Plasmodium and MIC2 in Toxoplasma. Here we show that the I domain of TRAP is essential for sporozoite gliding motility, mosquito salivary gland invasion and mouse infection. Its replacement with the I domain from Toxoplasma MIC2 fully restores tissue invasion and parasite transmission, while replacement with the aX I domain from human integrins still partially restores liver infection. Mutations around the ligand binding site allowed salivary gland invasion but led to inefficient transmission to the rodent host. These results suggest that apicomplexan parasites appropriated polyspecific I domains in part for their ability to engage with multiple ligands and to provide traction for emigration into diverse organs in distant phyla.Inserted (I) domains function as ligand-binding domains in adhesins that support cell adhesion and migration in many eukaryotic phyla. These adhesins include integrin αβ heterodimers in metazoans and single subunit transmembrane proteins in apicomplexans such as TRAP in  Plasmodium. Humans and other animals with backbones – such as birds, reptiles and rodents – can become hosts for these parasites if an infected female mosquito feeds on their blood. Likewise, healthy mosquitoes can in turn become infected with Plasmodium if they feed on the blood of an infected animal.Malaria is an infectious disease caused by single-celled parasites known as Plasmodium parasites within a mosquito must become spore-like cells called sporozoites. These sporozoites are highly mobile and can get into the mosquitoes’ salivary glands, meaning they can be passed on to a new host when the insect feeds. During a mosquito bite the sporozoites are spat into the skin of the potential host, where they then need to migrate rapidly to enter the bloodstream. Once in the blood, the sporozoites can then get into liver cells and begin a new infection. One protein called TRAP, which is found on the surface of the sporozoites, is important for their migration and the infection of the salivary glands or liver. Yet it was not known how this happens at the level of the individual proteins involved.To complete their life cycle, Plasmodium I domain of TRAP with the I domain from a distantly related parasite called Toxoplasma gondii, which causes a condition known as toxoplasmosis. The I domain from Toxoplasma allowed the Plasmodium parasites to infect the host tissues again. This observation was unexpected because Toxoplasma and Plasmodium parasites have evolved separately over the last 800 million years and Toxoplasma does not infect insects.Klug et al. have now tested how a part of the TRAP protein, called the I domain, contributes to the infection process. In the experiments, the I domain of TRAP was deleted which showed that the sporozoites need this domain to be able to move around and get into the host tissues. Without the I domain the sporozoites were stuck and could not successfully infect either the mosquitoes, the livers of mice, or human liver cells grown in the laboratory. Klug et al. then replaced the These findings suggest that the I domain of TRAP evolved to bind several other proteins in different tissues and hosts. Future studies will investigate which other parasite proteins TRAP works with to guide sporozoites to the salivary glands or liver. Knowledge of how these proteins act together may lead to new approaches for treating or preventing malaria. For example, some treatments could stop sporozoites from entering liver cells. Domains with similar overall structures, initially described in von Willebrand factor A (VWA domains), are found in cell-surface proteins including integrins, extracellular matrix, and complement components, and mediate a diversity of functions including cell adhesion, migration, and signaling . Here, wToxoplasma gondii and Plasmodium spp. and are present in all known apicomplexans (Plasmodium (P.), a protein named CTRP containing six I domains is required for invasion of the mosquito midgut by the ookinete  and TLP (TRAP-like protein). These proteins are stored in secretory vesicles called micronemes, at the apical end of the highly polarized sporozoite . After fvenously . Mutatiovenously . HoweverPlasmodium spp. and a TRAP orthologue in Toxoplasma gondii, the micronemal protein 2 (MIC2), revealed the I domain in both open and closed conformations in association with a thrombospondin type-I repeat domain  parasites and show that the I domain is essential for motility and invasion. Parasites expressing TRAP with the I domain of MIC2 from the related apicomplexan Toxoplasma gondii, show rescued motility and invasion. By reversing the charges of amino acid residues around the MIDAS motif we could partially uncouple the function of TRAP during salivary gland invasion and rodent infection. Our results show that I domains have the capacity to be poly-specific and permit TRAP to function as an adhesin in both vertebrate and arthropod hosts.Crystal structures of the N-terminal portion of TRAP in t domain . The apie domain . As thise domain . Becausee domain . P. bergtrapΔI parasite line (trapΔI or trap(-) sporozoites could be observed within the mosquito salivary glands, whereas for trap wild type ~10,000 sporozoites were observed per mosquito  sporozoites. To determine whether mutant parasites retained the ability to migrate steadily on microscope slides, that is to glide in circles (trapΔI (trap(-) mutants  parasites  sporozoites. TRAP could also be observed on the surface of unpermeabilized trapΔI sporozoites indicating that micronemal secretion is not abolished in these parasites  and αL (CD11a)  . Sequencand MIC2 . The I dand MIC2 . Furtherand MIC2 , while tand MIC2 .fluo line, with eGFP constitutively expressed in all parasite stages and mCherry specifically expressed in sporozoites, was used to generate some of the respective transgenic parasite lines to simplify analysis of TRAP I domain replacements throughout the parasite life cycle (P. berghei TRAP I domain (fluo and non-fluo trap(-) parasite lines by homologous recombination   to ~200 (non-fluo) and for αL-I from ~30 (non-fluo) to ~90 (fluo) sporozoites per gland (trap(-) sporozoites and 0–800 for trapΔI. These small numbers might also be due to contaminants from the hemolymph and hence need to be treated with caution. Yet in all (8/8) αX-I mosquito infections sporozoites were detected in salivary glands, whereas this was only the case in 50% (4/8) of αL-I infections. In contrast for mosquitoes infected with trap(-) or trapΔI sporozoites were only detected in 13% (1/8) and 21% (3/14) experiments, respectively.  . Similarrozoites . MIC2-I rozoites , Table 1croscopy  that indTRAP-I but only ~4% of MIC2-I sporozoites were productively motile   and trapΔI. Injections of αX-I midgut sporozoites infected 2 out of 4 mice with a prepatent period of 8 days. In contrast no infections in mice could be observed when αL-I (0/2), trap(-) (0/4) or trapΔI (0/6) midgut sporozoites were injected  for rozoites , Table 3rozoites , Table 2ve tests . We therrozoites , Table 2es (0/8) , Table 2injected . To exclinjected . AdditioPlasmodium sporozoites can infect different types of cells they have a strong tropism for the liver. In contrast, Toxoplasma gondii can infect any nucleated cell from a warm-blooded animal  with seven charge reversal mutations  around the perimeter of the putative ligand binding site, distal from the MIDAS   , Table 2RevCharge mutant compared to TRAP-I. While after 2 hr 54% of TRAP-I sporozoites showed an intracellular localization only 3% of RevCharge sporozoites were observed inside cells  hemolymph sporozoites. These results suggest that the biological functions of TRAP are dependent on its I domain. It was therefore remarkable that TRAP function was largely restored by replacement of its I domain by its homologue from Toxoplasma gondi with only 28% amino acid sequence identity. Compared to cell surface receptors that engage in typical protein-protein interactions, that is those that are not dependent on Mg2+ ions, these sequence identities are substantially below the level of 40% to 50% identity generally required for members of the same receptor family to recognize the same ligand. The strength of the Mg2+ ion bond to the oxygen in the ligand, which has a bond distance of only 2 Å and is partially covalent, may overcome the lack of complementarity in other regions of the ligand binding site, and explain the ability of I domains of such remarkably low sequence identity to function in TRAP. In addition to the low sequence identity, there are six to seven sequence positions where residues are inserted or deleted in Toxoplasma MIC2 or human integrin αI domains compared to TRAP  sporozoites. The greater efficacy of αX-I than αL-I sporozoites in invasion of mosquito salivary glands, infection of mice, and productive motility in vivo confirmed our hypothesis that the polyreactive αX I domain would better support TRAP function than the highly specific αL I domain, albeit at low levels.We also examined replacement with two mammalian integrin I domains, from the promiscuous αXβ2 integrin and the much more selective αLβ2 integrin. Salivary gland invasion was decreased 100 to 400 and 500 to 1,000-fold in Toxoplasma and may be its orthologue. Each has an I domain that is inserted in an extensible β-ribbon in tandem with a TSR domain and a long-range disulfide bond that moves in allostery  than the highly specific αL (pI 5.6), αE (pI 4.6), and collagen-binding α1, α2, α10, and α11 I domains (pI 5.1–5.6). As the surfaces of all cells are negatively charged and almost all proteins are acidic, we tested whether the functions of the basic TRAP I domain (pI = 9.7) would be altered with seven substitutions that made it neutral (pI 6.8). This P, or αX . These rToxoplasma MIC2 that encounters different ligands as TRAP can replace its function in mediating salivary gland invasion and infection of mice strongly suggest that the I domain functions to provide traction for motility and cell invasion rather than tissue tropism. Binding of the TRAP I domain to multiple distinctive ligands is required by its function in mosquitos in migration into salivary glands and in vertebrate hosts in emigration from the dermis into the circulation and from the circulation into the liver, followed by productive invasion of liver parenchymal cells. The ligands in the mosquito must differ from those in the vertebrate; those encountered in the vertebrate in the dermis, endothelium and liver may also differ. Furthermore, Toxoplasma lives in epithelia in the gastrointestinal system of cats and other mammals, and lacks an arthropod host, yet its MIC2 I domain functions robustly in mosquito salivary gland invasion. Our results are consistent with TRAP binding to the many ligands that have been identified for it, including liver-specific fetuin-A   (http://genomes.urv.es/OPTIMIZER/) (n 26–34)  and multustalo/) . To chanIMIZER/) .trap(-)) parasites were generated with the PlasmoGem (trap(-) parasites were subsequently negatively selected with 5-fluorocytosine (1.0 mg/mL in the drinking water) to give rise to selection marker free trap(-) parasites (trapΔI parasites we made use of the Pb238 vector (trap 3’UTR (970 bp) was amplified with the primers P165/P166 and cloned (BamHI and EcoRV) downstream of the resistance cassette in the Pb238 vector. In a next step the coding sequence of the trap gene including the 5’ and 3’ UTR was amplified with the primers P508/P509 and cloned in the pGEM-T-Easy vector giving rise to the plasmid pGEM-TRAPfull. Subsequently the pGEM-TRAPfull plasmid was mutated with the primers P535/P536 and P537/P538 to introduce a restriction site for NdeI directly in front of the start codon ATG and a restriction site for PacI directly after the stop codon TAA. The mutated sequence was cloned (SacII and EcoRV) in the Pb238 intermediate vector that contained already the trap 3’UTR downstream of the selection marker and the resulting plasmid was named Pb238-TRAP-NdeI/PacI. The designed DNA sequence lacking the coding region of the I domain was codon modified for E. coli K12 and synthesized at GeneArt (Invitrogen). Subsequently the designed sequence was cloned (NdeI and PacI) in the Pb238-TRAP-NdeI/PacI by replacing the endogenous trap gene. Final DNA sequences were digested for linearization , purified and transfected into wild type parasites (wt) using standard protocols (TRAP knockout (lasmoGem  vector (lasmoGem . Isogeniarasites . For the8 vector . In a firotocols .trap gene (Plasmodium berghei ANKA strain) were exchanged with sequences from the micronemal protein 2 (MIC2) of Toxoplasma gondii , the integrin CD11c/αX  and the integrin CD11a/αL  of Homo sapiens. Chimeric sequences as well as the coding sequence of the wild type trap gene that served as a control, were codon modified for E. coli K12 to prevent incorrect homologous recombination with the trap coding sequence downstream of the I domain coding region and to avoid changes of the codon usage within the open reading frame caused by the exchanged I domain coding sequence. This enabled also simple differentiation between wild type and transgenic parasites by PCR. For the generation of RevCharge parasites seven mutations of non-conserved amino acids  were introduced into the codon modified wild type trap gene. These mutations were expected to shift the surface charge at the apical side of the I domain from a pI of 9.7 to 6.8 while leaving the MIDAS intact and the structural integrity of the domain unaffected. All designed sequences were synthesized at GeneArt (Invitrogen) and cloned in the Pb238-TRAP-NdeI/PacI vector (NdeI/PacI) that was already used to generate the trapΔI line. Constructs were digested for linearization (ScaI-HF) and transfected using standard protocols (trap(-) as well as in the fluorescent background line fluo to independently generate fluorescent (fluo) and non-fluorescent (non-fluo) sets of mutants  of the wild type rotocols . Transfe mutants .Isogenic parasite lines were generated by serial dilution of parental populations obtained from transfections. Per transfection one mouse was infected by intraperitoneal injection of ~200 µL frozen parasites of the parental population. To increase the number of transfected parasites within infected mice pyrimethamine (0.07 mg/mL) was given within the drinking water 24 hr post injection (hpi). Donor mice were bled two to three days post injection once parasitemia reached 0.5–1%. Parasites were diluted in phosphate buffered saline (PBS) to 0.7–0.8 parasites per 100 µL and the same volume was subsequently injected into 6–10 naive mice. Parasites were allowed to grow for 8–10 days until parasitemia reached 1.5–2%. Blood of infected mice was taken by cardiac puncture  and used to make parasite stocks (~200 µL infected blood) and to isolate genomic DNA with the Blood and Tissue Kit (Qiagen).7 parasites by intraperitoneal injection into two to three naive mice. Parasites within recipient mice were allowed to grow for further three to four days, depending on the number of exflagellation events observed. To determine the number of exflagellation events, and subsequently the number of male gametocytes, a drop of tail blood was placed on a microscope slide, covered with a coverslip and incubated for 10–12 min at room temperature (20–22°C). The number of exflagellation events was counted with a light microscope (Zeiss) and a counting grid by using 40-fold magnification with phase contrast. If at least one exflagellation event per field could be observed mice were fed to mosquitoes. Mosquitoes were starved overnight prior to the feeding to increase the number of biting mosquitoes. Per mosquito cage  two to three mice were used for feeding.Naive mice were infected with 100–200 µL frozen parasite stocks and parasites allowed to grow for four to five days. Infected mice were either directly fed to mosquitoes or used for a fresh blood transfer of 2 × 10Anopheles stephensi mosquitoes were dissected on day 13, 14, 18, and 22 post infection. For the collection of hemolymph sporozoites, infected A. stephensi mosquitoes were cut with a needle to remove the last segment of the abdomen. Subsequently the thorax was pierced with a finely drawn Pasteur pipette filled with RPMI/PS solution. While gently pressing the pipette the haemocoel cavity was flushed with solution which dripped off the abdomen. The sporozoite solution was collected on a piece of foil and transferred into a plastic reaction tube. To determine the number of midgut sporozoites, the abdomen of infected mosquitoes was dissected with two needles and the midgut was extracted. Isolated midguts were transferred into a plastic reaction tube containing 50 µL RPMI/PS solution. For the isolation of salivary gland sporozoites, the head of infected mosquitoes was gently pulled away with a needle while fixing the mosquito in place with a second needle. Ideally the salivary glands stayed attached to the head and could be easily isolated. Salivary glands were transferred into a plastic reaction tube with 50 µL RPMI/PS. To release sporozoites, pooled midguts and salivary glands were homogenized using a plastic pestle for 2 min. To count the parasites in each sample, 5–10 µL of the sporozoite solution (1:10 dilution) was applied on a hemocytometer. Sporozoites were counted using a light microscope (Zeiss) with 40-fold magnification and phase contrast. For each counting experiment at least 10 mosquitoes were dissected. However, the number was adapted depending on the infection rate of the mosquitoes and the experiment that was performed.To estimate the number of sporozoites, infected To determine the prepatency of parasite lines during sporozoite transmission two different routes of infection were tested. Female C57BL/6 mice were either exposed to infected mosquitoes or infected by intravenous injection of midgut, hemolymph or salivary gland sporozoites. To infect mice by mosquito bites, mosquitoes infected with fluorescent parasite lines were pre-selected for fluorescence of the abdomen by using a stereomicroscope  with an attached fluorescence unit. Subsequently parasite positive mosquitoes were separated in cups to 10 each and allowed to recover overnight. Approximately six hours prior to the experiment mosquitoes were starved by removing salt and sugar pads. Mice were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine (87.5 mg/kg ketamine and 12.5 mg/kg xylazine) and placed with the ventral side on the mosquito cups. Mosquitoes infected 17–24 days prior to the experiment were allowed to feed for approximately 15 min before mice were removed. During this time eyes of mice were treated with Bepanthen cream (Bayer) to prevent dehydration of the cornea. After the experiment mice were allowed to recover and tested for blood stage parasites on a daily basis by evaluation of Giemsa stained blood smears. If non-fluorescent parasite lines were tested infected mosquitoes were not pre-sorted. In these experiments midguts of mosquitoes that had taken a blood meal were dissected after the experiment and the number of midgut sporozoites was counted as described previously. Mice that were bitten by mosquitoes that contained no midgut sporozoites were excluded from the analysis.For injections hemolymph or salivary gland sporozoites were isolated either 13–16 days (hemolymph) or 17–24 days  post infection. Sporozoite solutions were diluted to the desired concentration with RPMI/PS  and injected intravenously into the tail vein of naive mice. The presence of blood stage parasites was evaluated on a daily basis.A. stephensi mosquitoes. To free the sporozoites from salivary glands, samples were grounded with a pestle. Subsequently salivary gland samples were centrifuged for 3 min at 1,000 rpm  to separate sporozoites from tissue. Afterwards ~40 µL of the supernatant was transferred into a new 1.5 mL plastic reaction tube and diluted with a variable volume of RPMI/PS depending on the planned number of assays and the sporozoite concentration resulting in a minimum of 50,000 sporozoites per well. For each assay about 50 µL of the sporozoite suspension was mixed with 50 µL RPMI medium containing 6% bovine serum albumin (BSA) to initiate activation. Subsequently sporozoites were allowed to attach to the bottom by centrifugation at 800 rpm for 3 min (Heraeus Multifuge S1). Using fluorescence microscopy (Axiovert 200M) with a 10x objective movies were recorded with one image every three seconds for 3 to 5 min depending on the experiment. Movies were analyzed manually using the Manual Tracking Plugin from ImageJ . Hemolymph and salivary gland sporozoites were obtained by dissecting infected m ImageJ  to deterFor the imaging of sporozoites within salivary glands infected mosquitoes were dissected 17–24 days post infection as described previously. Isolated salivary glands were transferred with a needle to a microscope slide containing a drop of Grace’s medium (Gibco) and carefully sealed with a cover slip. Samples were imaged with an Axiovert 200M (Zeiss) using 63x (N.A. 1.3) and 10x (N.A. 0.25) objectives.For immunofluorescence assays we made use of antibodies directed against the circumsporozoite protein (CSP) and the thrombospondin related anonymous protein (TRAP). In all assays the anti-CSP antibody mAb 3D11  was applTo visualize the expression and localization of TRAP in sporozoites, infected salivary glands were dissected as described previously and pooled in plastic reaction tubes containing 50 µL PBS or RPMI/PS. Afterwards salivary glands were mechanically grounded with a plastic pestle to release sporozoites from tissue. Immunofluorescence assays were performed by two different methods either fixing sporozoites in solution or on glass cover slips. To fix the parasites on glass, salivary glands were dissected in RPMI/PS and treated as described. Sporozoite solutions were transferred into 24-well plates containing round cover slips, activated with an equal volume RPMI/PS containing 6% BSA and allowed to glide for approximately 30 min at RT. Subsequently the supernatant was discarded and sporozoites were fixed with 4% PFA (in PBS) for 1 hr at RT. Fixed samples were washed three times with PBS for 5 min each. If immunofluorescence was performed on sporozoites in solution, salivary