exp1 gene  (Kamada)

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) Simplified schematic of DiCre-based conditional exp1 KO using SLI. Arrows indicate primers P1 and P2 (see for details). (B) IFA of compound 2-arrested condΔEXP1 late-stage schizonts show localization of EXP1*-HA at the PVM. α-MSP1 (MSP1) labels the PPM. Nuclei were stained with DAPI; scale bars, 5 μm. (C) Strategy for depletion of EXP1 from the PVM of synchronized condΔEXP1 ring stages divided into a culture with rapalog (“rap”) and one without rapalog (“control”). Top: schematic: green boxes and green line signify PVM with EXP1. Mid: PCR with primers P1 and P2 from gDNA 24 and 48 hours after addition of rapalog. Original: band for intact exp1 locus (1,919 bp); excised: band after excision of exp1* (1,326 bp). Bottom: western blot probed with α-HA to detect EXP1*-HA and α-BIP as loading control. Asterisk: unskipped (first T2A) product that is present before excision and hence has no impact on parasites. (D) Quantification of levels of wtEXP1-HA in the first ("24 h") and second ("48 h") cycle after addition of rapalog based on densitometric analysis of anti-HA immunoblots (mean of n = 2 independent experiments) of which one is shown in (C). HA signal was normalized to BIP. Error bars indicate SD. (E) FC growth curves of synchronous ring stage condΔEXP1 parasites starting in cycle 2 (ΔEXP1 parasites) as shown in (C). One representative of n = 5 independent experiments. (F) Stage distribution of condΔEXP1 parasites in Giemsa smears of synchronous parasites grown with (+) and one without (−) rapalog (“rap”) at different time points (average time post invasion) after adding rapalog. Light blue arrowheads show blebs. One of n = 4 independent experiments is shown. Blue arrow in (C–F) indicates start of a new cycle without EXP1. 2A, T2A skip peptide; BIP, binding immunoglobulin protein; DIC, differential interference contrast; DiCre, Dimerizable Cre; EXP1, exported protein 1; EXP1*, recodonized exp1 ; FC, flow cytometry; gDNA, genomic DNA; HA, triple hemagglutinin tag; KO, knockout; MSP1, merozoite surface protein 1; PPM, parasite plasma membrane; PVM, parasitophorous vacuolar membrane; SLI, selection-linked integration; SP, signal peptide; TM, transmembrane domain; wt, wild-type.

Article Snippet: A codon changed exp1 gene was synthesized (Genscript) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment.

Techniques: Staining, Control, Western Blot, Binding Assay, Flow Cytometry, Knock-Out, Clinical Proteomics, Membrane, Selection

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) Time-lapse imaging of condΔEXP1 parasites starting a cycle with (control) and without EXP1 (rapalog) imaged side by side. Single DIC z-sections of selected time points of typical phenotypes are shown: top row: slow development; mid row: failure to reach full trophozoite stage; bottom row: cell showing extensive hugging. Arrowheads: white, ring stages after reinvasion; blue, amoeboid ring; light blue, blebs; red, hugging. (B) Number of hours (after start of the time lapse experiment) to reach young trophozoite stage in n = 24 control and N = 27 ΔEXP1 parasites. (C) Frequency of the indicated events in individual ring stages observed by time lapse microscopy (presence or absence of event was scored every hour); n = 25 parasites each for ΔEXP1 and control. (D, E) Left, live cell images of control and ΔEXP1 (rapalog) parasites expressing Lyn-mCherry (PPM marker) (D) or labelled with Bodipy TR ceramide (BODIPY) (E). Light blue arrowheads, blebs; yellow arrowheads, TVN. Graphs: quantification of number of blebs per cell (controls N = 25, 73 for (D) and (E), respectively; ΔEXP1 (rapalog) n = 28 and 156 for (D) and (E), respectively. (F) Left, IFA of control and ΔEXP1 (rapalog) late-stage gametocytes using α-HA to detect EXP1*-HA and α-Pfg377 (late-stage gametocyte marker). Right, PCR using primers P1 and P2 confirms excision of exp1 in late gametocytes. (G) Left, gametocytemia of control and ΔEXP1 parasites early after induction (Pfs16 positive cells) and 8 days later (Pfg377 positive cells) based on IFAs. Right, fold reduction in the number of early (Pfs16) and late (Pfg377) ΔEXP1 gametocytes versus control; n = 3. (D, E, and F); scale bars, 5 μm. In (F), nuclei were stained with DAPI. In (B–E and G), green lines indicate mean and error bars (SD); two-tailed unpaired t test, P values indicated. DIC, differential interference contrast; EXP1, exported protein 1; HA, triple hemagglutinin tag; IFA, immunofluorescence assay; PPM, parasite plasma membrane.

