Exploiting trypanosomes as models for pathogenicity and core biology research

Model systems are commonly used to investigate biological processes and understand molecular mechanisms. The choice of a model cell or organism is informed by the scope of the question being asked. Decades of research using predominantly animal and fungal models form the bulk of our knowledge of eukaryotic processes. The diversity of eukaryotic organisms, however, indicates that this may not be representative of what exists in more diverged groups, and key processes like cell division vary across the larger group. In the past, non-traditional models including Tetrahymena have been applied to answer biological questions because of peculiar traits that make them suitable for investigation. African trypanosomes offer an excellent opportunity as model organisms because of their divergence from common models and importance to human health and livelihood. Particularly Trypanosoma brucei, with several genetic tools available to study them, has become an interesting model to study processes like cell division, cytoskeleton and antigenic variation. Here, I present work exploring the application of two species of African trypanosomes as models to investigate common biology of eukaryotic kinesins and specific biology of animal African trypanosomiasis. The genetic tractability of T. brucei and its cytoskeletal structure, which is predominantly made up of tubulin, makes it a good model to study functional differentiation between kinesin families. I show cellular localisation and characterise the effect of RNAi depletion of representative kinesin families. The data suggest that Kinesin-2, -3 and 13-1 perform similar roles in T. brucei as seen in other systems, and can therefore be candidates for investigation. On the contrary, loss of both alleles of T. brucei Kinesin-1 does not affect in vitro growth, suggesting possible functional redundancy in trypanosomes. Trypanosoma congolense is the most significant cause of animal African trypanosomiasis, a wasting disease of livestock that results in huge economic losses to agriculture particularly in Africa. Disease presentation differs from T. brucei, and the lack of genetic tools makes it difficult to study the biology of T. congolense, specifically its pathogenesis in animal trypanosomiasis. I show the progressive development and application of genetic tools for endogenous locus tagging, RNA interference and high-complexity mutant library production. I demonstrate that loci on the minichromosome can be targeted for transgene integration, give good expression and are regulatable. Genes targeted by RNAi could only be depleted to about 50% of original levels, with similar proportion of cells expressing mutant phenotype, suggesting that T. congolense has lower RNAi penetrance than T. brucei. The development and optimization of a system that uses a homing endonuclease to increase DNA breaks at integration sites consistently increased the number of positive transformants to ~ 50,000 per transfection. This T. congolense line maintained stable transfection efficiency even after cycles of freeze-thawing. Finally, I demonstrate for the first time, a genome-wide technology in T. congolense. Using all the tools developed, I create two RNAi libraries containing > 5x105 independent transformants. The method of library generation by direct RNAi fragment sequencing (DRiF-Seq) allowed detection of very small changes in gene fitness cost due to RNAi. This enabled fitness screen of > 9000 genes in T. congolense even at a low RNAi penetrance. Analysis of differential biology between T. congolense and T. brucei gives some glimpse into similarities and differences in processes like endocytosis and quorum sensing. I also demonstrate application of the T. congolense RNAi libraries in a drug screen to investigate isometamidium mode of action and resistance

A conserved trypanosomatid differentiation regulator controls substrate attachment and morphological development in Trypanosoma congolense

Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in that parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunoreactive proteins in experimental bovine infections, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission

Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions

Exploiting trypanosomes as models for pathogenicity and core biology research

Model systems are commonly used to investigate biological processes and understand molecular mechanisms. The choice of a model cell or organism is informed by the scope of the question being asked. Decades of research using predominantly animal and fungal models form the bulk of our knowledge of eukaryotic processes. The diversity of eukaryotic organisms, however, indicates that this may not be representative of what exists in more diverged groups, and key processes like cell division vary across the larger group. In the past, non-traditional models including Tetrahymena have been applied to answer biological questions because of peculiar traits that make them suitable for investigation. African trypanosomes offer an excellent opportunity as model organisms because of their divergence from common models and importance to human health and livelihood. Particularly Trypanosoma brucei, with several genetic tools available to study them, has become an interesting model to study processes like cell division, cytoskeleton and antigenic variation. Here, I present work exploring the application of two species of African trypanosomes as models to investigate common biology of eukaryotic kinesins and specific biology of animal African trypanosomiasis. The genetic tractability of T. brucei and its cytoskeletal structure, which is predominantly made up of tubulin, makes it a good model to study functional differentiation between kinesin families. I show cellular localisation and characterise the effect of RNAi depletion of representative kinesin families. The data suggest that Kinesin-2, -3 and 13-1 perform similar roles in T. brucei as seen in other systems, and can therefore be candidates for investigation. On the contrary, loss of both alleles of T. brucei Kinesin-1 does not affect in vitro growth, suggesting possible functional redundancy in trypanosomes. Trypanosoma congolense is the most significant cause of animal African trypanosomiasis, a wasting disease of livestock that results in huge economic losses to agriculture particularly in Africa. Disease presentation differs from T. brucei, and the lack of genetic tools makes it difficult to study the biology of T. congolense, specifically its pathogenesis in animal trypanosomiasis. I show the progressive development and application of genetic tools for endogenous locus tagging, RNA interference and high-complexity mutant library production. I demonstrate that loci on the minichromosome can be targeted for transgene integration, give good expression and are regulatable. Genes targeted by RNAi could only be depleted to about 50% of original levels, with similar proportion of cells expressing mutant phenotype, suggesting that T. congolense has lower RNAi penetrance than T. brucei. The development and optimization of a system that uses a homing endonuclease to increase DNA breaks at integration sites consistently increased the number of positive transformants to ~ 50,000 per transfection. This T. congolense line maintained stable transfection efficiency even after cycles of freeze-thawing. Finally, I demonstrate for the first time, a genome-wide technology in T. congolense. Using all the tools developed, I create two RNAi libraries containing > 5x105 independent transformants. The method of library generation by direct RNAi fragment sequencing (DRiF-Seq) allowed detection of very small changes in gene fitness cost due to RNAi. This enabled fitness screen of > 9000 genes in T. congolense even at a low RNAi penetrance. Analysis of differential biology between T. congolense and T. brucei gives some glimpse into similarities and differences in processes like endocytosis and quorum sensing. I also demonstrate application of the T. congolense RNAi libraries in a drug screen to investigate isometamidium mode of action and resistance

