Michael Weig

The Cell Wall of the Human Pathogen Candida glabrata: Differential Incorporation of Novel Adhesin-Like Wall Proteins

ABSTRACT The cell wall of the human pathogen Candida glabrata governs initial host-pathogen interactions that underlie the establishment of fungal infections. With the aim of identifying species-specific features that may directly relate to its virulence, we have investigated the cell wall of C. glabrata using a multidisciplinary approach that combines microscopy imaging, biochemical studies, bioinformatics, and tandem mass spectrometry. Electron microscopy revealed a bilayered wall structure in which the outer layer is packed with mannoproteins. Biochemical studies showed that C. glabrata walls incorporate 50% more protein than Saccharomyces cerevisiae walls and, consistent with this, have a higher mannose/glucose ratio. Evidence is presented that C. glabrata walls contain glycosylphosphatidylinositol (GPI) proteins, covalently bound to the wall 1,6-β-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to 1,3-β-glucan. A comprehensive genome-wide in silico inspection showed that in comparison to other fungi, C. glabrata contains an exceptionally large number, 67, of genes encoding adhesin-like GPI proteins. Phylogenetically these adhesin-like proteins form different clusters, one of which is the lectin-like EPA family. Mass spectrometric analysis identified 23 cell wall proteins, including 4 novel adhesin-like proteins, Awp1/2/3/4, and Epa6, which is involved in adherence to human epithelia and biofilm formation. Importantly, the presence of adhesin-like proteins in the wall depended on the growth stage and on the genetic background used, and this was reflected in alterations in adhesion capacity and cell surface hydrophobicity. We propose that the large repertoire of adhesin(-like) genes of C. glabrata contributes to its adaptability and virulence.

Adhesins in Human Fungal Pathogens: Glue with Plenty of Stick

ABSTRACT Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.

cyp51A-Based Mechanisms of Aspergillus fumigatus Azole Drug Resistance Present in Clinical Samples from Germany

ABSTRACT Since the mid-1990s, a steady increase in the occurrence of itraconazole-resistant Aspergillus fumigatus isolates has been observed in clinical contexts, leading to therapeutic failure in the treatment of aspergillosis. This increase has been predominantly linked to a single allele of the cyp51A gene, termed TR/L98H, which is thought to have arisen through the use of agricultural azoles. Here, we investigated the current epidemiology of triazole-resistant A. fumigatus and underlying cyp51A mutations in clinical samples in Germany. From a total of 527 samples, 17 (3.2%) showed elevated MIC0 values (the lowest concentrations with no visible growth) for at least one of the three substances (itraconazole, voriconazole, and posaconazole) tested. The highest prevalence of resistant isolates was observed in cystic fibrosis patients (5.2%). Among resistant isolates, the TR/L98H mutation in cyp51A was the most prevalent, but isolates with the G54W and M220I substitutions and the novel F219C substitution were also found. The isolate with the G54W substitution was highly resistant to both itraconazole and posaconazole, while all others showed high-level resistance only to itraconazole. For the remaining six isolates, no mutations in cyp51A were found, indicating the presence of other mechanisms. With the exception of the strains carrying the F219C and M220I substitutions, many itraconazole-resistant strains also showed cross-resistance to voriconazole and posaconazole with moderately increased MIC0 values. In conclusion, the prevalence of azole-resistant A. fumigatus in our clinical test set is lower than that previously reported for other countries. Although the TR/L98H mutation frequently occurs among triazole-resistant strains in Germany, it is not the only resistance mechanism present.

Identification and Characterization of Four Azole-Resistant erg3 Mutants of Candida albicans

ABSTRACT Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol Δ5,6-desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, ≥256, 16, 16, 8, and 1 μg ml−1, respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 μg ml−1); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 μg ml−1). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14α-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.

A Clinical Isolate of Candida albicans with Mutations in ERG11 (Encoding Sterol 14α-Demethylase) and ERG5 (Encoding C22 Desaturase) Is Cross Resistant to Azoles and Amphotericin B

ABSTRACT A clinical isolate of Candida albicans was identified as an erg5 (encoding sterol C22 desaturase) mutant in which ergosterol was not detectable and ergosta 5,7-dienol comprised >80% of the total sterol fraction. The mutant isolate (CA108) was resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole (MIC values, 64, 8, 2, 1, and 2 μg ml−1, respectively); azole resistance could not be fully explained by the activity of multidrug resistance pumps. When susceptibility tests were performed in the presence of a multidrug efflux inhibitor (tacrolimus; FK506), CA108 remained resistant to azole concentrations higher than suggested clinical breakpoints for C. albicans (efflux-inhibited MIC values, 16 and 4 μg ml−1 for fluconazole and voriconazole, respectively). Gene sequencing revealed that CA108 was an erg11 erg5 double mutant harboring a single amino acid substitution (A114S) in sterol 14α-demethylase (Erg11p) and sequence repetition (10 duplicated amino acids), which nullified C22 desaturase (Erg5p) function. Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 μg ml−1). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic.

