Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense

Isometamidium chloride has remained a very important prophylactic and therapeutic drug against trypanosomosis in cattle since its introduction into the market in the 1950s with, unfortunately, a concomitant development of resistance in trypanosomosis endemic areas. Amplified Fragment Length Polymorphism (AFLP) was used to compare two isogenic clones of Trypanosoma congolense. The parent clone, sensitive to isometamidium, has a CD50 (the curative dose that gives complete cure in 50% of the animals) in the mouse of 0.018 mg/kg and its derivative exposed to increasing doses of isometamidium, has a CD50 that is 94-fold higher. Sixty-four combinations of eight Eco RI and eight Mse I primers were used in comparative AFLP analysis to detect subtle genetic differences between the two clones. Thirty-five polymorphic fragments of DNA that were observed only in the resistant clone were purified and then sequenced. The nucleotide sequences were used in searching the GeneDB T. congolense database to find surrounding sequences upstream of an open reading frame and downstream to a stop codon. The sequences of the open reading frames were subsequently compared to the sequences in the genomic databases. A predicted gene coding for an 854 amino acids protein was thus identified. The protein contains a putative ATP binding site, Walker B and LSGG motifs and eight predicted trans-membrane domains. The gene in the resistant strain of T. congolense has a triplet insertion coding for an extra lysine. Using polymerase chain reaction-restriction fragment length polymorphism, the insertion was sought in the genomes of 35 T. congolense strains isolated from different geographic origins and whose response to isometamidium chloride had been determined through single dose mouse tests. The presence of the insertion, specifying an extra codon was found to always be present in the genomes of T. congolense clones that were resistant to isometamidium chloride.     

Effect of adjuvants on the humoral immune response to congopain in mice and cattle

ABSTRACT: BACKGROUND: We investigated several adjuvants for their effects on the humoral immune response in both mice and cattle using the central domain of congopain (C2), the major cysteine protease of Trypanosoma congolense, as a model for developing a vaccine against animal trypanosomosis. The magnitude and sustainability of the immune response against C2 and the occurrence of a booster effect of infection, an indirect measure of the presence of memory cells, were determined by ELISA, while spectrofluorometry was used to determine and measure the presence of enzyme- inhibiting antibodies. RESULTS: Mice immunized with recombinant C2 in AdjuphosTM, TiterMaxTM, purified saponin Quil ATM or GERBUTM showed the best response according to the evaluation criteria and these three were chosen for the cattle vaccination study. The animals were challenged with T. congolense four and a half months after the last booster. Cattle immunized with recombinant C2 in purified saponin Quil ATM showed the best antibody response according to the measured parameters. CONCLUSIONS: We identified purified saponin Quil ATM as a good adjuvant for immunizations with C2. The results from this study will be useful in future attempts to develop an effective anti-disease vaccine against African trypanosomosis.     

The effect of trypanosomosis on pregnancy in trypanotolerant Orma Boran cattle

A study was undertaken to investigate the influence of trypanosomosis on the outcome of pregnancy in trypanotolerant Orma Boran (Bos indicus) exposed to natural tsetse challenge in an area of Kenya infested predominantly with Glossina pallidipes. Of 73 pregnant Orma heifers, 58 (79.5%) produced live calves at term, 13 (17.8%) aborted and 2 (2.7%) died of trypanosomosis. Of the 71 surviving animals, 22 (31%) were infected with Trypanosoma vivax , 21 (29.6%) T. congolense, and 26 (36.6%) had mixed infections with T. vivax and T. congolense. These results suggest that in areas of high trypanosomosis risk reproductive function is affected even in trypanotolerant cattle, and that both T. vivax and T. congolense can be responsible for the abortions observed in the field. It is suggested that maintenance of pregnancy in the face of trypanosome challenge was dependent on individual variation among the Orma cattle, but as challenge increased beyond the limits of effectiveness of trypanotolerance, disruption of pregnancy occurred.     

