Diffraction data were collected on beamline 9C2 at Stanford Synchrotron Radiation Laboratory (SSRL) at a wavelength of 0.9795 ?. MISP360 structure data processing, solution, and refinement Diffraction data to 1 1.82 ? resolution were processed using Imosflm [97] and Scala [98] in the CCP4 suite of programs [99]. homologs. Expression of the different isoforms was normalized to the expression of AnTat 1.1 cultured bloodstream forms (BSF), procyclic cultured form (PCF), midgut procyclic form (PF), proventricular mesocyclic form (MSC), and metacyclic form (MCF) immunostained with anti-MISP polyclonal antibody (cyan); DAPI (magenta); merged and differential interference contrast (DIC); scale bars = 5 mm.(TIF) ppat.1011269.s007.tif (1.3M) GUID:?6D3C622C-7091-49BE-AA7A-3F2F10515DB3 S8 Fig: Expression of EP-procyclins in proventricular trypanosomes. Parasites extracted from infected PV at Ctsd 30 d.p.i. Mesocyclics (MSC), long epimastigote forms (LEMF) and short epimastigote forms (SEMF) probed with anti-EP monoclonal antibody (yellow) and DAPI (blue). Nuclei (N) and kinetoplastids (K) noted in the blue channel. Scale bars: 5 m.(TIF) ppat.1011269.s008.tif (592K) GUID:?A89AE750-CD34-43B5-B4E9-19E43A95FA09 S9 Fig: Mean relative fluorescence intensity of anti-body staining. Anti-MISP (blue) and anti-BARP (orange) staining in attached epimastigote forms (EMF) and metacyclic forms (MCF); bars represent mean values; dots represent individual cell values. error bars indicate S.D.(TIF) ppat.1011269.s009.tif (245K) GUID:?993FC5F4-049D-48F7-9387-E11042C02AAA S10 Fig: Trypanosome populations in infected salivary glands. Cell type distribution (percentage) in SG-extracted parasites used for the quantification of mean fluorescence intensities of MISP (A) and BARP (B). Metacyclic forms (MCF), pre-metacyclic forms (P-MCF), epimastigotes with variable kDNA (K) and nuclei (N) numbers (E(1K1N), E(2K1N), E(2K2N)), multi-nucleated epimastigotes (E(multi)) and abnormal cells. C, Distribution (percentage) of types of localization of MISP and BARP (D), either flagellar (flagellum) or on the whole cell surface (surface).(TIF) ppat.1011269.s010.tif (212K) GUID:?FA39731D-3B0D-46F8-BF2D-A986BCF1AF98 S11 Fig: Characterization of salivary gland forms. A, Schematic of a metacyclic and an epimastigote cell indicating the measurements in B: total cell length (red), nucleus to posterior end (orange), nucleus to kDNA (green), and kDNA to posterior end (black); anterior end (A), posterior end (P), kDNA (K), nucleus (N). B, Length measurements (mm) described in A in metacyclic (n = 30; gray) and epimastigote cells (n = 30; blue) isolated from tsetse infected salivary glands (from 3 different biological replicates); circles indicate individual data points; thick horizontal line indicates mean value; error bars indicate SD; asterisks indicate significance (*** p<0.001); ns indicates nonsignificant differences; from one-sided SG stages. Immunostaining of epimastigotes (EMF), pre-metacyclics (P-MCF) and metacyclics (MCF) from tsetse infected SG with polyclonal anti-CRD antibody. Nuclei (N) and kinetoplastids (K) noted in the magenta channel. Differential Glucokinase activator 1 interference contrast (DIC), DAPI (magenta) and anti-CRD (blue). Scale bars: 5 m.(TIF) ppat.1011269.s012.tif (696K) GUID:?0F0E385E-DA05-499D-932B-CCB607DA7A02 S13 Fig: Ectopic localization of tagged MISP360. A, DNA construct encoding for the ectopic tagged MISP360. B, Immunoblotting to detect ectopic (HA-tagged) MISP360 expressed by the mutant HA-GFPMISP360 PCF cell line, either with the transgene uninduced (-) or tet-induced (+), probed with anti-HA (top). PVDF membrane stained with nigrosine after film exposure for sample loading control. C, Cellular localization of the HA-tagged ectopic MISP360 in AnTat 1.1 90:13 procyclic (PCF) and metacyclic form (MCF), detected by immunostaining on PFA-fixed non-permeabilized PCF cells with either anti-HA plus anti-MISP (co-localization of ectopic tag with MISP), or with anti-HA plus anti-PFR (flagellar marker) in methanol-fixed cells. Scale bars: 5 m.(TIF) ppat.1011269.s013.tif (1.4M) GUID:?BE6F44D1-2D55-4909-A283-0841E191A550 S14 Glucokinase activator 1 Fig: Immunodetection of MISP in live SG trypanosomes. Representative images of live immunostaining of SG parasites using anti-MISP. Epimastigote (EMF) and metacyclic form (MCF) stained with anti-MISP (cyan) and DAPI (magenta). Nuclei (N) and kinetoplasts (K) noted in the blue channel. Scale bars: 5m. DIC: differential interference contrast.(TIF) ppat.1011269.s014.tif (442K) GUID:?4853C731-605C-4547-BA7C-555D2C8A5098 S15 Fig: Immunodetection of MISP expression in TSW196 stain trypanosome. Representative metacyclic cell (MCF) obtained from infected tsetse salivary glands immunostained with polyclonal anti-MISP antibody (cyan); DAPI (magenta); merge, and differential interference contrast (DIC); scale bar = 5 m. TSW-196 epimastigote and pre-metacyclic cells were also detected by the anti-MISP antibody. Salivary gland stages obtained from three impartial tsetse infections with TSW-196 strain were performed and successfully immunostained with anti-MISP polyclonal antibody.(TIF) ppat.1011269.s015.tif (390K) GUID:?4D872F12-3507-4678-8682-CE8674AE1CFE S16 Fig: Purification of recombinant MISP and BARP proteins. A, SDS-PAGE (Coomassie blue-stained) of SEC elution fractions of the recombinant MISP360 ectodomain (ladder displaying relative molecular weight on the left (kDa). B, Complete SDS-PAGE with fraction of rMISP360 used to determine its crystal structure. C, Size exclusion chromatogram of rBARP used for crystallography (peaks for free TRX tag and rBARP monomer indicated). D, SDS-PAGE (Coomassie-stained) of rBARP fractions.(TIF) Glucokinase activator 1 ppat.1011269.s016.tif (1.8M) GUID:?5F757A5A-339A-406D-B8F3-5B7C60CAF4F3 S17 Fig: Comparison of the crystal structure of MISP360 N-terminus with other trypanosome surface proteins identified as structural analogues. MISP360 (PDB: 5VTL), BARP (high confidence model), GARP (PDB: Y44), TcHpHbR (PDB: 4E40), TbHpHbR (PDB: 4X0J), VSG 221 monomer (PDB: 1VSG). Structures colored from blue (N-terminus) to red (C-terminus); molecular surface in semi-transparent grey.(TIF) ppat.1011269.s017.tif (1.1M) GUID:?733F9329-243C-4A5C-8445-25A3AA7B3E4C S18 Fig: Suggested models of MISP C-terminal domains. A, Height comparison between the.