Niche-Specific Requirement for Hyphal Wall protein 1 in Virulence of Candida albicans

Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa.     

Intermittent altitude exposures improve muscular performance at 4,300 m.

Chronic altitude residence improves muscular performance at altitude, but the effect of intermittent altitude exposures (IAE) on muscular performance at altitude has not been defined. The purpose of this study was to determine the effects of 3 wk of IAE, in combination with rest and cycle training, on muscular performance at altitude. Six lowlanders (23 +/- 2 yr, 77 +/- 6 kg; means +/- SE) completed a cycle time trial and adductor pollicis endurance test at sea level and during a 30-h acute exposure to 4,300 m altitude equivalent (barometric pressure = 446 mmHg) once before (pre-IAE) and once after (post-IAE) a 3-wk period of IAE (4 h/day, 5 days/wk, 4,300 m). During each IAE, three subjects cycled for 45-60 min/day at 60%-70% of maximal O2 uptake and three subjects rested. Cycle training during each IAE did not appear to affect muscular performance at altitude. Thus data from all six subjects were combined. Three weeks of IAE resulted in 1) a 21 +/- 6% improvement (P < 0.05) in cycle time-trial performance (min) from pre-IAE (32.8 +/- 3.7) to post-IAE (24.8 +/- 1.2), 2) a 63 +/- 26% improvement (P < 0.05) in adductor pollicis endurance (min) from pre-IAE (9.2 +/- 2.8) to post-IAE (14.8 +/- 4.2), and 3) a 10 +/- 4% increase (P < 0.05) in resting arterial O2 saturation (%) from pre-IAE (82 +/- 2) to post-IAE (90 +/- 1). These improvements in muscular performance after IAE correlated strongly with increases in resting arterial O2 saturation and were comparable to those reported previously after chronic altitude residence. IAE may therefore be used as an alternative to chronic altitude residence to facilitate improvements in muscular performance in athletes, soldiers, mountaineers, shift workers, and others that are deployed to altitude.     

Evaluating the effectiveness of retention forestry to enhance biodiversity in production forests of Central Europe using an interdisciplinary,multi‐scale approach

Retention forestry, which retains a portion of the original stand at the time of harvesting to maintain continuity of structural and compositional diversity, has been originally developed to mitigate the impacts of clear‐cutting. Retention of habitat trees and deadwood has since become common practice also in continuous‐cover forests of Central Europe. While the use of retention in these forests is plausible, the evidence base for its application is lacking, trade‐offs have not been quantified, it is not clear what support it receives from forest owners and other stakeholders and how it is best integrated into forest management practices. The Research Training Group ConFoBi (Conservation of Forest Biodiversity in Multiple‐use Landscapes of Central Europe) focusses on the effectiveness of retention forestry, combining ecological studies on forest biodiversity with social and economic studies of biodiversity conservation across multiple spatial scales. The aim of ConFoBi is to assess whether and how structural retention measures are appropriate for the conservation of forest biodiversity in uneven‐aged and selectively harvested continuous‐cover forests of temperate Europe. The study design is based on a pool of 135 plots (1 ha) distributed along gradients of forest connectivity and structure. The main objectives are (a) to investigate the effects of structural elements and landscape context on multiple taxa, including different trophic and functional groups, to evaluate the effectiveness of retention practices for biodiversity conservation; (b) to analyze how forest biodiversity conservation is perceived and practiced, and what costs and benefits it creates; and (c) to identify how biodiversity conservation can be effectively integrated in multi‐functional forest management. ConFoBi will quantify retention levels required across the landscape, as well as the socio‐economic prerequisites for their implementation by forest owners and managers. ConFoBi's research results will provide an evidence base for integrating biodiversity conservation into forest management in temperate forests.     

Genome-wide transcriptional profiling of the cyclic AMP-dependent signaling pathway during morphogenic transitions of Candida albicans

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.     

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

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.     

