RESUMO
Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.
Assuntos
Movimento Celular , Entamoeba histolytica , Fibronectinas , Entamoeba histolytica/metabolismo , Entamoeba histolytica/fisiologia , Fibronectinas/metabolismo , Humanos , Movimento Celular/fisiologia , Entamebíase/parasitologia , Entamebíase/metabolismo , Actinas/metabolismo , Podossomos/metabolismo , Adesão Celular/fisiologia , Proteínas de Protozoários/metabolismoRESUMO
Entamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis.
Assuntos
Entamoeba histolytica , Animais , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Humanos , Lipopolissacarídeos , Fagocitose , Pinocitose , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trofozoítos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The protozoan parasite Entamoeba histolytica is the microbial agent of amoebiasis - an infection that is endemic worldwide and is associated with high morbidity and mortality rates. As the disease develops, virulent E. histolytica deplete the mucus layer, interact with the intestinal epithelium, and then degrade the colonic mucosa and disrupt the extracellular matrix (ECM). Our research demonstrated that virulent parasites with an invasive phenotype display rapid, highly specific changes in their transcriptome (notably for essential factors involved in carbohydrate metabolism and the processing of glycosylated residues). Moreover, combined activation of parasite and host lytic enzymes leads to the destruction of the intestinal parenchyma. Together, these enzymes degrade the mucus layer and the ECM, and trigger the inflammatory response essential to the development of amoebiasis.
Assuntos
Amebíase/parasitologia , Entamoeba histolytica/patogenicidade , Interações Hospedeiro-Parasita , Mucosa Intestinal/fisiologia , Mucosa Intestinal/parasitologia , Transdução de Sinais , Amebíase/fisiopatologia , Animais , Colo/citologia , Colo/parasitologia , Genoma Bacteriano , Humanos , Inflamação , TranscriptomaRESUMO
Entamoeba histolytica is the pathogenic amoeba responsible for amoebiasis, an infectious disease targeting human tissues. Amoebiasis arises when virulent trophozoites start to destroy the muco-epithelial barrier by first crossing the mucus, then killing host cells, triggering inflammation and subsequently causing dysentery. The main goal of this study was to analyse pathophysiology and gene expression changes related to virulent (i.e. HM1:IMSS) and non-virulent (i.e. Rahman) strains when they are in contact with the human colon. Transcriptome comparisons between the two strains, both in culture conditions and upon contact with human colon explants, provide a global view of gene expression changes that might contribute to the observed phenotypic differences. The most remarkable feature of the virulent phenotype resides in the up-regulation of genes implicated in carbohydrate metabolism and processing of glycosylated residues. Consequently, inhibition of gene expression by RNA interference of a glycoside hydrolase (ß-amylase absent from humans) abolishes mucus depletion and tissue invasion by HM1:IMSS. In summary, our data suggest a potential role of carbohydrate metabolism in colon invasion by virulent E. histolytica.
Assuntos
Colo/parasitologia , Disenteria Amebiana/parasitologia , Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/patogenicidade , Fatores de Virulência/genética , Adulto , Sequência de Aminoácidos , Animais , Clonagem Molecular , Colo/patologia , Cricetinae , Disenteria Amebiana/genética , Entamoeba histolytica/genética , Interações Hospedeiro-Parasita/genética , Humanos , Masculino , Mesocricetus , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fatores de Virulência/metabolismo , beta-Amilase/genética , beta-Amilase/metabolismoRESUMO
Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.
Assuntos
Proteínas de Bactérias/metabolismo , Colo/metabolismo , Limosilactobacillus reuteri/metabolismo , Mucina-2/metabolismo , Muco/metabolismo , Sequência de Aminoácidos , Animais , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Sobrevivência Celular , Colo/microbiologia , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/química , Glicosilação , Cobaias , Células HT29 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imuno-Histoquímica , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/fisiologia , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Muco/microbiologia , Ligação Proteica , Coelhos , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The extracellular matrix (ECM) and its role in the outcome of infectious diseases have been poorly investigated. In this study, we determined the impact of the collagen fibres architecture on the invasive process of the enteric parasite Entamoeba histolytica. The behaviour of E. histolytica wild-type and silenced for the cysteine protease A5 (CP-A5) were compared on a three-dimensional collagen matrix and within human colon fragments for fibrillar collagen cleavage and migration. The interstitial collagen fibres within the connective tissue of the human colon, visualized by multiphoton and second harmonic generation signals imaging, presented a dense scaffold at the subepithelial level and a loose meshwork within the chorion. To penetrate the tissue, E. histolytica migrated on the dense scaffold that remained intact, reached the crypt of Lieberkhün, migrated along and then disorganized the loose scaffold to escape into the mucosa. Interestingly, in vitro, CP-A5 was not required for collagenase activity and migration through the matrix but was necessary within the tissue environment for collagen meshwork remodelling and subsequent invasion. The data point out that further step of invasion relay with ECM destruction that requires human components induced or activated in the presence of CP-A5.
