5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes.

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (Plasmodium yoelii) and human (Plasmodium falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knockout of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in the knockout rodent malaria parasites. Restoration of the nsun2 gene in the knockout parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.

TLR7 enhancing follicular helper T (Tfh) cells response in C57BL/6 mice infected with Plasmodium yoelii NSM TLR7 mediated Tfh cells in P. yoelii infected mice.

Toll-like receptors (TLRs) play an important role in inducing innate and acquired immune responses against infection. However, the effect of Toll-like receptor 7 (TLR7) on follicular helper T (Tfh) cells in mice infected with Plasmodium is still not clear. The results showed that the splenic CD4+ CXCR5+ PD-1+ Tfh cells were accumulated after Plasmodium yoelii NSM infection, the content of splenic Tfh cells was correlated to parasitemia and/or the red blood cells (RBCs) counts in the blood. Moreover, the expression of TLR7 was found higher than TLR2, TLR3 and TLR4 in splenic Tfh cells of the WT mice. TLR7 agonist R848 and the lysate of red blood cells of infected mice (iRBCs) could induce the activation and differentiation of splenic Tfh cells. Knockout of TLR7 leads to a decrease in the proportion of Tfh cells, down-regulated expression of functional molecules CD40L, IFN-γ, IL-21 and IL-10 in Tfh cells; decreased the proportion of plasma cells and antibody production and reduces the expression of STAT3 and Ikzf2 in Tfh cells. Administration of R848 could inhibit parasitemia, enhance splenic Tfh cell activation and increase STAT3 and Ikzf2 expression in Tfh cells. In summary, this study shows that TLR7 could regulate the function of Tfh cells, affecting the immune response in the spleen of Plasmodium yoelii NSM-infected mice.

A protein palmitoylation cascade regulates microtubule cytoskeleton integrity in Plasmodium.

Morphogenesis of many protozoans depends on a polarized establishment of cytoskeletal structures. In malaria-causing parasites, this can be observed when a round zygote develops into an elongated motile ookinete within the mosquito stomach. This morphogenesis is mediated by the pellicle cytoskeletal structures, including the inner membrane complex (IMC) and the underlying subpellicular microtubules (SPMs). How the parasite maintains the IMC-SPM connection and establishes a dome-like structure of SPM to support cell elongation is unclear. Here, we show that palmitoylation of N-terminal cysteines of two IMC proteins (ISP1/ISP3) regulates the IMC localization of ISP1/ISP3 and zygote-to-ookinete differentiation. Palmitoylation of ISP1/ISP3 is catalyzed by an IMC-residing palmitoyl-S-acyl-transferase (PAT) DHHC2. Surprisingly, DHHC2 undergoes self-palmitoylation at C-terminal cysteines via its PAT activity, which controls DHHC2 localization in IMC after zygote formation. IMC-anchored ISP1 and ISP3 interact with microtubule component ß-tubulin, serving as tethers to maintain the proper structure of SPM during zygote elongation. This study identifies the first PAT-substrate pair in malaria parasites and uncovers a protein palmitoylation cascade regulating microtubule cytoskeleton.

Experimental determination of the force of malaria infection reveals a non-linear relationship to mosquito sporozoite loads.

Plasmodium sporozoites are the infective stage of the malaria parasite. Though this is a bottleneck for the parasite, the quantitative dynamics of transmission, from mosquito inoculation of sporozoites to patent blood-stage infection in the mammalian host, are poorly understood. Here we utilize a rodent model to determine the probability of malaria infection after infectious mosquito bite, and consider the impact of mosquito parasite load, blood-meal acquisition, probe-time, and probe location, on infection probability. We found that infection likelihood correlates with mosquito sporozoite load and, to a lesser degree, the duration of probing, and is not dependent upon the mosquito's ability to find blood. The relationship between sporozoite load and infection probability is non-linear and can be described by a set of models that include a threshold, with mosquitoes harboring over 10,000 salivary gland sporozoites being significantly more likely to initiate a malaria infection. Overall, our data suggest that the small subset of highly infected mosquitoes may contribute disproportionally to malaria transmission in the field and that quantifying mosquito sporozoite loads could aid in predicting the force of infection in different transmission settings.

Puf3 participates in ribosomal biogenesis in malaria parasites.

