Knockout studies reveal an important role of <i>plasmodium</i> lipoic acid protein ligase a1 for asexual blood stage parasite survival

Lipoic acid (LA) is a dithiol-containing cofactor that is essential for the function of a-keto acid dehydrogenase complexes. LA acts as a reversible acyl group acceptor and 'swinging arm' during acyl-coenzyme A formation. The cofactor is post-translationally attached to the acyl-transferase subunits of the multienzyme complexes through the action of octanoyl (lipoyl): &lt;i&gt;N&lt;/i&gt;-octanoyl (lipoyl) transferase (LipB) or lipoic acid protein ligases (LplA). Remarkably, apicomplexan parasites possess LA biosynthesis as well as scavenging pathways and the two pathways are distributed between mitochondrion and a vestigial organelle, the apicoplast. The apicoplast-specific LipB is dispensable for parasite growth due to functional redundancy of the parasite's lipoic acid/octanoic acid ligases/transferases. In this study, we show that &lt;i&gt;LplA1&lt;/i&gt; plays a pivotal role during the development of the erythrocytic stages of the malaria parasite. Gene disruptions in the human malaria parasite &lt;i&gt;P.falciparum&lt;/i&gt; consistently were unsuccessful while in the rodent malaria model parasite &lt;i&gt;P. berghei&lt;/i&gt; the &lt;i&gt;LplA1&lt;/i&gt; gene locus was targeted by knock-in and knockout constructs. However, the &lt;i&gt;LplA1&lt;/i&gt; &lt;sup&gt;(-)&lt;/sup&gt; mutant could not be cloned suggesting a critical role of LplA1 for asexual parasite growth &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. These experimental genetics data suggest that lipoylation during expansion in red blood cells largely occurs through salvage from the host erythrocytes and subsequent ligation of LA to the target proteins of the malaria parasite

Nonlinearity of Mechanochemical Motions in Motor Proteins

The assumption of linear response of protein molecules to thermal noise or structural perturbations, such as ligand binding or detachment, is broadly used in the studies of protein dynamics. Conformational motions in proteins are traditionally analyzed in terms of normal modes and experimental data on thermal fluctuations in such macromolecules is also usually interpreted in terms of the excitation of normal modes. We have chosen two important protein motors - myosin V and kinesin KIF1A - and performed numerical investigations of their conformational relaxation properties within the coarse-grained elastic network approximation. We have found that the linearity assumption is deficient for ligand-induced conformational motions and can even be violated for characteristic thermal fluctuations. The deficiency is particularly pronounced in KIF1A where the normal mode description fails completely in describing functional mechanochemical motions. These results indicate that important assumptions of the theory of protein dynamics may need to be reconsidered. Neither a single normal mode, nor a superposition of such modes yield an approximation of strongly nonlinear dynamics.Comment: 10 pages, 6 figure

Phosphoenolpyruvate carboxylase dentified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in thePlasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

Host Cell Egress and Invasion Induce Marked Relocations of Glycolytic Enzymes in Toxoplasma gondii Tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K+] experienced during egress and invasion, a signal that requires changes of [Ca2+]c in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites

Pre-Clinical Assessment of Novel Multivalent MSP3 Malaria Vaccine Constructs

BACKGROUND: MSP3 has been shown to induce protection against malaria in African children. The characterization of a family of Plasmodium falciparum merozoite surface protein 3 (MSP3) antigens sharing a similar structural organization, simultaneously expressed on the merozoite surface and targeted by a cross-reactive network of protective antibodies, is intriguing and offers new perspectives for the development of subunit vaccines against malaria. METHODS: Eight recombinant polyproteins containing carefully selected regions of this family covalently linked in different combinations were all efficiently produced in Escherichia coli. The polyproteins consisted of one monovalent, one bivalent, one trivalent, two tetravalents, one hexavalent construct, and two tetravalents incorporating coiled-coil repeats regions from LSA3 and p27 vaccine candidates. RESULTS: All eight polyproteins induced a strong and homogeneous antibody response in mice of three distinct genotypes, with a dominance of cytophilic IgG subclasses, lasting up to six months after the last immunization. Vaccine-induced antibodies exerted a strong monocyte-mediated in vitro inhibition of P. falciparum growth. Naturally acquired antibodies from individuals living in an endemic area of Senegal recognized the polyproteins with a reactivity mainly constituted of cytophilic IgG subclasses. CONCLUSIONS: Combination of genetically conserved and antigenically related MSP3 proteins provides promising subunit vaccine constructs, with improved features as compared to the first generation construct employed in clinical trials (MSP3-LSP). These multivalent MSP3 vaccine constructs expand the epitope display of MSP3 family proteins, and lead to the efficient induction of a wider range of antibody subclasses, even in genetically different mice. These findings are promising for future immunization of genetically diverse human populations

