Isogenic strain construction and gene mapping in Candida albicans.

Summary: High-affinity iron acquisition is mediated by siderophore-dependent pathways in the majority of pathogenic and nonpathogenic bacteria and fungi. Considerable progress has been made in characterizing and understanding mechanisms of siderophore synthesis, secretion, iron scavenging, and siderophore-delivered iron uptake and its release. The regulation of siderophore pathways reveals multilayer networks at the transcriptional and posttranscriptional levels. Due to the key role of many siderophores during virulence, coevolution led to sophisticated strategies of siderophore neutralization by mammals and (re)utilization by bacterial pathogens. Surprisingly, hosts also developed essential siderophore-based iron delivery and cell conversion pathways, which are of interest for diagnostic and therapeutic studies. In the last decades, natural and synthetic compounds have gained attention as potential therapeutics for iron-dependent treatment of infections and further diseases. Promising results for pathogen inhibition were obtained with various siderophore-antibiotic conjugates acting as “Trojan horse” toxins and siderophore pathway inhibitors. In this article, general aspects of siderophore-mediated iron acquisition, recent findings regarding iron-related pathogen-host interactions, and current strategies for iron-dependent pathogen control will be reviewed. Further concepts including the inhibition of novel siderophore pathway targets are discussed.

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Siderophore-Based Iron Acquisition and Pathogen Control

Aspergillus species are globally ubiquitous saprophytes found in a variety of ecological niches. Almost 200 species of aspergilli have been identified, less than 20 of which are known to cause human disease. Among them, Aspergillus fumigatus is the most prevalent and is largely responsible for the increased incidence of invasive aspergillosis (IA) in the immunocompromised patient population. IA is a devastating illness, with mortality rates in some patient groups reaching as high as 90%. Studies identifying and assessing the roles of specific factors of A. fumigatus that contribute to the pathogenesis of IA have traditionally focused on single-gene deletion and mutant characterization. In combination with recent large-scale approaches analyzing global fungal responses to distinct environmental or host conditions, these studies have identified many factors that contribute to the overall pathogenic potential of A. fumigatus. Here, we provide an overview of the significant findings regarding A. fumigatus pathogenesis as it pertains to invasive disease.

Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis

Fungi produce a multitude of low-molecular-mass compounds known as secondary metabolites, which have roles in a range of cellular processes such as transcription, development and intercellular communication. In addition, many of these compounds now have important applications, for instance, as antibiotics or immunosuppressants. Genome mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated because many of the fungal secondary metabolite biosynthesis gene clusters are silent under standard cultivation conditions. In this Review, I describe our current understanding of the regulatory elements that modulate the transcription of genes involved in secondary metabolism. I also discuss how an improved knowledge of these regulatory elements will ultimately lead to a better understanding of the physiological and ecological functions of these important compounds and will pave the way for a novel avenue to drug discovery through targeted activation of silent gene clusters.

Regulation of fungal secondary metabolism

The Class III PI3K (phosphoinositide 3-kinase), Vps34 (vacuolar protein sorting 34), was first described as a component of the vacuolar sorting system in Saccharomyces cerevisiae and is the sole PI3K in yeast. The homologue in mammalian cells, hVps34, has been studied extensively in the context of endocytic sorting. However, hVps34 also plays an important role in the ability of cells to respond to changes in nutrient conditions. Recent studies have shown that mammalian hVps34 is required for the activation of the mTOR (mammalian target of rapamycin)/S6K1 (S6 kinase 1) pathway, which regulates protein synthesis in response to nutrient availability. In both yeast and mammalian cells, Class III PI3Ks are also required for the induction of autophagy during nutrient deprivation. Finally, mammalian hVps34 is itself regulated by nutrients. Thus Class III PI3Ks are implicated in the regulation of both autophagy and, through the mTOR pathway, protein synthesis, and thus contribute to the integration of cellular responses to changing nutritional status.

