Steven G. Nyanjom

Odorant and Gustatory Receptors in the Tsetse Fly Glossina morsitans morsitans

Tsetse flies use olfactory and gustatory responses, through odorant and gustatory receptors (ORs and GRs), to interact with their environment. Glossina morsitans morsitans genome ORs and GRs were annotated using homologs of these genes in Drosophila melanogaster and an ab initio approach based on OR and GR specific motifs in G. m. morsitans gene models coupled to gene ontology (GO). Phylogenetic relationships among the ORs or GRs and the homologs were determined using Maximum Likelihood estimates. Relative expression levels among the G. m. morsitans ORs or GRs were established using RNA-seq data derived from adult female fly. Overall, 46 and 14 putative G. m. morsitans ORs and GRs respectively were recovered. These were reduced by 12 and 59 ORs and GRs respectively compared to D. melanogaster. Six of the ORs were homologous to a single D. melanogaster OR (DmOr67d) associated with mating deterrence in females. Sweet taste GRs, present in all the other Diptera, were not recovered in G. m. morsitans. The GRs associated with detection of CO2 were conserved in G. m. morsitans relative to D. melanogaster. RNA-sequence data analysis revealed expression of GmmOR15 locus represented over 90% of expression profiles for the ORs. The G. m. morsitans ORs or GRs were phylogenetically closer to those in D. melanogaster than to other insects assessed. We found the chemoreceptor repertoire in G. m. morsitans smaller than other Diptera, and we postulate that this may be related to the restricted diet of blood-meal for both sexes of tsetse flies. However, the clade of some specific receptors has been expanded, indicative of their potential importance in chemoreception in the tsetse.

Phylogenetic Relationships among Whiteflies in the Bemisia tabaci (Gennadius) Species Complex from Major Cassava Growing Areas in Kenya

Whiteflies, Bemisia tabaci (Gennadius) are major insect pests that affect many crops such as cassava, tomato, beans, cotton, cucurbits, potato, sweet potato, and ornamental crops. Bemisia tabaci transmits viral diseases, namely cassava mosaic and cassava brown streak diseases, which are the main constraints to cassava production, causing huge losses to many small-scale farmers. The aim of this work was to determine the phylogenetic relationships among Bemisia tabaci species in major cassava growing areas of Kenya. Surveys were carried out between 2013 and 2015 in major cassava growing areas (Western, Nyanza, Eastern, and Coast regions), for cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Mitochondrial cytochrome oxidase I (mtCOI-DNA) was used to determine the genetic diversity of B. tabaci. Phylogenetic trees were constructed using Bayesian methods to understand the genetic diversity across the study regions. Phylogenetic analysis revealed two B. tabaci species present in Kenya, sub-Saharan Africa 1 and 2 comprising five distinct clades (A–E) with percent sequence similarity ranging from 97.7 % to 99.5%. Clades B, C, D, and E are predominantly distributed in the Western and Nyanza regions of Kenya whereas clade B is dominantly found along the coast, the eastern region, and parts of Nyanza. Our B. tabaci clade A groups with sub-Saharan Africa 2-(SSA2) recorded a percent sequence similarity of 99.5%. In this study, we also report the identification of SSA2 after a 15 year absence in Kenya. The SSA2 species associated with CMD has been found in the Western region of Kenya bordering Uganda. More information is needed to determine if these species are differentially involved in the epidemiology of the cassava viruses.

Comparative analysis of salivary gland proteomes of two Glossina species that exhibit differential hytrosavirus pathologies

Glossina pallidipes salivary gland hypertrophy virus (GpSGHV; family Hytrosaviridae) is a dsDNA virus exclusively pathogenic to tsetse flies (Diptera; Glossinidae). The 190 kb GpSGHV genome contains 160 open reading frames and encodes more than 60 confirmed proteins. The asymptomatic GpSGHV infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (SGH). Flies with SGH show reduced general fitness and reproductive dysfunction. Although the occurrence of SGH is an exception rather than the rule, G. pallidipes is thought to be the most susceptible to expression of overt SGH symptoms compared to other Glossina species that are largely asymptomatic. Although Glossina salivary glands (SGs) play an essential role in GpSGHV transmission, the functions of the salivary components during the virus infection are poorly understood. In this study, we used mass spectrometry to study SG proteomes of G. pallidipes and G. m. morsitans, two Glossina model species that exhibit differential GpSGHV pathologies (high and low incidence of SGH, respectively). A total of 540 host proteins were identified, of which 23 and 9 proteins were significantly up- and down-regulated, respectively, in G. pallidipes compared to G. m. morsitans. Whereas 58 GpSGHV proteins were detected in G. pallidipes F1 progenies, only 5 viral proteins were detected in G. m. morsitans. Unlike in G. pallidipes, qPCR assay did not show any significant increase in virus titers in G. m. morsitans F1 progenies, confirming that G. m. morsitans is less susceptible to GpSGHV infection and replication compared to G. pallidipes. Based on our results, we speculate that in the case of G. pallidipes, GpSGHV employs a repertoire of host intracellular signaling pathways for successful infection. In the case of G. m. morsitans, antiviral responses appeared to be dominant. These results are useful for designing additional tools to investigate the Glossina-GpSGHV interactions.

