Patrick A. Sullivan

Utilisation of galacto-oligosaccharides as selective substrates for growth by lactic acid bacteria including Bifidobacterium lactis DR10 and Lactobacillus rhamnosus DR20

Two probiotic strains of bacteria Bifidobacterium lactis DR10 and Lactobacillus rhamnosus DR20 were tested for their ability to utilise and grow on galacto-oligosaccharides present in a commercial hydrolysed lactose milk powder. The results clearly demonstrated that B. lactis DR10 preferentially utilises tri- and tetra-saccharides whereas Lb. rhamnosus DR20 prefers sugars with a lower degree of polymerisation, i.e., disaccharides and monosaccharides. Since galacto-oligosaccharides are non-digestible oligosaccharides, this in vitro data suggest that galacto-oligosaccharides present in milk powders are likely to promote growth of DR10 and DR20 in vivo if these strains are consumed in combination with the milk powder. Fifty four strains of lactic acid bacteria, including members from the genera Bifidobacterium and Lactobacillus, were studied for their ability to utilise lactose derived oligosaccharides. A perfect correlation was observed between the ability of a strain to utilise oligosaccharide and the presence of the lactose hydrolysing enzyme β-galactosidase. Based on these observations, a mechanism for the utilisation of galacto-oligosaccharides in genus bifidobacteria is proposed that may help to explain the ability of these organisms to out-compete other bacteria in the ecosystem of the human gastro-intestinal tract.

The secreted aspartate proteinase of Candida albicans: physiology of secretion and virulence of a proteinase-deficient mutant.

It was established that Candida albicans grew rapidly in a simple medium containing yeast extract (0.2%, w/v) plus glucose (2%, w/v). These cultures were in or near to a state of nitrogen limitation and the concentration of secreted aspartate proteinase increased rapidly (within 3-4 h) on addition of BSA. Synthesis and secretion were apparently controlled both positively (induction by albumin or, more probably, the peptides produced from it) and negatively (repression by NH4Cl). A small intracellular pool of the enzyme was detected during production of the enzyme and this pool decreased with the cessation of synthesis and secretion. A stable mutant, IR24, was isolated which secreted less than 0.3% of the amount of the proteinase exported by the parent strain ATCC 10261. The LD50 values for mutant IR24 and the parent strain administered intravenously to mice were greater than 1.0 x 10(9) and 1.6 x 10(6) c.f.u. kg-1 respectively.

The crystal structure of a major secreted aspartic proteinase from Candida albicans in complexes with two inhibitors

BACKGROUNDnInfections caused by Candida albicans, a common fungal pathogen of humans, are increasing in incidence, necessitating development of new therapeutic drugs. Secreted aspartic proteinase (SAP) activity is considered an important virulence factor in these infections and might offer a suitable target for drug design. Amongst the various SAP isozymes, the SAP2 gene product is the major form expressed in a number of C. albicans strains.nnnRESULTSnThe three-dimensional structures of SAP2 complexed with the tight-binding inhibitor A70450 (a synthetic hexapeptide analogue) and with the general aspartic proteinase inhibitor pepstatin A (a microbial natural product) have been determined to 2.1 A and 3.0 A resolution, respectively. Although the protein structure retains the main features of a typical aspartic proteinase, it also shows some significant differences, due mainly to several sequence insertions and deletions (as revealed by homology modelling), that alter the shape of the binding cleft. There is also considerable variation in the C-terminal structural domain.nnnCONCLUSIONSnThe differences in side chains, and in the conformations adopted by the two inhibitors, particularly at their P4, P3 and P2 positions (using standard notation for protease-inhibitor residues), allows the A70450 structure to complement, more accurately, that of the substrate-binding site of SAP2. Some differences in the binding clefts of other SAP isoenzymes may be deduced from the SAP2 structure.

The Production and Growth Characteristics of Yeast and Mycelial Forms of Candida albicans in Continuous Culture

The growth characteristics of Candida albicans CM145,348 have been examined under aerobic conditions in continuous culture. At different steady states the environment was controlled with respect to the concentrations of dissolved oxygen, carbon and nitrogen, the pH, and the temperature. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release and respiration quotient were examined as a function of the dilution rate. The morphology depended on the carbon source. Maltose produced a mycelial morphology, whereas with lactate a yeast culture was obtained. With fructose or glucose as a carbon source a mixed morphology of yeast, pseudo-mycelial and mycelial forms was produced. A larger number of different growth conditions were examined in batch culture but a mixed morphology was always obtained.

Regulation of chitin synthesis during germ-tube formation in Candida albicans.

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely l-glutamine-d-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate, UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (Ki =1.2μM) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenzyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.

An exo-β-(1,3)-glucanase of Candida albicans : purification of the enzyme and molecular cloning of the gene

A nucleotide sequence encoding an exo-beta-(1,3)-glucanase was cloned from a library of genomic DNA of Candida albicans ATCC 10261. The sequenced gene encodes a protein of 438 amino acid residues. The amino terminal and an internal peptide sequence of the enzyme matched with deduced sequences within the cloned gene. Analysis of the sequence indicated that the nascent protein is processed during secretion by the signal peptidase and a Kex2-like proteinase, yielding a predicted mature enzyme of 400 residues. There is 58% identity and 85% similarity between the amino acid sequences of this exoglucanase and the homologous enzyme of Saccharomyces cerevisiae. An antiserum to the purified exoglucanase cross-reacted with the S. cerevisiae exoglucanase and a similar protein secreted by other C. albicans strains and Candida species. There are no sites for N-linked glycosylation in the sequence and this is consistent with the carbohydrate content of the secreted enzyme. Putative upstream promoter elements are associated with the gene. Southern analysis of the gene indicated that it was present at one copy per genome and that the diploid genome of C. albicans ATCC 10261 is heterozygous at this locus for a BglII RFLP. A 2.5 kb mRNA transcript was detected by Northern analysis and gene expression, as monitored by Northern and Western blots, reflected the growth rates of the cultures.

