RESUMEN
NEU-4438 is a lead for the development of drugs against Trypanosoma brucei, which causes human African trypanosomiasis. Optimized with phenotypic screening, targets of NEU-4438 are unknown. Herein, we present a cell perturbome workflow that compares NEU-4438's molecular modes of action to those of SCYX-7158 (acoziborole). Following a 6 h perturbation of trypanosomes, NEU-4438 and acoziborole reduced steady-state amounts of 68 and 92 unique proteins, respectively. After analysis of proteomes, hypotheses formulated for modes of action were tested: Acoziborole and NEU-4438 have different modes of action. Whereas NEU-4438 prevented DNA biosynthesis and basal body maturation, acoziborole destabilized CPSF3 and other proteins, inhibited polypeptide translation, and reduced endocytosis of haptoglobin-hemoglobin. These data point to CPSF3-independent modes of action for acoziborole. In case of polypharmacology, the cell-perturbome workflow elucidates modes of action because it is target-agnostic. Finally, the workflow can be used in any cell that is amenable to proteomic and molecular biology experiments.
RESUMEN
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 â¼26 h) but dropped significantly in the absence of GPI-PLC (t1/2 of â¼36 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.
Asunto(s)
Antígenos de Protozoos/inmunología , Estadios del Ciclo de Vida , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/fisiología , Antígenos de Protozoos/genética , Línea Celular , Endocitosis , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genéticaRESUMEN
Extracellular vesicles (EVs) are produced by invading pathogens and also by host cells in response to infection. The origin, composition, and function of EVs made during infection are diverse and provide effective vehicles for localized and broad dissimilation of effector molecules in the infected host. Extracellular pathogens use EVs to communicate with each other by sensing the host environment contributing to social motility, tissue tropism, and persistence of infection. Pathogen-derived EVs can also interact with host cells to influence the adhesive properties of host membranes and to alter immune recognition and response. Intracellular pathogens can affect both the protein and RNA content of EVs produced by infected host cells. Release of pathogen-induced host EVs can affect host immune responses to infection. In this review, we will describe both the biogenesis and content of EVs produced by a number of diverse pathogens. In addition, we will examine the pathogen-induced changes to EVs produced by infected host cells.
Asunto(s)
Vesículas Extracelulares/fisiología , Interacciones Huésped-Patógeno , Infecciones/fisiopatología , Infecciones/microbiología , Proteínas/metabolismo , ARN/metabolismoRESUMEN
Parasitic unicellular eukaryotes use extracellular vesicles (EVs) as vehicles for intercellular communication and host manipulation. By using various mechanisms to generate EVs and by transferring a wide range of molecules through EVs, pathogenic protozoans are able to establish infective niches, modulate the immune system of the host and cause disease. In addition to effects on the host, EVs are able to transfer virulence factors, drug-resistance genes and differentiation factors between parasites. In this Progress article, we explore recent insights into the biology of EVs from human infectious protozoan parasites, including Trichomonas vaginalis, Plasmodium spp. and kinetoplastids, such as Trypanosoma spp. and Leishmania spp.
Asunto(s)
Comunicación Celular , Vesículas Extracelulares/fisiología , Parásitos/fisiología , Animales , Transporte Biológico , Interacciones Huésped-Parásitos , Humanos , Leishmania/inmunología , Leishmania/patogenicidad , Leishmania/fisiología , Parásitos/inmunología , Parásitos/patogenicidad , Plasmodium/inmunología , Plasmodium/patogenicidad , Plasmodium/fisiología , Trichomonas vaginalis/inmunología , Trichomonas vaginalis/patogenicidad , Trichomonas vaginalis/fisiología , Trypanosoma/inmunología , Trypanosoma/patogenicidad , Trypanosoma/fisiología , Factores de Virulencia/fisiologíaRESUMEN
Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion, and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence, and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes, allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes, resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling, inducing anemia.
Asunto(s)
Vesículas Extracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei rhodesiense/citología , Trypanosoma brucei rhodesiense/inmunología , Tripanosomiasis Africana/patología , Tripanosomiasis Africana/parasitología , Factores de Virulencia/metabolismo , Anemia/patología , Animales , Eritrocitos/parasitología , Flagelos/metabolismo , Humanos , Evasión Inmune , Ratones , Proteoma/metabolismo , Rodaminas/análisis , Trypanosoma brucei rhodesiense/metabolismo , Trypanosoma brucei rhodesiense/patogenicidadRESUMEN
Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N'-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis.
