RESUMEN
Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Animales , Ratones , Humanos , Receptor Toll-Like 4/genética , Receptor PAR-2/genética , Enfermedad de Chagas/genética , Enfermedad de Chagas/parasitología , Antivirales/farmacología , Inhibidores de Serina Proteinasa/farmacología , Inflamación , Serina , Serina Endopeptidasas/genéticaRESUMEN
The drug discovery pipeline for leishmaniasis and trypanosomiasis has been filling with novel chemical entities with known mechanisms of action. González et al. and Braillard et al. report a cytochrome bc1 complex inhibitor as another promising preclinical candidate for visceral leishmaniasis (VL) and, in combination with benznidazole, for chronic Chagas' disease (CCD).
Asunto(s)
Enfermedad de Chagas , Leishmaniasis Visceral , Leishmaniasis , Trypanosoma cruzi , Tripanosomiasis , Humanos , Enfermedad de Chagas/tratamiento farmacológico , Leishmaniasis/tratamiento farmacológico , Leishmaniasis Visceral/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Leishmania mexicana , Leishmaniasis , Parásitos , Receptor Toll-Like 3 , Animales , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Leishmania mexicana/metabolismo , Leishmaniasis/metabolismo , Leishmaniasis/parasitología , Ratones , Parásitos/metabolismo , Proteínas Quinasas/metabolismo , Receptor Toll-Like 3/metabolismoRESUMEN
Macrophages play critical roles in inflammation and defense against pathogens, as well as in the return to tissue homeostasis. Macrophage subpopulations displaying antagonistic phenotypes are generally classified as proinflammatory M1, implicated in antipathogen and antitumoral activities, or as anti-inflammatory M2, associated with tissue repair. Granulocytic and monocytic myeloid-derived suppressor cells recruited from the bone marrow to tissues and phagocytosis of apoptotic neutrophils can attenuate macrophage microbicidal activity. Here, we showed that bone marrow neutrophils, but not thioglycollate-recruited neutrophils, directly suppress the responses of macrophages that were previously committed to an inflammatory phenotype. Cocultures of inflammatory macrophages with bone marrow CD11b+Ly6Ghi granulocytes led to reduced release of IL-1ß, TNF-α, and IL-6 by macrophages after lipopolysaccharide stimulation. The suppressive activity was unrelated to granulocyte apoptosis or to secreted factors and required cell-to-cell contact. The suppressive effect was paralleled by reduction in the nuclear levels of the NF-κB p65 subunit, but not of the p50 subunit. Furthermore, bone marrow granulocytes decreased the phagocytic activity of macrophages and their capacity to kill intracellular Escherichia coli. Taken together, these results show that bone marrow granulocytes can function as suppressors of the proinflammatory activity and microbial-killing responses of macrophages.
Asunto(s)
Médula Ósea , Macrófagos , Granulocitos , Humanos , Inflamación , FagocitosisRESUMEN
Visceral leishmaniasis is associated with hepato-splenomegaly and altered immune and hematological parameters in both preclinical animal models and humans. We studied mouse experimental visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani in BALB/c mice using dual RNA-seq to investigate the transcriptional response of host and parasite in liver and spleen. We identified only 4 species-specific parasite expressed genes (SSPEGs; log2FC >1, FDR <0.05) in the infected spleen, and none in the infected liver. For the host transcriptome, we found 789 differentially expressed genes (DEGs; log2FC >1, FDR <0.05) in the spleen that were common to both infections, with IFNγ signaling and complement and coagulation cascade pathways highly enriched, and an additional 286 and 186 DEGs that were selective to L. donovani and L. infantum infection, respectively. Among those, there were network interactions between genes of amino acid metabolism and PPAR signaling in L. donovani infection and increased IL1ß and positive regulation of fatty acid transport in L. infantum infection, although no pathway enrichment was observed. In the liver, there were 1,939 DEGs in mice infected with either L. infantum or L. donovani in comparison to uninfected mice, and the most enriched pathways were IFNγ signaling, neutrophil mediated immunity, complement and coagulation, cytokine-chemokine responses, and hemostasis. Additionally, 221 DEGs were selective in L. donovani and 429 DEGs in L. infantum infections. These data show that the host response for these two visceral leishmaniasis infection models is broadly similar, and â¼10% of host DEGs vary in infections with either parasite species. IMPORTANCE Visceral leishmaniasis (VL) is caused by two species of Leishmania parasites, L. donovani in the Old World and L. infantum in the New World and countries bordering the Mediterranean. Although cardinal features such as hepato-splenomegaly and alterations in blood and immune function are evident, clinical presentation may vary by geography, with for example severe bleeding often associated with VL in Brazil. Although animal models of both L. donovani and L. infantum have been widely used to study disease pathogenesis, a direct side-by-side comparison of how these parasites species impact the infected host and/or how they might respond to the stresses of mammalian infection has not been previously reported. Identifying common and distinct pathways to pathogenesis will be important to ensure that new therapeutic or prophylactic approaches will be applicable across all forms of VL.
