While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens. The mitochondrial tricarboxylic acid (TCA) cycle is one of the core metabolic pathways of eukaryotic cells, which contributes to cellular energy generation and provision of essential intermediates for macromolecule synthesis. Apicomplexan parasites possess the complete sets of genes coding for the TCA cycle. However, they lack a key mitochondrial enzyme complex that is normally required for production of acetyl-CoA from pyruvate, allowing further oxidation of glycolytic intermediates in the TCA cycle. This study unequivocally resolves how acetyl-CoA is generated in the mitochondrion using a combination of genetic, biochemical and metabolomic approaches. Specifically, we show that T. gondii and P. bergei utilize a second mitochondrial dehydrogenase complex, BCKDH, that is normally involved in branched amino acid catabolism, to convert pyruvate to acetyl-CoA and further catabolize glucose in the TCA cycle. In T. gondii, loss of BCKDH leads to global defects in glucose metabolism, increased gluconeogenesis and a marked attenuation of growth in host cells and virulence in animals. In P. bergei, loss of BCKDH leads to a defect in parasite proliferation in mature red blood cells, although the mutant retains the capacity to proliferate within 'immature' reticulocytes, highlighting the role of host metabolism/physiology on the development of Plasmodium asexual stages.

The glideosome is an actomyosin-based machinery that powers motility in Apicomplexa and participates in host cell invasion and egress from infected cells. The central component of the glideosome, myosin A (MyoA), is a motor recruited at the pellicle by the acylated gliding-associated protein GAP45. In Toxoplasma gondii, GAP45 also contributes to the cohesion of the pellicle, composed of the inner membrane complex (IMC) and the plasma membrane, during motor traction. GAP70 was previously identified as a paralog of GAP45 that is tailored to recruit MyoA at the apical cap in the coccidian subgroup of the Apicomplexa. A third member of this family, GAP80, is demonstrated here to assemble a new glideosome, which recruits the class XIV myosin C (MyoC) at the basal polar ring. MyoC shares the same myosin light chains as MyoA and also interacts with the integral IMC proteins GAP50 and GAP40. Moreover, a central component of this complex, the IMC-associated protein 1 (IAP1), acts as the key determinant for the restricted localization of MyoC to the posterior pole. Deletion of specific components of the MyoC-glideosome underscores the installation of compensatory mechanisms with components of the MyoA-glideosome. Conversely, removal of MyoA leads to the relocalization of MyoC along the pellicle and at the apical cap that accounts for residual invasion. The two glideosomes exhibit a considerable level of plasticity to ensure parasite survival. Toxoplasma gondii can infect most warm-blooded animals, and is an important opportunistic pathogen for humans. This obligate intracellular parasite is able to invade virtually all nucleated cells, and as with most parasites of the Apicomplexa phylum, relies on a substrate-dependent gliding motility to actively penetrate into host cells and egress from infected cells. The conserved molecular machine (named glideosome) powering motility is located at the periphery of the parasite and involves the molecular motor, myosin A (MyoA). The glideosome exists in three flavors, exhibiting the same overall organization and sharing some common components while being spatially restricted to the central IMC, the apical cap and the basal pole of the parasite, respectively. The central and apical glideosomes are associated with MyoA (MyoA-glideosome) whereas the basal complex recruits myosin C (MyoC). Deleting components of the MyoC-glideosome uncovers the existence of complementary and compensatory mechanisms that ensure successful establishment of infection. This study highlights a higher degree of complexity and plasticity of the gliding machinery.

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens. Microsporidia are obligate intracellular parasites that cause disease in immune suppressed individuals such as those with HIV/AIDS and recipients of organ transplants. The microsporidia are defined by a unique invasion organelle, the polar tube. The formation of this organelle and its role in the mechanism of infection remain unknown. Herein, we have identified a role for Encephalitozoon hellem polar tube protein 4 (PTP4) in infection demonstrating that PTP4 can bind to the host cell surface via the host transferrin receptor 1 (TfR1) protein. Interfering with the interaction of PTP4 and TfR1 causes a significant decrease in microsporidian infection of host cells. These data suggest that PTP4 functions as an important microsporidian protein during host cell infection by this pathogen.