Variant Surface Glycoprotein Genes Research Articles

DNA damage drives antigen diversification through mosaic Variant Surface Glycoprotein (VSG) formation in Trypanosoma brucei.

Antigenic variation, using large genomic repertoires of antigen-encoding genes, allows pathogens to evade host antibody. Many pathogens, including the African trypanosome Trypanosoma brucei, extend their antigenic repertoire through genomic diversification. While evidence suggests that T. brucei depends on the generation of new variant surface glycoprotein (VSG) genes to maintain a chronic infection, a lack of experimentally tractable tools for studying this process has obscured its underlying mechanisms. Here, we present a highly sensitive targeted sequencing approach for measuring VSG diversification. Using this method, we demonstrate that a Cas9-induced DNA double-strand break within the VSG coding sequence can induce VSG recombination with patterns identical to those observed during infection. These newly generated VSGs are antigenically distinct from parental clones and thus capable of facilitating immune evasion. Together, these results provide insight into the mechanisms of VSG diversification and an experimental framework for studying the evolution of antigen repertoires in pathogenic microbes.

Parasitological and molecular investigation of Trypanosoma evansi in dromedaries from Greater Cairo, Egypt.

In Egypt, camel trypanosomiasis is widespread. From October 2021 to March 2022, we collected 181 blood samples from apparently healthy one-humped camels (Camelus dromedarius) in Cairo and Giza Governates. The objective of this study was to assess infection rates of trypanosomes using blood smear examination and PCR-sequencing assays. Trypanosomes were detected in 8.3% (15/181) of camels by blood smear and in 23.8% (43/181) by PCR targeting the internal transcribed spacer (ITS). Based on blood smear and ITS-PCR results, and the absence of tsetse flies in the study area, we hypothesized that the Trypanosoma species was likely T. evansi. Validation using PCR based on the variant surface glycoprotein (VSG) of T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 (RoTat 1.2 VSG gene) on ITS-PCR-positive samples (n=43) confirmed that 88.4% (38/43) were RoTat 1.2 T. evansi, while 11.6% (5/43) were non-RoTat 1.2 T. evansi. This marks the second report of non-RoTat 1.2 T. evansi in dromedary camels in Egypt. Considering the underestimated zoonotic risk of T. evansi in Egypt, there is a potential threat to humans, underscoring the need for a "One Health" approach to safeguard animal and human health.

PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes

African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes.

African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation in Trypanosoma brucei.

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis, and centromere function. Increasing evidence suggests that RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: They can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialized telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide colocalization with DNA breaks and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG and that mutation of RAD51 alters RNA-DNA hybrid abundance at 70 bp repeats both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.

A structural classification of the variant surface glycoproteins of the African trypanosome.

Long-term immune evasion by the African trypanosome is achieved through repetitive cycles of surface protein replacement with antigenically distinct versions of the dense Variant Surface Glycoprotein (VSG) coat. Thousands of VSG genes and pseudo-genes exist in the parasite genome that, together with genetic recombination mechanisms, allow for essentially unlimited immune escape from the adaptive immune system of the host. The diversity space of the "VSGnome" at the protein level was thought to be limited to a few related folds whose structures were determined more than 30 years ago. However, recent progress has shown that the VSGs possess significantly more architectural variation than had been appreciated. Here we combine experimental X-ray crystallography (presenting structures of N-terminal domains of coat proteins VSG11, VSG21, VSG545, VSG558, and VSG615) with deep-learning prediction using Alphafold to produce models of hundreds of VSG proteins. We classify the VSGnome into groups based on protein architecture and oligomerization state, contextualize recent bioinformatics clustering schemes, and extensively map VSG-diversity space. We demonstrate that in addition to the structural variability and post-translational modifications observed thus far, VSGs are also characterized by variations in oligomerization state and possess inherent flexibility and alternative conformations, lending additional variability to what is exposed to the immune system. Finally, these additional experimental structures and the hundreds of Alphafold predictions confirm that the molecular surfaces of the VSGs remain distinct from variant to variant, supporting the hypothesis that protein surface diversity is central to the process of antigenic variation used by this organism during infection.

