Animal work statement

Experiments were performed strictly according to the Swiss Act on animal protection (TSchG; Animal Rights Laws). They were approved by the animal experimentation commission of the canton Bern (Permit Numbers BE86/19 and BE118/22). The research staff received their training at Certified education and training programs from the Federal European Laboratory Animal Science Associations (FELASA). The mice (C57BL/6 and BALB/c) were between six and ten weeks old and were either bred in-house or supplied from Janvier Labs (France). The mice were monitored daily in the mice facility of the Institute of Cell Biology at the University of Bern.

Transgenic eBFP2 and TagBFP2 parasite lines

To generate the BFP-reporter cassettes, the previously generated mCherry-reporter cassette pL1694 was used [29, 30]. The pL1694 construct contains the 5’hsp70-mCherry-3’hsp70 cassette, which allows mCherry expression under the heat shock protein 70 (Hsp70) promoter. The mCherry was removed using BamH1/Not1 and replaced by polymerase chain reaction (PCR)-amplified eBFP2 to generate the 5’hsp70-eBFP2-3’hsp70 cassette. The same approach was used to generate the 5’hsp70-mTagBFP2-3’hsp70 cassette. The eBFP2 was amplified from the pEBFP2-Nuc (Addgene #14893), and mTagBFP2 from pHAGE-BL-mTagBFP2-SopF from Richard Youle Laboratory [31]. The eBFP2 and mTagBFP2 were amplified using primers as indicated in Table 2 and subcloned into pJet1.2/blunt. The vectors were transfected into the GIMO (Gene Insertion/Marker Out) ANKA parasite mother line (GIMO_PbANKA,1596cl1, RMgm-687) as described [29].

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Table 2. List of the primers.

https://doi.org/10.1371/journal.pone.0308055.t002

Transgenic parasites transfection and selection

To transfect the eBFP2 and mTagBFP2 vectors into the GIMO ANKA parasite line, a schizont culture approach was used as previously described [29, 32–34]. A mouse was pre-infected intraperitoneally (IP) with the P. berghei GIMO mother line. The blood of the infected mouse was intravenously (IV) passaged three days later into a new naive mouse. When the passaged mouse’s parasitemia reached 1–3%, the schizont culture was prepared. For every transfection, a schizont culture medium was produced using 50 mL of Roswell Park Memorial Institute medium (RPMI1640), 12.5 mL of fetal calf serum (FCS), and 100 μL of Gentamycin. After two to three hours of incubation at 36.5°C, the infected mouse was euthanized with CO2, and a heart puncture was used to collect the blood. Subsequently, 2.5 mL of the schizont culture were transferred into a Falcon tube. The blood-medium suspension was centrifuged for 8 minutes at 450 g. The supernatant was removed under the hood, and the pellet was resuspended with 2 mL of warm Schizont culture medium. The suspension was then transferred carefully and slowly into a schizont culture flask with 5% CO₂, 5% O₂ and 90% N₂ for 90 seconds. The culture was then incubated with slow rotation overnight at 36.5°C, as described previously [35].

The next day, 10 mL of a 60% Nycodenz-PBS mixture (27.7 mL Nycodenz and 22.5 mL 1 x PBS) were carefully pipetted below the schizont culture. The Falcon tubes were then centrifuged for 20 minutes at 450 g without acceleration or breaks. After centrifugation, a gradient was observed (top layer: schizont medium, middle layer: schizonts, lower layer: Nycodenz-PBS mix, bottom: pellet of RBCs and other parasite stages). Using a pipette, the schizont layer was carefully removed and transferred into a new Falcon tube. After an additional centrifugation at 11 000 g without acceleration or breaks, the cells were resuspended in transfection mix composed of 5 μg of digested DNA construct (5’hsp70-eBFP2-3’hsp70 and 5’hsp70-mTagBFP-3’hsp70) were filled up to 100 μL with Nucleofector transfection buffer from the Amaxa® Human T cell Nucleofector® Kit. The solution was pipetted into an electroporation cuvette and placed into the Amaxa Nucleofector device. After electroporation using the U33 program, the solution was immediately transferred to an Eppendorf tube containing 100 μL of RPMI1640 without serum. 200 μL of the transfected schizonts were IV injected into naive mice.