Phylogenetic Variants of Rickettsia africae, and Incidental Identification of "Candidatus Rickettsia Moyalensis" in Kenya.

Background Rickettsia africae, the etiological agent of African tick bite fever, is widely distributed in sub-Saharan Africa. Contrary to reports of its homogeneity, a localized study in Asembo, Kenya recently reported high genetic diversity. The present study aims to elucidate the extent of this heterogeneity by examining archived Rickettsia africae DNA samples collected from different eco-regions of Kenya. Methods To evaluate their phylogenetic relationships, archived genomic DNA obtained from 57 ticks a priori identified to contain R. africae by comparison to ompA, ompB and gltA genes was used to amplify five rickettsial genes i.e. gltA, ompA, ompB, 17kDa and sca4. The resulting amplicons were sequenced. Translated amino acid alignments were used to guide the nucleotide alignments. Single gene and concatenated alignments were used to infer phylogenetic relationships. Results Out of the 57 DNA samples, three were determined to be R. aeschlimanii and not R. africae. One sample turned out to be a novel rickettsiae and an interim name of “Candidatus Rickettsia moyalensis” is proposed. The bonafide R. africae formed two distinct clades. Clade I contained 9% of the samples and branched with the validated R. africae str ESF-5, while clade II (two samples) formed a distinct sub-lineage. Conclusions This data supports the use of multiple genes for phylogenetic inferences. It is determined that, despite its recent emergence, the R. africae lineage is diverse. This data also provides evidence of a novel Rickettsia species, Candidatus Rickettsia moyalensis.

Green tea proanthocyanidins cause impairment of hormone-regulated larval development and reproductive fitness via repression of juvenile hormone acid methyltransferase, insulin-like peptide and cytochrome P450 genes in Anopheles gambiae sensu stricto

Successful optimization of plant-derived compounds into control of nuisance insects would benefit from scientifically validated targets. However, the close association between the genotypic responses and physiological toxicity effects mediated by these compounds remains underexplored. In this study, we evaluated the sublethal dose effects of proanthocyanidins (PAs) sourced from green tea (Camellia sinensis) on life history traits of Anopheles gambiae (sensu stricto) mosquitoes with an aim to unravel the probable molecular targets. Based on the induced phenotypic effects, genes selected for study targeted juvenile hormone (JH) biosynthesis, signal transduction, oxidative stress response and xenobiotic detoxification in addition to vitellogenesis in females. Our findings suggest that chronic exposure of larval stages (L3/L4) to sublethal dose of 5 ppm dramatically extended larval developmental period for up to 12 days, slowed down pupation rates, induced abnormal larval-pupal intermediates and caused 100% inhibition of adult emergence. Further, females exhibited significant interference of fecundity and egg hatchability relative to controls (p < 0.001). Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), our findings show that PA-treated larvae exhibited significant repression of AgamJHAMT (p < 0.001), AgamILP1 (p < 0.001) and AgamCYP6M2 (p < 0.001) with up-regulation of Hsp70 (p < 0.001). Females exposed as larvae demonstrated down-regulation of AgamVg (p = 0.03), AgamILP1 (p = 0.009), AgamCYP6M2 (p = 0.05) and AgamJHAMT (p = 0.02). Our findings support that C. sinensis proanthocyanidins affect important vectorial capacity components such as mosquito survival rates and reproductive fitness thus could be potentially used for controlling populations of malaria vectors.

