Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense.

Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.

Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol.

The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.

The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets.

The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.

In silico prediction of antimalarial drug target candidates.

The need for new antimalarials is persistent due to the emergence of drug resistant parasites. Here we aim to identify new drug targets in Plasmodium falciparum by phylogenomics among the Plasmodium spp. and comparative genomics to Homo sapiens. The proposed target discovery pipeline is largely independent of experimental data and based on the assumption that P. falciparum proteins are likely to be essential if (i) there are no similar proteins in the same proteome and (ii) they are highly conserved across the malaria parasites of mammals. This hypothesis was tested using sequenced Saccharomycetaceae species as a touchstone. Consecutive filters narrowed down the potential target space of P. falciparum to proteins that are likely to be essential, matchless in the human proteome, expressed in the blood stages of the parasite, and amenable to small molecule inhibition. The final set of 40 candidate drug targets was significantly enriched in essential proteins and comprised proven targets (e.g. dihydropteroate synthetase or enzymes of the non-mevalonate pathway), targets currently under investigation (e.g. calcium-dependent protein kinases), and new candidates of potential interest such as phosphomannose isomerase, phosphoenolpyruvate carboxylase, signaling components, and transporters. The targets were prioritized based on druggability indices and on the availability of in vitro assays. Potential inhibitors were inferred from similarity to known targets of other disease systems. The identified candidates from P. falciparum provide insight into biochemical peculiarities and vulnerable points of the malaria parasite and might serve as starting points for rational drug discovery.

Genome-wide identification of molecular mimicry candidates in parasites.

Among the many strategies employed by parasites for immune evasion and host manipulation, one of the most fascinating is molecular mimicry. With genome sequences available for host and parasite, mimicry of linear amino acid epitopes can be investigated by comparative genomics. Here we developed an in silico pipeline for genome-wide identification of molecular mimicry candidate proteins or epitopes. The predicted proteome of a given parasite was broken down into overlapping fragments, each of which was screened for close hits in the human proteome. Control searches were carried out against unrelated, free-living eukaryotes to eliminate the generally conserved proteins, and with randomized versions of the parasite proteins to get an estimate of statistical significance. This simple but computation-intensive approach yielded interesting candidates from human-pathogenic parasites. From Plasmodium falciparum, it returned a 14 amino acid motif in several of the PfEMP1 variants identical to part of the heparin-binding domain in the immunosuppressive serum protein vitronectin. And in Brugia malayi, fragments were detected that matched to periphilin-1, a protein of cell-cell junctions involved in barrier formation. All the results are publicly available by means of mimicDB, a searchable online database for molecular mimicry candidates from pathogens. To our knowledge, this is the first genome-wide survey for molecular mimicry proteins in parasites. The strategy can be adopted to any pair of host and pathogen, once appropriate negative control organisms are chosen. MimicDB provides a host of new starting points to gain insights into the molecular nature of host-pathogen interactions.

Species-specific typing of DNA based on palindrome frequency patterns.

DNA in its natural, double-stranded form may contain palindromes, sequences which read the same from either side because they are identical to their reverse complement on the sister strand. Short palindromes are underrepresented in all kinds of genomes. The frequency distribution of short palindromes exhibits more than twice the inter-species variance of non-palindromic sequences, which renders palindromes optimally suited for the typing of DNA. Here, we show that based on palindrome frequency, DNA sequences can be discriminated to the level of species of origin. By plotting the ratios of actual occurrence to expectancy, we generate palindrome frequency patterns that allow to cluster different sequences of the same genome and to assign plasmids, and in some cases even viruses to their respective host genomes. This finding will be of use in the growing field of metagenomics.