The BFP constructs were integrated into the silent 230P genomic locus of the modified P. berghei GIMO mother line. The 230P locus contains a fusion gene of the following selectable markers: human dihydrofolate reductase, yeast cytosine deaminase, and uridyl phosphoribosyl transferase (hdhfr::yfcu). Double-crossover homologous recombination was used to replace the selectable marker cassettes at the target locations to introduce the BFP-containing constructs. Negative selection was then carried out a day after the transfection with 5-fluorocytosine (Abcam, ab141197) in drinking water to select for the BFP transgenic parasites that have the BFP-reporter cassette introduced into the 230p locus and the hdhfr::yfcu marker removed (S1 Fig). The correct integration of the BFP constructs into the P. berghei genome was further confirmed by integration PCR (S1 Fig, S1 Raw images). The primers used and their specific binding positions are listed in Table 2 and S1 Fig.

Isolation of eBFP2 transgenic line

To generate the transgenic eBFP2 parasite line, a limiting dilution was carried out as described [36, 37]. Briefly, a naive mouse was pre-infected with the transfected BFP P. berghei parasite lines. When the parasitemia reached 0.2–1%, around 2 μL of blood was collected from the tail vein of the pre-infected mouse and put into an Eppendorf tube with 1 mL of RPMI1640. The tube was gently inverted several times, and the blood mixture was transferred to a Falcon tube with 9 mL of RPMI 1640. Afterwards, 10 μL were taken from the solution, and the erythrocytes were counted with a Neubauer cell counting chamber. The volume and the dilution factor for limiting injection were determined by calculating the average number of erythrocytes per blood parasite. A dilution of 1:100 in RPMI1640 was made. Per mouse, 200 μL of the final dilution was IV-injected using a 31G insulin syringe. A total of ten pre-warmed naive mice were infected, each with an average of one single parasite. The parasitemia of the infected mice was checked every day for seven days post-infection. The positive mice were euthanized with CO₂, and the blood was collected by heart puncture, from which the genomic DNA was isolated, and subsequently, the parasite lines were confirmed by integration PCR.

Oocyst size and volume measurements

To assess the performance of the transgenic BFP parasite lines in the sexual mosquito stage, naive mice were pre-infected and passaged as described above. Three days post-passage, the parasitemia was checked, and when the gametocytes reached 0.75 to 1%, the infected mice were anaesthetized and used to feed Anopheles stephensi mosquitoes as previously described [38, 39]. At days 5, 7, 11, and 14 after feeding, ten mosquitoes were dissected and their midguts were collected in 1 x PBS, placed on a microscopy slide coated with 21-wells, and covered with a coverslip. Subsequently, they were imaged using the 2.5x and 63x oil objectives on a Leica DM6000B, equipped with an EL6000 lamp. The number of oocysts was accessed using the find maxima plugin in Fiji, and the oocyst volumes were calculated from area measurements in Fiji using the formula V = 4/3πr³, where V represents the volume and r is the radius [40]. The experiments were carried out in triplicate for each of the parasite lines.

Sporozoite motility assay

The sporozoite motility was measured 18 days post-feeding when the sporozoites were supposed to be released from the oocysts into the salivary glands of the infected mosquitoes as previously described [41]. In total, ten mosquitoes were dissected, and the salivary glands were collected in Iscove’s Modified Dulbecco’s Media (IMDM). The sporozoites got activated with Minimum Essential Media (MEM) (BioConcept, 1-31F01-I) supplemented with Earle’s salts and 10% heat-inactivated fetal calf serum (FCS, GE Healthcare), referred to as the completed medium. This solution was centrifuged for 5 min at 11’000 g, the supernatant was discarded, and the pellet was resuspended with 40 μL MEM. 20 μL of this solution were transferred into a 96-well glass bottom plate (Cellvis). The plate was spun down for 5 minutes at 200 g and finally incubated for 10 min at 37°C before being imaged using an inverted Leica DM5500B microscope equipped with an EL6000 lamp. Maximum-intensity z-projections were used to visualize the sporozoite motility pathways in Fiji. Furthermore, the sporozoite velocity was measured using the MTrack2 plugin from Nico Stuurman [40, 42]. After processing the image sequence into a binary image, this plugin measured the sporozoite speed by tracking the sporozoite tip frame by frame to calculate the approximate trajectory during a given time range.