Asunto(s)
Leishmania donovani , Leishmania infantum , Leishmaniasis Visceral , Parásitos , Animales , Leishmania donovani/genética , Leishmania infantum/genética , Leishmaniasis Visceral/parasitología , Mamíferos/genética , Ratones , Ratones Endogámicos BALB C , Parásitos/genética , RNA-Seq , EsplenomegaliaRESUMEN
Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, provoking liver and spleen tissue destruction that is lethal unless treated. The parasite replicates in macrophages and modulates host microbicidal responses. We have previously reported that neutrophil elastase (NE) is required to sustain L. donovani intracellular growth in macrophages through the induction of interferon beta (IFN-ß). Here, we show that the gene expression of IFN-ß by infected macrophages was reduced by half when TLR4 was blocked by pre-treatment with neutralizing antibodies or in macrophages from tlr2-/- mice, while the levels in macrophages from myd88-/- mice were comparable to those from wild-type C57BL/6 mice. The neutralization of TLR4 in tlr2-/- macrophages completely abolished induction of IFN-ß gene expression upon parasite infection, indicating an additive role for both TLRs. Induction of type I interferon (IFN-I), OASL2, SOD1, and IL10 gene expression by L. donovani was completely abolished in macrophages from NE knock-out mice (ela2-/-) or from protein kinase R (PKR) knock-out mice (pkr-/-), and in C57BL/6 macrophages infected with transgenic L. donovani expressing the inhibitor of serine peptidase 2 (ISP2). Parasite intracellular growth was impaired in pkr-/- macrophages but was fully restored by the addition of exogenous IFN-ß, and parasite burdens were reduced in the spleen of pkr-/- mice at 7 days, as compared to the 129Sv/Ev background mice. Furthermore, parasites were unable to grow in macrophages lacking TLR3, which correlated with lack of IFN-I gene expression. Thus, L. donovani engages innate responses in infected macrophages via TLR2, TLR4, and TLR3, via downstream PKR, to induce the expression of pro-survival genes in the host cell, and guarantee parasite intracellular development.
Asunto(s)
Interferón-alfa/metabolismo , Interferón beta/metabolismo , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Macrófagos Peritoneales/inmunología , Transducción de Señal/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Femenino , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Técnicas de Inactivación de Genes , Interferón-alfa/genética , Interferón beta/genética , Leishmaniasis Visceral/parasitología , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/genética , Elastasa de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Sulfonamidas/farmacología , Receptor Toll-Like 2/genética , Receptor Toll-Like 3/genética , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/inmunología , eIF-2 Quinasa/genéticaRESUMEN
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.
Asunto(s)
Proteínas Protozoarias , Trypanosoma brucei rhodesiense , Tripanosomiasis Africana , Animales , Ratones , Ratones Endogámicos C57BL , Trypanosoma brucei rhodesiense/patogenicidad , Tripanosomiasis Africana/parasitología , Virulencia , Proteínas Protozoarias/metabolismoRESUMEN
Cruzipains are the main papain-like cysteine proteases of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. Encoded by a multigenic family, previous studies have estimated the presence of dozens of copies spread over multiple chromosomes in different parasite strains. Here, we describe the complete gene repertoire of cruzipain in three parasite strains, their genomic organization, and expression pattern throughout the parasite life cycle. Furthermore, we have analyzed primary sequence variations among distinct family members as well as structural differences between the main groups of cruzipains. Based on phylogenetic inferences and residue positions crucial for enzyme function and specificity, we propose the classification of cruzipains into two families (I and II), whose genes are distributed in two or three separate clusters in the parasite genome, according with the strain. Family I comprises nearly identical copies to the previously characterized cruzipain 1/cruzain, whereas Family II encompasses three structurally distinct sub-types, named cruzipain 2, cruzipain 3, and cruzipain 4. RNA-seq data derived from the CL Brener strain indicates that Family I genes are mainly expressed by epimastigotes, whereas trypomastigotes mainly express Family II genes. Significant differences in the active sites among the enzyme sub-types were also identified, which may play a role in their substrate selectivity and impact their inhibition by small molecules.
