNAs are detected in both the yeast and hyphal forms of C. albicans as judged by Northern hybridization. Growth of wild-type (sensitive) C. albicans cells is inhibited at 1 microg of mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenases, per ml, whereas transformants hosting a plasmid with the IMH3 gene are resistant to MPA levels of up to at least 40 microg/ml. The resistance of cells to MPA is gene dosage dependent and suggests that IMH3 can be used as a dominant selection marker in C. albicans.

The SLA RNA gene of Trypanosoma brucei is organized in a tandem array which encodes several small RNAs.

We have recently identified the Spliced Leader Associated RNA (SLA RNA) which is implicated in pre-messenger RNA splicing in Trypanosoma brucei by virtue of its interaction with the 5' splice site of the trans spliced spliced leader RNA (SL RNA) in vivo. Southern analyses reveal that the SLA RNA gene is found in a tandem array of 10-11 copies per haploid genome in T. brucei. Each repeat unit in the array encodes three additional small RNAs of unknown function. RNA polymerase inhibition studies are consistent with transcription of all four genes by the same polymerase, but do not clearly differentiate between RNA polymerases II and III. The SLA RNA has homologs in other kinetoplastid protozoa and we have determined the sequence from two additional species. Trypanosoma cruzi and Crithidia fasciculata. Features of both secondary structure and sequence are conserved in these organisms. One conserved element, 5'-UGUAGUG-3', has the potential to base-pair to the SL RNA upstream of the 5' splice site. This potential interaction is consistent with the sites of SL RNA to SLA RNA psoralen cross-linking in vivo [1].

Human and fungal 3' splice sites are used by Trypanosoma brucei for trans splicing.

In Trypanosoma brucei, pre-mRNAs are joined to a 5' 39 nt spliced leader sequence by trans splicing, a process that has not been well characterized. We have asked whether the 3' splice site regions of human and yeast introns are able to substitute in vivo for the 3' spliced leader acceptor regions of trypanosome pre-mRNA sequences. The ability of heterologous sequences to participate in trans splicing in trypanosomes was assayed by chloramphenicol acetyltransferase (CAT) enzyme activity and/or the detection of spliced CAT mRNA. Four out of the six heterologous 3' splice site regions (human beta-globin intervening sequence (IVS)2, human c-myc IVS2, human factor-VIII IVS1, and yeast actin IVS) functioned as 3' spliced leader acceptor regions in T. brucei, while two did not show significant or detectable levels of CAT activity (human beta-globin IVS1 and human c-myc IVS1). In the case of the human beta-globin IVS1 however, lengthening of the polypyrimidine tract as a result of single purine to pyrimidine transversions produced an active acceptor in which the spliced leader addition site coincides with the 3' splice site of the beta-globin exon 2. These studies indicate that some, but not all 3' acceptor regions in humans can function as spliced leader addition sites in trypansomes.

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique.

We have developed a nonradioactive branched DNA (bDNA)-based assay for the diagnosis of the African trypanosomiases in simple buffy coat preparations of human blood. Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets of the bDNA assay, a technique which amplifies the signal from a target molecule rather than the target itself. Comparable sensitivities were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes, and bloodstream trypomastigotes. The results of bDNA analysis of human blood samples from Côte d'Ivoire (n = 50) showed excellent agreement with those of buffy coat microscopy. The bDNA technology offers certain advantages over alternative molecular biological techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from crosscontamination artifacts. We have successfully applied the bDNA technique to the detection of T. brucei in clinical samples from regions where T. brucei infection is endemic; to our knowledge, this is the first report of the molecular detection of T. brucei in human blood.

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.

Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors.

For the pathogenic yeast Candida albicans, secreted aspartyl proteinase (Sap) activity has been correlated with virulence. A family consisting of at least eight SAP genes can be drawn upon to produce Sap enzymatic activity. In this study, the levels of Sap1, Sap2, and Sap3 isoenzymes were monitored under a variety of growth conditions for several strains, including strain WO-1, which alternates between two switch phenotypes, white (W) and opaque (O). When cultured under proteinase-inducing conditions, most strains and W cells produce Sap2, while O cells produce Sap1, Sap2, and Sap3. Both W and O cells of strain WO-1 produce Saps in enriched and defined media that do not induce Saps from other strains. The specific Sap isoenzyme that is produced is determined by the cell type, while the level of Sap production is determined by environmental factors. The levels and temporal regulation of the SAP mRNAs as determined by Northern (RNA) analysis were consistent with Sap protein levels and with previous results. S1 analysis showed that SAP6 is the predominant SAP gene transcribed during hyphal induction at neutral pH. These studies define the culture conditions which control the levels of SAP mRNAs and Sap proteins, and they indicate that both the yeast/hyphal transition and phenotypic switching can determine which of the Sap isoenzymes is produced.

Isolation and characterization of species-specific DNA probes from Taenia solium and Taenia saginata and their use in an egg detection assay.

