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Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.

ABSTRACT: The apicoplast housekeeping machinery, specifically apicoplast DNA replication, transcription, and translation, was targeted by ciprofloxacin, thiostrepton, and rifampin, respectively, in the in vitro cultures of four Babesia species. Furthermore, the in vivo effect of thiostrepton on the growth cycle of Babesia microti in BALB/c mice was evaluated. The drugs caused significant inhibition of growth from an initial parasitemia of 1% for Babesia bovis, with 50% inhibitory concentrations (IC(50)s) of 8.3, 11.5, 12, and 126.6 ?M for ciprofloxacin, thiostrepton, rifampin, and clindamycin, respectively. The IC(50)s for the inhibition of Babesia bigemina growth were 15.8 ?M for ciprofloxacin, 8.2 ?M for thiostrepton, 8.3 ?M for rifampin, and 206 ?M for clindamycin. The IC(50)s for Babesia caballi were 2.7 ?M for ciprofloxacin, 2.7 ?M for thiostrepton, 4.7 ?M for rifampin, and 4.7 ?M for clindamycin. The IC(50)s for the inhibition of Babesia equi growth were 2.5 ?M for ciprofloxacin, 6.4 ?M for thiostrepton, 4.1 ?M for rifampin, and 27.2 ?M for clindamycin. Furthermore, an inhibitory effect was revealed for cultures with an initial parasitemia of either 10 or 7% for Babesia bovis or Babesia bigemina, respectively. The three inhibitors caused immediate death of Babesia bovis and Babesia equi. The inhibitory effects of ciprofloxacin, thiostrepton, and rifampin were confirmed by reverse transcription-PCR. Thiostrepton at a dose of 500 mg/kg of body weight resulted in 77.5% inhibition of Babesia microti growth in BALB/c mice. These results implicate the apicoplast as a potential chemotherapeutic target for babesiosis.

SUBMITTER: Aboulaila M

PROVIDER: S-EPMC3370714 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.

Aboulaila Mahmoud M Munkhjargal Tserendorj T Sivakumar Thillaiampalam T Ueno Akio A Nakano Yuki Y Yokoyama Miki M Yoshinari Takeshi T Nagano Daisuke D Katayama Koji K El-Bahy Nasr N Yokoyama Naoaki N Igarashi Ikuo I

Antimicrobial agents and chemotherapy 20120305 6

The apicoplast housekeeping machinery, specifically apicoplast DNA replication, transcription, and translation, was targeted by ciprofloxacin, thiostrepton, and rifampin, respectively, in the in vitro cultures of four Babesia species. Furthermore, the in vivo effect of thiostrepton on the growth cycle of Babesia microti in BALB/c mice was evaluated. The drugs caused significant inhibition of growth from an initial parasitemia of 1% for Babesia bovis, with 50% inhibitory concentrations (IC(50)s) ...[more]

PMID: 22391527

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Project description:Babesia orientalis is an obligate intraerythrocytic protozoan parasite of the buffalo (Bubalus bubalis, Linnaeus, 1758) transmitted by the tick Rhipicephalus heamaphysaloides. It is the causative agent of water buffalo babesiosis, one of the most important pathogens of water buffalo in central and southern China. As a member of the phylum Apicomplexa, B. orientalis possesses a relatively independent and alga originated organelle the apicoplast. Apicoplasts in other apicomplexa parasites are involved in the biosynthesis of haem, fatty acids, iron-sulphur clusters and isoprenoids. Some of these metabolic pathways were shown to be essential for parasite survival, therefore can serve as potential drug targets.30 pairs of primers were designed based on the full genome sequence of B. orientalis (unpublished data) and by aligning reported apicoplast genomes of Babesia bovis and Theileria parva. Conventional PCRs was performed to obtain overlapped fragments to cover the whole apicoplast genome. Then the apicoplast genome of B.orientalis was sequenced, assembled and aligned with reported apicoplast genomes of B. bovis and T. parva. The obtained apicoplast genome was annotated by using Artemis and comparing with published apicomplexan apicoplast genomes. The SSU and LSU nucleotide sequences generated were used in a phylogenetic analysis using the maximum likelihood implemented in MAGE 6.0.We have obtained and analyzed the complete genome sequence of the B. orientalis apicoplast. It consisted of a 33.2 kb circular DNA (78.9 % A?+?T). The apicoplast genome unidirectionally encodes one large and one small subunit ribosomal RNAs, 24 tRNA genes, 4 DNA-dependent RNA polymerase beta subunits (rpoB, rpoC1, rpoC2a and rpoC2b), 17 ribosomal proteins, one EF-Tu elongation factor, 2 Clp protease chaperones, and 14 hypothetical proteins. In addition, it includes two copies of the clpC gene. The structure and organization of the B. orientalis apicoplast genome are most similar to those of the B. bovis apicoplast.This is the first report of the complete sequence of the B. orientalis apicoplast genome. This information should be useful in the development of safe and efficient treatment against buffalo babesiosis.

