Janet F. Staab

Clinical Significance of Azole Antifungal Drug Cross-Resistance in Candida glabrata

ABSTRACT Candida glabrata, which can become resistant to fluconazole, is a common cause of bloodstream infection. This study was performed to determine the significance of cross-resistance to new azole drugs among C. glabrata isolates recovered as a cause of infection in azole-treated hematopoietic stem cell transplant (HSCT) recipients. Seven cases of invasive candidiasis caused by C. glabrata occurred in HSCT recipients who were receiving azole therapy between January 2000 and December 2004 in our institution. Case characteristics were ascertained. Sequential colonizing and invasive isolates were examined to determine susceptibilities to fluconazole, itraconazole, and voriconazole, and molecular relatedness by restriction fragment length polymorphism (RFLP) analysis. Twenty-three C. glabrata isolates were recovered from 4 patients who developed candidemia while receiving fluconazole and three patients who developed candidemia while receiving voriconazole. The mode MICs of fluconazole, itraconazole, and voriconazole for these isolates were ≥64 μg/ml (range, 4 to ≥64 μg/ml), 2 μg/ml (range, 0.25 to ≥16 μg/ml), and 1 μg/ml (range, 0.03 to ≥16 μg/ml), respectively. Kendall tau b correlation coefficients demonstrated significant associations between the MICs of voriconazole with fluconazole (P = 0.005) and itraconazole (P = 0.008). Colonizing and invasive isolates exhibiting variable susceptibilities had similar RFLP patterns. These observations suggest that C. glabrata exhibits considerable clinically significant cross-resistance between older azole drugs (fluconazole and itraconazole) and voriconazole. Caution is advised when considering voriconazole therapy for C. glabrata candidemia that occurs in patients with extensive prior azole drug exposure.

Reevaluation of the Role of HWP1 in Systemic Candidiasis by Use of Candida albicans Strains with Selectable Marker URA3 Targeted to the ENO1 Locus

ABSTRACT Previous evaluation of HWP1 in systemic candidiasis in CBA/J mice was done with Candida albicans strains with differing genetic locations of URA3 as a result of Ura-blaster mutagenesis. In this study, the presence of HWP1 and the location of URA3 contributed to the severity of murine systemic candidiasis in BALB/c mice.

Genetic organization and sequence analysis of the hypha-specific cell wall protein gene HWP1 of Candida albicans.

A previously isolated partial cDNA encoding a cell wall protein antigen found on hyphal surfaces of the opportunistic fungal pathogen, Candida albicans (Staab et al., 1996) was used to clone the complete hyphal wall protein 1 gene (HWP1). Hyphal forms of C. albicans invade mucosal surfaces of immunocompromised patients such as those with AIDS. HWP1 consisted of an open reading frame predicting an acidic protein (pI of 3·37) with a calculated molecular size of 61,122. The antigenic domain was located in the N‐terminal third of the protein. The remainder of the protein contained abundant hydroxy amino acids, and terminated with a string of 15 amino acids typical of sequences specifying post‐translational modification with glycosylphosphatidylinositol (6PI). The analyses suggested that Hwp1 is a glucan‐linked protein with serine/threonine‐rich regions that are predicted to function in extending a ligand‐binding domain into the extracellular space. The nucleotide sequence reported in this paper has been submitted to GenBank/EMBL databank with Accession Number U64206.

URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes

The ability to generate isogenic sets of strains with mutations in a gene of interest but not in other genes by repeated use of the URA3 marker (Ura-blaster methodology) has advanced our understanding of the relationships between gene structure and function in Candida albicans. Common applications of Ura-blaster technology result in different genomic positions for the URA3 gene in strains complemented for the gene of interest compared with mutant strains. Studies using animal models of systemic candidiasis pointed to possible differences in URA3 gene expression, depending on its genomic location, which confounded interpretation of the role of the gene of interest in lethality. Positional effects on URA3 expression can be avoided by placement at a common locus in all strains used for comparison.

MyD88 Signaling Contributes to Early Pulmonary Responses to Aspergillus fumigatus

