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- All Subjects: Plant proteomics
- Creators: Chandler, Douglas
Description
Some cyanobacteria, referred to as boring or euendolithic, are capable of excavating tunnels into calcareous substrates, both mineral and biogenic. The erosive activity of these cyanobacteria results in the destruction of coastal limestones and dead corals, the reworking of carbonate sands, and the cementation of microbialites. They thus link the biological and mineral parts of the global carbon cycle directly. They are also relevant for marine aquaculture as pests of mollusk populations. In spite of their importance, the mechanism by which these cyanobacteria bore remains unknown. In fact, boring by phototrophs is geochemically paradoxical, in that they should promote precipitation of carbonates, not dissolution. To approach this paradox experimentally, I developed an empirical model based on a newly isolated euendolith, which I characterized physiologically, ultrastructurally and phylogenetically (Mastigocoleus testarum BC008); it bores on pure calcite in the laboratory under controlled conditions. Mechanistic hypotheses suggesting the aid of accompanying heterotrophic bacteria, or the spatial/temporal separation of photosynthesis and boring could be readily rejected. Real-time Ca2+ mapping by laser scanning confocal microscopy of boring BC008 cells showed that boring resulted in undersaturation at the boring front and supersaturation in and around boreholes. This is consistent with a process of uptake of Ca2+ from the boring front, trans-cellular mobilization, and extrusion at the distal end of the filaments (borehole entrance). Ca2+ disequilibrium could be inhibited by ceasing illumination, preventing ATP generation, and, more specifically, by blocking P-type Ca2+ ATPase transporters. This demonstrates that BC008 bores by promoting calcite dissolution locally at the boring front through Ca2+ uptake, an unprecedented capacity among living organisms. Parallel studies using mixed microbial assemblages of euendoliths boring into Caribbean, Mediterranean, North and South Pacific marine carbonates, demonstrate that the mechanism operating in BC008 is widespread, but perhaps not universal.
ContributorsRamírez-Reinat, Edgardo L (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Chandler, Douglas (Committee member) / Farmer, Jack (Committee member) / Neuer, Susanne (Committee member) / Arizona State University (Publisher)
Created2010
Description
Coccidioidomycosis (valley fever) is caused by inhalation of arthrospores from soil-dwelling fungi, Coccidioides immitis and C. posadasii. This dimorphic fungus and disease are endemic to the southwestern United States, central valley in California and Mexico. The Genome of Coccidioidies has been sequenced but proteomic studies are absent. To address this gap in knowledge, we generated proteome of Spherulin (lysate of Spherule phase) using LC-MS/MS and identified over 1300 proteins. We also investigated lectin reactivity to spherules in human lung tissue based on the hypothesis that coccidioidal glycosylation is different from mammalian glycosylation, and therefore certain lectins would have differential binding properties to fungal glycoproteins. Lectin-based immunohistochemistry using formalin-fixed paraffin-embedded human lung tissue from human coccidioidomycosis patients demonstrated that Griffonia simplificonia lectin II (GSL II) and succinylated wheat germ agglutinin (sWGA) bound specifically to endospores and spherules in infected lungs, but not to adjacent human tissue. GSL II and sWGA-lectin affinity chromatography using Spherulin, followed by LC-MS/MS was used to isolate and identify 195 proteins that bind to GSL-II lectin and 224 proteins that bind to sWGA lectin. This is the first report that GSL II and sWGA lectins bind specifically to Coccidioides endospores and spherules in infected human tissues. Our list of proteins from spherulin (whole and GSL-II and sWGA binding fraction) may also serve as a Coccidioidal Rosetta-Stone generated from mass spectra to identify proteins from 3 different databases: The Broad Institutes Coccidioides Genomes project, RefSeq and SwissProt. This also serves as a viable avenue for proteomics based diagnostic test development for valley fever. Using lectin chromatography and LC MS/MS, we identified over 100 proteins in plasma of two patients and six proteins in urine of one patient. We also identified over eighty fungal proteins isolated from spherules from biopsied infected lung tissue. This, to the best of our knowledge, is the first such example of detecting coccidioidal proteins in patient blood and urine and provides a foundation for development of a proteomics based diagnostic test as opposed to presently available but unreliable serologic diagnostic tests reliant on an antibody response in the host.
ContributorsKaushal, Setu (Author) / Lake, Douglas (Thesis advisor) / Magee, Dewey Mitchell (Committee member) / Chandler, Douglas (Committee member) / Rawls, Jeffery (Committee member) / Arizona State University (Publisher)
Created2015