Showing papers by "Wilhelm Schäfer published in 2000"

Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members.

Abstract: Extracellular lipolytic activity enabled the human pathogen Candida albicans to grow on lipids as the sole source of carbon. Nine new members of a lipase gene family (LIP2–LIP10) with high similarities to the recently cloned lipase gene LIP1 were cloned and characterised. The ORFs of all ten lipase genes are between 1281 and 1416 bp long and encode highly similar proteins with up to 80% identical amino acid sequences. Each deduced lipase sequence has conserved lipase motifs, four conserved cysteine residues, conserved putative N-glycosylation sites and similar hydrophobicity profiles. All LIP genes, except LIP7, also encode an N-terminal signal sequence. LIP3–LIP6 were expressed in all media and at all time points of growth tested as shown by Northern blot and RT-PCR analyses. LIP1, LIP3, LIP4, LIP5, LIP6 and LIP8 were expressed in medium with Tween 40 as a sole source of carbon. However, the same genes were also expressed in media without lipids. Two other genes, LIP2 and LIP9, were only expressed in media lacking lipids. Transcripts of most lipase genes were detected during the yeast-to-hyphal transition. Furthermore, LIP5, LIP6, LIP8 and LIP9 were found to be expressed during experimental infection of mice. These data indicate lipid-independent, highly flexible in vitro and in vivo expression of a large number of LIP genes, possibly reflecting broad lipolytic activity, which may contribute to the persistence and virulence of C. albicans in human tissue.

Cloning and targeted disruption of two polygalacturonase genes in Penicillium olsonii.

Abstract: The filamentous fungus Penicillium olsonii secretes several polygalacturonases (PGs) with molecular masses of about 47 kDa. These enzymes consist of several basic and acidic isoforms, with dominant activities at pI 4.5 and pI 7.9. Two polygalacturonase genes, pg1 and pg2, have been cloned. The corresponding enzymes, PG1 and PG2, consist of 370 and 380 amino acids, respectively, and show significant similarities to endo-polygalacturonases from other filamentous fungi. Targeted disruption of pg1 resulted in the elimination of all basic PG isoforms. In contrast, disruption of pg2 reduced, but did not eliminate the acidic PG activities. The PGs of P. olsonii must therefore be encoded by a gene family of at least three genes. Induction studies with various carbon sources revealed that the acidic and basic isoforms are differentially regulated. Pectin is the best inducer of the acidic PG isoforms. The basic isoforms, however, are best induced by monosaccharides like glucose, α-L-rhamnose and α-L-arabinose.

Extracellular hydrolytic enzymes and their relevance during Candida albicans infections

Abstract: Candida albicans cannot only infect skin and mucosa, but can also cause life threatening systemic candidosis. While natural barriers and the immune system of healthy individuals normally prevent such infections, virulence factors exist that enable C. albicans to survive on surfaces and the permit the fungus to invade tissues and organs in immunocompromised patients. Adhesions factors, morphological flexibility and hydrolytic enzymes belong to this group of virulence factors.C.albicans appears to be able to use these specific virulence attributes at distinct stages of an infection or in different types of candidosis. For example, distinct adhension factors are important for the persistence of C. albicans on mucosal epithelial cells, while other factors are necessary for the adhesion to endothelial tissue. The differential expression of specific virulence factors at different stages of an infection could be the reason why C. albicans not only has single genes for extracellular hydrolytic enzymes, but gene families. Both secreted aspartate proteinases (Saps) and secreted lipases (Lips) from C. albicans are encoded by at least 10 different genes. This high number of similar genes might empower C. albicans with the ability to secrete a specific and appropriate enzymatic response at distinct stages of an infection. For both gene families differential expression has been shown in vitro and in vivo, which would be reasonable for such an adaptation. Expression studies revealed that distinct SAP and LIP genes were expressed under conditions when potential subtrates ( proteins or lipids) were not present in the growth medium. Such expression patterns would imply that these genes may have functions other than simply providing nutrients for the fungus. The specific transcription of single SAP genes during the course of an infection suggests that these genes may have specific functions during different stages of an infection. In fact, inhibition studies and the use of mutants with targeted gene disruptions showed that distinct SAP genes (SAP1-3) are important durning infections of skin and mucosa, while others (SAP4-6) are most relevant for systemic infections.