Studies on trypanosomatid flagellates with special reference to antigenic variation and kinetoplast DNA

The results presented in this thesis concern two aspects of the biology of trypanosomatids. First, the process of antigenic variation in the African trypanosomes and second the structure and function of the kinetoplast DNA of trypanosomatids. Part I. Trypanosomes were cyclically transmitted by the insect vector, Glossina morsitans, and the expression of variable antigen types (VATs) in the metacyclic populations from the salivary glands and the first bloodstream populations in metacyclic initiated infections in mice were analysed. Tsetse flies were fed on the blood of mice containing any one of 5 VATs of Trypanosoma brucei of the ANTAR 1 serodeme. The VATS of the metacyclic trypanosomes subsequently detected in the flies' saliva probes vere investigated using monospecific antisera to AnTAK 1 VaTs in indirect immunofluorescence and trypanolysis reactions; these sera included 5 raised against AnTats 1.6, and 1.45, previously idnetified as components of the metacyclic population (M-VATs), and against the 5 VATs originally ingested by the flies. The percentage of metacyclics reacting with a particular M-VAT antiserum remained more or less constant (AnTat 1.6, 6.0-8.3% AnTat 1.30, 13.7-18.2%; AnTat 1.45, 2.0-8.0%), regardless of the age of the fly or the ingested VaT. AS these 3 VATS account for no more than one-third of the metacyclic population, the existence of at least one more VAT is envisaged. The ingested VAT could not be detected among the AnTAR 1 metacyclic trypanosomes. Metacyclic trypanosomes from the salivary glands of infected tsetse flies were also used to initiate infections in mice. Immunofluorescence and trypanolysis reactions employing 24 monospecific antisera were used to analyse the VATS present in the mice following cyclical transmission. Regardless of the VAT used to infect tsetse flies, the first VATS detectable in the bloodstream were those previously identified as M-VATS. These were present until at least 5 days after infection, at which time lytic antibodies against at least 2 of the M-VATs were detectable in the blood of infected mice. In mice immunosuppressed by X-irradiation the M-VATs were detectable in the bloodstream for longer periods, but the percentage of the population labelled with anti-metacyclic sera showed a decrease on day 5 as in non-irradiated animals. The VAT ingested by the tsetse was always detectable early during the first parasitaemia following cyclical transmission and was usually the first VAT detectable after the M-VATs. Neutralization of selected M-VATs before infecting mice resulted in elimination of the neutralized M-VAT from the first parasitaemia but had no effect on the expression of other VATs in the early infection. Part II. In studies on the structure and function of the kinetoplast DNA (KDNA) of trypanosomatids I have examined the KDNA structure and mitochondrial activity of two species of Herpetomonas and also a stock of T. brucei which has lost the ability to activate its mitochondrion during syringe passaging in laboratory rodents.The structure of the KDNA of Herpetomonas muscarum and Herpetomonas inrenoplastis was compared by electron microscopy, restriction endonuclease digestion and hydridization with cloned portions of the maxi-circle from T. brucei 427. The KDNA of both H. musearum and H.ingenoplasits has a buoyant density of 1 .69B g/cm3; however, the KDNA of H. ingenoplastis represents 31% of the total cellular DNA as compared with 8% for H. muscarum KDNA. The KDNA network of H. muscarum consists of thousands of mini-circles of 0.6 to 0.7 x 106 daltons and a few large circular molecules, maxi-circles, of 21 x 10 daltons. The mini-circles of H. muscarum show sequence heterogeneity while maxi-circles of H. muscarum have a unique nucleotide sequence. The KDNA of H. ingenoplastis completely lacks mini-circle size molecules and the network is composed 6 6 6 entirely of large circular molecules of 11 x 106, 15.5 x 106 and 24 x 106 daltons. The 11 x 106 and 15.5 x 106 dalton molecules show sequence heterogeneity and are the major component of the KDNA. Hybridization studies with cloned fragments of T. brucei maxi-circle suggest that the 24 X 106 dalton component of H. ingenoplastis kDNA is functionally equivalent to the maxi-circle of other trypanosomatids. It was concluded that the 11 x 106 and 15.5 x 106 dalton circles of H. in enoplastis are functionally similar to mini-circles of other tryoanosomatids and that the maxi-circles of H. ingenoplastis differ from those of T. brucei and H. muscarum in major nucleotide sequences. The structure and activity of the mitochondrion from H.ingenoplastis and H. muscarum have been studied by electron microscopy, respiration studies with different substrates and inhibitors, analysis of oligomycin- sensitive ATPase activity and low-temperature difference spectra of respiratory chain cytochromes. Certain differences in the two species can be correlated with alterations in the maxi-circle of H. ingenoplastis described in the preceding paper

