Endoplasmic reticulum localized TMEM33 domain‐containing protein is crucial for all life cycle stages of the malaria parasite

Endoplasmic reticulum (ER) plays a pivotal role in the regulation of stress responses in multiple eukaryotic cells. However, little is known about the effector mechanisms that regulate stress responses in ER of the malaria parasite. Herein, we aimed to identify the importance of a transmembrane protein 33 (TMEM33)‐domain‐containing protein in life cycle of the rodent malaria parasite Plasmodium berghei. TMEM33 is an ER membrane‐resident protein that is involved in regulating stress responses in various eukaryotic cells. A C‐terminal tagged TMEM33 was localized in the ER throughout the blood and mosquito stages of development. Targeted deletion of TMEM33 confirmed its importance for asexual blood stages and ookinete development, in addition to its essential role for sporozoite infectivity in the mammalian host. Pilot scale analysis shows that the loss of TMEM33 results in the initiation of ER stress response and induction of autophagy. Our findings conclude an important role of TMEM33 in the development of all life cycle stages of the malaria parasite, which indicates its potential as an antimalarial target.

IRE1 and ATF6, are absent in Plasmodium spp.genome (Fennell et al., 2009;Ward et al., 2004), the role of UPR and its activation by PERK homolog, PK4, in Artemisinin-induced parasite death has been reported (Bridgford et al., 2018).Moreover, other important UPR regulators like GRP78 and eIF2α have also been investigated for antimalarial agent development (Chen et al., 2018;Zhang et al., 2017).It is known that in higher eukaryotes, UPR activation in response to ER stress may lead to the induction of apoptosis, autophagy, or resistance to chemotherapeutic drugs (Kim et al., 2008;Kroemer et al., 2010;Ogata et al., 2006;Qing et al., 2023), and to some extent in parasitic protozoa as well (Dolai & Adak, 2014;Goldshmidt et al., 2010;Lee et al., 2007;Tsiatsiani et al., 2011).Therefore, identification of new molecular players which regulate the ER functioning may help in the development of novel antimalarial therapy.
Transmembrane protein 33 (TMEM33) protein family, conserved from zebrafish to mammals, contains three transmembrane domains and is localized to ER in all higher eukaryotes investigated (Chadrin et al., 2010).This protein was identified as an important regulator of intracellular calcium homeostasis through interaction with polycistine-2 that results in translocation of cathepsins and sensitization to apoptosis in renal tubular epithelial cells (Arhatte et al., 2019).TMEM33 plays important role in Vascular Endothelial Growth Factor-a (VEGFa) mediated calcium translocation and promotes angiogenesis in both human and zebrafish epithelial cells (Savage et al., 2019).A recent study shows that TMEM33-PKM2 axis regulates SREBPs activation and lipid metabolism in cancer cells (Liu et al., 2021).TMEM33 is also identified as a prognostic marker for cervical cancer (Chen et al., 2022).Its role has also been identified in UPR regulation and induction of apoptosis and autophagy in breast cancer cells (Sakabe et al., 2015).In addition, TMEM33 was shown to be a reticulon-binding protein, suggesting a potential role in shaping the ER membrane structure (Urade et al., 2014).
Although the biological significance of TMEM33 is gradually revealed in various aspects of many eukaryotic cells, its biological role in Plasmodium spp. is totally unknown.Considering the importance of TMEM33 in regulating ER stress responses, the identification of its significance and function in the malaria parasite may help to develop TMEM33 as a potential target of the machinery regulating parasite stress responses.
Here in this study, we performed gene targeting studies of TMEM33 domain-containing protein (PlasmoDB ID: PBANKA_1003300) (https:// plasm odb.org/ ) in rodent malaria parasite Plasmodium berghei by generating a TMEM33 knockout strain [Pbtmem33(−)], and a transgenic strain expressing C-terminal tagged TMEM33-mNeonGreen fusion protein  to identify the role and cellular localization of this protein in different life cycle stages of the malaria parasite.Additionally, we have also established an enhanced GFP (eGFP) expressing wild-type (WT)-like strain, P.bergheip230p(−), as a model WT-like strain, by using similar approach to our previous P.yoeliip230p (−) as WT-like model strain (Hart et al., 2014).

| Phylogenetic analysis reveals that TMEM33 is highly conserved among all Plasmodium species
First, we checked the amino acid sequence similarity between PbTMEM33 of different malaria parasite species and other eukaryotes.Genome-wide analysis showed that TMEM33 is conserved in all malaria parasite species and is also conserved in other higher eukaryotes including human, plants, and fungi.The PbTMEM33 proteins shows ≥90% amino acid identity with other rodent malaria parasites and ≥65% similarity with human malaria parasites.However, with other studied eukaryotes including other Apicomplexa, mammals, plants, and fungi, PbTMEM33 showed low level conservation (Figure 1a).Domain analysis showed that P. falciparum and P. vivax, have four transmembrane domains like P. berghei; however, P. malariae, P. ovale, and P. knowlesi contain five transmembrane domains (Figure 1b).

