Gérald Larcher

Purification and characterization of a fibrinogenolytic serine proteinase from Aspergillus fumigatus culture filtrate

A fibrinogenolytic proteinase has been isolated from Aspergillus fumigatus culture filtrate by ammonium sulfate precipitation followed by successive chromatographics on Sephadex G‐75 and immobilized phenylalanine. The purified proteinase exhibited a molecular weight of about 33 kDa. When analysed by SDS‐polyacrylamide gels containing co‐polymerized fibrinogen, the proteinase appeared as a broad band at the top of the gels, which could correspond to polymerization of the enzyme, as suggested by SDS‐PAGE analysis of the unboiled eluate. The isoelectric point was 8.75 and the enzyme was not glycosylated. Proteinase activity was optimum at pH 9 and between 37 and 42°C, although a decrease in activity was observed above 37°C. PMSF and chymostatin markedly inhibited the proteinase activity, and good kinetic constants were obtained for the synthetic substrate, N‐Suc‐Ala‐Ala‐Pro‐Phe‐pNA. These results provide direct evidence that this enzyme belongs to the chymotrypsin‐like serine proteinase group.

Mechanisms of Azole Resistance in a Clinical Isolate of Candida tropicalis

ABSTRACT Azole resistance has been insufficiently investigated in the yeast Candida tropicalis. Here we determined the molecular mechanisms responsible for azole resistance in a clinical isolate of this pathogenic yeast. Antifungal susceptibility testing performed by a disk diffusion method showed resistance or markedly decreased susceptibility to azoles, which was confirmed by determination of MICs. Considering the relationship between azole susceptibility and the respiration reported for other yeast species, the respiratory activity of this isolate was investigated. Flow cytometry using rhodamine 123 and oxygraphy demonstrated an increased respiratory activity, which was not linked to an overexpression or increased number of copies of the mitochondrial genome. Among previously described resistance mechanisms, an increased activity of efflux pumps was investigated by flow cytometry using rhodamine 6G. However, the efflux of rhodamine 6G was lower in the resistant isolate than in susceptible ones. Likewise, real-time reverse transcription-PCR quantification of the expression of C. tropicalis MDR1 (CtMDR1), which encodes an efflux protein belonging to the major facilitator superfamily, did not show overexpression of this gene. In contrast, the resistant isolate overexpressed the CtERG11 gene coding for lanosterol 14α-demethylase. This was in agreement with the larger amount of ergosterol found in this isolate. Moreover, sequencing of CtERG11 showed a point mutation leading to a tyrosine substitution in the protein sequence, which might lead to decreased binding affinity for azoles. In conclusion, overexpression of CtERG11 associated with a missense mutation in this gene seemed to be responsible for the acquired azole resistance of this clinical isolate.

Synthesis, structure and biological activity of nickel(II) complexes of 5-methyl 2-furfural thiosemicarbazone.

5-Methyl 2-furfuraldehyde thiosemicarbazone (M5HFTSC) with nickel(II) leads to three types of complexes: [Ni(M5HFTSC)(2)X(2)], [Ni(M5FTSC)(2)] and [Ni(M5FTSC)(2)] x 2DMF. In the first type the ligand remains in thione form, while in the two other, the anionic thiolato form is involved. The species [Ni(M5HFTSC)(2)X(2)] has been characterized spectroscopically. The structures of [Ni(M5FTSC)(2)] x 2DMF and [Ni(M5FTSC)(2)] have been solved using X-ray diffraction. Biological studies of [Ni(M5HFTSC)(2)Cl(2)] have been carried out in vitro for antifungal activity on human pathogenic fungi, Aspergillus fumigatus and Candida albicans, and in vivo for toxicity on mice. The results are compared to those of the ligand, the metal salt and a similar copper complex [Cu(M5HFTSC)Cl(2)].