A conserved trypanosomatid differentiation regulator controls substrate attachment and morphological development in Trypanosoma congolense.

Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in that parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunoreactive proteins in experimental bovine infections, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission

MOESM1 of Trends in paediatric and adult bloodstream infections at a Ghanaian referral hospital: a retrospective study

Additional file 1. The RECORD statement—checklist of items, extended from the STROBE statement that should be reported in observational studies using routinely collected health data

Trichomonas vaginalis infection and the diagnostic significance of detection tests among Ghanaian outpatients

Abstract Background There is little data on Trichomonas vaginalis infection in Ghana. This study evaluated the prevalence of trichomoniasis using different diagnostic methods and determined the risk factors for infection in patients. Methods A structured questionnaire was administered. Vaginal swabs, urethral swabs and urine specimens were obtained from consenting patients; and the samples processed following standard protocols. The presence of T. vaginalis was determined using wet mount microscopy and polymerase chain reaction (PCR) as gold standard. We also assessed the diagnostic performance the JD’s Trichomonas V® rapid antigen test to inform clinical practice. Results The PCR assay detected T. vaginalis positivity in 64 of 150 patients (42.6, 95%CI:35.0, 50.6) including all positive samples of wet mount microscopy and JD’s Trichomonas V® test. Wet mount microscopy showed low sensitivity (31.6%), high specificity (100%), moderate positive predictive value (75.0%), moderate positive likelihood ratio (3.0), and weak agreement (Cohen’s kappa, 0.283) with PCR assay. The JD’s Trichomonas V® test displayed lower sensitivity (25.0%), specificity (83.3%), and weaker measure of agreement (Cohen’s kappa, 0.233) with PCR. In multivariate analysis, the strongest independent predictor for T. vaginalis was female gender [adjusted odds ratio (AOR), 24.89; 95% confidence interval (CI): 10.58, 51.21; P-value< 0.001]. Knowledge of STI showed a protective effect against infection with the parasite (AOR, 0.13; 95%CI: 0.07, 0.29; P-value< 0.017). Conclusion The sensitivity of wet mount microscopy was low for T. vaginalis screening in our region. The JD’s Trichomonas V® test should not be considered as an alternative test. We recommend mandatory PCR assay for confirmation of negative wet mount results

Trichomoniasis and associated co-infections of the genital tract among pregnant women presenting at two hospitals in Ghana

Abstract Background Trichomonas vaginalis (TV) infection is the most prevalent non-viral sexually transmitted pathogen worldwide. Among pregnant women, the infection may cause adverse birth outcomes such as premature rupture of membranes and premature labour. In view of the paucity of information relating to TV among Ghanaian pregnant women, this study investigated its prevalence and associated co-infections among pregnant women. Methods High vaginal swabs were obtained from 99 pregnant women using sterile cotton swab sticks. Wet preparation, Grams staining, culturing, coagulase and sensitivity testing were carried out to determine the presence of TV and associated microorganisms. Results The prevalence of TV among the pregnant women was found to be 20.2% (n = 20). Concurring with Trichomoniasis, 75% (n = 15) of participants had other infections such as Candida with prevalence of 53% (n = 8), Proteus infection - 20% (n = 3), Streptococcus infection - 13% (n = 2) and other GNRs and Gonococci having 7% each (n = 1). Moreover, there was 86.9% (n = 86) prevalence of Staphylococcus spp. among study participants. There was statistically significant correlation between TV and Gonococci infection at a correlation co-efficient of 0.107 (P < 0.05) as well as significant correlation between TV and Proteus spp. at a correlation co-efficient of 0.189 (P < 0.05). TV infection was high (60%) among the most sexually active age group (19 to 29 yrs). Conclusion There was 20.2% prevalence of TV among the pregnant women presenting at the hospitals, with Gonococci and Proteus infections being statistically significant associated infections