Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy.

Aspergillus fumigatus and Aspergillosis

SUMMARY Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy.

Aspergillus fumigatus and aspergillosis.

The cell wall of Saccharomyces cerevisiae is an elastic structure that provides osmotic and physical protection and determines the shape of the cell. The inner layer of the wall is largely responsible for the mechanical strength of the wall and also provides the attachment sites for the proteins that form the outer layer of the wall. Here we find among others the sexual agglutinins and the flocculins. The outer protein layer also limits the permeability of the cell wall, thus shielding the plasma membrane from attack by foreign enzymes and membrane-perturbing compounds. The main features of the molecular organization of the yeast cell wall are now known. Importantly, the molecular composition and organization of the cell wall may vary considerably. For example, the incorporation of many cell wall proteins is temporally and spatially controlled and depends strongly on environmental conditions. Similarly, the formation of specific cell wall protein–polysaccharide complexes is strongly affected by external conditions. This points to a tight regulation of cell wall construction. Indeed, all five mitogen-activated protein kinase pathways in bakers’ yeast affect the cell wall, and additional cell wall-related signaling routes have been identified. Finally, some potential targets for new antifungal compounds related to cell wall construction are discussed.

Dynamics of cell wall structure in Saccharomyces cerevisiae.

An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.

Cell Wall Assembly in Saccharomyces cerevisiae

The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis.

Cell Wall and Secreted Proteins of Candida albicans: Identification, Function, and Expression

Formation of branched glucan, glucan-glucan cross links, and glucan-chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor β-1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor β-1,3 glucan, yielding a linear glucan containing a β-1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429–441], An apparent Km of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent Km of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H2O competing for reaction at the activated donor site, thus resulting in hydrolysis.



However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for β-1,3 glucan as acceptor, in as much as no transfer was detected when either hexa-N-acetyl-chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of β-1,3 glucan into the previously existing cell wall structure.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1995.tb20399.x

Kinetics of beta-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene.

Clarithromycin is a recently approved macrolide with improved pharmacokinetics, antibacterial activity, and efficacy in treating bacterial infections including those caused by Helicobacter pylori, an agent implicated in various forms of gastric disease. We successfully isolated ribosomes from H. pylori and present the results of a study of their interaction with macrolides. Kinetic data were obtained by using 14C-labeled macrolides to probe the ribosomal binding site. Clarithromycin, its parent compound erythromycin, and its 14-(R)-hydroxy metabolite all bound tightly to H. pylori ribosomes. Kd values were in the range of 2 x 10(-10) M, which is the tightest binding interaction observed to date for a macrolide-ribosome complex. This tight binding was due to very slow dissociation rate constants of 7.07 x 10(-4), 6.83 x 10(-4), and 16.6 x 10(-4) min-1 for clarithromycin, erythromycin, and 14-hydroxyclarithromycin, respectively, giving half-times of dissociation ranging from 7 to 16 h, the slowest yet measured for a macrolide-ribosome complex. These dissociation rate constants are 2 orders of magnitude slower than the dissociation rate constants of macrolides from other gram-negative ribosomes. [14C]clarithromycin was bound stoichiometrically to 50S ribosomal subunits following incubation with 70S ribosomes and subsequent separation of the 30S and 50S subunits by sucrose density gradient centrifugation. These data predict that the lower MIC of clarithromycin compared with that of erythromycin for H. pylori is likely due to a faster rate of intracellular accumulation, possibly because of increased hydrophobicity.

Tight binding of clarithromycin, its 14-(R)-hydroxy metabolite, and erythromycin to Helicobacter pylori ribosomes.

Clarithromycin, the 6-O-methyl derivative of erythromycin, is approved for treatment of Mycobacterium avium infections and for prophylaxis in patients at risk. Since clarithromycin is more active against mycobacteria than the parent compound, erythromycin, we evaluated the interaction of erythromycin and clarithromycin with cells and ribosomes isolated from M. avium and Mycobacterium smegmatis. The MIC of clarithromycin was 32 and 64 times lower than that of erythromycin for M. smegmatis and M. avium, respectively. The cellular uptake rate for clarithromycin was two- to five-fold faster than for erythromycin, and cell-associated clarithromycin reached a plateau two-fold higher than that of erythromycin after 3 h. Energy was not required for uptake. Fractionation of cell-associated clarithromycin yielded 12% in the walls, 21% bound to ribosomes, with the remainder being lost during work-up. In addition, three- to six-fold more clarithromycin was associated with the isolated cell integument compared with erythromycin. The Kd for clarithromycin binding to ribosomes was 2.9- and 3.5-fold tighter for M. smegmatis and M. avium, respectively, than for erythromycin, due mainly to a slower off-rate. The log partition coefficients of the non-ionized form (log Pu) for clarithromycin and erythromycin were 3.24 and 2.92, respectively. Thus clarithromycin is more hydrophobic than erythromycin. This would favour more rapid diffusion within and across hydrophobic regions of the cell integument, since once a solute saturates a membrane the net flux across the membrane must equal the net flux within the membrane as dictated by diffusion. We conclude that the lower MIC of clarithromycin for M. avium and M. smegmatis is due to a combination of increased cellular uptake, the major factor, possibly through a peripheral hydrophobic layer, and increased binding affinity to ribosomes.

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Molecular basis of clarithromycin activity against Mycobacterium avium and Mycobacterium smegmatis.

Design, synthesis and in vitro evaluation of pyridinium ion based cyclase inhibitors and antifungal agents.

The N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpyridinium and N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1- ylpicolinium cations were evaluated for their ability to inhibit 2,3-oxidosqualene-lanosterol cyclase activity in Candida albicans. Both compounds inhibited fungal growth, were fungicidal, and resulted in the accumulation of squalene epoxide concurrent with a decrease in ergosterol, monomethyl sterols, and lanosterol, as was expected for the specific inhibition of 2,3-oxidosqualene-lanosterol cyclase activity. These compounds are electron-poor aromatic mimics of a monocyclized transition state or high-energy intermediate formed from oxidosqualene, which may explain their selective action.

Dorothy Zakula

Papers

Kinetics of beta-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene.

Tight binding of clarithromycin, its 14-(R)-hydroxy metabolite, and erythromycin to Helicobacter pylori ribosomes.

Molecular basis of clarithromycin activity against Mycobacterium avium and Mycobacterium smegmatis.

Design, synthesis and in vitro evaluation of pyridinium ion based cyclase inhibitors and antifungal agents.

Inhibition of 2,3-oxidosqualene-lanosterol cyclase in Candida albicans by pyridinium ion-based inhibitors.