Neuropilin-1 identifies a subset of highly activated CD8+ T cells during parasitic and viral infections.

Neuropilin-1 (Nrp-1) expression on CD8+ T cells has been identified in tumor-infiltrating lymphocytes and in persistent murine gamma-herpes virus infections, where it interferes with the development of long-lived memory T cell responses. In parasitic and acute viral infections, the role of Nrp-1 expression on CD8+ T cells remains unclear. Here, we demonstrate a strong induction of Nrp-1 expression on CD8+ T cells in Plasmodium berghei ANKA (PbA)-infected mice that correlated with neurological deficits of experimental cerebral malaria (ECM). Likewise, the frequency of Nrp-1+CD8+ T cells was significantly elevated and correlated with liver damage in the acute phase of lymphocytic choriomeningitis virus (LCMV) infection. Transcriptomic and flow cytometric analyses revealed a highly activated phenotype of Nrp-1+CD8+ T cells from infected mice. Correspondingly, in vitro experiments showed rapid induction of Nrp-1 expression on CD8+ T cells after stimulation in conjunction with increased expression of activation-associated molecules. Strikingly, T cell-specific Nrp-1 ablation resulted in reduced numbers of activated T cells in the brain of PbA-infected mice as well as in spleen and liver of LCMV-infected mice and alleviated the severity of ECM and LCMV-induced liver pathology. Mechanistically, we identified reduced blood-brain barrier leakage associated with reduced parasite sequestration in the brain of PbA-infected mice with T cell-specific Nrp-1 deficiency. In conclusion, Nrp-1 expression on CD8+ T cells represents a very early activation marker that exacerbates deleterious CD8+ T cell responses during both, parasitic PbA and acute LCMV infections.

A Candidate Bacterial-Type Amino Acid Decarboxylase Is Essential for Male Gamete Exflagellation and Mosquito Transmission of the Malaria Parasite.

A frequent side effect of chemotherapy against malaria parasite blood infections is a dramatic induction of the sexual blood stages, thereby enhancing the risk of future malaria transmissions. The polyamine biosynthesis pathway has been suggested as a candidate target for transmission-blocking anti-malarial drug development. Herein, we describe the role of a bacterial-type amino acid decarboxylase (AAD) in the life cycle of the malaria model parasite Plasmodium yoelii. Hallmarks of AAD include a conserved catalytic lysine residue and high-level homology to arginine/lysine/ornithine decarboxylases of pathogenic bacteria. By targeted gene deletion, we show that AAD plays an essential role in the exflagellation of microgametes, resulting in complete absence of sporozoites in the mosquito vector. These data highlight the central role of the biosysthesis of polyamines in the final steps of male gamete sexual development of the malaria parasite and, hence, onward transmission to mosquitoes.

TLR9 signalling inhibits Plasmodium liver infection by macrophage activation.

Recognition of pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs) plays a pivotal role in first-line pathogen defense. TLRs are also likely triggered during a Plasmodium infection in vivo by parasite-derived components. However, the contribution of innate responses to liver infection and to the subsequent clinical outcome of a blood infection is not well understood. To assess the potential effects of enhanced TLR-signalling on Plasmodium infection, we systematically examined the effect of agonist-primed immune responses to sporozoite inoculation in the P. berghei/C57Bl/6 murine malaria model. We could identify distinct stage-specific effects on the course of infection after stimulation with two out of four TLR-ligands tested. Priming with a TLR9 agonist induced killing of pre-erythrocytic stages in the liver that depended on macrophages and the expression of inducible nitric oxide synthase (iNOS). These factors have previously not been recognized as antigen-independent effector mechanisms against Plasmodium liver stages. Priming with TLR4 and -9 agonists also translated into blood stage-specific protection against experimental cerebral malaria (ECM). These insights are relevant to the activation of TLR signalling pathways by adjuvant systems of antimalaria vaccine strategies. The protective role of TLR4-activation against ECM might also explain some unexpected clinical effects observed with pre-erythrocytic vaccine approaches.

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells.

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

A lipocalin mediates unidirectional heme biomineralization in malaria parasites.

During blood-stage development, malaria parasites are challenged with the detoxification of enormous amounts of heme released during the proteolytic catabolism of erythrocytic hemoglobin. They tackle this problem by sequestering heme into bioinert crystals known as hemozoin. The mechanisms underlying this biomineralization process remain enigmatic. Here, we demonstrate that both rodent and human malaria parasite species secrete and internalize a lipocalin-like protein, PV5, to control heme crystallization. Transcriptional deregulation of PV5 in the rodent parasite Plasmodium berghei results in inordinate elongation of hemozoin crystals, while conditional PV5 inactivation in the human malaria agent Plasmodium falciparum causes excessive multidirectional crystal branching. Although hemoglobin processing remains unaffected, PV5-deficient parasites generate less hemozoin. Electron diffraction analysis indicates that despite the distinct changes in crystal morphology, neither the crystalline order nor unit cell of hemozoin are affected by impaired PV5 function. Deregulation of PV5 expression renders P. berghei hypersensitive to the antimalarial drugs artesunate, chloroquine, and atovaquone, resulting in accelerated parasite clearance following drug treatment in vivo. Together, our findings demonstrate the Plasmodium-tailored role of a lipocalin family member in hemozoin formation and underscore the heme biomineralization pathway as an attractive target for therapeutic exploitation.

