Ken Haynes

Dectin-1 is required for beta-glucan recognition and control of fungal infection.

β-Glucan is one of the most abundant polysaccharides in fungal pathogens, yet its importance in antifungal immunity is unclear. Here we show that deficiency of dectin-1, the myeloid receptor for β-glucan, rendered mice susceptible to infection with Candida albicans. Dectin-1-deficient leukocytes demonstrated significantly impaired responses to fungi even in the presence of opsonins. Impaired leukocyte responses were manifested in vivo by reduced inflammatory cell recruitment after fungal infection, resulting in substantially increased fungal burdens and enhanced fungal dissemination. Our results establish a fundamental function for β-glucan recognition by dectin-1 in antifungal immunity and demonstrate a signaling non–Toll-like pattern-recognition receptor required for the induction of protective immune responses.

Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals.

Atypical oral Candida isolates were recovered from 60 HIV-infected and three HIV-negative individuals. These organisms were germ-tube-positive and produced abundant chlamydospores which were frequently arranged in triplets or in contiguous pairs. They belonged to C. albicans serotype A and had atypical carbohydrate assimilation profiles. Fingerprinting the genomic DNA of a selection of these organisms with the C. albicans-specific probe 27A and five separate oligonucleotides, homologous to eukaryotic microsatellite repeat sequences, demonstrated that they had a very distinct genomic organization compared to C. albicans and C. stellatoidea. This was further established by random amplified polymorphic DNA (RAPD) and karyotype analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from nine atypical isolates and the corresponding sequences determined from C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that they atypical organisms formed a homogeneous cluster (100% similarity) that was significantly different from the other Candida species analysed, but was most closely related to C. albicans and C. stellatoidea. These genetic data combined with the phenotypic characteristics of these atypical organisms strongly suggest that they constitute a novel species within the genus Candida for which the name Candida dubliniensis is proposed.

Siderophore Biosynthesis But Not Reductive Iron Assimilation Is Essential for Aspergillus fumigatus Virulence

The ability to acquire iron in vivo is essential for most microbial pathogens. Here we show that Aspergillus fumigatus does not have specific mechanisms for the utilization of host iron sources. However, it does have functional siderophore-assisted iron mobilization and reductive iron assimilation systems, both of which are induced upon iron deprivation. Abrogation of reductive iron assimilation, by inactivation of the high affinity iron permease (FtrA), has no effect on virulence in a murine model of invasive aspergillosis. In striking contrast, A. fumigatus l-ornithine-N 5-monooxygenase (SidA), which catalyses the first committed step of hydroxamate-type siderophore biosynthesis, is absolutely essential for virulence. Thus, A. fumigatus SidA is an essential virulence attribute. Combined with the absence of a sidA ortholog—and the fungal siderophore system in general—in mammals, these data demonstrate that the siderophore biosynthetic pathway represents a promising new target for the development of antifungal therapies.

Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant ΔsidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.

Candida dubliniensis: phylogeny and putative virulence factors.

Candida dubliniensis is a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to, Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubliniensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubliniensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The C. dubliniensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced using N-acetylglucosamine. Oral C. dubliniensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The C. dubliniensis isolates were sensitive to fluconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested C. albicans secretory aspartyl proteinase (SAP) genes were detected in C. dubliniensis by Southern analysis. In vivo virulence assays using a systemic mouse model suggest that C. dubliniensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of C. dubliniensis and suggest that this species may be particularly adapted to colonization of the oral cavity.

Sub-telomere directed gene expression during initiation of invasive aspergillosis

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.

Virulence in Candida species

Candida species other than Candida albicans now account for up to 50% of deep candidiasis cases, yet little attention has been paid to the virulence attributes of these fungi. Adherence to host tissues, response to environmental changes and the secretion of hydrolases are all thought to be important in Candida virulence. The identification of virulence attributes unique to a particular Candida species could provide powerful insights into the pathogenic process but will require the use of genome-wide approaches such as transcript profiling, signature-tagged mutagenesis and in vivo expression technology.

Value of antigen detection in predicting invasive pulmonary aspergillosis.

Two ELISAs were used to detect serum and urinary aspergillus antigen in 121 patients who were profoundly neutropenic after leukaemia therapy or bone marrow transplantation. The presence of antigen correctly predicted development of invasive pulmonary aspergillosis (IPA) in 16 patients. In 2 other cases antigen appeared after the clinical diagnosis had been made, while in only 1 case was antigen not detected. In 11 of 13 episodes of clinically suspected fungal infection antigen was detected before clinical diagnosis was made. By contrast, antigen was detected in only 1 of 90 patients who had no evidence of IPA. Both ELISAs gave positive and negative predictive values for IPA of greater than 95%, demonstrating the value of antigen detection in early diagnosis of aspergillus infection and the assays ability to predict subsequent development of IPA. We conclude that neutropenic patients should be screened for aspergillus antigen, and propose that initial detection of fungal antigen justifies commencement of empirical antifungal therapy. Such an approach should improve the survival of patients who are at risk of developing this usually fatal infection.

Nitrosative and oxidative stress responses in fungal pathogenicity.

Fungal pathogenicity has arisen in polyphyletic manner during evolution, yielding fungal pathogens with diverse infection strategies and with differing degrees of evolutionary adaptation to their human host. Not surprisingly, these fungal pathogens display differing degrees of resistance to the reactive oxygen and nitrogen species used by human cells to counteract infection. Furthermore, whilst evolutionarily conserved regulators, such as Hog1, are central to such stress responses in many fungal pathogens, species-specific differences in their roles and regulation abound. In contrast, there is a high degree of commonality in the cellular responses to reactive oxygen and nitrogen species evoked in evolutionarily divergent fungal pathogens.

The Aspergillus fumigatus transcriptional activator CpcA contributes significantly to the virulence of this fungal pathogen

We have cloned and characterized the Aspergillus fumigatus cpcA gene encoding the transcriptional activator of the cross‐pathway control system of amino acid biosynthesis. cpcA encodes a functional orthologue of Saccharomyces cerevisiae Gcn4p. The coding sequence of the 2.2 kb transcript is preceded by two short upstream open reading frames, the larger one being well conserved among Aspergilli. Deletion strains in which either the coding sequence or the entire locus are replaced by a bifunctional dominant marker are impaired in their cross‐pathway control response upon amino acid starvation, as demonstrated by analyses of selected reporter genes and specific enzymatic activities. In a murine model of pulmonary aspergillosis, cpcAΔ strains display attenuated virulence. Pathogenicity is restored to wild‐type levels in strains with reconstitution of the genomic locus. Competitive mixed infection experiments additionally demonstrate that cpcAΔ strains are less able to survive in vivo than their wild‐type progenitor. Our data suggest that specific stress conditions are encountered by A. fumigatus within the mammalian host and that the fungal cross‐pathway control system plays a significant role in pulmonary aspergillosis.