Atomic view into Plasmodium actin polymerization, ATP hydrolysis, and fragmentation.

Plasmodium actins form very short filaments and have a noncanonical link between ATP hydrolysis and polymerization. Long filaments are detrimental to the parasites, but the structural factors constraining Plasmodium microfilament lengths have remained unknown. Using high-resolution crystallography, we show that magnesium binding causes a slight flattening of the Plasmodium actin I monomer, and subsequent phosphate release results in a more twisted conformation. Thus, the Mg-bound monomer is closer in conformation to filamentous (F) actin than the Ca form, and this likely facilitates polymerization. A coordinated potassium ion resides in the active site during hydrolysis and leaves together with the phosphate, a process governed by the position of the Arg178/Asp180-containing A loop. Asp180 interacts with either Lys270 or His74, depending on the protonation state of the histidine, while Arg178 links the inner and outer domains (ID and OD) of the actin protomer. Hence, the A loop acts as a switch between stable and unstable filament conformations, the latter leading to fragmentation. Our data provide a comprehensive model for polymerization, ATP hydrolysis and phosphate release, and fragmentation of parasite microfilaments. Similar mechanisms may well exist in canonical actins, although fragmentation is much less favorable due to several subtle sequence differences as well as the methylation of His73, which is absent on the corresponding His74 in Plasmodium actin I.

Molecular Characterization and Phylogenetic Analysis of Theileria spp. and Babesia spp. Isolated from Various Ticks in Southeastern and Northwestern Regions of Iran.

INTRODUCTION: Piroplasms are hemoprotozoa comprising heterogeneous tick-borne parasites, which are differentiated into three genera, namely Babesia, Theileria, and Cytauxzoon. The aim of this study was to determine the prevalence, molecular identification, and phylogenetic relationship of both Theileria spp. and Babesia spp. in tick species isolated from different domestic animals from two different geographical locations of Iran. MATERIALS AND METHODS: A total of 930 ticks collected from goats, sheep, and cattle were examined for the presence of Theileria spp. and Babesia spp. using PCR targeting 18S rRNA gene followed by sequencing. Sequence analysis was performed based on the data published in the GenBank on Theileria spp. and Babesia spp. isolates using bioinformatic tools, such as the standard nucleotide BLAST. RESULTS: A 390 or 430 base pair fragment of 18S rRNA gene of Theileria and Babesia species was successfully amplified in 17.2% of the examined ticks (16of 93). Genome of Theileria or Babesia species was detected in 4 ticks collected in Heris, including 3 Dermacentor marginatus and 1 Rhipicephalus sanguineus, and also in 12 ticks collected in Chabahar, including 10 R. sanguineus and 2 D. marginatus. Partial analysis of 18S rRNA gene sequence of the four D. marginatus, collected from goats and sheep in Heris, showed that they were infected with Theileria spp. that were 95-97% identical to Iranian Theileria ovis present in the GenBank database (GenBank acc. no. KP019206.1). While the five R. sanguineus, collected from sheep and goats in Chabahar, were infected with Babesia spp. that were 91-97% identical to Iranian Babesia ovis present in the GenBank database (GenBank acc. no. AY362829.1: KT587794.1). CONCLUSION: The prevalence of Babesia and Theileria is different in southeastern and northwestern parts of Iran, with higher prevalence of babesiosis in the southeastern region and that of theileriosis in the northwestern region. Sequence analysis of 18S rRNA gene revealed that T. ovis and B. ovis are genetically polymorphic in these regions.

Comparative insight into nucleotide excision repair components of Plasmodium falciparum.

