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1.
Artigo em Inglês | MEDLINE | ID: mdl-32401956

RESUMO

Paracoccidioides species cause paracoccidioidomycosis (PCM), a systemic mycosis highly prevalent in Brazil. Therapy of PCM has some issues that make studies for new therapeutic and vaccine targets relevant, such as the P. brasiliensis 60-kDa-heat-shock protein (PbHsp60), an immunogenic antigen that induces protection in experimental mice infection. Here, we investigated the relative expression of mRNA for PbHsp60 in P. brasiliensis in the different morphotypes of P. brasiliensis and in morphological transition phases. In addition, antibodies to rPbHsp60 were produced and used to analyze the location of PbHsp60 in yeast and hyphae by electron microscopy. The analyses showed a substantial increase in the relative amounts of HSP60 mRNA in yeast when compared to mycelium and an intermediate expression in transitional forms. Regarding the cell location, immunoelectron microscopy analysis revealed that PbHsp60 is within the cell wall. These observations suggest that this protein may be involved in the maintenance of the cell wall integrity and the interaction with the host for colonization, infection and pathogenesis.

2.
Methods Mol Biol ; 2132: 139-149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306322

RESUMO

Studies on the effects of components derived from the human pathogenic fungi Paracoccidioides brasiliensis have identified paracoccin (PCN), as a bifunctional protein with lectin (GlcNAc-binding) and enzymatic (chitinase) activities, able to induce modulation of host immune response. Endogenous PCN acts as a fungal virulence factor, whereas exogenous purified PCN, administered to the host, confers protective immunity in a murine model of paracoccidioidomycosis. The immunomodulation induced by purified-PCN injection has characterized it as an agent applicable in the therapy and vaccine against paracoccidioidomycosis. This section describes methods for PCN purification and validation of its lectin and enzymatic activities. It includes detailed protocols to obtain homogeneous PCN from P. brasiliensis yeasts, as well as to purify recombinant PCN from transformed heterologous microorganisms.

3.
Methods Mol Biol ; 2132: 349-358, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306342

RESUMO

The immunomodulatory activity of plant lectins has been evaluated because of their high selectivity for glycans linked to receptors on innate and adaptative immune cells. ArtinM is a mannosyl-binding lectin, obtained from the seeds of Artocarpus heterophyllus, that induces the differentiation of CD4+ T cells and macrophages by interacting with CD3 and TLR2/CD14, respectively. This ArtinM property ultimately favors the combat of intracellular pathogens, opening new perspectives on the lectins application as immunomodulatory agents. The current section describes protocols for purification and evaluation of ArtinM biological activity. The purification is based on the ArtinM-D-mannose affinity. The effect of inducing IL-12 production by murine macrophages cell line is adopted to evaluate the ArtinM biological activity.

4.
Methods Mol Biol ; 2132: 379-389, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306345

RESUMO

Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.

5.
Methods Mol Biol ; 2132: 391-400, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306346

RESUMO

Some lectins of pathogens interact with host cells through the recognition of specific carbohydrates displayed on the mammals' cell surface. The microneme protein 1 (MIC1) from Toxoplasma gondii has a lectin domain that specifically binds sialic acid residues, often found in the terminal positions of N-glycans of mammalian cells. The necessary studies on the MIC1 biological roles have been limited initially by the laborious purification of the protein from T. gondii tachyzoites and the low yields verified. Then Escherichia coli has been transformed with a construct containing the MIC1 gene, and the obtained recombinant MIC1 (rMIC1) has been purified from the inclusion bodies. Herein, we detail the methodology of heterologous production and purification of rMIC1 and protocols to assay the rMIC1 lectin ability.

