RESUMO
Leishmania parasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasite's developmental changes remain elusive. Here, we report that Leishmania TORC1 is a key environmental sensor for parasite proliferation and differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and LST8, and identify new parasite-specific proteins that interact in this complex. We investigate TORC1 function by conditional deletion of RPTOR1, where under nutrient-rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites are unable to respond to nutrients to differentiate into proliferative retroleptomonads, which are required for their blood-meal induced amplification in sand flies and enhanced mammalian infectivity. We additionally show that RPTOR1-/- metacyclic promastigotes develop into amastigotes but do not proliferate in the mammalian host to cause pathology. RPTOR1-dependent TORC1 functionality represents a critical mechanism for driving parasite growth and proliferation.
Assuntos
Leishmania , Phlebotomus , Psychodidae , Animais , Psychodidae/parasitologia , Phlebotomus/parasitologia , Nutrientes , Proliferação de Células , MamíferosRESUMO
Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.
Assuntos
Flagelos/metabolismo , Cinesinas/metabolismo , Leishmania mexicana/patogenicidade , Leishmaniose/metabolismo , Virulência/fisiologia , Animais , Proliferação de Células , Leishmania mexicana/fisiologia , Camundongos , Morfogênese , Proteínas de Protozoários/metabolismo , PsychodidaeRESUMO
Selection and internalization of cargo via clathrin-mediated endocytosis requires adaptor protein complexes. One complex, AP-2, acts during cargo selection at the plasma membrane. African trypanosomes lack all components of the AP-2 complex, except for a recently identified orthologue of the AP-2-associated protein kinase 1, AAK1. In characterized eukaryotes, AAK1 phosphorylates the µ2 subunit of the AP-2 complex to enhance cargo recognition and uptake into clathrin-coated vesicles. Here, we show that kinetoplastids encode not one, but two AAK1 orthologues: one (AAK1L2) is absent from salivarian trypanosomes, while the other (AAK1L1) lacks important kinase-specific residues in a range of trypanosomes. These AAK1L1 and AAK1L2 novelties reinforce suggestions of functional divergence in endocytic uptake within salivarian trypanosomes. Despite this, we show that AAK1L1 null mutant Trypanosoma brucei, while viable, display slowed proliferation, morphological abnormalities including swelling of the flagellar pocket, and altered cargo uptake. In summary, our data suggest an unconventional role for a putative pseudokinase during endocytosis and/or vesicular trafficking in T. brucei, independent of AP-2.
Assuntos
Parasitos , Trypanosoma brucei brucei , Animais , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Clatrina/metabolismo , Parasitos/metabolismo , Endocitose/fisiologia , Membrana CelularAssuntos
Caspases/classificação , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/classificação , Proteínas de Plantas/classificação , Terminologia como Assunto , Animais , Caspases/química , Caspases/metabolismo , Consenso , Humanos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/química , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The protozoan parasite Leishmania (L.) amazonensis infects and replicates inside host macrophages due to subversion of the innate host cell response. In the present study, we demonstrate that TLR3 is required for the intracellular growth of L. (L.) amazonensis. We observed restricted intracellular infection of TLR3-/- mouse macrophages, reduced levels of IFN1ß and IL-10, and increased levels of IL-12 upon L. (L.) amazonensis infection, compared with their wild-type counterparts. Accordingly, in vivo infection of TLR3-/- mice with L. (L.) amazonensis displayed a significant reduction in lesion size. Leishmania (L.) amazonensis infection induced TLR3 proteolytic cleavage, which is a process required for TLR3 signaling. The chemical inhibition of TLR3 cleavage or infection by CPB-deficient mutant L. (L.) mexicana resulted in reduced parasite load and restricted the expression of IFN1ß and IL-10. Furthermore, we show that the dsRNA sensor molecule PKR (dsRNA-activated protein kinase) cooperates with TLR3 signaling to potentiate the expression of IL-10 and IFN1ß and parasite survival. Altogether, our results show that TLR3 signaling is engaged during L. (L.) amazonensis infection and this component of innate immunity modulates the host cell response.
Assuntos
Leishmania mexicana , Leishmaniose , Parasitos , Receptor 3 Toll-Like , Animais , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Leishmania mexicana/metabolismo , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Camundongos , Parasitos/metabolismo , Proteínas Quinases/metabolismo , Receptor 3 Toll-Like/metabolismoRESUMO
Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.
