Opportunistic Human Fungal Pathogen Research Articles

Characterisation and expression of phospholipases B from the opportunistic fungus Aspergillus fumigatus

The phospholipase B family (PLB) are enzymes sharing phospholipase (PL), lysophospholipase (LPL) and lysophospholipase-transacylase (LPTA) activities. They have been shown to be important virulence factors in several human fungal pathogens including Candida albicans and Cryptococcus neoformans. Aspergillus fumigatus, a human opportunistic fungal pathogen leading to a high rate of mortality in immunosuppressed patients is known to possess an extracellular phospholipase B activity. In this paper, we report the molecular characterisation of three PLB genes from A. fumigatus (af plb) using degenerate primers in PCR amplification and data from the A. fumigatus genome project. They are expressed at 37 °C, and two of them (af plb1 and af plb3) are induced by lecithin. They encode proteins of 633, 588 and 630 amino acids, respectively, presenting together a T-Coffee score of 81. They also possess the amino acid triad responsible for enzymatic activity in the mammalian cytosolic PLA 2 and other fungal PLBs. AfPLB1 and afPLB3 are secreted with a cleaved signal peptide. The complete cDNA sequences were obtained by RACE-PCR for the two secreted afPLBs and probably account for the extracellular phospholipase activity previously reported in the culture media of A. fumigatus.

Mutation of the regulator of G protein signaling Crg1 increases virulence in Cryptococcus neoformans.

The regulator of G protein signaling homolog Crg1 was found to be a key regulator of pheromone-responsive mating in the opportunistic human fungal pathogen Cryptococcus neoformans. A mutation in the CRG1 gene has greatly increased virulence in the prevalently distributed MATalpha strains of the fungus. Mouse survival time was shortened by 40%, and the lethal dosage was 100-fold less than that of wild-type strains. In addition, the increased virulence of crg1 mutant strains was dependent on the transcription factor homolog Ste12alpha but not on the mitogen-activated protein kinase homolog Cpk1. The enhanced mating due to CRG1 mutation, however, was still dependent on Cpk1. Interestingly, crg1 mutants of MATalpha cells produced dark melanin pigment under normally inhibitory conditions, which may relate to the mechanism for increased virulence.

Role of phospholipomannan in Candida albicans escape from macrophages and induction of cell apoptosis through regulation of bad phosphorylation.

Candida albicans, the most common opportunistic fungal pathogen of humans is a part of the normal microbial flora. To investigate host-parasite interaction related to the commensal-pathogen switch of this yeast we compared the response of macrophages to C. albicans and to the non-pathogenic yeast Saccharomyces cerevisiae. In contrast to S. cerevisiae, C. albicans survived within macrophages. This escape from macrophages was associated with qualitative differences in the sequential phosphorylation of MEK, ERK1/2, and p90RSK during phagocytosis. Decreased activation of this pathway was observed with C. albicans and was associated with a species-specific overexpression of the MEK phosphatase, MKP-1. Dysregulation of the ERK1/2/p90RSK signal transduction pathway by C. albicans was associated downstream with reduction in Bad phosphorylation, specifically at Ser-112, and disappearance of free Bcl-2. This ended at apoptosis of cells that have ingested C. albicans, as revealed by staining of phosphatidylserine exposure in the macrophage outer membrane. The role of phospholipomannan (PLM), a phylogenetically unique glycolipid with a phytoceramide moiety expressed at the surface of and shed by C. albicans, was examined. Addition of PLM to macrophages led to dysregulation similar to that observed with live C. albicans and promoted the survival of the sensitive S. cerevisiae within the cells. Evidence of externalization of membranous phosphatidylserine, loss of mitochondrial integrity, and DNA fragmentation after incubation of macrophages with PLM suggest that this molecule supported the activities observed with C. albicans yeast cells.

Binding of Candida albicans enolase to plasmin(ogen) results in enhanced invasion of human brain microvascular endothelial cells.

Infection by the human opportunistic fungal pathogen Candida albicans has been increasing over recent years. In an attempt to understand the molecular mechanism of Candida invasion across host tissues, the relationship of C. albicans enolase to human plasminogen/plasmin was investigated. C. albicans enolase is a cell-surface protein and an immunodominant antigen in infected patients' sera. Plasminogen is an abundant plasma protein. Several lines of evidence support the binding of C. albicans enolase to human plasminogen. Firstly, it was found that various Candida strains were able to bind to plasminogen and its active form, plasmin. Secondly, recombinant Candida enolase was retained in a nickel-chelating affinity column matrix that can bind (125)I-labelled plasminogen or plasmin in a dose-dependent manner. Plasmin(ogen)-specific inhibitors, such as epsilon -aminocaproic acid and aprotinin, can effectively block plasmin-binding activity. Thirdly, as with many plasminogen receptors, binding of Candida enolase to plasmin(ogen) is lysine-dependent, whereas little inhibition occurred with arginine, aspartate and glutamate. Fourthly, immobilized enolase enhanced plasminogen's affinity for streptokinase at least tenfold, as demonstrated by its activation of plasmin activity. To elucidate the biological significance of this result, it was demonstrated that the plasmin(ogen)-bound Candida cells were able to induce fibrinolysis activity in a matrix-gel assay. Furthermore, plasmin-bound Candida cells had an increased ability to cross an in vitro blood-brain barrier system. The results given here indicate that Candida enolase is a plasminogen- and plasmin-binding protein and that the interaction of C. albicans enolase with the plasminogen system may contribute to invasion of the tissue barrier.