6.
Odontology ; 2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076883

RESUMO

Previous studies have shown that topical application of lectin Artin-M accelerates wound healing in the rat oral mucosa. The aim of this study was to evaluate, by means of histology and immunohistochemistry (IHC) the effects of Artin-M on wound healing in the palatal mucosa in dogs. Three full thickness wounds of 6 mm diameter were surgically created in the palatal mucosa of twenty dogs and randomly divided into three groups according to one of the treatment assigned: Group C-Control (coagulum); Group A-Artin-M gel; Group V-Vehicle (carboxymethylcellulose 3%). Each animal received all the three experimental treatments. Afterwards, four animals were killed at 2, 4, 7, 14 and 21 days post-surgery. Wounded areas were photographed and scored for macroscopic evaluation. Biopsies were harvested and used for descriptive histological analysis, proliferating cell nuclear antigen IHC and measurement of myeloperoxidase activity. The results demonstrated faster wound closure in group A in comparison to the other groups in all the periods evaluated. Histological analyses exhibited improved re-epithelialization and collagen fiber formation resulting in faster maturation of granulation tissue in group A compared to the other groups by day 14. Treatment with Artin-M gel significantly induced cell proliferation and increased volumetric density of fibroblasts at day 2 and 4 (p < 0.05). Neutrophil infiltration in group A was significantly higher than the other groups (p < 0.05) at the same time points. Collectively, our findings demonstrated that Artin-M may potentially favor wound healing on palatal mucosa lesions via recruitment of neutrophils and promotion of cell proliferation.

8.
Int J Mol Sci ; 20(20)2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658592

RESUMO

The microneme organelles of Toxoplasma gondii tachyzoites release protein complexes (MICs), including one composed of the transmembrane protein MIC6 plus MIC1 and MIC4. In this complex, carbohydrate recognition domains of MIC1 and MIC4 are exposed and interact with terminal sialic acid and galactose residues, respectively, of host cell glycans. Recently, we demonstrated that MIC1 and MIC4 binding to the N-glycans of Toll-like receptor (TLR) 2 and TLR4 on phagocytes triggers cell activation and pro-inflammatory cytokine production. Herein, we investigated the requirement for TLR2 heterodimerization and co-receptors in MIC-induced responses, as well as the signaling molecules involved. We used MICs to stimulate macrophages and HEK293T cells transfected with TLR2 and TLR1 or TLR6, both with or without the co-receptors CD14 and CD36. Then, the cell responses were analyzed, including nuclear factor-kappa B (NF-κB) activation and cytokine production, which showed that (1) only TLR2, among the studied factors, is crucial for MIC-induced cell activation; (2) TLR2 heterodimerization augments, but is not critical for, activation; (3) CD14 and CD36 enhance the response to MIC stimulus; and (4) MICs activate cells through a transforming growth factor beta-activated kinase 1 (TAK1)-, mammalian p38 mitogen-activated protein kinase (p38)-, and NF-κB-dependent pathway. Remarkably, among the studied factors, the interaction of MIC1 and MIC4 with TLR2 N-glycans is sufficient to induce cell activation, which promotes host protection against T. gondii infection.

9.
PLoS Pathog ; 15(6): e1007871, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31226171

RESUMO

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.


Assuntos
Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Macrófagos/imunologia , Proteínas de Protozoários/imunologia , Ácidos Siálicos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Interleucina-12/imunologia , Camundongos , Camundongos Knockout , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Toxoplasmose Animal/genética
10.
mSphere ; 4(2)2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-31019001

RESUMO

The thermodimorphic pathogenic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii are the etiologic causes of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Galectin-3 (Gal-3), an animal ß-galactoside-binding protein, modulates important roles during microbial infections, such as triggering a Th2-polarized immune response in PCM. Herein, we demonstrate that Gal-3 also plays other important roles in P. brasiliensis infection. We verified that Gal-3 levels are upregulated in human and mice infections and established that Gal-3 inhibited P. brasiliensis growth by inhibiting budding. Furthermore, Gal-3 affected disruption and internalization of extracellular vesicles (EVs) from P. brasiliensis by macrophages. Our results suggest important protective roles for Gal-3 in P. brasiliensis infection, indicating that increased Gal-3 production during P. brasiliensis infection may affect fungal growth and EV stability, thus promoting beneficial effects that could influence the course of PCM. The finding that Gal-3 has effects against P. brasiliensis together with previously reported effects against Cryptococcus neoformans suggests that molecule has a general antifungal role in innate defenses against fungal pathogens.IMPORTANCE Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Although the immune mechanisms to control PCM are still not fully understood, several events of the host innate and adaptive immunity are crucial to determine the progress of the infection. Mammalian ß-galactoside-binding protein galectin-3 (Gal-3) plays significant roles during microbial infections and has been studied for its immunomodulatory roles, but it can also have direct antimicrobial effects. We asked whether this protein plays a role in Paracoccidioides brasiliensis We report herein that Gal-3 indeed has direct effects on the fungal pathogen, inhibiting fungal growth and reducing extracellular vesicle stability. Our results suggest a direct role for Gal-3 in P. brasiliensis infection, with beneficial effects for the mammalian host.


