Biology subjects

Mulindwa, J.

Publications and source records attributed to Mulindwa, J..

8 recordsLinked to original sources

bioRxiv2024

GENETIC DIVERSITY OF T. B. RHODESIENSE SERUM RESISTANCE ASSOCIATED GENE IN MALAWIAN ISOLATES

BackgroundHuman African Trypanosomiasis (HAT) is a health burden in most remote areas of Sub-Saharan Africa. Only 2 species of the Trypanosome parasites, namely, T. b. rhodesiense and T. b. gambiense can establish infection in humans whereas other trypanosome parasites are lysed by human serum APOL-1 protein. The mechanism of T. b. gambiense resistance to APOL-1 activity is complex and involves several parasite factors. On the other hand, T. b. rhodesiense evades the lytic activity of APOL-1 by intracellular expression of a Serum Resistance Associated (SRA) gene that binds to APOL-1 when uptaken by the parasite thereby disabling APOL-1 from causing cellular membrane rupture. APOL-1 has 2 variants, namely, APOL-1 G1 and APOL-1 G2 with the later having mutations on the SRA binding sites which restores APOL-1 lytic activity in parasite lysis assays. This phenomenon remains elusive in clinical setting as limited data is available. In the present study we investigated the genetic diversity of T. b. rhodesiense SRA gene and APOL-1 genotypes in Malawian r-HAT clinical phenotypes. MethodsT. b. rhodesiense SRA gene from Malawi endemic HAT samples (n= 77) as well as from Zambia and Uganda (n= 13) was amplified by PCR and PCR products were commercially sequenced. APOL-1 variants were identified by restriction fragment length polymorphism (RFLP) after a PCR amplification (n= 61). Results and conclusionSequencing data revealed a heterozygosity of the SRA gene within Malawi T. b. rhodesiense isolates. Malawian SRA gene was genetically different from some isolates in Uganda and Zambia. Contrary to the current understanding that APOL-1 G2 variants are immune to T. b. rhodesiense infection, severe cases of r-HAT in G2 individuals were identified. This study has brought new insight in understanding the determinants of r-HAT severity.

genomics↗

bioRxiv2023

Transcriptome profiles of T.b. rhodesiense parasites in Malawi reveal focus specific gene expression Profiles associated with pathology

BackgroundSleeping sickness caused by T.b. rhodesiense is a fatal disease and endemic in Southern and Eastern Africa. There is an urgent need to develop novel diagnostic and control tools in order to achieve elimination of rhodesiense sleeping sickness which might be achieved through a better understanding of trypanosome gene expression and genetics using endemic isolates. Here, we describe transcriptome profiles and population structure of endemic T. b. rhodesiense isolates in human blood in Malawi. MethodologyBlood samples of r-HAT cases from Nkhotakota and Rumphi foci were collected in PaxGene tubes for RNA extraction before initiation of r-HAT treatment. 100 million reads were obtained per sample, reads were initially mapped to the human genome reference GRCh38 using HiSat2 and then the unmapped reads were mapped against Trypanosoma brucei reference transcriptome (TriTrypDB54_TbruceiTREU927) using HiSat2. Differential gene expression analysis was done using the DeSeq2 package in R. SNPs calling from reads that were mapped to the T. brucei genome was done using GATK in order to identify T.b. rhodesiense population structure. Results24 samples were collected from r-HAT cases of which 8 were from Rumphi and 16 from Nkhotakota foci. The isolates from Nkhotakota were enriched with transcripts for cell cycle arrest and stumpy form markers, whereas isolates in Rumphi focus were enriched with transcripts for folate biosynthesis and antigenic variation pathways. These parasite focus-specific transcriptome profiles are consistent with the more virulent disease observed in Rumphi and a more silent disease in Nkhotakota associated with the non-dividing stumpy form. Interestingly, the Malawi T.b. rhodesiense isolates expressed genes enriched for reduced cell proliferation compared to the Uganda T.b. rhodesiense isolates. PCA analysis using SNPs called from the RNAseq data showed that T. b. rhodesiense parasites from Nkhotakota are genetically distinct from those collected in Rumphi. ConclusionOur results have added new insights on how clinical phenotypes of r-HAT in Malawi might be associated with differences in gene expression profiles and population structure of T. b. rhodesiense from its two major endemic foci of Rumphi and Nkhotakota. Author SummaryA better understanding of T. b. rhodesiense gene expression profiles and population structure using endemic isolate may fast track the current search for novel diagnostic and control tools for rhodesiense sleeping sickness. Here, we analysed T. b. rhodesiense transcriptome profiles from endemic isolated from peripheral blood in Nkhotakota and Rumphi foci in Malawi. In Nkhotakota focus, T. b. rhodesiense transcripts were enriched for cell cycle arrest and stumpy marker whereas in Rumphi focus, the isolates were enriched for antigenic variation and folate biosynthesis biological pathways. Furthermore, we also found that T. b. rhodesiense population structure in Nkhotakota focus is different from Rumphi focus. The differences in trypanosome gene expression profiles and population structure are consistent with a less severe and acute sleeping sickness clinical profiles in Nkhotakota and Rumphi foci respectively.