Characterization of the Penicillium chrysogenum antifungal protein PAF.

The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF ( Penicillium antifungal protein). In this study, the antifungal activity of PAF is described. PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected. PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching. Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi. In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations.

Genome-wide expression profile analysis reveals coordinately regulated genes associated with stepwise acquisition of azole resistance in Candida albicans clinical isolates.

Candida albicans is an opportunistic human fungal pathogen and a causative agent of oropharyngeal candidiasis (OPC), the most frequent opportunistic infection among patients with AIDS. Fluconazole and other azole antifungal agents have proven effective in the management of OPC; however, with increased use of these agents treatment failures have occurred. Such failures have been associated with the emergence of azole-resistant strains of C. albicans. In the present study we examined changes in the genome-wide gene expression profile of a series of C. albicans clinical isolates representing the stepwise acquisition of azole resistance. In addition to genes previously associated with azole resistance, we identified many genes whose differential expression was for the first time associated with this phenotype. Furthermore, the expression of these genes was correlated with that of the known resistance genes CDR1, CDR2, and CaMDR1. Genes coordinately regulated with the up-regulation of CDR1 and CDR2 included the up-regulation of GPX1 and RTA3 and the down-regulation of EBP1. Genes coordinately regulated with the up-regulation of CaMDR1 included the up-regulation of IFD1, IFD4, IFD5, IFD7, GRP2, DPP1, CRD2, and INO1 and the down-regulation of FET34, OPI3, and IPF1222. Several of these appeared to be coordinately regulated with both the CDR genes and CaMDR1. Many of these genes are involved in the oxidative stress response, suggesting that reduced susceptibility to oxidative damage may contribute to azole resistance. Further evaluation of the role these genes and their respective gene products play in azole antifungal resistance is warranted.

Ras1 controls pheromone expression and response during mating in Cryptococcus neoformans

The Cryptococcus neoformans Ras1 signal transduction pathway controls mating, hyphal differentiation, and the ability of this opportunistic human fungal pathogen to grow at elevated temperatures. To further elucidate how Ras1 signals in this organism, the RAS1 gene was disrupted in the congenic serotype D strain background. Genetic epistasis experiments indicated that Ras1 regulates the mating response through the MAP kinase/pheromone response pathway. In fact, Ras1 is required for the transcriptional induction of elements of the pheromone response pathway. However, the ability of C. neoformans Ras1 to allow growth at 37 °C is mediated by a separate signaling pathway. Therefore a single Ras protein may differentially activate distinct downstream targets in response to different signals within the same organism. This conserved signaling motif has been coopted in C. neoformans to regulate mating and morphogenesis in addition to being required for its pathogenic potential.

A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene.

Aspergillus fumigatus is an important opportunistic human pathogenic fungus. In severely immunocompromised patients, the fungus causes life-threatening diseases, such as pneumonia and invasive aspergillosis. In order to obtain a better understanding of the key elements involved in A. fumigatus virulence and for identifying possible drug targets, it is essential to be able to generate gene-deletion strains. Until recently, the molecular techniques available did not provide a rapid method for gene deletion. A novel method described for A. nidulans was adapted for A. fumigatus. This method is quick and produces an increased homologous recombination efficiency. By using an Escherichia coli strain expressing the lambda red operon, it is possible to induce an in vivo recombination of a PCR fragment flanked by >50-bp regions with a cosmid containing the gene of interest. This produces cosmids in which the gene of interest has been replaced by a bi-functional marker. Such cosmids have large flanking regions surrounding the selectable marker pyrG of A. fumigatus used here, which result in high recombination efficiencies in A. fumigatus. Here, we identified a new ABC transporter-encoding gene in A. fumigatus, designated abcA. By using this method, an A. fumigatus knock-out mutant was generated, providing evidence that this method of generating gene deletions can also be used in A. fumigatus and significantly broadens our repertoire of molecular techniques to study A. fumigatus.

Ura-status-dependent adhesion of Candida albicans mutants

Gene disruptions in the diploid opportunistic human fungal pathogen Candida albicans are usually created using multiple rounds of targeted integration called the ‘ura-blaster’ method. Resulting heterozygous and homozygous null mutants can be auxotrophic (Ura−) or prototrophic (Ura+) for uracil biosynthesis. Here we demonstrate that the Ura-status of otherwise isogenic mutants affected the adhesion of C. albicans. Moreover the effect of Ura-status on adhesion was also dependent on the null mutant background, the nature of the underlying surface and the carbon source for growth. Therefore the Ura-status is not neutral in determining adhesive properties of C. albicans mutants that are generated via the ura-blaster protocol.

