RESUMO
Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily its COOH-terminal region (Cyspep). This work aimed to track the fate of Cyspep by surface plasmon resonance (SPR) of promastigotes and amastigotes to gain a greater understanding of the adaptation of this parasite in both hosts. This strategy consisted of antibody immobilization on a COOH1 surface, followed by interaction with parasite proteins and epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64). Pro-CPB and Cyspep were detected using specific polyclonal antibodies against a recombinant Cyspep in both parasite forms. The parasitic supernatants from amastigotes and promastigotes exhibited higher anti-Cyspep recognition compared with that in the subcellular fractions. As the supernatant of the promastigote cultures exhibited resonance unit values indicative of an effective with to E-64, this result was assumed to be Pro-CPB detection. Finally, after using three sequential SPR assay steps, we propose that amastigotes and promastigotes release Cyspep into the extracellular environment, but only promastigotes release this polypeptide as Pro-CPB.
Assuntos
Adaptação Fisiológica/fisiologia , Cisteína Proteases/metabolismo , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/patologia , Animais , Anticorpos Antiprotozoários/imunologia , Cisteína Proteases/imunologia , Inibidores de Cisteína Proteinase/farmacologia , Imunoglobulina G/imunologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Ressonância de Plasmônio de SuperfícieRESUMO
The present study demonstrates that the Leishmania (Viannia) braziliensis strain MCAN/BR/1998/R619 is composed of multiple subpopulations with measurable distinctions. Single parasites were separated from a culture of promastigotes in stationary phase by cell sorting and then cultivated as subpopulations. Subsequently, these subpopulations were evaluated for features of in vitro growth, infectivity to murine macrophages and proteinase gene expression. The first evidence of distinct characteristics was observed during the in vitro cultivation of isolated subpopulations, as distinct clusters of patterns were formed among the cultures, indicating the existence of quantifiable fluctuations in metrics. Further, when infecting murine macrophages, the subpopulations induced distinct patterns of production of immune response mediators. While some subpopulations mainly induced the production of IL-1ß, IL-6 and TNF-α, others induced the production of IL-12p70 and nitric oxide. Finally, amastigotes of these subpopulations had higher expression of proteinase genes than promastigotes. Additionally, cysteine proteinase, serine proteinase, metalloproteinase and aspartic proteinases were differentially expressed in promastigote and amastigote forms. These data suggest the existence of distinct profiles for the L. (V.) braziliensis MCAN/BR/1998/R619 strain and subpopulations that could drive the success of parasite adaptation to the environments that they inhabit.
Assuntos
Leishmania braziliensis/crescimento & desenvolvimento , Animais , Humanos , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Leishmania braziliensis/classificação , Leishmania braziliensis/genética , Leishmania braziliensis/isolamento & purificação , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: Leishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite. OBJECTIVES: This works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features. METHODS: Leishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations. FINDINGS: We identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates. MAIN CONCLUSION: Altogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.
Assuntos
Variação Genética , Leishmania braziliensis/genética , Metaloendopeptidases/genética , Conformação Proteica , Cromossomos , Humanos , Modelos MolecularesRESUMO
BACKGROUND Leishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite. OBJECTIVES This works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features. METHODS Leishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations. FINDINGS We identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates. MAIN CONCLUSION Altogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.
Assuntos
Humanos , Leishmania braziliensis/genética , Metaloendopeptidases/genética , Conformação Proteica , Variação Genética , Modelos MolecularesRESUMO
Leishmania parasites cause human tegumentary and visceral infections that are commonly referred to as leishmaniasis. Despite the high incidence and prevalence of cases, leishmaniasis has been a neglected disease because it mainly affects developing countries. The data obtained from the analysis of patients' biological samples and from assays with animal models confirm the involvement of an array of the parasite's components in its survival inside the mammalian host. These components are classified as virulence factors. In this review, we focus on studies that have explored the role of proteinases as virulence factors that promote parasite survival and immune modulation in the mammalian host. Additionally, the direct involvement of proteinases from the host in lesion evolution is analyzed. The gathered data shows that both parasite and host proteinases are involved in the clinical manifestation of leishmaniasis. It is interesting to note that although the majority of the classes of proteinases are present in Leishmania spp., only cysteine-proteinases, metalloproteinases and, to a lesser scale, serine-proteinases have been adequately studied. Members from these classes have been implicated in tissue invasion, survival in macrophages and immune modulation by parasites. This review reinforces the importance of the parasite proteinases, which are interesting candidates for new chemo or immunotherapies, in the clinical manifestations of leishmaniasis.
Assuntos
Leishmania/enzimologia , Leishmaniose/parasitologia , Mamíferos , Peptídeo Hidrolases/metabolismo , Fatores de Virulência/metabolismo , Animais , HumanosRESUMO
Leishmania is inoculated, by the bite of an infected sandfly, into the skin of the host, where the promastigotes are phagocyted by dermal macrophages. The dermal region comprises cells and abundant extracellular matrix. Studies show that matrix metalloproteinases play an important role in host defense responses against pathogens in mammals and that their activities lead to the production of antimicrobial peptides. The aim of this study is to evaluate the changes in the distribution of fibronectin and laminin as well as in the elastic system fibres during the course of infection caused by Leishmania amazonensis in mice with distinct genetic backgrounds of susceptibility to this parasite. The results showed that BALB/c presented an enhancement of fibronectin during the course of infection when compared to their control group while the infected or non-infected C3H.He showed a decrease of this protein at end of the experiment. Laminin, on the other hand, remained unaltered in both strains. Also in both BALB/c and C3H.He mice the elastic and elaunin fibres remained unchanged while the oxytalan fibres decreased along the experiment. Ninety days after the infection C3H.He mice had recovered their capacity to produce oxytalan fibres.
Assuntos
Tecido Elástico/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/imunologia , Leishmaniose/imunologia , Animais , Suscetibilidade a Doenças , Matriz Extracelular/parasitologia , Feminino , Colágenos Fibrilares/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Leishmania/fisiologia , Leishmaniose/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Microscopia ConfocalRESUMO
One of the manifestations of leishmaniases is eye injuries which main characteristics are the injury of the anterior chamber of the eye and the resistance to specific treatments. The retinal pigment epithelial (RPE) cells participate in pathogen-induced intraocular inflammatory processes. We investigated Leishmania amazonensis-RPE cells relationship and its impact in laminin and fibronectin production. Using RPE cell (ARPE-19), we demonstrated that L. amazonensis adhere to these cells in the first hour of infection, whereas parasite internalization was only observed after 6 h. Seventy-two hours after infection, vacuoles with parasites debris were observed intracellularly, and no parasite were observed intra- or extracellularly at the 96 h, suggesting that Leishmania can infect ARPE-19 cells although this cells are able to clear the infection. Fibronectin and laminin were associated with L. amazonensis-ARPE-19 interaction. Confocal analysis showed no substantial alterations in fibronectin presence in ARPE-19-infected or ARPE-19-noninfected cells, whereas laminin levels increased three times 10 h after L. amazonensis infection. After this time, laminin levels decreased in infected cells. These results suggest that L. amazonensis-ARPE-19 infection induces increased production of laminin in the beginning of infection which may facilitate parasite-host cell interactions.