C. M. R. Turner

High frequency of antigenic variation in Trypanosoma brucei rhodesiense infections

Rates at which Trypanosoma brucei rhodesiense trypanosomes switch from expression of one variable antigen type (VAT) to that of another have been determined in cloned populations that have been recently tsetse-fly transmitted. Switching rates have been determined between several, specific pairs of VATs in each population. High rates of switching were observed in 2 cloned trypanosome lines, each derived from a separate cyclical transmission of the same parental stock and each expressing a different major VAT. Five estimates of the switching rate between one particular pair of VATs were consistently high (approximately 1 x 10(-3) switches/cell/generation). These high switching rates were similar both in bloodstream populations of mice and in populations confined to subcutaneously implanted growth chambers in mice, thus indicating that the interaction of the bloodstream population with other trypanosome populations in the lymphatics or extravascular sites in systemic infections did not influence the estimates of the rate of switching. Fourteen estimates were made of VAT-specific switching rates in bloodstream infections involving 8 combinations from among 6 VATs. Switching rate estimates were VAT-specific and showed considerable variation between different combinations of VATs--from 1.9 x 10(-6) to 6.9 x 10(-3) switches/cell/generation. The rates of switching to different metacyclic-VATs were, however, very similar. Summation of between 3 and 5 VAT-specific switching rate values in each of 4 experiments conducted in bloodstream infections has provided minimum estimates of the overall rate of antigenic variation: 2.0-9.3 x 10(-3) switches/cell/generation. These values are between 20 and 66,000-fold higher than previously published estimates.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that the mechanism of gene exchange in Trypanosoma brucei involves meiosis and syngamy.

All pairwise combinations of three cloned stocks of Trypanosoma brucei (STIB 247L, STIB 386AA and TREU 927/4) were co-transmitted through tsetse flies (Glossina morsitans) and screened for the production of hybrid trypanosomes. Clones of metacyclic and bloodstream trypanosomes from flies harbouring mature infections containing hybrid trypanosomes were established and screened for several isoenzyme and restriction fragment length polymorphisms. For each of the three combinations of parents, some progeny clones were observed to be of a phenotype and genotype indicating that genetic exchange had occurred during development of the trypanosomes in flies. These hybrid clones shared three salient features: (1) where the parents were homozygous variants the progeny were heterozygous, (2) where one of the parents was heterozygous, allelic segregation was observed and (3) the progeny clones were shown to be recombinant when two or more markers for which one of the parents was heterozygous were examined. These results are consistent with the progeny being an F1 in a diploid mendelian genetic system involving meiosis and syngamy. Our observations show that all possible combinations of the three stocks may undergo genetic exchange. A marker analysis of a series of clones each derived from single metacyclic trypanosomes showed that individual flies transmit a mixture of trypanosome genotypes corresponding to F1 progeny and to parental types, indicating that genetic exchange was a non-obligatory event in the life-cycle of the trypanosome. In addition, a preliminary analysis of the phenotype of procyclic stage trypanosomes derived from flies infected with two stocks, indicates that genetic exchange is unlikely to occur at this stage.

Mechanism of Trypanosoma brucei gambiense (group 1) resistance to human trypanosome lytic factor

Human innate immunity against most African trypanosomes, including Trypanosoma brucei brucei, is mediated by a minor subclass of toxic serum HDL, called trypanosome lytic factor-1 (TLF-1). This HDL contains two primate specific proteins, apolipoprotein L-1 and haptoglobin (Hp)-related protein, as well as apolipoprotein A-1. These assembled proteins provide a powerful defense against trypanosome infection. Trypanosoma brucei rhodesiense causes human African sleeping sickness because it has evolved an inhibitor of TLF-1, serum resistance-associated (SRA) protein. Trypanosoma brucei gambiense lacks the SRA gene, yet it infects humans. As transfection of T. b. gambiense (group 1) is not possible, we initially used in vitro-selected TLF-1–resistant T. b. brucei to examine SRA-independent mechanisms of TLF-1 resistance. Here we show that TLF-1 resistance in T. b. brucei is caused by reduced expression of the Hp/Hb receptor gene (TbbHpHbR). Importantly, T. b. gambiense (group 1) also showed a marked reduction in uptake of TLF-1 and a corresponding decrease in expression of T. b. gambiense Hp/Hb receptor (TbgHpHbR). Ectopic expression of TbbHpHbR in TLF-1–resistant T. b. brucei rescued TLF-1 uptake, demonstrating that decreased TbbHpHbR expression conferred TLF-1 resistance. Ectopic expression of TbgHpHbR in TLF-1–resistant T. b. brucei failed to rescue TLF-1 killing, suggesting that coding sequence changes altered Hp/Hb receptor binding affinity for TLF-1. We propose that the combination of coding sequence mutations and decreased expression of TbgHpHbR directly contribute to parasite evasion of human innate immunity and infectivity of group 1 T. b. gambiense.