ABSTRACT Toll-like receptors and the β-glucan receptor, dectin-1, mediate macrophage inflammatory responses to Aspergillus fumigatus through MyD88-dependent and -independent signaling mechanisms; however, pulmonary inflammatory responses in MyD88-deficient mice challenged with A. fumigatus are poorly defined. The role of MyD88 signaling in early pulmonary inflammation and fungal clearance was evaluated in C57BL/6J wild-type (WT) and MyD88-deficient (MyD88−/−) mice. Early (<48 h) after infection, MyD88−/− mice had higher fungal burdens than those of WT mice, although fungal burdens rapidly declined (>72 h) in both. MyD88−/− mice had less consolidated inflammation, with fewer NK cells, in lung tissue early (24 h) after infection than did WT mice. At the latter time point, MyD88−/− mouse lungs were characterized by a large amount of necrotic cellular debris and fibrin, while WT lungs had organized inflammation. Although there were equivalent numbers of macrophages in WT and MyD88−/− mouse lung tissues, MyD88−/− cells demonstrated delayed uptake of green fluorescent protein-expressing A. fumigatus (GFP-Af293); histologically, MyD88−/− mouse lungs had more hyphal invasion of terminal airways and vessels, the appearance of bronchiolar epithelial cell necrosis, and necrotizing vasculitis. MyD88−/− lung homogenates contained comparatively decreased amounts of interleukin-1β (IL-1β), IL-6, KC, and gamma interferon and paradoxically increased amounts of tumor necrosis factor alpha and macrophage inflammatory protein 1α. These data indicate that the MyD88-dependent pathway mediates acute pulmonary fungal clearance, inflammation, and tissue injury very early after infection. Resolution of abnormalities within a 3-day window demonstrates the importance of redundant signaling pathways in mediating pulmonary inflammatory responses to fungi.

Increased high‐affinity phosphodiesterase PDE2 gene expression in germ tubes counteracts CAP1‐dependent synthesis of cyclic AMP, limits hypha production and promotes virulence of Candida albicans

Frequent interconversion between yeasts, pseudohyphae and true hyphae is a hallmark of Candida albicans growth in mammalian tissues. The requirement for transient CAP1‐dependent pulses of cAMP for generating true hyphae, Hwp1 and virulence raises questions about the role of yeast and pseudohyphal forms in the pathogenesis of candidiasis. In this study, hyperfilamentous mutants, limited in their capacity to produce buds, were generated by disrupting the high‐affinity phosphodiesterase gene PDE2. Degradation of cAMP by the PDE2 gene product was confirmed by higher basal cAMP levels in the pde2/pde2 mutant and by accumulation of cAMP to levels permitting germ tube formation upon disrupting PDE2 in the cap1/cap1 mutant. Similar phenotypes of the C. albicans and Saccharomyces cerevisiae pde2/pde2 mutants were found, including sensitivity to nutritional starvation and exogenous cAMP and defective entry into stationary phase. Importantly, the hyperfilamentous mutants were as avirulent as hypofilamentous mutants in a systemic model of candidiasis. Growth in a multiplicity of forms appears to be a virulence attribute that is controlled by tight coupling of cAMP synthesis and degradation. Delayed increases in PDE2 mRNA in cAMP‐deficient cap1/cap1 mutants during germ tube‐inducing conditions suggested a mechanism of control involving cAMP‐dependent induction of PDE2 mRNA.

Cystic Fibrosis Transmembrane Conductance Regulator Regulates Epithelial Cell Response to Aspergillus and Resultant Pulmonary Inflammation

RATIONALE Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. OBJECTIVES To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. METHODS A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. MEASUREMENTS AND MAIN RESULTS Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR(-/-) mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. CONCLUSIONS Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease.

Genome-Wide Transcriptional Profiling of the Cyclic AMP-Dependent Signaling Pathway during Morphogenic Transitions of Candida albicans

ABSTRACT Candida albicans is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment, C. albicans transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of C. albicans by studying genes encoding adenylate cyclase-associated protein (CAP1) and high-affinity phosphodiesterase (PDE2) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. Microbiol. 50:391-409, 2003; and Y. S. Bahn and P. Sundstrom, J. Bacteriol. 183:3211-3223, 2001). However, little is known about the downstream targets of the cAMP signaling pathway that are responsible for morphological transitions and the expression of virulence factors. Here, microarrays were probed with RNA from strains with hypoactive (cap1/cap1 null mutant), hyperactive (pde2/pde2 null mutant), and wild-type cAMP signaling pathways to provide insight into the molecular mechanisms of virulence that are regulated by cAMP and that are related to the morphogenesis of C. albicans. Genes controlling metabolic specialization, cell wall structure, ergosterol/lipid biosynthesis, and stress responses were modulated by cAMP during hypha formation. Phenotypic traits predicted to be regulated by cAMP from the profiling results correlated with the relative strengths of the mutants when tested for resistance to azoles and subjected to heat shock stress and oxidative/nitrosative stress. The results from this study provide important insights into the role of the cAMP signaling pathway not only in morphogenic transitions of C. albicans but also for adaptation to stress and for survival during host infections.

Aspergillus Section Fumigati Typing by PCR-Restriction Fragment Polymorphism

ABSTRACT Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding β-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati.

A 368-Base-Pair cis-Acting HWP1 Promoter Region, HCR, of Candida albicans Confers Hypha-Specific Gene Regulation and Binds Architectural Transcription Factors Nhp6 and Gcf1p

ABSTRACT To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene HWP1 of Candida albicans, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the HWP1 control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous ENO1 promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in Saccharomyces cerevisiae, and another with weak similarity to an HMG-like condensation factor from Physarum polycephalum implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in C. albicans and suggest a new model for hypha-specific gene regulation.