Assuntos
Galectina 3/genética , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/imunologia , Animais , Antifúngicos , Modelos Animais de Doenças , Vesículas Extracelulares , Galectina 3/imunologia , Humanos , Imunidade Inata , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Viabilidade Microbiana , Regulação para Cima
11.
Molecules ; 23(9)2018 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-30216978

RESUMO

ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing Toll-like receptor (TLR)2 and cluster of differentiation (CD)14 N-glycans, induces cytokine production, and promotes type 1 T helper (Th1) immunity, a process that plays an assisting role in the combat against fungal infections. We recently demonstrated that ArtinM stimulates CD4⁺ T cells to produce interleukin (IL)-17 through direct interaction with CD3. Here, we further investigated the effects of ArtinM on the production of IL-17 by B cell activation. We showed that ArtinM activates murine B cells, increasing IL-17 and IL-12p40 production. The direct effect of ArtinM was sufficient to induce IL-17 production in B cells, and we did not find differences in the levels of IL-17 between the B cells purified from the wild-type (WT) and knockout (KO) mice for TLR2 or CD14 in the presence of ArtinM. Thus, the effects of ArtinM on splenic B cells through carbohydrate recognition may contribute to Th17 immunity; however, the mechanism involved is not associated with the interaction of ArtinM with TLR2 and CD14. The current work represents a pioneering effort in the understanding of the induction of IL-17 by lectins in B cells.


Assuntos
Linfócitos B/efeitos dos fármacos , Interleucina-17/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lectinas de Plantas/farmacologia , Receptor 2 Toll-Like/metabolismo , Animais , Artocarpus/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Receptores de Lipopolissacarídeos/genética , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Receptor 2 Toll-Like/genética
12.
Front Microbiol ; 9: 1780, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30186241

RESUMO

Salmonella enterica infection is a major public health concern worldwide, particularly when associated with other medical conditions. The serovars Typhimurium and Enteritidis are frequently associated with an invasive illness that primarily affects immunocompromised adults and children with HIV, malaria, or malnutrition. These serovars can also cause infections in a variety of animal hosts, and they are the most common isolates in poultry materials. Here, we described S. Enteritidis mutants, where hupA and hupB genes were deleted, and evaluated their potential use as live-attenuated vaccine candidates. In vitro, the mutants behaved like S. Typhimurium described previously, but there were some particularities in macrophage invasion and survival experiments. The virulence and immunogenicity of the mutant lacking both hupA and hupB (PT4ΔhupAB) were evaluated in a BALB/c mice model. This mutant was highly attenuated and could, therefore, be administrated at doses higher than 109 CFU/treatment, which was sufficient to protect all treated mice challenged with the wild-type parental strain with a single dose. Additionally, the PT4ΔhupAB strain induced production of specific IgG and IgA antibodies against Salmonella and TH1-related cytokines (IFN-γ and TNF-α), indicating that this strain can induce systemic and mucosal protection in the murine model. Additional studies are needed to better understand the mechanisms that lead to attenuation of the double-mutant PT4ΔhupAB and to elucidate the immune response induced by immunization using this strain. However, our data allow us to state that hupAB mutants could be potential candidates to be explore as live-attenuated vaccines.

13.
Front Microbiol ; 9: 867, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29780375

RESUMO

Infections caused by fungi are prominent in our environment and can be potentially fatal. paracoccidioidomycosis (PCM), caused by fungi of the Paracoccidioides genus, is the most frequent systemic mycosis in Brazil and the main cause of death among immunocompetent individuals. The antifungal therapy for PCM is usually effective but side effects and relapses are often reported. The latter could be avoided with alternative or complementary therapies aimed at boosting the immune response to combat this pathogen. Recent reports have pointed at the importance of an effective cellular immune response, with the participation of Th1 cells, in the resistance to and control of Paracoccidioides infection. The ArtinM lectin, extracted from jackfruit (Artocarpus heterophyllus) seeds, exhibits immunomodulatory activity against several intracellular pathogens, including Paracoccidioides brasiliensis, by promoting the development of a Th1 immune response. The aim of this work was to characterize the effect of ArtinM on peripheral blood cells of patients with PCM and on those of control individuals infected with fungal yeasts cells in vitro. Our results demonstrate that ArtinM activates human neutrophils in vitro, leading to an increase in cytokine production and CD54 expression. ArtinM activated P. brasiliensis-infected neutrophils from both healthy individuals and patients with PCM. This activation was not dependent on the dectin-1 receptor, because pre-incubation with laminarin, a dectin-1 receptor blocker, did not reverse the activated state of the cells. ArtinM also stimulated human peripheral blood mononuclear cells to secrete pro-inflammatory Th1-related cytokines, which are protective against Paracoccidioides infection. These data support the immunostimulatory action of ArtinM and encourage new studies using the lectin for the immunotherapy of PCM.

