Alain Boulangé

Congopain from Trypanosoma congolense: Drug target and vaccine candidate

Abstract Trypanosomes are the etiological agents of human sleeping sickness and livestock trypanosomosis (nagana), which are major diseases in Africa. Their cysteine proteases (CPs), which are members of the papain family, are expressed during the infective stages of the parasites life cycle. They are suspected to act as pathogenic factors in the mammalian host, where they also trigger prominent immune responses. Trypanosoma congolense, a major pathogenic species in livestock, possesses at least two families of closely related CPs, named CP1 and CP2. Congopain, a CP2-type of enzyme, shares structural and functional resemblances with cruzipain from T. cruzi and with mammalian cathepsin L. Like CPs from other Trypanosomatids, congopain might be an attractive target for trypanocidal drugs. Here we summarise the current knowledge in the two main areas of research on congopain: first, the biochemical properties of congopain were characterised and its substrate specificity was determined, as a first step towards drug design; second, the possibility was being explored that inhibition of congopain by hostspecific antibodies may mitigate the pathology associated with trypanosome infection.

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: Targetting the disease rather than the parasite

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique to Trypanosoma congolense

ABSTRACT Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis.

Identification of total and differentially expressed excreted-secreted proteins from Trypanosoma congolense strains exhibiting different virulence and pathogenicity.

Animal trypanosomosis is a major constraint to livestock productivity in the tropics and has a significant impact on the life of millions of people globally (mainly in Africa, South America and south-east Asia). In Africa, the disease in livestock is caused mainly by Trypanosoma congolense, Trypanosoma vivax, Trypanosoma evansi and Trypanosoma brucei brucei. The extracellular position of trypanosomes in the bloodstream of their host requires consideration of both the parasite and its naturally excreted-secreted factors (secretome) in the course of pathophysiological processes. We therefore developed and standardised a method to produce purified proteomes and secretomes of African trypanosomes. In this study, two strains of T. congolense exhibiting opposite properties of both virulence and pathogenicity were further investigated through their secretome expression and its involvement in host-parasite interactions. We used a combined proteomic approach (one-dimensional SDS-PAGE and two-dimensional differential in-gel electrophoresis coupled to mass spectrometry) to characterise the whole and differentially expressed protein contents of secretomes. The molecular identification of differentially expressed trypanosome molecules and their correlation with either the virulence process or pathogenicity are discussed with regard to their potential as new diagnostic or therapeutic tools against animal trypanosomosis.

Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense.

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.

Procongopain from Trypanosoma congolense Is Processed at Basic pH: An Unusual Feature among Cathepsin L-Like Cysteine Proteases

Abstract Congopain, the major cysteine protease from Trypanosoma congolense, is synthesized as an inactive zymogen, and further converted into its active form after removal of the proregion, most probably via an autocatalytic mechanism. Processing of recombinant procongopain occurs via an apparent one-step or a multistep mechanism depending on the ionic strength. The auto-activation is pH-dependent, with an optimum at pH 4.0, and no activation observed at pH 6.0. After addition of dextran sulfate (10 ug/ml), an approx. 20-fold increase of processing (expressed as enzymatic activity) is observed. Furthermore, in the presence of dextran sulfate, procongopain can be processed at pH 8.0, an unusual feature among papainlike enzymes. Detection of procongopain and trypanosomal enzymatic activity in the plasma of T. congolenseinfected cattle, together with the capacity of procongopain to be activated at weakly basic pH, suggest that procongopain may be extracellularly processed in the presence of blood vessel glycosaminoglycans, supporting the hypothesis that congopain acts as a pathogenic factor in host-parasite relationships.

Serodiagnosis of bovine trypanosomosis based on HSP70/BiP inhibition ELISA

Animal trypanosomosis is a serious constraint to livestock productivity in tropical and sub-tropical countries. The pathogenic trypanosomes in bovidae are Trypanosoma congolense, T. vivax, T. brucei and T. evansi. Current serological tests to detect trypanosome infections are based on the use of whole trypanosome lysates; their potential is limited by antigen instability, lack of reproducibility and lack of test specificity due to the antibodys long persistence after treatment. The development of new tests based on recombinant technology that could be standardized and applied on a large scale at low cost would be very helpful. The major invariant antigen recognized by T. congolense infected cattle belongs to the heat shock protein (HSP) 70 family and is closely related to mammalian Immunoglobulin Binding Protein (BiP). To improve the initial ELISA based on a recombinant fragment of HSP70/BiP, we developed an inhibition ELISA using an anti-BiP monoclonal antibody and a full-length fusion protein expressed in E. coli. Here we report on the development of the test and provide an initial assessment of its performance using sets of sera from experimental infections and from naturally infected cattle maintained in tsetse infested areas of Africa. The HSP70/BIP-based inhibition ELISA shows a good sensitivity in cattle experimentally infected with T. congolense, with an improved sensitivity in secondary infections. One major advantage, particularly for its further application in national laboratories, is that one single set of reagents and one single procedure are sufficient to apply on different mammalian host species infected with different trypanosome species.

Recombinant and native Tvi CATL from Trypanosoma vivax : Enzymatic characterisation and evaluation as a diagnostic target for animal African trypanosomosis

African animal trypanosomosis (nagana) is caused by tsetse-transmitted protozoan parasites. Their cysteine proteases are potential chemotherapeutic and diagnostic targets. The N-glycosylated catalytic domain of Trypanosoma vivax cathepsin L-like cysteine protease, rTviCATLcat, was recombinantly expressed and purified from culture supernatants while native TviCATL was purified from T. vivax Y486 parasite lysates. Typical of Clan CA, family C1 proteases, TviCATL activity is sensitive to E-64 and cystatin and substrate specificity is defined by the S2 pocket. Leucine was preferred in P2 and basic and non-bulky, hydrophobic residues accepted in P1 and P3 respectively. Reversible aldehyde inhibitors, antipain, chymostatin and leupeptin, with Arg in P1 and irreversible peptidyl chloromethylketone inhibitors with hydrophobic residues in P2 inhibited TviCATL activity. TviCATL digested host proteins: bovine haemoglobin, serum albumin, fibrinogen and denatured collagen (gelatine) over a wide pH range, including neutral to slightly acidic pH. The recombinant catalytic domain of TviCATL showed promise as a diagnostic target for detecting T. vivax infection in cattle in an indirect antibody detection ELISA.