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.

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-␤-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to 1,3-␤-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 adhesinlike 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.

The key to therapeutic success with yeast infections is an early onset of antifungal treatment with an appropriate drug regimen. To do this, yeast species identification is necessary, but conventional biochemical and morphological approaches are time-consuming. The recent arrival of biophysical methods, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), in routine diagnostic laboratories holds the promise of significantly speeding up this process. In this study, two commercially available MALDI-TOF MS species identification systems were evaluated for application in clinical diagnostics, using a geographically diverse collection of 1192 clinical yeast and yeast-like isolates. The results were compared with those of the classical differentiation scheme based on microscopic and biochemical characteristics. For 95.1% of the isolates, all three procedures consistently gave the correct species identification, but the rate of misclassification was greatly reduced in both MALDI-TOF MS systems. Furthermore, several closely related species (e.g. Candida orthopsilosis/metapsilosis/parapsilosis or Candida glabrata/bracarensis) could be resolved by both MALDI-TOF MS systems, but not by the biochemical approach. A significant advantage of MALDI-TOF MS over biochemistry in the recognition of isolates novel to the system was observed. Although both MALDI-TOF MS systems employed different approaches in the database structure and showed different susceptibilities to errors in database entries, these were negligible in terms of clinical usefulness. The time-saving benefit of MALDI-TOF MS over biochemical identification will substantially improve fungal diagnostics and patient treatment.

Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol ⌬ 5,6 -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 g 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 g ml ؊1 ); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 g ml ؊1 ). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14␣-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.

The incidence of azole resistance in species has increased over the past years, most importantly for. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.