Genetic exchange in <i>Trypanosoma brucei</i>: evidence for mating prior to metacyclic stage development

It is well established that genetic exchange occurs between Trypanosoma brucei parasites when two stocks are used to infect tsetse flies under laboratory conditions and a number of such crosses have been undertaken. Both cross and self-fertilisation can take place and, with the products of mating being the equivalent of F1 progeny in a Mendelian system and. Recently, analysis of a large collection of independent progeny using a series of polymorphic micro and minisatellite markers, has formally demonstrated that the allelic segregation at loci on each of the 11-megabase chromosomes conforms to ratios predicted for a classical diploid genetic system involving meiosis as well as independent assortment of markers on different chromosomes. Further extensive analysis of these F1 progeny, using a large panel of micro and minisatellite markers, has led to the construction of a genetic map of one parasite stock A. MacLeod, A. Tweedie and S. McLellan et al., The genetic map of Trypanosoma brucei, Nucleic Acids Res 33 (2005), pp. 6688–6693. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (10)

Genetics and genomics converge on the human blood fluke

A genetic map of the human infective blood fluke Schistosoma mansoni offers new approaches for research on this important parasitic worm

Allelic segregation and independent assortment in <i>T. brucei</i> crosses: proof that the genetic system is Mendelian and involves meiosis

The genetic system on Trypanosoma brucei has been analysed by generating large numbers of independent progeny clones from two crosses, one between two cloned isolates of Trypanosoma brucei brucei and one between cloned isolates of T. b. brucei and Trypanosoma brucei gambiense, Type 2. Micro and minisatellite markers (located on each of the 11 megabase housekeeping chromosomes) were identified, that are heterozygous in one or more of the parental strains and the segregation of alleles at each locus was then determined in each of the progeny clones. The results unequivocally show that alleles segregate in the predicted ratios and that alleles at loci on different chromosomes segregate independently. These data provide statistically robust proof that the genetic system is Mendelian and that meiosis occurs. Segregation distortion is observed with the minisatellite locus located on chromosome I of T. b. gambiense Type 2 and neighboring markers, but analysis of markers further along this chromosome did not show distortion leading to the conclusion that this is due to selection acting on one part of this chromosome. The results obtained are discussed in relation to previously proposed models of mating and support the occurrence of meiosis to form haploid gametes that then fuse to form the diploid progeny in a single round of mating

Molecular surveillance of Theileria parasites of livestock in Oman

Background: Theileriosis is one of the most prevalent infectious diseases of livestock in the Arabian Peninsula, and causes high rates of mortality and morbidity in sheep and cattle. However, there is a paucity of information on the distribution of Theileria spp. over the whole region and their impact on different hosts. The present study carried out a country-wide molecular survey for Theileria spp. of livestock in Oman across four governorates. The aim of the survey was to define the prevalence of Theileria spp. in cattle, sheep and goats, highlight risk factors for infection and identify the main tick species involved in parasite transmission. Material and methods: A total of 2020 animals were examined in the survey consisting of sheep [n = 592], goats [n = 981] and cattle [n = 447]. All three species were raised and co-grazed on the same farms. Theileria parasites were detected using PCR-RFLP and RLB of the 18S rRNA gene. Cloning and sequencing of the 18S rRNA was carried out on 11 T. lestoquardi isolates from Ash-Sharqiyah, and Ad-Dhahira governorates, and phylogenetic relationships were inferred using additional sequences of T. lestoquardi, T. annulata and T. ovis available in GenBank. Results: Theileria spp. prevalence was 72.3%, 36.7% and 2.7% among cattle, sheep and goats, respectively. Strong similarity in results was obtained using RLB and PCR-RFLP for detection of Theileria spp. however, RLB detected a higher rate of mixed infection than PCR-RFPL (P &lt; 0.001). Theileria annulata was the only parasite detected in cattle, while sheep and goats carried T. ovis, T. lestoquardi and T. annulata as well as Theileria spp. OT1. Of the four Theileria spp. detected in small ruminants, overall T. ovis was most prevalent (sheep [33.4%], goats [2.0%]), whereas T. lestoquardi was less prevalent (sheep [22.0%], goats [0.5%]). A large proportion of infected sheep (19%) carried mixed infection of T. ovis and T. lestoquardi. However, single T. lestoquardi infections (3.0%) were less prevalent than T. ovis infections (14.5%). Risk of Theileria spp. infection was significantly higher for exotic breeds, relative to native breeds, of cattle (p = 0.00002) and sheep (p = 0.005). Phylogenetic analysis placed T. lestoquardi in Oman in the same clade as other T. lestoquardi strains isolated from the same regional area (Iraq and Iran). The main tick species, identified on the examined animals, Hyalomma anatolicum, was widely distributed and was found in all of the surveyed governorates. Conclusion: Theileria spp. are widespread in Oman with variable prevalence detected in different regions. Two economically important hosts, cattle and sheep are at high risk from virulent T. annulata and T. lestoquardi, respectively. The survey indicates extensive exposure to ticks and transmission of infection that has a significant economic impact. The higher prevalence of T. lestoquardi as mixed rather than single infection requires further investigation

Population genetics of trypanosoma brucei rhodesiense: clonality and diversity within and between foci

African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda) and Southern (Malawi) Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics

Discovery of mating in the major African livestock pathogen Trypanosoma congolense

The protozoan parasite, Trypanosoma congolense, is one of the most economically important pathogens of livestock in Africa and, through its impact on cattle health and productivity, has a significant effect on human health and well being. Despite the importance of this parasite our knowledge of some of the fundamental biological processes is limited. For example, it is unknown whether mating takes place. In this paper we have taken a population genetics based approach to address this question. The availability of genome sequence of the parasite allowed us to identify polymorphic microsatellite markers, which were used to genotype T. congolense isolates from livestock in a discrete geographical area of The Gambia. The data showed a high level of diversity with a large number of distinct genotypes, but a deficit in heterozygotes. Further analysis identified cryptic genetic subdivision into four sub-populations. In one of these, parasite genotypic diversity could only be explained by the occurrence of frequent mating in T. congolense. These data are completely inconsistent with previous suggestions that the parasite expands asexually in the absence of mating. The discovery of mating in this species of trypanosome has significant consequences for the spread of critical traits, such as drug resistance, as well as for fundamental aspects of the biology and epidemiology of this neglected but economically important pathogen

T. brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

Aims: African trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response. Methods and results: Using confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events. Conclusion: These data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function

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

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct