Biochemical characterisation of trans-sialidases from Trypanosoma congolense

The work compiled here aimed at biochemically characterising trans-sialidase (TS) genes from Trypanosoma congolense, the most prevalent causative agent of animal African trypanosomiasis, also called nagana. Using published partial TS sequences as starting queries, database at the Welcome Trust Sanger Institute (WTSI) was queried and TS1 genes identified. This is a multi-copy gene group comprising 11 members. Members were cloned and recombinant protein expressed in fibroblasts and purified proteins assayed for enzyme activities (Publication 1/Chapter 1). All TS1 variants were found to be active TS enzymes, transferring Sia from donor fetuin to the lactose acceptor substrate and could resialylate asialofetuin up to 50%. Use of synthetic substrates revealed poor specific activities. Further searches on WTSI using TconTS1 gene variants as queries revealed 3 other TS genes, TconTS2, TconTS3 and TconTS4, all conserving the critical amino acids required in activity. A further 3 distantly related genes, this latter group is presumed inactive due to lack of conservation of critical amino acids (Publication 2/Chapter 3). TS/sialidase ratios confirmed the former to be active trans-sialidases but with differing specific activities. A mouse monoclonal antibody raised from native proteins probably containing other TconTS and not only TconTS1 reacted with both TconTS1 and TconTS2, two of the proteins with the highest specific activities. Incidentally, the epitope for the antibody is localised on the lectin domain (LD) in both enzymes. Phylogenetic analysis using the LD grouped TconTS1 and TconTS2 together while the use of catalytic domain (CD) grouped them separately. This suggests a possible role for the LD in enzyme activities and hence pathology of nagana. To gain knowledge on the role of TS in pathogenesis of nagana, blood glycoconjugates were employed as substrates and activities of TS gene characterised (Manuscript/Chapter 4). All TconTS proteins transferred Sia from serum-bound glycoconjugates to lactose. In absence of lactose, TconTS1 and TconTS2 released free Sia from the serum-glycoconjugates, possibly explaining the high amounts of free Sia observed in blood and serum of animals suffering nagana. TconTS3 showed an unidentified product peak in presence of serum-bound Sia and lactose, while 3 unidentified signals probably representing serum-glycoconjugates inherent in serum were altered by TconTS2. Collectively, the above indicate the possibility of acceptor and donor preferences and show that expressing more than one TS gene at a time could be beneficial to the parasite. Since TS genes are expressed in two different hosts systems; the Glossina insect vector and the mammalian hosts with different pH systems, pH optima for the enzymes was studied. TconTS2 exhibited a wide pH optima that would make it active in both host systems

Biochemical characterisation of trans-sialidases from Trypanosoma congolense

The work compiled here aimed at biochemically characterising trans-sialidase (TS) genes from Trypanosoma congolense, the most prevalent causative agent of animal African trypanosomiasis, also called nagana. Using published partial TS sequences as starting queries, database at the Welcome Trust Sanger Institute (WTSI) was queried and TS1 genes identified. This is a multi-copy gene group comprising 11 members. Members were cloned and recombinant protein expressed in fibroblasts and purified proteins assayed for enzyme activities (Publication 1/Chapter 1). All TS1 variants were found to be active TS enzymes, transferring Sia from donor fetuin to the lactose acceptor substrate and could resialylate asialofetuin up to 50%. Use of synthetic substrates revealed poor specific activities. Further searches on WTSI using TconTS1 gene variants as queries revealed 3 other TS genes, TconTS2, TconTS3 and TconTS4, all conserving the critical amino acids required in activity. A further 3 distantly related genes, this latter group is presumed inactive due to lack of conservation of critical amino acids (Publication 2/Chapter 3). TS/sialidase ratios confirmed the former to be active trans-sialidases but with differing specific activities. A mouse monoclonal antibody raised from native proteins probably containing other TconTS and not only TconTS1 reacted with both TconTS1 and TconTS2, two of the proteins with the highest specific activities. Incidentally, the epitope for the antibody is localised on the lectin domain (LD) in both enzymes. Phylogenetic analysis using the LD grouped TconTS1 and TconTS2 together while the use of catalytic domain (CD) grouped them separately. This suggests a possible role for the LD in enzyme activities and hence pathology of nagana. To gain knowledge on the role of TS in pathogenesis of nagana, blood glycoconjugates were employed as substrates and activities of TS gene characterised (Manuscript/Chapter 4). All TconTS proteins transferred Sia from serum-bound glycoconjugates to lactose. In absence of lactose, TconTS1 and TconTS2 released free Sia from the serum-glycoconjugates, possibly explaining the high amounts of free Sia observed in blood and serum of animals suffering nagana. TconTS3 showed an unidentified product peak in presence of serum-bound Sia and lactose, while 3 unidentified signals probably representing serum-glycoconjugates inherent in serum were altered by TconTS2. Collectively, the above indicate the possibility of acceptor and donor preferences and show that expressing more than one TS gene at a time could be beneficial to the parasite. Since TS genes are expressed in two different hosts systems; the Glossina insect vector and the mammalian hosts with different pH systems, pH optima for the enzymes was studied. TconTS2 exhibited a wide pH optima that would make it active in both host systems

