RESUMEN
Congenital transmission of Trypanosoma cruzi is an important source of new Chagas infections worldwide. The mechanisms of congenital transmission remain poorly understood, but there is evidence that parasite factors are involved. Investigating changes in parasite strain diversity during transmission could provide insight into the parasite factors that influence the process. Here we use amplicon sequencing of a single copy T. cruzi gene to evaluate the diversity of infection in clinical samples from Chagas positive mothers and their infected infants. Several infants and mothers were infected with multiple parasite strains, mostly of the same TcV lineage, and parasite strain diversity was higher in infants than mothers. Two parasite haplotypes were detected exclusively in infant samples, while one haplotype was never found in infants. Together, these data suggest multiple parasites initiate a congenital infection and that parasite factors influence the probability of vertical transmission.
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Enfermedad de Chagas , Parásitos , Trypanosoma cruzi , Femenino , Animales , Humanos , Lactante , Trypanosoma cruzi/genética , Enfermedad de Chagas/congénito , Madres , Transmisión Vertical de Enfermedad InfecciosaRESUMEN
Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). In the mammalian host, the parasite lives entirely extracellularly, in both the blood and interstitial spaces in tissues. Although most T. brucei research has focused on the biology of blood- and central nervous system (CNS)-resident parasites, a number of recent studies have highlighted parasite reservoirs in the dermis and adipose tissue, leading to a renewed interest in tissue-resident parasite populations. In light of this renewed interest, work describing tissue-resident parasites can serve as a valuable resource to inform future investigations of tissue-resident T. brucei. Here, we review this body of literature, which describes infections in humans, natural hosts, and experimental animal models, providing a wealth of information on the distribution and biology of extravascular parasites, the corresponding immune response in each tissue, and resulting host pathology. We discuss the implications of these studies and future questions in the study of extravascular T. brucei.
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Interacciones Huésped-Parásitos/fisiología , Trypanosoma brucei brucei/parasitología , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/parasitología , Animales , HumanosRESUMEN
African trypanosomes use an extreme form of antigenic variation to evade host immunity, involving the switching of expressed variant surface glycoproteins by a stochastic and parasite-intrinsic process. Parasite development in the mammalian host is another feature of the infection dynamic, with trypanosomes undergoing quorum sensing (QS)-dependent differentiation between proliferative slender forms and arrested, transmissible, stumpy forms. Longstanding experimental studies have suggested that the frequency of antigenic variation and transmissibility may be linked, antigen switching being higher in developmentally competent, fly-transmissible, parasites ("pleomorphs") than in serially passaged "monomorphic" lines that cannot transmit through flies. Here, we have directly tested this tenet of the infection dynamic by using 2 experimental systems to reduce pleomorphism. Firstly, lines were generated that inducibly lose developmental capacity through RNAi-mediated silencing of the QS signaling machinery ("inducible monomorphs"). Secondly, de novo lines were derived that have lost the capacity for stumpy formation by serial passage ("selected monomorphs") and analyzed for their antigenic variation in comparison to isogenic preselected populations. Analysis of both inducible and selected monomorphs has established that antigen switch frequency and developmental capacity are independently selected traits. This generates the potential for diverse infection dynamics in different parasite populations where the rate of antigenic switching and transmission competence are uncoupled. Further, this may support the evolution, maintenance, and spread of important trypanosome variants such as Trypanosoma brucei evansi that exploit mechanical transmission.
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Antígenos de Protozoos/inmunología , Trypanosoma brucei brucei/inmunología , Animales , Antígenos de Protozoos/genética , Interferencia de ARN , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive.
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Secuencias Repetitivas de Ácidos Nucleicos/genética , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Variación Antigénica/genética , Variación Antigénica/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Duplicación de Gen , Genómica , Secuencias Repetitivas de Ácidos Nucleicos/inmunología , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunologíaRESUMEN
Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite's surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.