Immunofluorescence assay (IFA) of fixed HeLa cells

To access the development of the BFP-expressing parasites in the mammalian cells, 40’000 HeLa cells were seeded into a 24-well plate on a coverslip containing MEM-completed medium. The next day, the cells were infected with 20’000 sporozoites. At specific hours post-infection (6 hpi, 24 hpi, 30 hpi, 48 hpi, and 56 hpi), the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature as previously described [43]. After two washes with 1 x PBS, the cells were blocked with 10% FCS in 1 x PBS for 20 min at room temperature. Subsequently, the cells were incubated with the first antibodies diluted in 10% FCS for 1 hour at room temperature. The primary antibodies used for the cells infected with the BFP-expressing parasites were Rabbit anti-UIS4 (1:1’000, P-Sinnis, Baltimore) and Mouse anti-LAMP1 (1:1’000, Developmental Hybridoma Bank, clone H4A3, 1:1’000) while Rabbit anti-LAMP1 (1:1000, Cell Signaling, CS9091P) and Chicken anti-UIS4 (1:500, Proteogenix) were used for the cell infected with the mCherry-expressing parasites. After another washing step, the secondary antibodies, anti-Rabbit Cy5 (1:1’000, Jackson Immuno Research), anti-Mouse Alexa 594 (1:1’000, Invitrogen Molecular Probes, A11032), and anti-chicken Cy5 (1:1’000, Jackson Immuno Research) and anti-rabbit Alexa 350 (1:1’000, Invitrogen Molecular Probes, A11069), were added to the BFP and mCherry expressing parasite-infected cells, respectively. Single-stain controls have been performed to ensure that bleed-through was negligible. Incubation was carried out protected from light for 1 hour before staining the actin polymerization with phalloidin Alexa Fluor 488 (1:1’000, Invitrogen Molecular Probes, A123779). Finally, the coverslips were mounted with 5 μL ProLong™ Gold Antifade Mounting (Invitrogen, P36930) and imaged using the 60X objective of a Nikon Crest V3 spinning disc microscope in widefield mode.

The host lysosome interaction with the parasite PVM was determined using the 3D and 4D image analysis software Imaris (Imaris X64, 9.9.0 [March 11, 2022]). In order to measure the number of lysosomes attached to the parasite PVM (0 to 0.5 μm) in infected HeLa cells, the PVM stained with anti-UIS4 was used to compute the isosurface, and the lysosomal vesicles stained with anti-LAMP1 were used to compute isospots as previously described [38].

Parasite size measurement and detached cell formation

The parasite size in the HeLa cells was measured as previously described [2, 44]. Briefly, 40’000 HeLa cells per well were seeded in triplicate in 96-well plates. The next day, each replicate was infected with 20’000 sporozoites from the different parasite lines. After 2 hours of incubation at 37°C, the cells from one well were seeded into 8 wells of two 96-well plates (i.e., 4 wells per plate). At specific time points post-infection (6 hpi, 24 hpi, 48 hpi, and 56 hpi), one 96-well plate was sequentially imaged with the 10x objective of an InCell Analyzer 2000 microscope. Analysis using the InCell Developer tool allowed individual parasites to be separated from the background by defining parameters such as kernel size, sensitivity, and the range for parasite size. Based on the size of each parasite, parasite growth patterns were measured. The absolute number of parasites at specific time points was used to calculate the percentage of parasite survival over time (6 hpi = 100%). To measure the formation of detached cells, they (together with thousands of merozoites inside of them) were harvested 65 hpi from the second plate by gently pipetting the supernatant of each well up and down three times. Finally, the solution containing the detached cells was transferred to another 96-well plate, and only the mature detached cells were counted under the microscope. The number of detached cells per triplicate was divided by the number of parasites present at 48 hpi to obtain the detached cell formation rate in percentage.

Live cell imaging (movie) of the parasite development in HeLa cells

For live cell imaging of parasite development in HeLa cells, the cells were infected as described above. After 2 h of incubation at 37°C, they were reseeded on a dark 24-well glass bottom plate (Cellvis) to image the cells under a 40X objective of Nikon W1 LIPSI spinning disk microscope or 63X objective of a DMI 6000B Leica inverted microscope. Using the “Mark and find” function of the LEICA software, each invaded parasite could be marked and pictured every hour to finally simulate a movie showing the parasite’s development within the living HeLa cell over time, as described previously [19].