Assuntos
Leishmania/fisiologia , Leishmaniose/parasitologia , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Animais , Feminino , Humanos , Leishmaniose/metabolismo , Leishmaniose/patologia , Camundongos , Conformação Proteica , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.
Assuntos
Citoesqueleto/metabolismo , Flagelos/fisiologia , Interações Hospedeiro-Parasita , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Morfogênese , Psychodidae/parasitologia , Animais , Membrana Celular , Citocinese , Feminino , Flagelos/ultraestrutura , Leishmania/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.
Assuntos
Ciclo Celular , Enzimas Desubiquitinantes/metabolismo , Leishmania mexicana/enzimologia , Proteínas de Protozoários/metabolismo , Ubiquitinação , Animais , Enzimas Desubiquitinantes/genética , Feminino , Deleção de Genes , Leishmania mexicana/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genéticaRESUMO
Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.
Assuntos
Doença de Chagas/tratamento farmacológico , Kinetoplastida/efeitos dos fármacos , Kinetoplastida/enzimologia , Leishmaniose/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Pirimidinas/farmacologia , Triazóis/farmacologia , Tripanossomíase Africana/tratamento farmacológico , Animais , Doença de Chagas/parasitologia , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Concentração Inibidora 50 , Leishmaniose/parasitologia , Camundongos , Estrutura Molecular , Terapia de Alvo Molecular , Inibidores de Proteassoma/efeitos adversos , Inibidores de Proteassoma/classificação , Pirimidinas/efeitos adversos , Pirimidinas/química , Pirimidinas/uso terapêutico , Especificidade da Espécie , Triazóis/efeitos adversos , Triazóis/química , Triazóis/uso terapêutico , Tripanossomíase Africana/parasitologiaRESUMO
Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.
Assuntos
Cílios/fisiologia , Flagelos/fisiologia , Leishmania/fisiologia , Leishmania/patogenicidade , Psychodidae/parasitologia , Animais , Membrana Celular/metabolismo , Cílios/genética , Cílios/ultraestrutura , Endocitose , Flagelos/genética , Flagelos/ultraestrutura , Deleção de Genes , Interações Hospedeiro-Parasita , Junções Intercelulares , Leishmania/genética , Leishmania/ultraestrutura , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Virulência/genéticaRESUMO
The protozoan Trypanosoma brucei rhodesiense causes Human African Trypanosomiasis, also known as sleeping sickness, and penetrates the central nervous system, leading to meningoencephalitis. The Cathepsin L-like cysteine peptidase of T. b. rhodesiense has been implicated in parasite penetration of the blood-brain barrier and its activity is modulated by the chagasin-family endogenous inhibitor of cysteine peptidases (ICP). To investigate the role of ICP in T. b. rhodesiense bloodstream form, ICP-null (Δicp) mutants were generated, and lines re-expressing ICP (Δicp:ICP). Lysates of Δicp displayed increased E-64-sensitive cysteine peptidase activity and the mutant parasites traversed human brain microvascular endothelial cell (HBMEC) monolayers in vitro more efficiently. Δicp induced E-selectin in HBMECs, leading to the adherence of higher numbers of human neutrophils. In C57BL/6 mice, no Δicp parasites could be detected in the blood after 6 days, while mice infected with wild-type (WT) or Δicp:ICP displayed high parasitemia, peaking at day 12. In mice infected with Δicp, there was increased recruitment of monocytes to the site of inoculation and higher levels of IFN-γ in the spleen. At day 14, mice infected with Δicp exhibited higher preservation of the CD4+, CD8+, and CD19+ populations in the spleen, accompanied by sustained high IFN-γ, while NK1.1+ populations receded nearly to the levels of uninfected controls. We propose that ICP helps to downregulate inflammatory responses that contribute to the control of infection.
Assuntos
Proteínas de Protozoários , Trypanosoma brucei rhodesiense , Tripanossomíase Africana , Animais , Camundongos , Camundongos Endogâmicos C57BL , Trypanosoma brucei rhodesiense/patogenicidade , Tripanossomíase Africana/parasitologia , Virulência , Proteínas de Protozoários/metabolismoRESUMO
Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major, named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-ß production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE (ela-/-), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela-/- mice were reduced and accompanied by increased NO and decreased TGF-ß production. Expression of ISP2 was not detected in L. donovani, and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela-/- macrophages displayed significantly lower IFN-ß mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-ß. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN-ß necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon-ß.