Article Snippet: A codon changed exp1 gene was synthesized (Genscript) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment.

Techniques: Imaging, Control, Time-lapse Microscopy, Expressing, Marker, Staining, Two Tailed Test, Immunofluorescence, Clinical Proteomics, Membrane

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) IFA images of condΔEXP1 schizont stages expressing EXP1 wt-Ty probed with α-HA and α-Ty show localization of EXP1*-HA and EXP1 wt-Ty in the PVM. α-MSP1 (MSP1) labels the PPM. DAPI, nuclei; scale bars, 5 μm. Right: immunoblot of extracts of cell line on the left probed with α-Ty to detect EXP1wt-Ty and α-SBP1 as control for a TM protein. Saponin was used to separate the parasite pellet (“P”) from the supernatant (“SN”) containing PV and host cell content. See S3 Fig for IFAs and immunoblots of all complementation constructs. (B) Relative activity of the EXP1 complementation constructs indicated. Except where otherwise indicated, constructs were expressed under the nmd3 (mid) promoter. The complementation capacity of every tested construct was calculated as relative activity to the EXP1wt-Ty construct under the nmd3 promoter, which was set as 100% (right dotted green line). ΔEXP1 was set to 0% (left dotted green line). Each data point (red dot) shows growth of rapalog-treated versus unexcised parasites at the end of a 5-day growth assay relative to the growth of the wt construct; n ≥ 4 independent experiments per cell line. Error bars: SD. See for activity of all complementation constructs. (C) Immunoblot of lysates of condΔEXP1 parasites expressing EXP1wt low , EXP1 mid , and EXP1 high probed with α-Ty (EXP1wt-Ty), α-HA (EXP1*-HA), and α-BIP (loading control). (D) Densitometric analysis of EXP1wt expression levels (C) under low, mid, and high promoters relative to the mid promoter (green). Mean of 3 independent experiments. Error bars: SD. (E) Immunoblot of extracts of +/−formaldehyde (PFA) treated cell lines expressing the indicated constructs probed with α-Ty. Single asterisk: monomer; double asterisk: dimer. BIP, binding immunoglobulin protein; DIC, differential interference contrast; EXP1, exported protein 1; HA, triple hemagglutinin tag; IFA, immunofluorescence assay; MSP1, merozoite surface protein 1; PPM, parasite plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuolar membrane; SBP1, skeleton binding protein 1; TM, transmembrane; wt, wild type.

Article Snippet: A codon changed exp1 gene was synthesized (Genscript) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment.

Techniques: Expressing, Western Blot, Control, Construct, Activity Assay, Growth Assay, Binding Assay, Immunofluorescence, Clinical Proteomics, Membrane

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) Schematic of the region of EXP1 with the proposed catalytic site of the GST activity and of the mutations introduced. (B) Relative activity of the EXP1 complementation constructs indicated. Green lines: activity of EXP1wt ( nmd3 , mid) (set as 100%) and absence of activity (ΔEXP1) set as 0%; n ≥ 4 independent experiments per cell line. (C) Live cell images of condΔEXP1 parasites incubated with CM-H 2 DCFDA. Scale bars: 5 μm. (D) Fluorescence intensity of matching stages of control and ΔEXP1 parasites (rapalog) incubated with CM-H 2 DCFDA (C). Results from 3 independent experiments with a total of n = 55 control and n = 53 ΔEXP1 cells. Green line, mean; error bars, SD. (E) FC growth curves of synchronous condΔEXP1 parasites and the complementation cell line EXP1 wt low after one cycle with (red) and without (black) rapalog (see ) grown in RPMI alone or supplemented with the compounds indicated. One representative of n = 3 independent biological replicas. (F) Left, growth curves of EXP1wt low parasites ± E64 starting after one growth cycle ± rapalog (see ). Right, Giemsa smears of parasites on day 2 (after incubation with E64) and day 3 (after removal). Arrowheads: swollen food vacuole. One representative of n = 3 biological replicas. (G) Effect of E64 treatment on survival of DHA and rapalog-treated EXP1wt low parasites versus untreated. Mean of n = 3 independent experiments. (H) Left, dose-response curves of the parasite lines indicated treated with DHA (0–50 nM). Right, DHA IC 50 values for these cell lines ± rapalog. Mean of n ≥ 3 experiments. (I) RSAs of the indicated complementation cell lines and a Kelch13 C580Y mutant line. Data points are percent survival of DHA treated versus untreated parasites ± rapalog. Mean of n ≥ 3 per cell line. (D, G, and H), two-tailed unpaired t test; P values indicated; (B, D, and G), error bars, SD. CM-H 2 DCFDA,; DHA, dihydroartemisinin; DIC, differential interference contrast; EXP1, exported protein 1; FC, flow cytometry; GST, glutathione S-transferase; IC 50 , half maximal inhibitory concentration; RPMI, Roswell Park Memorial Institute; RSA, ring-stage survival assay; wt, wild type.