Asunto(s)
Dominio Catalítico , Cisteína Endopeptidasas/genética , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estadios del Ciclo de Vida/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Trypanosoma brucei rhodesiense is one of the causative agents of Human African Trypanosomiasis (HAT), known as sleeping sickness. The parasite invades the central nervous system and causes severe encephalitis that is fatal if left untreated. We have previously identified ecotin-like inhibitors of serine peptidases, named ISPs, in trypanosomatid parasitic protozoa. Here, we investigated the role of ISP2 in bloodstream form T. b. rhodesiense. We generated gene-deficient mutants lacking ISP2 (Δisp2), which displayed a growth profile in vitro similar to that of wild-type (WT) parasites. C57BL/6 mice infected with Δisp2 displayed lower blood parasitemia, a delayed hind leg pathological phenotype and survived longer. The immune response was examined at two time-points that corresponded with two peaks of parasitemia. At 4 days, the spleens of Δisp2-infected mice had a greater percentage of NOS2+ myeloid cells, IFN-γ+-NK cells and increased TNF-α compared to those infected with WT and parasites re-expressing ISP2 (Δisp2:ISP2). By 13 days the increased NOS2+ population was sustained in Δisp2-infected mice, along with increased percentages of monocyte-derived dendritic cells, as well as CD19+ B lymphocytes, and CD8+ and CD4+ T lymphocytes. Taken together, these findings indicate that ISP2 contributes to T. b. rhodesiense virulence in mice and attenuates the inflammatory response during early infection.
Asunto(s)
Inhibidores de Serina Proteinasa/metabolismo , Trypanosoma brucei rhodesiense/genética , Trypanosoma brucei rhodesiense/patogenicidad , Tripanosomiasis Africana/inmunología , Animales , Animales Modificados Genéticamente , Anticuerpos Monoclonales , Femenino , Inflamación , Ratones Endogámicos C57BL , Inhibidores de Serina Proteinasa/genética , Bazo/parasitología , VirulenciaRESUMEN
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.
Asunto(s)
Interferón beta/metabolismo , Leishmania donovani/patogenicidad , Leishmaniasis Visceral/etiología , Elastasa de Leucocito/metabolismo , Animales , Animales Modificados Genéticamente , Leishmania donovani/genética , Leishmania donovani/fisiología , Leishmaniasis Visceral/metabolismo , Leishmaniasis Visceral/parasitología , Elastasa de Leucocito/deficiencia , Elastasa de Leucocito/genética , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
There has been a very limited number of high-throughput screening campaigns carried out with Leishmania drug targets. In part, this is due to the small number of suitable target genes that have been shown by genetic or chemical methods to be essential for the parasite. In this perspective, we discuss the state of genetic target validation in the field of Leishmania research and review the 200 Leishmania genes and 36 Trypanosoma cruzi genes for which gene deletion attempts have been made since the first published case in 1990. We define a quality score for the different genetic deletion techniques that can be used to identify potential drug targets. We also discuss how the advances in genome-scale gene disruption techniques have been used to assist target-based and phenotypic-based drug development in other parasitic protozoa and why Leishmania has lacked a similar approach so far. The prospects for this scale of work are considered in the context of the application of CRISPR/Cas9 gene editing as a useful tool in Leishmania.
Asunto(s)
Antiprotozoarios/aislamiento & purificación , Descubrimiento de Drogas/métodos , Leishmania/efectos de los fármacos , Leishmania/fisiología , Proteínas Protozoarias/metabolismo , Antiprotozoarios/farmacología , Descubrimiento de Drogas/tendencias , Eliminación de Gen , Leishmania/genética , Proteínas Protozoarias/genética , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologíaRESUMEN
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.
