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The major surface antigen of procyclic and epimastigote forms of Trypanosoma congolense in the tsetse fly is GARP (glutamic acid/alanine-rich protein), which is thought to be the analogue of procyclin/PARP in Trypanosoma brucei. We have studied two T.congolense GARP loci (the 4.3 and 4.4 loci) whose transcription is alpha-amanitin sensitive. Whilst a transcriptional gap 5' of the first GARP gene in the cloned region of the 4.4 locus could not be detected, such a gap was present in the 5' flank of the first GARP gene in the 4.3 locus. We have located a GARP transcription start site and, using reporter gene constructs containing a putative GARP promoter region in transient transfection studies, we have demonstrated promoter activity for the test region in T.congolense. There are species-specific differences in sequences regulating expression of the two major surface antigens, GARP and procyclin/PARP: the GARP promoter is inactive in T.brucei while the procyclin/PARP promoter is inactive in T.congolense. We have defined the splice acceptor site for the 4.3 GARP gene by sequencing and by 5' RT-PCR and demonstrated microheterogeneity in GARP polyadenylation by 3' RT-PCR. It appears that some GARP and procyclin/PARP RNA processing signals, although similar, are also species-specific.

Five monoclonal antibodies (mAb) were raised that bound to the surface of procyclic stage Trypanosoma congolense with high intensity in immunofluorescence. Immunoblot analysis of trypanosome lysates using 3 of these mAb revealed a diffuse SDS-PAGE band of 36-40 kDa. The purified antigen did not react with Coomassie Blue or silver stains, but did stain blue with Stains-allTM, indicating acidity. For the one mAb tested, the epitope was periodate-sensitive and therefore probably glycan. Although this antigen shares properties with procyclin/PARP, which forms a surface coat on procyclic Trypanosoma brucei, a search in T. congolense for homologues of a procyclin/PARP gene revealed only non-coding sequence of partial similarity. Using a differential screen, a procyclic stage T. congolense cDNA clone was isolated that encoded a putative 256-amino acid protein containing 2 peptides chemically sequenced independently by Beecroft et al. [36]. The protein, termed glutamate and alanine-rich protein (GARP), has potential hydrophobic leader and tail sequences (the latter with potential for replacement by aglycosyl phosphoinositol anchor) and no potential N-linked glycosylation sites. It has no significant sequence homology with known proteins. Antibodies against a translational fusion of GARP bound specifically in Western blots to a band very similar to that detected by the mAb and also to the purified antigen. Immunogold electron microscopy revealed a dense packing of the antigen on the cell surface. It appears that procyclic T. brucei and T. congolense have major surface proteins with structural analogy, but with no sequence homology.

Expression site-associated genes (ESAGs) of Trypanosoma brucei are found upstream of variant surface glycoprotein (VSG) genes in bloodstream expression sites. There are at least 6 different ESAGs in each of these expression sites, and each ESAG is repetitive in the genome. ESAGs are believed to reside only in VSG expression sites and to be co-transcribed with the VSG gene from a common alpha-amanitin-insensitive promoter. Our results show that this is not always true. The transcriptionally active 1.22 metacyclic expression site contains no ESAGs, but ESAGs are highly transcribed in these cells. The level of transcription indicates that more than one copy of each of these genes is active. Furthermore, some of these genes are transcribed, to produce steady state RNA, in procyclic culture cells which do not express the VSG gene: there is differential expression of ESAGs between the bloodstream and procyclic phases of the trypanosome life cycle. Thus ESAGs can be transcribed outwith an active VSG gene expression site and in the absence of expression of the VSG.

We describe the use of repetitive DNA probes to characterise the relationships between different stocks of African trypanosomes representing the subspecies of Trypanosoma brucei. Probes derived from the ribosomal RNA genes (coding region and nontranscribed spacer) and another repetitive DNA sequence were used to characterise trypanosome stocks by Southern blotting. Numerical taxonomy methods applied to the resulting restriction enzyme patterns were used to derive a dendrogram depicting the relationships between the stocks examined. We show that three groups of West African human infective stocks can be distinguished: firstly, a group containing exclusively T. b. gambiense; secondly, a group which is indistinguishable from animal isolates in West Africa; and thirdly, a single stock which is indistinguishable from East African T. b. rhodesiense. In addition, we observe that T. b. rhodesiense stocks from East Africa are indistinguishable from animal isolates from the same area. Finally, we show that a group of T. b. rhodesiense stocks, isolated from a 1978 sleeping sickness outbreak in Zambia, are probably derived from a single parasite strain, and that this strain is distinct from T. b. rhodesiense parasites from Kenya and Uganda.

