RESUMO
Ticks host different pathogens as endosymbiont and nonpathogenic microorganisms and play an important role in reproductive fitness and nutrient provision. However, the bacterial microbiomes of white-tailed deer ticks have received minimal attention. This study aimed to examine the bacterial microbiome of ticks collected from Odocoileus virginianus on the Mexico-United States border to assess differences in microbiome diversity in ticks of different species, sexes, and localities. Five different tick species were collected: Rhipicephalus microplus, Dermacentor nitens, Otobius megnini, Amblyomma cajennense, and A. maculatum. The tick microbiomes were analyzed using next-generation sequencing. Among all tick species, the most predominant phylum was Proteobacteria, followed by Actinobacteria and Firmicutes. The ticks from Tamaulipas and Nuevo León presented the highest bacterial species diversity. Acinetobacter johnsonii and A. lwoffii were the common bacterial species in the microbiome of all ticks, Coxiella were present in R. microplus, and Dermacentor nitens also exhibited a Francisella-like endosymbiont. The microbiome of most females in D. nitens was less diverse than that of males, whereas R. microplus occurs in females, suggesting that microbiome diversity is influenced by sex. In the bacterial communities of A. maculatum and O. megnini, Candidatus Midichloria massiliensis, and Candidatus Endoecteinascidia fumentensis were the most predominant endosymbionts. These results constitute the initial report on these bacteria, and this is also the first study to characterize the microbiome of O. megnini.
Assuntos
Cervos , Microbiota , Rhipicephalus , Animais , Feminino , Masculino , México , Microbiota/genéticaRESUMO
Enolase proteins play a significant role as moonlighting proteins. In their role as surface-associated enolase, they have multiple functions as they interact with extracellular matrix proteins. Type I and III collagens are the major constituents of this extracellular matrix, and collagen is one of the targets of interaction with the enolase of many pathogens, thereby helping the colonization process and promoting the subsequent invasion of the host. This work aimed to determine the participation of non-typeable H. influenzae enolase as a collagen-binding protein. In this study, through the use of in vitro tests it was demonstrated that recombinant enolase of non-typeable H. influenzae (rNTHiENO) strongly binds to type I collagen. Using molecular docking, the residues that could take part in the interaction of non-typeable H. influenzae enolase-type I collagen (NTHiENO-Cln I) and non-typeable H. influenzae enolase-type III collagen (NTHiENO-Cln III) were identified. However, in vitro assays show that NTHiENO has a better affinity to interact with Cln I, concerning type Cln III. The interaction of NTHiENO with collagen could play a significant role in the colonization process; this would allow H. influenzae to increase its virulence factors and strengthen its pathogenesis.
Assuntos
Infecções por Haemophilus , Haemophilus influenzae , Humanos , Fosfopiruvato Hidratase/genética , Colágeno Tipo I , Simulação de Acoplamento Molecular , Colágeno/metabolismo , Matriz Extracelular/metabolismoRESUMO
To infect the human host, Entamoeba histolytica carries out processes requiring cytoskeleton remodeling, which involves reorganizing the actin fibers. However, little is known about the external influence factors, e.g., the pH, on the parasite's cytoskeleton remodeling or cell morphology. Such influence becomes relevant given the pH gradient that the amoeba cope with when going through the human colonic mucus during infection. Therefore, we analyzed the proliferation, the reorganization of the actin fibers, and other actin structures and cell shape during adhesion to fibronectin and erythrophagocytosis in trophozoites at different external pH conditions (6.0, 6.5, 6.8, 7.5, 8.0). We found that the best condition of external pH to perform such functions was 6.8. At acid pH, the trophozoites presented better-defined actin fibers that formed a more compact network, while at alkaline pH, the fibers reorganized, forming a looser and less defined network. Similarly, the number of actin dots also changed from acid to alkaline pH. In conclusion, the external pH alters the proliferation of the amoebas and promotes the dynamic restructuration of their cytoskeleton, allowing them to carry out their functions.