Project description:BackgroundThe Babesia microti-like parasite is an emerging tick-borne piroplasm that has been detected in a range of hosts worldwide. Babesia vulpes, which is found in dogs and foxes, has been reclassified from B. microti-like parasites. The relationships among these B. microti-like parasites and B. vulpes with respect to host range and geographical origin have not been elucidated.MethodsBlood samples were collected from 27 raccoon dogs in South Korea and used to screen for B. microti-like parasites based on a PCR assay targeting the 18S rRNA gene of Babesia. For comparative purposes, in addition to 18S rRNA sequences from nine raccoon dogs, we also analyzed 18S rRNA sequences from B. microti-like parasites infecting hosts in different geographical regions worldwide obtained from the GenBank database, giving 123 sequences in total. The genetic variation and evolutionary relationships among these sequences were examined based on analyses using DnaSP, MEGA, Arlequine, and BEAST software.ResultsBabesia microti-like parasites were identified in nine raccoon dogs and found to be related to B. vulpes obtained from Spanish dogs. Among the 123 sequences from 14 countries and various hosts, we identified 43 haplotypes with high genetic variance. Based on the genetic variance and phylogenetic analyses, we established that the B. microti-like parasites isolated in different geographical regions and from hosts belonging to five orders showed higher among-population variation than within-population variation. Babesia vulpes parasites infecting carnivore hosts, including raccoon dogs, foxes, skunks and dogs, appear to be genetically distinct from B. microti-like parasites infecting hosts belonging to the other orders.ConclusionsOur study demonstrated the genetic variation and evolutionary relationships among 18S rRNA sequences obtained from blood samples collected from various hosts and different geographical regions. Babesia vulpes was identified from raccoon dogs in South Korea. In addition, higher genetic variations were observed among populations of different hosts and geographical origins and, in particular, low connectivity was observed among host populations in the order Carnivora and those in other orders. These results suggest the B. vulpes, a piroplasmid species pathogenic in domestic dogs and wild canines, is genetically and evolutionarily different from B. microti-like parasites.

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Project description:BackgroundBabesiosis is an economically important disease caused by tick-borne apicomplexan protists of the genus Babesia. Most apicomplexan parasites, including Babesia, have a plastid-derived organelle termed an apicoplast, which is involved in critical metabolic pathways such as fatty acid, iron-sulphur, haem and isoprenoid biosynthesis. Apicoplast genomic data can provide significant information for understanding and exploring the biological features, taxonomic and evolutionary relationships of apicomplexan parasites, and identify targets for anti-parasitic drugs. However, there are limited data on the apicoplast genomes of Babesia species infective to small ruminants.MethodsPCR primers were designed based on the previously reported apicoplast genome sequences of Babesia motasi Lintan and Babesia sp. Xinjiang using Illumina technology. The overlapped apicoplast genomic fragments of six ovine Babesia isolates were amplified and sequenced using the Sanger dideoxy chain-termination method. The full-length sequences of the apicoplast genomes were assembled and annotated using bioinformatics software. The gene contents and order of apicoplast genomes obtained in this study were defined and compared with those of other apicomplexan parasites. Phylogenetic trees were constructed on the concatenated amino acid sequences of 13 gene products using MEGA v.6.06.ResultsThe results showed that the six ovine Babesia apicoplast genomes consisted of circular DNA. The genome sizes were 29,916-30,846 bp with 78.7-81.0% A + T content, 29-31 open reading frames (ORF) and 23-24 transport RNAs. The ORFs encoded four DNA-directed RNA polymerase subunits (rpoB, rpoCl, rpoC2a and rpoC2b), 13 ribosomal proteins, one elongation factor TU (tufA), two ATP-dependent Clp proteases (ClpC) and 7-11 hypothetical proteins. Babesia sp. has three more genes than Babesia motasi (rpl5, rps8 and rpoB). Phylogenetic analysis showed that Babesia sp. is located in a separate clade. Babesia motasi Lintan/Tianzhu and B. motasi Ningxian/Hebei were divided into two subclades.ConclusionsTo our knowledge, this study is the first to elucidate the whole apicoplast genomic structural features of six Babesia isolates infective to small ruminants in China using Sanger sequencing. The data provide useful information confirming the taxonomic relationships of these parasites and identifying targets for anti-apicomplexan parasite drugs.

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Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.

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Apicoplast-targeting antibacterials inhibit the growth of Babesia parasites.

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