Cysticercosis results from ingestion of the eggs of the tapeworm Taenia solium. Reduction of the incidence of human and swine cysticercosis requires identification and treatment of individuals who carry the adult tapeworm. T. solium and Taenia saginata eggs cannot be differentiated on the basis of morphology; thus, in order to improve existing methods for the diagnosis of taeniasis, we have developed highly sensitive, species-specific DNA probes which differentiate T. solium and T. saginata. Recombinant clones containing repetitive DNA sequences which hybridize specifically with genomic DNAs from either species were isolated and characterized. T. solium-specific DNA sequences contained complete and truncated forms of a tandemly repeated 158-bp DNA sequence. An unrelated T. saginata DNA sequence was also characterized and shown to encode a portion of the mitochondrial cytochrome c oxidase I gene. T. solium- and T. saginata-specific DNA probes did not hybridize in dot blot assays either with genomic DNA from the platyhelminths Taenia hydatigena, Taenia pisiformis, Taenia taeniaeformis, Echinococcus granulosus, and Schistosoma mansoni or with genomic DNA from other eukaryotes, including Saccharomyces cerevisiae, Candida albicans, Cryptosporidium parvum, Entamoeba histolytica, Trypanosoma gambiense, Trypanosoma brucei, and Giardia lamblia, Caenorhabditis elegans, and human DNA. By using these T. solium and T. saginata DNA probes, a rapid, highly sensitive and specific dot blot assay for the detection of T. solium eggs was developed.

The "universal" leucine codon CTG in the secreted aspartyl proteinase 1 (SAP1) gene of Candida albicans encodes a serine in vivo.

A number of Candida species possess a tRNA(Ser)-like species that recognizes CTG codons that normally specify leucine (Leu) in the universal code of codon usage. Mass spectrometry and Edman sequencing of peptides from the secreted aspartyl proteinase isoenzyme (Sap1) demonstrate that positions specified by the CTG codon contain a nonmodified serine (Ser) in Candida albicans.

Nuclear-encoded mitochondrial tRNAs of Trypanosoma brucei have a modified cytidine in the anticodon loop.

The mitochondrial genome of Trypanosoma brucei does not appear to encode any tRNA genes. Isolated organellar tRNAs hybridize to nuclear DNA, suggesting that they are synthesized in the nucleus and subsequently imported into the mitochondrion. Most imported tRNAs have cytosolic counterparts, showing identical mobility on two-dimensional polyacrylamide gels. We have compared three nuclear-encoded mitochondrial tRNAs (tRNA(Lys), tRNA(Leu), tRNA(Tyr)) with their cytosolic isoforms by direct enzymatic sequence analysis. Our findings indicate that the primary sequences of the mitochondrial and the corresponding cytosolic tRNAs are identical. However, we have identified a mitochondrion-specific nucleotide modification of each tRNA which is localized to a conserved cytidine residue at the penultimate position 5' of the anticodon. The modification present in mature mitochondrial tRNA(Tyr) was not found in a mutant tRNA(Tyr) defective in splicing in either cytosolic or mitochondrial fractions. The mutant tRNA(Tyr) has been expressed in transformed cells and its import into mitochondria has been demonstrated, suggesting that the modified cytidine residue is not required for import and therefore may be involved in adapting imported tRNAs to specific requirements of the mitochondrial translation machinery.

A common core structure for U3 small nucleolar RNAs.

U3 nucleolar small RNA (snRNA) is involved in early processing of the primary rRNA transcript. A secondary structure model for the unusually small Trypanosoma brucei U3 snRNA was deduced by chemical modification and enzymatic cleavage of U3 snRNA in deproteinized and ribonucleoprotein (RNP) forms. Comprehensive alignment of U3 snRNAs from vertebrate, plant, fungal and protozoan species clearly delineated conserved and divergent features. The 5' domain of the T. brucei U3 snRNA appears to form one small, flexible 5' stem loop structure followed by a long single-stranded region; this model is a variation on 5' domain structures proposed for other U3 snRNAs which do not conform to a single model. The 3' domain of T. brucei U3 snRNA contains four single-stranded sequences conserved between U3 snRNAs. Of these, structural probing determined that the configurations of GAU region and box B and C sequences are altered by protein interactions in U3 snRNP. Conspicuously, the 3' domains of trypanosomal U3 snRNAs lack stem loops II and III, indicating that these structures are not required for conserved U3 snRNA functions.

Characterization of putative small nuclear RNAs from Giardia lamblia.

Small nuclear RNAs (snRNAs), which have roles in RNA metabolism, have been found in all eukaryotic cells. Candidate snRNAs were identified in the primitive protozoan parasite, Giardia lamblia, by either immunoprecipitation of total RNA with antiserum directed against the 2,2,7-trimethylguanosine cap structure characteristic of snRNAs or by Northern hybridization with oligonucleotide probes complementary to snRNA consensus sequences. Isolated putative snRNAs include eight 2,2,7-trimethylguanosine-capped species: RNAs A through H, and one non-2,2,7-trimethylguanosine-capped species: RNA J. Single copy genes encoding RNA D, RNA H and RNA J were cloned and sequenced, and the 5' end of each RNA was determined by primer extension analysis. Certain characteristics suggest tentative identification of these Giardia RNAs as nucleolar RNAs with possible roles in rRNA processing.