genomics↗

bioRxiv2023

Transcriptome analysis of peripheral blood of Schistosoma mansoni infected children from the Albert Nile region in Uganda reveals genes implicated in fibrosis pathology.

Over 290 million people are infected by schistosomes worldwide. Schistosomiasis control efforts focus on mass drug treatment with praziquantel (PZQ), a drug that kills the adult worm of all Schistosoma species. Nonetheless, re-infections have continued to be detected in endemic areas with individuals living in the same area presenting with varying infection intensities. Our objective was to characterize the transcriptome profiles in peripheral blood of children between 10 - 15 years with varying intensities of Schistosoma mansoni infection living along the Albert Nile in Uganda. RNA extracted from peripheral blood collected from 44 S. mansoni infected (34 high and 10 low by circulating anodic antigen [CAA] level) and 20 uninfected children was sequenced using Illumina NovaSeq S4 and the reads aligned to the GRCh38 human genome. Differential gene expression analysis was done using DESeq2 and enriched pathways in differentially expressed genes (DEGs) were identified using REACTOME. Principal component analysis revealed clustering of gene expression by gender when S. mansoni infected children were compared with uninfected children. In addition, we identified 14 DEGs between S. mansoni infected and uninfected individuals, 56 DEGs between children with high infection intensity and uninfected individuals, 33 DEGs between those with high infection intensity and low infection intensity and no DEGs between those with low infection and uninfected individuals. We also observed upregulation and downregulation of some DEGs that are associated with fibrosis and its regulation. These data suggest expression of fibrosis associated genes as well as genes that regulate fibrosis in S. mansoni infection. The relatively few significant DEGS observed in children with schistosomiasis suggests that chronic S. mansoni infection is a stealth infection that does not stimulate a strong immune response. Author SummarySchistosomiasis is a neglected tropical disease transmitted via an intermediate snail host through contact with contaminated fresh water. Even with routine Mass Drug Administration for treatment of the infection, re-infections are still common and variations in infection intensity and pathology are still observed in individuals in the same location. These may be due to differences in individuals response to S. mansoni infection. In this study, we used RNAseq to identify differentially expressed genes associated with S. mansoni infection in children between 10-15 years. We conducted comparisons between phenotypes including infection intensities measured by circulating anodic antigen, wasting by body mass index and stunting by height-for-age z score. Our data showed very low numbers of significant differentially expressed genes in all comparisons. Some of the few differentially expressed genes that were observed were associated with fibrosis which is the cause of pathology in humans and has been observed in late stages of S. mansoni infection in murine studies.

genomics↗

bioRxiv2022

Distinct Differences in Gene Expression Profiles in Early and Late Stage Rhodesiense HAT Individuals in Malawi

T. b. rhodesiense is the causative agent of rhodesian Human African trypanosomiasis (r-HAT) in Malawi. Clinical presentation of r-HAT in Malawi varies between the different foci and differs from East African HAT clinical phenotypes. The purpose of this study was to gain more insights into the transcriptomic profiles of patients with early stage 1 and late stage 2 HAT disease in Malawi. Whole blood from individuals infected with T. b. rhodesiense was used for RNA-Seq. Control samples were from healthy trypanosome negative individuals matched on sex, age range, and disease focus. Illumina sequence FASTQ reads were aligned to the GRCh38 release 84 human genome sequence using HiSat2 and differential analysis was done in R using the DESeq2 package. XGR, ExpressAnalyst and InnateDB algorithms were used for functional annotation and gene enrichment analysis of significant differentially expressed genes. RNA-seq was done on 25 healthy controls and 23 r-HAT case samples of which 3 case samples were excluded for downstream analysis as outliers. 4519 genes were significantly differentially expressed (p adjusted <0.05) in individuals with early stage 1 r-HAT disease (n = 12) and 1824 genes in individuals with late stage 2 r-HAT disease (n = 8). Enrichment of innate immune response genes through neutrophil activation was identified in individuals with both early and late stages of the disease. Additionally, lipid metabolism genes were enriched in late stage 2 disease. We further identified uniquely upregulated genes (log2 Fold Change 1.4 - 2.0) in stage 1 (ZNF354C) and stage 2 (TCN1 and MAGI3) blood. Our data brings new insight into the human transcriptome landscape during T. b. rhodesiense infection. We have further identified key biological pathways and transcripts during stage 1 and stage 2 r-HAT. Lastly, we have identified potential diagnostic biomarkers that may be used for staging of r-HAT disease.

genetics↗

bioRxiv2021

The effect of in vitro culture on unicellular eukaryotes: adaptation of Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes.

Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were obtained from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains. MAK65 and MAK98, which have undergone only 3 rodent passages since isolation from Ugandan cattle. High-coverage sequencing revealed that adaptation of the parasites to culture was accompanied by changes in gene copy numbers. T. brucei has so far been considered to be uniformly diploid, but we also found trisomy of chromosome 5 not only in one Lister 427 culture, but also in the MAK98 field isolate. Trisomy of chromosome 6, and increased copies of other chromosome segments, were also seen in established cultured lines. The two new T. brucei strains should be useful to researchers interested in trypanosome differentiation and pathogenicity. Initial results suggested that the two strains have differing infection patterns in rodents. MAK65 is uniformly diploid and grew more reproducibly in bloodstream-form culture than MAK98.

genomics↗

bioRxiv2017

Genomic evidence for population specific selection in Nilo-Saharan and Niger-Congo linguistic groups in Africa

BackgroundThere are over 2000 genetically diverse ethno-linguistic groups in Africa that could help decipher human evolutionary history and the genetic basis of phenotypic variation. We have sequenced 300 genomes from Niger-Congo populations from six sub-Saharan African countries (Uganda, Democratic Republic of Congo, Cameroon, Zambia, Ivory Coast, Guinea) and a Nilo-Saharan population from Uganda. Of these, we analysed 289 samples for population structure, genetic admixture, population history and signatures of selection. These samples were collected as part of the TrypanoGEN consortium project [1].\n\nResultsThe population genetic structure of the 289 individuals revealed four clusters, which correlated with ethno-linguistic group and geographical latitude. These were the West African Niger-Congo A, Central African Niger-Congo B, East African Niger-Congo B and the Nilo-Saharan. We observed a spatial distribution of positive natural selection signatures in genes previously associated with AIDS, Tuberculosis, Malaria and Human African Trypanosomiasis among the TrypanoGEN samples. Having observed a marked difference between the Nilo-Saharan Lugbara and Niger-Congo populations, we identified four genes (APOBEC3G, TOP2B, CAPN9, LANCL2), which are highly differentiated between the two ethnic groups and under positive selection in the Lugbara population (_iHS -log p > 3.0, Rsb -log p > 3.0, Fst > 0.1 bonferroni p > 1.8x10e4).\n\nConclusionThe signatures that differentiate ethnically distinct populations could provide information on the specific ecological adaptations with respect to disease history and susceptibility/resistance. For instance in this study we identified APOBEC3G which is believed to be involved in the susceptibility of the Nilo-Saharan Lugbara population to Hepatitis B virus infection.

genomics↗

bioRxiv2017

Candidate genes-based investigation of susceptibility to Human African Trypanosomiasis in Côte d’Ivoire

