Global screening of potential Candida albicans biofilm-related transcription factors via network comparison.

BACKGROUND: Candida albicans is a commonly encountered fungal pathogen in humans. The formation of biofilm is a major virulence factor in C. albicans pathogenesis and is related to antidrug resistance of this organism. Although many factors affecting biofilm have been analyzed, molecular mechanisms that regulate biofilm formation still await to be elucidated. RESULTS: In this study, from the gene regulatory network perspective, we developed an efficient computational framework, which integrates different kinds of data from genome-scale analysis, for global screening of potential transcription factors (TFs) controlling C. albicans biofilm formation. S. cerevisiae information and ortholog data were used to infer the possible TF-gene regulatory associations in C. albicans. Based on TF-gene regulatory associations and gene expression profiles, a stochastic dynamic model was employed to reconstruct the gene regulatory networks of C. albicans biofilm and planktonic cells. The two networks were then compared and a score of relevance value (RV) was proposed to determine and assign the quantity of correlation of each potential TF with biofilm formation. A total of twenty-three TFs are identified to be related to the biofilm formation; ten of them are previously reported by literature evidences. CONCLUSIONS: The results indicate that the proposed screening method can successfully identify most known biofilm-related TFs and also identify many others that have not been previously reported. Together, this method can be employed as a pre-experiment screening approach that reveals new target genes for further characterization to understand the regulatory mechanisms in biofilm formation, which can serve as the starting point for therapeutic intervention of C. albicans infections.

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation.

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.

Think globally, act locally, and collaborate internationally: global health sciences at the University of California, San Francisco.

The University of California, San Francisco (UCSF) established Global Health Sciences (GHS) as a campus-wide initiative in 2003. The mission of GHS is to facilitate UCSF's engagement in global health across its four schools by (1) creating a supportive environment that promotes UCSF's leadership role in global health, (2) providing education and training in global health, (3) convening and coordinating global health research activities, (4) establishing global health outreach programs locally in San Francisco and California, (5) partnering with academic centers, especially less-well-resourced institutions in low- and middle-income countries, and (6) developing and collaborating in international initiatives that address neglected global health issues.GHS education programs include a master of science (MS) program expected to start in September 2008, an introduction to global health for UCSF residents, and a year of training at UCSF for MS and PhD students from low- and middle-income countries that is "sandwiched" between years in their own education program and results in a UCSF Sandwich Certificate. GHS's work with partner institutions in California has a preliminary focus on migration and health, and its work with academic centers in low- and middle-income countries focuses primarily on academic partnerships to train human resources for health. Recognizing that the existing academic structure at UCSF may be inadequate to address the complexity of global health threats in the 21st century, GHS is working with the nine other campuses of the University of California to develop a university-wide transdisciplinary initiative in global health.

Fungicidal activity of a phospholipase-A2-derived synthetic peptide variant against Candida albicans.

pEM-2, a 13-mer synthetic peptide variant derived from myotoxin II, a phospholipase A(2) homologue present in Bothrops asper snake venom, has shown potent bactericidal activity in previous studies due to the combination of cationic and hydrophobic amino acids, including three tryptophan-substituted residues in its sequence. This study reports that pEM-2 also exerts potent fungicidal activity against a variety of clinically relevant Candida species, killing 100% of yeasts at concentrations near 10 mg/l (5 microM), as indicated by plate counting assays. Thus, this peptide displays a broad-spectrum antimicrobial activity, in the absence of hemolytic activity. The fungicidal action of pEM-2 against Candida can be partially inhibited by increasing concentrations of extracellular divalent cations (Ca(+2) or Mg(+2)), in agreement with its proposed membrane-permeabilizing mechanism of action.

Candida albicans modulates host defense by biosynthesizing the pro-resolving mediator resolvin E1.

Candida albicans is an opportunistic fungal pathogen of humans that resides commensally on epithelial surfaces, but can cause inflammation when pathogenic. Resolvins are a class of anti-inflammatory lipids derived from omega-3 polyunsaturated fatty acids (PUFA) that attenuate neutrophil migration during the resolution phase of inflammation. In this report we demonstrate that C. albicans biosynthesizes resolvins that are chemically identical to those produced by human cells. In contrast to the trans-cellular biosynthesis of human Resolvin E1 (RvE1), RvE1 biosynthesis in C. albicans occurs in the absence of other cellular partners. C. albicans biosynthesis of RvE1 is sensitive to lipoxygenase and cytochrome P450 monoxygenase inhibitors. We show that 10nM RvE1 reduces neutrophil chemotaxis in response to IL-8; 1nM RvE1 enhanced phagocytosis of Candida by human neutrophils, as well as intracellular ROS generation and killing, while having no direct affect on neutrophil motility. In a mouse model of systemic candidiasis, RvE1 stimulated clearance of the fungus from circulating blood. These results reveal an inter-species chemical signaling system that modulates host immune functions and may play a role in balancing host carriage of commensal and pathogenic C. albicans.