Novel African trypanocidal agents: membrane rigidifying peptides

The bloodstream developmental forms of pathogenic African trypanosomes are uniquely susceptible to killing by small hydrophobic peptides. Trypanocidal activity is conferred by peptide hydrophobicity and charge distribution and results from increased rigidity of the plasma membrane. Structural analysis of lipid-associated peptide suggests a mechanism of phospholipid clamping in which an internal hydrophobic bulge anchors the peptide in the membrane and positively charged moieties at the termini coordinate phosphates of the polar lipid headgroups. This mechanism reveals a necessary phenotype in bloodstream form African trypanosomes, high membrane fluidity, and we suggest that targeting the plasma membrane lipid bilayer as a whole may be a novel strategy for the development of new pharmaceutical agents. Additionally, the peptides we have described may be valuable tools for probing the biosynthetic machinery responsible for the unique composition and characteristics of African trypanosome plasma membranes

Hemoglobin Is a Co-Factor of Human Trypanosome Lytic Factor

Trypanosome lytic factor (TLF) is a high-density lipoprotein (HDL) subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr) and apolipoprotein L-1 (ApoL-1), have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr–Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites

Nucleotide Sequences within the U5 Region of the Viral RNA Genome Are the Major Determinants for an Human Immunodeficiency Virus Type 1 to Maintain a Primer Binding Site Complementary to tRNAHis

AbstractThe initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires cellular tRNALys,3as a primer and occurs at a site in the viral RNA genome, designated as the primer binding site (PBS), which is complementary to the 3′-terminal 18 nucleotides of tRNALys,3. We previously described an HIV-1 virus [designated as HXB2(His-AC)], which contained a sequence within the U5 region complementary to the anticodon region of tRNAHisin addition to a PBS complementary to the 3′-terminal 18 nucleotides of the tRNAHis. That virus maintained a PBS complementary to tRNAHisafter extendedin vitroculture (Wakefieldet al., J. Virol.70, 966–975, 1996). In the present study, we report that subcloning a 200-base-pair DNA fragment encompassing the U5 and PBS regions from an integrated provirus of HXB2(His-AC) back into the wild-type genome (pHXB2) resulted in an infectious virus, designated as HXB2(His-AC-gac), which again stably maintained a PBS complementary to tRNAHis. DNA sequence analysis of the 200-base-pair region revealed only three nucleotide changes from HXB2(His-AC): a T-to-G change at nucleotide 174, a G-to-A change at nucleotide 181, and a T-to-C change at nucleotide 200. The new mutant virus replicated in CD4+Sup T1 cells similarly to the wild-type virus. Comparison of the nucleotide sequence of nucleocapsid gene of the wild-type and HXB2 (His-AC-gac) virus revealed no differences. Although we found numerous mutations in the reverse transcriptase gene in proviral clones derived from HXB2 (His-AC-gac), no common mutations were found among the 13 clones examined. Comparison of the virion-associated tRNAs of HXB2(His-AC-gac) with those of the wild type revealed that both viruses incorporated a similar subset of cellular tRNAs, with tRNALys,3being the predominant tRNA found within virions. There was no selective enrichment for tRNAHiswithin virions of HXB2(His-AC-gac) virus which selectively use tRNAHisto initiate reverse transcription. The results of these studies suggest that the U5 and PBS regions in the viral RNA genome are important determinants for HXB2(His-AC) viruses in the selective use of tRNAHisto initiate reverse transcription