Human African Trypanosomiasis (HAT) or sleeping sickness is a Neglected Tropical Disease. Long regarded as an invariably fatal disease, there is increasing evidence that infection by T. b. gambiense can result in a wide range of clinical outcomes, including latent infections, which are long lasting infections with no parasites detectable by microscopy. The determinants of this clinical diversity are not well understood but could be due in part to parasite or host genetic diversity in multiple genes, or their interactions. A candidate gene association study was conducted in Cote dIvoire using a case-control design which included a total of 233 subjects (100 active HAT cases, 100 controls and 33 latent infections). All three possible pairwise comparisons between the three phenotypes were tested using 96 SNPs in16 candidate genes (IL1, IL4, IL4R, IL6, IL8, IL10, IL12, IL12R, TNFA, INFG, MIF, APOL1, HPR, CFH, HLA-A and HLA-G). Data from 77 SNPs passed quality control. There were suggestive associations at three loci in IL6 and TNFA in the comparison between active cases and controls, one SNP in each of APOL1, MIF and IL6 in the comparison between latent infections and active cases and seven SNP in IL4, HLA-G and TNFA between latent infections and controls. No associations remained significant after Bonferroni correction, but the Benjamini Hochberg false discovery rate test indicated that there were strong probabilities that at least some of the associations were genuine.\n\nThe excess of associations with latent infections despite the small number of samples available suggests that these subjects form a distinct genetic cluster different from active HAT cases and controls, although no clustering by phenotype was observed by principle component analysis. This underlines the complexity of the interactions existing between host genetic polymorphisms and parasite diversity.\n\nAuthor summarySince it was first identified, human African trypanosomiasis (HAT) or sleeping sickness has been described as invariably fatal. Recent data however suggest that infection by T. b. gambiense can result in a wide range of clinical outcomes in its human host including long lasting infections, that can be detected by the presence of antibodies, but in which parasites cannot be seen by microscopy; these cases are known as latent infections. While the factors determining this varied response have not been clearly characterized, the effectors of the immune responses have been partially implicated as key players. We collected samples from people with active HAT, latent infections and controls in endemic foci in the Cote dIvoire. We tested the role of single nucleotide polymorphisms (SNPs) in 16 genes on susceptibility/resistance to HAT by means of a candidate gene association study. There was some evidence that variants of the genes for IL4, IL6, APOL1, HLAG, MIF and TNFA modified the risk of developing HAT. These proteins regulate the inflammatory response to many infections or are directly involved in killing the parasites. In this study, the results were statistically weak and would be inconclusive on their own, however other studies have also found associations in these genes, increasing the chance that the variants that we have identified play a genuine role in the response to trypanosome infection in Cote DIvoire.

genetics↗

bioRxiv2017

No evidence for association with APOL1 kidney disease risk alleles and Human African Trypanosomiasis in two Ugandan populations

BackgroundHuman African trypanosomiasis (HAT) manifests as an acute form caused by Trypanosoma brucei rhodesiense (Tbr) and a chronic form caused by Trypanosoma brucei gambiense (Tbg). Previous studies have suggested a host genetic role in infection outcomes, particularly for APOL1. We have undertaken a candidate gene association studies (CGAS) in a Ugandan Tbr and a Tbg HAT endemic area, to determine whether polymorphisms in IL10, IL8, IL4, HLAG, TNFA, TNX4LB, IL6, IFNG, MIF, APOL1, HLAA, IL1B, IL4R, IL12B, IL12R, HP, HPR, and CFH have a role in HAT.\n\nMethodology and resultsWe included 238 and 202 participants from the Busoga Tbr and Northwest Uganda Tbg endemic areas respectively. Single Nucleotide Polymorphism (SNP) genotype data were analysed in the CGAS. The study was powered to find odds ratios > 2 but association testing of the SNPs with HAT yielded no positive associations i.e. none significant after correction for multiple testing. However there was strong evidence for no association with Tbr HAT and APOL1 G2 of the size previously reported in the Kabermaido district.\n\nConclusions/significanceA recent study in the Soroti and Kaberamaido focus in Central Uganda found that the APOL1 G2 allele was strongly associated with protection against Tbr HAT (odds ratio = 0.2). However, in our study no effect of G2 on Tbr HAT was found, despite being well powered to find a similar sized effect. It is possible that the G2 allele is protective from Tbr in the Soroti/Kabermaido focus but not in the Iganga district of Busoga, which differ in ethnicity and infection history. Mechanisms underlying HAT infection outcome and virulence are complex and might differ between populations, and likely involve several host, parasite or even environmental factors.\n\nAuthor SummaryHuman African Trypanosomiasis (HAT) occurs in two distinct disease forms; the acute form and the chronic form which are caused by microscopically indistinguishable hemo-parasites, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense respectively. Uganda is the only country where both forms of the disease are found, though in geographically distinct areas. Recent studies have shown that host genetic factors play a role in HAT resistance and/or susceptibility, particularly by genes involved in the immune response. In this study, we identified single nucleotide polymorphisms in selected genes involved in immune responses and carried out a case-control candidate gene association study in Ugandan participants from the two endemic areas. We were unable to detect any polymorphisms that were robustly associated with either Tbr or Tbg HAT. However, our findings differ from recent studies carried out in the Tbr HAT another endemic area of Uganda that showed the APOL1 (Apolipoprotein 1) G2 allele to be protective against the disease which merits further investigation. Larger studies such as genome wide association studies (GWAS) by the TrypanoGEN network that has >3000 cases and controls covering seven countries (Cameroon, Cote dIvoire, DRC, Malawi, Uganda, Zambia) using the H3Africa customized chip reflective of African genetic diversity will present novel association targets.

genetics↗