Transcriptomics reveal potential vaccine antigens and a drastic increase of upregulated genes during Theileria parva development from arthropod to bovine infective stages

Theileria parva is a protozoan parasite transmitted by the brown ear tick Rhipicephalus appendiculatus that causes East Coast fever (ECF) in cattle, resulting in substantial economic losses in the regions of southern, eastern and central Africa. The schizont form of the parasite transforms the bovine host lymphocytes into actively proliferating cancer-like cells. However, how T. parva causes bovine host cells to proliferate and maintain a cancerous phenotype following infection is still poorly understood. On the other hand, current efforts to develop improved vaccines have identified only a few candidate antigens. In the present paper, we report the first comparative transcriptomic analysis throughout the course of T. parva infection. We observed that the development of sporoblast into sporozoite and then the establishment in the host cells as schizont is accompanied by a drastic increase of upregulated genes in the schizont stage of the parasite. In contrast, the ten highest gene expression values occurred in the arthropod vector stages. A comparative analysis showed that 2845 genes were upregulated in both sporozoite and schizont stages compared to the sporoblast. In addition, 647 were upregulated only in the sporozoite whereas 310 were only upregulated in the schizont. We detected low p67 expression in the schizont stage, an unexpected finding considering that p67 has been reported as a sporozoite stage-specific gene. In contrast, we found that transcription of p67 was 20 times higher in the sporoblast than in the sporozoite. Using the expression profiles of recently identified candidate vaccine antigens as a benchmark for selection for novel potential vaccine candidates, we identified three genes with expression similar to p67 and several other genes similar to Tp1—Tp10 schizont vaccine antigens. We propose that the antigenicity or chemotherapeutic potential of this panel of new candidate antigens be further investigated. Structural comparisons of the transcripts generated here with the existing gene models for the respective loci revealed indels. Our findings can be used to improve the structural annotation of the T. parva genome, and the identification of alternatively spliced transcripts.

Expression Levels of Odorant Receptor Genes in the Savanna Tsetse Fly, Glossina morsitans morsitans

Abstract Tsetse flies (Glossina) are vectors of African trypanosomiasis. Olfaction plays a critical role in Glossina behavior, including larviposition, feeding, and reproduction. Odorant receptors (ORs) are important in insect chemoreception as they bind volatile odorants and transport them to olfactory receptor neurons to elicit behavioral response.To better understand Glossina chemoreception, we used quantitative polymerase chain reaction to examine the expression levels of ORs in female and male Glossina morsitans morsitans Wiedemann, 1850 (Diptera: Glossinidae) antennae and legs. Results showed that G. m. morsitans ORs code for a transmembrane domain and are involved in odorant binding. The ORs had homologs in Drosophila, mosquitoes, other Glossina species, and the reduced number of tsetse ORs could be linked to its restricted blood-feeding diet. The OR genes were more highly expressed in antennae than the legs with GmmOR33 and GmmOR45 transcript levels being high in the female and male antennae, respectively, whereas GmmOR26 and GmmOR34 levels were high in female and male G. m. morsitans legs, respectively. These findings identified sex- and tissue-specific G. m. morsitans ORs. The expression levels of OR genes in female and male G. m. morsitans could be conserved in function with the antenna being the main olfactory organ.Thus, this study provides a blueprint to explore the functional roles of tsetse ORs with the potential to identify molecular targets that can be used to control the vector based on disruption of its chemosensory system.

Target-similarity search using Plasmodium falciparum proteome identifies approved drugs with anti-malarial activity and their possible targets.

Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 μM to 50 μM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens.

In Silico Analysis of Occurrence of Tricorn Protease and Its Homologs

Tricorn protease is an archaeal protease acting downstream of the proteasome and together with its interacting aminopeptidases, degrades oligopeptides to free amino acids thus playing an important role in protein turnover. This study reports a wide distribution of tricorn protease and its homologs in archaea and bacteria. The homologs were identified through a combination of PSI-BLAST, orthology clustering and domain predictions. Functionally important sites were identified through multiple sequence alignment conducted by MAFFT v. 7. The aligned sequences were used to predict the phylogenetic relationship of tricorn protease and its homologs using MEGA v. 7. The functional associations of tricorn protease were predicted through STRING network v.10.0. This study identified several tricorn protease homologs in archaea and in all the bacterial phyla complete with β-propeller, PDZ and catalytic domains. However, in eukaryotes, tricorn protease-like homologs seemed limited to viridiplantae, stramenopile and in a basal metazoa and were classified as non-peptidase homologs with unknown functions. Conserved domain architecture retrieval revealed detectable homology of tricorn protease C-terminal half with the carboxyl-terminal proteases with similar PDZ domains. Therefore, this study predicts functional conservation of tricorn core catalytic domain in prokaryotes and given its role in cellular functions, targeting this protein or its functional homologs in prokaryotic pathogens could lead to development of alternative therapeutic agents.