Replication, differentiation, growth and the virulence of Trypanosoma brucei infections

This study had 2 objectives: first, to investigate how the processes of slender form replication, of differentiation from dividing slender to non-dividing stumpy forms, and of stumpy mortality, combine to determine the initial (acute-phase) growth rate of Trypanosoma brucei populations; second, to determine how acute-phase growth rates influence parasite densities during the subsequent, chronic phase of infection. During the acute phase, slender and stumpy populations both grew approximately exponentially, the latter more slowly than the former. Mathematical models showed how this difference in slender and stumpy growth rates can be explained in terms of heterogeneous replication and differentiation rates. Stumpy life-expectancy was determined for one stock and found to be age-dependent with a half-life of 48-72 h, much larger than observed population doubling times of 5-10 h. A comparison of cloned stocks showed that the highest parasite densities during the chronic phase were associated with the highest acute-phase growth rates of both the whole parasite population and of the subpopulation of slender forms. By contrast, high chronic-phase parasitaemias artificially produced following rapid syringe passage were associated with low acute-phase growth rates of slender forms. Syringe-passaging is a laboratory procedure which selects for virulent parasites, but these parasites behave differently from naturally virulent stocks.

An estimate of the size of the metacyclic variable antigen repertoire of Trypanosoma brucei rhodesiense

A group of 27 variable antigen type (VAT)-specific monoclonal antibodies (McAbs) have been made against metacyclic forms of a cloned stock of Trypanosoma brucei rhodesiense. In combination, these labelled in immunofluorescence 99.3% of trypanosomes in salivary probes from tsetse flies. The 0.7% of unlabelled trypanosomes were believed to be uncoated forms. The ability of a mixture of antibodies to kill metacyclics in vitro by complement-mediated lysis, thus neutralizing their infectivity for mice, was tested. The antibody mixture consisted of 24 McAbs plus 3 VAT-specific rabbit antisera. In 12 replicate experiments this mixture of antibodies prevented infection of mice. Parallel controls showed that neutralization was probably antibody-mediated and VAT specific. However, we have not been able to repeat these results on a long-term basis; this may be due to a loss of neutralizing activity by one of the McAbs. The successful neutralization experiments indicate that the number of VATs in the metacyclic repertoire of one stock of T. b. rhodesiense is limited to at most 27.

Loss of variable antigen during transformation of Trypanosoma brucei rhodesiense from bloodstream to procyclic forms in the tsetse fly

A pleomorphic line of Trypanosoma brucei rhodesiense expressing a single variable antigen was used to quantify the rate of loss of the surface coat from bloodstream forms transforming to procyclics in the tsetse fly, Glossina morsitans, and in in vitro culture. Loss of variable antigen occurred at similar rates in the crop and anterior portion of the midgut of tsetse flies and in in vitro culture, but in the posterior portion of the fly midgut it occurred 2–3 times faster. The posterior portion of the midgut is the most important site for transformation of bloodstream-form trypanosomes to procyclics, and the dynamics of at least one component of this process are therefore not accurately paralleled in vitro.