glands were dissected in PBS and treated as described previously. Sporozoite solutions were directly fixed by adding 1 mL of 4% PFA (in PBS) for 1 hr at RT. After fixation samples were washed as described for samples fixed on glass while samples in solution had to be additionally pelleted after each step by centrifugation for 3 min at 10,000 rpm . Subsequently sporozoites were blocked (PBS containing 2% BSA) or blocked and permeabilized (PBS containing 2% BSA and 0.5% Triton X-100) over night at 4°C or for 1 hr at RT, respectively. Samples were incubated with primary antibody solutions for 1 hr at RT in the dark and subsequently washed three times with PBS. After the last washing step, samples were treated with secondary antibody solutions and incubated for 1 hr at RT in the dark. Stained samples were washed three times in PBS and the supernatant was discarded. If the immunofluorescence assay was performed in solution, sporozoite pellets were resuspended in 50 μL of remaining PBS, carefully pipetted on microscopy slides and allowed to settle for 10–15 min at RT. Remaining liquid was removed with a soft tissue and samples were covered with cover slips which had been prepared with 7 μL of mounting medium . If the immunofluorescence assay was performed on sporozoites that were fixed on glass, cover slips were removed with forceps, carefully dabbed on a soft tissue and placed on microscopy slides that had been prepared with 7 μL of mounting medium. Samples were allowed to set overnight at RT and kept at 4°C or directly examined. Images were acquired with a spinning disc confocal microscope (Nikon Ti series) with 60-fold magnification .Salivary glands of infected mosquitoes were dissected in 100 µL PBS on ice and subsequently smashed with a pestle to release sporozoites. For the isolation of hemolymph sporozoites infected mosquitoes were flushed with PBS as previously described. Sporozoite solutions were kept on ice, counted using a haemocytometer and distributed to 30,000 sporozoites per reaction tube. Samples were centrifuged for 1 min with 13,000 rpm  at 4°C to pellet sporozoites. Subsequently the supernatant was discarded, and pellets were lysed in 20 µL RIPA buffer  supplemented with protease inhibitors  for ≥1 hr on ice. Lysates were mixed with Laemmli buffer, heated for 10 min at 95°C and centrifuged for 1 min at 13,000 rpm . Samples were separated on precast 4–15% SDS-PAGE gels  and blotted on nitrocellulose membranes with the Trans-Blot Turbo Transfer System (Bio-Rad). Blocking was performed by incubation in PBS containing 0.05% Tween20% and 5% milk powder for 1 hr at RT. Afterwards, the solution was refreshed and antibodies directed against TRAP  or the loading control CSP  were added. Membranes were washed three times (PBS with 0.05% Tween20) and secondary anti-rabbit antibodies (Immun-Star (GAR)-HRP, Bio-Rad) or anti-mouse antibodies  conjugated to horse radish peroxidase were applied for 1 hr  at room temperature. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). After the detection of TRAP, blots were treated with stripping buffer  for 15 min prior to incubation with anti-CSP antibodies used as loading control.HepG2 cells were a gift from our virology department who had obtained the cells from ATCC. Cell line identity was regularly confirmed by SNP sequencing and visual observations of cell morphology. Cells were routinely tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit . We culture cells for a maximum of 10 passages.PbHSP70 1:300 in 10% FBS/PBS for 2 hr at 37°C was succeeded by two washing steps with 1% FBS/PBS. Incubation with secondary antibody α-mouse Alexa Fluor 488 1:300 in 10% FBS/PBS was performed for 1 hr at 37°C. Hoechst 33342 was added and incubated for 5 min at RT followed by two washing steps with 1% FBS/PBS. The assay was mounted in 50% glycerol and sealed using a glass cover slip. Samples were imaged with an Axiovert 200M (Zeiss) microscope and subsequently analyzed using ImageJ . On day zero salivary glands were isolated from infected female mosquitoes and collected in RPMI medium within a 1.5 mL reaction tube. Sporozoites were released by mechanically disrupting the salivary glands with a polypropylene pestle. The solution was centrifuged in a tabletop centrifuge at 1,000 rpm for 3 min at RT and the supernatant was transferred to a new 1.5 mL reaction tube. The salivary gland pellet was resuspended in 100 µL RPMI medium, smashed again using a pestle, centrifuged  and the supernatant was pooled with the first one. A 1:10 dilution of the sporozoite solution was counted in a Neubauer counting chamber and 10,000 salivary gland sporozoites were used to infect HepG2 cells per well. After 1.5 hr wells were washed twice with complete DMEM medium and HepG2 cells were allowed to grow in complete DMEM medium supplemented with 1x Antibiotic-Antimycotic (Thermo Fisher Scientific). At 24 hr and 48 hr post infection cells were fixed using ice cold methanol for 10 min at RT, followed by blocking with 10% FBS/PBS overnight at 4°C. Staining with primary antibody α-g ImageJ . In briePbCSP 1:100 in 10% FBS/PBS (2 hr 37°C), two washing steps with 1% FBS/PBS and incubation with secondary antibodies α-mouse Alexa Fluor 488 1:300 in 10% FBS/PBS (1 hr 37°C). After two washing steps with 1% FBS/PBS, cells were permeabilized by addition of ice cold methanol and incubation at RT for 10 min. Blocking with 10% FBS/PBS (4°C and overnight) was followed by an incubation with primary antibodies α-PbCSP 1:100 in 10% FBS/PBS (2 hr at 37°C), two washing steps with 1% FBS/PBS and incubation with primary antibodies α-mouse Alexa Fluor 546 1:300 in 10% FBS/PBS (1 hr at 37°C). The assay was mounted in 50% glycerol after two washing steps with 1% FBS/PBS.Two days prior to the experiment 180,000 HepG2 cells/well were seeded in an 8-well Permanox Lab-Tek chamber slide (Nunc). Sporozoites were isolated as described above and 10,000 sporozoites/well were used to infect HepG2. At 1.5 hr post infection cells were washed twice with complete DMEM medium and fixed using 4% PFA/PBS 20 min at RT. Blocking was performed with 10% FBS/PBS o/n at 4°C followed by incubation with primary antibodies α-Plasmodium berghei 18S rRNA was used to quantify parasites and mouse specific GAPDH was utilized as housekeeping gene for normalization. Subsequently the ∆cT was plotted as mean of all replicates per parasite strain and harvested organ . Subsequently RNA content of generated samples was measured using a NanoDrop and liver, intestine, spleen and lung samples were pooled in equal amounts of RNA to generate single samples for each parasite line and harvested organ. RNA pools were used to synthetize cDNA using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative RT-PCR was performed in triplicates on an ABI7500 (Applied Biosystems) using a 2x SYBR green Mastermix (Applied Biosystems). ed organ .Plasmodium berghei ANKA background (trap(-) and fluo). Parasites were cultivated in NMRI mice while transmission experiments with sporozoites were performed in C57Bl/6 mice only.For all experiments female 4–6 week-old Naval Medical Research Institute (NMRI) mice or C57BL/6 mice obtained from Janvier laboratories were used. Transgenic parasites were generated in the ckground  either dStatistical analysis was performed using GraphPad Prism 5.0 . Data sets were either tested with a one-way ANOVA or a Student’s t test. A value of p<0.05 was considered significant. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The thrombospondin related anonymous protein (TRAP) is an important adhesin expressed by the sporozoite stage of the malaria parasite, required for sporozoite motility and invasion of the salivary gland in the mosquito and of the liver in the mammalian host. To gain insight into the contribution of integrin I-domain of TRAP, the authors performed deletion as well as creative swap between Plasmodium, Toxoplasma and mammalian integrin I-domains. These technically challenging experiments established that the I domains have the versatile capacity to confer adhesive properties to TRAP both in vertebrate and arthropod hosts.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "Cationic charge and polyspecificity of an integrin domain regulates infectivity of malaria parasites" for consideration by eLife at this stage.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered for publication in As you will see, the three reviewers are broadly supportive of this work, which addresses an important topic. However, they were especially concerned that the conclusions drawn from the a-L and a-X swapped mutants are not sufficiently supported by the data. Importantly they raised three main issues that will take some time to address (more than two months) and in consequence, we have together opted for a rejection with a strong encouragement for re-submission. If you are able to address these issues we will consider a newly submitted form of this paper that we will treat as a revised manuscript. If you choose this course of action please submit a separate cover letter detailing the changes you have made.Main points to be addressed:1) Establish if the two human integrin I-domain swaps have the same phenotype as the trap null allele. At the moment the authors imply that they are strong hypomorphs but two reviewers point out there is no side-by-side quantitative comparison with the null. If the human integrin I-domain swaps do phenocopy the null then the wording in the manuscript must be changed significantly.2) Make a direct link between I-domain ligand binding and phenotype rather than implying this is the reason for the observed effects. The suggestion of performing the gliding assays on different substrates, and especially on known ligands of the swapped I-domains, is a good one and would be informative.3) Establish that the I-domains retain their folding and binding functions in the context of the chimeric trap.Reviewer #1:In this study, Klug and colleagues report on the role of the integrin I-domain of TRAP, an important adhesin expressed by the sporozoite stage of the malaria parasite. Previous studies have shown that TRAP is required for sporozoite motility and invasion of the salivary gland in the mosquito and of the liver in the mammalian host. A metal ion-dependent adhesion site (MIDAS) within the I-domain of TRAP is known to play a role in sporozoite infectivity, however the functional and adhesive properties of the I-domain remain undefined. More generally, the mechanisms of sporozoite infection are poorly understood at the molecular level, so this study addresses an important topic.P. berghei and performed robust phenotype analysis including gliding motility assays and analysis of sporozoite infectivity in the mosquito  and in the mouse (invasion of the liver). The work is very well performed and includes a large body of data, combining genetic approaches, in vitro and in vivo experiments. The paper is clearly written and the conclusions drawn from the results generally justified.To investigate the function of TRAP I-domain, the authors generated a number of mutants in The data convincingly show