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Modelos Moleculares , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Trypanosoma brucei brucei/fisiología , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Evasión Inmune , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformación Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análisis de Supervivencia , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Factores de Transcripción/genética , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología , Tripanosomiasis Africana/fisiopatología , VirulenciaRESUMEN
Trypanosoma brucei is a master of antigenic variation and immune response evasion. Utilizing a genomic repertoire of more than 1000 Variant Surface Glycoprotein-encoding genes (VSGs), T. brucei can change its protein coat by "switching" from the expression of one VSG to another. Each active VSG is monoallelically expressed from only one of approximately 15 subtelomeric sites. Switching VSG expression occurs by three predominant mechanisms, arguably the most significant of which is the non-reciprocal exchange of VSG containing DNA by duplicative gene conversion (GC). How T. brucei orchestrates its complex switching mechanisms remains to be elucidated. Recent work has demonstrated that an exogenous DNA break in the active site could initiate a GC based switch, yet the source of the switch-initiating DNA lesion under natural conditions is still unknown. Here we investigated the hypothesis that telomere length directly affects VSG switching. We demonstrate that telomerase deficient strains with short telomeres switch more frequently than genetically identical strains with long telomeres and that, when the telomere is short, switching preferentially occurs by GC. Our data supports the hypothesis that a short telomere at the active VSG expression site results in an increase in subtelomeric DNA breaks, which can initiate GC based switching. In addition to their significance for T. brucei and telomere biology, the findings presented here have implications for the many diverse pathogens that organize their antigenic genes in subtelomeric regions.
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Variación Antigénica/genética , Variación Genética , Telómero/genética , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , ADN Protozoario/genética , Conversión Génica , Duplicación de Gen , Humanos , Fenotipo , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismo , Homeostasis del Telómero/genética , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
The signals required to generate long-lived plasma cells remain unresolved. One widely cited model posits that long-lived plasma cells derive from germinal centers (GCs) in response to T cell-dependent (TD) Ags. Thus, T cell-independent (TI) Ags, which fail to sustain GCs, are considered ineffective at generating long-lived plasma cells. However, we show that long-lived hapten-specific plasma cells are readily induced without formation of GCs. Long-lived plasma cells developed in T cell-deficient mice after a single immunization with haptenated LPS, a widely used TI Ag. Long-lived plasma cells also formed in response to TD Ag when the GC response was experimentally prevented. These observations establish that long-lived plasma cells are induced in both TI and TD responses, and can arise independently of B cell maturation in GCs.
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Células de la Médula Ósea/inmunología , Diferenciación Celular/inmunología , Epítopos de Linfocito T/inmunología , Células Plasmáticas/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Linaje de la Célula/inmunología , Supervivencia Celular/inmunología , Epítopos de Linfocito T/metabolismo , Femenino , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Trypanosoma cruzi is the causative agent of Chagas disease, which causes 10,000 deaths per year. Despite the high mortality associated with Chagas, relatively few parasite genomes have been assembled to date, with genome assemblies unavailable even for some commonly used laboratory strains. This is at least partially due to T. cruzi's highly complex and highly repetitive genome, which defies investigation using traditional short-read sequencing methods. In this study, we have generated a high-quality whole-genome assembly of the hybrid Tulahuen strain, a commercially available type VI strain, using long-read Nanopore sequencing without short-read scaffolding. The assembled genome contains 25% repeat regions, 17% variable multigene family members, and 27% transposable elements (TEs) and is of comparable quality with T. cruzi genome assemblies that utilized both long- and short-read data. Notably, we find that regions with TEs are significantly enriched for multicopy surface proteins, and that surface proteins are, on average, closer to TEs than to other coding regions. This finding suggests that mobile genetic elements such as transposons may drive recombination within surface protein gene families. This work demonstrates the feasibility of Nanopore sequencing to resolve complex regions of T. cruzi genomes, and with these resolved regions, provides support for a possible mechanism for genomic diversification.
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Elementos Transponibles de ADN , Genoma de Protozoos , Secuenciación de Nanoporos , Trypanosoma cruzi , Trypanosoma cruzi/genética , Secuenciación de Nanoporos/métodos , Genómica/métodos , Secuenciación Completa del Genoma/métodos , Anotación de Secuencia MolecularRESUMEN
Introduction: Growing evidence from animal models indicates that the myocardium hosts a population of B cells that play a role in the development of cardiomyopathy. However, there is minimal data on human myocardial B cells in the context of cardiomyopathy. Methods: We integrated single-cell and single-nuclei datasets from 45 healthy human hearts, 70 hearts with dilated cardiomyopathy (DCM), and 8 hearts with Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC). Interactions between B cells and other cell types were investigated using the CellChat Package. Differential gene expression analysis comparing B cells across conditions was performed using DESeq2. Pathway analysis was performed using Ingenuity, KEGG, and GO pathways analysis. Results: We identified 1,100 B cells, including naive B cells and plasma cells. B cells showed an extensive network of interactions within the healthy myocardium that included outgoing signaling to macrophages, T cells, endothelial cells, and pericytes, and incoming signaling from endothelial cells, pericytes, and fibroblasts. This niche relied on ECM-receptor, contact, and paracrine interaction; and changed significantly in the context of cardiomyopathy, displaying disease-specific features. Differential gene expression analysis showed that in the context of DCM both naive and plasma B cells upregulated several pathways related to immune activation, including upregulation of oxidative phosphorylation, upregulation of leukocyte extravasation, and, in naive B cells, antigen presentation. Discussion: The human myocardium contains naive B cells and plasma cells, integrated into a diverse and dynamic niche that has distinctive features in healthy myocardium, DCM, and ARVC. Naive myocardial-associated B cells likely contribute to the pathogenesis of human DCM.
