Cell cycle, growth and differentiation in Trypanosoma brucei and Leishmania species
Kinetoplastid protozoans have unusual cell cycles. Three unitary organelles need to be replicated and segregated to the daughter cells at each cell division; the nucleus, kinetoplast and basal body, associated with the flagellum The replication and segregation of these organelles requires to be co-ordinated. Inthis study the timing of nuclear and kinetoplast cell cycle events was examined in two species of kinetoplastids, Trypanosoma brucei and Leishmania mexicana, which are major pathogens of humans, to ascertain the degree of co-ordination between the two organelles in the cell cycle. Particular attention was paid to three main features; the duration of S-phase for each organelle, the relative timing of mitosis and kinetoplast division and the lengths of the post-mitotic/division cytokinesis periods. Two life cycle stages were examined for each species i.e. procyclic and bloodstream forms of T. brucei and promastigote and amastigote forms of L. mexicana. The objectives of this study were to establish firstly whether a similar pattern of events occurs for both stages within each species examined and secondly, whether or not T. brucei and L. mexicana share common features to their cell cycles. Studies of cell cycle events were conducted using immunofluorescent labelling to detect S-phase in both organelles, enabled by use of an anti-bromo-deoxyuridine (BrdU) antibody as an S-phase marker. Division of organelles was identified by staining the cells with DAPI, a DNA intercalating dye. Initial studies confirmed that the cell cycles of both species were consistent with the general pattern of events of the eukaryotic nuclear cycle. Also, as has been described previously in kinetoplastids, the kinetoplast DNA (containing mitochondrial DNA) has a pattern of discretely separated phases of replication and division analagous to the nuclear cycle. Analysis was made of the relative timing of S-phase, mitosis/division and cytokinesis for each life cycle stage, producing data which were statistically testable. For both life cycle stages of T. brucei a similar pattern of events was observed. Three populations of BrdU labelled cells were identified; cells which were BrdU labelled in the nucleus only, labelled in the kinetoplast only and labelled for both organelles. These observations indicate that there was a non-co-ordinate start and finish to S-phase, with an overlap in the timing of the S-phase periods. Kinetoplast division was initiated and completed before the start of mitosis in the nucleus and an extended cytokinesis period for each organelle was identified, although of longer duration in the kinetoplast. For both life cycle stages of L. mexicana, three populations of cells labelled with BrdU were observed as in T. brucei, again indicating that there was a non-co-ordinate start and finish to S-phase, with an overlap in the timing of both periods. The sequence of division for the organelles for both life cycle stages differed to that observed in T brucei. The nucleus divided before the kinetoplast and there was a much shorter cytokinesis period for both organelles in comparison to T brucei. These differences in the pattern of events may reflect either a difference in the control of cell cycle timing in each species or in the morphology of each cell type. Trypanosomes have to re-arrange their organelles, such that they occupy sites on either side of the division furrow, hence the extended cytokinesis phase. In L. mexicana, however, this requirement for organelle re-positioning is reduced or absent and therefore the cytokinesis periods are much shorter. Antigenic variation is the key strategy which allows African trypanosomes to evade the effects of the host's immune response. The switching from expression of one variant surface glycoprotein (VSG) to that of another has been indirectly linked to the cell cycle, as bloodstream slender forms divide and undergo antigenic switching, whereas bloodstream stumpy forms are non-dividing and do not appear to switch. An attempt to examine directly the proposed link between antigenic switching and the cell cycle was made using cell cycle markers (anti-BrdU antibody as an S-phase marker and DAPI staining to determine the configurations of the organelles), as well as VAT specific antibodies against VSGs expressed in the cloned lines studied. Unfortunately, trypanosomes expressing two VSG coats simultaneously and therefore undergoing antigenic switch, could not be detected in any of the cloned lines examined. This unresolved difficulty was attributed to a deficiency in the detection system employed. Differentiation in kinetoplastids is thought to be linked to the cell cycle, as progression through the life cycle involves transitions from proliferative to nonproliferative forms. The differentiation of Leishmania major promastigote forms, from dividing non-infective stages to non-dividing infective meta cyclic forms, involves major molecular and morphological changes. Using three metacyclic-specific markers (non-agglutination by peanut agglutinin, a monoclonal antibody (3F12) against metacyclic-specific epitopes of the major surface molecule lipophosphoglycan (LPG) and a rabbit anti-serum (ab336) against a metacyclic-specific surface protein, the Gene B protein) metacyclic production in in vitro culture was examined. Observation of population growth CUIVes suggests that differentiation is in part intrinsically programmed within the parasite but may also be inducible by environmental changes. Comparison of these data with mathematical models indicated that the promastigote population was likely to be heterogeneous, containing sub-populations that replicate and differentiate at different rates. Two major assumptions of the heterogeneous model are that meta cyclic forms are both non-dividing and incapable of dedifferentiation. These two possibilities were tested experimentally and indicated that meta cyclic forms are indeed non-dividing, but are also capable of de-differentiation, albeit at a very low rate. Examination of meta cyclic production at the cellular level also indicated that the event which causes commitment to differentiation occurs at least one cell division before symmetrical production of two daughter meta cyclic forms. It has been found that in the presence of a chronic infection the growth of a secondary infection is significantly inhibited (Turner et al., 1996). The available data suggests that there appears to be an overall down-regulation in the growth of the entire mixed population. The aim of this component of the project was to select for 'growth' mutant trypanosomes i.e. mutants which overcome growth inhibition. Bloodstream trypanosomes which had been mutagenised in vitro with ethyl methane sulphonate (EMS) were inoculated into mice in the presence of a pre-existing chronic infection. Populations of mutagenised trypanosomes which grew significantly in comparison to controls where only low rates of growth occurred, were selected by optical cloning. This rationale led to the generation of growth mutant clones which stably expressed the altered growth phenotype. These studies may permit the development of methods for the investigation of the regulation of growth and virulence in these parasites. In conclusion, the analysis of the relative timing of cell cycle events in T. brucei and L. mexicana has highlighted both common and novel features to the cell cycles of these kinetoplastids. The study of meta cyclic production in L. major has also revealed the intrinsic and extrinsic nature of regulation of differentiation in these parasites, as well as the commitment to differentiate at the cellular level. Furthermore, the generation of 'growth' mutant trypanosomes provides an additional tool for the future study of growth regulation in trypanosome infections.