Trypanosoma brucei, the parasite causing human sleeping sickness, relies on the tsetse fly for its transmission. In the insect, EP and GPEET procyclins are the major surface glycoproteins of procyclic (midgut) forms of the parasite, with GPEET predominating in the early procyclic form and two isoforms of EP in the late procyclic form. EP procyclins were previously detected on salivary gland trypanosomes, presumably epimastigotes, by immunoelectron microscopy. However, no procyclins could be detected by mass spectrometry when parasites were isolated from infected glands. We have used qualitative and quantitative RT-PCR to analyse the procyclin mRNAs expressed by trypanosomes in the tsetse midgut and salivary glands at different time points after infection. The coding regions of the three EP isoforms (EP1, EP2 and EP3) are extremely similar, but their 3′ untranslated regions contain unique sequences that make it possible to assign the cDNAs amplified by this technique. With the exception of EP2, we found that the spectrum of procyclin mRNAs expressed in the midgut mirrors the protein repertoire of early and established procyclic forms. Surprisingly, procyclin mRNAs, including that of GPEET, are present at relatively high levels in salivary gland trypanosomes, although the proteins are rarely detected by immunofluorescence. Additional experiments using transgenic trypanosomes expressing reporter genes or mutant forms of procyclin point to a mechanism of translational or post-translational control, involving the procyclin coding regions, in salivary gland trypanosomes. It is widely accepted that T. brucei always has a coat of either variant surface glycoprotein or procyclin. It has been known for many years that the epimastigote form does not have a variant surface glycoprotein coat. The finding that this life cycle stage is usually negative for procyclin as well is new, and means that the paradigm will need to be revised., Synopsis The tropical parasite Trypanosoma brucei relies on the tsetse fly for its transmission between mammals. The parasite first establishes an infection in the fly midgut then migrates and colonizes the salivary glands. A paradigm in trypanosome biology is that parasites in contact with the mammalian host are covered by variant surface glycoproteins, enabling them to evade the immune system, while all other life cycle stages are covered by repetitive glycoproteins known as procyclins. In this publication the authors investigated the expression pattern of procyclins in all life cycle stages in infected tsetse flies, including epimastigote and metacyclic forms in the salivary glands. These stages are usually neglected as, in contrast to trypanosomes from the midgut, they cannot be cultured. The researchers showed that the vast majority of trypanosomes in the salivary glands have no procyclin coat although the levels of procyclin messenger RNAs are comparatively high. Experiments with reporter genes indicated that procyclin expression in the glands is regulated by the coding region. The lack of procyclins on salivary gland trypanosomes leads to the prediction that the epimastigote form possesses a novel surface coat that may be responsible for the tight binding of the parasite to gland tissue.

Four types of proteolytic activity were detected in the bloodstream form of each of the four Trypanosoma species: (i) HPAase, active on hide powder azure and detected on polyacrylamide gels containing denatured haemoglobin; (ii) AZCase, active on azocasein; (iii) type 1, active on the chromogenic peptide N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine p-nitroanilide in the presence of dithiothreitol, and (iv) type 2, active against several nitroanilide derivatives in the absence of dithiothreitol. Studies of the pH optimum, dithiothreitol requirement and inhibitor sensitivities of the proteolytic activities suggested that: (a) HPAase and type 1 activities could be due to the same enzymes, probably a family of cysteine proteinases; (b) AZCase had some characteristics of a cysteine proteinase, but was not identical to HPAase, and (c) type 2 activity could be due to a serine proteinase. Procyclic T. brucei contained relatively low cysteine proteinase activities (HPAase, AZCase and type 1) but high type 2 activity. Their proteolytic enzymes thus were apparently more similar to those in Crithidia fasciculata and Leishmania tarentolae promastigotes than those in T. brucei bloodstream forms.

A cDNA for a Trypanosoma brucei cysteine proteinase has been cloned and sequenced. The deduced protein can be divided into four domains, based on homologies with other cysteine proteinases: the pre-, pro- and central regions show considerable homology to the cathepsin L class of mammalian enzymes, whilst the long C-terminal extension distinguishes the trypanosome enzyme from all mammalian cysteine proteinases reported. This 108 amino acid extension, which includes 9 contiguous prolines near the junction with the central domain, appears likely to be processed in part to produce the mature enzyme, and may be involved in targetting the protein within the cell. The trypanosome genome contains more than 20 copies of the cysteine proteinase gene arranged in a long tandem array.

The identification of antigen types in tsetse salivary gland metacyclic populations of Trypanosoma brucei requires the production of monospecific antisera to the corresponding bloodstream variable antigen types. Monospecific antisera against clones from cyclically transmitted populations are difficult to prepare, however, owing to the antigenic lability of such clones. This problem has been overcome by isolating an antigenically stable clone from a syringe-infected rabbit at a time when its serum showed incipient activity towards metacyclic trypanosomes. Monospecific antisera raised against this clone reacted with up to 20% metacyclics in trypanolysis and immunofluorescence tests, confirming that a clone-derived metacyclic population of T. brucei is heterogeneous with respect to variable antigen type.