In vivo observation of amyloid-like fibrils produced under stress.

The participation of amyloids in neurodegenerative diseases and functional processes has triggered the quest for methods allowing their direct detection in vivo. Despite the plethora of data, those methods are still lacking. The autofluorescence from the extended ß-sheets of amyloids is here used to track fibrillation of S. cerevisiae Golgi Reassembly and Stacking Protein (Grh1). Grh1 has been implicated in starvation-triggered unconventional protein secretion (UPS), and here its participation also in heat shock response (HSR) is suggested. Fluorescence Lifetime Imaging (FLIM) is used to detect fibril autofluorescence in cells (E. coli and yeast) under stress (starvation and higher temperature). The formation of Grh1 large complexes under stress is further supported by size exclusion chromatography and ultracentrifugation. The data show for the first time in vivo detection of amyloids without the use of extrinsic probes as well as bring new perspectives on the participation of Grh1 in UPS and HSR.

Yeast Expressing Gp43 Protein as a Vaccine Against Paracoccidioides brasiliensis Infection.

Paracoccidioidomycosis (PCM) represents the most frequent systemic mycosis in Latin American. The disease is caused by the pathogenic thermally dimorphic fungus Paracoccidioides brasiliensis, and is initially characterized by pulmonary lesions, which can subsequently disseminate to other organs, resulting in secondary injuries. Although its high incidence, there is no commercially available vaccine against fungal diseases. A novel strategy, using Saccharomyces cerevisiae yeast as a vehicle for immunization against PCM, was recently successfully described. Herein, we describe strategies for the construction of the suitable S. cerevisiae vaccine, and protocols of administration and evaluation of the efficacy of the vaccine against experimental PCM.

Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays.

We profiled the global immunoglobulin response against fungal infection by using yeast protein microarrays. Groups of CD-1 mice were infected systemically with human fungal pathogens (Coccidioides posadasii, Candida albicans, or Paracoccidioides brasiliensis) or inoculated with PBS as a control. Another group was inoculated with heat-killed yeast (HKY) of Saccharomyces cerevisiae. After 30 days, serum from mice in the groups were collected and used to probe S. cerevisiae protein microarrays containing 4800 full-length glutathione S-transferase (GST)-fusion proteins. Antimouse IgG conjugated with Alexafluor 555 and anti-GST antibody conjugated with Alexafluor 647 were used to detect antibody-antigen interactions and the presence of GST-fusion proteins, respectively. Serum after infection with C. albicans reacted with 121 proteins: C. posadasii, 81; P. brasiliensis, 67; and after HKY, 63 proteins on the yeast protein microarray, respectively. We identified a set of 16 antigenic proteins that were shared across the three fungal pathogens. These include retrotransposon capsid proteins, heat shock proteins, and mitochondrial proteins. Five of these proteins were identified in our previous study of fungal cell wall by mass spectrometry (Ann. N. Y. Acad. Sci. 2012, 1273, 44-51). The results obtained give a comprehensive view of the immunological responses to fungal infections at the proteomic level. They also offer insight into immunoreactive protein commonality among several fungal pathogens and provide a basis for a panfungal vaccine.

Transformation of Candida albicans with a synthetic hygromycin B resistance gene.

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2-containing plasmids or single-copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT-1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild-type C. albicans. We used PCR to fuse CaHygB or SAT-1 to approximately 1 kb of 5' and 3' noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild-type C. albicans strains. Homologous targeting frequencies were approximately 50-70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates.

A novel mitochondrial protein, Tar1p, is encoded on the antisense strand of the nuclear 25S rDNA.

In eukaryotes, it is widely assumed that genes coding for proteins and structural RNAs do not overlap. Using a transposon-tagging strategy to globally analyze the Saccharomyces cerevisiae genome for expressed genes, we identified multiple insertions in an open reading frame that is contained fully within and transcribed antisense to the 25S rRNA gene in the nuclear rDNA repeat region on Chromosome XII. Expression of this gene, TAR1 (Transcript Antisense to Ribosomal RNA), can be detected at the RNA and protein levels, and the primary sequence of the corresponding 124-amino-acid protein is conserved in several yeast species. Tar1p was found to localize to mitochondria, and overexpression of the protein suppresses the respiration-deficient petite phenotype of a point mutation in mitochondrial RNA polymerase that affects mitochondrial gene expression and mtDNA stability. These findings indicate that coding information for protein and structural RNAs can overlap, raising issues regarding the coevolution of such complex genes, and also suggest that rDNA transcription and mitochondrial function are coordinately regulated in eukaryotic cells.