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Introduction: Growing evidence from animal models indicates that the myocardium hosts a population of B cells that play a role in the development of cardiomyopathy. However, there is minimal data on human myocardial B cells in the context of cardiomyopathy. Methods: We integrated single-cell and single-nuclei datasets from 45 healthy human hearts, 70 hearts with dilated cardiomyopathy (DCM), and 8 hearts with arrhythmogenic right ventricular cardiomyopathy (ARVC). Interactions between B cells and other cell types were investigated using the CellChat Package. Differential gene expression analysis comparing B cells across conditions was performed using DESeq2. Pathway analysis was performed using Ingenuity, KEGG, and GO pathways analysis. Results: We identified 1,100 B cells, including naive B cells and plasma cells. Cells showed an extensive network of interactions within the healthy myocardium that included outgoing signaling to macrophages, T cells, endothelial cells, and pericytes, and incoming signaling from endothelial cells, pericytes, and fibroblasts. This niche relied on ECM-receptor, contact, and paracrine interactions; and changed significantly in the context of cardiomyopathy, displaying disease-specific features. Differential gene expression analysis showed that in the context of DCM both naive and plasma B cells upregulated several pathways related to immune activation, including upregulation of oxidative phosphorylation, upregulation of leukocyte extravasation, and, in naive B cells, antigen presentation. Discussion: The human myocardium contains naive B cells and plasma cells, integrated into a diverse and dynamic niche that has distinctive features in healthy, DCM, and ARVC. Naive myocardial-associated B cells likely contribute to the pathogenesis of human DCM.
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Displasia Ventricular Derecha Arritmogénica , Linfocitos B , Cardiomiopatía Dilatada , Miocardio , Humanos , Cardiomiopatía Dilatada/inmunología , Cardiomiopatía Dilatada/genética , Displasia Ventricular Derecha Arritmogénica/genética , Displasia Ventricular Derecha Arritmogénica/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Miocardio/metabolismo , Miocardio/inmunología , Miocardio/patología , Masculino , Femenino , Comunicación Celular/inmunología , Perfilación de la Expresión Génica , Persona de Mediana Edad , Adulto , Transcriptoma , Regulación de la Expresión GénicaAsunto(s)
Interacciones Huésped-Patógeno/inmunología , Evasión Inmune/inmunología , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Africana/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunología , Animales , Variación Genética , Interacciones Huésped-Patógeno/genética , Evasión Inmune/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genéticaRESUMEN
Drs. Monica Mugnier and Chi-Min Ho work in the field of parasitology. In this mSphere of Influence article, they share their experience as cochairs of the Young Investigators in Parasitology (YIPs) meeting, a 2-day biennial meeting for new PIs in parasitology. Setting up a new lab can be a daunting task. YIPS is designed to make the transition a little easier. YIPs is both a crash course in the skills needed to run a successful research lab and a way to build community among new group leaders in parasitology. In this perspective, they describe YIPs and the benefit it has had on the molecular parasitology community. They also provide some tips for building and running a meeting like YIPs, in the hopes that other fields might replicate their model.