The regulation and function of Class III PI3Ks: novel roles for Vps34

The phosphatidylinositol (PI) 3-kinase Vps34p of Candida albicans has lipid kinase and autophosphorylation activity and is involved in virulence and vesicular protein transport. In order to characterize the roles of lipid kinase activity, a chimeric Vps34 protein was created which lacks lipid kinase but retains autophosphorylation activity. To this end, six amino acids within the putative lipid-binding site of Vps34p were replaced by the homologous region of the PI 3-kinase-like C. albicans Tor protein. The resulting chimeric Vps34T protein was recombinantly expressed in Escherichia coli and shown to lack lipid kinase activity. The corresponding chimeric VPS34TOR gene was inserted into the genome of C. albicans, and this lipid-kinase-defective strain had a distinctive phenotype compared to those of the wild-type strain SC5314 and the vps34 null mutant. The lipid-kinase-defective strain was non-virulent, and showed altered hyphal growth, reduced adherence, as well as defective vacuole morphology and endosomal vesicle transport. These results demonstrate an important role for the lipid kinase activity of Vps34p in virulence and vesicular protein transport. On the other hand, the lipid-kinase-defective strain and the vps34 null mutant differ in their temperature- and osmotic-stress response. This indicates a possible role for activities different from the lipid kinase function of Vps34p.

Generation and functional in vivo characterization of a lipid kinase defective phosphatidylinositol 3-kinase Vps34p of Candida albicans.

The impact of sitosterol formulation particle size on the intestinal sterol absorption and the sterol status in various tissues in Dunkin Hartley guinea pigs was investigated. Three groups of animals (six each) were fed a basal diet ("control") or a basal diet containing either customary sitosterol ("customary", particle size: 10 000-90 000 nm) or nanoscale sitosterol ("nanoscale", particle size: 200-300 nm). The average daily sitosterol intake was 21 +/- 7 mg (control), 154 +/- 8 mg (customary), and 127 +/- 18 mg (nanoscale) for 2 weeks. Sitosterol and cholesterol were analyzed in samples of plasma, blood cells, bile, liver, kidney, jejunal mucosa/serosa, cecum, colon and feces. Concentrations of sitosterol in all analyzed matrices increased significantly in the supplemented groups when compared to control group. No differences in the sitosterol concentrations in analyzed matrices occurred between nanoscale and customary group. The cholesterol concentrations in tissues remained unchanged. Fecal fatty acid and sterol distributions were modified during sitosterol intervention. Both particle sizes equally increased sitosterol levels in cholesterol-metabolizing compartments in the guinea pig. No differences in body compartment accumulation and intestinal absorption of the different sitosterol particle sizes were observed.

Nanoscale and customary non-esterified sitosterols are equally enriched in different body compartments of the guinea pig

The phosphatidylinositol (PtdIns) 3-kinase Vps34p of the human pathogenic yeast Candida albicans participates in virulence and in protein transport. In order to dissect these two functions, a search for proteins interacting with C. albicans Vps34p was performed using a yeast two-hybrid system. This study demonstrates the physical interaction between Vps34p and Ade5,7p, which is the bifunctional enzyme of the de novo purine nucleotide biosynthetic pathway. The interaction initially observed in a yeast two-hybrid system was confirmed in vitro with recombinant proteins. Given the complex formation between Ade5,7p and the virulence-regulating Vps34p, it was of interest to characterize the function of Ade5,7p in C. albicans. To this end, ade5,7 null mutants were generated. The resulting mutants were adenine deficient, and sensitive to the presence of metal ions. In addition, the ade5,7 null mutants were avirulent in a mouse model of systemic candidiasis, and showed reduced hyphal growth in an agar matrix under embedded conditions. In summary, Ade5,7p interacts with the multifunctional virulence regulator PtdIns 3-kinase Vps34p, and ade5,7 and vps34 null mutant strains show similar phenotypes regarding sensitivity to metal ions, hyphal growth and virulence.

Albert Härtl

Papers

Generation and functional in vivo characterization of a lipid kinase defective phosphatidylinositol 3-kinase Vps34p of Candida albicans.

Nanoscale and customary non-esterified sitosterols are equally enriched in different body compartments of the guinea pig

Role of the Vps34p-interacting protein Ade5,7p in hyphal growth and virulence of Candida albicans