Analysis of wall glucans from yeast, hyphal and germ-tube forming cells of Candida albicans.

Acid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germ-tube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained beta(1----3)- and beta(1----6)-chains but no mixed intra-chain beta(1----3)/(1----6) linkages. Methylation and 13C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (67.0% to 76.6%) of beta(1----6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29.6% to 38.9% beta(1----3) and 43.3% to 53.2% beta(1----6) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67.0% beta(1----3) and 14% beta(1----6) linkages. Branch points accounted for 6.7%, 12.3% and 17.4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively.

Analysis of secreted aspartic proteinases from Candida albicans: purification and characterization of individual Sap1, Sap2 and Sap3 isoenzymes.

The recently discovered secreted aspartic proteinase multi-gene (SAP) family in Candida albicans has complicated assessment of proteolytic activity as a factor in the onset and development of Candida infections. Differential expression of the SAP genes under various conditions, as well as possible variation in the properties of the individual isoenzymes, have consequences for immunological detection, for targeted drug design and possibly for pathogenicity. It is therefore important to be able to monitor Sap isoenzyme profiles in different strains of C. albicans cultures, and to know the biochemical properties of each isoenzyme. We have employed a simple purification protocol based on strong anion exchange chromatography for the direct analysis of C. albicans Sap isoenzymes from culture filtrates, as well as recovery of individual Sap1, Sap2 and Sap3 products. In the case of Sap1, this involved development of an overexpression system using the pEMBLyex4 vector transformed into Saccharomyces cerevisiae. The C. albicans strains ATCC 10231 and 10261 were shown to produce different ratios of Sap2 and Sap3 under the same conditions. Analysis of all three purified proteins by gel electrophoresis, immunoblotting and proteinase assays which were designed to evaluate pH dependence, thermal stability and substrate specificity revealed similar but distinct properties for each isoenzyme. Although Sap3 was shown to be antigenically more similar to Sap2 than was Sap1, it was less similar in terms of thermal stability and activity at low pH, being more stable and more active.

Evidence for the occurrence of calmodulin in the yeasts Candidas albicans and Saccharomyces cerevisiae

Calmodulin was originally characterised as the calcium-dependent activator of brain cyclic nucleotide phosphodiesterase. Subsequently it was shown to be the modulator of a variety of calcium-dependent cellular activities (reviews [l-3]). Calmodulin is now recognised as being a highly conserved and widely distributed protein, having been demonstrated in many vertebrates and invertebrates [3,4], plants and higher fungi [5,6] as well as several unicellular eukaryotes; e.g., Dictyostelium discoideum [7], Blastocladiella emersonii [8], Euglena gracilis and Amoeba proteus [9]. Prokaryotes, in contrast,appear to lack calmodulin [6,7] although (exogenous) calmodulin has been shown to activate the adenylate cyclase of Bordetella pertussis [lo]. The observed distribution of calmodulin has led to the suggestion that it is ubiquitous in eukaryotes [3,5]. However, the inability of several laboratories to detect calmodulin in yeast [6,7,1 l] is in conflict with this assertion. This paper reports the presence of a calmodulln-like protein in extracts of the yeasts Candida albicans and Saccharomyces cerevisiae. The demonstration of this protein appears to be dependent on the use of the protease inhibitor, PMSF.

Identification of Glu-330 as the Catalytic Nucleophile of Candida albicans Exo-β-(1,3)-glucanase

The exo-β-(1,3)-glucanase from Candida albicans hydrolyzes cell wall β-glucans via a double-displacement mechanism involving a glycosyl enzyme intermediate. Reaction of the enzyme with 2′,4′-dinitrophenyl-2-deoxy-2-fluoro-β-D-glucopyranoside resulted in the time-dependent inactivation of this enzyme via the accumulation of a 2-deoxy-2-fluoro-glycosyl-enzyme intermediate as monitored also by electrospray mass spectrometry. The catalytic competence of this intermediate is demonstrated by its reactivation through hydrolysis (kreact = 0.0019 min−1) and by transglycosylation to benzyl thio-β-D-glucopyranoside (kreact = 0.024 min−1; Kreact = 56 mM). Peptic digestion of the labeled enzyme followed by tandem mass spectrometric analysis in the neutral loss mode allowed detection of two glycosylated active site peptides, the sequences of which were identified as NVAGEW and NVAGEWSAA. A crucial role for Glu-330 is confirmed by site-directed mutagenesis at this site and kinetic analysis of the resultant mutant. The activity of the Glu-330 → Gln mutant is reduced over 50,000-fold compared to the wild type enzyme. The glutamic acid, identified in the exoglucanase as Glu-330, is completely conserved in this family of enzymes and is hereby identified as the catalytic nucleophile.