Malaria parasite evades mosquito immunity by glutaminyl cyclase-mediated posttranslational protein modification.

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.

Combinatorial Tim-3 and PD-1 activity sustains antigen-specific Th1 cell numbers during blood-stage malaria.

AIMS: Co-inhibitory receptors play a major role in controlling the Th1 response during blood-stage malaria. Whilst PD-1 is viewed as the dominant co-inhibitory receptor restricting T cell responses, the roles of other such receptors in coordinating Th1 cell activity during malaria are poorly understood. METHODS AND RESULTS: Here, we show that the co-inhibitory receptor Tim-3 is expressed on splenic antigen-specific T-bet+ (Th1) OT-II cells transiently during the early stage of infection with transgenic Plasmodium yoelii NL parasites expressing ovalbumin (P yoelii NL-OVA). We reveal that co-blockade of Tim-3 and PD-L1 during the acute phase of P yoelii NL infection did not improve the Th1 cell response but instead led to a specific reduction in the numbers of splenic Th1 OT-II cells. Combined blockade of Tim-3 and PD-L1 did elevate anti-parasite IgG antibody responses. Nevertheless, co-blockade of Tim-3 and PD-L1 did not affect IFN-γ production by OT-II cells and did not influence parasite control during P yoelii NL-OVA infection. CONCLUSION: Thus, our results show that Tim-3 plays an unexpected combinatorial role with PD-1 in promoting and/ or sustaining a Th1 cell response during the early phase of blood-stage P. yoelii NL infection but combined blockade does not dramatically influence anti-parasite immunity.

Chimeric Plasmodium falciparum parasites expressing Plasmodium vivax circumsporozoite protein fail to produce salivary gland sporozoites.

BACKGROUND: Rodent malaria parasites where the gene encoding circumsporozoite protein (CSP) has been replaced with csp genes from the human malaria parasites, Plasmodium falciparum or Plasmodium vivax, are used as pre-clinical tools to evaluate CSP vaccines in vivo. These chimeric rodent parasites produce sporozoites in Anopheles stephensi mosquitoes that are capable of infecting rodent and human hepatocytes. The availability of chimeric P. falciparum parasites where the pfcsp gene has been replaced by the pvcsp would open up possibilities to test P. vivax CSP vaccines in small scale clinical trials using controlled human malaria infection studies. METHODS: Using CRISPR/Cas9 gene editing two chimeric P. falciparum parasites, were generated, where the pfcsp gene has been replaced by either one of the two major pvcsp alleles, VK210 or VK247. In addition, a P. falciparum parasite line that lacks CSP expression was also generated. These parasite lines have been analysed for sporozoite production in An. stephensi mosquitoes. RESULTS: The two chimeric Pf-PvCSP lines exhibit normal asexual and sexual blood stage development in vitro and produce sporozoite-containing oocysts in An. stephensi mosquitoes. Expression of the corresponding PvCSP was confirmed in oocyst-derived Pf-PvCSP sporozoites. However, most oocysts degenerate before sporozoite formation and sporozoites were not found in either the mosquito haemocoel or salivary glands. Unlike the chimeric Pf-PvCSP parasites, oocysts of P. falciparum parasites lacking CSP expression do not produce sporozoites. CONCLUSIONS: Chimeric P. falciparum parasites expressing P. vivax circumsporozoite protein fail to produce salivary gland sporozoites. Combined, these studies show that while PvCSP can partially complement the function of PfCSP, species-specific features of CSP govern full sporozoite maturation and development in the two human malaria parasites.

The subcellular location of ovalbumin in Plasmodium berghei blood stages influences the magnitude of T-cell responses.

Model antigens are frequently introduced into pathogens to study determinants that influence T-cell responses to infections. To address whether an antigen's subcellular location influences the nature and magnitude of antigen-specific T-cell responses, we generated Plasmodium berghei parasites expressing the model antigen ovalbumin (OVA) either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). For cytosolic expression, OVA alone or conjugated to mCherry was expressed from a strong constitutive promoter (OVAhsp70 or OVA::mCherryhsp70); for PVM expression, OVA was fused to HEP17/EXP1 (OVA::Hep17hep17). Unexpectedly, OVA expression in OVAhsp70 parasites was very low, but when OVA was fused to mCherry (OVA::mCherryhsp70), it was highly expressed. OVA expression in OVA::Hep17hep17 parasites was strong but significantly less than that in OVA::mCherryhsp70 parasites. These transgenic parasites were used to examine the effects of antigen subcellular location and expression level on the development of T-cell responses during blood-stage infections. While all OVA-expressing parasites induced activation and proliferation of OVA-specific CD8(+) T cells (OT-I) and CD4(+) T cells (OT-II), the level of activation varied: OVA::Hep17hep17 parasites induced significantly stronger splenic and intracerebral OT-I and OT-II responses than those of OVA::mCherryhsp70 parasites, but OVA::mCherryhsp70 parasites promoted stronger OT-I and OT-II responses than those of OVAhsp70 parasites. Despite lower OVA expression levels, OVA::Hep17hep17 parasites induced stronger T-cell responses than those of OVA::mCherryhsp70 parasites. These results indicate that unconjugated cytosolic OVA is not stably expressed in Plasmodium parasites and, importantly, that its cellular location and expression level influence both the induction and magnitude of parasite-specific T-cell responses. These parasites represent useful tools for studying the development and function of antigen-specific T-cell responses during malaria infection.