Ura-status-dependent adhesion of Candida albicans mutants.

Gene disruptions in the diploid opportunistic human fungal pathogen Candida albicans are usually created using multiple rounds of targeted integration called the 'ura-blaster' method. Resulting heterozygous and homozygous null mutants can be auxotrophic (Ura(-)) or prototrophic (Ura(+)) for uracil biosynthesis. Here we demonstrate that the Ura-status of otherwise isogenic mutants affected the adhesion of C. albicans. Moreover the effect of Ura-status on adhesion was also dependent on the null mutant background, the nature of the underlying surface and the carbon source for growth. Therefore the Ura-status is not neutral in determining adhesive properties of C. albicans mutants that are generated via the ura-blaster protocol.

Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase.

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.

An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant.

Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25 degrees P. marneffei exhibits true filamentous growth, while at 37 degrees P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12alpha genes, form a distinct subclass of STE12 homologs that have a C2H2 zinc-finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number of mutants in the P. marneffei-type strain and, in combination with selectable markers, developed a highly efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei. Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle.

Membrane and cell wall targets in Aspergillus fumigatus.

Antifungal drugs directed against the human opportunistic fungal pathogen Aspergillus fumigatus are limited in number and ergosterol-targeted: the polyenes bind to the membrane ergosterol and the azoles block the ergosterol biosynthesis pathway. The efficacy of the drugs currently available for clinical use (amphotericin B and itraconazole) is limited and the frequent occurrence of therapeutic failures in the treatment of invasive aspergillosis emphasizes the need for the development of new agents. Cell wall biosynthetic pathways have been recognized for a long time as essential and unique specific drug targets. Recent studies of the chemical organization of the cell wall of A. fumigatus together with comparative analysis of yeast cell wall data have shown that beta 1-3 glucan branching and chitin-beta 1-3 glucan binding are essential exocellular enzymatic steps in cell wall biosynthesis. The enzymes involved in the biosynthesis and remodeling of cell wall polysaccharides especially in A. fumigatus are reviewed.

Diploid Strains of the Pathogenic Basidiomycete Cryptococcus neoformans Are Thermally Dimorphic

Sia, R. A., Lengeler, K. B., and Heitman, J. 2000. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATα and MATa strains. At 37°C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATα and MATa loci. At 24°C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.

Cloning and characterization of PRA1, a gene encoding a novel pH-regulated antigen of Candida albicans.

Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of C. albicans was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designated PRA1, for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The PRA1 protein was homologous to surface antigens of Aspergillus spp. which react with serum from aspergillosis patients, suggesting that the PRA1 protein may have a role in the host-parasite interaction during candidal infection.

Relatedness ofPseudallescheria, ScedosporiumandGraphium pro partebased on SSU rDNA sequences

Genetic relatedness of 10 strains of opportunistic human pathogenic fungi in the genera Pseudallescheria and Scedosporium and related fungi including Petriella setifera, Graphium tectonae and Ceratocystis fimbriata, were tested by sequencing the entire SSU rDNA. From these molecular data, the imperfect genus Scedosporium can be divided in two, S. prolificans being separable from the S. apiospermum complex. Nearest sexual relatives of S. prolificans seem to be in the microascalean genus Petriella. Strains of Ps. boydii and S. apiospermum, although differing by their previously known DNA/DNA reassociation values, were not separable from each other once a 380 nucleotide long insertion had been removed from the sequence of the two S. apiospermum strains tested. The SSU rDNA of Ps. ellipsoidea is identical to that of Ps. boydii. Fungi which have been known to have Graphium anamorphs can be divided in at least three groups, one of which is related to Petriella and includes Graphium tectonae.

A role for complement receptor-like molecules in iron acquisition by Candida albicans.

Candida albicans, an opportunistic fungal pathogen of humans, is dependent upon iron for growth. Consequently, human serum inhibits C. albicans growth due to the presence of high affinity iron-binding proteins that sequester serum iron, making it unavailable for use by the organism. We report that in the inhibitory environment of human serum, the growth of C. albicans can be restored by the addition of exogenous hemoglobin or heme, but not by protoporphyrin IX, the heme precursor that does not contain iron. We further report that C. albicans can utilize cell surface proteins that are homologues of the mammalian complement receptors (CR) to rosette complement-coated red blood cells (RBC) and obtain RBC-derived iron for growth. The ability of Candida to acquire RBC-derived iron under these conditions is dependent upon Candida-RBC rosetting mediated by CR-like molecules. Unopsonized RBC do not support Candida growth in serum, and restoration of Candida growth in serum by complement-opsonized RBC is inhibited by monoclonal antibodies to the human CR type 3 (CR3). In addition, activation of the human alternative pathway of complement by Candida leads to "bystander" deposition of C3 fragments on the surface of autologous, unopsonized RBC, generating the ligands necessary for Candida-RBC rosetting. These results suggest that C. albicans has evolved a unique strategy for acquiring iron from the host, which exploits the host complement system, and which may contribute to the pathogenic potential of the organism.