Biochemische charakterisierung der trans-Sialidase von Trypanosoma congolense

The work compiled here aimed at biochemically characterising trans-sialidase (TS) genes from Trypanosoma congolense, the most prevalent causative agent of animal African trypanosomiasis, also called nagana. Using published partial TS sequences as starting queries, database at the Welcome Trust Sanger Institute (WTSI) was queried and TS1 genes identified. This is a multi-copy gene group comprising 11 members. Members were cloned and recombinant protein expressed in fibroblasts and purified proteins assayed for enzyme activities (Publication 1/Chapter 1). All TS1 variants were found to be active TS enzymes, transferring Sia from donor fetuin to the lactose acceptor substrate and could resialylate asialofetuin up to 50%. Use of synthetic substrates revealed poor specific activities. Further searches on WTSI using TconTS1 gene variants as queries revealed 3 other TS genes, TconTS2, TconTS3 and TconTS4, all conserving the critical amino acids required in activity. A further 3 distantly related genes, this latter group is presumed inactive due to lack of conservation of critical amino acids (Publication 2/Chapter 3). TS/sialidase ratios confirmed the former to be active trans-sialidases but with differing specific activities. A mouse monoclonal antibody raised from native proteins probably containing other TconTS and not only TconTS1 reacted with both TconTS1 and TconTS2, two of the proteins with the highest specific activities. Incidentally, the epitope for the antibody is localised on the lectin domain (LD) in both enzymes. Phylogenetic analysis using the LD grouped TconTS1 and TconTS2 together while the use of catalytic domain (CD) grouped them separately. This suggests a possible role for the LD in enzyme activities and hence pathology of nagana. To gain knowledge on the role of TS in pathogenesis of nagana, blood glycoconjugates were employed as substrates and activities of TS gene characterised (Manuscript/Chapter 4). All TconTS proteins transferred Sia from serum-bound glycoconjugates to lactose. In absence of lactose, TconTS1 and TconTS2 released free Sia from the serum-glycoconjugates, possibly explaining the high amounts of free Sia observed in blood and serum of animals suffering nagana. TconTS3 showed an unidentified product peak in presence of serum-bound Sia and lactose, while 3 unidentified signals probably representing serum-glycoconjugates inherent in serum were altered by TconTS2. Collectively, the above indicate the possibility of acceptor and donor preferences and show that expressing more than one TS gene at a time could be beneficial to the parasite. Since TS genes are expressed in two different hosts systems; the Glossina insect vector and the mammalian hosts with different pH systems, pH optima for the enzymes was studied. TconTS2 exhibited a wide pH optima that would make it active in both host systems

Molecular screening of tsetse flies and cattle reveal different Trypanosoma species including T. grayi and T. theileri in northern Cameroon

Abstract Background African trypanosomes are mainly transmitted through the bite of tsetse flies (Glossina spp.). The present study investigated the occurrence of pathogenic trypanosomes in tsetse flies and cattle in tsetse fly-infested areas of Northern Cameroon. Results Trypanosomes were identified using nested polymerase chain reaction (PCR) analysis of internal transcribed spacer 1 (ITS1) region, both by size estimation and sequencing of PCR products. Apparent density indices recorded in Gamba and Dodeo were 3.1 and 3.6 tsetse flies per trap and day, respectively. Trypanosoma prevalence infection rate for the tsetse fly gut (40%) and proboscis (19%) were recorded. Among the flies where trypanosomes were detected in the gut, 41.7% were positive for T. congolense and 14.6% for T. brucei ssp., whereas in the proboscis 36% harboured T. congolense and 62% contained T. vivax. T. grayi was highly prevalent in tsetse fly gut (58%). The most common mixed infections were the combination of T. congolense and T. grayi. Trypanosome prevalence rate in cattle blood was 6%. Among these, T. vivax represented 26%, T. congolense 35%, T. brucei ssp. 17% and T. theileri 17% of the infections. Surprisingly, in one case T. grayi was found in cattle. The mean packed cell volume (PCV) of cattle positive for trypanosomes was significantly lower (24.1 ± 5.6%; P < 0.05) than that of cattle in which trypanosomes were not detected (27.1 ± 4.9%). Interestingly, the occurrence of T. theileri or T. grayi DNA in cattle also correlated with low PCV at pathological levels. Conclusion This molecular epidemiological study of Trypanosoma species in Northern Cameroon revealed active foci of trypanosomes in Dodeo and Gamba. These findings are relevant in assessing the status of trypanosomosis in these regions and will serve as a guide for setting the priorities of the government in the control of the disease

Tryapanosoma prevalence in tsetse proboscis (Glossina sp.) collected in protected and tsetse invested areas in Nigeria