Light and electron microscopical observations of the effects of high-density lipoprotein on growth of Plasmodium falciparum in vitro

Author Posting. © Cambridge University Press, 2004. This article is posted here by permission of Cambridge University Press for personal use, not for redistribution. The definitive version was published in Parasitology 128 (2004): 577-584, doi:10.1017/S0031182004005025.Human serum high-density lipoprotein (HDL) is necessary and sufficient for the short-term maintenance of Plasmodium falciparum in in vitro culture. However, at high concentrations it is toxic to the parasite. A heat-labile component is apparently responsible for the stage-specific toxicity to parasites within infected erythrocytes 12–42 h after invasion, i.e. during trophozoite maturation. The effects of HDL on parasite metabolism (as determined by nucleic acid synthesis) are evident at about 30 h after invasion. Parasites treated with HDL show gross abnormalities by light and electron microscopy.Professor Hajduk was supported by NIH. Professor Day was supported by a Research Leave Fellowship from The Wellcome Trust. Dr Imrie and Ms Carter were supported by Programme Grant funding awarded to Professor Day from The Wellcome Trust. Dr Ferguson was supported by an equipment grant from The Wellcome Trust

In vivo analysis of trypanosome mitochondrial RNA function by artificial site-specific RNA endonuclease-mediated knockdown

Trypanosomes possess a unique mitochondrial genome called the kinetoplast DNA (kDNA). Many kDNA genes encode pre-mRNAs that must undergo guide RNA-directed editing. In addition, alternative mRNA editing gives rise to diverse mRNAs and several kDNA genes encode open reading frames of unknown function. To better understand the mechanism of RNA editing and the function of mitochondrial RNAs in trypanosomes, we have developed a reverse genetic approach using artificial site-specific RNA endonucleases (ASREs) to directly silence kDNA-encoded genes. The RNA-binding domain of an ASRE can be programmed to recognize unique 8-nucleotide sequences, allowing the design of ASREs to cleave any target RNA. Utilizing an ASRE containing a mitochondrial localization signal, we targeted the extensively edited mitochondrial mRNA for the subunit A6 of the F0F1 ATP synthase (A6) in the procyclic stage of Trypanosoma brucei. This developmental stage, found in the midgut of the insect vector, relies on mitochondrial oxidative phosphorylation for ATP production with A6 forming the critical proton half channel across the inner mitochondrial membrane. Expression of an A6-targeted ASRE in procyclic trypanosomes resulted in a 50% reduction in A6 mRNA levels after 24 h, a time-dependent decrease in mitochondrial membrane potential (ΔΨm), and growth arrest. Expression of the A6-ASRE, lacking the mitochondrial localization signal, showed no significant growth defect. The development of the A6-ASRE allowed the first in vivo functional analysis of an edited mitochondrial mRNA in T. brucei and provides a critical new tool to study mitochondrial RNA biology in trypanosomes

Oxidative phosphorylation is required for powering motility and development of the sleeping sickness parasite Trypanosoma brucei in the tsetse fly vector

The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS). This process involves respiratory chain complexes and F1Fo-ATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly. IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought

Turnover of variant surface glycoprotein in Trypanosoma brucei is a bimodal process