Assuntos
Colo/patologia , Colo/parasitologia , Entamoeba histolytica/patogenicidade , Colágenos Fibrilares/metabolismo , Movimento Celular , Tecido Conjuntivo/parasitologia , Tecido Conjuntivo/patologia , Humanos , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Microscopia de Fluorescência por Excitação MultifotônicaRESUMO
The method proposed in this paper is a robust combination of multi-task learning and unsupervised domain adaptation for segmenting amoeboid cells in microscopy. A highlight of this work is the manner in which the model's hyperparameters are estimated. The detriments of ad-hoc parameter estimation are well known, but this issue remains largely unaddressed in the context of CNN-based segmentation. Using a novel min-max formulation of the segmentation cost function our proposed method analytically estimates the model's hyperparameters, while simultaneously learning the CNN weights during training. This end-to-end framework provides a consolidated mechanism to harness the potential of multi-task learning to isolate and segment clustered cells from low contrast brightfield images, and it simultaneously leverages deep domain adaptation to segment fluorescent cells without explicit pixel-level re- annotation of the data. Experimental validations on multi-cellular images strongly suggest the effectiveness of the proposed technique, and our quantitative results show at least 15% and 10% improvement in cell segmentation on brightfield and fluorescence images respectively compared to contemporary supervised segmentation methods.
Assuntos
Amoeba , Microscopia , Algoritmos , Processamento de Imagem Assistida por Computador/métodosRESUMO
Amoebae found in aquatic and terrestrial environments encompass various pathogenic species, including the parasite Entamoeba histolytica and the free-living Acanthamoeba castellanii. Both microorganisms pose significant threats to public health, capable of inducing life-threatening effects on humans. These amoebae exist in two cellular forms: trophozoites and cysts. The trophozoite stage is the form used for growth and reproduction while the cyst stage is the resistant and disseminating form. Cysts occur after cellular metabolism slowdown due to nutritional deprivation or the appearance of environmental conditions unfavourable to the amoebae's growth and division. The initiation of encystation is accompanied by the activation of stress responses, and scarce data indicate that encystation shares factors and mechanisms identified in stress responses occurring in trophozoites exposed to toxic compounds derived from human immune defence. Although some "omics" analyses have explored how amoebae respond to diverse stresses, these studies remain limited and rarely report post-translational modifications that would provide knowledge on the molecular mechanisms underlying amoebae-specific stress responses. In this review, we discuss ubiquitin-like proteins associated with encystation and cell survival during oxidative damage. We aim to shed light on the signalling pathways involved in amoebic defence mechanisms, with a focus on their potential clinical implications against pathogenic amoebae, addressing the pressing need for effective therapies.
RESUMO
Physical forces are essential to biological function, but their impact at the tissue level is not fully understood. The gut is under continuous mechanical stress because of peristalsis. To assess the influence of mechanical cues on enteropathogen invasion, we combine computational imaging with a mechanically active gut-on-a-chip. After infecting the device with either of two microbes, we image their behavior in real time while mapping the mechanical stress within the tissue. This is achieved by reconstructing three-dimensional videos of the ongoing invasion and leveraging on-manifold inverse problems together with viscoelastic rheology. Our results show that peristalsis accelerates the destruction and invasion of intestinal tissue by Entamoeba histolytica and colonization by Shigella flexneri. Local tension facilitates parasite penetration and activates virulence genes in the bacteria. Overall, our work highlights the fundamental role of physical cues during host-pathogen interactions and introduces a framework that opens the door to study mechanobiology on deformable tissues.