Comparison of the effects of immune killing mechanisms on Trypanosoma brucei parasites of slender and stumpy morphology

Trypanosoma brucei slender forms predominate over stumpy forms as the parasite population grows but at the peak of a parasitaemic wave and during remission of infection stumpy forms predominate. To determine whether this change in predominance might be caused by selective killing of slender forms, the fates of slender and stumpy form trypanosomes in two in vitro assays of immune‐mediated killing were compared. Parasite populations in which > 90% of cells were of slender morphology were observed to be killed by antibody‐dependent complement‐mediated lysis approximately five times faster than populations in which < 15% of cells were slender and most were of intermediate or stumpy morphology. Quantification of the relationship between the proportion of slender forms in the population and the rate of lysis indicated that slender forms were killed approximately 7.3 times faster than other forms. In an opsonization assay, no differences were observed between slender and stumpy forms in the extent to which they attached to macrophages in an antibody‐dependent manner. These results suggest that the change in proportions of slender and stumpy forms at the peak of a parasitaemic wave results from slender forms being more susceptible to complement‐mediated killing as the antibody response develops.

Trypanosoma vivax displays a clonal population structure

African animal trypanosomiasis, or Nagana, is a debilitating and economically costly disease with a major impact on animal health in sub-Saharan Africa. Trypanosoma vivax, one of the principal trypanosome species responsible for the disease, infects a wide host range including cattle, goats, horses and donkeys and is transmitted both cyclically by tsetse flies and mechanically by other biting flies, resulting in a distribution covering large swathes of South America and much of sub-Saharan Africa. While there is evidence for mating in some of the related trypanosome species, Trypanosoma brucei, Trypanosoma congolense and Trypanosoma cruzi, very little work has been carried out to examine this question in T. vivax. Understanding whether mating occurs in T. vivax will provide insight into the dynamics of trait inheritance, for example the spread of drug resistance, as well as examining the origins of meiosis in the order Kinetoplastida. With this in mind we have identified orthologues of eight core meiotic genes within the genome, the presence of which imply that the potential for mating exists in this species. In order to address whether mating occurs, we have investigated a sympatric field population of T. vivax collected from livestock in The Gambia, using microsatellite markers developed for this species. Our analysis has identified a clonal population structure showing significant linkage disequilibrium, homozygote deficits and disagreement with Hardy-Weinberg predictions at six microsatellite loci, indicative of a lack of mating in this population of T. vivax.

Human infectivity trait in Trypanosoma brucei: stability, heritability and relationship to sra expression

Some Trypanosoma brucei lines infect humans whereas others do not because the parasites are lysed by human serum. We have developed a robust, quantitative in vitro assay based on differential uptake of fluorescent dyes by live and dead trypanosomes to quantify the extent and kinetics of killing by human serum. This method has been used to discriminate between 3 classes of human serum resistance; sensitive, resistant and intermediate. TREU 927/4, the parasite used for the T. brucei genome project, is intermediate. The phenotype is expressed in both bloodstream and metacyclic forms, is stably expressed during chronic infections and on cyclical transmission through tsetse flies. Trypanosomes of intermediate phenotype are distinguished from sensitive populations of cells by the slower rate of lysis and by the potential to become fully resistant to killing by human serum as a result of selection or long-term serial passaging in mice, and to pass on full resistance phenotype to its progeny in a genetic cross. The sra gene has been shown previously to determine human serum resistance in T. brucei but screening for the presence and expression of this gene indicated that it is not responsible for the human serum resistance phenotype in the trypanosome lines that we have examined, indicating that an alternative mechanism for HSR exists in these stocks. Examination of the inheritance of the phenotype in F1 hybrids for both bloodstream and metacyclic stages from 2 genetic crosses demonstrated that the phenotype is co-inherited in both life-cycle stages in a manner consistent with being a Mendelian trait, determined by only one or a few genes.

Independent expression of the metacyclic and bloodstream variable antigen repertoires of Trypanosoma brucei rhodesiense

The variable antigen repertoire expressed by metacyclic Trypanosoma brucei rhodesiense is not influenced by the anamnestic expression whereby the variable antigen type (VAT) ingested by a tsetse fly is present at high levels in early bloodstream populations of fly-infected mice. This has been demonstrated by feeding to Glossina morsitans a trypanosome line expressing a VAT which is normally a component of the metacyclic repertoire. The VAT did not constitute a significantly increased proportion of the resultant metacyclic population which would have occurred had anamnestic expression and metacyclic expression been linked. Five other metacyclic VATs were also present at control levels. We conclude that the mechanisms of expression of VATs in the fly and in the mammal are independently controlled.