Stable interaction of the cargo receptor VIP36 with molecular chaperone BiP

VIP36 is an intracellular lectin that cycles between the endoplasmic reticulum (ER) and the Golgi apparatus, and is thought to act as a cargo receptor in the transport and sorting of glycoproteins. Here we sought to identify the proteins that interact with VIP36 during the quality control of secretory proteins. VIP36 was crosslinked and immunoprecipitated from HEK293 cells that expressed Myc-tagged VIP36. An approximately 80 kDa protein coprecipitated with VIP36 and LC/MS/MS analysis revealed it to be immunoglobulin-binding protein (BiP), a major protein of the Hsp70 chaperone family. A VIP36 mutant with defective lectin activity was also proficient for the coimmunoprecipitation of an equivalent amount of BiP, indicating that the interaction between VIP36 and BiP was carbohydrate-independent. Immunoelectron microscopy experiment demonstrated that the interaction between VIP36 and BiP occurred in the ER. However, the VIP36 coprecipitated with BiP was resistant to endo beta-N-acetylglucosaminidase H treatment. A pulse-chase experiment revealed that the amount of BiP interacting with VIP36 did not change over more than 2 h. These results suggest that the interaction of VIP36 and BiP is not due to chaperone-substrate complex. Surface plasmon resonance analysis using recombinant proteins confirmed these binding characteristics of VIP36 and BiP in vitro. The interaction between recombinant soluble VIP36 and BiP is dependent on divalent cations but not on ATP. This mode of interaction is also different from that observed between BiP and its chaperone substrates. These observations suggest a new role for VIP36 in the quality control of secretory proteins.     

JPDI,a novel endoplasmic reticulum-resident protein containing both a BiP-interacting J-domain and thioredoxin-like motifs

Several endoplasmic reticulum (ER)-resident luminal proteins have a characteristic ER retrieval signal, KDEL, or its variants at their C terminus. Our previous work searching EST databases for proteins containing the C-terminal KDEL motif predicted some novel murine proteins, one of which designated JPDI (J-domain-containing protein disulfide isomerase-like protein) is characterized in this study. The primary structure of JPDI is unique, because in addition to a J-domain motif adjacent to the N-terminal translocation signal sequence, four thioredoxin-like motifs were found in a single polypeptide. As examined by Northern blotting, the expression of JPDI was essentially ubiquitous in tissues and almost independent of ER stress. A computational prediction that JPDI is an ER-resident luminal protein was experimentally supported by immunofluorescent staining of epitope-tagged JPDI-expressing cells together with glycosylation and protease protection studies of this protein. JPDI probably acts as a DnaJ-like partner of BiP, because a recombinant protein carrying the J-domain of JPDI associated with BiP in an ATP-dependent manner and enhanced its ATPase activity. We speculate that for the folding of some proteins in the ER, chaperoning by BiP and formation of proper disulfide bonds may synchronously occur in a JPDI-dependent manner.     

Trypanosoma vivax: a novel method for purification from experimentally infected sheep blood

Trypanosoma vivax is the principal etiological agent of bovine trypanosomosis, a widely disseminated disease in tropical and subtropical regions. Here, we present a simple and reproducible method for the purification of T. vivax from experimentally infected and immunosuppressed sheep, using an isopycnic Percoll gradient, followed by DEAE-cellulose chromatography, with an estimated yield of 11-15%. This method could be used for the purification of T. vivax geographical isolates from various locations and from different natural hosts.     