In memoriam: Theodor Hiepe (1929-2022)-great German scholar of parasitology.

Theodor Hiepe (1929-2022) was an outstanding researcher, a world-renowned scientist, a dedicated teacher and a great mentor. During his scientific career, which spanned over 60 years, he made major contributions to many different fields of parasitology. With the passing of Dr. h.c. mult. Theodor Hiepe in September 2022 the scientific community suffered a great loss.

Molecular characterization of Schellackia parasites in an urban population of sand lizards (Lacerta agilis) from Berlin, Germany.

Lizards are hosts of several taxa of unicellular parasites of the phylum Apicomplexa, including Karyolysus, Schellackia, Lankesterella, and Hepatozoon. Parasite prevalence and the impact of infections on lizard biology remain largely unexplored. In this study, blood parasite infections were investigated in sand lizards (Lacerta agilis) from Berlin, Germany. Eighty-three individuals were investigated, and the detected blood parasites were identified as Schellackia sp. The combination of microscopic and molecular screening revealed a prevalence of 14.5%. Parasitemia values were low and most infections were subpatent. Phylogenetic analysis recovered a close relationship of the Schellackia parasites of this study with Schellackia sp. parasites of different Lacerta and Podarcis lizard species from Spain. Monitoring of Schellackia parasite infections in free-ranging lizards contributes to a better understanding of the distribution, diversity, and phylogenetic relationships of the neglected parasite taxon.

A Plasmodium cysteine protease required for efficient transition from the liver infection stage.

The transitions between developmental stages are critical points in the Plasmodium life cycle. The development of Plasmodium in the livers of their mammalian hosts bridges malaria transmission and the onset of clinical symptoms elicited by red blood cell infection. The egress of Plasmodium parasites from the liver must be a carefully orchestrated process to ensure a successful switch to the blood stage of infection. Cysteine protease activity is known to be required for liver-stage Plasmodium egress, but the crucial cysteine protease(s) remained unidentified. Here, we characterize a member of the papain-like cysteine protease family, Plasmodium berghei serine repeat antigen 4 (PbSERA4), that is required for efficient initiation of blood-stage infection. Through the generation PbSERA4-specific antisera and the creation of transgenic parasites expressing fluorescently tagged protein, we show that PbSERA4 is expressed and proteolytically processed in the liver and blood stages of infection. Targeted disruption of PbSERA4 results in viable and virulent blood-stage parasites. However, upon transmission from mosquitoes to mice, Pbsera4(-) parasites displayed a reduced capacity to initiate a new round of asexual blood-stage replication. Our results from cultured cells indicate that this defect results from an inability of the PbSERA4-deficient parasites to egress efficiently from infected cells at the culmination of liver-stage development. Protection against infection with wildtype P. berghei could be generated in animals in which Pbsera4(-) parasites failed to establish infection. Our findings confirm that liver-stage merozoite release is an active process and demonstrate that this parasite-encoded cysteine protease contributes to parasite escape from the liver.

An apicoplast-resident folate transporter is essential for sporogony of malaria parasites.

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.

Of membranes and malaria: phospholipid asymmetry in Plasmodium falciparum-infected red blood cells.

Malaria is a vector-borne parasitic disease with a vast impact on human history, and according to the World Health Organisation, Plasmodium parasites still infect over 200 million people per year. Plasmodium falciparum, the deadliest parasite species, has a remarkable ability to undermine the host immune system and cause life-threatening disease during blood infection. The parasite's host cells, red blood cells (RBCs), generally maintain an asymmetric distribution of phospholipids in the two leaflets of the plasma membrane bilayer. Alterations to this asymmetry, particularly the exposure of phosphatidylserine (PS) in the outer leaflet, can be recognised by phagocytes. Because of the importance of innate immune defence numerous studies have investigated PS exposure in RBCs infected with P. falciparum, but have reached different conclusions. Here we review recent advancements in our understanding of the molecular mechanisms which regulate asymmetry in RBCs, and whether infection with the P. falciparum parasite results in changes to PS exposure. On the balance of evidence, it is likely that membrane asymmetry is disrupted in parasitised RBCs, though some methodological issues need addressing. We discuss the potential causes and consequences of altered asymmetry in parasitised RBCs, particularly for in vivo interactions with the immune system, and the role of host-parasite co-evolution. We also examine the potential asymmetric state of parasite membranes and summarise current knowledge on the parasite proteins, which could regulate asymmetry in these membranes. Finally, we highlight unresolved questions at this time and the need for interdisciplinary approaches to uncover the machinery which enables P. falciparum parasites to hide in mature erythrocytes.

