Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization

Recently developed tissue-hydrogel methods for specimen expansion now enable researchers to perform super-resolution microscopy with ∼65 nm lateral resolution using ordinary microscopes, standard fluorescent probes, and inexpensive reagents. Here we use the combination of specimen expansion and the optical super-resolution microscopy technique structured illumination microscopy (SIM) to extend the spatial resolution to ∼30 nm. We apply this hybrid method, which we call ExSIM, to study the cytoskeleton of the important human pathogen <i>Giardia lamblia</i> including the adhesive disc and flagellar axonemes. We determined the localization of two recently identified disc-associated proteins, including DAP86676, which localizes to disc microribbons, and the functionally unknown DAP16263, which primarily localizes to dorsal microtubules of the disc overlap zone and the paraflagellar rod of ventral axonemes. Based on its strong performance in revealing known and unknown details of the ultrastructure of <i>Giardia</i>, we find that ExSIM is a simple, rapid, and powerful super-resolution method for the study of fixed specimens, and it should be broadly applicable to other biological systems of interest

Identification and Validation of Small-Gatekeeper Kinases as Drug Targets in <i>Giardia lamblia</i>

<div><p>Giardiasis is widely acknowledged to be a neglected disease in need of new therapeutics to address toxicity and resistance issues associated with the limited available treatment options. We examined seven protein kinases in the <i>Giardia lamblia</i> genome that are predicted to share an unusual structural feature in their active site. This feature, an expanded active site pocket resulting from an atypically small gatekeeper residue, confers sensitivity to “bumped” kinase inhibitors (BKIs), a class of compounds that has previously shown good pharmacological properties and minimal toxicity. An initial phenotypic screen for biological activity using a subset of an in-house BKI library found that 5 of the 36 compounds tested reduced trophozoite growth by at least 50% at a concentration of 5 μM. The cellular localization and the relative expression levels of the seven protein kinases of interest were determined after endogenously tagging the kinases. Essentiality of these kinases for parasite growth and infectivity were evaluated genetically using morpholino knockdown of protein expression to establish those that could be attractive targets for drug design. Two of the kinases were critical for trophozoite growth and attachment. Therefore, recombinant enzymes were expressed, purified and screened against a BKI library of >400 compounds in thermal stability assays in order to identify high affinity compounds. Compounds with substantial thermal stabilization effects on recombinant protein were shown to have good inhibition of cell growth in wild-type <i>G</i>. <i>lamblia</i> and metronidazole-resistant strains of <i>G</i>. <i>lamblia</i>. Our data suggest that BKIs are a promising starting point for the development of new anti-giardiasis therapeutics that do not overlap in mechanism with current drugs.</p></div

Initial set of eight putative <i>Giardia lamblia</i> small gatekeeper kinases.

<p>BLASTP against the <i>Giardia</i> genome database using the <i>T</i>. <i>gondii</i> CDPK1 core kinase domain as a probe found 8 kinases with substantial sequence similarity (E < 10⁻²⁴) to the probe whose alignment indicated a small gatekeeper residue (threonine or smaller). Sequences near the small gatekeeper are shown with small gatekeeper amino acids in red.</p

Structural differences between small gatekeeper ATP binding sites and large gatekeeper ATP binding sites.

<p>Structural differences form the basis for BKIs to selectively act on the <i>Giardia</i> kinases described in this study. <b>(a)</b> The ATP binding pocket of protein kinases typically contains a large amino acid gatekeeper residue (e.g. methionine). <b>(b)</b> BKIs do not bind to the ATP binding site of typical kinases because of a clash between the “bump” and the side chain of the gatekeeper. <b>(c)</b> A small amino acid in the gatekeeper position has no implications for ATP access to the ATP binding site. <b>(d)</b> In contrast, the small gatekeeper residue permits the BKI “bump” to extend into the hydrophobic subpocket and outcompete ATP.</p

BKI 1213 mimics defects observed in knockdown of kinases Gl50803_ 8445 and Gl50803_16034.