Competition among variants is predictable and contributes to the antigenic variation dynamics of African trypanosomes.

Several persistent pathogens employ antigenic variation to continually evade mammalian host adaptive immune responses. African trypanosomes use variant surface glycoproteins (VSGs) for this purpose, transcribing one telomeric VSG expression-site at a time, and exploiting a reservoir of (sub)telomeric VSG templates to switch the active VSG. It has been known for over fifty years that new VSGs emerge in a predictable order in Trypanosoma brucei, and differential activation frequencies are now known to contribute to the hierarchy. Switching of approximately 0.01% of dividing cells to many new VSGs, in the absence of post-switching competition, suggests that VSGs are deployed in a highly profligate manner, however. Here, we report that switched trypanosomes do indeed compete, in a highly predictable manner that is dependent upon the activated VSG. We induced VSG gene recombination and switching in in vitro culture using CRISPR-Cas9 nuclease to target the active VSG. VSG dynamics, that were independent of host immune selection, were subsequently assessed using RNA-seq. Although trypanosomes activated VSGs from repressed expression-sites at relatively higher frequencies, the population of cells that activated minichromosomal VSGs subsequently displayed a competitive advantage and came to dominate. Furthermore, the advantage appeared to be more pronounced for longer VSGs. Differential growth of switched clones was also associated with wider differences, affecting transcripts involved in nucleolar function, translation, and energy metabolism. We conclude that antigenic variants compete, and that the population of cells that activates minichromosome derived VSGs displays a competitive advantage. Thus, competition among variants impacts antigenic variation dynamics in African trypanosomes and likely prolongs immune evasion with a limited set of antigens.

UMSBP2 is chromatin remodeler that functions in regulation of gene expression and suppression of antigenic variation in trypanosomes.

Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of kinetoplastids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and to play an essential role in chromosome end protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1. DNA decondensation is mediated via protein-protein interactions between TbUMSBP2 and these histones, independently of its previously described DNA binding activity. Silencing of the TbUMSBP2 gene resulted in a significant decrease in the disassembly of nucleosomes in T. brucei chromatin, a phenotype that could be reverted, by supplementing the knockdown cells with TbUMSBP2. Transcriptome analysis revealed that silencing of TbUMSBP2 affects the expression of multiple genes in T. brucei, with a most significant effect on the upregulation of the subtelomeric variant surface glycoproteins (VSG) genes, which mediate the antigenic variation in African trypanosomes. These observations suggest that UMSBP2 is a chromatin remodeling protein that functions in the regulation of gene expression and plays a role in the control of antigenic variation in T. brucei.

Chemical Inhibition of Bromodomain Proteins in Insect-Stage African Trypanosomes Perturbs Silencing of the Variant Surface Glycoprotein Repertoire and Results in Widespread Changes in the Transcriptome.