This work was supported by United States Public Health Service grant R01 AI035739 and funds from the Jacobs School of Medicine and Biomedical Sciences to J.D.B. and from United States Public Health Service grant 1S10OD021719-01A1 to the University of Georgia, which purchased the ImageStreamX Mk II. This work was also supported by the Wellcome Trust (grant 094476/Z/10/Z) for funding the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility.African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246-257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG+ extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [t1/2] of ∼26 h) but dropped significantly in the absence of GPI-PLC (t1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis. IMPORTANCE African trypanosomes, the protozoan agent of human African trypanosomaisis, avoid the host immune system by switching expression of the variant surface glycoprotein (VSG). VSG is a long-lived protein that has long been thought to be turned over by hydrolysis of its glycolipid membrane anchor. Recent work demonstrating the shedding of VSG-containing extracellular vesicles has led us to reinvestigate the mode of VSG turnover. We found that VSG is shed in part by glycolipid hydrolysis but also in approximately equal part by direct shedding of protein with intact lipid anchors. Shedding of exocytic vesicles made a very minor contribution to overall VSG turnover. These results indicate that VSG turnover is a bimodal process and significantly alter our understanding of the "life cycle" of this critical virulence factor.Publisher PDFPeer reviewe

Individual variation in levels of haptoglobin-related protein in children from Gabon

Background: Haptoglobin related protein (Hpr) is a key component of trypanosome lytic factors (TLF), a subset of highdensity lipoproteins (HDL) that form the first line of human defence against African trypanosomes. Hpr, like haptoglobin (Hp) can bind to hemoglobin (Hb) and it is the Hpr-Hb complexes which bind to these parasites allowing uptake of TLF. This unique form of innate immunity is primate-specific. To date, there have been no population studies of plasma levels of Hpr, particularly in relation to hemolysis and a high prevalence of ahaptoglobinemia as found in malaria endemic areas. Methods and Principal Findings: We developed a specific enzyme-linked immunosorbent assay to measure levels of plasma Hpr in Gabonese children sampled during a period of seasonal malaria transmission when acute phase responses (APR), malaria infection and associated hemolysis were prevalent. Median Hpr concentration was 0.28 mg/ml (range 0.03-1.1). This was 5-fold higher than that found in Caucasian children (0.049 mg/ml, range 0.002-0.26) with no evidence of an APR. A general linear model was used to investigate associations between Hpr levels, host polymorphisms, parasitological factors and the acute phase proteins, Hp, C-reactive protein (CRP) and albumin. Levels of Hpr were associated with Hp genotype, decreased with age and were higher in females. Hpr concentration was strongly correlated with that of Hp, but not CRP

Differences between <i>Trypanosoma brucei gambiense</i> groups 1 and 2 in their resistance to killing by Trypanolytic factor 1

&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; The three sub-species of &lt;i&gt;Trypanosoma brucei&lt;/i&gt; are important pathogens of sub-Saharan Africa. &lt;i&gt;T. b. brucei&lt;/i&gt; is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. &lt;i&gt;T. b. rhodesiense&lt;/i&gt; and &lt;i&gt;T. b. gambiense&lt;/i&gt; are able to resist lysis by TLF. There are two distinct sub-groups of &lt;i&gt;T. b. gambiense&lt;/i&gt; that differ genetically and by human serum resistance phenotypes. Group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (&lt;i&gt;HpHbR&lt;/i&gt;)) gene. Here we investigate if this is also true in group 2 parasites.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methodology:&lt;/b&gt; Isogenic resistant and sensitive group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the &lt;i&gt;HpHbR&lt;/i&gt; gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to &lt;i&gt;T. b. brucei&lt;/i&gt;. Both resistant and sensitive group 2, as well as group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt;, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Our data indicate that, despite group 1 &lt;i&gt;T. b. gambiense&lt;/i&gt; avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 &lt;i&gt;T. b. gambiense&lt;/i&gt; variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of &lt;i&gt;HpHbR&lt;/i&gt;. Thus there are differences in the mechanism of human serum resistance between &lt;i&gt;T. b. gambiense&lt;/i&gt; groups 1 and 2.&lt;/p&gt