Flow cytometry

To measure the parasitemia ratio of the infected mice, a few drops (around 2 μl) of blood from the tail vein of the mouse were added to 500 μL of 1 x PBS and measured by flow cytometry using the MoFlo ASTRIOS EQ as previously described [45]. mCherry (Excitation 561–614) and BFP (Excitation 405–450) lasers were used to respectively select the mCherry and BFP transgenic expressing parasites in infected erythrocytes. The data were analyzed using the NanoSight NS300 and FCS Express Flow Cytometry Analysis Software (version 7.22.0006).

Data analysis

The data were analyzed with GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, USA (www.graphpad.com). The experiments were carried out in triplicate. The statistical comparison tests were carried out using one-way ANOVA with Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). The microscope images were analyzed using FIJI software. When specified, the images were deconvolved with Huygens Professional version [Huygens Remote Manager v3.8] (Scientific Volume Imaging, The Netherlands).

Generation of the eBFP2 and mTagBFP2 transgenic parasite lines

To generate the two cytosolic BFP-expressing P. berghei parasite lines, the eBFP2 and mTagBFP2-reporter cassettes were individually transfected into the GIMO acceptor 1596 parasite mother line [29, 30]. In this process, constructs containing the 5’hsp70-eBFP2-3’hsp70 and 5’hsp70-mTagBFP2-3’hsp70 cassettes were integrated via double-crossover homologous recombination into the modified P. berghei 230p locus, which already expressed the hdhfr:yfcu selectable marker cassette [34]. This selectable maker cassette facilitated enrichment of the PbeBFP2- or PbmTagBFP2-transfected parasites by administering the pro-drug 5-fluorocytosine as a negative selection agent (S1A Fig). The PbmTagBFP2-infected erythrocytes were selectively isolated from a mixed population by FACS (S1B Fig). Conversely, the PbeBFP2 transgenic parasite lines were isolated through limiting dilution (S1C Fig). Out of ten initially infected mice, six tested positive, and integration PCR confirmed the transgenic populations for four out of these six mice. Integration PCRs targeting the 5’ and 3’ UTR as well as the entire gene construct were also performed on the mTagBFP2 transgenic parasite line, verifying the correct integration of the constructs in the genome (S1D and S1E Fig). Following the successful generation of these two transgenic reporter lines, we verified the BFP visibility during blood stage development. In both parasite lines the BFP was clearly visible by fluorescence microscopy in all stages in blood: ring (R), trophozoite (T), gametocyte (G), and schizont (Sc) stages (Fig 1A and 1B). The mean fluorescent intensity of the BFP signals were compared by flow cytometry (Fig 1C and 1D) and revealed that mTagBFP was about 4.5 times stronger than in eBFP2 parasites (Fig 1E and 1F).

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Fig 1.

mTagBFP2 and eBFP2 transgenic parasite were visible during blood stage development. (A) and (B) display widefield microscope images showing the development of the PbmTagBFP2 and PbeBFP2 parasite lines in erythrocytes, highlighting ring (R), trophozoite (T), gametocyte (G), and schizont (Sc) stages of the BFP-expressing parasites. Scale bars = 10 μm. (C) Flow cytometry profile of PbmTagBFP2-infected mice indicating a parasitemia of 0.19%. (D) Flow cytometry profile of PbeBFP2-infected mice showing a parasitemia of 0.34%. (E) and (F) depict the intensity histograms for PbmTagBFP2 and PbeBFP2, respectively. The mean fluorescence intensity for PbmTagBFP2 is 1948.05 a.u., while for PbeBFP2 it is 442.29 a.u.

https://doi.org/10.1371/journal.pone.0308055.g001

Investigation of the PbeBFP2 and PbmTagBFP2 reporter parasite lines development within the mosquito midgut