Phospholipase A2 and phospholipase B activities in fungi.

As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A(2) (PLA(2)) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host's immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA(2) activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host.

Role of calcineurin in stress resistance, morphogenesis, and virulence of a Candida albicans wild-type strain.

By generating a calcineurin mutant of the Candida albicans wild-type strain SC5314 with the help of a new recyclable dominant selection marker, we confirmed that calcineurin mediates tolerance to a variety of stress conditions but is not required for the ability of C. albicans to switch to filamentous growth in response to hypha-inducing environmental signals. While calcineurin was essential for virulence of C. albicans in a mouse model of disseminated candidiasis, deletion of CMP1 did not significantly affect virulence during vaginal or pulmonary infection, demonstrating that the requirement for calcineurin for a successful infection depends on the host niche.

Inactivation of the phospholipase B gene PLB5 in wild-type Candida albicans reduces cell-associated phospholipase A2 activity and attenuates virulence.

Phospholipases are critical for modification and redistribution of lipid substrates, membrane remodeling and microbial virulence. Among the many different classes of phospholipases, fungal phospholipase B (Plb) proteins show the broadest range of substrate specificity and hydrolytic activity, hydrolyzing acyl ester bonds in phospholipids and lysophospholipids and further catalyzing lysophospholipase-transacylase reactions. The genome of the opportunistic fungal pathogen Candida albicans encodes a PLB multigene family with five putative members; we present the first characterization of this group of potential virulence determinants. CaPLB5, the third member of this multigene family characterized herein is a putative secretory protein with a predicted GPI-anchor attachment site. Real-time RT-PCR gene expression analysis of CaPLB5 and the additional CaPLB gene family members revealed that filamentous growth and physiologically relevant environmental conditions are associated with increased PLB gene activity. The phenotypes expressed by null mutant and revertant strains of CaPLB5 indicate that this lipid hydrolase plays an important role for cell-associated phospholipase A(2) activity and in vivo organ colonization.

Genome-wide transcription profiling of the early phase of biofilm formation by Candida albicans.

The ability to adhere to surfaces and develop as a multicellular community is an adaptation used by most microorganisms to survive in changing environments. Biofilm formation proceeds through distinct developmental phases and impacts not only medicine but also industry and evolution. In organisms such as the opportunistic pathogen Candida albicans, the ability to grow as biofilms is also an important mechanism for persistence, facilitating its growth on different tissues and a broad range of abiotic surfaces used in medical devices. The early stage of C. albicans biofilm is characterized by the adhesion of single cells to the substratum, followed by the formation of an intricate network of hyphae and the beginning of a dense structure. Changes in the transcriptome begin within 30 min of contact with the substrate and include expression of genes related to sulfur metabolism, in particular MET3, and the equivalent gene homologues of the Ribi regulon in Saccharomyces cerevisiae. Some of these changes are initiated early and maintained throughout the process; others are restricted to the earliest stages of biofilm formation. We identify here a potential alternative pathway for cysteine metabolism and the biofilm-associated expression of genes involved in glutathione production in C. albicans.

A human-curated annotation of the Candida albicans genome.

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry.

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.

The functional basis of mycophenolic acid resistance in Candida albicans IMP dehydrogenase.