Pbtmem33-mNG and PbWT
To determine the role of TMEM33 and p230p in parasite life cycle, a reverse genetics approach of gene knockout was used to generate TMEM33 and p230p deficient parasites in P. berghei ANKA.The PbTMEM33 and Pbp230p deficient parasites along with Pbtmem33-mNG tag parasites were generated using double crossover homologous recombination strategy.A donor plasmid, pAA20, containing the eGFP fluorescence protein cassette, and the human DHFR drug selection marker cassette, was used to clone the 5′ and 3′ UTR regions of P230p (Figure 2a) and TMEM33 (Figure 2b) genes.
Similarly, pAA32 plasmid that contains mNeonGreen fluorescence protein cassette, and the human DHFR drug selection marker cassette was used to clone downstream coding sequence and 3′ UTR regions of TMEM33 (Figure 2c).To confirm the genomic integration of knockout and knock-in constructs in the parasite genomes, and so the parasite cloning, diagnostic PCRs were performed.The diagnostic PCRs using gene-specific primers that amplify the 5′ and 3′ integration sites of knockout/knock-in constructs confirmed the integration of knockout/knock-in constructs into their chromosomal loci (Figure 2).
To quantitatively determine the effect of TMEM33 and P230p genes in asexual erythrocytic stage growth, we compared the blood stage parasitemia of Pbp230p(−), Pbtmem33(−) and Pbtmem33-mNG tagged parasite strains to PbWT parasites in mice.Groups of CD1 mice (n = 5 mice per genotype) were intravenously injected with 20,000 of Pbp230p(−), Pbtmem33(−), Pbtmem33-mNG, and PbWT parasite-infected erythrocytes (iRBC).Parasitemia was recorded daily by Giemsa-stained thin blood smears for 10 days postinfection (pi), since all infected mice except the mice infected with Pbtmem33(−) were dead after 10 days pi.All Mice infected with these different strains showed detectable blood-stage parasitemia by Giemsa-stained thin blood smears starting from day two pi.

| Pbtmem33(−) parasites are non-lethal to mice
Since Pbtmem33(−) parasites were severely growth attenuated, the next goal was to check its effect on the survival of infected mice.
Groups of CD1 mice (n = 5 mice per genotype) were intravenously injected with 20,000 of Pbp230p(−), Pbtmem33(−), Pbtmem33-mNG, and PbWT parasite-infected erythrocytes (iRBC), and the survival of mice was followed till Day 30 pi.The survival curve (Figure 3b) agrees with the parasitemia curve, where all PbWT, Pbp230p(−), and Pbtmem33-mNG injected mice survived maximum of 10-, 11-, and 12day pi, respectively.In contrast, all mice infected with Pbtmem33(−) survived till Day 23 pi.One mouse died on 23 days pi and two mice died on 24 days pi.The other two mice survived with positive parasitemia well past 30 days pi.

| TMEM33 does not affect gametocytogenesis and male gamete exflagellation in P. berghei
Though Pbtmem33(−) blood stages are growth attenuated, still their growth in blood stage made it possible to identify the role of TMEM33 in gametocyte formation, maturation, and gametogenesis.
For this phenotypic characterization, groups of mice (n = 4 mice for each genotype) were intravenously injected with 1.5 × 10 7 iRBC from Pbp230p(−), Pbtmem33(−), Pbtmem33-mNG, and PbWT.Percentage of male and female gametocytes in all four genotypes was recorded by Giemsa-stained thin blood smears at Day 3 pi.The male gamete exflagellation events were counted by diluting blood from the same mice with incomplete ookinete medium (containing RPMI, sodium F I G U R E 1 Conservation of TMEM33 in the malaria parasite and in higher eukaryotes.(a) Phylogenetic analysis and amino acid identity percentages of TMEM33 in different apicomplexan parasites and higher eukaryotes shows that TMEM33 is highly conserved among all malaria parasite species, but different than other parasitic protozoa or higher eukaryotes.(b) Schematic representation of number and position of TMEM33 transmembrane domains.