Article Snippet: A codon changed exp1 gene was synthesized (Genscript) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment.

Techniques: Activity Assay, Construct, Incubation, Fluorescence, Control, Mutagenesis, Two Tailed Test, Flow Cytometry, Concentration Assay, Clonogenic Cell Survival Assay

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) IFA images of control and ΔEXP1 parasites (rapalog) probed with α-HA (EXP1*-HA) and α-KAHRP. Graph to the right shows quantification of export ( n = 20 cells). See for other exported proteins. (B) Live cell imaging of control and ΔEXP1 parasites (rapalog) expressing SBP1-mScarlet. Graph to the right shows quantification of export ( n = 20 cells). (C) Live cell images of a ΔEXP1 trophozoite expressing SP-mScarlet liberated from its host RBC. (D) Left, current recorded from liberated control and ΔEXP1 parasites (rapalog) at 30 mV applied potential to the pipette electrode. Scale bar shows time in seconds and current in pA. The dotted line indicates a current reference level. At 30 mV, the PVM channels have an open probably of about one-half, therefore the flicker is offset in the control example as multiple channels are in the recording. The shown recordings are representative of the experiments done in each condition and show 1-second details from longer recordings to resolve the typical channel flicker in print. Right, probability of detecting at least one PVM channel per sealed patch (f chan ) in ΔEXP1 parasites (rapalog, n = 14) and controls ( n = 12). Fischer's exact test was used to estimate P value. Error bars indicate SD. In (A) and (B), nuclei were stained with DAPI; scale bars: 5 μm. DIC, differential interference contrast; EXP1, exported protein 1; HA, triple hemagglutinin tag; KAHRP, knob-associated histidine-rich protein; pA, Picoampere; PSAC, parasite surface anion channel; PVM, parasitophorous vacuolar membrane; RBC, red blood cell; SBP1, skeleton binding protein 1.

Article Snippet: A codon changed exp1 gene was synthesized (Genscript) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment.

Techniques: Control, Live Cell Imaging, Expressing, Transferring, Staining, Membrane, Binding Assay

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) Live cell images of ΔEXP1 (rapalog) and control trophozoites expressing EXP2-GFP. Light blue arrowhead, loop-like protrusions. (B) Quantification of the phenotype of the cells from (A). Green, signal around the parasite; black, aberrant distribution of signal. Mean of a total of n = 109 control and n = 99 ΔEXP1 cells derived from 4 biological replicas. (C) Live cell images of Bodipy-TR-ceramide labelled ΔEXP1 (rapalog) and control parasites expressing EXP2-GFP. (D) IFA images of ΔEXP1 (rapalog) and control parasites probed with α-HA to detect EXP1*-HA and α-EXP2 for endogenous EXP2. (E) Quantification of the phenotype of the cells from (D). Red, signal around the parasite; black, aberrant distribution of signal. Mean of a total of N = 108 control and N = 135 ΔEXP1 cells derived from 3 biological replicas. (F–H) IFA images of ΔEXP1 (rapalog) and control ring stages (F) or trophozoites (G, H) probed with α-HA (EXP1*-HA) and α-ETRAMP10.1 (F), α-GFP (EXP2-GFP) with α-ETRAMP4 (G), or with α-ETRAMP5 (H). (I) IFA images of ΔEXP2 (rapalog) and control trophozoites expressing EXP1-Ty probed with α-Ty and α-HA to detect EXP1-Ty and EXP2-HA, respectively. (J) Quantification of phenotypes of the parasites shown in (I). Red, signal around the parasite; black, aberrant distribution of signal. Mean of a total of n = 86 control and n = 110 ΔEXP1 cells derived from 3 biological replicas. (K) IFA images of ΔEXP2 (rapalog) and control parasites probed with α-HA and α-ETRAMP5. In (I) and (K), α-HA detects full (control) or truncated (rapalog) EXP2-HA. In (A, C, D, F–I, K), scale bar: 5 μm. DAPI, nuclei. (l) Western blot of a co-IP experiment in the cell line condΔEXP1 expressing EXP2-GFP (IP of EXP1*-HA with α-HA). α-HA detects EXP1*-HA (monomer: asterisk; dimer: double asterisk); α-GFP, EXP2-GFP (arrowhead); α-SERP, soluble PV protein; α-ETRAMP4, integral PVM protein; α-aldolase, cytosolic parasite protein. Input (I): total lysate before IP; post IP lysate (PI); Eluate (E). One representative of n = 3 independent biological replicas. In (B, E, and J), P values were calculated with a Fischer’s exact test. P < 0.05, significant. ETRAMP, early transcribed membrane protein; EXP1, exported protein 1; GFP, green fluorescent protein; HA, triple hemagglutinin tag; IFA, immunofluorescence assay; IP, immunoprecipitation; PV, parsitophorous vacuole; PVM, parasitophorous vacuolar membrane; SERP, serine-rich antigen also known as serine repeat antigen 5 (SERA5).

