Bernhard Hube

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans

The opportunistic fungal pathogen Candida albicans produces secretory aspartyl proteinases, which are believed to be virulence factors in infection. We have studied the in vitro expression of seven known members of the SAP gene family in a range of strains and serotypes by Northern analysis. SAP1 and SAP3 were regulated during phenotypic switching between the white and opaque forms of the organism. The SAP2 mRNA, which was the dominant transcript in the yeast form, was found to be autoinduced by peptide products of Sap2 activity and to be repressed by amino acids. The expression of the closely related SAP4‐SAP6 genes was observed only at neutral pH during serum‐induced yeast to hyphal transition. No SAP7 mRNA was detected under any of the conditions or in any of the strains tested. Our data suggest that the various members of the SAP gene family may have distinct roles in the colonization and invasion of the host.

Multiplicity of genes encoding secreted aspartic proteinases in Candida species

The secreted aspartic proteinases (SAP) of Candida sp. are presumed to be potential virulence factors. In the opportunistic pathogen Candida albicans the proteinase genes identified to date, SAP1, SAP2, SAP3 and SAP4, constitute a multigene family. Before addressing the possible role of each proteinase in virulence, we sought to isolate all the members of this multigene family by screening a genomic library with a SAP1 probe for additional C. albicans SAP genes using low‐stringency hybridization conditions. Three putative new members, SAP5, SAP6 and SAP7 were isolated and sequenced. The N‐terminal segments of the deduced amino acid sequences of SAP5 and SAP6 contained secretion signal sequences similar to those of other Candida SAPs. Upon comparison and alignment with the other reported SAP amino acid sequences, SAP7 is not only the most divergent protein but also exhibits a much longer putative pro‐sequence with a single Lys‐Lys putative processing site. Using SAP1 to SAP7 as probes, the overall number of SAP genes in C. albicans was tentatively estimated by low‐stringency hybridization to EcoRI‐digested genomic DNA. While each isolated SAP gene could be assigned to distinct EcoRI bands, the existence of two additional genes not isolated after screening of the C. albicans gene library was inferred. Furthermore, evidence was obtained for the existence of SAP muttigene families in other Candida species such as C. tropicalis, C. parapsilosis and C. guiller‐mondii.

Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis.

Secreted aspartic proteinases (Saps) are important virulence factors during Candida albicans mucosal or disseminated infections. A differential expression of individual SAP genes has been shown previously in a model of oral candidosis based on reconstituted human epithelium (RHE), and in the oral cavity of patients. In this study, the ultrastructural localization of distinct groups of Sap isoenzymes expressed during RHE infection was shown by immunoelectron microscopy using specific polyclonal antibodies directed against the gene products of SAP1‐3 and SAP4‐6. Large amounts of Sap1‐3 antigen were found within C. albicans yeast and hyphal cell walls, often predominantly in close contact with epithelial cells, whereas lower quantities of Sap4‐6 were detected in hyphal cells. To elucidate the relevance of the expressed Saps during oral infections, we examined the effect of the aspartic proteinase inhibitor, pepstatin A, during infection of the RHE. The extent of lesions caused by the strain SC5314 was found to be strongly reduced by the inhibitor, indicating that proteinase activity contributes to tissue damage in this model. To clarify which of the SAP genes are important for tissue necrosis, the histology of RHE infection with Δsap1, Δsap2, Δsap3, Δsap4‐6 and three Δsap1/3 double mutants were examined. Although tissue damage was not blocked completely with these mutants, an attenuated phenotype was observed for each of the single sap null mutants, and was more strongly attenuated in the Δsap1/3 double null mutants. In contrast, the lesions caused by the Δsap4‐6 triple mutant were at least as severe as those caused by SC5314. During infection with the mutants, we observed that the SAP gene expression pattern of the Δsap1 and the Δsap1/3 mutants was altered in comparison with the wild‐type strain. Expression of SAP5 was observed only during infection with the Δsap1/3 mutant, whereas upregulation of SAP2 and SAP8 transcripts was observed in the Δsap1 and the Δsap1/3 mutants. These results suggest that Sap1‐3, but not Sap4‐6, contribute to tissue damage in this model. Furthermore, C. albicans may compensate for the deletion of certain SAP genes by upregulation of alternative SAP genes.

Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity

Candida albicans, an opportunistic pathogen in humans, secretes secretory aspartyl proteinases (Saps), which have been correlated with virulence. We examined the temporal regulation of the mRNA expression of seven known members of the SAP gene family by reverse transcription polymerase chain reaction (RT–PCR) in (i) an in vitro model of oral candidosis based on reconstituted human epithelium (RHE); and (ii) clinical samples from patients with oral candidosis. SAP1 and SAP3 transcripts were first detected 42u2003h after inoculation of RHE, while at the same time, slight morphological alterations in the epithelium were documented by light microscopy. SAP6 expression occurred 6u2003h later concomitantly with germ tube formation of some infecting Candida cells and severe lesions of the epithelial tissue. SAP2 and SAP8 RT–PCR products were first detected 60u2003h after infection, while SAP4 and SAP5 transcripts were never discovered. Thus, a temporal progression of SAP expression in the order SAP1 and SAP3u2003>u2003SAP6u2003>u2003SAP2 and SAP8 was observed at the same time as increasing RHE damage occurred. At the protein level, Sap antigen was found within the C. albicans yeast cells and the epithelial cells by immunoelectron microscopy using an anti‐Sap murine monoclonal antibody directed against the gene products Sap1–3. Expression of SAP1–3 and 6 was also detected by RT–PCR in samples from patients suffering from oral candidosis. Our results suggest that the pathogenesis of experimental and clinical oral candidosis is associated with the differential and temporal regulation of SAP gene expression.