Interaction between Aspergillus fumigatus and basement membrane laminin: Binding and substrate degradation

Summary— Aspergillus fumigatus, the causative agent of human aspergillosis, binds to and degrades basement membrane laminin. Using immunoelectron microscopy, laminin binding appeared to be associated with the cell wall expansions of resting conidia, and progressively extended to the outer electron dense layer of the conidial wall during the germination process. Labeling of thin sections revealed numerous binding sites in the cytoplasm, whereas the inner cell wall and the plasma membrane were not labeled. Attachment of A fumigatus conidia on microtiter plates coated with laminin and its fragments P1 and E8 was also investigated. Conidia cells showed good adhesion to wells coated with laminin. As indicated by inhibition experiments, the interaction was specific and fragment P1 represented the major binding site on the laminin molecule. In addition, since A fumigatus produced an extracellular serine protease, we determined the susceptibility of laminin to this enzyme. We demonstrated that a crude protease extract was capable to degrade laminin in solution as well as in tissue sections. The laminin cleavage products were detected by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. All the three chains were extensively degraded within 1 h. Treatment of the crude protease extract with the enzyme inhibitors, phenylmethylsulfonyl‐fluoride and chymostatin, blocked the degradation of laminin, indicating a chymotrypsin‐like serine protease activity. Immunofluorescence microscopy of cryostat sections of mouse and rat kidneys treated with the protease extract showed widespread loss of laminin epitopes from basement membranes. Enzyme treatment also removed immunoreactivity from lungs as observed after immunoperoxidase performed on paraffin sections. Binding and proteolytic degradation of laminin may together facilitate initial interaction of A fumigatus with the host tissues.

Purification and Partial Characterization of a 32-Kilodalton Sialic Acid-Specific Lectin from Aspergillus fumigatus

ABSTRACT Adherence of the opportunistic fungus Aspergillus fumigatus to the extracellular matrix components is considered a crucial step in the establishment of the infection. Given the high carbohydrate content of these glycoproteins and the role of carbohydrate-protein interactions in numerous adherence processes, the presence of a lectin in A. fumigatus was investigated. Different fungal extracts obtained by sonication or grinding in liquid nitrogen from resting or swollen conidia, as well as from germ tubes and mycelium, were tested by hemagglutination assays using rabbit erythrocytes. A lectin activity was recovered in all the extracts tested. However, sonication of resting conidia resulted in the highest specific activity. Purification of the lectin was achieved by gel filtration followed by ion-exchange and hydrophobic-interaction chromatographies. Analysis of the purified lectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an apparent molecular mass of 32 kDa, which is similar to that of the alkaline protease already identified from different strains of A. fumigatus. However, as evidenced by the use of an alkaline protease-deficient mutant, the two activities were supported by distinct proteins. In addition, hemagglutination inhibition experiments using different saccharides and glycoproteins demonstrated the specificity of the lectin for sialic acid residues. Together these results suggest that this lectin may contribute to the attachment of conidia to the extracellular matrix components through the recognition of the numerous terminal sialic acid residues of their carbohydrate chains.

The search for virulence determinants in Aspergillus fumigatus

The development of aspergillosis in an immunodeficient host depends on interactions between fungal and host components. The recognition by Aspergillus fumigatus of fibrinogen and laminin, and the secretion of extracellular proteinases and ribonucleotoxin have been suggested to mediate adherence to mucosal surfaces and subsequently to bring about host-tissue invasion.

A nonsense mutation in the ERG6 gene leads to reduced susceptibility to polyenes in a clinical isolate of Candida glabrata

ABSTRACT Unlike the molecular mechanisms that lead to azole drug resistance, the molecular mechanisms that lead to polyene resistance are poorly documented, especially in pathogenic yeasts. We investigated the molecular mechanisms responsible for the reduced susceptibility to polyenes of a clinical isolate of Candida glabrata. Sterol content was analyzed by gas-phase chromatography, and we determined the sequences and levels of expression of several genes involved in ergosterol biosynthesis. We also investigated the effects of the mutation harbored by this isolate on the morphology and ultrastructure of the cell, cell viability, and vitality and susceptibility to cell wall-perturbing agents. The isolate had a lower ergosterol content in its membranes than the wild type, and the lower ergosterol content was found to be associated with a nonsense mutation in the ERG6 gene and induction of the ergosterol biosynthesis pathway. Modifications of the cell wall were also seen, accompanied by increased susceptibility to cell wall-perturbing agents. Finally, this mutation, which resulted in a marked fitness cost, was associated with a higher rate of cell mortality. Wild-type properties were restored by complementation of the isolate with a centromeric plasmid containing a wild-type copy of the ERG6 gene. In conclusion, we have identified the molecular event responsible for decreased susceptibility to polyenes in a clinical isolate of C. glabrata. The nonsense mutation detected in the ERG6 gene of this isolate led to a decrease in ergosterol content. This isolate may constitute a useful tool for analysis of the relevance of protein trafficking in the phenomena of azole resistance and pseudohyphal growth.

Synthesis, structure and antifungal activity of thiophene-2,3-dicarboxaldehyde bis(thiosemicarbazone) and nickel(II), copper(II) and cadmium(II) complexes: Unsymmetrical coordination mode of nickel complex

The reaction of nickel(II), copper(II) chlorides and cadmium(II) chloride and bromide with thiophene-2,3-dicarboxaldehyde bis(thiosemicarbazone) (2,3BTSTCH2) leads to a series of new complexes: [Ni(2,3BTSTCH)]Cl, [Cu(2,3BTSTC)], [CdCl2(2,3BTSTCH2)] and [CdBr2(2,3BTSTCH2)]. The crystal structures of the ligand and of [Ni(2,3BTSTCH)]Cl complex have been determined. In this case, we remark an unusual non-symmetrical coordination mode for the two functional groups: one acting as a thione and the second as a deprotonated thiolate. All compounds have been tested for their antifungal activity against human pathogenic fungi: Candida albicans, Candida glabrata and Aspergillus fumigatus, the cadmium complexes exhibit the highest antifungal activity. Cytotoxicity was evaluated using two biological methods: human MRC5 cultured cells and brine shrimp Artemia salina bioassay.

Molecular Mechanisms of Resistance to 5-Fluorocytosine in Laboratory Mutants of Candida glabrata

Resistance to 5-fluorocytosine (5-FC) has been poorly investigated in the yeast Candida glabrata. This study was conducted on laboratory mutants obtained by exposure of a wild-type isolate to 5-FC. Based on their susceptibility to 5-fluorouracil (5-FU), two of these mutants were selected for further analysis of the molecular mechanisms of 5-FC resistance. One mutant, resistant to both compounds, exhibited a missense mutation in the gene coding the cytosine deaminase and a decrease in the expression level of the gene coding the uridine monophosphate pyrophosphorylase. The other mutant that showed a reduced susceptibility to 5-FC and 5-FU exhibited an overexpression of the genes coding the thymidylate synthase and a cytosine permease, associated with a missense mutation in the last gene. Thus, beside mutations in the FUR1 gene which represent the most common cause of resistance to 5-FC, other mechanisms may also occur in C. glabrata.

Hydroxamate, a key pharmacophore exhibiting a wide range of biological activities

Naturally occurring hydroxamic acid derivatives are biosynthesized by microorganisms (siderophores) and plants (benzoxazinoids). Recent developments in drug discovery have highlighted the numerous biological and pharmacological properties that the hydroxamic acid function may possess, leading to therapeutic applications. These properties may be explained by its ability to chelate metals via the presence of two oxygen atoms. Their pharmacological activities can be divided into three groups. The first concerns the ability of these hydroxamic acid derivatives to scavenge metals (particularly iron), which leads to antioxidant, antimicrobial and metal detoxification activities. The latter is largely used to treat iron overload in patients. The second group of activities is related to their ability to inhibit metallo-enzymes, which gives them a wide range of pharmacological effects: antimicrobial, anti-inflammatory and antitumor. The third group is linked to the capacity of these compounds to generate nitric oxide, which confers hypotensive activity. However, hydroxamates exhibit relatively low stability in vivo, which can be overcome by the synthesis of appropriately designed analogs. For this purpose, many different strategies have been proposed. In this review, we compare and discuss the various synthetic pathways used to obtain the most complex of them, the N-substituted hydroxamic acids. We conclude that among numerous protocols reported so far, the direct N-substitution of hydroxamic acids, the acylation of the appropriate N-O derivative and the direct oxidation of the corresponding amide allow for the synthesis of a wide range of new biologically active compounds.