Possible Chemotherapeutic Potential of Inhibiting N-Alpha Terminal Acetylation Activities to Combat Trypanosome Infections

New anti-trypanosome drugs focusing on N-alpha terminal acetylation (Nt-acetylation) interference are necessary scientific inputs because currently, many of the drugs in use are unacceptably toxic; moreover, resistance is emerging. Nt-acetylation transfers an acetyl molecule to the N-alpha terminal of a protein by enzymes called N-alpha terminal acetyltransferases (Nats). Nats are grouped according to their amino acid sequence at the N-terminus where they acetylate. It is conserved in all kingdoms of life, and in humans, approximately 80% of proteins are thought to be Nt-acetylated. NatA-NatF and NatH identified in humans, and NatG has been observed in plants. Nats play critical roles in several cellular processes and integrity and have been suggested as possible drug targets to control different cancer diseases. NatA and NatC have been partially characterized in trypanosomes and shown to be essential for parasite viability. Biologically, the way parasites program their lives is embedded in their unique organelles, metabolic pathways, gene regulation, epigenetic gene activities, and many virulence factors including surface molecules. These characteristics and the different protein-coding genes involved could be Nt-acetylated, and the inhibition of Nats can deny the ability of trypanosomes to survive in any environment because many proteins can be simultaneously affected

Characterization of evolutionarily conserved Trypanosoma cruzi NatC and NatA- N-terminal acetyltransferase complexes

Protein N-terminal acetylation is a co- and post-translational modification, conserved among eukaryotes. It determines the functional fate of many proteins including their stability, complex formation and subcellular localization. N-terminal acetyltransferases (NATs) transfer an acetyl group to the N-termini of proteins, and the major NATs in yeast and humans are NatA, NatB and NatC. In this study, we characterized the Trypanosoma cruzi (T. cruzi) NatC and NatA protein complexes, each consisting of one catalytic subunit and predicted auxiliary subunits. The proteins were found to be expressed in the three main life cycle stages of the parasite, formed stable complexes in vivo, and partially co-sedimented with the ribosome in agreement with a co-translational function. An in vitro acetylation assay clearly demonstrated that the acetylated substrates of the NatC catalytic subunit from T. cruzi were similar to those of yeast and human NatC, suggesting evolutionary conservation of function. An RNAi knockdown of the Trypanosome brucei (T. brucei) NatC catalytic subunit indicated that reduced NatC-mediated N-terminal acetylation of target proteins reduce parasite growth

Trypanosoma cruzi genome plasticity and evolution

Trypanosoma cruzi, a protozoan from the Kinetoplastidae family, is the etiologic agent of Chagas disease, a major public health problem affecting mostly the poorest areas of Latin America. Due to the complex nature of the parasite’s genome it has been impossible to produce a complete reference genome sequence, thus hampering the implementation of post- genomic approaches to unveil the mechanisms of generation of antigenic variation and the identification of new drug targets. My doctoral studies have focused on the application of combined genome sequencing and computational methods to produce a complete reference T. cruzi genome sequence and perform comparative analyses to better understand the mechanisms that allow T. cruzi to evade the mammalian host immune system and to briskly adapt to novel environments. In paper I and II, different genome assembly strategies and second generation sequencing technologies were implemented to perform comparative analyses to identify elements of virulence between T. cruzi and two trypanosomatids that are non-pathogenic to humans: Trypanosoma cruzi marinkellei, a bat-restricted sub-species of the T. cruzi clade and the human avirulent species Trypanosoma rangeli. The studies reveal the expansion of T. cruzi- specific genomic traits specialised in the invasion of mammalian cells. In paper III, using third-generation, PacBio sequencing data it was possible to assemble the complete reference genome sequence of a Trypanosoma cruzi isolate from the DTU-I clade. This breakthrough allowed us - for the first time - to explore in detail the genome architecture of the subtelomeric areas where many parasite virulence factors are encoded. One of the most interesting discoveries was the overrepresentation of interspersed retrotransposons and microsatellites in tandem gene arrays coding for surface molecules, hinting at a retrotransposon-driven mechanism of recombination for generating new sequence variants. Whole genome sequencing of 35 T. cruzi DTU-I isolates, collected from different locations in the American continent, made possible to identify and characterise the mechanisms of adaptability employed by the parasite. Finally, paper IV analyses the mechanisms of genomic hybridisation in T. cruzi and the evolution over time of the hybrid offspring. The analysis revealed that during hybrid formation, the parasite integrates genetic material from each parental strains with the aid of retrotransposons and microsatellites, and the genome of these hybrid isolates moves quickly from a tetraploid to a diploid state. As a result, the hybrid strain has more genetic material, mostly in the subtelomeres, providing the parasite with a pool of new surface molecule genes with the potential to possibly increase its fitness in a new environment. In conclusion, the work presented here has advanced the understanding of parasite biology and provided a genomic resource to be exploited for the identification of drug targets and vaccine candidates