Asunto(s)
Membrana Celular/metabolismo , Lipoproteínas HDL/farmacología , Presión Osmótica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Trypanosoma brucei brucei/metabolismo , Apolipoproteína L1 , Apolipoproteínas/metabolismo , Apolipoproteínas/farmacología , Membrana Celular/genética , Radicales Libres/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Lipoproteínas HDL/metabolismo , Oxidación-Reducción/efectos de los fármacos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
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.
Asunto(s)
Endonucleasas/metabolismo , Técnicas de Silenciamiento del Gen , ARN Protozoario/genética , ARN/genética , Trypanosoma brucei brucei/genética , Animales , Edición de ARN , ARN MitocondrialRESUMEN
Haptoglobin-related protein (Hpr) is a component of a minor subspecies of high density lipoproteins (HDL) that function in innate immunity. Here we show that assembly of Hpr into HDL is mediated by its retained N-terminal signal peptide, an unusual feature for a secreted protein and the major difference between Hpr and the soluble acute phase protein haptoglobin (Hp). The 18-amino acid signal peptide is necessary for binding to HDL and interacts directly with the hydrocarbon region of lipids. Utilizing model liposomes, we show that the rate of assembly and steady-state distribution of Hpr in lipid particles is mediated by the physical property of lipid fluidity. Dye release assays reveal that Hpr interacts more rapidly with fluid liposomes. Conversely, steady-state binding assays indicate that more rigid lipid compositions stabilize Hpr association. Lipid association also plays a role in facilitating hemoglobin binding by Hpr. Our data may offer an explanation for the distinct distribution of Hpr among HDL subspecies. Rather than protein-protein interactions mediating localization, direct interaction with phospholipids and sensitivity to lipid fluidity may be sufficient for localization of Hpr and may represent a mechanism of HDL subspeciation.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Haptoglobinas/metabolismo , Lipoproteínas HDL/metabolismo , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Anisotropía , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Apolipoproteínas/química , Apolipoproteínas/metabolismo , Western Blotting , Membrana Celular/química , Membrana Celular/metabolismo , Células HEK293 , Haptoglobinas/química , Haptoglobinas/genética , Hemoglobinas/química , Hemoglobinas/metabolismo , Células Hep G2 , Humanos , Lipoproteínas HDL/química , Liposomas/química , Liposomas/metabolismo , Fluidez de la Membrana , Microscopía Fluorescente , Datos de Secuencia Molecular , Fosfolípidos/química , Fosfolípidos/metabolismo , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.
Asunto(s)
Ciclo del Ácido Cítrico , ADN de Cinetoplasto/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/enzimología , Células Cultivadas , Citocinesis , Replicación del ADN , Estabilidad de Enzimas , Flagelos/metabolismo , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/fisiología , Potencial de la Membrana Mitocondrial , Mitocondrias/enzimología , Mitocondrias/genética , Unión Proteica , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Interferencia de ARN , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
BACKGROUND: Haptoglobin related protein (Hpr) is a key component of trypanosome lytic factors (TLF), a subset of high-density 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. CONCLUSIONS/SIGNIFICANCE: Individual variation in Hpr levels was related to Hp level, Hp genotype, demographics, malaria status and the APR. The strong correlations between plasma levels of Hp and Hpr suggest that they are regulated by similar mechanisms. These population-based observations indicate that a more dynamic view of the relative roles of Hpr and Hpr-Hb complexes needs to be considered in understanding innate immunity to African trypanosomes and possibly other pathogens including the newly discovered Plasmodium spp of humans and primates.
Asunto(s)
Antígenos de Neoplasias , Haptoglobinas/metabolismo , Malaria , Polimorfismo Genético , Adolescente , Adulto , Animales , Antígenos de Neoplasias/sangre , Antígenos de Neoplasias/genética , Proteína C-Reactiva/metabolismo , Niño , Femenino , Gabón , Genotipo , Haptoglobinas/genética , Hemoglobinas/química , Hemoglobinas/metabolismo , Humanos , Lipoproteínas HDL/sangre , Lipoproteínas HDL/química , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Malaria/sangre , Malaria/transmisión , Masculino , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
Trypanosome lytic factors (TLFs) are powerful, naturally occurring toxins in humans that provide sterile protection against infection by several African trypanosomes. These trypanocidal complexes predominantly enter the parasite by binding to the trypanosome haptoglobin/hemoglobin receptor (HpHbR), trafficking to the lysosome, causing membrane damage and, ultimately, cell lysis. Despite TLF-mediated immunity, the parasites that cause human African Trypanosomiasis (HAT), Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense, have developed independent mechanisms of resistance to TLF killing. In this review we describe the parasite defenses that allow trypanosome infections of humans and discuss how targeting these apparent strengths of the parasite may reveal their Achilles' heel, leading to new approaches in the treatment of HAT.
