Silvia Slesiona

Identifying infection-associated genes of Candida albicans in the postgenomic era

The human pathogenic yeast Candida albicans can cause an unusually broad range of infections reflecting a remarkable potential to adapt to various microniches within the human host. The exceptional adaptability of C. albicans is mediated by rapid alterations in gene expression in response to various environmental stimuli and this transcriptional flexibility can be monitored with tools such as microarrays. Using such technology it is possible to (1) capture a genome-wide portrait of the transcriptome that mirrors the environmental conditions, (2) identify known genes, signalling pathways and transcription factors involved in pathogenesis, (3) identify new patterns of gene expression and (4) identify previously uncharacterized genes that may be associated with infection. In this review, we describe the molecular dissection of three distinct stages of infections, covering both superficial and invasive disease, using in vitro, ex vivo and in vivo infection models and microarrays.

Embryonated eggs as an alternative infection model to investigate Aspergillus fumigatus virulence.

ABSTRACT Infection models are essential tools for studying microbial pathogenesis. Murine models are considered the “gold standard” for studying in vivo infections caused by Aspergillus species, such as A. fumigatus. Recently developed molecular protocols allow rapid construction of high numbers of fungal deletion mutants, and alternative infection models based on cell culture or invertebrates are widely used for screening such mutants to reduce the number of rodents in animal experiments. To bridge the gap between invertebrate models and mice, we have developed an alternative, low-cost, and easy-to-use infection model for Aspergillus species based on embryonated eggs. The outcome of infections in the egg model is dose and age dependent and highly reproducible. We show that the age of the embryos affects the susceptibility to A. fumigatus and that increased resistance coincides with altered chemokine production after infection. The progress of disease in the model can be monitored by using egg survival and histology. Based on pathological analyses, we hypothesize that invasion of embryonic membranes and blood vessels leads to embryonic death. Defined deletion mutant strains previously shown to be fully virulent or partially or strongly attenuated in a mouse model of bronchopulmonary aspergillosis showed comparable degrees of attenuation in the egg model. Addition of nutrients restored the reduced virulence of a mutant lacking a biosynthetic gene, and variations of the infectious route can be used to further analyze the role of distinct genes in our model. Our results suggest that embryonated eggs can be a very useful alternative infection model to study A. fumigatus virulence and pathogenicity.

Small but Crucial: The Novel Small Heat Shock Protein Hsp21 Mediates Stress Adaptation and Virulence in Candida albicans

Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity. Hsp21 therefore represents the first reported example of a small heat shock protein functioning as a virulence factor in a eukaryotic pathogen.

Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages.

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts.

Murine Infection Models for Aspergillus terreus Pulmonary Aspergillosis Reveal Long-term Persistence of Conidia and Liver Degeneration

Aspergillus terreus is emerging as a causative agent of life-threatening invasive aspergillosis. Prognosis for affected patients is often worse than for A. fumigatus infections. To study A. terreus-mediated disease, we developed 3 infection models. In embryonated hens eggs and leucopenic mice, the outcome of invasive aspergillosis was similar to that described for A. fumigatus. However, 10(2)- and 10(3)-fold higher conidia concentrations were required for 100% lethality. In corticosteroid-treated mice, only 50% mortality was observed, although bioluminescence imaging revealed transient disease in all infected animals. In surviving animals, we observed persistence of ungerminated but viable conidia. Cytokine levels in these mice were comparable to uninfected controls. In contrast to A. fumigatus infections, all mice infected with A. terreus developed fatty liver degeneration, suggesting the production of toxic secondary metabolites. Thus, at least in mice, persistence and subclinical liver damage are unique features of A. terreus infections.