In this study, we characterized the Puf family gene member Puf3 in the malaria parasites Plasmodium falciparum and Plasmodium yoelii Secondary structure prediction suggested that the RNA-binding domains of the Puf3 proteins consisted of 11 pumilio repeats that were similar to those in the human Puf-A (also known as PUM3) and Saccharomyces cerevisiae Puf6 proteins, which are involved in ribosome biogenesis. Neither P. falciparum (Pf)Puf3 nor P. yoelii (Py)Puf3 could be genetically disrupted, suggesting they may be essential for the intraerythrocytic developmental cycle. Cellular fractionation of PfPuf3 in the asexual stages revealed preferential partitioning to the nuclear fraction, consistent with nuclear localization of PfPuf3::GFP and PyPuf3::GFP as detected by immunofluorescence. Furthermore, PfPuf3 colocalized with the nucleolar marker PfNop1, demonstrating that PfPuf3 is a nucleolar protein in the asexual stages. We found, however, that PyPuf3 changed its localization from being nucleolar to being present in cytosolic puncta in the mosquito and liver stages, which may reflect alternative functions in these stages. Affinity purification of molecules that associated with a PTP-tagged variant of PfPuf3 revealed 31 proteins associated with the 60S ribosome, and an enrichment of 28S rRNA and internal transcribed spacer 2 sequences. Taken together, these results suggest an essential function for PfPuf3 in ribosomal biogenesis.

Rapid identification of genes controlling virulence and immunity in malaria parasites.

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.

Synthesis, Biological Evaluation, and Molecular Modeling Studies of Chiral Chloroquine Analogues as Antimalarial Agents.

In a focused exploration, we designed, synthesized, and biologically evaluated chiral conjugated new chloroquine (CQ) analogues with substituted piperazines as antimalarial agents. In vitro as well as in vivo studies revealed that compound 7c showed potent activity (in vitro 50% inhibitory concentration, 56.98 nM for strain 3D7 and 97.76 nM for strain K1; selectivity index in vivo [up to at a dose of 12.5 mg/kg of body weight], 3,510) as a new lead antimalarial agent. Other compounds (compounds 6b, 6d, 7d, 7h, 8c, 8d, 9a, and 9c) also showed moderate activity against a CQ-sensitive strain (3D7) and superior activity against a CQ-resistant strain (K1) of Plasmodium falciparum Furthermore, we carried out docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of all in-house data sets (168 molecules) of chiral CQ analogues to explain the structure-activity relationships (SAR). Our new findings specify the significance of the H-bond interaction with the side chain of heme for biological activity. In addition, the 3D-QSAR study against the 3D7 strain indicated the favorable and unfavorable sites of CQ analogues for incorporating steric, hydrophobic, and electropositive groups to improve the antimalarial activity.

The malaria parasite RhopH protein complex interacts with erythrocyte calmyrin identified from a comprehensive erythrocyte protein library.

Malaria merozoite apical organelles; microneme and rhoptry secreted proteins play functional roles during and following invasion of host erythrocytes. Among numerous proteins, the rhoptries discharge high molecular weight proteins known as RhopH complex. Recent reports suggest that the RhopH complex is essential for growth and survival of the malaria parasite within erythrocytes. However, an in-depth understanding of the host-parasite molecular interactions is indispensable. Here we utilized a comprehensive mouse erythrocyte protein library consisting of 443 proteins produced by a wheat germ cell-free system, combined with AlphaScreen technology to identify mouse erythrocyte calmyrin as an interacting molecule of the rodent malaria parasite Plasmodium yoelii RhopH complex (PyRhopH). The PyRhopH interaction was dependent on the calmyrin N-terminus and divalent cation capacity. The finding unveils a recommendable and invaluable usefulness of our comprehensive mouse erythrocyte protein library together with the AlphaScreen technology in investigating a wide-range of host-parasite molecular interactions.

Synthesis and Evaluation of Chirally Defined Side Chain Variants of 7-Chloro-4-Aminoquinoline To Overcome Drug Resistance in Malaria Chemotherapy.

A novel 4-aminoquinoline derivative [(S)-7-chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

The Clinically Tested Gardos Channel Inhibitor Senicapoc Exhibits Antimalarial Activity.