Shotgun Sequencing Analysis of Trypanosoma cruzi I Sylvio X10/1 and Comparison with T. cruzi VI CL Brener

Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite

Mapping the genome and characterization of an acetyltransferase of Trypanosoma cruzi

Trypanosoma cruzi (T.cruzi) is a flagellate protozoan parasite that causes Chagas' disease and is most common in Latin American countries. The disease currently infects over 13 million persons and about 100 million more are at risk. Despite basic research for many years, there is neither a vaccine nor useful drugs available to combat the disease. We were involved in sequencing the genome of this parasite, launched by WHO in order to increase knowledge on the molecular biology of the parasite and disease pathology. In the first part of this thesis, we tried to elucidate the molecular karyotype (actual numbers and sizes of chromosomes in a cell) of T.cruzi, which was an essential part of the genome project, newly published in Science together with two other kinetoplastid genomes. For the karyotyping study, three strains were used. CL-Brener (lineage IIe), chosen as a reference strain in the genome project and two T. cruzi lineage 1 strains, Sylvio X 10/7 and CAI/72 for comparative purposes. Since the T cruzi chromosomes do not condense during cell division, we separated the intact chromosomes by PFGE using three different running conditions. We hybridized 239 cDNA markers (probes) to the PFGE separated chromosomes. Using densitometry analysis- the total number of chromosomes for CL-Brener was estimated to be 55 and 57 in the two other strains. Size differences between homologous chromosomes, especially for CL-Brener are very high, up to 173%, and may be due to chromosome rearrangements. Forty markers distributed into 15 linkage groups, were found to identify specific chromosomes or chromosome pairs, hence showing conservation of gene order (synteny) between strains. This data provides valuable information for the finishing of the CL- Brener genome sequence. In the second part of this thesis, a Trypanosoma cruzi acetyltransferase (TcAT) protein that was annotated in our laboratory during the genome sequencing was characterized as a first step to evaluate its future potential as a drug target. Acetyltransferases (ATs) in general are enzymes that transfer acetyl-Coenzyme A (AcCo A) to the Nterminal of a protein or a peptide. In principle, acetylation is a posttranslational modification present in a majority of eukaryotes, altering the properties of proteins in different ways. The consequences of acetylation include effects on protein stability, protein-protein interaction and DNA binding. Our analysis of TcAT shows that the gene is single copy. The TcAT motifs match the GCN5-related acetyltransferase (GNAT) family. Orthologous proteins are present in the other two kinetoplastids (Trypanosoma brucei and Leishmania). The protein appears to be more closely related to N-terminal acetyltransferases (ATs) than to histone acetyltransferases (HATs). The native protein has a cytosolic location. It is expressed in the three different life-cycle stages of the parasite, determined by western blot. The recombinant protein has an autoacetylation activity. The 3D topology model prediction for TcAT is typical of the GNAT super family of ATs. The N-terminal of the human ortholog (HYPO-HUMAN) is elongated and there are three extra alpha helices predicted. Interestingly, kinetoplastids also have three extra helices but not in the same positions as in human. Some of the predicted functional sites where Acetyl CoA binds, and ligand binding sites were conserved in Kinetoplastids compared to other organisms including human. Proteins that interact with TcAT, native substrate specificity and metabolic pathway are not yet known. Despite that fact, the predicted protein structures of Kinetoplastids, amino acid, functional and ligand binding sites seem to differ from that of human counterpart and could be exploited and used in drug design against Trypanosomes and Leishmania