This data set includes the results of nested PCR-analysis of tsetse (Glossina sp.) proboscis samples with primers described by Adams et.al. (2006) and Hamilton et al (2004) from a tsetse (Glossina sp.) collection carried out in Nigeria between 2014 and 2016. Traps were employed for one to three days in four different National Parks, one Game Reserve and two tsetse invested areas. Each tsetse was identified morphologically and dissected in the field. DNA was crudely extracted from the proboscis by Proteinase K digest. Presence of trypanosomes was investigated by amplification of Internal Transcribed Spacer-1 (ITS1). Identification of the respective trypanosome species was done based on lengths polymorphism of the ITS1 region by estimating the approximate size of the DNA amplicon based on electrophoretic separation in agarose gels, as well as sequencing of selected ITS1 amplicons and additional glycosomal Glyceraldehyde-3-phosohate dehydrogenase

Environmental data and tsetse numbers per trap in protected and tsetse invested areas in Nigeria

This data set includes data from a tsetse (Glossina sp.) collection carried out in Nigeria between 2014 and 2016. Traps were employed for one to three days in four different National Parks, one Game Reserve and two tsetse invested areas. The dataset includes temperature and relative humidity at the set traps, and the number of total trapped and dissected Glossina sp

Sialidase assay and analysis of free sialic acid in tsetse (Glossina sp.) collected in protected and tsetse invested areas in Nigeria

This data set includes results of a sialidase assay applied in tsetse gut tissue from a tsetse (Glossina sp.) collection carried out in Nigeria between 2014 and 2016. Traps were employed for one to three days in four different National Parks, one Game Reserve and two tsetse invested areas. Each tsetse was identified morphologically and dissected in the field, all tissues preserved and used for further analysis. The whole gut of each tsetse was homogenized in the field and partially preserved for DNA analysis and used to investigate for sialidase activity with a fluorescent substrate and a natural substrate. Analysis for sialidase activity was done by fluorescent measurement and reverse phase HPLC of processed gut samples

Collected tsetse tissue and tsetse species (Glossina sp.) collected in protected and tsetse invested areas in Nigeria

This data set includes data from a tsetse (Glossina sp.) collection carried out in Nigeria between 2014 and 2016. Traps were employed for one to three days in four different National Parks, one Game Reserve and two tsetse invested areas. Each tsetse was identified morphologically and dissected in the field. Wings were removed and stored dry. Legs were immersed in a DNA-preservation agent. Salivary glands and ovaries were dissected and stored in a DNA-preservation agent. The whole gut was dissected, homogenized in the field and partially preserved for DNA analysis and used to investigate for sialidase activity. The data table summarizes the available tissue, sex and species information for each dissected specimen. Species collected were G. morsitans submorsitans, G. tachinoides, G. palpalis palpalis and several unidentified Glossina species

Tryapanosoma prevalence in tsetse (Glossina sp.) gut collected in protected and tsetse invested areas in Nigeria

This data set includes the results of nested PCR-analysis of tsetse (Glossina sp.) gut samples with primers described by Adams et.al. (2006) and Hamilton et al (2004) from a tsetse (Glossina sp.) collection carried out in Nigeria between 2014 and 2016. Traps were employed for one to three days in four different National Parks, one Game Reserve and two tsetse invested areas. Each tsetse was identified morphologically and dissected in the field. The gut samples were homogenized in the field. DNA was purified from preserved homogenized gut using the Qiagen Blood and Tissue Kit. Presence of trypanosomes was investigated by amplification of Internal Transcribed Spacer-1 (ITS1). Identification of the respective trypanosome species was done based on lengths polymorphism of the ITS1 region by estimating the approximate size of the DNA amplicon based on electrophoretic separation in agarose gels, as well as sequencing of selected ITS1 amplicons and additional glycosomal Glyceraldehyde-3-phosohate dehydrogenase

Molecular and morphological characterization of Glossina sp. and identification of associated Trypanosoma sp. in Nigeria including analysis for sialidase activity

These data sets include morphological and molecular data generated from a tsetse (Glossina sp.) collection carried out in Nigeria between 2014 and 2016. Traps were employed for one to three days in four different National Parks (Kainji Lake National Park, Old Oyo National Park, Cross River National Park, Gashaka Gumti National Park), one Game Reserve (Yankari Game Reserve) and two tsetse invested areas (Ijah Gwari, Tuongo). Each tsetse was identified morphologically and dissected in the field, all tissues preserved and used for further analysis. Selected tsetse wings were mounted and photographed to be used for morphometric analysis. The whole gut of each tsetse was homogenized in the field and partially preserved for DNA analysis and used to investigate for sialidase activity with a fluorescent substrate and a natural substrate. Analysis for sialidase activity was done by fluorescent measurement and reverse phase HPLC of processed gut samples. DNA was purified from preserved homogenized gut and used for phylogenetic analysis of tsetse by amplification of Cytochrome Oxidase C SU1 and Internal Transcribed Spacer-1 (ITS1). Presence of trypanosomes was investigated in gut and crudely extracted DNA from proboscis by amplification of ITS1. Identification of the respective trypanosome species was done based on lengths polymorphism of the ITS1 region as well as sequencing of selected ITS1 amplicons and additional glycosomal Glyceraldehyde-3-phosohate dehydrogenase