| TMEM33 plays an important role in ookinete development
The role of TMEM33 in P. berghei ookinete development was followed by in vitro ookinete development assay.Here the 1.5 × 10 7 iRBC injected mice were bled and cultured into complete ookinete medium at 20-21°C for 24 h.The developed ookinetes were harvested and counted using hemocytometer.No significant difference in the ookinete numbers was observed between Pbp230p(−), Pbtmem33-mNG, and PbWT cultured parasites; however, Pbtmem33(−) parasites showed a roughly 50% reduction in ookinete numbers in comparison to PbWT (Figure 4a).This result shows that TMEM33 is crucial in ookinete development.Hence it is very important for mosquitostage infection of P. berghei ANKA.The mNeonGreen tagged parasites were generated by double cross over homologous recombination to introduce fragment containing the coding sequence of targeted gene (minus stop codon) followed by mNeonGreen-Twin strep tag.Right hand panel shows PCR genotyping which confirmed the site-specific integration and generation of recombinant parasites.PCR amplification using 3′ and 5′ integration-specific primers confirmed the integration of gene deletion and tagging constructs to generate Pbp230p(−), Pbtmem33(−), and Pbtmem33-mNG Tag parasites, respectively.Locations of PCR products on the target loci were shown as black lines on the left-hand panel.No bands were amplified from WT gDNA using integration-specific primers (KO: Knockout, KN: Knock-in or tagging).

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gland sporozoites of P. berghei.Overnight starved female Anopheles stephensi mosquitoes were blood-fed on highly exflagellated Pbp230p(−), Pbtmem33(−), Pbtmem33-mNG, and PbWT parasites-infected mice and the pervasiveness of mosquito stage infection was determined.The number of oocyst and oocyst sporozoites were counted on 15th day, whereas the number of salivary glands sporozoites were counted on 21st day post blood feeding.Here, the results agreed with the ookinete numbers, as the number of oocyst, oocyst sporozoites, and salivary gland sporozoites showed a nonsignificant difference among the Pbp230p(−), Pbtmem33-mNG, and PbWT parasites-infected mosquitoes.
However, the oocyst, oocyst sporozoites, and salivary gland sporozoite numbers were highly reduced in Pbtmem33(−) parasite-infected mosquitoes as compared to PbWT and WT-like parasites-infected mosquitoes (Figure 4b-d).Collectively, these results confirmed that TMEM33 is crucial in mosquito-stage infection of P. berghei.However, this significant reduction in the numbers of these stages could be due to the highly reduced numbers of ookinetes, not due to the crucial biological role of TMEM33 in these stages.

| TMEM33 plays an important role in mice infection by salivary gland sporozoites
To assess the impact of TMEM33 deletion on sporozoite infectivity, mice were intravenously injected with Day 21 salivary gland sporozoites.On Day 21 postinfectious blood feeding, salivary glands of infected mosquitoes for all three genotypes (Pbp230p(−), Pbtmem33(−), and PbWT parasite strains) were dissected, salivary gland sporozoites were extracted, and intravenously injected into CD1 mice in two different doses: 20,000 and 50,000 sporozoites, in 150 μL of incomplete RPMI media supplemented with 3%BSA.
Sporozoite infectivity was determined by evaluating the blood F I G U R E 3 Targeted deletion of TMEM33 caused severe reduction in asexual blood stage growth and abolished pathogenicity but did not affect gametocytogenesis or male gamete exflagellation.(a) CD1 mice (5 mice per group) were intravenously injected with 20,000 infected erythrocytes (iRBC).The parasitemia was followed by Giemsa staining of thin blood smears until death of PbWT-infected mice.The 10 days follow-up of parasitemia confirmed that TMEM33 deletion affected the blood stage growth of Plasmodium berghei ANKA; however, P230p deletion and TMEM33 tagging did not have any significant effect on blood stage parasitemia as compared to WT.  1).The results show that TMEM33 plays an important role in sporozoite infectivity of P. berghei to the mammalian host.