Asunto(s)
Inmunidad Innata , Trypanosoma brucei brucei/inmunología , Tripanosomiasis/inmunología , Tripanosomiasis/parasitología , Animales , Evolución Biológica , Humanos , Lipoproteínas HDL/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
The haptoglobin-hemoglobin receptor (HpHbR) of African trypanosomes plays a critical role in human innate immunity against these parasites. Localized to the flagellar pocket of the veterinary pathogen Trypanosoma brucei brucei this receptor binds Trypanosome Lytic Factor-1 (TLF-1), a subclass of human high-density lipoprotein (HDL) facilitating endocytosis, lysosomal trafficking and subsequent killing. Recently, we found that group 1 Trypanosoma brucei gambiense does not express a functional HpHbR. We now show that loss of the TbbHpHbR reduces the susceptibility of T. b. brucei to human serum and TLF-1 by 100- and 10,000-fold, respectively. The relatively high concentrations of human serum and TLF-1 needed to kill trypanosomes lacking the HpHbR indicates that high affinity TbbHpHbR binding enhances the cytotoxicity; however, in the absence of TbbHpHbR, other receptors or fluid phase endocytosis are sufficient to provide some level of susceptibility. Human serum contains a second innate immune factor, TLF-2, that has been suggested to kill trypanosomes independently of the TbbHpHbR. We found that T. b. brucei killing by TLF-2 was reduced in TbbHpHbR-deficient cells but to a lesser extent than TLF-1. This suggests that both TLF-1 and TLF-2 can be taken up via the TbbHpHbR but that alternative pathways exist for the uptake of these toxins. Together the findings reported here extend our previously published studies and suggest that group 1 T. b. gambiense has evolved multiple mechanisms to avoid killing by trypanolytic human serum factors.
Asunto(s)
Lipoproteínas HDL/inmunología , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/inmunología , Suero/inmunología , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Africana/inmunología , Animales , Humanos , Proteínas Protozoarias/genética , Receptores de Superficie Celular/genética , Suero/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei gambiense/genética , Trypanosoma brucei gambiense/inmunología , Tripanosomiasis Africana/parasitologíaRESUMEN
Human high-density lipoproteins (HDLs) play an important role in human innate immunity to infection by African trypanosomes with a minor subclass, Trypanosome Lytic Factor-1 (TLF-1), displaying highly selective cytotoxicity to the veterinary pathogen Trypanosoma brucei brucei but not against the human sleeping sickness pathogens Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. T. b. rhodesiense has evolved the serum resistance associated protein (SRA) that binds and confers resistance to TLF-1 while T. b. gambiense lacks the gene for SRA indicating that these parasites have diverse mechanisms of resistance to TLF-1. Recently, we have shown that T. b. gambiense (group 1) resistance to TLF-1 correlated with the loss of the haptoglobin/hemoglobin receptor (HpHbR) expression, the protein responsible for high affinity binding and uptake of TLF-1. In the course of these studies we also examined TLF-1 resistant T. b. brucei cell lines, generated by long-term in vitro selection. We found that changes in TLF-1 susceptibility in T. b. brucei correlated with changes in variant surface glycoprotein (VSG) expression in addition to reduced TLF-1 binding and uptake. To determine whether the expressed VSG or expression site associated genes (ESAGs) contribute to TLF-1 resistance we prepared a TLF-1 resistant T. b. brucei with a selectable marker in a silent bloodstream expression site (BES). Drug treatment allowed rapid selection of trypanosomes that activated the tagged BES. These studies show that TLF-1 resistance in T. b. brucei is largely independent of the expressed VSG or ESAGs further supporting the central role of HpHbR expression in TLF-1 susceptibility in these cells.