The goal of this study was to create BFP-expressing parasite lines capable of normal development throughout their entire life cycle, spanning from the sexual stage in mosquitoes to the asexual stages in the liver and blood. To achieve this, both BFP-expressing parasite lines and as a control mCherry-expressing parasites [30] were fed to female Anopheles mosquitoes. To assess parasite development within the mosquito midgut at 7 and 14 days post-feeding, ten midguts were dissected and examined using low magnification (2.5X objective) widefield microscopy (Fig 2A and 2B), and the oocyst number was quantified at 7days post-feeding (Fig 2D). The PbeBFP2 and PbmTagBFP2 reporter parasite lines exhibited oocyst counts comparable to the PbmCherry control parasites. Typically, Plasmodium oocysts are oval or spherical [46]. To determine if BFP-expressing parasites maintained this characteristic oocyst form, we analyzed oocyst morphology at 11 days post-feeding (Fig 2C). Additionally, the volume of individual oocysts was measured (Fig 2E). No significant differences were observed in the size or morphology of oocysts between the control and the BFP-expressing parasites. The intensity of the BFP reporter expression under the Hsp70 promoter within the mosquito midgut was also evaluated by measuring the pixel intensity of oocysts intensities of both BFP-expressing parasites (Fig 2F). The PbmTagBFP2 oocysts displayed the brightest fluorescent signals compared to those of PbeBFP2 at 7 and 11 post-feeding (Fig 2F). Despite this, these findings confirm the normal progression of the BFP-expressing parasites throughout the mosquito stage.

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Fig 2.

mTagBFP2 and eBFP2 transgenic parasite development in the mosquito midgut. (A) and (B) display images of mosquito midguts containing oocysts expressing BFP or mCherry 7 and 14 days post-feed (acquired using a 2.5x widefield microscope objective). The images of mosquito midguts containing oocysts in (C) were imaged eleven days post-feed with a 63x objective widefield microscope. (D) At day 7 post-feed, the total number of oocysts per midgut was computed using the “find maxima” plugin in Fiji. The mean oocyst number in triplicate was 680, 560, and 670, respectively, for the PbmTagBFP2, PbeBFP2, and PbmCherry parasite lines, with no significant differences. (E) shows the volume of the oocysts at eleven days post-feed. The average oocyst volume was between 11000 and 13000 μm³ for all the parasite lines, with no significant differences. (F) represents the pixel intensity (a.u., arbitrary units) of the oocysts at seven and eleven days post-feeding. At day 7 post-feed, pixel intensity averages were 51 and 13.3, respectively, for the PbmTagBFP2 and PbeBFP2, and 61.2 and 47.7, respectively, for the PbmTagBFP2, and PbeBFP2 at day 14 post-feed. (D), (E), and (F) were color-coded: blue = PbmTagBFP; cyan = PbeBFP2; and red = PbmCherry. The comparisons of oocyst number, volume, and pixel intensity between the parasite lines were conducted using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Image scale bars: (A) and (B) = 500 μm; (C) = 10 μm.

https://doi.org/10.1371/journal.pone.0308055.g002

Quantification of the PbeBFP2 and PbmTagBFP2 sporozoite motility and exo-erythrocytic form development