14.
Nat Commun ; 8(1): 1968, 2017 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-29213074

RESUMO

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian ß-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host.


Assuntos
Antifúngicos/imunologia , Antifúngicos/farmacologia , Criptococose/tratamento farmacológico , Criptococose/imunologia , Cryptococcus neoformans/efeitos dos fármacos , Galectina 3/imunologia , Galectina 3/farmacologia , Imunidade Adaptativa , Animais , Cápsulas Bacterianas/efeitos dos fármacos , Encéfalo/imunologia , Criptococose/microbiologia , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Galectina 3/sangue , Galectina 3/genética , Expressão Gênica , Humanos , Pulmão/imunologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia
15.
PLoS One ; 12(10): e0187151, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29084277

RESUMO

Toll-like receptors (TLR) contain N-glycans, which are important glycotargets for plant lectins, to induce immunomodulation. The lectin ArtinM obtained from Artocarpus heterophyllus interacts with TLR2 N-glycans to stimulate IL-12 production by antigen-presenting cells and to drive the immune response toward the Th1 axis, conferring resistance against intracellular pathogens. This immunomodulatory effect was demonstrated by subcutaneously injecting (s.c.) ArtinM (0.5 µg) in infected mice. In this study, we evaluated the systemic implications of ArtinM administration in naïve BALB/c mice. The mice were s.c. injected twice (7 days interval) with ArtinM (0.5, 1.0, 2.5, or 5.0 µg), LPS (positive control), or PBS (negative control) and euthanized after three days. None of the ArtinM-injected mice exhibited change in body weight, whereas the relative mass of the heart and lungs diminished in mice injected with the highest ArtinM dose (5.0 µg). Few and discrete inflammatory foci were detected in the heart, lung, and liver of mice receiving ArtinM at doses ≥2.5 µg. Moreover, the highest dose of ArtinM was associated with increased serum levels of creatine kinase MB isoenzyme (CK-MB) and globulins as well as an augmented presence of neutrophils in the heart and lung. IL-12, IFN-γ, TNF-α, and IL-10 measurements in the liver, kidney, spleen, heart, and lung homogenates revealed decreased IL-10 level in the heart and lung of mice injected with 5.0 µg ArtinM. We also found an augmented frequency of T helper and B cells in the spleen of all ArtinM-injected naïve mice, whereas the relative expressions of T-bet, GATA-3, and ROR-γt were similar to those in PBS-injected animals. Our study demonstrates that s.c. injection of high doses of ArtinM in naïve mice promotes mild inflammatory lesions and that a low immunomodulatory dose is innocuous to naïve mice.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Lectinas/administração & dosagem , Animais , Peso Corporal/efeitos dos fármacos , Cromatografia de Afinidade , Citocinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Tamanho do Órgão/efeitos dos fármacos
16.
PLoS One ; 12(8): e0184010, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28846733

RESUMO

Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-ß-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72-96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation.


Assuntos
Proteínas Fúngicas/metabolismo , Paracoccidioides/metabolismo , Paracoccidioides/crescimento & desenvolvimento , Aglutininas do Germe de Trigo/metabolismo
17.
Sci Rep ; 7(1): 7083, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28765651