Effects of Trypanosoma vivax and Trypanosoma congolense infections on the reaction time and semen characteristics of Zebu (Bunaji) x Friesian crossbred bulls

The effect of trypanosomosis on reaction time and semen characteristics of 12 Zebu (Bunaji) x Friesian crossbred bulls aged between 3 and 5 years was studied for a duration of 12 weeks. Four of the bulls were infected with Trypanosoma vivax, another four with Trypanosoma congolense and the remaining four bulls served as controls. Rectal temperatures and haematological parameters were monitored twice weekly. The pre-infection mean value of the rectal temperature was 38.3 degrees C, and this rose to a mean of between 40.5 and 41.1 degrees C in the infected animals. Concurrently, the infected animals exhibited signs of anaemia shown by pale mucous membranes and decreased packed cell volume (PCV), weight loss, lethargy, weakness and dullness.The reaction time (ejaculation time) of semen collection significantly increased from a pre-infection mean value of 20.46-25.14 s to a mean of 290.33-301.15 s within 12 weeks post-infection. Semen characteristics deteriorated progressively within the same period in the infected bulls. There were highly significant and drastic decreases in sperm concentration and volume of semen and increases in sperm morphological defects. By the third week, all the infected bulls were unfit for breeding because of very poor semen characteristics. Deterioration, also characterized by oligospermia at 6 weeks post-infection in all bulls which later culminated in azoospermia in two bulls infected with T. vivax and two bulls infected with T. congolense continued to the end of the investigation. The present results indicate that trypanosomosis due to T. vivax and T. congolense infections is very pathogenic and devastating in its effect on the reaction time (ejaculation time) and semen characteristics which resulted in very poor semen quality. The practical implication is infertility and sterility in Zebu x Friesian crossbred bulls in trypanosome endemic areas.     

In vitro binding of Trypanosoma congolense to erythrocytes.

Trypanosoma congolense Broden, an intravascular parasite, binds to vessel walls and erythrocytes of infected hosts. In an attempt to characterize T. congolense adhesion to host cells, an in vitro assay was devised. It was shown in the in vitro experiments that T. congolense binds to bovine, sheep, and goat erythrocytes, but not always to erythrocytes of rats, mice, rabbits, horses or humans. Only the anterior part of live trypanosomes adheres to erythrocytes, and the attachment site on the trypanosomes is destroyed by trypsin and chymotrypsin-trypanosomes did not adhere to bovine erythrocytes that had been incubated with neuraminidase, sodium periodate and poly-L-lysine. The foregoing experiments suggest that the surface of T. congolense contains a protein-associated site which binds to sialic acid of some host cells. This surface site is most likely responsible for attachment to blood vessels in vivo.     

牛病毒性腹泻病毒E^rns-E2融合蛋白在果蝇S2细胞中的表达与鉴定

目的：利用果蝇S2细胞表达牛病毒性腹泻病毒（BVDV）Erns-E2融合蛋白,并对其抗体结合能力进行鉴定。方法：用RT-PCR方法扩增BVDV NADL株Erns和E2蛋白的编码基因,利用（G4-S）3柔性15肽基因将扩增的2个基因连接,再与昆虫表达载体pMT/BiP/V5-His连接构建重组表达载体pMT/BiP/V5-His-Erns-E2,将后者与筛选质粒pCoBlast共转染果蝇S2细胞后表达Erns-E2融合蛋白,并对表达产物进行鉴定。结果：SDS-PAGE结果表明,融合蛋白相对分子质量为76800;Western blotting检测表明,该融合蛋白具有与BVDV抗体良好的结合能力。结论：BVDV的Erns-E2融合蛋白能在果蝇S2细胞中进行表达;经鉴定,表达产物具有良好的抗体结合能力,可用于抗原检测。     

Analysis of molecular interactions in heat-induced aggregation of a non-inhibitory serpin ovalbumin using a molecular chaperone

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a Km of 165 microM and a Vmax of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a Kd of 0.52 microM by BiP, lower than that for RCMLA (Kd = 1.1 microM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (Kd = 21 microM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.     

Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l⁻¹ in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER.     

Isolation of proteins that speifically interact with the ATPase domain of mammalian ER chaperone,BiP

BiP, immunoglobulin binding protein, is an ER homologue of Hsp 70. However, unlike other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demonstrated the presence of potential regulatory proteins for BiP using a pull-down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull-down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein. Eight proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp 170 and BiP where identified, while the others were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for Bip was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.     