<p><b>a)</b> Fluorescent microscopy images of cells 48 hours after morpholino knockdown or treatment with BKI 1213 demonstrate a defect in cytokinesis compared to wild type control cells; cells complete nuclear division but not cytokinesis. <b>b)</b> Quantification of percent of cells that failed to complete cytokinesis. In each treatment, more than 67% of cells were stuck in cytokinesis; 68% of Gl50803_8445, 69.5% of Gl50803_16034 and 77.5% of cells treated with BKI 1213 were unable to complete cytokinesis. <b>c)</b> Attachment assays confirmed that when protein is depleted or cells are treated with BKI 1213, the ability for cells to attach is greatly reduced. Less than 20% of kinase 50803_8445 cells were able to maintain attachment, 43.8% of kinase 50803_16034 cells were able to maintain attachment and only 13% of cells treated with 5 μM BKI 1213 maintained attachment. Experiments were replicated a minimum of three times and significance was evaluated by t-test, ** = p<0.01, *** = p<0.001, **** = p<0.0001.</p

Integration of 3xHA tag allows visualization of protein expression and localization.

<p><b>(a)</b> Western blot analysis showing kinases Gl50803_8445, Gl50803_9421, Gl50803_11364, Gl50803_13215 and Gl50803_16034 are expressed in trophozoites (red). Actin (green) was used as a loading control. <b>(b)</b> Immunofluorescence imaging of trophozoites expressing the designated protein: HA (grayscale) and DAPI (blue). Images were acquired and scaled identically. Representative images are shown from a minimum of 150 cells examined for each line. Scale bar = 5 μm.</p

Kinase depletion interferes with growth.

<p>The five tagged-kinases shown to express in trophozoites were knocked down using anti-sense morpholinos. <b>(a)</b> Knockdown was measured and quantified by Western blot analysis, HA (green) and actin as loading control (red) <b>(b)</b> Cell growth was assessed after morpholino knockdown. Experiments were replicated a minimum of three times and significance was evaluated by t-test, * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Thermal shift assays identify bumped kinase inhibitors that bind to purified proteins.

<p>Kinases Gl50803_8445 and Gl50803_16034 were purified from <i>E</i>. <i>coli</i>. <b>(a)</b> SDS-PAGE was used to estimate protein purity. Gl50803_8445 was 91% pure and Gl50803_16034 was 98% pure. Note that minor impurities are not expected to affect ΔTm determination. <b>(b, c)</b> Melting curves for the BKIS showing the largest ΔTm are shown for Gl50803_8445 (b) and Gl50803_16034 (c). <b>(d)</b> The name, chemical structure, ΔTm, and EC₅₀ value obtained for 7 BKIs prioritized for phenotypic characterization of anti-growth activity (Also see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005107#pntd.0005107.s004" target="_blank">S3 Fig</a>).</p

Bumped kinase inhibitors impact <i>Giardia</i> trophozoite growth.

<p>A subset ~10% of the existing BKI library was screened for impairment of <i>Giardia</i> growth at a concentration of 5 μM and results from selected compounds are shown. The general kinase inhibitor staurosporine reduced growth by 95%. Five bumped kinase inhibitors reduced growth by at least 50% (1483, 1511, Rm-1-89, 1210 and 1213.) The results are averaged from 3 biological replicates that were normalized to the control, DMSO 0.1%. Significance was evaluated by t-test, * = p<0.05, ** = p<0.01.</p

BKIs are effective against metronidazole-resistant <i>Giardia</i> cells.

<p><b>(a)</b> Wild-type <i>G</i>. <i>lamblia</i> clone <i>WBC6</i> and <b>(b)</b> clone <i>713</i> are inhibited by metronidazole (EC₅₀ of 3.8 μM and 2.7μM, respectively), but (<b>c)</b> metronidazole-resistant clone <i>713-M3</i> is not. Clones <b>(d)</b> <i>WBC6</i>, <b>(e)</b> <i>713</i> and <b>(f)</b> <i>713-M3</i> are each inhibited by BKI 1213 (EC₅₀ of 0.8 μM, 1.2 μM, and 0.6 μM, respectively).</p