Asunto(s)
Evasión Inmune/genética , Lipoproteínas HDL/farmacología , Trypanosoma brucei brucei/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Secuencia de Bases , Cinamatos/farmacología , Interacciones Huésped-Parásitos , Humanos , Higromicina B/análogos & derivados , Higromicina B/farmacología , Lipoproteínas HDL/química , Datos de Secuencia Molecular , Unión Proteica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de ADN , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
BACKGROUND: The three sub-species of Trypanosoma brucei are important pathogens of sub-Saharan Africa. T. b. brucei is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. T. b. rhodesiense and T. b. gambiense are able to resist lysis by TLF. There are two distinct sub-groups of T. b. gambiense that differ genetically and by human serum resistance phenotypes. Group 1 T. b. gambiense have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 T. b. gambiense 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 (HpHbR)) gene. Here we investigate if this is also true in group 2 parasites. METHODOLOGY: Isogenic resistant and sensitive group 2 T. b. gambiense were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the HpHbR gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to T. b. brucei. Both resistant and sensitive group 2, as well as group 1 T. b. gambiense, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed. CONCLUSIONS: Our data indicate that, despite group 1 T. b. gambiense avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 T. b. gambiense is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 T. b. gambiense variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of HpHbR. Thus there are differences in the mechanism of human serum resistance between T. b. gambiense groups 1 and 2.
Asunto(s)
Apolipoproteínas/farmacología , Productos Biológicos/farmacología , Lipoproteínas HDL/farmacología , Trypanosoma brucei gambiense/efectos de los fármacos , Apolipoproteína L1 , Apolipoproteínas/inmunología , Supervivencia Celular/efectos de los fármacos , Resistencia a Medicamentos , Humanos , Lipoproteínas HDL/inmunología , Pruebas de Sensibilidad Parasitaria , Suero/inmunología , Suero/parasitología , Trypanosoma brucei gambiense/clasificación , Trypanosoma brucei gambiense/inmunología , Trypanosoma brucei gambiense/fisiologíaRESUMEN
We present the first functional analysis of the small GTPase, TbRab7, in Trypanosoma brucei. TbRab7 defines discrete late endosomes closely juxtaposed to the terminal p67(+) lysosome. RNAi indicates that TbRab7 is essential in bloodstream trypanosomes. Initial rates of endocytosis were unaffected, but lysosomal delivery of cargo, including tomato lectin (TL) and trypanolytic factor (TLF) were blocked. These accumulate in a dispersed internal compartment of elevated pH, likely derived from the late endosome. Surface binding of TL but not TLF was reduced, suggesting that cellular distribution of flagellar pocket receptors is differentially regulated by TbRab7. TLF activity was reduced approximately threefold confirming that lysosomal delivery is critical for trypanotoxicity. Unexpectedly, delivery of endogenous proteins, p67 and TbCatL, were unaffected indicating that TbRab7 does not regulate biosynthetic lysosomal trafficking. Thus, unlike mammalian cells and yeast, lysosomal trafficking of endocytosed and endogenous proteins occur via different routes and/or are regulated differentially. TbRab7 silencing had no effect on a cryptic default pathway to the lysosome, suggesting that the default lysosomal reporters p67ΔTM, p67ΔCD and VSGΔGPI do not utilize the endocytic pathway as previously proposed. Surprisingly, conditional knockout indicates that TbRab7 may be non-essential in procyclic insect form trypanosomes.
Asunto(s)
Endocitosis , Endosomas/fisiología , Lisosomas/fisiología , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/fisiología , Proteínas de Unión al GTP rab/metabolismo , Endosomas/metabolismo , Técnicas de Silenciamiento del Gen , Lectinas/metabolismo , Lisosomas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Trypanosoma brucei rhodesiense is the causative agent of human African sleeping sickness. While the closely related subspecies T. brucei brucei is highly susceptible to lysis by a subclass of human high-density lipoproteins (HDL) called trypanosome lytic factor (TLF), T. brucei rhodesiense is resistant and therefore able to establish acute and fatal infections in humans. This resistance is due to expression of the serum resistance-associated (SRA) gene, a member of the variant surface glycoprotein (VSG) gene family. Although much has been done to establish the role of SRA in human serum resistance, the specific molecular mechanism of SRA-mediated resistance remains a mystery. Thus, we report the trafficking and steady-state localization of SRA in order to provide more insight into the mechanism of SRA-mediated resistance. We show that SRA traffics to the flagellar pocket of bloodstream-form T. brucei organisms, where it localizes transiently before being endocytosed to its steady-state localization in endosomes, and we demonstrate that the critical point of colocalization between SRA and TLF occurs intracellularly.