Assuntos
Entamoeba histolytica , Actinas/metabolismo , Animais , Proliferação de Células , Citoesqueleto/metabolismo , Entamoeba histolytica/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Trofozoítos/metabolismoRESUMO
Leishmaniasis is a disease caused by an intracellular protozoan parasite of the genus Leishmania. Current treatments for leishmaniasis are long, toxic, and expensive and are not available in some endemic regions. Attempts to develop an effective vaccine are feasible, but no vaccine is in active clinical use. In this study, the LmxMBA gene of Leishmania mexicana was selected as a possible vaccine candidate using the reverse vaccinology approach, and the prophylactic effect generated by DNA vaccination with this gene in a murine model of cutaneous leishmaniasis was evaluated. The results showed that prophylactic vaccination with pVAX1::LmxMBA significantly reduced the size of the lesion and the parasitic load on the footpad, compared to the control groups. At a histological level, a smaller number of parasites were evident in the dermis, as well as the absence of connective tissue damage. Mice immunized with plasmid pVAX1::LmxMBA induced immunity characterized by an increase in the IgG2a/IgG1 > 1 ratio and a higher rate of lymphocyte proliferation. In this study, immunization with the plasmid promoted an improvement in the macroscopic and microscopic clinical manifestations of the experimental infection by L. mexicana, with a T helper 1 response characterized by an IgG2a/IgG1 > 1 ratio and high lymphoproliferative response. These findings support immunization with the plasmid pVAX1::LmxMBA as a preventive strategy against cutaneous infection of L. mexicana.
Assuntos
Fosfatase Ácida/genética , Leishmania mexicana/fisiologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/imunologia , Proteínas de Protozoários/genética , Pele/patologia , Células Th1/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/sangue , Leishmaniose Cutânea/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Vacinação , Vacinas de DNARESUMO
INTRODUCTION AND OBJECTIVES: Free and conjugated bile acids (BA's) cannot cross cell membranes; therefore, a particular transport system is required by the cell. Members of the family of ABC (ATP-binding proteins) transporters transfer bile acids in and out of the cell, preventing their accumulation. High intracellular concentrations of bile acids, such as those observed in cholestasis, have been related to oxidative stress and apoptosis, which in many cases are the leading causes of hepatocyte damage. MRP3 and MRP4 (multidrug resistance-associated protein 3 and 4) proteins belong to the ABC subfamily C, and are transporters of the hepatocyte's basolateral membrane with a compensatory role. Both transporters' increased expression constitutes an essential role in the protective and adaptive responses of bile acid overload, such as cholestasis. This work aimed to analyze both transporters' mRNA and protein expression in an in vitro model of cholestasis using HepG2 cell line treated with main bile acids. METHODS: The expression of transporters was investigated through confocal microscopy immunofluorescence, Western Blot, and RT-qPCR after the main bile acids in HepG2 line cells. RESULTS: The results showed the relation between confluence and expression of both transporters in the plasma membrane. MRP3 showed atypical and heterogeneous distribution in this cell line. CDCA (chenodeoxycholic acid) at low concentrations induced the expression of mRNA of both transporters. In contrast, protein expression was induced by CA (cholic acid) at high concentrations. CONCLUSION: Primary bile acids (CDCA and CA) induce overexpression of the MRP4 and MRP3 transporters in the HepG2 cell line.
Assuntos
Ácidos e Sais Biliares/farmacologia , Colestase/genética , Colestase/patologia , Fármacos Gastrointestinais/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Técnicas de Cultura de Células , Colestase/metabolismo , Células Hep G2 , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , RNA Mensageiro/metabolismoRESUMO
Chagas disease is a major public health problem in Latin America. The mixed Th1/Th2 immune response is required against Trypanosoma cruzi. Electrolyzed oxidizing water (EOW) has been shown to have germicidal efficacy. The objective of this study was to evaluate the EOW effectiveness in T. cruzi-infected BALB/c mice clinically, immunologically, and histologically. The severity of the infection was assessed by parasitaemia, general health condition, mortality, mega syndromes, and histological lesions. IgG, TNF-alpha, IFN-gamma, and IL-1 beta levels were quantified. The EOW administration showed a beneficial effect on parasitaemia, general physical condition, and mortality. High levels of IgG1 at 50 days postinfection were observed. Prophylactic EOW treatment was able to induce a predominantly TH1 immune response based on an IgG2a levels increase at the late acute phase, and a 10-fold increase of INF-gamma in whole acute phase. EOW was able to control the acute phase infection as effectively as benznidazole. Splenomegaly was caused by EOW treatment and lymphadenopathy was stimulated by T. cruzi infection in all groups. Severe tissue damage was not prevented by EOW treatments. Moderate efficacy may be due to immunomodulatory properties and not to a direct toxic effect on the parasite.