During Plasmodium development, liver infection is a critical bottleneck in the parasite’s life cycle [47]. The successful invasion of hepatocytes is dependent on the gliding motility of the sporozoites [48, 49]. To assess the motility of the sporozoites from the newly generated parasite lines, salivary gland sporozoites (day 18 post-feeding) from infected mosquitoes were placed on glass bottom dishes and imaged by wide-field microscopy. Both the movement and velocity of the sporozoites were quantified using live cell imaging (S1–S3 Movies). The BFP-expressing parasite lines exhibited a normal circular movement pattern similar to that observed in the PbmCherry parasites (Fig 3A, S1 to S3 Movies). The average velocity recorded was approximately 1.5 μm/s (Fig 3B) and is consistent with previous findings [48–51]. Following the assessment of gliding motility, the development of the parasites into exo-erythrocytic forms within HeLa cells was evaluated at multiple post-infection time points (6, 24, 30, 48, and 56 hpi). After fixing the cells, an immunofluorescence assay (IFA) was conducted by staining polymerized host cell actin with phalloidin to identify and delineate the border of individual cells. A hallmark of normal infection, the formation of a parasitophorous vacuole membrane (PVM), was visualized using IFA with antibodies targeting the parasite protein upregulated in infective sporozoite gene 4 (UIS4). As depicted in Fig 3C–3E, both BFP-expressing parasite lines demonstrated prominent UIS4 staining, akin to the mCherry control line, indicating typical infection patterns. Furthermore, live cell imaging of infected HeLa cells demonstrated normal liver stage development of the parasites (S4–S8 Movies). To evaluate the expression level of the two BFP reporters under the Hsp70 promoter during liver stage development, the pixel intensities of live cell images were measured at 48 hpi (Fig 3F). PbmTagBFP2 parasites showed significantly higher brightness than the PbeBFP2. Additionally, parasite size and survival were quantitatively assessed in host cells at various post-infection intervals. No significant differences were noted in size or survival between the BFP-expressing parasite lines and the PbmCherry line (Fig 4A and 4B). Finally, to determine if the two BFP-expressing parasite lines could fully complete the hepatic stage in vitro, detached cell formation rates and the parasites survival were determined at 65 hpi. No significant differences were observed between the BPF-expressing lines and control parasites (Fig 4C). These results demonstrate that the PbeBFP2 and PbmTagBFP2 parasite lines develop comparably to the PbmCherry control line throughout the P. berghei liver stage.

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Fig 3. The BFP transgenic parasites develop normally throughout the liver stage.

(A) illustrates the sporozoite motility projection. Transgenic sporozoites expressing BFP and mCherry were isolated from mosquito salivary glands and imaged live using a wide-field microscope. FIJI software was used to project the circular motility pathways of the parasites. (B) The velocities of the sporozoites were measured and compared between the BFP-expressing transgenic parasites and the PbmCherry control. The mean motility rates were 1.4, 1.38, and 1.5 μm/s for PbmCherry, PbeBFP2, and PbmTagBFP2, respectively. (C), (D), and (E) display images from an immunofluorescence assay of fixed parasite-infected HeLa cells, showing sporozoite infection at 6 hpi (hours post infection), trophozoite stages at 24 hpi, schizogony at 30 and 48 hpi, and the cytomere stage at 56 hpi. Polymerized host cell actin was stained with phalloidin dye conjugated to Alexa Fluor 488, and the parasite parasitophorous vacuole membrane (PVM) was stained with anti-UIS4 (upregulated in infective sporozoite gene 4) antiserum. (F) compares the BFP signal intensities of PbmTagBFP2 and PbeBFP2 parasites in live HeLa cells at 48 hpi, captured using confocal microscopy with a 40x objective. The average pixel intensity for PbmTagBFP2 was 420.65 a.u., while for PbeBFP2 it was 238.81 a.u. The bars were color-coded as indicated in the graphs (red: PbmCherry, blue: PbmTagBFP2, cyan: PbeBFP2). Mean pixel intensities were compared using a two-tailed t-test and mean velocities were compared using one-way ANOVA with Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). All experiments were conducted in triplicate. Scale bars = 10μm.

https://doi.org/10.1371/journal.pone.0308055.g003

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Fig 4. Newly generated BFP parasite lines exhibit a normal exoerythrocytic development.

(A) represents the size measurement of liver stage parasites. 20000 sporozoites were used to infect HeLa cells. The sizes of both BFP transgenic parasites and PbmCherry were calculated in HeLa cells over a time course of 6, 24, and 48 hpi using one-way ANOVA with Tukey’s multiple comparison test. The bar plots depict the mean size of the parasites along with their standard deviation. At 6 hpi, the mean size was approximately 50 μm2, increasing to 100 μm2 at 24 hpi, 200 μm2 at 48 hpi, and 300 μm2 at 56 hpi in the PbmCherry, PbeBFP2, and PbmTagBFP2 parasite lines with no significant difference. (B) shows the ratio of normalized survival data for parasites at 48 hpi. The survival ratio of the parasites was calculated based on the absolute number of infected parasites at 6 hpi. Approximately 50% of the PbmCherry and BFP transgenic parasites developed through the normal Plasmodium liver stage. Based on the survival ratio of parasites at 48 hpi, the ratio of detached cell formation was calculated at 65 hpi, as depicted in Panel (C). No significant difference was observed in the detachment cell formation of the three transgenic parasites. On average, approximately 20% of the surviving parasites in the three transgenic parasite lines formed detached cells. Each experiment was carried out in triplicate. The statistical comparison tests were carried out using one-way ANOVA with Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Scale bars = 10 μm.