Assuntos
Entamoeba histolytica , Peristaltismo , Dispositivos Lab-On-A-Chip , Simulação por Computador , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Entamoeba histolytica is the protozoan parasite responsible for human amoebiasis. During invasive amoebiasis, migration is an essential process and it has previously been shown that the pro-inflammatory compound tumour necrosis factor (TNF) is produced and that it has a migratory effect on E. histolytica. This paper focuses on the analysis of parasite signalling and cytoskeleton changes leading to directional motility. TNF-induced signalling was PI3K-dependent and could lead to modifications in the polarization of certain cytoskeleton-related proteins. To analyse the effect of TNF signalling on gene expression, we used microarray analysis to screen for genes encoding proteins that were potentially important during chemotaxis towards TNF. Interestingly, we found that elements of the galactose/N-acetylgalactosamine lectin (Gal/GalNAc lectin) were upregulated during chemotaxis as well as genes encoding proteins involved in cytoskeleton dynamics. The alpha-actinin protein appeared to be an important candidate to link the Gal/GalNAc lectin to the cytoskeleton during chemotaxis signalling. Dominant negative parasites blocked for Gal/GalNAc lectin signalling were no longer able to chemotax towards TNF. These results have given us an insight on how E. histolytica changes its cytoskeleton dynamics during chemotaxis and revealed the capital role of PI3K and Gal/GalNAc lectin signalling in chemotaxis.
Assuntos
Quimiotaxia , Entamoeba histolytica/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Fatores de Necrose Tumoral/imunologia , Acetilgalactosamina/metabolismo , Androstadienos/farmacologia , Animais , Citoesqueleto/química , Citoesqueleto/metabolismo , Entamoeba histolytica/imunologia , Entamoeba histolytica/metabolismo , Galactose/metabolismo , Expressão Gênica , Humanos , Lectinas/imunologia , Lectinas/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas de Protozoários/análise , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , WortmaninaRESUMO
Cell motility is governed by a complex molecular machinery that converts physico-chemical cues into whole-cell movement. Understanding the underlying biophysical mechanisms requires the ability to measure physical quantities inside the cell in a simple, reproducible and preferably non-invasive manner. To this end, we developed BioFlow, a computational mechano-imaging method and associated software able to extract intracellular measurements including pressure, forces and velocity everywhere inside freely moving cells in two and three dimensions with high spatial resolution in a non-invasive manner. This is achieved by extracting the motion of intracellular material observed using fluorescence microscopy, while simultaneously inferring the parameters of a given theoretical model of the cell interior. We illustrate the power of BioFlow in the context of amoeboid cell migration, by modelling the intracellular actin bulk flow of the parasite Entamoeba histolytica using fluid dynamics, and report unique experimental measures that complement and extend both theoretical estimations and invasive experimental measures. Thanks to its flexibility, BioFlow is easily adaptable to other theoretical models of the cell, and alleviates the need for complex or invasive experimental conditions, thus constituting a powerful tool-kit for mechano-biology studies. BioFlow is open-source and freely available via the Icy software.
Assuntos
Modelos Teóricos , Imagem Molecular , Software , Algoritmos , Movimento Celular , Fenômenos Mecânicos , Microscopia de Fluorescência , Imagem Molecular/métodos , Fenômenos FísicosRESUMO
During amebiasis, E. histolytica motility is a key factor to achieve its progression across tissues. The pathogenicity of E. histolytica includes its capacity to phagocyte human cells. Motility requires polarization of E. histolytica that involves protrusion of a pseudopod containing actin and associated proteins [myosin IB, ABP-120 and a p21-activated kinase (PAK)] and whole-cell propulsion after contraction of the rear of the cell, where myosin II and F-actin are concentrated. An interesting characteristic of this parasite is the presence of two unique myosins (myosin II and unconventional myosin IB), in contrast to several actin genes. Little is known about the regulation of the actin-myosin cytoskeleton dynamics of E. histolytica, and a better understanding of signaling pathways that stimulate and coordinate regulators protein and cytoskeleton elements will provide new insight into the cell biology of the parasite and in amebiasis. Here we summarize the pleiotropic functions described for myosin II and PAK in E. histolytica. We propose that survival and pathogenicity of E. histolytica require an active actin-myosin cytoskeleton to cap surface receptors, to adhere to host components, to migrate through tissues, to phagocyte human cells and to form liver abscesses.