Assuntos
Interferon beta/metabolismo , Leishmania donovani/patogenicidade , Leishmaniose Visceral/etiologia , Elastase de Leucócito/metabolismo , Animais , Animais Geneticamente Modificados , Leishmania donovani/genética , Leishmania donovani/fisiologia , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Elastase de Leucócito/deficiência , Elastase de Leucócito/genética , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9-RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome-wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryotes.
Assuntos
Proteínas de Ciclo Celular/genética , Enzimas Reparadoras do DNA/genética , Endonucleases/genética , Genoma de Protozoário , Leishmania major/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Biologia Computacional/métodos , Meios de Cultura/química , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Endonucleases/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Deleção de Genes , Regulação da Expressão Gênica , Engenharia Genética , Variação Genética , Instabilidade Genômica , Histonas/genética , Histonas/metabolismo , Leishmania major/metabolismo , Sequenciamento Completo do GenomaRESUMO
All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities.
Assuntos
Reparo do DNA , Genoma de Protozoário , Proteínas Quinases/genética , Proteínas de Protozoários/genética , Interferência de RNA , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Dano ao DNA/efeitos dos fármacos , Evolução Molecular , Metanossulfonato de Metila/análogos & derivados , Metanossulfonato de Metila/toxicidade , Mutagênicos/toxicidade , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major. Significant infiltrates of neutrophils and monocytes occurred at 1-4 d and 2-4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-γ in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.-Goundry, A., Romano, A., Lima, A. P. C. A., Mottram, J. C., Myburgh, E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells.
Assuntos
Células Dendríticas/imunologia , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Monócitos/imunologia , Serina Endopeptidases/metabolismo , Pele/parasitologia , Fatores de Virulência/metabolismo , Animais , Células Cultivadas , Feminino , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pele/imunologiaRESUMO
The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.
Assuntos
Apolipoproteínas/sangue , Apolipoproteínas/metabolismo , Lipoproteínas HDL/sangue , Lipoproteínas HDL/metabolismo , Trypanosoma brucei gambiense/fisiologia , África , Animais , Animais Geneticamente Modificados , Apolipoproteína L1 , Apolipoproteínas/antagonistas & inibidores , Apolipoproteínas/toxicidade , Membrana Celular/química , Membrana Celular/metabolismo , Cisteína Proteases/metabolismo , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Hemólise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos , Lipoproteínas HDL/antagonistas & inibidores , Lipoproteínas HDL/química , Lipoproteínas HDL/toxicidade , Parasitos/patogenicidade , Parasitos/fisiologia , Estrutura Secundária de Proteína , Soro/química , Soro/parasitologia , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/patogenicidade , Tripanossomíase Africana/parasitologia , Glicoproteínas Variantes de Superfície de Trypanosoma/química , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismoRESUMO
Cysteine peptidases play a central role in the biology of Leishmania. In this work, we sought to further elucidate the mechanism(s) by which the cysteine peptidase CPB contributes to L. mexicana virulence and whether CPB participates in the formation of large communal parasitophorous vacuoles induced by these parasites. We initially examined the impact of L. mexicana infection on the trafficking of VAMP3 and VAMP8, two endocytic SNARE proteins associated with phagolysosome biogenesis and function. Using a CPB-deficient mutant, we found that both VAMP3 and VAMP8 were down-modulated in a CPB-dependent manner. We also discovered that expression of the virulence-associated GPI-anchored metalloprotease GP63 was inhibited in the absence of CPB. Expression of GP63 in the CPB-deficient mutant was sufficient to down-modulate VAMP3 and VAMP8. Similarly, episomal expression of GP63 enabled the CPB-deficient mutant to establish infection in macrophages, induce the formation of large communal parasitophorous vacuoles, and cause lesions in mice. These findings implicate CPB in the regulation of GP63 expression and provide evidence that both GP63 and CPB are key virulence factors in L. mexicana.
Assuntos
Regulação da Expressão Gênica/fisiologia , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/metabolismo , Metaloendopeptidases/biossíntese , Proteínas de Protozoários/metabolismo , Animais , Western Blotting , Cisteína/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Peptídeo Hidrolases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Virulência , Fatores de Virulência/metabolismoRESUMO
Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/ß/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys(147), resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys(147) to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca(2+) for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.
Assuntos
Proteínas de Bactérias/química , Bacteroidaceae/enzimologia , Cisteína Proteases/química , Microbioma Gastrointestinal , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.