The eukaryotic protozoan parasite Trypanosoma brucei is transmitted by the tsetse fly to both humans and animals, where it causes a fatal disease called African trypanosomiasis. While the parasite lacks canonical DNA sequence-specific transcription factors, it does possess histones, histone modifications, and proteins that write, erase, and read histone marks. Chemical inhibition of chromatin-interacting bromodomain proteins has previously been shown to perturb bloodstream specific trypanosome processes, including silencing of the variant surface glycoprotein (VSG) genes and immune evasion. Transcriptomic changes that occur in bromodomain-inhibited bloodstream parasites mirror many of the changes that occur as parasites developmentally progress from the bloodstream to the insect stage. We performed transcriptome sequencing (RNA-seq) time courses to determine the effects of chemical bromodomain inhibition in insect-stage parasites using the compound I-BET151. We found that treatment with I-BET151 causes large changes in the transcriptome of insect-stage parasites and also perturbs silencing of VSG genes. The transcriptomes of bromodomain-inhibited parasites share some features with early metacyclic-stage parasites in the fly salivary gland, implicating bromodomain proteins as important for regulating transcript levels for developmentally relevant genes. However, the downregulation of surface procyclin protein that typically accompanies developmental progression is absent in bromodomain-inhibited insect-stage parasites. We conclude that chemical modulation of bromodomain proteins causes widespread transcriptomic changes in multiple trypanosome life cycle stages. Understanding the gene-regulatory processes that facilitate transcriptome remodeling in this highly diverged eukaryote may shed light on how these mechanisms evolved. IMPORTANCE The disease African trypanosomiasis imposes a severe human and economic burden for communities in sub-Saharan Africa. The parasite that causes the disease is transmitted to the bloodstream of a human or ungulate via the tsetse fly. Because the environments of the fly and the bloodstream differ, the parasite modulates the expression of its genes to accommodate two different lifestyles in these disparate niches. Perturbation of bromodomain proteins that interact with histone proteins around which DNA is wrapped (chromatin) causes profound changes in gene expression in bloodstream-stage parasites. This paper reports that gene expression is also affected by chemical bromodomain inhibition in insect-stage parasites but that the genes affected differ depending on life cycle stage. Because trypanosomes diverged early from model eukaryotes, an understanding of how trypanosomes regulate gene expression may lend insight into how gene-regulatory mechanisms evolved. This could also be leveraged to generate new therapeutic strategies.

Structural similarities between the metacyclic and bloodstream form variant surface glycoproteins of the African trypanosome.

During infection of mammalian hosts, African trypanosomes thwart immunity using antigenic variation of the dense Variant Surface Glycoprotein (VSG) coat, accessing a large repertoire of several thousand genes and pseudogenes, and switching to antigenically distinct copies. The parasite is transferred to mammalian hosts by the tsetse fly. In the salivary glands of the fly, the pathogen adopts the metacyclic form and expresses a limited repertoire of VSG genes specific to that developmental stage. It has remained unknown whether the metacyclic VSGs possess distinct properties associated with this particular and discrete phase of the parasite life cycle. We present here three novel metacyclic form VSG N-terminal domain crystal structures (mVSG397, mVSG531, and mVSG1954) and show that they mirror closely in architecture, oligomerization, and surface diversity the known classes of bloodstream form VSGs. These data suggest that the mVSGs are unlikely to be a specialized subclass of VSG proteins, and thus could be poor candidates as the major components of prophylactic vaccines against trypanosomiasis.

DOCUMENTATION OF TRYPANOSOMA EVANSI IN CAPTIVE TIGERS AND LIONS IN PUNJAB (2016-2018), PAKISTAN.

Trypanosoma evansi is an important hemoparasite of a variety of animal species worldwide. This parasite is a threat to the health of domestic animals as well as wild animals, particularly those managed in captivity. The current study investigated the presence of T. evansi in captive tigers (Panthera tigris tigris) and lions (Panthera leo) in Pakistan. In total, 24 blood samples from 11 tigers and 3 lions (n = 14) were collected during the course of roughly 3 yr (2016-2018). Eighteen samples were subjected to both microscopic and molecular evaluation for the presence of T. evansi; the remaining 6 samples were processed for PCR only. Of the 18 samples tested by both methods, 3 (16%) and 8 (44%) were positive by microscopy and PCR, respectively. This highlights the higher sensitivity of PCR over microscopy for detection of trypanosomes. Of the 24 total samples evaluated by PCR, 12 (50%) were positive. The three sequences obtained showed 99% identity with variant surface glycoprotein genes of the different isolates of T. evansi. The sensitivity, specificity, positive predictive value, and negative predictive value of microscopy in identifying T. evansi was 37.5, 100, 100, and 66.7%, respectively, considering PCR as the gold standard. We recommend rigorous monitoring of captive tigers and lions for hemoparasites, particularly in winter and early spring in areas with high infection rate of this parasite, preferably via PCR.

Identifying Antigenic Switching by Clonal Cell Barcoding and Nanopore Sequencing in Trypanosoma brucei.

