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T Brandhorst

Publications and source records attributed to T Brandhorst.

6 recordsLinked to original sources
PubMed2001

The WI-1 adhesin blocks phagocyte TNF-alpha production, imparting pathogenicity on Blastomyces dermatitidis.

The WI-1 adhesin is indispensable for pathogenicity of Blastomyces dermatitidis and is thought to promote pulmonary infection by fixing yeast to lung tissue and cells. Recent findings suggest that WI-1 confers pathogenicity by mechanisms in addition to adherence. Here, we investigated whether WI-1 modulates host immunity by altering production of pro-inflammatory cytokines. Production of TNF-alpha in lung alveolar fluids of mice infected with B. dermatitidis was severalfold higher for WI-1 knockout yeast compared with wild-type yeast, and in vitro coculture of unseparated lung cells with these isogenic yeast disclosed similar differences. Upon coculture with purified macrophages and neutrophils, wild-type yeast blocked TNF-alpha production, yet WI-1 knockout yeast stimulated production. Coating knockout yeast with purified WI-1 converted them from stimulating TNF-alpha production to inhibiting production. Addition of purified WI-1 into stimulated phagocyte cultures led to concentration-dependent inhibition of TNF-alpha production. Neutralization of TNF-alpha in vivo exacerbated experimental pulmonary infection, particularly for the nonpathogenic WI-1 knockout yeast. Inducing increased TNF-alpha levels in the lung by adenovirus-vectored gene therapy controlled infection with wild-type yeast. Thus, the WI-1 adhesin on yeast modulates host immunity through blocking TNF-alpha production by phagocytes, which fosters progression of pulmonary infection.

Adenoviridae↗
PubMed2001

The effect of fungal ribosome inactivating proteins upon feeding choice in C. freemani, and indications of a mutualistic relationship with A. restrictus. Environmental mycology.

Carpophilus freemani beetles' feeding on the fungus Aspergillus nidulans was substantially inhibited when A. nidulans was transformed and induced to secrete the ribosome inactivating protein, restrictocin (genetic source: Aspergillus restrictus). No inhibition of feeding was observed when A. nidulans was transformed and induced to produce an inactive form of restrictocin with a single amino-acid substitution in the active site. Similarly, there was no inhibition of feeding upon transgenic strains when the production of restrictocin was not induced. Feeding inhibition of C. freemani by restrictocin requires that the ribonuclease be active and is not due to other characteristics of the protein or the transgenic host fungus.

Allergens↗
PubMed2000

Cell wall biogenesis of Blastomyces dermatitidis. Evidence for a novel mechanism of cell surface localization of a virulence-associated adhesin via extracellular release and reassociation with cell wall chitin.

Pathogenic yeast of Blastomyces dermatitidis express a surface protein adhesin, WI-1. Due to the crucial role of WI-1 in adherence and disease pathogenesis, we investigated how the protein localizes to the surface of B. dermatitidis. WI-1 released extracellularly by wild-type yeast coated the surfaces of co-cultured knockout yeast within 3 h of incubation, implying that secreted WI-1 provides a pathway for loading the protein onto the yeast cell wall. In radioligand binding assays, purified WI-1 bound saturably, specifically, and with high affinity (K(d) = 8.3 x 10(-9)) to the cell surface of knockout yeast devoid of WI-1. WI-1 added exogenously, in vitro, to knockout yeast was indistinguishable from native cell surface WI-1 by fluorescence staining and restored adhesivity to the knockout yeast in macrophage binding and phagocytosis assays. Analysis of interactions between WI-1 and elements of the yeast cell wall identified chitin as the anchor point for WI-1. This interaction was shown to hinge on the 24-amino acid tandem repeat sequence of WI-1. Efforts to extract surface WI-1 from the yeast demonstrated that it is fastened to the wall by non-covalent interactions and covalent links between cysteine residues. We conclude that the yeast cell surface adhesin WI-1 localizes to the cell wall, in part, through extracellular release followed by high affinity binding back onto exposed chitin fibrils. These findings point to a novel pathway of cell wall biogenesis in yeast and an unanticipated role for chitin in anchoring and displaying a surface adhesin and virulence determinant.

Animals↗
PubMed1995

Effects of leader sequences upon the heterologous expression of restrictocin in Aspergillus nidulans and Aspergillus niger.

The effects of altered leader sequences on the secretion and localization of restrictiocin expression in Aspergillus nidulans and Aspergillus niger were investigated. The region encoding the leader sequence of the Aspergillus restrictus restrictocin (res) gene was altered and variants were expressed under the glucoamylase (glaA) promoter in A. nidulans and A. niger. The entire restrictocin leader sequence was replaced by the glaA leader sequence in one variant. In another, the signal sequence of restrictocin was replaced with the glaA signal, leaving a hybrid with the putative restrictocin pro region in place of the glaA pro region. The putative pro region was deleted from the restrictocin leader of a third variant. Toxic effects, such as reduced transcript levels and cellular lysis, were minimal when restrictocin was expressed with the native leader sequence, but became more pronounced as the leader sequence was varied. These toxic effects were inversely proportional to the level of restrictocin secreted. In all transformed strains, restrictocin secretion appeared at the periphery of colonies and was observed to occur at the tips of hyphae. Localization of restrictocin to differentiated structures (conidiophores), as occurs in A. restrictus, was observed only in transformed strains containing the complete restrictocin leader sequence.

Allergens↗
PubMed1994

Heterologous expression of the cytotoxin restriction in Aspergillus nidulans and Aspergillus niger.

The cDNA clone of restrictocin was placed under the control of the glucoamylase promoter from Aspergillus awamori and was transformed into Aspergillus nidulans and Aspergillus niger. Site-specific changes were introduced into cDNA constructs and these were transformed into A. nidulans. The secretion signal sequence was deleted from one form of the gene and three mutations introduced single amino acid substitutions into the protein. Culture conditions were optimized for maximum expression levels of restrictocin. The activities of the expressed proteins were characterized with an in vitro rabbit reticulocyte assay. Protein synthesis in this assay was inhibited 50% by 2.5 ng/ml wild-type restrictocin, 3.5 ng/ml E95G, 30 ng/ml E95C, and 600 ng/ml H136L. Toxic effects of restrictocin were observed in the A. nidulans expression system with reduced levels of cellular protein and messenger RNA upon induction of restrictocin expression as well as the formation of the alpha-fragment product of ribosomal RNA cleavage. Toxic effects were most highly pronounced in strains expressing restrictocin without the signal sequence, less so in strains expressing native restrictocin, and absent in strains expressing H136L restrictocin.

Allergens↗