Clinical manifestations and diagnosis of invasive aspergillosis in immunocompromised children.

Abstract. Invasive aspergillosis (IA) is a serious life-threatening complication in immunocompromised children. The commonest risk groups are children with acquired immunodeficiency syndrome, leukaemia, corticosteroid and other immunosuppressive therapy, chronic granulomatous disease and severe combined immunodeficiency as well as neonates. The clinical manifestations are heterogeneous and many organ systems can be involved. Diagnosis based on the clinical presentation alone is cumbersome. Innovative and sensitive laboratory test systems which detect fungal antigens or DNA in clinical specimens have been recently developed. Specific Aspergillus antibody detection using recombinant antigen technique has also been introduced. Although each individual technique has drawbacks, the combined use of culture with antigen and antibody ELISA as well as PCR should result in an earlier and more definitive diagnosis of IA in children presenting with clinical and/or radiological signs of aspergillosis. In high risk children these methods are valuable for serial screening and early detection of Aspergillus infection. The implementation of accurate diagnostic criteria and standardised diagnostic flow charts in children at risk will lead to a better outcome of IA in the future. Conclusion: definite, well-timed early diagnosis and sufficient therapy is elementary for a successful outcome of invasive aspergillosis in immunocompromised children. To date, the diagnosis of invasive aspergillosis remains a combination of clinical presentation, radiology and microbiological tests.

Rapid Discrimination of Salmonella enterica Serovar Typhi from Other Serovars by MALDI-TOF Mass Spectrometry

Systemic infections caused by Salmonella enterica are an ongoing public health problem especially in Sub-Saharan Africa. Essentially typhoid fever is associated with high mortality particularly because of the increasing prevalence of multidrug-resistant strains. Thus, a rapid blood-culture based bacterial species diagnosis including an immediate sub-differentiation of the various serovars is mandatory. At present, MALDI-TOF based intact cell mass spectrometry (ICMS) advances to a widely used routine identification tool for bacteria and fungi. In this study, we investigated the appropriateness of ICMS to identify pathogenic bacteria derived from Sub-Saharan Africa and tested the potential of this technology to discriminate S. enterica subsp. enterica serovar Typhi (S. Typhi) from other serovars. Among blood culture isolates obtained from a study population suffering from febrile illness in Ghana, no major misidentifications were observed for the species identification process, but serovars of Salmonella enterica could not be distinguished using the commercially available Biotyper database. However, a detailed analysis of the mass spectra revealed several serovar-specific biomarker ions, allowing the discrimination of S. Typhi from others. In conclusion, ICMS is able to identify isolates from a sub-Saharan context and may facilitate the rapid discrimination of the clinically and epidemiologically important serovar S. Typhi and other non-S. Typhi serovars in future implementations.

Human Deep Tissue Infection with an Entomopathogenic Beauveria Species

ABSTRACT Beauveria spp. are ubiquitous fungal entomopathogens that are commercially distributed as biological insecticides worldwide. In this paper we describe the clinical manifestation, diagnosis, and therapy of the first documented human deep tissue infection with an entomopathogenic Beauveria species in a patient receiving immunosuppressive therapy and describe the morphological and molecular characterization of the mold.

Clinical aspects and pathogenesis of Candida infection.

Abstract The Interdisciplinary Forum on Candidosis , organized by the DGHM section Eukaryotic Pathogens , was held at the University of Wurzburg, Germany, 17–18 April 1998.

Analysis of the major proteins secreted by the human opportunistic pathogen Aspergillus fumigatus under in vitro conditions

Although secreted proteins of pathogenic microorganisms often represent potential virulence factors, so far only limited information has been available on the proteins secreted by Aspergillus fumigatus. We therefore analysed supernatant proteins after growth in different media. In serum-free cell culture medium A. fumigatus growth was limited and no protein secretion was detectable, whereas distinct protein patterns were detectable after growth in either aspergillus minimal medium (AMM) or the more complex yeast glucose medium (YG). The three major proteins secreted under these conditions were identified as the ribotoxin mitogillin, a chitosanase and the aspergillopepsin i. Mitogillin and chitosanase were secreted in AMM, whereas aspergillopepsin i was especially prominent after growth in YG. When the AMM cultures reached stationary phase, seven additional major proteins were detectable. Two of them were identified as the chitinase chiB1 and a beta(1-3) endoglucanase. Conditioned medium containing mitogillin and chitosanase did not have a detectable cytotoxic effect on A549 and Vero cells. Using recombinant mitogillin and chitosanase we detected anti-chitosanase and antimitogillin antibodies in sera of patients suffering from invasive aspergillosis or aspergilloma, but not in control sera of healthy individuals. This suggests that chitosanase, like mitogillin, is expressed during infection and might therefore be of diagnostic relevance.