https://doi.org/10.1371/journal.pone.0308055.g004

Interaction of host cell lysosomes with intracellular PbeBFP2 and PbmTagBFP2 parasites during Plasmodium hepatic stage development

Recent observations have shown that lysosomes are recruited to the parasite parasitophorous vacuole membrane (PVM), as evidenced by the incorporation of Lysosomal-associated membrane protein 1 (LAMP1) into the PVM [9, 38, 52]. To verify whether the two BFP-expressing parasite lines similarly recruit host cell lysosomes to the PVM as observed with the PbmCherry control line, parasite-infected HeLa cells were fixed and labeled with antibodies detecting LAMP1 and the PVM marker UIS4 (S2A–S2C Fig). Our analysis revealed that approximately 40% of the host cell lysosomes are located within a close proximity of less than 0.5 μm to the PVM. No significant differences are observed between the BFP-expressing parasites and the PbmCherry line (S2D Fig).

Transition from the hepatic to the blood stage is not affected in PbeBFP2 and PbmTagBFP2 parasites in vivo

To confirm that the development of the PbeBFP2 and PbmTagBFP2 parasites is not adversely affected in vivo, three mice per parasite line were intravenously injected with 5,000 salivary gland sporozoites each. For the co-infection, the mice were infected with 2500 sporozoites from each parasite clones. The progression of the parasite’s blood-stage infection was monitored daily from the day of infection by flow cytometry (Fig 5A, 5C and 5E) until day 5 post infection (Fig 5B, 5D and 5F). All mice were tested clearly positive from day 3 onwards, without significant growth difference between the two BFP-expressing lines and the PbmCherry control line, confirming the full viability of the new parasite lines (Fig 5B). Coinfections of BFP-expressing sporozoites together with the control sporozoites showed no negative impact on either parasite line (Fig 5C–5F). Additionally, the PbmCherry parasites exhibited a higher brightness than the BFP-expressing parasites during the asexual blood stage development. With mean fluorescence intensities of 14116, PbmCherry parasites were 7.5 times brighter than PbmTagBFP2, and these in turn were also about 4.5 times brighter than PbeBFP2 parasites (Figs 1E, 1F and 5C and 5E). It is important to note that different lasers were used to detect the mCherry and BFP fluorescent signals and that a fluorescent protein’s brightness can also be affected by factors such as the laser power and sensitivity.

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Fig 5. Blood stage PbmTagBFP2 and PbeBFP2 parasite lines develop normally in vivo.

Development of BFP-expressing blood stage parasites in infected C57Bl/6 mice. (A) Flow cytometry profile of the blood from naive mice used as a control. The selection thresholds for the BFP (excitation 405–450) and mCherry (excitation 561–614) lasers are indicated by the outlined boxes. (B) The parasitemia of the infected mice was measured daily at the same hour. When the parasitemia reached between 4 and 5%, the mice were sacrificed. Each transgenic-line infection was carried out in triplicate. No significant difference was observed in the in vivo development from day 1 to 5 days post-infection of the BFP-expressing parasites compared to the PbmCherry control. (C) Flow cytometry profile of the blood from mice co-infected with PbmCherry and PbmTagBFP2 on day 3, showing parasitemia levels of 0.14% for PbmCherry and 0.10% for PbmTagBFP2. Due to the higher mean intensity of mTagBFP2 compared to eBFP2, the selection thresholds for mTagBFP2 in (C) were set higher than those for PbeBFP2 in (A) and (E). (E) shows the flow cytometry profile of the blood from mice co-infected with PbmCherry and PbeBFP2 on day 3, with parasitemia levels of 0.17% for PbmCherry and 0.19% for PbmTagBFP2. (D) and (F) represent the parasitemia progression in the blood of mice co-infected with PbmCherry and either PbeBFP2 or PbmTagBFP2 parasites. No significant differences were observed in the in vivo development from day 0 to 5 days post-infection between the co-infected BFP and PbmCherry-expressing parasites.

https://doi.org/10.1371/journal.pone.0308055.g005