A versatile Plasmodium falciparum reporter line expressing NanoLuc enables highly sensitive multi-stage drug assays.

Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial drugs effective against multiple stages of the parasite, we generate a P. falciparum reporter parasite that constitutively expresses NanoLuciferase (NanoLuc) throughout its whole life cycle. The NanoLuc-expressing P. falciparum reporter parasite shows a quantitative NanoLuc signal in the asexual blood, gametocyte, mosquito, and liver stages. We also establish assay systems to evaluate the anti-malarial activity of compounds at the asexual blood, gametocyte, and liver stages, and then determine the 50% inhibitory concentration (IC50) value of several anti-malarial compounds. Through the development of this robust high-throughput screening system, we identify an anti-malarial compound that kills the asexual blood stage parasites. Our study highlights the utility of the NanoLuc reporter line, which may advance anti-malarial drug development through the improved screening of compounds targeting the human malarial parasite at multiple stages.

Three members of the 6-cys protein family of Plasmodium play a role in gamete fertility.

The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates.

Experimentally controlled downregulation of the histone chaperone FACT in Plasmodium berghei reveals that it is critical to male gamete fertility.

Human FACT (facilitates chromatin transcription) consists of the proteins SPT16 and SSRP1 and acts as a histone chaperone in the (dis)assembly of nucleosome (and thereby chromatin) structure during transcription and DNA replication. We identified a Plasmodium berghei protein, termed FACT-L, with homology to the SPT16 subunit of FACT. Epitope tagging of FACT-L showed nuclear localization with high expression in the nuclei of (activated) male gametocytes. The gene encoding FACT-L could not be deleted indicating an essential role during blood-stage development. Using a 'promoter-swap' approach whereby the fact-l promoter was replaced by an 'asexual blood stage-specific' promoter that is silent in gametocytes, transcription of fact-l in promoter-swap mutant gametocytes was downregulated compared with wild-type gametocytes. These mutant male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility was strongly reduced while female gamete fertility was unaffected; residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. Therefore FACT is critically involved in the formation of fertile male gametes and parasite transmission. 'Promoter swapping' is a powerful approach for the functional analysis of proteins in gametocytes (and beyond) that are essential during asexual blood-stage development.

Creation and preclinical evaluation of genetically attenuated malaria parasites arresting growth late in the liver.

Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study.

Screening of viral-vectored P. falciparum pre-erythrocytic candidate vaccine antigens using chimeric rodent parasites.

To screen for additional vaccine candidate antigens of Plasmodium pre-erythrocytic stages, fourteen P. falciparum proteins were selected based on expression in sporozoites or their role in establishment of hepatocyte infection. For preclinical evaluation of immunogenicity of these proteins in mice, chimeric P. berghei sporozoites were created that express the P. falciparum proteins in sporozoites as an additional copy gene under control of the uis4 gene promoter. All fourteen chimeric parasites produced sporozoites but sporozoites of eight lines failed to establish a liver infection, indicating a negative impact of these P. falciparum proteins on sporozoite infectivity. Immunogenicity of the other six proteins (SPELD, ETRAMP10.3, SIAP2, SPATR, HT, RPL3) was analyzed by immunization of inbred BALB/c and outbred CD-1 mice with viral-vectored (ChAd63 or ChAdOx1, MVA) vaccines, followed by challenge with chimeric sporozoites. Protective immunogenicity was determined by analyzing parasite liver load and prepatent period of blood stage infection after challenge. Of the six proteins only SPELD immunized mice showed partial protection. We discuss both the low protective immunogenicity of these proteins in the chimeric rodent malaria challenge model and the negative effect on P. berghei sporozoite infectivity of several P. falciparum proteins expressed in the chimeric sporozoites.

Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity.

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.

Egress of Plasmodium berghei gametes from their host erythrocyte is mediated by the MDV-1/PEG3 protein.

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes - the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV-1/PEG3, like its Plasmodium falciparum orthologue, is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Deltamdv-1/peg3 parasites in which MDV-1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV-1/PEG3-dependent mechanism that is distinct from mechanisms used by asexual parasites.

Generation of a Genetically Modified Chimeric Plasmodium falciparum Parasite Expressing Plasmodium vivax Circumsporozoite Protein for Malaria Vaccine Development.

Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP.

The Plasmodium TRAP/MIC2 family member, TRAP-Like Protein (TLP), is involved in tissue traversal by sporozoites.