that:a) Sporozoites expressing a truncated TRAP lacking the I-domain are non-motile and do not invade the mosquito salivary glands and the mouse liver.b) Replacement of TRAP I-domain with the I-domain from Toxoplasma MIC2 results in normal invasion of salivary glands and normal sporozoite infectivity in mice, although gliding motility is reduced.c) Replacement of TRAP I-domain with the I-domain from human aX or aL integrin results in a severe reduction of sporozoite infectivity and gliding motility, although residual infectivity was observed in both mosquito and mouse.d) Introduction of 7 point mutations in the I-domain of TRAP, to replace positively charged residues (H/K/R) by negatively charged residues (E/D), results in normal invasion of mosquito salivary glands yet reduced motility and slightly reduced infectivity in the mouse.Based on the presented data, the authors conclude that the adhesive properties of the I-domain of TRAP can be partially fulfilled by the I-domain from other organisms, and that the cationic charge and the polyspecificity of the I-domain regulates sporozoite infectivity. However, the main limitation of the study is that adhesion and ligand binding were not directly assessed, so the aforementioned conclusions are based on indirect evidence or speculation. It is important to experimentally address the adhesive properties of the chimeric TRAP proteins expressed in the mutant parasites.Major points1) The authors conclude that the observed phenotypic defects are due to altered ligand binding properties of mutated TRAP proteins. However this was not addressed experimentally. Other modifications in the mutants, such as an alteration of the conformation of TRAP, could also contribute to the observed phenotype, unrelated with TRAP-I adhesive properties. In the absence of direct experimental evidence, it seems too speculative to conclude that the polyspecificity of the I-domain regulates sporozoite infectivity. The data clearly shows that MIC2-I almost completely complement TRAP-I functions, whilst aX-I and aL-I are poorly functional in this context. TRAP-I is basic (like aX-I), but MIC2-I is acidic (like aL-I). The data therefore show no correlation between infectivity and charges, and thus do not support an important role of the I-domain charges. Other factors may explain why MIC2-I is functionally superior to the I-domain from humain integrins, including the size of the domain size . The authors cannot exclude that a smaller I-domain may affect the conformation of TRAP protein, including other parts of the proteins such as the β ribbon. Surprisingly, the data also show no direct correlation between gliding motility and infectivity, since MIC2 and RevCharge mutants show severely reduced motility yet invade almost normally the mosquito and the mouse. This questions the relevance of the gliding assay. How do sporozoites with reduced gliding motility invade mosquito salivary glands and mouse cells?Maybe a simple way to address these issues would be to perform gliding assays not merely on glass slides, as performed here, but on specific substrates, including known ligands of MIC2, aX or aL integrins. This would strengthen the conclusion that the effects of the I-domain modifications are really due to changes in the adhesional properties of the chimeric proteins. For example, do sporozoites expressing the aL-I domain recover gliding motility on ICAM1?2) The phenotype of the RevCharge mutant should be more thoroughly investigated to better document a defect in sporozoite infectivity for the liver. Figure 6F shows a partial reduction of parasite fitness in the mouse, which could be quantified more precisely. In vitro assays in HepG2 cultures, as performed with the MIC2 mutant, should be included. This is important to strengthen or invalidate the hypothesis that charges in the I-domain play an important role in sporozoite infectivity.E. coli were used, which may differentially affect the level of expression of the proteins. Why using E. coli optimized sequences?3) The authors checked by immunofluorescence that TRAP is expressed in the mutant sporozoites. However, they should also provide evidence that the level of expression of TRAP is unaffected in the mutants, ideally using Western Blot as shown for the RevCharge mutant. This is particularly important as sequences codon-optimized for Reviewer #2:T. gondii) ligand, and two from human integrins. Plasmodium parasites lacking either the entire TRAP locus or just the I-domain of TRAP do not move, or invade the vector salivary glands. The authors find that the exogenous I domains from different sources can, to varying degrees, complement TRAP function resulting in the partial rescue of some motility, and ability to invade. The authors notice that the surface charges of TRAP in the rodent parasite P. berghei are cationic and to determine if this is important for function, mutate these residues to acidic amino acids and show again that the functions of TRAP are impaired.The manuscript by Klug et al. investigates the biochemical and functional properties of I-domains by genetically replacing the I-domain within an important malaria parasite ligand called TRAP with I domains taken from another parasite  and that the human integrin I -domain replacement parasites are very strong hypomorphs ; however, there does not appear to be a direct side-by-side comparison – e.g. injecting 25,000 HL sporozoites of the TRAP I-domain deletion alongside the integrin replacements in Figure 3—figure supplement 3B, for example. Also, are the authors able to demonstrate that the inserted I-domains retain their functions from their original proteins – is there a known ligand for the inserted integrin I-domains?For the revCharge mutant, the authors imply that the loss of motility and ability to infect mice is due to the alteration in I-domain ligand binding, but could not an alternative explanation be that a proportion of the protein no longer folds correctly or that interactions with other parasite proteins necessary for full function are altered? The authors suggest the role of the I-domain is to interact with the host substrate, but these are not defined in the manuscript making it conceptually a rather large leap to link the mutations made in the protein to the phenotypes seen in the parasite.Reviewer #3:In this study the authors swap integrin "I" domains from MIC2 and mammalian integrins with the I-domain of TRAP, the key motility adhesin of malaria sporozoites. Though the domain structure of the extracellular portion of TRAP has long been known to include an integrin-like I-domain, previous mutational studies of this domain were not that informative and the idea to perform domain-swapping experiments between Plasmodium, Toxoplasma and mammalian integrin I-domains is creative and potentially informative. Overall, the MIC2 swapped TRAP and the charge-altered TRAP are the most informative data while the conclusions drawn from the a-L and a-X swapped mutants are not justified by the data shown.1) The human integrin I-domain swapped mutants essentially behave like the TRAP KO. There is an elegant explanation in the Discussion as to why the a-L and a-X domains may not open and close in the same way as the I-domains of TRAP and MIC2. However, in the face of no data to support this or another hypothesis, it is equally plausible that there is some significant misfolding on the TRAP with a mammalian I domain. It would be important to show that this is not the case. Since there are commercially available antibodies to human integrins, can they be used, especially ones specific for conformational epitopes, to confirm the overall folding of the a-L and a-X domains.2) The data shown for the a-L and a-X mutants do not support the conclusions. These mutants have less than 200 sporozoites in their salivary glands, the a-L mutant is not motile and not infectious while the a-X mutant is minimally motile and very poorly infectious after IV inoculation. These data look like the TRAP KO , where they observed 100 to 200 sporozoites in the salivary glands and had a few infected mice when sporozoites were inoculated IV. Given this, how can they state: "Parasites expressing TRAP with I-domains from other organisms including humans can have nearly intact motility and invasion”. Or "…I-domain of TRAP is required to mediate adhesional properties which can be partially preserved when the native I-domain is replaced by I-domains from human integrins…". These statements are in the Introduction and Abstract but additionally, a central theme of the Discussion is that the a-L and a-X mutants are sufficiently intact to draw meaningful conclusions.3) TRAP expression in mutant lines is verified by immunofluorescence that is not quantified. Since decreased levels of mutant TRAP could have phenotypes due to the mutation or to expression level, its critical to have western blot verification of mutant TRAP expression compared to WT TRAP expression, in all lines analyzed.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Dominique Soldati-Favre as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Plasmodium adhesin TRAP retains function when its ligand-binding domain replaced by evolutionarily distant I domains" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in Specifically, the interpretation of the human I domain swaps needs to be carefully worded to avoid over-interpreting the data, which seems to be the case here.Summary:Toxoplasma gondii ligand MIC2 and mammalian integrins with the I-domain of TRAP, the key motility adhesin of malaria sporozoites. Though the domain structure of the extracellular portion of TRAP has long been known to include an integrin-like I-domain, previous mutational studies of this domain were not that informative and the idea to perform domain-swapping experiments between Plasmodium, Toxoplasma and mammalian integrin I-domains is creative and potentially informative. Overall, the MIC2 swapped TRAP and the charge-altered TRAP are the most informative data while the conclusions drawn from the a-L and a-X swapped mutants are not justified by the data shown.In this study, Klug and colleagues report on the role of the integrin I-domain of TRAP, an important adhesin expressed by the sporozoite stage of the malaria parasite. Previous studies have shown that TRAP is required for sporozoite motility and invasion of the salivary gland in the mosquito and of the liver in the mammalian host. The authors swap integrin "I" domains from the Revisions for this paper:One major reservations of the previous iteration of this study was that the conclusions drawn from the parasites in which the TRAP I-domain was replaced by the human a-L and a-X integrin domains were not justified by the data shown and this has not been corrected in the revised manuscript. Indeed, salivary gland sporozoite numbers of the a-L and a-X mutants are in the same range as the TRAPΔI and TRAP KO parasites, with only the prevalence of infection somewhat increased in the a-X parasites. Infectivity studies demonstrate that the a-L parasites are similar to the TRAPΔI and TRAP KO parasites. With numerous