Inferior T cell immunogenicity of a Plasmodium berghei model liver stage antigen expressed throughout pre-erythrocytic maturation.

Sporozoite antigens are the basis of a number of malaria vaccines being tested, but the contribution of antigens expressed during subsequent liver stage development to pre-erythrocytic stage immunity is poorly understood. We previously showed that, following immunisation with radiation attenuated sporozoites (RAS), a model epitope embedded in a sporozoite surface protein elicited robust CD8+ T cell responses, whilst the same epitope in a liver stage antigen induced inferior responses. Since RAS arrest early in their development in host hepatocytes, we hypothesised that extending parasite maturation in the liver could considerably improve the epitope-specific CD8+ T cell response. Here, we employed a late liver stage arrested parasite model, azithromycin prophylaxis alongside live sporozoites, to increase expression of the model epitope until full liver stage maturation. Strikingly, this alternative immunisation strategy, which has been shown to elicit superior protection, failed to improve the resulting epitope-specific CD8+ T cell responses. Our findings support the notion that liver stage antigens are poorly immunogenic and provide additional caution about prioritising antigens for vaccine development based solely on immunogenicity.

Molecular characterization of Polychromophilus parasites of Scotophilus kuhlii bats in Thailand.

Parasites of the haemosporidian genus Polychromophilus have exclusively been described in bats. These parasites belong to the diverse group of malaria parasites, and Polychromophilus presents the only haemosporidian taxon that infects mammalian hosts in tropical as well as in temperate climate zones. This study provides the first information of Polychromophilus parasites in the lesser Asiatic yellow bat (Scotophilus kuhlii) in Thailand, a common vespertilionid bat species distributed in South and Southeast Asia. The gametocyte blood stages of the parasites could not be assigned to a described morphospecies and molecular analysis revealed that these parasites might represent a distinct Polychromophilus species. In contrast to Plasmodium species, Polychromophilus parasites do not multiply in red blood cells and, thus, do not cause the clinical symptoms of malaria. Parasitological and molecular investigation of haemosporidian parasites of wildlife, such as the neglected genus Polychromophilus, will contribute to a better understanding of the evolution of malaria parasites.

Importance of the Immunodominant CD8+ T Cell Epitope of Plasmodium berghei Circumsporozoite Protein in Parasite- and Vaccine-Induced Protection.

The circumsporozoite protein (CSP) builds up the surface coat of sporozoites and is the leading malaria pre-erythrocytic-stage vaccine candidate. CSP has been shown to induce robust CD8+ T cell responses that are capable of eliminating developing parasites in hepatocytes, resulting in protective immunity. In this study, we characterized the importance of the immunodominant CSP-derived epitope SYIPSAEKI of Plasmodium berghei in both sporozoite- and vaccine-induced protection in murine infection models. In BALB/c mice, where SYIPSAEKI is efficiently presented in the context of the major histocompatibility complex class I (MHC-I) molecule H-2-Kd, we established that epitope-specific CD8+ T cell responses contribute to parasite killing following sporozoite immunization. Yet, sterile protection was achieved in the absence of this epitope, substantiating the concept that other antigens can be sufficient for parasite-induced protective immunity. Furthermore, we demonstrated that SYIPSAEKI-specific CD8+ T cell responses elicited by viral-vectored CSP-expressing vaccines effectively targeted parasites in hepatocytes. The resulting sterile protection strictly relied on the expression of SYIPSAEKI. In C57BL/6 mice, which are unable to present the immunodominant epitope, CSP-based vaccines did not confer complete protection, despite the induction of high levels of CSP-specific antibodies. These findings underscore the significance of CSP in protection against malaria pre-erythrocytic stages and demonstrate that a significant proportion of the protection against the parasite is mediated by CD8+ T cells specific for the immunodominant CSP-derived epitope.

Identification of clustered organellar short (cos) RNAs and of a conserved family of organellar RNA-binding proteins, the heptatricopeptide repeat proteins, in the malaria parasite.