RESUMO

Agonist interaction with Toll-like receptors (TLRs) induces T cell-mediated immunity, which is effective against intracellular pathogens. Consequently, TLR agonists are being tried as immunomodulatory agents. The lectin ArtinM targets TLR2 N-glycans on macrophages, induces cytokines production, and promotes T helper-1 immunity, a process that culminates in resistance to several parasitic and fungal infections in vivo. Because co-receptors influence agonist binding to TLRs, we investigated whether CD14 is required for macrophage activation induced by ArtinM. Macrophages from wild-type mice stimulated by ArtinM not only produced cytokines but also had the following activation profile: (i) expression of M1 polarization markers; (ii) nitrite oxide production; (iii) cellular migration; (iv) enhanced phagocytic and fungicide activity; (v) modulation of TLR2 expression; and (vi) activation of NF-κB pathway. This activation profile induced by ArtinM was evaluated in macrophages lacking CD14 that showed none of the ArtinM effects. We demonstrated by immunoprecipitation and sugar inhibition assays the physical interaction of ArtinM, TLR2, and CD14, which depends on recognition of the trimannoside that constitutes the core of N-glycans. Thus, our study showed that CD14 is critical for ArtinM-induced macrophage activation, providing fundamental insight into the design of anti-infective therapies based on carbohydrate recognition.


Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Ativação de Macrófagos/fisiologia , Receptor 2 Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Polissacarídeos/metabolismo
18.
mBio ; 8(4)2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28720727

RESUMO

Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, several recent studies have employed the methodology of gene modulation in P. brasiliensis using antisense RNA (AsRNA) and Agrobacterium tumefaciens-mediated transformation (ATMT) to identify proteins that influence fungus virulence. Our previous observations suggested that paracoccin (PCN), a multidomain fungal protein with both lectin and enzymatic activities, may be a potential P. brasiliensis virulence factor. To explore this, we used AsRNA and ATMT methodology to obtain three independent PCN-silenced P. brasiliensis yeast strains (AsPCN1, AsPCN2, and AsPCN3) and characterized them with regard to P. brasiliensis biology and pathogenicity. AsPCN1, AsPCN2, and AsPCN3 showed relative PCN expression levels that were 60%, 40%, and 60% of that of the wild-type (WT) strain, respectively. PCN silencing led to the aggregation of fungal cells, blocked the morphological yeast-to-mycelium transition, and rendered the yeast less resistant to macrophage fungicidal activity. In addition, mice infected with AsPCN1, AsPCN2, and AsPCN3 showed a reduction in fungal burden of approximately 96% compared with those inoculated with the WT strain, which displayed a more extensive destruction of lung tissue. Finally, mice infected with the PCN-silenced yeast strains had lower mortality than those infected with the WT strain. These data demonstrate that PCN acts as a P. brasiliensis contributory virulence factor directly affecting fungal pathogenesis.IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis, the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens-mediated transformation system, provides new strategies for studying P. brasiliensis Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis, we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis.


Assuntos
Proteínas Fúngicas/metabolismo , Inativação Gênica , Lectinas/metabolismo , Paracoccidioides/patogenicidade , Fatores de Virulência/metabolismo , Animais , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Lectinas/genética , Masculino , Camundongos Endogâmicos BALB C , Micélio/citologia , Micélio/crescimento & desenvolvimento , Paracoccidioides/citologia , Paracoccidioides/genética , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/patologia , Análise de Sobrevida , Virulência , Fatores de Virulência/genética
19.
Int J Mol Sci ; 18(7)2017 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-28665310

RESUMO

The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4⁺ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4⁺ and CD8⁺ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4⁺ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4⁺ and CD8⁺ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1ß production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia.


Assuntos
Morte Celular/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Lectinas de Ligação a Manose/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
20.
Methods Mol Biol ; 1625: 159-167, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28584990

RESUMO

Adjuvants and immunomodulatory molecules could be included in the treatment of P. brasiliensis infection. In this context, we reported that the therapeutic and/or prophylactic administration of Th1-inducing agents, such as immunomodulatory lectins and adjuvants, was able to provide protection against experimental paracoccidioidomycosis. Then, we described the protocols to investigate the effect of immunomodulatory agents on the course of P. brasiliensis infection. In this sense, we detailed the measurement of fungal burden and cytokine production, and the histopathological analysis used to evaluate the most effective administration regime.


Assuntos
Blastomyces/imunologia , Fatores Imunológicos/farmacologia , Paracoccidioidomicose/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Adjuvantes Imunológicos , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Imunomodulação/efeitos dos fármacos , Masculino , Camundongos , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/prevenção & controle , Paracoccidioidomicose/terapia , Células Th1/metabolismo
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