Senicapoc, a Gardos channel inhibitor, prevented erythrocyte dehydration in clinical trials of patients with sickle cell disease. We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits Plasmodium growth. Senicapoc inhibited in vitro growth of human and primate plasmodia during the clinical blood stage. Senicapoc treatment suppressed P. yoelii parasitemia in vivo in C57BL/6 mice. The reassuring safety and biochemical profile of senicapoc encourage its use in antimalarial development.

Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver-stage development.

Malaria parasites scavenge nutrients from their host but also harbour enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver-stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbour genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic acid synthesis. Our research shows that apicoplast-targeted Plasmodium yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver-stage development and deletion of the encoding genes resulted in late liver-stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite life cycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver-stage maturation.

Hybridization and pre-zygotic reproductive barriers in Plasmodium.

Sexual reproduction is an obligate step in the life cycle of many parasites, including the causative agents of malaria (Plasmodium). Mixed-species infections are common in nature and consequently, interactions between heterospecific gametes occur. Given the importance of managing gene flow across parasite populations, remarkably little is understood about how reproductive isolation between species is maintained. We use the rodent malaria parasites P. berghei and P. yoelii to investigate the ecology of mixed-species mating groups, identify proteins involved in pre-zygotic barriers, and examine their evolution. Specifically, we show that (i) hybridization occurs, but at low frequency; (ii) hybridization reaches high levels when female gametes lack the surface proteins P230 or P48/45, demonstrating that these proteins are key for pre-zygotic reproductive isolation; (iii) asymmetric reproductive interference occurs, where the fertility of P. berghei gametes is reduced in the presence of P. yoelii and (iv) as expected for gamete recognition proteins, strong positive selection acts on a region of P230 and P47 (P48/45 paralogue). P230 and P48/45 are leading candidates for interventions to block malaria transmission. Our results suggest that depending on the viability of hybrids, applying such interventions to populations where mixed-species infections occur could either facilitate or hinder malaria control.

Two Plasmodium 6-Cys family-related proteins have distinct and critical roles in liver-stage development.

The 10 Plasmodium 6-Cys proteins have critical roles throughout parasite development and are targets for antimalaria vaccination strategies. We analyzed the conserved 6-cysteine domain of this family and show that only the last 4 positionally conserved cysteine residues are diagnostic for this domain and identified 4 additional "6-Cys family-related" proteins. Two of these, sequestrin and B9, are critical to Plasmodium liver-stage development. RT-PCR and immunofluorescence assays show that B9 is translationally repressed in sporozoites and is expressed after hepatocyte invasion where it localizes to the parasite plasma membrane. Mutants lacking B9 expression in the rodent malaria parasites P. berghei and P. yoelii and the human parasite P. falciparum developmentally arrest in hepatocytes. P. berghei mutants arrest in the livers of BALB/c (100%) and C57BL6 mice (>99.9%), and in cultures of Huh7 human-hepatoma cell line. Similarly, P. falciparum mutants while fully infectious to primary human hepatocytes abort development 3 d after infection. This growth arrest is associated with a compromised parasitophorous vacuole membrane a phenotype similar to, but distinct from, mutants lacking the 6-Cys sporozoite proteins P52 and P36. Our results show that 6-Cys proteins have critical but distinct roles in establishment and maintenance of a parasitophorous vacuole and subsequent liver-stage development.

Identification of a new export signal in Plasmodium yoelii: identification of a new exportome.

Development of the erythrocytic malaria parasite requires targeting of parasite proteins into multiple compartments located within and beyond the parasite confine. Beyond the PEXEL/VTS pathway and its characterized players, increasing amount of evidence has highlighted the existence of proteins exported using alternative export-signal(s)/pathway(s); hence, the exportomes currently predicted are incomplete. The nature of these exported proteins which could have a prominent role in most of the Plasmodium species remains elusive. Using P. yoelii variant proteins, we identified a signal associated to lipophilic region that mediates export of P. yoelii proteins. This non-PEXEL signal termed PLASMED is defined by semi-conserved residues and possibly a secondary structure. In vivo characterization of exported-proteins indicated that PLASMED is a bona fide export-signal that allowed us to identify an unseen P. yoelii exportome. The repertoire of the newly predicted exported proteins opens up perspectives for unravelling the remodelling of the host-cell by the parasite, against which new therapies could be elaborated.