Many organisms alternate the expression of genes from large gene sets or gene families to adapt to environmental cues or immune pressure. The single-celled protozoan pathogen Trypanosoma brucei spp. periodically changes its homogeneous surface coat of variant surface glycoproteins (VSGs) to evade host antibodies during infection. This pathogen expresses one out of ~2,500 VSG genes at a time from telomeric expression sites (ESs) and periodically changes their expression by transcriptional switching or recombination. Attempts to track VSG switching have previously relied on genetic modifications of ES sequences with drug-selectable markers or genes encoding fluorescent proteins. However, genetic modifications of the ESs can interfere with the binding of proteins that control VSG transcription and/or recombination, thus affecting VSG expression and switching. Other approaches include Illumina sequencing of the VSG repertoire, which shows VSGs expressed in the population rather than cell switching; the Illumina short reads often limit the distinction of the large set of VSG genes. Here, we describe a methodology to study antigenic switching without modifications of the ES sequences. Our protocol enables the detection of VSG switching at nucleotide resolution using multiplexed clonal cell barcoding to track cells and nanopore sequencing to identify cell-specific VSG expression. We also developed a computational pipeline that takes DNA sequences and outputs VSGs expressed by cell clones. This protocol can be adapted to study clonal cell expression of large gene families in prokaryotes or eukaryotes. Key features • This protocol enables the analysis of variant surface glycoproteins (VSG) switching in T. brucei without modifying the expression site sequences. • It uses a streamlined computational pipeline that takes fastq DNA sequences and outputs expressed VSG genes by each parasite clone. • The protocol leverages the long reads sequencing capacity of the Oxford nanopore sequencing technology, which enables accurate identification of the expressed VSGs. • The protocol requires approximately eight to nine days to complete.

Stage-specific transcription activator ESB1 regulates monoallelic antigen expression in Trypanosoma brucei

Variant surface glycoprotein (VSG) coats bloodstream form Trypanosoma brucei parasites, and monoallelic VSG expression underpins the antigenic variation necessary for pathogenicity. One of thousands of VSG genes is transcribed by RNA polymerase I in a singular nuclear structure called the expression site body (ESB), but how monoallelic VSG transcription is achieved remains unclear. Using a localization screen of 153 proteins we found one, ESB-specific protein 1 (ESB1), that localized only to the ESB and is expressed only in VSG-expressing life cycle stages. ESB1 associates with DNA near the active VSG promoter and is necessary for VSG expression, with overexpression activating inactive VSG promoters. Mechanistically, ESB1 is necessary for recruitment of a subset of ESB components, including RNA polymerase I, revealing that the ESB has separately assembled subdomains. Because many trypanosomatid parasites have divergent ESB1 orthologues yet do not undergo antigenic variation, ESB1 probably represents an important class of transcription regulators.

Transcriptomic profiling of Trypanosoma congolense mouthpart parasites from naturally infected flies

BackgroundAnimal African trypanosomiasis, or nagana, is a veterinary disease caused by African trypanosomes transmitted by tsetse flies. In Africa, Trypanosoma congolense is one of the most pathogenic and prevalent causes of nagana in livestock, resulting in high animal morbidity and mortality and extensive production losses. In the tsetse fly, parasites colonise the midgut and eventually reach the mouthparts, from where they can be transmitted as the fly feeds on vertebrate hosts such as cattle. Despite the extreme importance of mouthpart-form parasites for disease transmission, very few global expression profile studies have been conducted in these parasite forms.MethodsHere, we collected tsetse flies from the Shimba Hills National Reserve, a wildlife area in southeast Kenya, diagnosed T. congolense infections, and sequenced the transcriptomes of the T. congolense parasites colonising the mouthparts of the flies.ResultsWe found little correlation between mouthpart parasites from natural and experimental fly infections. Furthermore, we performed differential gene expression analysis between mouthpart and bloodstream parasite forms and identified several surface-expressed genes and 152 novel hypothetical proteins differentially expressed in mouthpart parasites. Finally, we profiled variant antigen expression and observed that a variant surface glycoprotein (VSG) transcript belonging to T. congolense phylotype 8 (i.e. TcIL3000.A.H_000381200), previously observed to be enriched in metacyclic transcriptomes, was present in all wild-caught mouthpart samples as well as bloodstream-form parasites, suggestive of constitutive expression.ConclusionOur study provides transcriptomes of trypanosome parasites from naturally infected tsetse flies and suggests that a phylotype 8 VSG gene is constitutively expressed in metacyclic- and bloodstream-form parasites at the population level.Graphical

VEX1 Influences mVSG Expression During the Transition to Mammalian Infectivity in Trypanosoma brucei.