In the apicomplexan protozoans motility and cell invasion are mediated by the TRAP/MIC2 family of transmembrane proteins, members of which link extracellular adhesion to the intracellular actomyosin motor complex. Here we characterize a new member of the TRAP/MIC2 family, named TRAP-Like Protein (TLP), that is highly conserved within the Plasmodium genus. Similar to the Plasmodium sporozoite protein, TRAP, and the ookinete protein, CTRP, TLP possesses an extracellular domain architecture that is comprised of von Willebrand factor A (vWA) and thrombospondin type 1 (TSP1) domains, plus a short cytoplasmic domain. Comparison of the vWA domain of TLP genes from multiple Plasmodium falciparum isolates showed relative low sequence diversity, suggesting that the protein is not under selective pressures of the host immune system. Analysis of transcript levels by quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that TLP is predominantly expressed in salivary gland sporozoites of P. falciparum and P. berghei. Targeted disruption of P. berghei TLP resulted in a decreased capacity for cell traversal by sporozoites, and reduced infectivity of sporozoites in vivo, whereas in vitro sporozoite motility and hepatocyte invasion were unaffected. These results indicate a role of TLP in cell traversal by sporozoites.

Author Correction: The Plasmodium falciparum male gametocyte protein P230p, a paralog of P230, is vital for ookinete formation and mosquito transmission.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

A P. falciparum NF54 Reporter Line Expressing mCherry-Luciferase in Gametocytes, Sporozoites, and Liver-Stages.

Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic Plasmodium falciparum (Pf) NF54 line was generated that expresses a fusion of mCherry and luciferase genes under the control of the Pf etramp10.3 gene promoter (line mCherry-luc@etramp10.3). Pf etramp10.3 is related to rodent Plasmodium uis4 and the uis4 promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive gapdh and eef1a promoters. The mCherry-luc@etramp10.3 parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in in vitro assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology.

Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium.

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as 'Hit and Run' and 'vector recycling' which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.

Expression of full-length Plasmodium falciparum P48/45 in P. berghei blood stages: A method to express and evaluate vaccine antigens.

The transmission-blocking vaccine candidate Pfs48/45 from the human malaria parasite Plasmodium falciparum is known to be difficult to express in heterologous systems, either as full-length protein or as correctly folded protein fragments that retain conformational epitopes. In this study we express full-length Pfs48/45 in the rodent parasite P. berghei. Pfs48/45 is expressed as a transgene under control of the strong P. berghei schizont-specific msp1 gene promoter (Pfs48/45@PbMSP1). Pfs48/45@PbMSP1 schizont-infected red blood cells produced full-length Pfs48/45 and the structural integrity of Pfs48/45 was confirmed using a panel of conformation-specific monoclonal antibodies that bind to different Pfs48/45 epitopes. Sera from mice immunized with transgenic Pfs48/45@PbMSP1 schizonts showed strong transmission-reducing activity in mosquitoes infected with P. falciparum using standard membrane feeding. These results demonstrate that transgenic rodent malaria parasites expressing human malaria antigens may be used as means to evaluate immunogenicity and functionality of difficult to express malaria vaccine candidate antigens.

The Plasmodium falciparum male gametocyte protein P230p, a paralog of P230, is vital for ookinete formation and mosquito transmission.

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol.

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.

Plasmodium berghei alpha-tubulin II: a role in both male gamete formation and asexual blood stages.

Plasmodium falciparum contains two genes encoding different isotypes of alpha-tubulin, alpha-tubulin I and alpha-tubulin II. alpha-Tubulin II is highly expressed in male gametocytes and forms part of the microtubules of the axoneme of male gametes. Here we present the characterization of Plasmodium berghei alpha-tubulin I and alpha-tubulin II that encode proteins of 453 and 450 amino acids, respectively. alpha-Tubulin II lacks the well-conserved three amino acid C-terminal extension including a terminal tyrosine residue present in alpha-tubulin I. Investigation of transcription by Northern analysis and RT-PCR and analysis of promoter activity by GFP tagging showed that alpha-tubulin I is expressed in all blood and mosquito stages. As expected, alpha-tubulin II was highly expressed in the male gametocytes, but transcription was also observed in the asexual blood stages, female gametocytes, ookinetes and oocysts. Gene disruption experiments using standard transfection technologies did not produce viable parasites indicating that both alpha-tubulin isotypes are essential for the asexual blood stages. Targeted modification of alpha-tubulin II by the addition of the three C-terminal amino acids of alpha-tubulin I did not affect either blood stage development nor male gamete formation. Attempts to modify the C-terminal region by adding a TAP tag to the endogenous alpha-tubulin II gene were not successful. Introduction of a transgene, expressing TAP-tagged alpha-tubulin II, next to the endogenous alpha-tubulin II gene, had no effect on the asexual blood stages but strongly impaired formation of male gametes. These results show that alpha-tubulin II not only plays an important role in the male gamete but is also expressed in and essential for asexual blood stage development.