| Absence of TMEM33 does not affect sporozoite motility
To further analyze the effect of TMEM33 deletion on sporozoites, we observed the sporozoite motility of Pbtmem33(−) (Video S1) and PbWT (Video S2) parasites under the confocal microscope.Timelapse images showed that Pbtmem33(−) parasites were capable of gliding without any visible defect as compared to the wild-type P. berghei sporozoites (Figure 5).

| PbTMEM33 is localized to the endoplasmic reticulum
After identifying the importance of TMEM33 in different life cycle stages of the malaria parasite, our next goal was to check the expression and cellular localization of TMEM33 in P. berghei ANKA.
To examine the expression and assess the cellular localization of  -d) Graphs shows that the mosquito stage infectivity is significantly affected in parasites lacking TMEM33; however, P230p deletion did not show any significant effect on mosquito stage infectivity as compared to WT control.10-, 15-, and 21-days pi, mosquitoes were dissected, and average number of oocysts, oocyst sporozoites, and salivary gland sporozoites were counted, respectively.Statistical analysis was performed by one-way ANOVA, and statistical significance was set at p < 0.05.n > 30 mosquitoes for each data point.Nonsignificant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001 versus WT control.
antibody confirmed its presence in the integral membrane fraction, which further confirmed the ER localization (Figure S1).

| TMEM33 deficiency upregulates mRNA expression of autophagy-related genes
We first verified the absence of TMEM33 transcripts in Pbtmem33(-) parasites by RT-PCR (Figure S2a).To probe the effects of TMEM33 deficiency, we later checked the transcription levels of ER stress (BiP), autophagy (ATG5, ATG8, ATG12), and apoptosis (MCA1, MCA2, MCA3) related genes.We observed over two-fold increase on BiP, and almost three-fold increase on autophagy-related genes, but no significant changes in metacaspase genes (Figure S2b-d).

| DISCUSS ION
ER is a vital organelle for processing and synthesis of secretory and membrane proteins, lipid synthesis, and calcium storage which make it important to maintain ER homeostasis (Dolai & Adak, 2014).
Imbalance of ER homeostasis results in generation of ER stress, which consequently results in activation of two important pathways: Endoplasmic Reticulum Associated Death pathway (ERAD) and Unfolded Protein Response pathway (UPR) (Bukau et al., 2006).
UPR induction results in the increased expression of ER chaperone proteins which leads the degradation of misfolded proteins within the ER.In mammalian cells IRE1, PERK and ATF6 are the main regulators of UPR (Hwang & Qi, 2018).An important ER chaperone GRP78 (Glucose-regulated protein 78), also known as BiP (immunoglobulin heavy chain binding protein) regulates the UPR by either binding with IRE1, PERK and ATF6 in unstressed cells, or binding with unfolded or misfolded proteins in stressed cells (Bertolotti et al., 2000;Grootjans et al., 2016;Shen et al., 2002).
The role of ER stress and UPR as target pathways in antimalarial drug development has been reported by a number of studies.
Since ER localized proteins play important role in maintaining ER homeostasis, the identification of novel proteins that regulate ER functioning will help in the development of effective antimalarial therapy.Here in this study, we aimed to identify the importance of a new ER localized protein in P. berghei life cycle by generating Pbtmem33(−) parasitic strain.We explored the role of TMEM33 domain-containing protein, PBANKA_1003300, at different life cycle stages of parasites in mammalian and mosquito hosts.This protein is designated as TMEM33 in humans and other eukaryotes which is stress inducible ER transmembrane protein and plays important role in UPR regulation (Lu et al., 2021;Sakabe et al., 2015).
The TMEM33 expression has been identified as the regulator of UPR response elements where the overexpression of TMEM33 results in increased expression of p-eIF2α and p-IRE1α and their