High prevalence of Pfmdr-1 N86 and D1246 genotypes detected among febrile malaria outpatients attending Lira Regional Referral Hospital, Northern Uganda

Abstract Objective To determine the prevalence of Plasmodium falciparum multi-drug resistant gene-1 (Pfmdr-1) N86Y and D1246Y genotypes among febrile malaria outpatients attending Lira Regional Referral Hospital, Uganda. Results Overall, 92.3% (n = 48/52) and 90% (n = 45/50) of the parasites detected carried the wild type alleles 1246D and N86, respectively. Only 7.7% (n = 4/52) and 10% (n = 5/50) of these P. falciparum isolates carried the Pfmdr-1 mutant alleles 1246Y and 86Y, respectively. Our results show high prevalence of wild type alleles N86 and D1246 in P. falciparum isolates from Lira Regional Referral Hospital, which could translate to a decreased sensitivity to artemether-lumefantrine. Continued monitoring of prevalence of single nucleotide polymorphisms is warranted to timely inform malaria treatment policies and guidelines

Soil-transmitted helminths: A critical review of the impact of co-infections and implications for control and elimination.

Researchers have raised the possibility that soil-transmitted helminth (STH) infections might modify the host's immune response against other systemic infections. STH infections can alter the immune response towards type 2 immunity that could then affect the likelihood and severity of other illnesses. However, the importance of co-infections is not completely understood, and the impact and direction of their effects vary considerably by infection. This review synthesizes evidence regarding the relevance of STH co-infections, the potential mechanisms that explain their effects, and how they might affect control and elimination efforts. According to the literature reviewed, there are both positive and negative effects associated with STH infections on other diseases such as malaria, human immunodeficiency virus (HIV), tuberculosis, gestational anemia, pediatric anemia, neglected tropical diseases (NTDs) like lymphatic filariasis, onchocerciasis, schistosomiasis, and trachoma, as well as Coronavirus Disease 2019 (COVID-19) and human papillomavirus (HPV). Studies typically describe how STHs can affect the immune system and promote increased susceptibility, survival, and persistence of the infection in the host by causing a TH2-dominated immune response. The co-infection of STH with other diseases has important implications for the development of treatment and control strategies. Eliminating parasites from a human host can be more challenging because the TH2-dominated immune response induced by STH infection can suppress the TH1 immune response required to control other infections, resulting in an increased pathogen load and more severe disease. Preventive chemotherapy and treatment are currently the most common approaches used for the control of STH infections, but these approaches alone may not be adequate to achieve elimination goals. Based on the conclusions drawn from this review, integrated approaches that combine drug administration with water, sanitation and hygiene (WASH) interventions, hygiene education, community engagement, and vaccines are most likely to succeed in interrupting the transmission of STH co-infections. Gaining a better understanding of the behavior and relevance of STH co-infections in the context of elimination efforts is an important intermediate step toward reducing the associated burden of disease

Sibling Species Composition and Susceptibility Status of Anopheles gambiae s.l. to Insecticides Used for Indoor Residual Spraying in Eastern Uganda