RESUMO
Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a-better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified.
Assuntos
Movimento Celular , Extensões da Superfície Celular/fisiologia , Extensões da Superfície Celular/ultraestrutura , Entamoeba histolytica/fisiologia , Entamoeba histolytica/ultraestrutura , Trofozoítos/fisiologia , Trofozoítos/ultraestrutura , Extratos Celulares/isolamento & purificação , Extensões da Superfície Celular/efeitos dos fármacos , Entamoeba histolytica/efeitos dos fármacos , Eritrócitos/química , Fibronectinas/isolamento & purificação , Fibronectinas/metabolismo , Humanos , Microscopia , Microscopia Confocal , Microscopia Eletrônica de Varredura , Trofozoítos/efeitos dos fármacosRESUMO
Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host-pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica. Here, we reported that the EhPRL protein possessed the classical HCX5R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31-32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3-o-methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o-vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism.
Assuntos
Entamoeba histolytica/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA de Protozoário/química , Inibidores Enzimáticos/farmacologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Abscesso Hepático Amebiano/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Vanadatos/farmacologiaRESUMO
Chagas disease is a parasitic infection caused by the protozoan Trypanosoma cruzi, a flagellated organism that is transmitted mainly to humans through the infected feces of triatomine kissing bugs (vector transmission in endemic areas) or by transfusion of infected blood, donations of infected organ, or transmission from an infected mother to her child at birth. Chagas disease was first described in 1909 by the Brazilian physician Carlos Chagas, and due to the parasite's distribution throughout North, Central and South America, the disease is commonly known as American trypanosomiasis. However, this disease is now present in non-endemic countries such as Canada, the United States of America, and several countries in Europe (principally Spain). Moreover, Chagas disease was recently designated by the World Health Organization as one of the main neglected tropical diseases. The aim of this review is to summarize the research efforts recently described in studies conducted in Mexico on Chagas disease. In this country, there are no existing vector control programs. In addition, there is no consensus on the diagnostic methods for acute and chronic Chagas disease in maternity wards and blood banks, and trypanocidal therapy is not administered to chronic patients. The actual prevalence of the disease is unknown because no official reporting of cases is performed. Therefore, the number of people infected by different routes of transmission (vector, congenital, blood transfusion, organ transplantation, or oral) is unknown. We believe that by promoting education about Chagas disease in schools starting at the basic elementary level and including reinforcement at higher education levels will ensure that the Mexican population would be aware of this health problem and that the control measures adopted will have more acceptance and success. We hope that this review sensitizes the relevant authorities and that the appropriate measures to reduce the risk of infection by T. cruzi are undertaken to provide the Mexican people a better quality of life.
Assuntos
Doença de Chagas/epidemiologia , Animais , Doença de Chagas/prevenção & controle , Humanos , México/epidemiologia , Doenças Negligenciadas , Vacinas ProtozoáriasRESUMO
A 30-kDa surface collagen binding protein peroxiredoxin of Entamoeba histolytica (EhCBP30) was evaluated either alone or fused to the chaperone (CHP) or ATPase (ATP) domains of heat shock protein 70 of Trypanosoma cruzi (TcHSP70) as a vaccine candidate in a hamster model of experimental amoebic liver abscess (ALA) development. Three constructs were produced containing the EhCBP30 DNA sequence, one expressing EhCBP30 and two expressing EhCBP30 fused to either CHP or ATP domains of TcHSP70. High purity recombinant proteins rEhCBP30, rEhCBP30-CHP and rEhCBP30-ATP with N-terminal His tag were obtained by single step affinity purification. Hamsters were immunized without adjuvant with the antigenic recombinant proteins and then challenged intrahepatically with E. histolytica trophozoites. A 70% decrease in ALA development was detected in hamsters immunized with rEhCBP30 and rEhCBP30-CHP, while animals immunized with rEhCBP30-ATP did not show a statistically significant decrease in ALA formation compared with non-immunized animals. Histological analysis of liver tissue showed that the inflammatory infiltrate was discrete or moderate in hamsters immunized with rEhCBP30 or rEhCBP30-CHP compared with that observed in control hamsters or hamsters immunized with rEhCBP30-ATP. These results suggest that rEhCBP30 and rEhCBP30-CHP are able to induce an effective immune response that may protect hamsters against ALA development.