The Trypanosoma (T) brucei life cycle alternates between the tsetse fly vector and the mammalian host. In the insect, T. brucei undergoes several developmental stages until it reaches the salivary gland and differentiates into the metacyclic form, which is capable of infecting the next mammalian host. Mammalian infectivity is dependent on expression of the metacyclic variant surface glycoprotein genes as the cells develop into mature metacyclics. The VEX complex is essential for monoallelic variant surface glycoprotein expression in T. brucei bloodstream form, however, initiation of expression of the surface proteins genes during metacyclic differentiation is poorly understood. To better understand the transition to mature metacyclics and the control of metacyclic variant surface glycoprotein expression we examined the role of VEX1 in this process. We show that modulating VEX1 expression leads to a dysregulation of variant surface glycoprotein expression during metacyclogenesis, and that following both in vivo and in vitro metacyclic differentiation VEX1 relocalises from multiple nuclear foci in procyclic cells to one to two distinct nuclear foci in metacyclic cells - a pattern like the one seen in mammalian infective bloodstream forms. Our data suggest a role for VEX1 in the metacyclic differentiation process and their capacity to become infectious to the mammalian host.

N6-methyladenosine in poly(A) tails stabilize VSG transcripts.

RNA modifications are important regulators of gene expression1. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally2. N6-methyladenosine (m6A) has been detected in this parasite, but its function remains unknown3. Here we found that m6A is enriched in 342 transcripts using RNA immunoprecipitation, with an enrichment in transcripts encoding variant surface glycoproteins (VSGs). Approximately 50% of the m6A is located in the poly(A) tail of the actively expressed VSG transcripts. m6A residues are removed from the VSG poly(A) tail before deadenylation and mRNA degradation. Computational analysis revealed an association between m6A in the poly(A) tail and a 16-mer motif in the 3' untranslated region of VSG genes. Using genetic tools, we show that the 16-mer motif acts as a cis-acting motif that is required for inclusion of m6A in the poly(A) tail. Removal of this motif from the 3' untranslated region of VSG genes results in poly(A) tails lacking m6A, rapid deadenylation and mRNA degradation. To our knowledge, this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a post-transcriptional mechanism of gene regulation.

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages.

Early diverging lineages such as trypanosomes can provide clues to the evolution of sexual reproduction in eukaryotes. In Trypanosoma brucei, the pathogen that causes Human African Trypanosomiasis, sexual reproduction occurs in the salivary glands of the insect host, but analysis of the molecular signatures that define these sexual forms is complicated because they mingle with more numerous, mitotically-dividing developmental stages. We used single-cell RNA-sequencing (scRNAseq) to profile 388 individual trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies allowing us to identify tissue-specific cell types. Further investigation of salivary gland parasite transcriptomes revealed fine-scale changes in gene expression over a developmental progression from putative sexual forms through metacyclics expressing variant surface glycoprotein genes. The cluster of cells potentially containing sexual forms was characterized by high level transcription of the gamete fusion protein HAP2, together with an array of surface proteins and several genes of unknown function. We linked these expression patterns to distinct morphological forms using immunofluorescence assays and reporter gene expression to demonstrate that the kinetoplastid-conserved gene Tb927.10.12080 is exclusively expressed at high levels by meiotic intermediates and gametes. Further experiments are required to establish whether this protein, currently of unknown function, plays a role in gamete formation and/or fusion.