Assuntos
Entamoeba histolytica/fisiologia , Fagocitose , Sequência de Aminoácidos , Animais , Entamoeba histolytica/enzimologia , Dados de Sequência Molecular , Miosina Tipo II/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Homologia de Sequência de Aminoácidos , Quinases Ativadas por p21RESUMO
BACKGROUND: Entamoeba histolytica cell migration is essential for the development of human amoebiasis (an infectious disease characterized by tissue invasion and destruction). The tissue inflammation associated with tumour necrosis factor (TNF) secretion by host cells is a well-documented feature of amoebiasis. Tumour necrosis factor is a chemoattractant for E. histolytica, and the parasite may have a TNF receptor at its cell surface. METHODS: confocal microscopy, RNA Sequencing, bioinformatics, RNA antisense techniques and histological analysis of human colon explants were used to characterize the interplay between TNF and E. histolytica. RESULTS: an antibody against human TNF receptor 1 (TNFR1) stained the E. histolytica trophozoite surface and (on immunoblots) binds to a 150-kDa protein. Proteome screening with the TNFR1 sequence revealed a BspA family protein in E. histolytica that carries a TNFR signature domain and six leucine-rich repeats (named here as "cell surface protein", CSP, in view of its cellular location). Cell surface protein shares structural homologies with Toll-Like receptors, colocalizes with TNF and is internalized in TNF-containing vesicles. Reduction of cellular CSP levels abolished chemotaxis toward TNF and blocked parasite invasion of human colon. CONCLUSIONS: there is a clear link between TNF chemotaxis, CSP and pathogenesis.
RESUMO
This paper presents a segmentation and tracking method for quantitative analysis of cell dynamics from in vitro videomicroscopy data. The method is based on parametric active contours and includes several adaptations that address important difficulties of cellular imaging, particularly the presence of low-contrast boundary deformations known as pseudopods, and the occurence of multiple contacts between cells. First, we use an edge map based on the average intensity dispersion that takes advantage of relative background homogeneity to facilitate the detection of both pseudopods and interfaces between adjacent cells. Second, we introduce a repulsive interaction between contours that allows correct segmentation of objects in contact and overcomes the shortcomings of previously reported techniques to enforce contour separation. Our tracking technique was validated on a realistic data set by comparison with a manually defined ground-truth and was successfully applied to study the motility of amoebae in a biological research project.
Assuntos
Movimento Celular/fisiologia , Entamoeba histolytica/citologia , Entamoeba histolytica/fisiologia , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Vídeo/métodos , Algoritmos , Animais , Adesão Celular/fisiologia , Agregação Celular/fisiologia , Divisão Celular/fisiologia , Tamanho Celular/fisiologia , Células Cultivadas , Avaliação de Medicamentos/métodos , Entamoeba histolytica/classificação , Modelos Biológicos , Reconhecimento Automatizado de Padrão , Pseudópodes/fisiologia , Pseudópodes/ultraestrutura , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Intestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and -3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis.
Assuntos
Colo/metabolismo , Cisteína Proteases/genética , Entamoeba histolytica/patogenicidade , Precursores Enzimáticos/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Colo/patologia , Cisteína Proteases/metabolismo , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismoRESUMO
Variational deformable models have proven over the past decades a high efficiency for segmentation and tracking in 2-D sequences. Yet, their application to 3-D time-lapse images has been hampered by discretization issues, heavy computational loads and lack of proper user visualization and interaction, limiting their use for routine analysis of large data-sets. We propose here to address these limitations by reformulating the problem entirely in the discrete domain using 3-D active meshes, which express a surface as a discrete triangular mesh, and minimize the energy functional accordingly. By performing computations in the discrete domain, computational costs are drastically reduced, whilst the mesh formalism allows to benefit from real-time 3-D rendering and other GPU-based optimizations. Performance evaluations on both simulated and real biological data sets show that this novel framework outperforms current state-of-the-art methods, constituting a light and fast alternative to traditional variational models for segmentation and tracking applications.