Molecular epidemiology and virulence assessment of Aspergillus fumigatus isolates from white stork chicks and their environment

Aspergillus fumigatus is a common pathogen in poultry and captive wild birds and an emerging opportunistic fungal pathogen in immunocompromised humans. Although invasive aspergillosis is frequently reported in free-ranging wild birds, the incidence and epidemiology of the disease in a natural setting is unknown. We recently reported endemic outbreaks of invasive aspergillosis at white stork nesting sites close to human habitation in Germany with significant subsequent breeding losses. Therefore, we hypothesized that A. fumigatus strains with higher virulence in birds may have evolved in this environment and performed the first epidemiological analysis of invasive aspergillosis in free-ranging wild birds. Sixty-one clinical and environmental A. fumigatus isolates from six affected nesting sites were genotyped by microsatellite analysis using the STRAf-assay. The isolates showed a remarkable high genomic diversity and, contrary to the initial hypothesis, clinical and environmental isolates did not cluster significantly. Interestingly, storks were infected with two to four different genotypes and in most cases both mating types MAT-1.1 and MAT-1.2 were present within the same specimen. The majority of selected clinical and environmental strains exhibited similar virulence in an in vivo infection model using embryonated chicken eggs. Noteworthy, virulence was not associated with one distinct fungal mating type. These results further support the assumption that the majority of A. fumigatus strains have the potential to cause disease in susceptible hosts. In white storks, immaturity of the immune system during the first three weeks of age may enhance susceptibility to invasive aspergillosis.

The glycolytic enzyme enolase represents a plasminogen-binding protein on the surface of a wide variety of medically important fungal species.

Allergies are an increasing issue in human health and can, eventually, cause severe anaphylactic shock. Aspergillus fumigatus and Candida albicans are leading causes of life-threatening invasive fungal infections in immunocompromised patients, but can also cause severe allergic responses in otherwise healthy individuals. The glycolytic enzyme enolase is known as a major allergen despite its function in intracellular metabolism. Therefore, its presentation on surfaces of different fungal species was investigated by using antibodies raised against recombinant enolases from A. fumigatus and C. albicans. Examination of antibody specificity revealed cross-reactivity to cell-free extracts from Aspergillus terreus, Aspergillus flavus, Aspergillus nidulans and Candida glabrata, but not against any of the three human enolases. Antibody specificity was further confirmed by hybridization with other recombinant fungal enolases, where the antibodies recognized different subsets of fungal enolases. When surface presentation of enolase was tested on intact fungal cells, a positive staining was obtained with those antibodies that also recognized the enzyme from the respective cell-free extract. This implies a general surface presentation of this glycolytic enzyme among fungal species and provides hints for its predominant recognition as an allergen. Additionally, A. fumigatus and C. albicans enolase bound to human plasminogen, which remained accessible for the plasminogen activator uPA. This implies a potential role of enolase in the invasion and dissemination process during fungal infections.

Distinct roles of Candida albicans-specific genes in host-pathogen interactions

ABSTRACT Human fungal pathogens are distributed throughout their kingdom, suggesting that pathogenic potential evolved independently. Candida albicans is the most virulent member of the CUG clade of yeasts and a common cause of both superficial and invasive infections. We therefore hypothesized that C. albicans possesses distinct pathogenicity mechanisms. In silico genome subtraction and comparative transcriptional analysis identified a total of 65 C. albicans-specific genes (ASGs) expressed during infection. Phenotypic characterization of six ASG-null mutants demonstrated that these genes are dispensable for in vitro growth but play defined roles in host-pathogen interactions. Based on these analyses, we investigated two ASGs in greater detail. An orf19.6688Δ mutant was found to be fully virulent in a mouse model of disseminated candidiasis and to induce higher levels of the proinflammatory cytokine interleukin-1β (IL-1β) following incubation with murine macrophages. A pga16Δ mutant, on the other hand, exhibited attenuated virulence. Moreover, we provide evidence that secondary filamentation events (multiple hyphae emerging from a mother cell and hyphal branching) contribute to pathogenicity: PGA16 deletion did not influence primary hypha formation or extension following contact with epithelial cells; however, multiple hyphae and hyphal branching were strongly reduced. Significantly, these hyphae failed to damage host cells as effectively as the multiple hypha structures formed by wild-type C. albicans cells. Together, our data show that species-specific genes of a eukaryotic pathogen can play important roles in pathogenicity.