Yarrowia lipolytica cell wall architecture: interaction of Ywp1, a mycelial protein, with other wall components and the effect of its depletion.

Linkages of Ywp1 to other components of the Yarrowia lipolytica mycelial cell wall were studied by extraction with beta-mercaptoethanol and zymolyase (a beta-glucanase complex) and by the use of rabbit polyclonal antibody preparation raised against Ywp1. Ywp1 complexed with an N-glycosylated cell wall protein(s) to form supramolecular complexes through disulphide bridges (extractable with beta-mercaptoethanol) or bonded to beta-1,3-glucan (extractable with zymolyase). The lack of a specific morphological phenotype when YWP1 was knocked out by gene disruption might indicate that other proteins present in the cell wall of Y. lipolytica compensated for its loss. In this mutant, the electrophoretic pattern of proteins, detected with polyclonal antibodies against the entire cell wall, was different from that obtained with the parental strain, but sensitivity to calcofluor white, zymolyase and chitinase did not change. Quantitative analysis of fluorescence emitted by cells in the presence of fluorescent wheat germ agglutinin (FITC-WGA) indicated that chitin was organized in the cell wall of the mutant cells in a form different from that in the parental strain.

Expression of YWP1, a gene that encodes a specific Yarrowia lipolytica mycelial cell wall protein, in Saccharomyces cerevisiae.

The YWP1 gene encoding a specific mycelial cell wall protein of Yarrowia lipolytica has been cloned and expressed in Saccharomyces cerevisiae using different episomal plasmids. Because the plasmids pYAE35BB and pYAE35ES carrying the YWP1 gene (including the 5' noncoding promoter sequences) failed to express it, the YWP1 gene was cloned under the control of GAL/CYC or ACT S. cerevisiae promoters. A main band with an apparent molecular mass of 70 kDa was detected by immunoblotting in the cell wall fraction of transformants. Ywp1 processing and incorporation to the cell wall were similar in both Y. lipolytica and S. cerevisiae but not in its final localization in the cell wall. In Y. lipolytica Ywp1 is covalently bound to the cell wall (it is released only by Zymolyase digestion), whereas in S. cerevisiae it was not (it was released by boiling SDS solutions). These results suggest that the sequences involved in recognition, anchoring of a protein to the cell wall, or the catalytic activities implicated are different, at least for Ywp1, in Y. lipolytica and S. cerevisiae. Another possibility is that the target for attachment of Ywp1 is missing or cryptic in the cell wall of S. cerevisiae.

PRR1, a homolog of Aspergillus nidulans palF, controls pH-dependent gene expression and filamentation in Candida albicans.

The pH of the environment has been implicated in controlling the yeast-hypha transition and pathogenesis of Candida albicans. Several C. albicans genes, including PHR1 and PHR2, are pH dependent in their expression. To investigate the mechanism of pH-dependent expression, we have cloned and characterized PRR1 (for pH response regulator). PRR1 is homologous to palF, a component of the pH response pathway in Aspergillus nidulans. Expression of PRR1 was itself pH dependent, being maximal at acid pH but reduced severalfold at alkaline pH. In a prr1 null mutant the alkaline-induced expression of PHR1 was completely abolished. Conversely, expression of PHR2 was no longer repressed at alkaline pH. A prr1 null mutant exhibited no morphological abnormalities at either pH; however, it lost the ability to form hyphae on medium 199 and on 10% serum plates. The ability to filament on serum was not restored by forced expression of PHR1, indicating that additional PRR1-dependent genes are required for hyphal development. These developmental genes appear to be distinct from those controlled by the developmental regulator EFG1, since the EFG1-dependent gene HWP1 was expressed normally in the prr1 null mutant. We conclude that PRR1 encodes a component of the pH-dependent response pathway in C. albicans and that this pathway regulates the expression of multiple components of hyphal development.

Effect of environmental pH on morphological development of Candida albicans is mediated via the PacC-related transcription factor encoded by PRR2.