careful experiments they that show the a-X parasites are more infectious than the a-L, TRAPΔI and TRAP KO parasites, and this interesting finding suggests that the charge on a-X may enable the mutant TRAP to have some minimal activity. However, their data show that the a-X sporozoites are about 100-fold decreased in their infectivity compared to controls. It has been previously demonstrated that a one day delay in patency is equivalent to a 10-fold decrease in infection. When 10,000 hemolymph sporozoites are inoculated IV, 100% of control mice are infected with a prepatent period of 4 days. With the a-X parasites only 50% of mice are infected and the prepatent period is 6 days. Thus, even if one ignores the fact that half the mice were not infected, the ones that did get infected had a 100-fold lower functional inoculum.Characterization of this significant attenuation of infectivity of the a-X parasites in mosquito and mammalian hosts as "partial restoration" of TRAP function is misleading. Indeed given the phenotype they describe, it is clear that the a-X parasites cannot be naturally transmitted for a single life cycle. Thus, the paper should focus on the interesting phenotypes of the MIC2 and reverse charge sporozoites, and only compare the a-X parasites to the a-L and TRAP KO parasites where there is a small marginal increase in infectivity supporting the importance of charge in the TRAP I-domain.The following statements in the paper should be deleted, or changed to only include the MIC-2 mutant.Title:The current title implies that human I domains confer some functionality to TRAP which, given that these mutants have salivary gland sporozoite counts in the range of the TRAP KO, and infectivity levels that are 100-fold decreased, is misleading.Abstract:"…its replacement with I domains from Toxoplasma MIC2 or the human integrin αX-subunit substantially or partially restores these functions"."Results with TRAP, MIC2 and aX-I domains show remarkable interchangeability in their ability to engage with ligands in divergent tissues in arthropoa and chordata"."…results suggest that apicomplexan parasites appropriated I domains in part for their ability to recognize structurally diverse ligands""…permit TRAP to function as an adhesin with diverse ligands in both vertebrate and arthropod hosts"The ability to engage with ligands is not addressed here, so wording should be revised. Also, the aX-I domain is poorly functional in TRAP, so "remarkable interchangeability" is not correct in this case.Introduction:"Parasites expressing TRAP with I domains from other organisms, including humans show partially rescued motility and invasion."Results:"This could hint that the charge of αX could compensate for the long evolutionary distance between apicomplexa and mammals."Discussion:Homo sapiens with only 28% and 18% amino acid sequence identity, respectively.""It was therefore remarkable that TRAP function was largely or partially restored by replacement of its I domain by homologues from Toxoplasma gondi and "TRAP function was largely or partially restored by replacement of its I domain". This is true for Toxoplasma MIC2 domain but not human integrin I domains."…immunofluorescence showed normal localization in sporozoites, suggesting that they were properly folded".The immunofluorescence data provide no information on the folding of TRAP."Infectivity of hemolymph αX-I and αL-I sporozoites in mice was decreased at least 4-fold and 15-fold, respectively." Infectivity in the a-L sporozoites was similar to the TRAP KO, and in the a-X sporozoites was 100-fold decreased compared to controls."The findings that I domains from organisms that encounter none of the same ligands as TRAP can replace its function in mediating salivary gland invasion and infection of mice strongly suggest that the I domain functions to provide traction for motility and cell invasion rather than tissue tropism." This should specify that they are referring to replacement with MIC2."…the I domain of TRAP is required for gliding motility and invasion by sporozoites of mosquito salivary glands and mammalian liver, without a requirement for evolved specificity".The data with the human I domains show that any I domain cannot confer function in TRAP, so this statement is not correct.The authors attempted to analyze the motility of mutant sporozoites on various substrates. They indicate that they "were not able to measure a significant effect of a specific ligand".However, Figure 1 shows that aX-I and aL-I activated on BSA are mostly floating, while on the other substrates sporozoites are mostly attached or display unproductive motility. This could be an indication that the human I-domains retained some adhesive properties. However, it would be useful to have the same type of data with the I domain-deleted mutant, and the threshold, as indicated in A, should also be shown on the other panels. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]As you will see, the three reviewers are broadly supportive of this work, which addresses an important topic. However, they were especially concerned that the conclusions drawn from the a-L and a-X swapped mutants are not sufficiently supported by the data. Importantly they raised three main issues that will take some time to address (more than two months) and in consequence, we have together opted for a rejection with a strong encouragement for re-submission. If you are able to address these issues we will consider a newly submitted form of this paper that we will treat as a revised manuscript. If you choose this course of action please submit a separate cover letter detailing the changes you have made.Thank you for the constructive and encouraging evaluation of our manuscript. Please find in the following our comments describing the changes in the manuscript we introduced and the additional experiments we performed to address the comments from the reviewers. Please note that due to the extensive changes and the suggestion by reviewer 3 to remove part of the supplement two authors who participated in the deleted work were removed from the author list and two authors changed position reflecting their respective contributions since the last submission. In total we added data from over 30 mosquito cage infections to improve the infection data of some of the mutants and to provide adequate Western blots for all mutants, who did not enter the salivary glands from hemolymph sporozoites.Main points to be addressed:1) Establish if the two human integrin I-domain swaps have the same phenotype as the trap null allele. At the moment the authors imply that they are strong hypomorphs but two reviewers point out there is no side-by-side quantitative comparison with the null. If the human integrin I-domain swaps do phenocopy the null then the wording in the manuscript must be changed significantly.Thank you for this suggestion, which helped us clarifying a discussion amongst the authors when writing the manuscript. We now performed a side by side comparison with the null mutant and the I-domain deletion as recommended (Table 2 of the manuscript). The new results show that the aX line partially rescues TRAP function, as sporozoite injection led to the infection of around 50% of all mice (11/20) that received a sporozoite injection while no mice became blood stage patent if infected with the I-domain deletion (0/14) or the TRAP knockout (0/10); note that Table 3 summarizes these data for injections with hemolymph sporozoites. Only one mouse (from 20) could be infected with the aL line. Therefore we agree with the reviewers that the aL mutant is essentially not rescuing TRAP functions and changed the wording across the manuscript accordingly. The new data were incorporated into Table 2 and a Table 3 was added that sums up the key data from Table 2 for faster direct comparison between the mutant lines.2) Make a direct link between I-domain ligand binding and phenotype rather than implying this is the reason for the observed effects. The suggestion of performing the gliding assays on different substrates, and especially on known ligands of the swapped I-domains, is a good one and would be informative.Ligand binding by TRAP is indeed a key question for sporozoite biology. In the literature several potential ligands of TRAP are described such as proteoglycans and Fetuin-A on hepatocytes, saglin on salivary glands and an integrin in the skin. This broad range of potential ligands indicates already that TRAP interacts promiscuously with different proteins on different tissues even if the interaction might be specific in each case. It is also important to point out that these putative ligands are only important for TRAP mediated invasion but not needed for TRAP mediated motility although the data for the integrin in the skin suggest a minor effect on in vivo motility .While productive movement is dependent on TRAP and, as we show in this manuscript, also requires the presence of the I-domain within TRAP no specific TRAP-ligand is needed to activate sporozoites. In fact sporozoites do glide even in RPMI medium that contains only amino acids without protein additives or even in PBS with just added glucose. Hence, addition of a protein like bovine serum albumin (BSA) boosts activation and/or adhesion of sporozoites and increases the percentage of gliding parasites. However, other added proteins show similar effects. Therefore testing ligand specificity of TRAP in a motility assay is unfortunately not trivial; a rather disappointing observation that cost us many hairs over the years.To illustrate this, we include TRAP-I, MIC2-I, αX-I and αL-I sporozoites:Evaluating substrate specificity of A.stephensi mosquitoes as described in Materials and methods. In brief, salivary glands were dissected and transferred into a plastic reaction tube containing 50 µL RPMI/PS. Salivary glands were ground with a pestle and centrifuged for 3min at 1000rpm at room temperature to separate freed sporozoites from mosquito tissue. Subsequently approximately 40µL of the supernatant was transferred into a new reaction tube. For the collection of hemolymph sporozoites infected A. stephensi mosquitoes were cut with a needle to remove the last segment of the abdomen. Subsequently the thorax was pierced with a finely drawn Pasteur pipette filled with RPMI/PS solution. While gently pressing the pipette the haemocoel cavity was flushed with solution which dripped off the abdomen. The sporozoite solution was collected on a piece of foil and transferred into a plastic reaction tube. Sporozoite concentration of salivary gland and hemolymph sporozoites was subsequently calculated using a hemocytometer. Afterwards sporozoites were diluted with RPMI/PS to achieve a volume of 50 µL per tested condition containing not less than 5000 sporozoites. Depending on the condition 50µL of sporozoite suspension was mixed with either 50µL RPMI containing 6% bovine serum albumin (BSA) or with 50µL peptide solution for activation. Peptides were ordered as solid substrates and solubilized in water to generate stocks with a final concentration of 10mg/mL. Using RPMI/PS the