Background. Malaria remains one of the most critical disease causing morbidity and mortality in Uganda. Indoor residual spraying (IRS) and the use of insecticide-treated bed nets are currently the predominant malaria vector control interventions. However, the emergence and spread of insecticide resistance among malaria vectors threaten the continued effectiveness of these interventions to control the disease, particularly in high transmission areas. To inform decisions on vector control, the current study evaluated the Anopheles malaria vector species and their susceptibility levels to 0.1% bendiocarb and 0.25% pirimiphos-methyl insecticides used in IRS intervention program in Namutumba district, Eastern Uganda. Methods.Anopheles larvae were collected between March and May 2017 from different breeding sites in the parishes of Nsinze and Nawaikona in Nsinze sub-county and reared to adults to assess the susceptibility status of populations in the study area. Mosquitoes were identified using morphological keys and species-specific polymerase chain reaction (PCR) assays. Susceptibility tests were conducted on 2- to 5-day-old non-blood-fed adult female Anopheles that emerged using insecticide-impregnated papers with 0.1% bendiocarb and 0.25% pirimiphos-methyl following standard World Health Organization (WHO) insecticide susceptibility bioassays. A Log-probit regression model was used to derive the knock-down rates for 50% and 95% of exposed mosquitoes. Results. A total of 700 mosquito larvae were collected from different breeding sites. Morphological identification showed that 500 individuals that emerged belonged to Anopheles gambiae sensu lato (s.l.), the main malaria vector. The PCR results showed that the dominant sibling species under the A. gambiae complex was Anopheles arabiensis 99.5% (395/397). WHO bioassay tests revealed that the population of mosquitoes exhibited high levels of susceptibility (24-hour post-exposure mortality 98–100%) to both insecticides tested. The median knock-down time, KDT50, ranged from 6.6 to 81.4 minutes, while the KDT95 ranged from 21.6 to 118.9 minutes for 0.25% pirimiphos-methyl. The KDT50 for 0.1% bendiocarb ranged from 2.8 to 62.9 minutes, whereas the KDT95 ranged from 36.0 to 88.5 minutes. Conclusions. These findings indicate that bendiocarb and pirimiphos-methyl are still effective against the major malaria vector, A. arabiensis in Nsinze sub-county, Namutumba district, Uganda and can be effectively used for IRS. The study has provided baseline information on the insecticide susceptibility status on malaria vectors in the study area. However, routine continuous monitoring program of insecticide susceptibility and malaria vector composition is required so as to guide future decisions on insecticide use for IRS intervention toward malaria elimination and to track future changes in vector population

Hookworm Infection among Pregnant Women at First Antenatal Visit in Lira, Uganda: A Cross-Sectional Study

Background: Hookworm infection in expectant mothers has adverse health effects on both the mothers and their unborn babies. Foetal effects are known to include intrauterine growth retardation and physical and mental growth retardation, while the mothers may develop anemia which could potentially result in death. Unfortunately, little is known about factors that may predispose a pregnant woman to infection by hookworm. In this study, we strived to determine not only the prevalence of hookworm infection among pregnant women attending their first antenatal visit during the current pregnancy in a local health center in northern Uganda but also factors that might predispose them to hookworm infection. Method: This cross-sectional study was conducted among 346 pregnant women from Ogur Health Center IV located in Lira district, northern Uganda. Stool samples were collected from each study participant and analyzed for hookworms. The independent variables listed in this study (participant’s sociodemographic characteristics, preconception care, and sanitation factors) were obtained using a structured questionnaire. Data analysis, including calculation of adjusted ratios, was performed using STATA software (version 14). Results: Prevalence of hookworm infection among pregnant women who attended their first antenatal visit at Ogur Health Center IV was 11% (n = 38). After controlling for confounders, factors found to be significantly associated with this infection among pregnant women here were gardening barefooted (adjusted odds ratio (AOR), 3.4; 95% confidence interval (CI), 1.6 to 7.5; P < 0:001) and fetching unsafe water shared with animals for domestic uses (AOR, 2.8; 95% CI, 1.3 to 6.2; P value of 0.002). Conclusion: Hookworm infection among pregnant women at Ogur Health Center IV in Lira district, at 11%, is a public health concern and significantly associated with barefoot gardening and fetching water from unsafe sources shared with animals. We, therefore, recommend that special emphasis during routine prenatal health education be placed on the use of protective footwear during farming and fetching water for domestic use from protected safe sources. Author Summary: Hookworm infection is a parasitic condition that more often goes unnoticed, yet it presents immense detrimental effects, especially to pregnant women and their unborn children. It is a chronic disease with accruing effects of blood depletion resulting in anemia. Anemia is, by far, one of the major causes of maternal morbidity and mortality in Uganda. Pregnant women are more prone to hookworm infection by virtue of their compromised immunity, secondary to the physiological process of pregnancy. We demonstrated here that hookworm infection still exists among pregnant women in Uganda. We also showed that gardening barefooted and fetching water for domestic uses from unsafe sources shared with animals were major factors associated with this helminthic infection. This study provides evidence necessary to influence decision making on prevention of hookworm infection in the study area