Characterization of Two Malaria Parasite Organelle Translation Elongation Factor G Proteins: The Likely Targets of the Anti-Malarial Fusidic Acid

Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria

Compartmentation of Redox Metabolism in Malaria Parasites

Malaria, caused by the apicomplexan parasite Plasmodium, still represents a major threat to human health and welfare and leads to about one million human deaths annually. Plasmodium is a rapidly multiplying unicellular organism undergoing a complex developmental cycle in man and mosquito – a life style that requires rapid adaptation to various environments. In order to deal with high fluxes of reactive oxygen species and maintain redox regulatory processes and pathogenicity, Plasmodium depends upon an adequate redox balance. By systematically studying the subcellular localization of the major antioxidant and redox regulatory proteins, we obtained the first complete map of redox compartmentation in Plasmodium falciparum. We demonstrate the targeting of two plasmodial peroxiredoxins and a putative glyoxalase system to the apicoplast, a non-photosynthetic plastid. We furthermore obtained a complete picture of the compartmentation of thioredoxin- and glutaredoxin-like proteins. Notably, for the two major antioxidant redox-enzymes – glutathione reductase and thioredoxin reductase – Plasmodium makes use of alternative-translation-initiation (ATI) to achieve differential targeting. Dual localization of proteins effected by ATI is likely to occur also in other Apicomplexa and might open new avenues for therapeutic intervention

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

In cell-based drug development, researchers attempt to create drugs that kill a pathogen without necessarily understanding the details of how the drugs work. In contrast, target-based drug development entails the search for compounds that act on a specific intracellular target—often a protein known or suspected to be required for survival of the pathogen. The latter approach to drug development has been facilitated greatly by the sequencing of many pathogen genomes and the incorporation of genome data into user-friendly databases. The present paper shows how the database TDRtargets.org can identify proteins that might be considered good drug targets for diseases such as African sleeping sickness, Chagas disease, parasitic worm infections, tuberculosis, and malaria. These proteins may score highly in searches of the database because they are dissimilar to human proteins, are structurally similar to other “druggable” proteins, have functions that are easy to measure, and/or fulfill other criteria. Researchers can use the lists of high-scoring proteins as a basis for deciding which potential drug targets to pursue experimentally

Expression of a malarial Hsp70 improves defects in chaperone-dependent activities in ssa1 mutant yeast

Plasmodium falciparum causes the most virulent form of malaria and encodes a large number of molecular chaperones. Because the parasite encounters radically different environments during its lifecycle, many members of this chaperone ensemble may be essential for P. falciparum survival. Therefore, Plasmodium chaperones represent novel therapeutic targets, but to establish the mechanism of action of any developed therapeutics, it is critical to ascertain the functions of these chaperones. To this end, we report the development of a yeast expression system for PfHsp70-1, a P. falciparum cytoplasmic chaperone. We found that PfHsp70-1 repairs mutant growth phenotypes in yeast strains lacking the two primary cytosolic Hsp70s, SSA1 and SSA2, and in strains harboring a temperature sensitive SSA1 allele. PfHsp70-1 also supported chaperone-dependent processes such as protein translocation and ER associated degradation, and ameliorated the toxic effects of oxidative stress. By introducing engineered forms of PfHsp70-1 into the mutant strains, we discovered that rescue requires PfHsp70-1 ATPase activity. Together, we conclude that yeast can be co-opted to rapidly uncover specific cellular activities mediated by malarial chaperones. © 2011 Bell et al