Silent Trypanosoma evansi infection in humans from India revealed by serological and molecular surveys, and characterized by variable surface glycoprotein gene sequences

BackgroundThe importance of emerging atypical human trypanosomosis is gaining momentum due to increasing detection and its possible impact on human health. A cross sectional study of atypical human trypanosomosis due to Trypanosoma evansi was carried out in Kolkata and Canning area of West Bengal state of India where previously a death was reported. MethodsIn this study blood and serum samples from 173 individuals were collected during August to December 2014. To check the presence of antibodies against T. evansi, card agglutination test and for the presence of T. evansi specific DNA, PCR were conducted. ResultsT. evansi infection was identified in 5.2% (9/173) human blood samples by CATT serological test (Card agglutination test for trypanosomosis). PCR targeting VSG gene sequences suggested active T. evansi infection in 2.89% (5/173). VSG gene sequences herein determined for five isolates from human cases shared high similarity (89.4–100%). Phylogenetic inference clustered the human isolates with other isolates from different host species from India and other countries, forming a clade exclusive of Indian isolates (84.0 to 100% sequence similarity). ConclusionFirst report of symptomless human T. evansi infection detected by combined serological and PCR assays. First phylogenetic analysis of VSG gene sequences including human isolates of T. evansi in which Indian isolates of T. evansi from human and other hosts clustered in a single clade.

Signatures of hybridization in Trypanosoma brucei.

Genetic exchange among disease-causing micro-organisms can generate progeny that combine different pathogenic traits. Though sexual reproduction has been described in trypanosomes, its impact on the epidemiology of Human African Trypanosomiasis (HAT) remains controversial. However, human infective and non-human infective strains of Trypanosoma brucei circulate in the same transmission cycles in HAT endemic areas in subsaharan Africa, providing the opportunity for mating during the developmental cycle in the tsetse fly vector. Here we investigated inheritance among progeny from a laboratory cross of T. brucei and then applied these insights to genomic analysis of field-collected isolates to identify signatures of past genetic exchange. Genomes of two parental and four hybrid progeny clones with a range of DNA contents were assembled and analysed by k-mer and single nucleotide polymorphism (SNP) frequencies to determine heterozygosity and chromosomal inheritance. Variant surface glycoprotein (VSG) genes and kinetoplast (mitochondrial) DNA maxi- and minicircles were extracted from each genome to examine how each of these components was inherited in the hybrid progeny. The same bioinformatic approaches were applied to an additional 37 genomes representing the diversity of T. brucei in subsaharan Africa and T. evansi. SNP analysis provided evidence of crossover events affecting all 11 pairs of megabase chromosomes and demonstrated that polyploid hybrids were formed post-meiotically and not by fusion of the parental diploid cells. VSGs and kinetoplast DNA minicircles were inherited biparentally, with approximately equal numbers from each parent, whereas maxicircles were inherited uniparentally. Extrapolation of these findings to field isolates allowed us to distinguish clonal descent from hybridization by comparing maxicircle genotype to VSG and minicircle repertoires. Discordance between maxicircle genotype and VSG and minicircle repertoires indicated inter-lineage hybridization. Significantly, some of the hybridization events we identified involved human infective and non-human infective trypanosomes circulating in the same geographic areas.

First molecular survey of animal trypanosomes in Paraguayan horses.

Despite the epidemic situation of animal trypanosomosis caused by Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma vivax in South American countries, there are no reports for the prevalence of animal trypanosomes in Paraguay. In this study, 408 blood samples were obtained from apparently healthy horses from sixteen departments of Paraguay, for routine medical check-up from August to September 2019, and a polymerase chain reaction (PCR)-based cross-sectional study was carried out to identify trypanosome prevalence. The prevalence of Trypanozoon (T. evansi and T. equiperdum) and T. vivax was 7.11% (29/408) and 26.23% (107/408), respectively. Mixed infections were detected in 4.90% (20/408) of the samples. Some of the selected trypanosome positive samples were confirmed as T. vivax and T. evansi Type A by sequence analysis of the internal transcribe spacer region and RoTat1.2 variant surface glycoprotein gene, respectively. In conclusion, we found higher prevalence of T. vivax than Trypanozoon in Paraguayan horses. However, the genotypic variation should be verified in further studies.