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ParasitologíaRESUMEN
Trypanosoma brucei is a protozoan parasite that causes human and animal African trypanosomiases (HAT and AAT). Cardiac symptoms are commonly reported in HAT patients, and intracardiac parasites with accompanying myocarditis have been observed in both natural hosts and animal models of T. brucei infection. However, despite the importance of T. brucei as a cause of cardiac dysfunction and the dramatic socioeconomic impact of African trypanosomiases in sub-Saharan Africa, there are currently no reproducible murine models of T. brucei-associated cardiomyopathy. We present the first clinically relevant, reproducible murine model of cardiac dysfunction in chronic T. brucei infection. Similar to humans, mice showed histological evidence of myocarditis and elevation of serum NT-proBNP. Serum NT-proBNP levels were elevated prior to the development of severe ventricular dysfunction. On flow cytometry, myocarditis was associated with an increase of most myocardial immune cell populations, including multiple T cell and macrophage subsets, corroborating the notion that T. brucei-associated cardiac damage is an immune-mediated event. This novel mouse model represents a powerful and practical tool to investigate the pathogenesis of T. brucei-mediated heart damage and support the development of therapeutic options for T. brucei-associated cardiac disease.
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Trypanosoma cruzi is the causative agent of Chagas disease, which causes 10,000 deaths per year. Despite the high mortality caused by the pathogen, relatively few parasite genomes have been assembled to date; even some commonly used laboratory strains do not have publicly available genome assemblies. This is at least partially due to T. cruzi's highly complex and highly repetitive genome: while describing the variation in genome content and structure is critical to better understanding T. cruzi biology and the mechanisms that underlie Chagas disease, the complexity of the genome defies investigation using traditional short read sequencing methods. Here, we have generated a high-quality whole genome assembly of the hybrid Tulahuen strain, a commercially available Type VI strain, using long read Nanopore sequencing without short read scaffolding. Using automated tools and manual curation for annotation, we report a genome with 25% repeat regions, 17% variable multigene family members, and 27% transposable elements. Notably, we find that regions with transposable elements are significantly enriched for surface proteins, and that on average surface proteins are closer to transposable elements compared to other coding regions. This finding supports a possible mechanism for diversification of surface proteins in which mobile genetic elements such as transposons facilitate recombination within the gene family. This work demonstrates the feasibility of nanopore sequencing to resolve complex regions of T. cruzi genomes, and with these resolved regions, provides support for a possible mechanism for genomic diversification.
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Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a neglected parasitic disease that affects approximately 6 million individuals worldwide. Of those infected, 20-30% will go on to develop chronic Chagas cardiomyopathy (CCC), and ultimately many of these individuals will progress to advanced heart failure. The mechanism by which this progression occurs is poorly understood, as few studies have focused on early CCC. In this study, we sought to understand the physiologic changes associated with T. cruzi infection and the development of CCC. We analyzed gene expression in the peripheral blood of asymptomatic Chagas patients with early structural heart disease, Chagas patients without any signs or symptoms of disease, and Chagas-negative patients with and without early structural heart disease. Our analysis shows that early CCC was associated with a downregulation of various peripheral immune response genes, with gene expression changes suggestive of reduced antigen presentation and T cell activation. Notably, these genes and processes were distinct from those of early cardiomyopathy in Chagas-negative patients, suggesting that the processes mediating CCC may be unique from those mediating progression to other cardiomyopathies. This work highlights the importance of the immune response in early CCC, providing insight into the early pathogenesis of this disease. The changes we have identified may serve as biomarkers of progression and could inform strategies for the treatment of CCC in its early stages, before significant cardiac damage has occurred.
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UNLABELLED: Infantile cholestatic disorders arise in the context of progressively developing intrahepatic bile ducts. Biliary atresia (BA), a progressive fibroinflammatory disorder of extra- and intrahepatic bile ducts, is the most common identifiable cause of infantile cholestasis and the leading indication for liver transplantation in children. The etiology of BA is unclear, and although there is some evidence for viral, toxic, and complex genetic causes, the exclusive occurrence of BA during a period of biliary growth and remodeling suggests an importance of developmental factors. Interestingly, interferon-γ (IFN-γ) signaling is activated in patients and in the frequently utilized rhesus rotavirus mouse model of BA, and is thought to play a key mechanistic role. Here we demonstrate intrahepatic biliary defects and up-regulated hepatic expression of IFN-γ pathway genes caused by genetic or pharmacological inhibition of DNA methylation in zebrafish larvae. Biliary defects elicited by inhibition of DNA methylation were reversed by treatment with glucocorticoid, suggesting that the activation of inflammatory pathways was critical. DNA methylation was significantly reduced in bile duct cells from BA patients compared to patients with other infantile cholestatic disorders, thereby establishing a possible etiologic link between decreased DNA methylation, activation of IFN-γ signaling, and biliary defects in patients. CONCLUSION: Inhibition of DNA methylation leads to biliary defects and activation of IFN-γ-responsive genes, thus sharing features with BA, which we determine to be associated with DNA hypomethylation. We propose epigenetic activation of IFN-γ signaling as a common etiologic mechanism of intrahepatic bile duct defects in BA.