Importance of the Candida albicans cell wall during commensalism and infection

An imbalance of the normal microbial flora, breakage of epithelial barriers or dysfunction of the immune system favour the transition of the human pathogenic yeast Candida albicans from a commensal to a pathogen. C. albicans has evolved to be adapted as a commensal on mucosal surfaces. As a commensal it has also acquired attributes, which are necessary to avoid or overcome the host defence mechanisms. The human host has also co-evolved to recognize and eliminate potential fungal invaders. Many of the fungal genes that have been the focus of this co-evolutionary process encode cell wall components. In this review, we will discuss the transition from commensalism to pathogenesis, the key players of the fungal cell surface that are important for this transition, the role of the morphology and the mechanisms of host recognition and response.

Sequence of the Candida albicans gene encoding the secretory aspartate proteinase

The gene encoding the Candida albicans aspartate proteinase that is secreted by cells grown in protein-containing media was cloned from a C. albicans genomic bank. The base sequence of the insert shows a 1173 bp open-reading frame and indicates an amino acid sequence typical of aspartate proteinases, with amino acid sequence homology to other enzymes of this class and a putative signal peptide consisting of 50 amino acids upstream of the active enzyme.

Expression analysis of the Candida albicans lipase gene family during experimental infections and in patient samples

Secreted lipases of Candida albicans are encoded by a gene family with at least 10 members (LIP1-LIP10). The expression pattern of this multigene family was investigated using reverse transcription polymerase chain reaction in experimental infections and in samples of patients suffering from oral candidosis. The findings illustrate that individual lipase genes are differentially regulated in a mouse model of systemic candidosis with some members showing sustained expression and others being transiently expressed or even silent. The lipase gene expression profile depended on the stage of infection rather than on the organ localization. This temporal regulation of lipase gene expression was also detected in an experimental model of oral candidosis. Furthermore, the expression of candidal lipase genes in human specimens is shown for the first time.

The role and relevance of phospholipase D1 during growth and dimorphism of Candida albicans.

The phosphatidylcholine-specific phospholipase D1 (PLD1) in Saccharomyces cerevisiae is involved in vesicle transport and is essential for sporulation. The gene encoding the homologous phospholipase D1 from Candida albicans (PLD1) was used to study the role of PLD1 in this pathogenic fungus. In vitro and in vivo expression studies using Northern blots and reverse transcriptase-PCR showed low PLD1 mRNA levels in defined media supporting yeast growth and during experimental infection, while enhanced levels of PLD1 transcripts were detected during the yeast to hyphal transition. To study the relevance of PLD1 during yeast and hyphal growth, an essential part of the gene was deleted in both alleles of two isogenic strains. In vitro PLD1 activity assays showed that pld1 mutants produced no detectable levels of phosphatidic acid, the hydrolytic product of PLD1 activity, and strongly reduced levels of diacylglycerol, the product of lipid phosphate phosphohydrolase, suggesting no or a negligible background PLD1 activity in the pld1 mutants. The pld1 mutants showed no growth differences compared to the parental wild-type in liquid complex and minimal media, independent of the growth temperature. In addition, growth rates of pld1 mutants in media with protein as the sole source of nitrogen were similar to growth rates of the wild-type, indicating that secretion of proteinases was not reduced. Chlamydospore formation was normal in pld1 mutants. When germ tube formation was induced in liquid media, pld1 mutants showed similar rates of yeast to hyphal transition compared to the wild-type. However, no hyphae formation was observed on solid Spider medium, and cell growth on cornmeal/Tween 80 medium indicated aberrant morphogenesis. In addition, pld1 mutants growing on solid media had an attenuated ability to invade the agar. In a model of oral candidosis, pld1 mutants showed no attenuation of virulence. In contrast, the mutant was less virulent in two different mouse models. These data suggest that PLD1 is not essential for growth and oral infections. However, they also suggest that a prominent part of the phosphatidic acid and diacylglycerol pools is produced by PLD1 and that the level of these components is important for morphological transitions under certain conditions in C. albicans.

Microbial lipases as virulence factors

Abstract Up to now, lipases of microbial origin are known to be very useful in a palette of industrial applications. But it becomes more and more obvious that extracellular lipases also play a role during microbial infections. This review will focus on the virulent traits of these secreted enzymes from bacteria and fungi. Special emphasis will be laid on Candida albicans research. This human pathogenic fungus possesses a lipase gene family, which is expressed and differentially regulated under a variety of in vitro conditions. First results show that this isoenzyme family is also expressed during an experimental infection. In addition, putative functions of extracellular lipases of pathogenic micro-organisms will be discussed.