stock solution was diluted to an initial concentration of 2mg/mL to achieve a final dilution of 1mg/mL after addition of sporozoites. Alongside to BSA and peptides, six different coatings were tested for sporozoite motility. Coated wells were loaded with 50 µL sporozoite solution that was mixed with an equal volume of RPMI/PS. As a negative control sporozoites were allowed to glide in an uncoated well in RPMI/PS without any addition of peptides or BSA. To ensure the same conditions for each gliding assay, samples were prepared in 3min intervals. To allow sporozoites to attach to the optical bottom, samples were centrifuged at 800rpm for 3min before imaging. Using fluorescent microscopy  videos were recorded with three seconds per frame for 3min. Subsequently data were analyzed using ImageJ.We tested gliding motility in vitro for sporozoites of the four parasite lines TRAP-I, TRAP-MIC2, TRAP-αX and TRAP-αL in 96-well plates coated with heparin, ICAM-I, laminin, fibronectin and collagen. We included also a library of seven differently charged heptapeptides that we had available in our laboratory. As positive control we made use of 3% bovine serum albumin (BSA) and as negative control we tested RPMI medium mixed with penicillin and streptomycin that was used as base in all conditions. Hemolymph and salivary gland sporozoites were obtained by dissecting infected In contrast to the intuitive hypothesis that known ligands of the human integrin I-domains should activate sporozoite motility we were not able to measure a significant effect of a specific ligand. aL-I sporozoites and aX-I sporozoites isolated from the hemolymph were either not able to glide productively or showed gliding at very low percentages (1/100-200 sporozoites). Control sporozoites from salivary glands showed about 50% of actively gliding sporozoites in BSA while already about 10% of sporozoites were gliding in the negative control (RPMI medium only). In other conditions sporozoite activation was lower than with BSA but mostly above 10%. We couldn’t observe any significant differences in activation between different compounds other than BSA.While these results were disappointing at first they agree with published observation of sporozoite activation .3) Establish that the I-domains retain their folding and binding functions in the context of the chimeric trap.We agree with the reviewers that this is an important question. Naturally, verification of protein folding in vivo is a difficult task. We already considered the suggestion of reviewer 3 to do immunofluorescence with I-domain specific antibodies during our studies. Indeed there are antibodies targeting integrins available that target the I-domain of human integrin aX and aL available. Yet we feel that a proper positive and negative control would necessitate further parasite lines such as a TRAP-aX line with a targeted mutation that disrupts the conformation or epitope.As an alternative to generating more lines that could be less infective as the ones at hand, we performed more infection experiments. These showed that aX can rescue sporozoite infectivity to mice at a rather high level, while aL could not (only one infected mouse). In addition, we performed additional experiments with the RevCharge mutant, which revealed an interesting functionality: the RevCharge mutant infects salivary glands normally, yet it shows a severe impairment in gliding motility and infectivity of mice. This suggests that the folding of the protein must be correct yet, it cannot fully function in the mammalian host. In the aX line we observed invasion of salivary glands at very low rates as well as little (but clearly detectable) ability to actively glide in circles (Video 2). However, injected hemolymph sporozoites caused infections in more than 50% of all treated mice . This suggests that the I-domain is partly functional in this mutant. Hence, one domain (RevCharge) allows salivary gland invasion, while both allow some degree of mice infection. All these observations document a restored function of TRAP that could not be observed if the I-domain is not at least partially functional. We performed also western blot experiments with hemolymph sporozoites of all lines that could not enter the salivary gland showing that TRAP is expressed at comparable levels – we found similar results in midgut sporozoites but these experiments suffered from degradations and are thus not shown. These data are now added to Figure 3.Taken together, the data now suggest that TRAP is functionally folded in the TRAP-I, MIC-I, TRAP-RevCharge and the aX-I line while we cannot be sure if this is true for the aL line. We therefore changed the wording across the manuscript to point out that folding of the I-domain could have affected the phenotype observed in aL parasites.Reviewer #1:In this study, Klug and colleagues report on the role of the integrin I-domain of TRAP, an important adhesin expressed by the sporozoite stage of the malaria parasite. Previous studies have shown that TRAP is required for sporozoite motility and invasion of the salivary gland in the mosquito and of the liver in the mammalian host. A metal ion-dependent adhesion site (MIDAS) within the I-domain of TRAP is known to play a role in sporozoite infectivity, however the functional and adhesive properties of the I-domain remain undefined. More generally, the mechanisms of sporozoite infection are poorly understood at the molecular level, so this study addresses an important topic.To investigate the function of TRAP I-domain, the authors generated a number of mutants in P. berghei and performed robust phenotype analysis including gliding motility assays and analysis of sporozoite infectivity in the mosquito  and in the mouse (invasion of the liver). The work is very well performed and includes a large body of data, combining genetic approaches, in vitro and in vivo experiments. The paper is clearly written and the conclusions drawn from the results generally justified.The data convincingly show that:a) Sporozoites expressing a truncated TRAP lacking the I-domain are non-motile and do not invade the mosquito salivary glands and the mouse liver.b) Replacement of TRAP I-domain with the I-domain from Toxoplasma MIC2 results in normal invasion of salivary glands and normal sporozoite infectivity in mice, although gliding motility is reduced.c) Replacement of TRAP I-domain with the I-domain from human aX or aL integrin results in a severe reduction of sporozoite infectivity and gliding motility, although residual infectivity was observed in both mosquito and mouse.d) Introduction of 7 point mutations in the I-domain of TRAP, to replace positively charged residues (H/K/R) by negatively charged residues (E/D), results in normal invasion of mosquito salivary glands yet reduced motility and slightly reduced infectivity in the mouse.Based on the presented data, the authors conclude that the adhesive properties of the I-domain of TRAP can be partially fulfilled by the I-domain from other organisms, and that the cationic charge and the polyspecificity of the I-domain regulates sporozoite infectivity. However, the main limitation of the study is that adhesion and ligand binding were not directly assessed, so the aforementioned conclusions are based on indirect evidence or speculation. It is important to experimentally address the adhesive properties of the chimeric TRAP proteins expressed in the mutant parasites.Major points1) The authors conclude that the observed phenotypic defects are due to altered ligand binding properties of mutated TRAP proteins. However this was not addressed experimentally. Other modifications in the mutants, such as an alteration of the conformation of TRAP, could also contribute to the observed phenotype, unrelated with TRAP-I adhesive properties. In the absence of direct experimental evidence, it seems too speculative to conclude that the polyspecificity of the I-domain regulates sporozoite infectivity. The data clearly shows that MIC2-I almost completely complement TRAP-I functions, whilst aX-I and aL-I are poorly functional in this context. TRAP-I is basic (like aX-I), but MIC2-I is acidic (like aL-I). The data therefore show no correlation between infectivity and charges, and thus do not support an important role of the I-domain charges. Other factors may explain why MIC2-I is functionally superior to the I-domain from humain integrins, including the size of the domain size . The authors cannot exclude that a smaller I-domain may affect the conformation of TRAP protein, including other parts of the proteins such as the β ribbon. Surprisingly, the data also show no direct correlation between gliding motility and infectivity, since MIC2 and RevCharge mutants show severely reduced motility yet invade almost normally the mosquito and the mouse. This questions the relevance of the gliding assay. How do sporozoites with reduced gliding motility invade mosquito salivary glands and mouse cells?We agree with the reviewer that there is no clear correlation between the overall surface charge of the I-domains in general. However, the correlation between phenotype and charge becomes more significant in the RevCharge mutant where the charges in the perimeter of the MIDAS motif are altered in an otherwise unchanged TRAP. To avoid that the reader is misled we changed the wording along the manuscript to highlight this difference more clearly.The RevCharge mutant allowed some striking observations. This mutant shows a strong phenotype in gliding motility in vitro and an uncoupling of salivary gland and mouse infection. The RevCharge sporozoites enter normally into salivary glands but are much less infectious to mice. This highlights that the charge around the MIDAS must be important in the skin and/or liver and less so for salivary gland invasion. However, we refrain from overinterpreting this data towards explaining the results obtained with the other I-domain swaps.P. falciparum sporozoites do not glide in this standard assay used for rodent-infecting Plasmodium sporozoites, while their motility in the skin (ie in 3D) is very similar . This, however doesn’t diminish the use of the gliding assay. On the contrary, this simple and highly quantitative assay allows to detect minor but nevertheless interesting phenotypes that will help us to understand how the proteins of the parasite act together to ensure efficient gliding . Please think of the migration assays used for mammalian cells, most of which are similarly simple. They are used to dissect the molecular mechanisms governing cell migration. Often effects of a single gene deletion/mutation can lead to a better understanding of the mechanics while they have little consequence when observed in vivo.The value (and problem) of the gliding assay on glass is often misunderstood. The striking phenomenon that sporozoites cannot glide in vitro but invade just fine in vivo has most likely to do with the two-dimensionality of the gliding assay as we also discussed in Bane et al., 2016, where the coronin(-) mutant showed a similar phenotype: highly compromised gliding but no difference in skin