Groups Strains
No. of sporozoites

Blood stage infection
Pre-patent period downstream regulators in breast cancer cells (Sakabe et al., 2015).
Importantly ER stress response elements and UPR regulators are identified to play important role in blood stage infection and mosquito transmission of the malaria parasite.The role of mammalian PERK homolog, PK4 (member of eIF2α kinase family), is identified in red blood cell invasion of merozoites, and it was shown that PK4 deletion in P. berghei alleviates malaria symptoms (Zhang et al., 2012).
Another study showed that a PK4 inhibitor GSK2606414 inhibits the transformation of P. falciparum trophozoites into schizonts (Zhang et al., 2017).
As a transmembrane protein, TMEM33 is not well conserved across higher organisms.Amino acid identity matrix shows very low conservation levels even within Apicomplexa (Figure S3), with the exception of hydrophobic domains (Figure S4) which is also observed in many transmembrane proteins (Johnston et al., 1989;Sojo et al., 2016).Earlier studies confirmed the ER localization of TMEM33 in higher eukaryotic cells.TMEM33 was previously shown in heavy membrane fraction of breast cancer cell lysate (Sakabe et al., 2015), exogenous expression of TMEM33 showed it as an ER and nuclear envelope protein (Urade et al., 2014), Zebrafish TMEM33 protein expressed in ECL cells was shown to be localized in ER (Lu et al., 2021), and POM33 a member of the same family in yeast was identified as integral membrane protein (Chadrin et al., 2010).In consistent with these results, our cellular fractionation analysis of Pbtmem33-mNG showed TMEM33 as an integral membrane protein (Figure S1), and co-localization experiments by confocal microscopy showed the ER localization of TMEM33 in P. berghei.Recently, in a comprehensive Toxoplasma gondii proteome study, by using HyperLOPIT method, T.
gondii homolog of TMEM33 (TGME49_270270) was shown to cofractionate with ER1 class of proteins which are integral membrane proteins (Barylyuk et al., 2020).Collectively, these results support the idea that TMEM33 domain-containing protein of Plasmodium has the same localization, and possibly the similar roles with its eukaryotic counterparts as an integral ER membrane protein.
Considering the importance of ER homeostasis on parasite growth, the growth profile of Pbtmem33(−) parasites in blood might be due to the imbalance in ER homeostasis.It might also point out to preferential parasitization of reticulocytes.P. berghei has ̴ 150 times more preference to reticulocytes than erythrocytes (Cromer et al., 2006).Reticulocyte metabolome may help rescuing deficiencies in parasite metabolism (Srivastava et al., 2015).More experiments are required in the follow-up studies to probe these possibilities.In contrast with asexual growth, there was no apparent effect on Pbtmem33(−) parasite gametocytogenesis and gametogenesis.This might also be due to the reason that for the first 4 days of parasitemia analysis, there was no significant difference in the parasitemia between all 4 strains.The difference in parasitemia started after 5 day pi, where Pbtmem33(−) had grown slower than wild-type parasites.However, for the gametocyte analysis, the mice were inoculated with extremely high parasite numbers, and parasites were allowed to grow for only 3 days.
The effect of TMEM33 deletion on the development of ookinetes were evaluated by in vitro ookinete development assay, in which ookinete development was found to be significantly inhibited.