Asunto(s)
Células Dendríticas/inmunología , Leishmania major/crecimiento & desarrollo , Leishmaniasis Cutánea/parasitología , Monocitos/inmunología , Serina Endopeptidasas/metabolismo , Piel/parasitología , Factores de Virulencia/metabolismo , Animales , Células Cultivadas , Femenino , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Ratones , Ratones Endogámicos C57BL , Piel/inmunologíaRESUMEN
Alzheimer's disease (AD) is a disabling and highly prevalent neurodegenerative condition, for which there are no effective therapies. Soluble oligomers of the amyloid-ß peptide (AßOs) are thought to be proximal neurotoxins involved in early neuronal oxidative stress and synapse damage, ultimately leading to neurodegeneration and memory impairment in AD. The aim of the current study was to evaluate the neuroprotective potential of mesenchymal stem cells (MSCs) against the deleterious impact of AßOs on hippocampal neurons. To this end, we established transwell cocultures of rat hippocampal neurons and MSCs. We show that MSCs and MSC-derived extracellular vesicles protect neurons against AßO-induced oxidative stress and synapse damage, revealed by loss of pre- and postsynaptic markers. Protection by MSCs entails three complementary mechanisms: 1) internalization and degradation of AßOs; 2) release of extracellular vesicles containing active catalase; and 3) selective secretion of interleukin-6, interleukin-10, and vascular endothelial growth factor to the medium. Results support the notion that MSCs may represent a promising alternative for cell-based therapies in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Vesículas Extracelulares/metabolismo , Hipocampo/citología , Células Madre Mesenquimatosas/citología , Neuronas/metabolismo , Estrés Oxidativo , Sinapsis/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/química , Animales , Células Cultivadas , Técnicas de Cocultivo , Vesículas Extracelulares/genética , Hipocampo/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Neuronas/citología , Ratas , Ratas Wistar , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Chagasin-type inhibitors comprise natural inhibitors of papain-like cysteine proteases that are distributed among Protist, Bacteria and Archaea. Chagasin was identified in the pathogenic protozoa Trypanosoma cruzi as an approximately 11 kDa protein that is a tight-binding and highly thermostable inhibitor of papain, cysteine cathepsins and endogenous parasite cysteine proteases. It displays an Imunoglobulin-like fold with three exposed loops to one side of the molecule, where amino acid residues present in conserved motifs at the tips of each loop contact target proteases. Differently from cystatins, the loop 2 of chagasin enters the active-site cleft, making direct contact with the catalytic residues, while loops 4 and 6 embrace the enzyme from the sides. Orthologues of chagasin are named Inhibitors of Cysteine Peptidases (ICP), and share conserved overall tri-dimensional structure and mode of binding to proteases. ICPs are tentatively distributed in three families: in family I42 are grouped chagasin-type inhibitors that share conserved residues at the exposed loops; family I71 contains Plasmodium ICPs, which are large proteins having a chagasin-like domain at the C-terminus, with lower similarity to chagasin in the conserved motif at loop 2; family I81 contains Toxoplasma ICP. Recombinant ICPs tested so far can inactivate protozoa cathepsin-like proteases and their mammalian counterparts. Studies on their biological roles were carried out in a few species, mainly using transgenic protozoa, and the conclusions vary. However, in all cases, alterations in the levels of expression of chagasin/ICPs led to substantial changes in one or more steps of parasite biology, with higher incidence in influencing their interaction with the hosts. We will cover most of the findings on chagasin/ICP structural and functional properties and overview the current knowledge on their roles in protozoa.
