Gliding Motility of Babesia bovis Merozoites Visualized by Time-Lapse Video Microscopy

BACKGROUND: Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed "gliding motility". However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite. METHODOLOGY/PRINCIPAL FINDINGS: Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion. CONCLUSIONS/SIGNIFICANCE: This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding

The Cryptosporidium parvum Kinome

<p>Abstract</p> <p>Background</p> <p>Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the <it>Cryptosporidium parvum </it>kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.</p> <p>Results</p> <p>The <it>C</it>. <it>parvum </it>kinome comprises over 70 members, some of which may be promising drug targets. These <it>C. parvum </it>protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of <it>Cryptosporidium spp</it>. Comparison of specific kinases with their <it>Plasmodium falciparum </it>and <it>Toxoplasma gondii </it>orthologues revealed some distinct characteristics within the <it>C. parvum </it>kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening <it>Cp</it>CDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC₅₀values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of <it>Cp</it>CDPK1. In addition, structural analysis of <it>Cp</it>CDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.</p> <p>Conclusions</p> <p>Identification and comparison of the <it>C. parvum </it>protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.</p

Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility

Gene-Specific Signatures of Elevated Non-Synonymous Substitution Rates Correlate Poorly across the Plasmodium Genus

BACKGROUND: Comparative genome analyses of parasites allow large scale investigation of selective pressures shaping their evolution. An acute limitation to such analysis of Plasmodium falciparum is that there is only very partial low-coverage genome sequence of the most closely related species, the chimpanzee parasite P. reichenowi. However, if orthologous genes have been under similar selective pressures throughout the Plasmodium genus then positive selection on the P. falciparum lineage might be predicted to some extent by analysis of other lineages. PRINCIPAL FINDINGS: Here, three independent pairs of closely related species in different sub-generic clades (P. falciparum and P. reichenowi; P. vivax and P. knowlesi; P. yoelii and P. berghei) were compared for a set of 43 candidate ligand genes considered likely to be under positive directional selection and a set of 102 control genes for which there was no selective hypothesis. The ratios of non-synonymous to synonymous substitutions (dN/dS) were significantly elevated in the candidate ligand genes compared to control genes in each of the three clades. However, the rank order correlation of dN/dS ratios for individual candidate genes was very low, less than the correlation for the control genes. SIGNIFICANCE: The inability to predict positive selection on a gene in one lineage by identifying elevated dN/dS ratios in the orthologue within another lineage needs to be noted, as it reflects that adaptive mutations are generally rare events that lead to fixation in individual lineages. Thus it is essential to complete the genome sequences of particular species of phylogenetic importance, such as P. reichenowi

A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor

Background Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro. Methodology/Principal Findings Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace\u27s Insect Medium mediated their transformation into trypomastigotes. Grace\u27s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP3R) - an intracellular Ca2+ channel and a key molecule involved in Ca2+ signaling in the parasite - is important for the transformation process. Conclusion/Significance Our findings provide a new tool to identify the molecular mechanisms of the amastigote-totrypomastigote transformation, leading to a new strategy for drug development against Chagas disease

Molecular characterization of ribosomal DNA (rDNA) polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s.

The sequence of a 2.3 kb long DNA segment derived from the 5'-most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako

Identification of Plasmodium berghei Oocyst Rupture Protein 2 (ORP2) domains involved in sporozoite egress from the oocyst

Sporozoites are the infective form of malaria parasites which are transmitted from the mosquito salivary glands to a new host in a mosquito blood meal. The sporozoites develop inside the sporogonic oocyst and it is crucial for the continuation of the life cycle that the oocyst ruptures to release sporozoites. We recently described two Plasmodium Oocyst Rupture Proteins (ORP1 and ORP2), localized at the oocyst capsule, that are each essential for rupture of the oocysts. Both ORPs contain a histone fold domain implicated in the mechanism of oocyst rupture, possibly through the formation of a heterodimer between the two histone fold domains. To gain an understanding of the function of the different regions of the ORP2 protein, we generated deletion mutants. We monitored oocyst formation and rupture as well as sporozoites in the salivary gland. Our results show that different regions of ORP2 play independent roles in sporozoite egress. Deleting the N-terminal histone fold domain of ORP2 blocked sporozoite egress from the oocyst. Progressive deletions from the C-terminal resulted in no or significantly impaired sporozoite egress