Candida albicans is an important fungal pathogen of immunocompromised patients. In cell culture, C. albicans is sensitive to mycophenolic acid (MPA) and mizoribine, both natural product inhibitors of IMP dehydrogenase (IMPDH). These drugs have opposing interactions with the enzyme. MPA prevents formation of the closed enzyme conformation by binding to the same site as a mobile flap. In contrast, mizoribine monophosphate, the active metabolite of mizoribine, induces the closed conformation. Here, we report the characterization of IMPDH from wild-type and MPA-resistant strains of C. albicans. The wild-type enzyme displays significant differences from human IMPDHs, suggesting that selective inhibitors that could be novel antifungal agents may be developed. IMPDH from the MPA-resistant strain contains a single substitution (A251T) that is far from the MPA-binding site. The A251T variant was 4-fold less sensitive to MPA as expected. This substitution did not affect the k(cat) value, but did decrease the K(m) values for both substrates, so the mutant enzyme is more catalytically efficient as measured by the value of k(cat)/K(m). These simple criteria suggest that the A251T variant would be the evolutionarily superior enzyme. However, the A251T substitution caused the enzyme to be 40-fold more sensitive to mizoribine monophosphate. This result suggests that A251T stabilizes the closed conformation, and this hypothesis is supported by further inhibitor analysis. Likewise, the MPA-resistant strain was more sensitive to mizoribine in cell culture. These observations illustrate the evolutionary challenge posed by the gauntlet of chemical warfare at the microbial level.

Regulatory networks affected by iron availability in Candida albicans.

Iron, an essential element for almost every organism, serves as a regulatory signal for the expression of virulence determinants in many prokaryotic and eukaryotic pathogens. Using a custom Affymetrix GeneChip representing the entire Candida albicans genome, we examined the changes in genome-wide gene expression in this opportunistic pathogen as a function of alterations in environmental concentrations of iron. A total of 526 open reading frame (ORF) transcripts are more highly expressed when the levels of available iron are low, while 626 ORF transcripts are more highly expressed in high-iron conditions. The transcripts dominantly affected by iron concentration range from those associated with cell-surface properties to others which affect mitochondrial function, iron transport and virulence-related secreted hydrolases. Moreover gene expression as assayed in DNA microarrays confirms and extends reports of alterations in cell-surface antigens and drug sensitivity correlated with iron availability. To understand how these genes and pathways might be regulated, we isolated a gene designated SFU1 that encodes a homologue of the Ustilago maydis URBS1, a transcriptional repressor of siderophore uptake/biosynthesis. Comparisons between wild-type and SFU1-null mutant strains revealed 139 potential target genes of Sfu1p; many of which are iron-responsive. Together, these results not only expand our understanding of global iron regulation in C. albicans, but also provide insights into the potential role of iron availability in C. albicans virulence.

The diploid genome sequence of Candida albicans.

We present the diploid genome sequence of the fungal pathogen Candida albicans. Because C. albicans has no known haploid or homozygous form, sequencing was performed as a whole-genome shotgun of the heterozygous diploid genome in strain SC5314, a clinical isolate that is the parent of strains widely used for molecular analysis. We developed computational methods to assemble a diploid genome sequence in good agreement with available physical mapping data. We provide a whole-genome description of heterozygosity in the organism. Comparative genomic analyses provide important clues about the evolution of the species and its mechanisms of pathogenesis.

Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with active oral and vaginal infections.

The in vivo expression of Candida albicans secreted aspartyl proteinase (SAP1-SAP8) and phospholipase B (PLB1 and PLB2) genes was analyzed in 137 human subjects with oral and vaginal candidiasis or carriage. Total RNA was isolated from whole unstimulated saliva or vaginal swabs, and the expression of SAP1-8 and PLB1-2 was evaluated by reverse-transcriptase polymerase chain reaction using specific primer sets. A spectrum of SAP gene expression profiles was obtained from different C. albicans strains during symptomatic disease and asymptomatic carriage. SAP2 and SAP5 were the most common genes expressed during both infection and carriage. SAP1, SAP3, SAP4, SAP7, SAP8, and PLB1 expression was correlated with oral disease, whereas SAP1, SAP3, and SAP6-SAP8 expression was correlated with vaginal disease. Furthermore, SAP1, SAP3, and SAP8 were preferentially expressed in vaginal, rather than oral, infections. This study demonstrates the differential expression of the hydrolytic enzyme genes in humans and correlates the expression of specific Candida species virulence genes with active disease and anatomical location.

Inactivation of Kex2p diminishes the virulence of Candida albicans.

Deletion of the kexin gene (KEX2) in Candida albicans has a pleiotropic effect on phenotype and virulence due partly to a defect in the expression of two major virulence factors: the secretion of active aspartyl proteinases and the formation of hyphae. kex2/kex2 mutants are highly attenuated in a mouse systemic infection model and persist within cultured macrophages for at least 24 h without causing damage. Pathology is modest, with little disruption of kidney matrix. The infecting mutant cells are largely confined to glomeruli, and are aberrant in morphology. The complex phenotype of the deletion mutants reflects a role for kexin in a wide range of cellular processes. Taking advantage of the specificity of Kex2p cleavage, an algorithm we developed to scan the 9168 open reading frames in Assembly 6 of the C. albicans genome identified 147 potential substrates of Kex2p. These include all previously identified substrates, including eight secreted aspartyl proteinases, the exoglucanase Xog1p, the immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative Kex2p substrates identified include several adhesins, cell wall proteins, and hydrolases previously not implicated in pathogenesis. Kexins also process fungal mating pheromones; a modification of the algorithm identified a putative mating pheromone with structural similarities to Saccharomyces cerevisiae alpha-factor.

Metabolic specialization associated with phenotypic switching in Candidaalbicans.

Phase and antigenic variation are mechanisms used by microbial pathogens to stochastically change their cell surface composition. A related property, referred to as phenotypic switching, has been described for some pathogenic fungi. This phenomenon is best studied in Candida albicans, where switch phenotypes vary in morphology, physiology, and pathogenicity in experimental models. In this study, we report an application of a custom Affymetrix GeneChip representative of the entire C. albicans genome and assay the global expression profiles of white and opaque switch phenotypes of the WO-1 strain. Of 13,025 probe sets examined, 373 ORFs demonstrated a greater than twofold difference in expression level between switch phenotypes. Among these, 221 were expressed at a level higher in opaque cells than in white cells; conversely, 152 were more highly expressed in white cells. Affected genes represent functions as diverse as metabolism, adhesion, cell surface composition, stress response, signaling, mating type, and virulence. Approximately one-third of the differences between cell types are related to metabolic pathways, opaque cells expressing a transcriptional profile consistent with oxidative metabolism and white cells expressing a fermentative one. This bias was obtained regardless of carbon source, suggesting a connection between phenotypic switching and metabolic flexibility, where metabolic specialization of switch phenotypes enhances selection in relation to the nutrients available at different anatomical sites. These results extend our understanding of strategies used in microbial phase variation and pathogenesis and further characterize the unanticipated diversity of genes expressed in phenotypic switching.

Functional analysis of a vacuolar ABC transporter in wild-type Candida albicans reveals its involvement in virulence.

ATP-driven transport proteins belonging to the ATP-binding cassette (ABC) superfamily perform important functions in cell metabolism and detoxification. Compounds can be actively transported across membranes, including the plasma membrane or organellar membranes. The vacuole is an important organelle in fungal cells required for compartmentalization of metabolites as well as toxic substances. Sequestration into the vacuole is often energy-dependent. We present the first isolation and molecular analysis of a vacuolar ABC transporter gene in the opportunistic fungal pathogen Candida albicans. The protein encoded by the MLT1 gene is highly similar to Multiple Drug Resistance-associated Protein (MRP)-like transporters of yeast and higher organisms that form the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)/MRP subfamily of ABC transporters, a class of proteins so far not characterized in C. albicans. MLT1 expression is extensively growth phase-regulated, and gene transcripts are inducible by metabolic poisons. Gene replacement mutants generated in wild-type C. albicans with the dominant selection marker MPAR showed a profound reduction in virulence in a mouse peritonitis model that was reversed by complementation with an intact MLT1 gene. Hence, this report provides primary evidence for the involvement of vacuolar ABC transporters in fungal virulence.

A Trypanosoma brucei protein complex that binds G-overhangs and co-purifies with telomerase activity.

The chromosomal ends of Trypanosoma brucei, like those of most eukaryotes, contain conserved 5'-TTAGGG-3' repeated sequences and are maintained by the action of telomerase. Fractionated T. brucei cell extracts with telomerase activity were used as a source of potential regulatory factors or telomerase-associated components that might interact with T. brucei telomeres. Electrophoretic mobility shift assays and UV cross-linking were used to detect possible single-stranded telomeric protein.DNA complexes and to estimate the approximate size of the protein constituents. Three single-stranded telomeric protein.DNA complexes were observed. Complex C3 was highly specific for the G-strand telomeric repeat sequence and shares biochemical characteristics with G-rich, single-stranded telomeric binding proteins and with components of the telomerase holoenzyme described in yeast, ciliates, and humans. Susceptibility to RNase A or chemical nuclease (hydroxyl radical) pre-treatment showed that complex C3 was tightly associated with an RNA component. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to estimate the molecular mass of the peptides obtained by in-gel Lys-C digestion of low abundance C3-associated proteins. The molecular masses of the peptides showed no homologies with other proteins from trypanosomes or with any protein in the data bases screened.