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Conductos Biliares/anomalías , Conductos Biliares/crecimiento & desarrollo , Metilación de ADN/efectos de los fármacos , Adenosilhomocisteinasa/genética , Animales , Azacitidina/farmacología , Conductos Biliares/efectos de los fármacos , Atresia Biliar/etiología , Atresia Biliar/fisiopatología , Epigenómica , Humanos , Inflamación/fisiopatología , Interferón gamma/fisiología , Pez CebraRESUMEN
Trypanosoma brucei gambiense is the primary causative agent of human African trypanosomiasis (HAT), a vector-borne disease endemic to West and Central Africa. The extracellular parasite evades antibody recognition within the host bloodstream by altering its variant surface glycoprotein (VSG) coat through a process of antigenic variation. The serological tests that are widely used to screen for HAT use VSG as one of the target antigens. However, the VSGs expressed during human infection have not been characterized. Here, we use VSG sequencing (VSG-seq) to analyze the VSGs expressed in the blood of patients infected with T. b. gambiense and compared them to VSG expression in Trypanosoma brucei rhodesiense infections in humans as well as Trypanosoma brucei brucei infections in mice. The 44 VSGs expressed during T. b. gambiense infection revealed a striking bias toward expression of type B N termini (82% of detected VSGs). This bias is specific to T. b. gambiense, which is unique among T. brucei subspecies in its chronic clinical presentation and anthroponotic nature. The expressed T. b. gambiense VSGs also share very little similarity to sequences from 36 T. b. gambiense whole-genome sequencing data sets, particularly in areas of the VSG protein exposed to host antibodies, suggesting the antigen repertoire is under strong selective pressure to diversify. Overall, this work demonstrates new features of antigenic variation in T. brucei gambiense and highlights the importance of understanding VSG repertoires in nature. IMPORTANCE Human African trypanosomiasis is a neglected tropical disease primarily caused by the extracellular parasite Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their variant surface glycoprotein (VSG) coat. Despite the important role of VSGs in prolonging infection, VSG expression during human infections is poorly understood. A better understanding of natural VSG gene expression dynamics can clarify the mechanisms that T. brucei uses to alter its VSG coat. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that there are features of antigenic variation unique to human-infective T. brucei subspecies and that natural VSG repertoires may vary more than previously expected.
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Trypanosoma brucei brucei , Tripanosomiasis Africana , Humanos , Animales , Ratones , Tripanosomiasis Africana/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei gambiense/genética , Glicoproteínas de MembranaRESUMEN
Many pathogens evade host immunity by periodically changing the proteins they express on their surface - a phenomenon termed antigenic variation. An extreme form of antigenic variation, based around switching the composition of a Variant Surface Glycoprotein (VSG) coat, is exhibited by the African trypanosome Trypanosoma brucei, which causes human disease. The molecular details of VSG switching in T. brucei have been extensively studied over the last three decades, revealing in increasing detail the machinery and mechanisms by which VSG expression is controlled and altered. However, several key components of the models of T. brucei antigenic variation that have emerged have been challenged through recent discoveries. These discoveries include new appreciation of the importance of gene mosaics in generating huge levels of new VSG variants, the contributions of parasite development and body compartmentation in the host to the infection dynamics and, finally, potential differences in the strategies of antigenic variation and host infection used by the crucial livestock trypanosomes T. congolense and T. vivax. This review will discuss all these observations, which raise questions regarding how secure the existing models of trypanosome antigenic variation are. In addition, we will discuss the importance of continued mathematical modelling to understand the purpose of this widespread immune survival process.
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Parasites have long been known to influence host responses to infection through the secretion of virulence factors. Extracellular vesicles are emerging as important mediators of these manipulations, and a new study by Szempruch et al. suggests they could play a crucial role in host responses to African trypanosome infections.
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Interacciones Huésped-Parásitos/inmunología , Vesículas Secretoras/inmunología , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/parasitología , Factores de Virulencia/inmunología , Animales , Interacciones Huésped-Parásitos/genética , Humanos , Trypanosoma brucei brucei/inmunología , Factores de Virulencia/genéticaRESUMEN
Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.