Sialidases play a key role in infection and anaemia in Trypanosoma congolense animal trypanosomiasis

Animal African trypanosomiasis is a major constraint to livestock productivity and has an important impact on millions of people in developing African countries. This parasitic disease, caused mainly by Trypanosoma congolense, results in severe anaemia leading to animal death. In order to characterize potential targets for an anti-disease vaccine, we investigated a multigenic trans-sialidase family (TcoTS) in T. congolense. Sialidase and trans-sialidase activities were quantified for the first time, as well as the tightly regulated TcoTS expression pattern throughout the life cycle. Active enzymes were expressed in bloodstream form parasites and released into the blood during infection. Using genetic tools, we demonstrated a significant correlation between TcoTS silencing and impairment of virulence during experimental infection with T. congolense. Reduced TcoTS expression affected infectivity, parasitaemia and pathogenesis development. Immunization-challenge experiments using recombinant TcoTS highlighted their potential protective use in an anti-disease vaccine.     

Characterization of a lympho-inhibitory peptide produced by Mycoplasma bovis

Mycoplasma bovis is able to inhibit the mitogen-induced proliferation of bovine lymphocytes. Herein is described the isolation of an immuno-inhibitory peptide from M. bovis. Using size exclusion chromatography, three lympho-suppressive fractions were isolated from M. bovis free supernatant. MALDI-TOF analysis revealed a common peak throughout the suppressive fractions. The purest of these fractions was subjected to N-terminal sequencing, revealing an 84% homologous match with the C-terminus of the M. bovis surface protein VspL (variable surface protein-L). A recombinant of the 26 amino acid peptide was also able to suppress Concanavalin A (ConA)-induced proliferation of bovine lymphocytes. This describes a unique immunosuppressive peptide produced by the bovine respiratory pathogen, M. bovis.     

Does isometamidium chloride treatment protect tsetse flies from trypanosome infections during SIT campaigns?

African animal trypanosomosis is a major pathological constraint to cattle breeding across 10 million km2 of sub-Saharan West African countries infested by tsetse flies, their cyclic vectors. The release of sterile males (sterile insect technique [SIT]) is a potentially important control technique aimed at eliminating the vectors. Prior to release, tsetse are generally treated with isometamidium chloride, a trypanocide, to prevent them from transmitting parasites. The present study investigated the preventive action of isometamidium chloride (0.5 mg/L) on the subsequent susceptibility of tsetse released into the wild. A total of 1755 Glossina palpalis gambiensis Vanderplank and 744 Glossina tachinoides Westwood were released, of which 50 and 48, respectively, were recaptured 22-43 days after release. Their probosces were analysed by polymerase chain reaction to identify mature infections with three trypanosome species (Trypanosoma vivax, Trypanosoma brucei sensu lato and Trypanosoma congolense savannah type). Two mature infections with T. vivax and four with T. congolense were detected, indicating that the use of this treatment regimen in an SIT campaign would not totally prevent sterile males from transmitting trypanosomes.     

MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis.     

A nonradioactive 96-well plate assay for screening of trans-sialidase activity

Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2,3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbelliferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems.     

Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding

The production of recombinant proteins is a critical technology for biotechnology and biomedical research. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. The overexpression of chaperone binding protein (BiP) and disulfide bond isomerase (PDI) in Saccaromyces cerevisiae can effectively increase protein production levels of single-chain antibody (scFv) 4-4-20. These studies show that overexpression of BiP did not reduce the UPR activated by heterologous protein expression; however, overexpression of PDI or co-overexpression of BiP and PDI could reduce the UPR. We observed that co-overexpression of BiP and PDI led to the greatest secretion of scFv from the cell, but BiP and PDI appear to interact with the newly synthesized scFv at different stages in the folding process, as determined by pulse-chase analysis. We propose that BiP acts primarily to facilitate translocation and retain unfolded or partially folded scFv, and PDI actively folds the scFv through its functions as a catalyst, and/or an isomerase, of disulfide bonds. Free BiP is released when scFv is folded, stabilizing Ire1p, and leading to the reduced UPR.