title: Human serum resistance in Trypanosoma brucei
Creators: name: Lindergard, Louise Anna Gabriella
Publisher: name: University of Glasgow
Year: 1999
Subjects: subject: Parasitology.

description: T.b.brucei, T.b.rhodesiense and T.b.gambiense are all morphological identical and cannot be distinguished by microscopical observations. The original definition of these three subspecies was presented by Hoare (1970) taking into account their geographical distribution, their host specificity, their ability to infect humans and the pattern of disease they cause. By these criteria, T.b.brucei is defined as non-human infective and T.b.gambiense and T.b.rhodesiense as human infective since they have the ability to withstand lysis by human serum. The underlying mechanisms for human serum sensitivity/resistance are not yet fully understood and more than one factor in human serum may be involved. The overall aim of this thesis was to investigate the inheritance and molecular basis for human serum resistance/sensitivity. Inheritance studies were made possible because of the availability of recombinant progeny from a cross between a human serum resistant and a human serum sensitive trypanosome strain. A molecular approach based on differential display RT-PCR was also used to complement the more biochemical approaches adopted by others. A range of variables were examined and optimised to develop a reliable in vitro bloodstream form human serum sensitivity assay but it was clear that cell viability could not be estimated from morphological appearance alone. A statistically significant difference in mean percentage lysis was observed between stocks that correlated with human serum sensitivity but the data varied too much between individual assays for the assay to provide unambiguous results. Instead, a reliable in vivo human serum resistance assay, which involved incubating bloodstream trypanosomes with human serum, injecting them into mice and screening of the mice for the development of parasitaemia, was optimised and adopted. Genetic analysis and the heritability of human serum resistance/sensitivity was investigated with the aim of revealing the number of loci and alleles involved and determining the dominance relationships between alleles. From the pattern of inheritance a model for the inheritance of human serum resistance is suggested and a number of models for inheritance which are incompatible with the results are refuted. As a result of detecting F1 progeny clones of intermediate resistance, the simplest possible model is based on 3 alleles; codominant sensitive and resistant alleles and a recessive sensitive wild type allele, all at a single locus. A candidate gene determining resistance to human serum has been previously described; the serum resistance associated (SRA) gene (De Greef &amp; Hamers, 1994). To determine whether SRA expression was causing resistance in the present study, the presence of a genomic copy of the SRA gene was initially investigated by PCR in the cloned stocks STIB 386, STIB 247 and TREU 927. PCR products of the expected size were obtained from STIB 247 and TREU 927 but not from STIB 386. No PCR product could be amplified from STIB 386 whatever primer combination and PCR conditions were used implying that the SRA gene may be absent from this isolate. To further investigate the presence of a sequence related to SRA in STIB 386 and TREU 927, Southern blots were performed. Hybridisation with fragments of similar, but not identical, sizes were noted in all of the clones indicating that sequences with homology are present but that sequence variation occurs within the SRA gene. The expression of the SRA gene and its correlation with a HSR phenotype was further examined but its expression does not coincide with HSR in the stocks used in this study. A study was initiated to identify candidate genes determining resistance or sensitivity to human serum. To do this differential display RT-PCR was used in order to identify cDNA fragments expressed in either of the serum sensitive or serum resistant trypanosome lines. Nine differentially amplified products were identified, reamplified, cloned and sequenced and 16 clones obtained. Four of these clones showed expression patterns associated with human serum resistance or sensitivity, three were specifically expressed in human serum sensitive trypanosomes and one was specifically expressed in human serum resistant trypanosomes. Two of the sensitive specific clones were related to EST sequences from a cDNA library of a T.brucei bloodstream clone which have not been further characterised. The other sensitive specific clone was part of a gene for ribosomal RNA while the resistance specific clone was unrelated to any other sequences in the database. These data, taken together, suggest that human serum sensitivity in trypanosomes is determined by both a lack of resistance and a specific expression of gene product(s) conferring sensitivity.