The ability to respond to ambient pH is critical to the growth and virulence of the fungal pathogen Candida albicans. This response entails the differential expression of several genes affecting morphogenesis. To investigate the mechanism of pH-dependent gene expression, the C. albicans homolog of pacC, designated PRR2 (for pH response regulator), was identified and cloned. pacC encodes a zinc finger-containing transcription factor that mediates pH-dependent gene expression in Aspergillus nidulans. Mutants lacking PRR2 can no longer induce the expression of alkaline-expressed genes or repress acid-expressed genes at alkaline pH. Although the mutation did not affect growth of the cells at acid or alkaline pH, the mutants exhibited medium-conditional defects in filamentation. PRR2 was itself expressed in a pH-conditional manner, and its induction at alkaline pH was controlled by PRR1. PRR1 is homologous to palF, a regulator of pacC. Thus, PRR2 expression is controlled by a pH-dependent feedback loop. The results demonstrate that the pH response pathway of Aspergillus is conserved and that this pathway has been adapted to control dimorphism in C. albicans.

A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica.

A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector lambda gt 11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is solubilized following partial degradation of beta-glucan by Zymolyase digestion. The protein is localized in the outer surface on the tip of the growing mycelial cells and is found partially cryptic in sub-apical locations, suggesting that it participates directly in the mycelial wall architecture.

Serum aminotransferase levels and histological disease in chronic hepatitis C.

The reliability of serum aminotransferase (ASAT and ALAT) levels, currently used in deciding on performing liver biopsy and to assess interferon therapy in chronic hepatitis C has been questioned. In Belgium, interferon therapy is actually only reimbursed for treatment of chronic hepatitis C when serum aminotransferase levels are more than twice the upper limit of normal. The aim of the present study was to assess the relationship between serum aminotransferase levels and histological severity of chronic hepatitis C. Sixty-seven liver biopsies from 51 different patients with chronic hepatitis C and presenting with elevated ASAT and/or ALAT levels, were retrospectively evaluated using the original terminology (minimal hepatitis, chronic persistent hepatitis, chronic active hepatitis, cirrhosis), the Knodell score and the components of the Bianchi-Gudat score, where grading (portal inflammation, piecemeal necrosis, intra-acinar necrosis and inflammation) and staging components (fibrosis/ cirrhosis) are quantitated separately. The correlation between amino-transferase levels measured at or near to the biopsy date and histological criteria were evaluated using Spearman's rank correlation. About one third of the patients, including patients with chronic active hepatitis and cirrhosis, presented with ASAT and ALAT levels less than twice the upper limit of normal. ASAT levels correlated with originally determined histological severity, the numerical Knodell score and the numerical scores for piecemeal necrosis, for intra-acinar necrosis and inflammation and for fibrosis in the Bianchi-Gudat score. ALAT levels correlated only with intra-acinar necrosis and inflammation. It is concluded that limiting interferon therapy to patients with aminotransferase levels over twice the upper limit of normal excludes a large proportion of patients from potentially curative treatment. ASAT levels are more useful than ALAT to assess the histological severity of the disease, probably because this mitochondrial enzyme is present in higher quantities in the liver as compared to the cytosolic ALAT, and is more released when tissue damage is more severe.

Hepatotoxicity after a short course of low-dose pyrazinamide.

We report on a patient who developed extensive centrolobular liver necrosis after a treatment with low-dose (25 mg/kg/d) pyrazinamide for only 4 weeks, combined with rifampicin, 600 mg/d). In the past, the patient already developed severe aminotransferase elevations under isoniazid treatment. After prompt withdrawal of the drugs, a gradual decline of the aminotransferases was observed. No signs of hepatic failure developed. Pyrazinamide has to be used with caution, even in low-dose, especially when combined with rifampicin.

A rare cause of colitis--Brucella melitensis. Report of a case.

Documentation of gastrointestinal lesions in Brucella infections is sparse. A case of Brucella melitensis type 3 infection accompanied by erosive lesions of the colon, observed by endoscopy and histopathologic examination, is reported. Such gastrointestinal lesions have not been described since 1934. Before 1934 only postmortem observations are recorded.