migration. Also Maybe a simple way to address these issues would be to perform gliding assays not merely on glass slides, as performed here, but on specific substrates, including known ligands of MIC2, aX or aL integrins. This would strengthen the conclusion that the effects of the I-domain modifications are really due to changes in the adhesional properties of the chimeric proteins. For example, do sporozoites expressing the aL-I domain recover gliding motility on ICAM1?Completely agreed: Please do see our answer to point 2 above. We are currently working on establishing assays that use substrates of different stiffness in both 2D and 3D.2) The phenotype of the RevCharge mutant should be more thoroughly investigated to better document a defect in sporozoite infectivity for the liver. Figure 6F shows a partial reduction of parasite fitness in the mouse, which could be quantified more precisely. In vitro assays in HepG2 cultures, as performed with the MIC2 mutant, should be included. This is important to strengthen or invalidate the hypothesis that charges in the I-domain play an important role in sporozoite infectivity.We performed an invasion assay as well as a liver stage assay of the RevCharge mutant compared to the control line TRAP-I. The invasion capacity of the RevCharge mutant is severely impaired while liver stage development seems to be unaffected similar to the results obtained for the MIC2-I mutant. The new data are presented in Figure 6—figure supplement 1 . We also added more in vivo infection experiments with the RevCharge mutant, which are reported in Tables 2 and 3.E. coli were used, which may differentially affect the level of expression of the proteins. Why using E. coli optimized sequences?3) The authors checked by immunofluorescence that TRAP is expressed in the mutant sporozoites. However, they should also provide evidence that the level of expression of TRAP is unaffected in the mutants, ideally using Western Blot as shown for the RevCharge mutant. This is particularly important as sequences codon-optimized for We included now a western blot of hemolymph derived sporozoites from all chimeras that do not enter salivary glands in sufficient amounts in Figure 3C. We also include a Western blot comparing WT with MIC2 from salivary gland sporozoites . This shows that TRAP expression is similar to the control (TRAP-I). Please note that we could not detect any phenotypic difference between parasite lines expressing the codon-optimized TRAP (TRAP-I) and wild type sporozoites . We mentioned this now at several occasions in the manuscript.P. falciparum was published but not yet implemented in common online tools for codon usage modification. Therefore one possibility would have been to modify the introduced sequences from Homo sapiens and Toxoplasma manually according to the codon usage of P. falciparum leaving the remaining part of the open reading frame unchanged. We decided not to do so because we were unsure how similar the codon usage of P. falciparum and P. berghei really is. To avoid a change in codon usage from one species to another within a single ORF we codon optimized all chimeric sequences for E. coli K12 that we used to optimize genes in previous studies  observing no effect on protein expression. However, for some other genes, we noted that codon modification can have dramatic effects on expression levels . Hence the western blots are indeed essential.At the time the study was designed only the codon usage of Reviewer #2:The manuscript by Klug et al. investigates the biochemical and functional properties of I-domains by genetically replacing the I-domain within an important malaria parasite ligand called TRAP with I domains taken from another parasite (T. gondii) ligand, and two from human integrins. Plasmodium parasites lacking either the entire TRAP locus or just the I-domain of TRAP do not move, or invade the vector salivary glands. The authors find that the exogenous I domains from different sources can, to varying degrees, complement TRAP function resulting in the partial rescue of some motility, and ability to invade. The authors notice that the surface charges of TRAP in the rodent parasite P. berghei are cationic and to determine if this is important for function, mutate these residues to acidic amino acids and show again that the functions of TRAP are impaired.The manuscript is very nicely put together with high quality data, well-prepared figures, and the writing is clear. Overall, I found this a rather strange study where the authors have used the phenotyping available in the Plasmodium berghei rodent infection model to investigate general properties of I domains from different proteins and organisms.Plasmodium in order to discover specific functionality during the complex migrations of the sporozoite with the hope that this could help in defining receptor ligand interactions. We hence made use of those domains found in the distantly related apicomplexan parasite T. gondii and the very distant human integrins. Of course studying human integrin domains could be done more easily in mammalian cell culture.Thank you for this summary of our manuscript. We are glad that the reviewer recognizes our study as unique. The rationale of the experiments was to study the function of I-domains of Major pointsThe authors take several precautions to try to ensure the I-domains retain their function when transplanted in the Plasmodium TRAP sequence by taking structural considerations into account and showing that the proteins are detectable using anti-TRAP antibodies in the transgenic parasites. A key point in my mind is whether the human integrin I-domains really do complement the function of the I-domain in TRAP. In the manuscript, the authors show that the I-domain mutant is a null  and that the human integrin I -domain replacement parasites are very strong hypomorphs ; however, there does not appear to be a direct side-by-side comparison – e.g. injecting 25,000 HL sporozoites of the TRAP I-domain deletion alongside the integrin replacements in Figure 3—figure supplement 3B, for example. Also, are the authors able to demonstrate that the inserted I-domains retain their functions from their original proteins – is there a known ligand for the inserted integrin I-domains?We have now added these experiments as described above in our response to the editor.For the revCharge mutant, the authors imply that the loss of motility and ability to infect mice is due to the alteration in I-domain ligand binding, but could not an alternative explanation be that a proportion of the protein no longer folds correctly or that interactions with other parasite proteins necessary for full function are altered? The authors suggest the role of the I-domain is to interact with the host substrate, but these are not defined in the manuscript making it conceptually a rather large leap to link the mutations made in the protein to the phenotypes seen in the parasite.We also addressed this question in the response to the editor.Reviewer #3:In this study the authors swap integrin "I" domains from MIC2 and mammalian integrins with the I-domain of TRAP, the key motility adhesin of malaria sporozoites. Though the domain structure of the extracellular portion of TRAP has long been known to include an integrin-like I-domain, previous mutational studies of this domain were not that informative and the idea to perform domain-swapping experiments between Plasmodium, Toxoplasma and mammalian integrin I-domains is creative and potentially informative. Overall, the MIC2 swapped TRAP and the charge-altered TRAP are the most informative data while the conclusions drawn from the a-L and a-X swapped mutants are not justified by the data shown.We thank the reviewer for the constructive critique and hope that he/she agrees that the newly added data in Figure 6 and Table 3 improved the conclusions for both RevCharge and human integrin domains.1) The human integrin I-domain swapped mutants essentially behave like the TRAP KO. There is an elegant explanation in the Discussion as to why the a-L and a-X domains may not open and close in the same way as the I-domains of TRAP and MIC2. However, in the face of no data to support this or another hypothesis, it is equally plausible that there is some significant misfolding on the TRAP with a mammalian I domain. It would be important to show that this is not the case. Since there are commercially available antibodies to human integrins, can they be used, especially ones specific for conformational epitopes, to confirm the overall folding of the a-L and a-X domains.As described above in our response to reviewer 1, we have added additional infection experiments to Table 2 and the newly added Table 3 that support the folding and functionality of the integrin αX I domain. We did not use the specific antibodies as we feel that for proper controls we would have to generate new parasite lines with mutations in the respective epitopes. We also have preliminary data that conformationally trapped TRAP I-domains influence in vivo and in vitro motility and infectivity to similar degrees as those observed for aX I-domains. However, much more work needs to be done before these experiments will be conclusive and could be published and it would hence go beyond the scope of this manuscript to include those data.2) The data shown for the a-L and a-X mutants do not support the conclusions. These mutants have less than 200 sporozoites in their salivary glands, the a-L mutant is not motile and not infectious while the a-X mutant is minimally motile and very poorly infectious after IV inoculation. These data look like the TRAP KO , where they observed 100 to 200 sporozoites in the salivary glands and had a few infected mice when sporozoites were inoculated IV. Given this, how can they state: "Parasites expressing TRAP with I-domains from other organisms including humans can have nearly intact motility and invasion.” Or "…I-domain of TRAP is required to mediate adhesional properties which can be partially preserved when the native I-domain is replaced by I-domains from human integrins…". These statements are in the Introduction and Abstract but additionally, a central theme of the Discussion is that the a-L and a-X mutants are sufficiently intact to draw meaningful conclusions.Thanks for this remark, which partly reflects a long discussion we had while putting together the manuscript. Based on the new in vivo results (Tables 2 and 3) we agree with the reviewer that the aL-I domain is most likely nonfunctional and changed the wording across the manuscript accordingly; please also see our comments above.There are some differences between our study and Sultan et al., 1997 that could explain different results obtained for infectivity and numbers of sporozoites observed in the salivary glands. One explanation is the wild type ANKA background used in our study compared to the NK65 strain used by Sultan et al. Strain-specific differences have been often observed in malaria research and might also explain these subtle differences in salivary gland invasion and in vivo infectivity in absence of TRAP