Biology subjects

Nguyen, K.

Publications and source records attributed to Nguyen, K..

4 recordsLinked to original sources

bioRxiv2018

Giardia’s ventral disc is hyperstable and composed of over 80 disc-associated proteins

Giardia is a common protistan parasite that causes diarrheal disease worldwide. Motile trophozoites colonize the small intestine, attaching to the villi with the ventral disc, a unique and complex microtubule (MT) organelle. Attachment to the host epithelium allows Giardia to resist peristalsis during infection of the host gastrointestinal tract. Despite our emerging view of the complexity of ventral disc architecture, we are still in the very preliminary stages of understanding how specific structural elements contribute to disc stability or generate forces for attachment. The ventral disc is a large, dome-shaped, spiral MT array decorated with microribbon-crossbridge protein complexes (MR-CB) that extend upward into the cytoplasm. To find additional disc-associated proteins (DAPs), we used a modified method for disc biochemical fractionation in high salt followed by shotgun proteomic analyses and validation by GFP-tagging. Using this method in conjunction with an ongoing subcellular localization screen, we identified 54 new DAPs. Of the 87 DAPs confirmed to date, 54 localize only to the disc, and the remainder localize to additional structures including the flagella, basal bodies, or median body. Almost one third of the known DAPs lack any homology to proteins in other eukaryotes and another one third simply contain ankyrin repeat domains. Many DAPs localize to specific structural regions of the disc, including the ventral groove region and disc margin. Lastly, we show that spiral singlet MT array comprising the disc is hyperstable and lacks dynamic instability, and we attribute these unique properties to the presence of both novel DAPs as well conserved MAPs and MIPs that are known to stabilize ciliary doublet and triplet MTs.

microbiology↗

bioRxiv2018

Robust and stable transcriptional repression in Giardia using CRISPRi

Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of non-homologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disc MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than five days longer than transient morpholino-based knockdown. By expressing two gRNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disc defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows for rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

microbiology↗

bioRxiv2017

Agrochemical pollution increases risk of human exposure to schistosome parasites

Roughly 10% of the global population is at risk of schistosomiasis, a snail-borne parasitic disease that ranks among the most important water-based diseases of humans in developing countries1-3. Increased prevalence, infection intensity, and spread of human schistosomiasis to non-endemic areas has been consistently linked with water resource management related to agricultural expansion, such as dam construction, which has resulted in increased snail habitat1,4-6. However, the role of agrochemical pollution in human schistosome transmission remains unexplored, despite strong evidence of agrochemicals increasing snail-borne diseases of wildlife7-9 and a projected 2- to 5-fold increase in global agrochemical use by 205010 that will disproportionately occur in schistosome-endemic regions. Using a field mesocosm experiment, we show that environmentally relevant concentrations of fertilizer, the common herbicide atrazine, and the common insecticide chlorpyrifos, individually and as mixtures, increase densities of schistosome-infected snails by increasing the algae snails eat (fertilizer and atrazine) and decreasing densities of snail predators (chlorpyrifos). Epidemiological models indicate that these agrochemical effects can increase transmission of schistosomiasis. Hence, the rapid agricultural changes occurring in schistosome-endemic regions11,12 that are driving increased agrochemical use and pollution could potentially increase the burden of schistosomiasis in these areas. Identifying agricultural practices or agrochemicals that minimize disease risk will be critical to meeting growing food demands while improving human wellbeing13,14.

ecology↗

bioRxiv2017

Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an -tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.\n\nTable of Content (TOC) Highlight SummaryDifferent tubulin isotypes perform different functions in the regulation of MT structure and neurite growth, and missense mutations of tubulin genes have three types of distinct effects on MT stability and neurite growth. One -tubulin isotype appears to induce relative instability due to the lack of potential post-translational modification sites.

cell biology↗