Molecular imaging of amyloid beta peptides in mouse brain sections using mass spectrometry

In this paper, a reverse-phase high-performance liquid chromatographic method using a chemically modified electrode coupled with microdialysis was developed to study the effect of electromagnetic impulse (EMI) on monoamine neurotransmitter metabolism in nerve cells. To detect the monoamines and their metabolites, a poly (para-aminobenzoic acid) (P-pABA)-modified electrode was prepared. The modified electrode exhibited efficiently electrocatalytic oxidation for monoamines and their metabolites with relatively high sensitivity, stability, and long life. Nerve cells were primarily cultured. EMI was radiated to three experimental model nerve cells: (i) on mature nerve cells, (ii) on the culture medium, and (iii) on juvenile nerve cells for various periods of time. Then the levels of monoamines in the culture medium were detected by high-performance liquid chromatography-electrochemical detection. The data indicated that electromagnetic fields could influence neurotransmitter metabolism by direct effect on nerve cells or effect on the nutrient medium and that the effect was not only relevant with the length of radiation time, but also with the growing state of the nerve cells.     

The adhesin Hwp1 and the first daughter cell localize to the a/a portion of the conjugation bridge during Candida albicans mating

The cell wall protein Hwp1 was originally demonstrated to be expressed exclusively in hyphae of Candida albicans and cross-linked to human epithelium by mammalian transglutaminase. Hwp1 is expressed on the walls of hyphae formed by a/alpha, a/a, and alpha/alpha cells. Hence, it is expressed on hyphae independently of mating type. However, Hwp1 is selectively expressed on the wall of conjugation tubes formed by a/a cells, but not alpha/alpha cells, in the mating process. This was demonstrated in all possible crosses between four unrelated natural a/a strains and four unrelated alpha/alpha strains. In zygotes, Hwp1 is restricted to that portion of the wall of the conjugation bridge contributed by the a/a parent cell. Hwp1 staining further revealed that the first daughter bud that emerges from the conjugation bridge does so from the a/a-contributed portion. Hwp1 expression and localization during the mating process is, therefore, mating type specific, opaque phase specific, and alpha-pheromone induced. These results indicate that the mating type-specific contributions to the conjugation bridge during the mating process in C. albicans are qualitatively and functionally distinct and that the a/a portion of the bridge, which selectively contains Hwp1, bears the first daughter cell in the mating process.     

Healthy Human T-Cell Responses to Aspergillus fumigatus Antigens

Background

Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T_H1–T_H17) and destructive allergic (T_H2) immunity. How Aspergillus allergens (Asp f proteins) participate in the development of allergic sensitization is unknown.

Methodology/Principal Findings

To determine whether Asp f proteins are strictly associated with T_H2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-γ, IL-4 or IL-17) to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-γ more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T_H1-responses to Asp f3 (a putative peroxismal membrane protein), Asp f9/16 (cell wall glucanase), Asp f11 (cyclophilin type peptidyl-prolyl isomerase) and Asp f22 (enolase). Strong IFN-γ responses were reproduced in most subjects tested over 6 month intervals.

Conclusions

Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.     

Expression of transglutaminase substrate activity on Candida albicans germ tubes through a coiled, disulfide-bonded N-terminal domain of Hwp1 requires C-terminal glycosylphosphatidylinositol modification

By serving as a microbial substrate for epithelial cell transglutaminase, Hwp1 (Hyphal wall protein 1) of Candida albicans participates in cross-links with proteins on the mammalian mucosa. Biophysical properties of the transglutaminase substrate domain were explored using a recombinant protein representative of the N-terminal domain of Hwp1 and were similar to other transglutaminase substrates, the small proline-rich proteins of cornified envelopes found in stratified squamous epithelia. Recombinant Hwp1 lacks alpha and beta structures by circular dichroism and likely exists as a disulfide-cross-linked coiled-coil. The transglutaminase substrate property prompted a unique approach for investigating the features of surface Hwp1 on germ tubes. A lysine analog, 5-(biotinamido)pentylamine, was cross-linked to germ tubes catalyzed by transglutaminase 2 prior to cell fractionation, immunoprecipitation, and detection with streptavidin conjugates. The majority of the transglutaminase-modifiable Hwp1 was covalently attached to the beta-glucan of hyphae by the C terminus of Hwp1 via a glycosylphosphatidylinositol remnant anchor. A putative precursor of cell wall forms of Hwp1 was identified in the cell extract and in the culture medium. Hwp1 was modified by relatively short N-linked glycans, and the molecular size of the protein was reduced by hypomannosylation when expressed in O-glycosylation mutant strains. Hwp1 combines features of mammalian transglutaminase substrate proteins with characteristics of fungal cell wall proteins to form an unconventional adhesin at the hyphal wall of C. albicans.