Asunto(s)
Inhibidores de Cisteína Proteinasa/metabolismo , Plasmodium/enzimología , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Plasmodium/genética , Proteínas Protozoarias/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Trypanosoma cruzi/genéticaRESUMEN
BACKGROUND: Chagas disease, caused by the protozoan Trypanosoma cruzi (T. cruzi), is a complex disease endemic in Central and South America. It has been gathering interest due to increases in non-vectorial forms of transmission, especially in developed countries. The objective of this work was to investigate if adipose tissue-derived mesenchymal stromal cells (ASC) can alter the course of the disease and attenuate pathology in a mouse model of chagasic cardiomyopathy. METHODOLOGY/PRINCIPAL FINDINGS: ASC were injected intraperitoneally at 3 days post-infection (dpi). Tracking by bioluminescence showed that cells remained in the abdominal cavity for up to 9 days after injection and most of them migrated to the abdominal or subcutaneous fat, an early parasite reservoir. ASC injection resulted in a significant reduction in blood parasitemia, which was followed by a decrease in cardiac tissue inflammation, parasitism and fibrosis at 30 dpi. At the same time point, analyses of cytokine release in cells isolated from the heart and exposed to T. cruzi antigens indicated an anti-inflammatory response in ASC-treated animals. In parallel, splenocytes exposed to the same antigens produced a pro-inflammatory response, which is important for the control of parasite replication, in placebo and ASC-treated groups. However, splenocytes from the ASC group released higher levels of IL-10. At 60 dpi, magnetic resonance imaging revealed that right ventricular (RV) dilation was prevented in ASC-treated mice. CONCLUSIONS/SIGNIFICANCE: In conclusion, the injection of ASC early after T. cruzi infection prevents RV remodeling through the modulation of immune responses. Lymphoid organ response to the parasite promoted the control of parasite burden, while the heart, a target organ of Chagas disease, was protected from damage due to an improved control of inflammation in ASC-treated mice.
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Tejido Adiposo/citología , Cardiomiopatías/prevención & control , Enfermedad de Chagas/complicaciones , Células Madre Mesenquimatosas/inmunología , Miocardio/inmunología , Trypanosoma cruzi/inmunología , Tejido Adiposo/inmunología , Animales , Cardiomiopatías/etiología , Cardiomiopatías/inmunología , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Modelos Animales de Enfermedad , Femenino , Corazón/parasitología , Inmunidad , Interleucina-10/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Trypanosoma cruzi/fisiologíaRESUMEN
BACKGROUND: The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. METHODOLOGY/PRINCIPAL FINDINGS: Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. CONCLUSIONS: This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.
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Venenos de Crotálidos/farmacología , Leishmania mexicana/efectos de los fármacos , Proteínas de Reptiles/farmacología , Trypanosoma brucei rhodesiense/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Animales , Antiprotozoarios/farmacología , Proteínas Portadoras , Enfermedad de Chagas/tratamiento farmacológico , Crotalus/metabolismo , Citoplasma , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas con Dominio LIM , Leishmania , Leishmania mexicana/crecimiento & desarrollo , Ratones , Enfermedades Desatendidas/tratamiento farmacológico , Enfermedades Desatendidas/parasitología , Pruebas de Sensibilidad Parasitaria , Espectrometría de Masas en Tándem , Trypanosoma brucei rhodesiense/crecimiento & desarrollo , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr(-/-), tlr2(-/-), trif(-/-), and myd88(-/-) mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon ß (IFNß) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor-knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.-Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNß.
Asunto(s)
Interferón beta/inmunología , Leishmania major/inmunología , Elastasa de Leucocito/inmunología , Macrófagos/inmunología , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/inmunología , eIF-2 Quinasa/inmunología , Animales , Células Cultivadas , Leishmaniasis Cutánea/inmunología , Ratones , Ratones NoqueadosRESUMEN
Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.
Asunto(s)
Leishmania major/fisiología , Inhibidores de Proteasas/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Células Cultivadas , Flagelos/metabolismo , Flagelos/ultraestructura , Técnicas de Inactivación de Genes , Interacciones Huésped-Parásitos , Leishmania major/genética , Leishmania major/metabolismo , Leishmania major/ultraestructura , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos BALB C , Inhibidores de Proteasas/química , Transporte de Proteínas , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Vesículas Secretoras/metabolismo , Vesículas Secretoras/ultraestructura , Serina Proteasas/químicaRESUMEN
Protozoa of the genus Leishmania cause a wide variety of pathologies ranging from self-healing skin lesions to visceral damage, depending on the parasite species. The outcome of infection depends on the quality of the adaptive immune response, which is determined by parasite factors and the host genetic background. Innate responses, resulting in the generation of mediators with anti-leishmanial activity, contribute to parasite control and help the development of efficient adaptive responses. Among those, the potential contribution of members of the Toll-like receptors (TLRs) family in the control of Leishmania infections started to be investigated about a decade ago. Although most studies appoint a protective role for TLRs, there is growing evidence that in some cases, TLRs facilitate infection. This review highlights recent advances in TLR function during Leishmania infections and discusses their potential role in restraining parasite growth versus yielding disease.
RESUMEN
The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K(m), but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25°C but concealed at 37°C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25°C, but not at 37°C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.