Consequently, the number of oocysts, oocyst sporozoites, and salivary gland sporozoites were also significantly lower in Pbtmem33(−) parasites-infected mosquitoes as compared to PbWT parasitesinfected mosquitoes.ER stress-induced phosphorylation of eIF2α was shown in the global inhibition of translation that leads to gametocytogenesis (Chaubey et al., 2014), or gametocyte conversion into ookinetes in P. berghei (Duran-Bedolla et al., 2017).Reduced ookinete development in the absence of TMEM33 supports the connection between ER stress and TMEM33 deficiency.
Following mosquito stages, infectivity of Pbtmem33(−) sporozoites were evaluated in vivo by intravenous injection of extreme number of sporozoites into C57BL/6 mice which yielded no blood stage parasitemia.To understand strong liver stages attenuation of Pbtmem33(−) sporozoites, we subsequently observed sporozoite motility.Pbtmem33(−) sporozoites were completely capable of gliding motility as WT sporozoites.This suggests that the defect of liver stages infection is either in invasion of hepatocytes or in liver stages development.In addition to the potential role in ER homeostasis, TMEM33 was suggested to have a role in shaping the ER tubular structure as a reticulon-binding protein (Urade et al., 2014).During schizogony phase of liver stages, parasites undergo an extensive replication phase to produce up to 30,000 parasites in a very short window.In this replication phase, P. berghei ER generates extensive accumulations, forming intricate and dense networks of ER tubules possibly to cope with high levels of ER stress (Kaiser et al., 2016).
Considering both suggested roles of TMEM33, we suggest that the liver stages attenuation created by TMEM33 deficiency starts just before or during nuclear replications.
Since TMEM33 is localized into ER, and its absence is critical for blood stage parasitemia and mosquito stages, we performed a pilot scale analysis at mRNA level to get an idea about the effect of TMEM33 deletion on the generation of ER stress and regulation of autophagy and apoptosis.Though we have only evaluated the level of BiP mRNA, the upregulation in its expression can still be considered as an indicator of ER stress in Pbtmem33(−) parasites (Figure S2b).Since ER stress results in the activation of ERAD and UPR, which in turn regulates cell death pathways like apoptosis and autophagy (Peng et al., 2021), we checked the mRNA expression of autophagy (ATGs) and apoptosis (metacaspases) related genes (Kamil et al., 2019;Kamil, Atmaca, et al., 2022;Vandana et al., 2019).Our preliminary qPCR data showed that TMEM33 deletion results in the generation of ER stress which upregulated the expression of autophagy-related genes in the malaria parasites (Figure S2c); however, no effect was observed in the expression of apoptosis marker genes (Figure S2d).The overexpression of TMEM33 is shown to regulate UPR and induction of apoptosis and autophagy in breast cancer cells (Sakabe et al., 2015).Conversely, in our primary study, we found that TMEM33 deletion results in autophagy upregulation, There are different possibilities for this opposite results; mainly (i) Plasmodium lacks orthologues of important UPR regulators including XBP1, IRE1, ATF6, and ATF4, (Chaubey et al., 2014) and (ii) autophagy is a context-dependent pathway (Pandey et al., 2021).
In conclusion, with this study we highlighted the importance of TMEM33 at three important life cycle stages: (i) the blood stage parasitemia, (ii) rodent to mosquito host transmission, and (iii) mosquito to rodent host transmission.Our data showed that TMEM33 domain-containing protein of P. berghei is the Apicomplexan counterpart of human TMEM33 and has a significant effect on parasite survival with its vital role in ER homeostasis.Though here in this study, we did not directly dissect out the relationship between TMEM33, ERAD, and UPR, it will be interesting to identify TMEM33  Kamil, Deveci, et al., 2022).