Total and putative surface proteomics of malaria parasite salivary gland sporozoites.

Malaria infections of mammals are initiated by the transmission of Plasmodium salivary gland sporozoites during an Anopheles mosquito vector bite. Sporozoites make their way through the skin and eventually to the liver, where they infect hepatocytes. Blocking this initial stage of infection is a promising malaria vaccine strategy. Therefore, comprehensively elucidating the protein composition of sporozoites will be invaluable in identifying novel targets for blocking infection. Previous efforts to identify the proteins expressed in Plasmodium mosquito stages were hampered by the technical difficulty of separating the parasite from its vector; without effective purifications, the large majority of proteins identified were of vector origin. Here we describe the proteomic profiling of highly purified salivary gland sporozoites from two Plasmodium species: human-infective Plasmodium falciparum and rodent-infective Plasmodium yoelii. The combination of improved sample purification and high mass accuracy mass spectrometry has facilitated the most complete proteome coverage to date for a pre-erythrocytic stage of the parasite. A total of 1991 P. falciparum sporozoite proteins and 1876 P. yoelii sporozoite proteins were identified, with >86% identified with high sequence coverage. The proteomic data were used to confirm the presence of components of three features critical for sporozoite infection of the mammalian host: the sporozoite motility and invasion apparatus (glideosome), sporozoite signaling pathways, and the contents of the apical secretory organelles. Furthermore, chemical labeling and identification of proteins on live sporozoites revealed previously uncharacterized complexity of the putative sporozoite surface-exposed proteome. Taken together, the data constitute the most comprehensive analysis to date of the protein expression of salivary gland sporozoites and reveal novel potential surface-exposed proteins that might be valuable targets for antibody blockage of infection.

Antimalarial activity of cupredoxins: the interaction of Plasmodium merozoite surface protein 119 (MSP119) and rusticyanin.

The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed, and the 19-kDa C-terminal fragment (MSP119) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth, MSP119 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based on BiGGER is employed on MSP119-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP119 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria treatment and provide valuable information for drug design.

SSP3 is a novel Plasmodium yoelii sporozoite surface protein with a role in gliding motility.

Plasmodium sporozoites develop within oocysts in the mosquito midgut wall and then migrate to the salivary glands. After transmission, they embark on a complex journey to the mammalian liver, where they infect hepatocytes. Proteins on the sporozoite surface likely mediate multiple steps of this journey, yet only a few sporozoite surface proteins have been described. Here, we characterize a novel, conserved sporozoite surface protein (SSP3) in the rodent malaria parasite Plasmodium yoelii. SSP3 is a putative type I transmembrane protein unique to Plasmodium. By using epitope tagging and SSP3-specific antibodies in conjunction with immunofluorescence microscopy, we showed that SSP3 is expressed in mosquito midgut oocyst sporozoites, exhibiting an intracellular localization. In sporozoites derived from the mosquito salivary glands, however, SSP3 localized predominantly to the sporozoite surface as determined by immunoelectron microscopy. However, the ectodomain of SSP3 appeared to be inaccessible to antibodies in nonpermeabilized salivary gland sporozoites. Antibody-induced shedding of the major surface protein circumsporozoite protein (CSP) exposed the SSP3 ectodomain to antibodies in some sporozoites. Targeted deletion of SSP3 adversely affected in vitro sporozoite gliding motility, which, surprisingly, impacted neither their cell traversal capacity, host cell invasion in vitro, nor infectivity in vivo. Together, these data reveal a previously unappreciated complexity of the Plasmodium sporozoite surface proteome and the roles of surface proteins in distinct biological activities of sporozoites.

Arrest of nuclear division in Plasmodium through blockage of erythrocyte surface exposed ribosomal protein P2.

