Birkeland Shanda R.

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Shanda R.

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  • Article
    A new family of giardial cysteine-rich non-VSP protein genes and a novel cyst protein
    (Public Library of Science, 2006-12) Davids, Barbara J. ; Reiner, David S. ; Birkeland, Shanda R. ; Preheim, Sarah P. ; Cipriano, Michael J. ; McArthur, Andrew G. ; Gillin, Frances D.
    Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, “HCNCp” (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many “CxxC” tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple “CxC” motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp C-terminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing ≥20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine candidates, and subjects for further research into Giardia biology.
  • Article
    In vitro generation of human high-density-lipoprotein-resistant Trypanosoma brucei brucei
    (American Society for Microbiology, 2006-08) Faulkner, Sara D. ; Oli, Monika W. ; Kieft, Rudo ; Cotlin, Laura F. ; Widener, Justin ; Shiflett, April M. ; Cipriano, Michael J. ; Pacocha, Sarah E. ; Birkeland, Shanda R. ; Hajduk, Stephen L. ; McArthur, Andrew G.
    The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.
  • Preprint
    Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation
    ( 2006-12-06) Lauwaet, Tineke ; Davids, Barbara J. ; Torres-Escobar, Ascencion ; Birkeland, Shanda R. ; Cipriano, Michael J. ; Preheim, Sarah P. ; Palm, Daniel ; Svard, Staffan G. ; McArthur, Andrew G. ; Gillin, Frances D.
    The ability of Giardia lamblia to undergo two distinct differentiations in response to physiologic stimuli is central to its pathogenesis. The giardial cytoskeleton changes drastically during encystation and excystation. However, the signal transduction pathways mediating these transformations are poorly understood. We tested the hypothesis that PP2A, a highly conserved serine/threonine protein phosphatase, might be important in giardial differentiation. We found that in vegetatively growing trophozoites, gPP2A-C protein localizes to basal bodies/centrosomes, and to cytoskeletal structures unique to Giardia: the ventral disk, and the dense rods of the anterior, posterior-lateral, and caudal flagella. During encystation, gPP2A-C protein disappears from only the anterior flagellar dense rods. During excystation, gPP2A-C localizes to the cyst wall in excysting cysts but is not found in the wall of cysts with emerging excyzoites. Transcriptome and immunoblot analyses indicated that gPP2A-C mRNA and protein are upregulated in mature cysts and during the early stage of excystation that models passage through the host stomach. Stable expression of gPP2A-C antisense RNA did not affect vegetative growth, but strongly inhibited the formation of encystation secretory vesicles (ESV) and water-resistant cysts. Moreover, the few cysts that formed were highly defective in excystation. Thus, gPP2A-C localizes to universal cytoskeletal structures and to structures unique to Giardia. It is also important for encystation and excystation, crucial giardial transformations that entail entry into and exit from dormancy.
  • Article
    Long serial analysis of gene expression for gene discovery and transcriptome profiling in the widespread marine coccolithophore Emiliania huxleyi
    (American Society for Microbiology, 2006-01) Dyhrman, Sonya T. ; Haley, Sheean T. ; Birkeland, Shanda R. ; Wurch, Louie L. ; Cipriano, Michael J. ; McArthur, Andrew G.
    The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism.
  • Article
    Differential gene expression between fall- and spring-run Chinook salmon assessed by long serial analysis of gene expression
    (American Fisheries Society, 2008-09-15) Bernier, Jeremiah C. ; Birkeland, Shanda R. ; Cipriano, Michael J. ; McArthur, Andrew G. ; Banks, Michael A.
    Of all Pacific salmonids, Chinook salmon Oncorhynchus tshawytscha display the greatest variability in return times to freshwater. The molecular mechanisms of these differential return times have not been well described. Current methods, such as long serial analysis of gene expression (LongSAGE) and microarrays, allow gene expression to be analyzed for thousands of genes simultaneously. To investigate whether differential gene expression is observed between fall- and spring-run Chinook salmon from California's Central Valley, LongSAGE libraries were constructed. Three libraries containing between 25,512 and 29,372 sequenced tags (21 base pairs/tag) were generated using messenger RNA from the brains of adult Chinook salmon returning in fall and spring and from one ocean-caught Chinook salmon. Tags were annotated to genes using complementary DNA libraries from Atlantic salmon Salmo salar and rainbow trout O. mykiss. Differentially expressed genes, as estimated by differences in the number of sequence tags, were found in all pairwise comparisons of libraries (freshwater versus saltwater = 40 genes; fall versus spring = 11 genes; and spawning versus nonspawning = 51 genes). The gene for ependymin, an extracellular glycoprotein involved in behavioral plasticity in fish, exhibited the most differential expression among the three groupings. Reverse transcription polymerase chain reaction analysis verified the differential expression of ependymin between the fall- and spring-run samples. These LongSAGE libraries, the first reported for Chinook salmon, provide a window of the transcriptional changes during Chinook salmon return migration to freshwater and spawning and increase the amount of expressed sequence data.
  • Preprint
    Transcriptome analyses of the Giardia lamblia life cycle
    ( 2010-06-04) Birkeland, Shanda R. ; Preheim, Sarah P. ; Davids, Barbara J. ; Cipriano, Michael J. ; Palm, Daniel ; Reiner, David S. ; Svard, Staffan G. ; Gillin, Frances D. ; McArthur, Andrew G.
    We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of Giardia lamblia.