Gene expression in mitochondria of Plasmodium falciparum is essential for parasite survival. The molecular mechanisms of Plasmodium organellar gene expression remain poorly understood. This includes the enigmatic assembly of the mitochondrial ribosome from highly fragmented rRNAs. Here, we present the identification of clustered organellar short RNA fragments (cosRNAs) that are possible footprints of RNA-binding proteins (RBPs) in Plasmodium organelles. In plants, RBPs of the pentatricopeptide repeat (PPR) class produce footprints as a consequence of their function in processing organellar RNAs. Intriguingly, many of the Plasmodium cosRNAs overlap with 5'-ends of rRNA fragments. We hypothesize that these are footprints of RBPs involved in assembling the rRNA fragments into a functioning ribosome. A bioinformatics search of the Plasmodium nuclear genome identified a hitherto unrecognized organellar helical-hairpin-repeat protein family that we term heptatricopeptide repeat (HPR) proteins. We demonstrate that selected HPR proteins are targeted to mitochondria in P. berghei and that one of them, PbHPR1, associates with RNA, but not DNA in vitro. A phylogenetic search identified HPR proteins in a wide variety of eukaryotes. We hypothesize that HPR proteins are required for processing and stabilizing RNAs in Apicomplexa and other taxa.

Mycobacterium tuberculosis infection modulates adipose tissue biology.

Mycobacterium tuberculosis (Mtb) primarily resides in the lung but can also persist in extrapulmonary sites. Macrophages are considered the prime cellular habitat in all tissues. Here we demonstrate that Mtb resides inside adipocytes of fat tissue where it expresses stress-related genes. Moreover, perigonadal fat of Mtb-infected mice disseminated the infection when transferred to uninfected animals. Adipose tissue harbors leukocytes in addition to adipocytes and other cell types and we observed that Mtb infection induces changes in adipose tissue biology depending on stage of infection. Mice infected via aerosol showed infiltration of inducible nitric oxide synthase (iNOS) or arginase 1 (Arg1)-negative F4/80+ cells, despite recruitment of CD3+, CD4+ and CD8+ T cells. Gene expression analysis of adipose tissue of aerosol Mtb-infected mice provided evidence for upregulated expression of genes associated with T cells and NK cells at 28 days post-infection. Strikingly, IFN-γ-producing NK cells and Mtb-specific CD8+ T cells were identified in perigonadal fat, specifically CD8+CD44-CD69+ and CD8+CD44-CD103+ subpopulations. Gene expression analysis of these cells revealed that they expressed IFN-γ and the lectin-like receptor Klrg1 and down-regulated CD27 and CD62L, consistent with an effector phenotype of Mtb-specific CD8+ T cells. Sorted NK cells expressed higher abundance of Klrg1 upon infection, as well. Our results reveal the ability of Mtb to persist in adipose tissue in a stressed state, and that NK cells and Mtb-specific CD8+ T cells infiltrate infected adipose tissue where they produce IFN-γ and assume an effector phenotype. We conclude that adipose tissue is a potential niche for Mtb and that due to infection CD8+ T cells and NK cells are attracted to this tissue.

Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood- and mosquito-stage development.

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species.

The spatiotemporal dynamics and membranous features of the Plasmodium liver stage tubovesicular network.

For membrane-bound intracellular pathogens, the surrounding vacuole is the portal of communication with the host cell. The parasitophorous vacuole (PV) harboring intrahepatocytic Plasmodium parasites satisfies the parasites' needs of nutrition and protection from host defenses to allow the rapid parasite growth that occurs during the liver stage of infection. In this study, we visualized the PV membrane (PVM) and the associated tubovesicular network (TVN) through fluorescent tagging of two PVM-resident Plasmodium berghei proteins, UIS4 and IBIS1. This strategy revealed previously unrecognized dynamics with which these membranes extend throughout the host cell. We observed dynamic vesicles, elongated clusters of membranes and long tubules that rapidly extend and contract from the PVM in a microtubule-dependent manner. Live microscopy, correlative light-electron microscopy and fluorescent recovery after photobleaching enabled a detailed characterization of these membranous features, including velocities, the distribution of UIS4 and IBIS1, and the connectivity of PVM and TVN. Labeling of host cell compartments revealed association of late endosomes and lysosomes with the elongated membrane clusters. Moreover, the signature host autophagosome protein LC3 was recruited to the PVM and TVN and colocalized with UIS4. Together, our data demonstrate that the membranes surrounding intrahepatic Plasmodium are involved in active remodeling of host cells.

Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation.

Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression.

Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4(+) Foxp3(+) regulatory T cells.

Recently, we demonstrated elevated numbers of CD4(+) Foxp3(+) regulatory T (Treg) cells in Plasmodium yoelii-infected mice contributing to the regulation of anti-malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus-derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3(+) Treg cells expressing neuropilin-1 (Nrp-1) decreased at early time-points during P. yoelii infection, whereas percentages of Helios(+) Foxp3(+) Treg cells remained unchanged. Both Foxp3(+) Nrp-1(+) and Foxp3(+) Nrp-1(-) Treg cells from P. yoelii-infected mice exhibited a similar T-cell receptor Vß chain usage and methylation pattern in the Treg-specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3(-) T cells adoptively transferred to P. yoelii-infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3(+) Treg cells.

Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite.

Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs.