Jacqueline Sarfati

Comparison of restriction fragment length polymorphism, microsatellite length polymorphism, and random amplification of polymorphic DNA analyses for fingerprinting Aspergillus fumigatus isolates.

ABSTRACT Aspergillus fumigatus fingerprints generated by random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) upon hybridization with repeated DNA sequences, and PCR detection of microsatellite length polymorphism (MLP) were compared among 67 isolates. In contrast to RAPD, RFLP and MLP gave discriminating and significantly concordant genotyping results.

Comparison of an enzyme immunoassay and latex agglutination test for detection of galactomannan in the diagnosis of invasive aspergillosis

Aspergillus antigenemia was followed up in 215 consecutively observed bone marrow transplant (BMT) patients over a period of two years, using both a latex agglutination test and a sandwich immunocapture enzyme immunoassay (EIA) with a rat antigalactomannan monoclonal antibody as capture and detector antibody. For each patient, sequential sera (3 to 20) were obtained before and after BMT. No positivity was observed before BMT. After BMT, the EIA and latex agglutination test were positive in 19 and 4 patients respectively of 25 patients with confirmed aspergillosis and 14 and 7 of 15 patients with probable aspergillosis. In 19 of 25 patients with confirmed aspergillosis and 9 of 15 patients with probable aspergillosis, the EIA was more sensitive and detected infection earlier than the latex test. In all positive cases, antigenemia rapidly increased in sequential samples and remained strongly positive. In 31 of 169 (19%) BMT patients without clinical signs of aspergillosis, the EIA was occasionally positive in samples taken within the first month after BMT, giving a specificity of 81 % in these patients. In non-BMT patients suffering from other diseases (n=77), the specificity was 98.7%. The overall positive and negative predictive values for the EIA were 54% and 95% respectively. These results favour the use of EIA for early diagnosis and monitoring of aspergillosis in BMT patients, although the predictive value of transient positivity remains to be ascertained.

Prospective sandwich enzyme-linked immunosorbent assay for serum galactomannan: early predictive value and clinical use in invasive aspergillosis.

BACKGROUND The delay between the onset of invasive aspergillosis and the start of antifungal therapy is crucial for the patients recovery. Early diagnosis is difficult in cancer patients through lack of precocious specific signs. We have investigated the clinical usefulness of circulating Aspergillus antigen monitoring in pediatric hematology patients with a new sensitive sandwich enzyme-linked immunosorbent assay. METHODS A prospective study was conducted by assessing circulating galactomannan levels in high risk patients. Thirty-seven patients studied during an 18-month period were evaluated twice weekly during neutropenic phases with the sandwich enzyme-linked immunosorbent assay for serum Aspergillus galactomannan. RESULTS Twelve patients had one or more episodes of positive circulating galactomannan detection, 10 of whom developed presumptive invasive aspergillosis. The clinical and radiologic signs occurred at a mean of 13.4 days (range, 0 to 48) after circulating galactomannan detection and reversed in 6 patients treated with amphotericin B at the same time circulating galactomannan detection became negative. Reappearance of circulating galactomannan was observed during subsequent neutropenic periods in 3 patients. CONCLUSIONS The detection of galactomannan at concentrations as low as 1 ng/ml can be useful for the early initiation of antifungal therapy and monitoring treatment in clinically documented lung aspergillosis. This technique coupled with chest computed tomography could help to restrict the need of invasive diagnostic procedures in fragile patients.

Immune Sensing of Aspergillus fumigatus Proteins, Glycolipids, and Polysaccharides and the Impact on Th Immunity and Vaccination

The ability of the fungus Aspergillus fumigatus to activate, suppress, or subvert host immune response during life cycle in vivo through dynamic changing of cell wall structure and secretion implicates discriminative immune sensing of distinct fungal components. In this study, we have comparatively assessed secreted- and membrane-anchored proteins, glycolipids, and polysaccharides for the ability to induce vaccine-dependent protection in transplanted mice and Th cytokine production by human-specific CD4+ T cell clones. The results show that the different fungal components are endowed with the distinct capacity to activate Th cell responses in mice and humans, with secreted proteins inducing Th2 cell activation, membrane proteins Th1/Treg, glycolipids Th17, and polysaccharides mostly IL-10 production. Of interest, the side-by-side comparison revealed that at least three fungal components (a protease and two glycosylphosphatidylinositol-anchored proteins) retained their immunodominant Th1/Treg activating potential from mice to humans. This suggests that the broadness and specificity of human T cell repertoire against the fungus could be selectively exploited with defined immunoactive Aspergillus Ags.

Deletion of GEL2 encoding for a β(1-3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus

The first fungal glycosylphosphatidylinositol an‐chored β(1–3)glucanosyltranferase (Gel1p) has been described in Aspergillus fumigatus and its encoding gene GEL1 identified. Glycosylphosphatidylinositol‐anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. We characterize here GEL2, a homologue of GEL1. Both homologues share common characteristics: (i) GEL1 and GEL2 are constitutively expressed during over a range of growth conditions; (ii) Gel2p is also a putative GPI‐anchored protein and shares the same β(1–3)glucanosyltransferase activity as Gel1p and (iii) GEL2, like GEL1, is able to complement the Δgas1 deletion in Saccharomyces cerevisiae confirming that Gelp and Gasp have the same enzymatic activity. However, disruption of GEL1 did not result in a phenotype whereas a Δgel2 mutant and the double mutant Δgel1Δgel2 exhibit slower growth, abnormal conidiogenesis, and an altered cell wall composition. In addition, the Δgel2 and the Δgel1Δgel2 mutant have reduced virulence in a murine model of invasive aspergillosis. These data suggest for the first time that β(1–3)glucanosyltransferase activity is required for both morphogenesis and virulence in A. fumigatus.

Cell wall biogenesis in a double chitin synthase mutant (chsG−/chsE−) of Aspergillus fumigatus

Previous studies (Aufauvre-Brown et al., 1997; Mellado et al., 1996a,b ) have shown that only two genes of the Aspergillus fumigatus chitin synthase family, chsG and chsE, play a role in the morphogenesis of this fungal species. An A. fumigatus strain lacking both chsG (class III CHS) and chsE (class V CHS) genes was constructed by gene replacement of the chsE gene with a copy that has its conserved coding region interrupted by the hph resistance cassette in an A. fumigatus chsG- genetic background. Unexpectedly the double disruption was not lethal. The double mutant AfchsG-/chsE- strain (i) has reduced chitin synthase activity with or without trypsin stimulation, (ii) has a reduced colony radial growth rate, (iii) produces highly branched hyphae, (iv) exhibits aberrant features, such as periodic swellings along the length of the hyphae and a block in conidiation that can be partially restored by an osmotic stabilizer (v) shows alterations in the shape and germination capacity of the conidia, and (vi) has a cell wall that contains half the chitin of the parental strain and is, unexpectedly, highly enriched in alpha-(1-3) glucan.

The role of the sakA (Hog1) and tcsB (sln1) genes in the oxidant adaptation of Aspergillus fumigatus

The Hog1 MAP kinase pathway regulates stress adaptation in several fungi. To assess its role in stress adaptation in Aspergillus fumigatus, we constructed mutants in genes encoding the sensor histidine kinase (HK) tcsB as well as sakA, which are homologues of the Saccharomyces cerevisiae sln1 and Hog1, respectively. Compared to the wild type strain (Wt), growth of sakA (sakAtriangle up) mutant was reduced, and growth inhibition was increased when H(2)O(2), menadione, or SDS was added to the media. On the other hand, the tcsB mutant (tcsBtriangle up) was similar to the Wt strain in regard to growth and morphology, although a partial sensitivity to SDS was observed. Western blot analysis of Wt and the tcsBtriangle up strains indicated that when stressed with H(2)O(2), phosphorylation of Hog1p still occurs in the mutant. Since in Candida albicans, Hog1 regulates transcription of at least one histidine kinase, we performed RT-PCR of 6 histidine kinase genes as well as the ssk1 and skn7 response regulator genes of A. fumigatus. No significant differences in transcription were observed with the sakAtriangle up when compared to the Wt, indicating that the sakA does not regulate transcription of these genes. Our studies indicate that the A. fumigatus sakA is required for optimal growth of the organism with or without oxidant stress, while tcsB gene is dispensable.

Members of protein O‐mannosyltransferase family in Aspergillus fumigatus differentially affect growth, morphogenesis and viability

O‐mannosylation is an essential protein modification in eukaryotes. It is initiated at the endoplasmic reticulum by O‐mannosyltransferases (PMT) that are evolutionary conserved from yeast to humans. The PMT family is phylogenetically classified into PMT1, PMT2 and PMT4 subfamilies, which differ in protein substrate specificity and number of genes per subfamily. In this study, we characterized for the first time the whole PMT family of a pathogenic filamentous fungus, Aspergillus fumigatus. Genome analysis showed that only one member of each subfamily is present in A. fumigatus, PMT1, PMT2 and PMT4. Despite the fact that all PMTs are transmembrane proteins with conserved peptide motifs, the phenotype of each PMT deletion mutant was very different in A. fumigatus. If disruption of PMT1 did not reveal any phenotype, deletion of PMT2 was lethal. Disruption of PMT4 resulted in abnormal mycelial growth and highly reduced conidiation associated to significant proteomic changes. The double pmt1pmt4 mutant was lethal. The single pmt4 mutant exhibited an exquisite sensitivity to echinocandins that is associated to major changes in the expression of signal transduction cascade genes. These results indicate that the PMT family members play a major role in growth, morphogenesis and viability of A. fumigatus.

Glycosylphosphatidylinositol-Anchored Ecm33p Influences Conidial Cell Wall Biosynthesis in Aspergillus fumigatus

ABSTRACT ECM33 encodes a glycosylphosphatidylinositol-anchored protein whose orthologs in yeast are essential for sporulation. Aspergillus fumigatus Ecm33p is unique and has an apparent mass of 55 kDa. Disruption of A. fumigatus ECM33 results in a mutant with several morphogenetic aberrations, including the following: (i) a defect in conidial separation, (ii) an increase in the diameter of the conidia of the mutant associated with an increase in the concentration of the cell wall chitin, (iii) conidia that were sensitive to the absence of aeration during long-term storage, and (iv) conidia that were more resistant to killing by phagocytes, whereas the mycelium was more easily killed by neutrophils.

Longitudinal study of Aspergillus fumigatus strains isolated from cystic fibrosis patients.

The colonization over time of cystic fibrosis patients byAspergillus fumigatus was investigated using a DNA fingerprinting method.Aspergillus fumigatus isolates collected sequentially for more than one year from six patients with cystic fibrosis were typed by Southern blot hybridization with a repetitive DNA sequence. Each cystic fibrosis patient harbored several strains ofAspergillus fumigatus that were isolated recurrently over time. Isolates collected from a cystic fibrosis patient with aspergilloma displayed the same genotype, suggesting that the infection was due to a single strain. Continuous isolation of the same genotype in another cystic fibrosis patient, however, was not correlated clinically with anAspergillus infection.