| Analysis of amino acid sequences
PlasmoDB (Amos et al., 2022) and NCBI (Sayers et al., 2022) databases were used to acquire amino acid sequences, and BLAST tool (Altschul et al., 1990) was used for searching homologs.InterProScan database (Finn et al., 2016) was used for domain identification.
The sequences of the primer pairs for the preparation of donor DNA plasmid and for the integration-specific diagnostic PCR are listed in Table S1.PbWT were counted at 400x magnification (Deveci et al., 2022).

| Determination of blood stage development and male gamete exflagellation
At the same time, blood from tail puncture was thin smeared and stained with Giemsa to determine the ratio of male versus female gametocytes.

| In vitro ookinete quantification
Exflagellation-positive Pbp230p(−), Pbtmem33(−), Pbtmem33-mNG, and PbWT mice (n = 5) were bled by cardiac puncture, and 500 μL of blood was immediately mixed with 4.5 mL of complete ookinete culture medium in a 6-well tissue culture plate.The ookinete culture was incubated at 20-21°C for 24 h and the ookinetes were Development of oocysts, oocyst sporozoites, and salivary gland sporozoites are significantly reduced in Pbtmem33(−) Since ookinete development was significantly affected in Pbtmem33(−) parasites, the next goal was to check the role of TMEM33 in mosquito infection and development of oocyst, oocyst sporozoites, and salivary F I G U R E 2 Targeted deletion of P230p and TMEM33 and C-terminal tagging of TMEM33.Left-hand panels show schematic representation of gene targeting strategy.(a and b) Both gene loci were targeted for deletion by knockout (KO) plasmid constructs.After double cross over homologous recombination, the targeted gene's coding sequence was replaced by eGFP and hDHFR drug selection marker cassettes.(c) Cterminal mNeonGreen tagging of TMEM33.
(b) The Kaplan-Meier survival curve shows that the targeted deletion of TMEM33 in P. berghei ANKA severely impaired its pathogenicity in mice.Here the PbWT, Pbp230p(−) and Pbtmem33-mNG parasite iRBC injected mice survived maximum of 10-, 11-and 12-days pi, respectively, however, not a single death was observed in Pbtmem33(−) iRBC injected mice till Day 23 pi.(c-e) Average number of exflagellation centers per μL of mice blood, and gametocyte percentages of Pbtmem33(−), Pbp230p(−) and Pbtmem33-mNG shows that parasites undergo normal sexual blood stages differentiation compared to PbWT parasites.CD1 mice (4 mice per group) were intravenously injected with 1.5 × 10 7 iRBC.Sexual stages differentiation was observed for each genotype on Day 3 pi, which shows that TMEM33 or P230p deletion does not have any significant effect on sexual differentiation of P. berghei ANKA parasites.Statistical analysis was performed by one-way ANOVA, and statistical significance was set at p < 0.05.Nonsignificant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001 versus WT control.stage parasitemia by Giemsa stained thin blood smears starting at Day 3 pi.While PbWT and Pbp230p(−) infected mice showed parasitemia in thin blood smears on Day 4 pi, Pbtmem33(−) infected mice did not show parasitemia in any of the thin blood smears till Day 16 pi (Table TMEM33, we tagged C-terminal end of PbTMEM33 with mNeon-Green fluorescent protein containing twin-strep epitope tag and then analyzed mNeonGreen live fluorescence expression in blood and mosquito stages.A strong mNeonGreen signal was observed throughout all life cycle stages analyzed, with localization mostly appearing to be around the nucleus.Co-localization with ER-Tracker in sporozoites and with BIP in blood stages confirmed that TMEM33 is localized into the ER in both mosquito and blood stages of the parasite life cycle (Figure 6).Western blot analysis of the subcellular fractionation of blood stage parasites using anti-twin-strep monoclonal F I G U R E 4 Targeted Deletion of TMEM33 reduced mosquito stage infectivity.CD1 mice (5 mice per group) were intravenously injected with 1.5 × 10 7 infected erythrocytes (iRBC) from Pbtmem33(−), Pbp230p(−), Pbtmem33-mNG, and PbWT genotypes.Infected mice that showed the highest male gamete parasite exflagellation were either bled on Day 3 pi to do in vitro ookinete culture or fed to overnight starved mosquitos.(a) The hemocytometer counting of the in vitro cultured ookinetes showed that Pbtmem33(−) parasite strain is deficient in ookinete development, as the numbers of ookinetes were significantly reduced in Pbtmem33(−) in comparison to PbWT; however, in Pbp230p(−) and Pbtmem33-mNG, the ookinete numbers were similar to PbWT.(b Infectivity of genetically attenuated Pbtmem33(−)andPbp230p(−) salivary gland sporozoites using PbWT salivary gland sporozoites as a control.F I G U R E 5 TMEM33 deletion does not affect sporozoite motility.PbWT and Pbtmem33(−) salivary gland sporozoites were collected on Day 21.Sporozoites were incubated on ice for an hour, followed by at 37°C for 5 min in 3% BSA/DMEM.Sporozoites were subsequently imaged every second for 3 to 5 min under confocal microscope.No visible reduction was observed in the gliding motility abilities of sporozoites in time-lapse confocal microscopy imaging of Pbtmem33(−) as compared to WT.

F I G U R E 6
PbTMEM33 is localized in the Endoplasmic Reticulum (ER).The mNeonGreen fluorescent tagged Plasmodium berghei TMEM33 signals co-localized with ER stain ER-tracker red in sporozoites and BiP in blood stages, which confirmed the ER localization of PbTMEM33.Pbtmem33(−) and Pbp230p(−) were also used in immunofluorescence assay.Hoechst nuclear staining dye was used to stain the parasite nuclei (Scale bar 2 μm).

Frozen
stocks of Pbp230p(−), Pbtmem33(−), and    PbWT were injected intraperitoneally into three naive female CD1 as donor mice.The mice were bled, and 20,000 infected erythrocytes were prepared with incomplete RPMI media in doses of 150 μL, and each mouse (in groups of 5 mice for each genotype) received one dose intravenously.The parasitemia was monitored for 10 days postinfection (pi) by Giemsa stained thin blood smears and counting 50 fields (1000× magnification).To calculate exflagellation events, a group of 5 mice per genotype, each mouse was intravenously injected with 15,000,000 infected erythrocytes of Pbp230p(−), Pbtmem33(−), Pbtmem33-mNG and PbWT.On Day 3 pi, 2 μL of blood was collected from the tail vein of the mouse with a heparinized needle and immediately diluted (1:30) in incomplete ookinete medium (RPMI 1640 supplemented with 25 mM HEPES, 50 μg/mL hypoxanthine, 2 g/L NaHCO 3 , and 100 μM xanthurenic acid).The diluted blood samples were placed in a hemocytometer and incubated at room temperature for 10 min.The exflagellation centers in Pbp230p(−), Pbtmem33(−), and