Malaria parasites reside inside erythrocytes and the disease manifestations are linked to the growth inside infected erythrocytes (IE). The growth of the parasite is mostly confined to the trophozoite stage during which nuclear division occurs followed by the formation of cell bodies (schizogony). The mechanism and regulation of schizogony are poorly understood. Here we show a novel role for a Plasmodium falciparum 60S stalk ribosomal acidic protein P2 (PfP2) (PFC0400w), which gets exported to the IE surface for 6-8 hrs during early schizogony, starting around 26-28 hrs post-merozoite invasion. The surface exposure is demonstrated using multiple PfP2-specific monoclonal antibodies, and is confirmed through transfection using PfP2-GFP. The IE surface-exposed PfP2-protein occurs mainly as SDS-resistant P2-homo-tetramers. Treatment with anti-PfP2 monoclonals causes arrest of IEs at the first nuclear division. Upon removal of the antibodies, about 80-85% of synchronized parasites can be released even after 24 hrs of antibody treatment. It has been reported that a tubovesicular network (TVN) is set up in early trophozoites which is used for nutrient import. Anti-P2 monoclonal antibodies cause a complete fragmentation of TVN by 36 hrs, and impairs lipid import in IEs. These may be downstream causes for the cell-cycle arrest. Upon antibody removal, the TVN is reconstituted, and the cell division progresses. Each of the above properties is observed in the rodent malaria parasite species P. yoelii and P. berghei. The translocation of the P2 protein to the IE surface is therefore likely to be of fundamental importance in Plasmodium cell division.

Transfusion of stored blood impairs host defenses against Gram-negative pathogens in mice.

BACKGROUND: Although human red blood cell (RBC) units may be refrigerator stored for up to 42 days, transfusion of older RBCs acutely delivers a large bolus of iron to mononuclear phagocytes. Similarly, iron dextran circulates in plasma for hours to days and is progressively cleared by mononuclear phagocytes, which return iron to plasma. Finally, malaria infection continuously delivers iron to macrophages by intra- and extravascular hemolysis. Studies suggest that iron administration increases infectious risk. STUDY DESIGN AND METHODS: To assess the effects of increased iron availability on susceptibility to infection, we infected mice with model Gram-negative intracellular or extracellular pathogens (Salmonella typhimurium or Escherichia coli, respectively), accompanied by RBC transfusion, iron dextran administration, or malarial coinfection. RESULTS: In our mouse models, transfusion of older RBCs exacerbates infection with both Gram-negative pathogens. Although iron dextran exacerbates E. coli infection to a similar extent as transfusion of corresponding amounts of iron, higher iron doses are required to produce comparable effects with S. typhimurium. Coinfection of mice with Plasmodium yoelii and S. typhimurium produces overwhelming Salmonella sepsis. Finally, treating mice with antibiotics abrogates the enhancing effect on E. coli infection of both older RBC transfusion and iron dextran administration. CONCLUSIONS: Transfusion of older RBCs exacerbates Gram-negative infection to a similar extent as malaria coinfection or iron dextran administration. Appropriate antibiotic therapy abrogates the effect of older RBC transfusions on infection with E. coli. Iron delivery to macrophages may be an underappreciated mechanism mediating, at least some, adverse effects of RBC transfusions.

Perturbations of Plasmodium Puf2 expression and RNA-seq of Puf2-deficient sporozoites reveal a critical role in maintaining RNA homeostasis and parasite transmissibility.

Malaria's cycle of infection requires parasite transmission between a mosquito vector and a mammalian host. We here demonstrate that the Plasmodium yoelii Pumilio-FBF family member Puf2 allows the sporozoite to remain infectious in the mosquito salivary glands while awaiting transmission. Puf2 mediates this solely through its RNA-binding domain (RBD) likely by stabilizing or hastening the degradation of specific mRNAs. Puf2 traffics to sporozoite cytosolic granules, which are negative for several markers of stress granules and P-bodies, and disappear rapidly after infection of hepatocytes. In contrast to previously described Plasmodium berghei pbpuf2(-) parasites, pypuf2(-) sporozoites have no apparent defect in host infection when tested early in salivary gland residence, but become progressively non-infectious and prematurely transform into EEFs during prolonged salivary gland residence. The premature overexpression of Puf2 in oocysts causes striking deregulation of sporozoite maturation and infectivity while extension of Puf2 expression in liverstages causes no defect, suggesting that the presence of Puf2 alone is not sufficient for its functions. Finally, by conducting the first comparative RNA-seq analysis of Plasmodium sporozoites, we find that Puf2 may play a role in directly or indirectly maintaining the homeostasis of specific transcripts. These findings uncover requirements for maintaining a window of opportunity for the malaria parasite to accommodate the unpredictable moment of transmission from mosquito to mammalian host.