Nucleotide excision repair (NER) is one of the DNA repair pathways crucial for maintenance of genome integrity and deals with repair of DNA damages arising due to exogenous and endogenous factors. The multi-protein transcription initiation factor TFIIH plays a critical role in NER and transcription and is highly conserved throughout evolution. The malaria parasite Plasmodium falciparum has been a challenge for the researchers for a long time because of emergence of drug resistance. The availability of its genome sequence has opened new avenues for research. Antimalarial drugs like chloroquine and mefloquine have been reported to inhibit NER pathway mediated repair reactions and thus promote mutagenesis. Previous studies have validated existence and implied possible association of defective or altered DNA repair pathways with development of drug resistant phenotype in certain P. falciparum strains. We conjecture that a compromised NER pathway in combination with other DNA repair pathways might be conducive for the emergence and sustenance of drug resistance in P. falciparum. Therefore we decided to unravel the components of NER pathway in P. falciparum and using bioinformatics based approaches here we report a genome wide in silico analysis of NER components from P. falciparum and their comparison with the human host. Our results reveal that P. falciparum genome contains almost all the components of NER but we were unable to find clear homologue for p62 and XPC in its genome. The structure modeling of all the components further suggests that their structures are significantly conserved. Furthermore this study lays a foundation to perform similar comparative studies between drug resistant and drug sensitive strains of parasite in order to understand DNA repair-related mechanisms of drug resistance.

Plasmodium falciparum XPD translocates in 5' to 3' direction, is expressed throughout the blood stages, and interacts with p44.

XPD helicase, a TFIIH subunit, is essential for several processes including transcription, NER, cell cycle regulation, and apoptosis in eukaryotes. Another component of TFIIH, namely p44, is among the well-known interacting partners of XPD and is vital in regulating the helicase activities of latter. However, none of the above mentioned proteins have been functionally characterized in Plasmodium falciparum. Consequently, in this study, we performed detailed studies on XPD and its interacting partner, p44, from P. falciparum 3D7 strain. Accordingly, we expressed and purified recombinant PfXPD and its fragments and Pfp44 proteins and characterized the enzymatic activities of PfXPD and its fragments. The in vivo stage-specific expression and subcellular localizations of PfXPD and Pfp44 proteins were studied using the specific antibodies in the intraerythrocytic developmental stages of P. falciparum 3D7 strain. Our results suggest that PfXPD displays the characteristic ssDNA-dependent ATPase and 5'-3' DNA helicase activities. We also report the existence of two high molecular weight forms of p44 in P. falciparum 3D7 strain. Both PfXPD and Pfp44 colocalize in the nucleus and interact with each other, which suggest that they are most likely components of the same complex apparently, TFIIH. Furthermore, during trophozoite and schizont stages, both proteins exhibit a distinct cytoplasmic distribution pattern which implies that PfXPD and Pfp44 might also be involved in other functions. These studies will aid in understanding the basic biology of malaria parasite.

Mitochondrial DNA diversity in the populations of great gerbils, Rhombomys opimus, the main reservoir of cutaneous leishmaniasis.

Accurate identification of animal reservoirs of transmissible diseases is an absolute requirement to any epidemiological survey of zoonoses and is essential for predicting species-specific population outbreaks and therefore to develop accurate ecological control strategies. The systematic status of the great gerbil (Rhombomys opimus) remains unclear, despite the fundamental role of these rodents as the main known reservoir hosts of the protozoan parasite Leishmania major in the epidemiology of zoonotic cutaneous leishmaniasis (ZCL) in central and south Asia. In the present work, we represent molecular evidence supporting the identification of at least two major lineages (subspecies) within the species of R. opimus in Iran. Analysis of the mitochondrial cytochrome b (cyt b) gene, revealed a range of 1-10% genetic variation among populations, which were well associated with biogeographic origins and subspecies. Results of laboratory cross hybridization between the subspecies and finding sympatric haplotypes of the two subspecies suggested that no pre- or post-zygotic barriers exist between the subspecies indicating that they still belong to a single taxon. However, the amount of genetic variations between populations/subspecies is high enough to lead them to speciation in future. Implications of such findings on the eco-epidemiology of ZCL in Iran are discussed.