Assuntos
Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Imagem com Lapso de Tempo/métodos , Entamoeba histolytica/citologia , Entamoeba histolytica/fisiologia , Imageamento Tridimensional/métodos , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.
Assuntos
Colo/parasitologia , Entamoeba histolytica/patogenicidade , Entamebíase/parasitologia , Modelos Biológicos , Idoso , Idoso de 80 Anos ou mais , Animais , Colo/imunologia , Citocinas/imunologia , Entamoeba histolytica/genética , Entamoeba histolytica/imunologia , Entamebíase/imunologia , Feminino , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-IdadeRESUMO
In an analysis of the molecular factors triggering amebiasis, we investigated the chemotaxis of Entamoeba histolytica toward tumor necrosis factor (TNF) in vitro, using quantitative imaging techniques. Our findings enabled us to propose a hitherto unknown role for TNF as a chemokinetic and chemoattractant agent for this parasite.
Assuntos
Fatores Quimiotáticos/fisiologia , Entamoeba histolytica/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Quimiotaxia , Entamoeba histolytica/crescimento & desenvolvimento , Entamoeba histolytica/patogenicidade , Humanos , Processamento de Imagem Assistida por ComputadorRESUMO
Blocking expression of EhCaBP1, a calmodulin-like, four EF-hand protein from the protozoan parasite Entamoeba histolytica, resulted in inhibition of cellular proliferation. In this paper we report that EhCaBP1 is involved in dynamic changes of the actin cytoskeleton. Both endocytosis and phagocytosis were severely impaired in cells where EhCaBP1 expression was blocked by inducible expression of the antisense RNA. In wild-type cells both actin and EhCaBP1 were found to co-localize in phagocytic cups and in pseudopods. However, in antisense-blocked cells the phagocytic cup formation is affected. Analysis of the staining patterns in the presence and absence of actin dynamics inhibitors, jasplakinolide and cytochalasin D suggested that EhCaBP1 and polymerized F-actin co-localize on membrane protrusions. Direct interaction between soluble EhCaBP1 and F-actin was further demonstrated by a co-sedimentation assay. A variant of EhCaBP1 did not bind F-actin showing the specificity of the interaction between EhCaBP1 and actin. There is no significant change in the kinetics of in vitro polymerization of actin in presence of EhCaBP1, indicating that EhCaBP1 does not affect filament treadmilling. In addition, using atomic force microscopy; it was found that filaments of F-actin, polymerized in presence of EhCaBP1, were thinner. These results indicate that EhCaBP1 may be involved in dynamic membrane restructuring at the time of cell pseudopod formation, phagocytosis and endocytosis in a process mediated by direct binding of EhCaBP1 to actin, affecting the bundling of actin filaments.
Assuntos
Actinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citoesqueleto/metabolismo , Entamoeba histolytica/metabolismo , Animais , Biopolímeros , Endocitose , Imunofluorescência , Cinética , Microscopia de Força Atômica , Microscopia Confocal , Fagocitose , Ligação ProteicaRESUMO
Entamoeba histolytica migration is essential for the development of amoebiasis, a human disease characterised by invasion and destruction of tissues. Amoebic motility requires both polarisation of the cell and formation of a predominant pseudopod. As p21-activated kinases PAKs are known to regulate eukaryotic cell motility and morphology, we investigated the role of PAK in E. histolytica. We showed that the C-terminal domain of EhPAK comprised a constitutive kinase activity in vitro and that overproduction of this fragment, in E. histolytica, caused a significant reduction in amoeboid migration, as measured by dynamic image analysis, indicating an involvement of EhPAK in this process. A dramatic loss of polarity, as indicated by the increased number of membrane extensions all around E. histolytica, was also observed, suggesting that the N-terminal domain of EhPAK was necessary for maintenance of cell polarity. To support this view, we showed that despite the absence of the consensus motif to bind to Rac and Cdc42, the N-terminal domain of EhPAK bound to Rac1, suggesting that the N-terminal region was a regulatory domain. In addition, we also found an increased rate of human red blood cell phagocytosis, suggesting for the first time an active role for a PAK protein in this process. Taking together, the results suggest strongly that EhPAK is a key regulatory element in polarity, motility and phagocytosis of E. histolytica.