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It has long been speculated that porcine cathelin is an N-terminal fragment of a longer precursor protein which possesses antimicrobial activity. In an attempt to find such a precursor, a cDNA clone was recently isolated and sequenced by screening a cDNA library from porcine bone marrow. In order to identify the functional activity of the putative protein encoded by an open reading frame, we have synthesized various lengths of peptides that correspond to the C-terminal region of the protein and examined them for their antimicrobial activities. We found that a 13 amino acid tryptophan-rich region with the sequence of VRRFPWWWPFLRR had strong antimicrobial activity with a wide spectrum. It showed potency against Escherichia coli, Pseudomonas aeruginosa, Kiebsiella pneumonia, Staphylococcus epidermidis, Proteus mirabilis, and Streptococcus group D as well as Aspergillus fumigatus. The action of this peptide is bactericidal rather than bacteriostatic and this activity is completely inhibited by 2 mM MgC12. Our results indicate that the previously identified putative precursor encoded by the isolated cDNA indeed possesses a potent antimicrobial activity and that this 13 amino acid synthetic peptide is considered to be a potentially effective drug against various infections agents.

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By screening for the osmotically remediable phenotype, mutations in two genes (orlA and or1B) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, oriC and orlD, which affect cell wall j0-1,3-glucan content were also identified.Walls from strains carrying mutations in these genes exhibit normal amounts of a-1,3-glucan and chitin but reduced amounts of 03-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and I0-1,3-glucan are likely to contribute to the structural rigidity of the cell wall.The cell wall plays an important role in the growth and development of the fungi. In addition to its function as the primary osmotic barrier of the cell, the temporal and spatial regulation of wall polymer synthesis is critical to the morphogenesis of the cell types characteristic of many fungi. The architecture and synthesis of the cell walls of fungi are poorly understood. For a majority of the fungi, including most of those of medical and economic importance, chitin and/or P-1,3-glucans are believed to be the most important structural polysaccharides (3). Many studies of wall synthesis have concerned the enzymology of chitin synthase and, to a lesser extent, P-1,3-glucan synthase.In the yeasts or yeastlike fungi such as Saccharomyces cerevisiae, Candida albicans, and Schizosaccharomyces pombe, chitin is a relatively small fraction of the cell wall. In S. cerevisiae, chitin is found predominantly in the primary septa between cells (9, 10, 26). Three related genes (CHSJ, CHS2, and CALI) involved in chitin synthesis from S. cerevisiae (7,29,32) and one from C. albicans (1) have been sequenced. The finding that the CHSJ (chitin synthase 1) gene from S. cerevisiae, which codes for the major chitin synthase activity, could be disrupted without affecting chitin synthesis was unexpected (7). In addition, at least under some conditions, both the CHSJ and CHS2 genes can be disrupted without affecting the majority of chitin synthesis (5). Recently, a gene (CALl) with regions of homology to chitin synthases 1 and 2 and required for chitin synthase 3 activity was identified (32). Chitin synthase 3 was shown to be responsible for chitin synthesis in the portion of the wall not associated with the septum and to be responsible for the chitin ring formation which prec...

A cosmid carrying the orlA gene from Aspergillus nidulans was identified by complementation of an orlA1 mutant strain with DNA from the pKBY2 cosmid library. An orlA1 complementing fragment from the cosmid was sequenced. orlA encodes a predicted polypeptide of 227 amino acids (26360 Da) that is homologous to a 211-amino-acid domain from the polypeptide encoded by the Saccharomyces cerevisiae TPS2 gene and to almost the entire Escherichia coli otsB-encoded polypeptide. TPS2 and otsB each specify a trehalose-6-phosphate phosphatase, an enzyme that is necessary for trehalose synthesis. orlA disruptants accumulate trehalose-6-phosphate and have reduced trehalose-6-phosphatate phosphatase levels, indicating that the gene encodes a trehalose-6-phosphatate phosphatase. Disruptants have a nearly-wild-type morphology at 32 degrees C. When germinated at 42 degrees C, the conidia and hyphae from disruptants are chitin deficient, swell excessively, and lyse. The lysis is almost completely remedied by osmotic stabilizers and is partially remedied by N-acetylglucosamine (GlcNAc). The activity of glutamine:fructose-6-phosphate amido-transferase (GFAT), the first enzyme unique to aminosugar synthesis, is reduced and is labile in orlA disruption strains. The findings are consistent with the hypothesis that trehalose-6-phosphate reduces the temperature stability of GFAT and other enzymes of chitin metabolism at elevated temperatures. The results extend to filamentous organisms the observation that mutations in fungal trehalose synthesis are highly pleiotropic and affect aspects of carbohydrate metabolism that are not directly related to trehalose synthesis.