Asunto(s)
Endosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei rhodesiense/fisiología , Tripanosomiasis Africana/parasitología , Células Cultivadas , Flagelos/metabolismo , Humanos , Evasión Inmune , Lipoproteínas HDL/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Trypanosoma brucei rhodesiense/metabolismo , Tripanosomiasis Africana/inmunologíaRESUMEN
Trypanosoma brucei is the causative agent of both a veterinary wasting disease and human African trypanosomiasis, or sleeping sickness. The cell membrane of the developmental stage found within the mammalian host, the bloodstream form (BSF), is highly dynamic, exhibiting rapid rates of endocytosis and lateral flow of glycosylphosphatidylinositol-anchored proteins. Here, we show that the cell membrane of these organisms is a target for killing by small hydrophobic peptides that increase the rigidity of lipid bilayers. Specifically, we have derived trypanocidal peptides that are based upon the hydrophobic N-terminal signal sequences of human apolipoproteins. These peptides selectively partitioned into the plasma membrane of BSF trypanosomes, resulting in an increase in the rigidity of the bilayer, dramatic changes in cell motility, and subsequent cell death. No killing of the developmental stage found within the insect midgut, the procyclic form, was observed. Additionally, the peptides exhibited no toxicity toward mammalian cell lines and did not induce hemolysis. Studies with model liposomes indicated that bilayer fluidity dictates the susceptibility of membranes to manipulation by hydrophobic peptides. We suggest that the composition of the BSF trypanosome cell membrane confers a high degree of fluidity and unique susceptibility to killing by hydrophobic peptides and is therefore a target for the development of trypanocidal drugs.
Asunto(s)
Antiprotozoarios/farmacología , Apolipoproteínas/farmacología , Membrana Celular/metabolismo , Fluidez de la Membrana/efectos de los fármacos , Señales de Clasificación de Proteína , Trypanosoma brucei brucei/metabolismo , Animales , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/farmacología , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/metabolismoRESUMEN
Trypanosome lytic factor (TLF) is a subclass of human high density lipoprotein (HDL) that mediates an innate immune killing of certain mammalian trypanosomes, most notably Trypanosoma brucei brucei, the causative agent of a wasting disease in cattle. Mechanistically, killing is initiated in the lysosome of the target trypanosome where the acidic pH facilitates a membrane-disrupting activity by TLF. Here we utilize a model liposome system to characterize the membrane binding and permeabilizing activity of TLF and its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas non-lytic human HDL exhibits inefficient permeabilizing activity. Purified, delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic membranes. Analysis of the relative particle sizes of susceptible liposomes reveals distinctly different membrane-active behavior for native TLF and the delipidated protein components. We propose that lysosomal membrane damage in TLF-susceptible trypanosomes is initiated by the stable association of the TLF particle with the lysosomal membrane and that this is a property unique to this subclass of human HDL.
Asunto(s)
Inmunidad Innata/fisiología , Membrana Dobles de Lípidos/química , Lipoproteínas HDL/química , Liposomas/química , Lisosomas/química , Modelos Biológicos , Trypanosoma brucei brucei/química , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Apolipoproteína L1 , Apolipoproteínas/química , Apolipoproteínas/inmunología , Apolipoproteínas/metabolismo , Bovinos , Haptoglobinas/química , Haptoglobinas/inmunología , Haptoglobinas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Lipoproteínas HDL/inmunología , Lipoproteínas HDL/metabolismo , Lisosomas/inmunología , Lisosomas/metabolismo , Especificidad de la Especie , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Bovina/inmunología , Tripanosomiasis Bovina/metabolismoRESUMEN
The mitochondrial genome of trypanosomes is composed of thousands of topologically interlocked circular DNA molecules that form the kinetoplast DNA (kDNA). Most genes encoded by the kDNA require a posttranscriptional modification process called RNA editing to form functional mRNAs. Here, we show that alternative editing of the mitochondrial cytochrome c oxidase III (COXIII) mRNA in Trypanosoma brucei produces a novel DNA binding protein, alternatively edited protein 1 (AEP-1). AEP-1 localizes to the region of the cell between the kDNA and the flagellum and purifies with the tripartite attachment complex, a structure believed to physically link the kDNA and flagellar basal bodies. Expression of the DNA binding domain of AEP-1 results in aberrant kDNA structure and reduced cell growth, indicating that AEP-1 is involved in the maintenance of the kDNA. Perhaps most important, our studies show a gain of function through an alternatively edited mRNA and, for the first time, provide a link between the unusual structure of the kDNA and RNA editing in trypanosome mitochondria.
Asunto(s)
Empalme Alternativo , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Protozoarias/genética , Edición de ARN , ARN Guía de Kinetoplastida/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genética , Secuencia de Aminoácidos , Animales , Línea Celular , ADN Mitocondrial/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Modelos Moleculares , Datos de Secuencia Molecular , Fenotipo , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
RNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific approximately 90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (approximately 20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four- to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH--chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.