Article Snippet: A codon changed exp1 gene was synthesized (Genscript) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment.

Techniques: Control, Expressing, Derivative Assay, Western Blot, Co-Immunoprecipitation Assay, Membrane, Immunofluorescence, Immunoprecipitation

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) Live cell images of control and ΔEXP1 parasites (rapalog) incubated with 5-ALA. (B) Quantification of PPIX fluorescence in control and ΔEXP1 parasites (rapalog) using FC. Mean (green line) of n = 11 independent experiments. Error bars indicate SD. (C) Stage distribution of tightly synchronous parasite cell lines 24 h.p.i. (after an initial cycle ± rapalog) grown in amino acid–limited and complete medium. Mean of n = 3 independent experiments. (D) Percentage of rings at different time points post invasion (after 1 cycle ± rapalog) of condΔEXP1 and EXP1wt low parasites grown in amino acid–limited and complete medium. Mean of n = 3 independent experiments. (E) Growth on day 4 (after 2 cycles) of the indicated parasite lines and condition (±rapalog) in the presence of azide in proportion to growth of the same parasites in medium without azide (left, NaN 3 versus no NaN 3 ) or growth in amino acid–limited medium in proportion to the same parasites grown in complete medium (right, limiting versus complete medium). Growth of the control culture (medium without NaN 3 or complete medium) was set as 100%. Rapalog was added 1 cycle prior to the growth test to start with the corresponding KO parasites. Green line indicates mean of at least n = 5 independent experiments. In (B, C, and E), two-tailed unpaired t test, P values are indicated. 5-ALA, 5-aminolevulinic acid; DIC, differential interference contrast; EXP1, exported protein 1; FC, flow cytometry; h.p.i., hours post invasion; KO, knockout; PPIX, protoporphyrin IX; rapa, rapalog; wt, wild type.

Article Snippet: A codon changed exp1 gene was synthesized (Genscript) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment.

Techniques: Control, Incubation, Fluorescence, Two Tailed Test, Flow Cytometry, Knock-Out

Journal: PLoS Biology

Article Title: EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

doi: 10.1371/journal.pbio.3000473

Figure Lengend Snippet: (A) Transcript levels of EXP1, EXP2, and the PTEX components HSP101 and PTEX150 across the asexual intra-erythrocytic cycle. Values were obtained from a previous publication . (B) Schematics of the PVM of wild-type (left) and ΔEXP1 parasites (right) during the ring (upper panel) and trophozoite stage (lower panel). Depicted molecules are explained in the box. Note that early expressed EXP2 is in light green and late expressed EXP2 is in dark green. In wild-type rings, EXP2 is predominantly associated with PTEX components to promote protein export, a major function of this stage. Less EXP2 is in the nutrient-permeable channel complex that depends on EXP1 but does not contain other PTEX components. The proportion of the abundance of the two EXP2 complexes is reversed in the trophozoite stage when most of protein export has been completed but rapid parasite growth demands more nutrients. PTEX150 and HSP101 are not expressed in this stage, but PTEX translocons left over from the ring stage make up a minor proportion of EXP2 complexes to maintain protein export activity. The nutrient-permeable channel function of EXP2 is negatively affected by loss of EXP1 either through indirect effects (indicated as aggregated EXP2, left part of PVM enlargement of ΔEXP1 KO) or a direct defect of the complex (“defective” nutrient-permeable channel, right part of ΔEXP1 KO). EXP1, exported protein 1; FPKM, fragments per kilobase of exon model per million mapped reads; h.p.i., hours post invasion; HSP101, heat shock protein 101; KO, knockout; PTEX, Plasmodium translocon of exported proteins; PVM, parasitophorous vacuolar membrane.

Article Snippet: A codon changed exp1 gene was synthesized (Genscript) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment.

Techniques: Activity Assay, Knock-Out, Membrane