between different strains. Another explanation might be the different levels of infection from one insectary to another. Indeed, while we repeated the infections we obtained higher infection levels and in some of these infections some sporozoites could be seen in the salivary glands . The data is included in Table 2 and we modified our text accordingly.These lower numbers might also arise from the different design of the TRAP knockout lines. Sultan et al. generated a first knockout by single crossover integration into the coding sequence of TRAP. This design can cause the looping out of the selection marker thus restoring wild type parasites. As a consequence the authors might have observed low levels of salivary gland sporozoites (about 300-400 per infected mosquito). Indeed these sporozoites were able to infect mice and were shown by Southern blotting to be wild type parasites, i.e. they did indeed loop out the integrated sequence. To overcome this problem Sultan et al. created two additional knockout lines by double crossover integration to prevent unwanted recombination events. Unfortunately the homology arms that were chosen to initiate recombination are placed inside the open reading frame leaving the coding sequence for the N- and C-terminus of TRAP intact potentially leading to some activity that could explain why numbers for salivary gland sporozoites increased in both lines about 2-fold  compared to the single crossover knockout and why sporozoites are still infective if injected in high numbers. Alternatively, this difference is due to the variability of infection as we noticed. The important fact is that the lines show at least 90% reduction in salivary gland infection.In contrast to this approach the design for the TRAP knockout in our study excludes most of the coding sequence including the start codon to prevent any residual transcriptional activity. We couldn’t observe any productive motility and any infectivity of this line independent if 500k midgut sporozoites or 25k hemolymph sporozoites were injected.3) TRAP expression in mutant lines is verified by immunofluorescence that is not quantified. Since decreased levels of mutant TRAP could have phenotypes due to the mutation or to expression level, its critical to have western blot verification of mutant TRAP expression compared to WT TRAP expression, in all lines analyzed.To exclude that differences in protein expression between the different lines are affecting the phenotype we performed western blotting on hemolymph sporozoites of those lines that did not enter the salivary glands. This showed similar TRAP expression in all mutants. The new western blot results were implemented in Figure 3.[Editors’ note: what follows is the authors’ response to the second round of review.]Revisions for this paper:One major reservations of the previous iteration of this study was that the conclusions drawn from the parasites in which the TRAP I-domain was replaced by the human a-L and a-X integrin domains were not justified by the data shown and this has not been corrected in the revised manuscript. Indeed, salivary gland sporozoite numbers of the a-L and a-X mutants are in the same range as the TRAPΔI and TRAP KO parasites, with only the prevalence of infection somewhat increased in the a-X parasites. Infectivity studies demonstrate that the a-L parasites are similar to the TRAPΔI and TRAP KO parasites. With numerous careful experiments they that show the a-X parasites are more infectious than the a-L, TRAPΔI and TRAP KO parasites, and this interesting finding suggests that the charge on a-X may enable the mutant TRAP to have some minimal activity. However, their data show that the a-X sporozoites are about 100-fold decreased in their infectivity compared to controls. It has been previously demonstrated that a one day delay in patency is equivalent to a 10-fold decrease in infection. When 10,000 hemolymph sporozoites are inoculated IV, 100% of control mice are infected with a prepatent period of 4 days. With the a-X parasites only 50% of mice are infected and the prepatent period is 6 days. Thus, even if one ignores the fact that half the mice were not infected, the ones that did get infected had a 100-fold lower functional inoculum.Characterization of this significant attenuation of infectivity of the a-X parasites in mosquito and mammalian hosts as "partial restoration" of TRAP function is misleading. Indeed given the phenotype they describe, it is clear that the a-X parasites cannot be naturally transmitted for a single life cycle. Thus, the paper should focus on the interesting phenotypes of the MIC2 and reverse charge sporozoites, and only compare the a-X parasites to the a-L and TRAP KO parasites where there is a small marginal increase in infectivity supporting the importance of charge in the TRAP I-domain.We now reworded all remaining overstatements accordingly and also corrected one sentence that still overstated the rescue effect of aX.The following statements in the paper should be deleted, or changed to only include the MIC-2 mutant.Title:The current title implies that human I domains confer some functionality to TRAP which, given that these mutants have salivary gland sporozoite counts in the range of the TRAP KO, and infectivity levels that are 100-fold decreased, is misleading.We changed the title omitting the mention of human I domains and introducing the word “can” to show that not all I domains rescue, which now reads: Evolutionarily distant I domains can functionally replace the essential ligand-binding domain of Plasmodium TRAP. We also changed the Abstract to make sure that this is reflected. Please see that manuscript file with tracked changes.Abstract:"…its replacement with I domains from Toxoplasma MIC2 or the human integrin αX-subunit substantially or partially restores these functions".We completely rewrote the Abstract to put more emphasis. Doing so we removed “or the human integrin aX-subunit“."Results with TRAP, MIC2 and aX-I domains show remarkable interchangeability in their ability to engage with ligands in divergent tissues in arthropoa and chordata".This sentence only appeared in the initial submission and has already been corrected in the revised version. Yet, please note the re-written Abstract, where this statement is not included anymore."…results suggest that apicomplexan parasites appropriated I domains in part for their ability to recognize structurally diverse ligands"We removed “structurally diverse ligands” and modified the sentence/absrtract accordingly."…permit TRAP to function as an adhesin with diverse ligands in both vertebrate and arthropod hosts"The ability to engage with ligands is not addressed here, so wording should be revised. Also, the aX-I domain is poorly functional in TRAP, so "remarkable interchangeability" is not correct in this case.We removed “with diverse ligands”. The expression “remarkable interchangeability” is no longer part of the manuscript.Introduction:"Parasites expressing TRAP with I domains from other organisms, including humans show partially rescued motility and invasion."We removed “including humans” and modified the sentence accordingly.Results:"This could hint that the charge of αX could compensate for the long evolutionary distance between apicomplexa and mammals."This sentence was completely deleted.Discussion:Homo sapiens with only 28% and 18% amino acid sequence identity, respectively.""It was therefore remarkable that TRAP function was largely or partially restored by replacement of its I domain by homologues from Toxoplasma gondi and Homo sapiens” and rephrased the sentence accordingly.We removed “"TRAP function was largely or partially restored by replacement of its I domain". This is true for Toxoplasma MIC2 domain but not human integrin I domains.As before, this sentence was changed accordingly."…immunofluorescence showed normal localization in sporozoites, suggesting that they were properly folded".The immunofluorescence data provide no information on the folding of TRAP.We completely agree with the reviewer and changed “they were properly folded” to “trafficking was not impaired”."Infectivity of hemolymph αX-I and αL-I sporozoites in mice was decreased at least 4-fold and 15-fold, respectively." Infectivity in the a-L sporozoites was similar to the TRAP KO, and in the a-X sporozoites was 100-fold decreased compared to controls.We rephrased the sentence accordingly.“The findings that I domains from organisms that encounter none of the same ligands as TRAP can replace its function in mediating salivary gland invasion and infection of mice strongly suggest that the I domain functions to provide traction for motility and cell invasion rather than tissue tropism.” This should specify that they are referring to replacement with MIC2.We specified this sentence."…the I domain of TRAP is required for gliding motility and invasion by sporozoites of mosquito salivary glands and mammalian liver, without a requirement for evolved specificity".The data with the human I domains show that any I domain cannot confer function in TRAP, so this statement is not correct.We do think that this sentence is still correct considering that the common ancestor of Toxoplasma and Plasmodium is about 800 million year old. We did modify by including a “strong” such that it reads “…without a strong requirement…” to modulate our statement.The authors attempted to analyze the motility of mutant sporozoites on various substrates. They indicate that they "were not able to measure a significant effect of a specific ligand".However, Figure 1 shows that aX-I and aL-I activated on BSA are mostly floating, while on the other substrates sporozoites are mostly attached or display unproductive motility. This could be an indication that the human I-domains retained some adhesive properties. However, it would be useful to have the same type of data with the I domain-deleted mutant, and the threshold, as indicated in A, should also be shown on the other panels.Thanks for pointing this out. Indeed the differences in the amount of unproductive gliding sporozoites are interesting and could indicate minimal TRAP activity. We are planning to investigate this in more detail making further use of the TRAP mutant library we created. As explained in our last rebuttal, a proper analysis will require a functional linkage of the ligands to a surface. Therefore, we think that now testing of the I domain deletion mutant alone would be not advance our understanding significantly. Also, this would require a repeat of many experiments as the peptides would have to be reordered.We now include the threshold also in the panel showing gliding motility on different substrates with MIC2-I salivary gland sporozoites. We didn’t include a negative control (RPMI only) for gliding assays with hemolymph sporozoites because these sporozoites are difficult to activate even in the presence of the strong activating compound BSA . As a consequence a threshold is not indicated for these experiments.ReferencesJournal of Immunology , 152, pp. 4582–4589.Gao, C. et al. 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