RESUMO
Several epidemiological studies have identified the cigarette smoke as a risk factor for the infection and development of tuberculosis. Nicotine is considered the main immunomodulatory molecule of the cigarette. In the present study, we evaluated the effect of nicotine in the growth of M. tuberculosis. Lung epithelial cells and macrophages were infected with M. tuberculosis and/or treated with nicotine. The results show that nicotine increased the growth of M. tuberculosis mainly in type II pneumocytes (T2P) but not in airway basal epithelial cells nor macrophages. Further, it was observed that nicotine decreased the production of ß-defensin-2, ß-defensin-3, and the cathelicidin LL-37 in all the evaluated cells at 24 and 72 h post-infection. The modulation of the expression of antimicrobial peptides appears to be partially mediated by the nicotinic acetylcholine receptor α7 since the blockade of this receptor partially reverted the production of antimicrobial peptides. In summary, it was found that nicotine decreases the production of HBD-2, HBD-3, and LL-37 in T2P during the infection with M. tuberculosis promoting its intracellular growth.
Assuntos
Células Epiteliais Alveolares/microbiologia , Mycobacterium tuberculosis/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Tuberculose Pulmonar/microbiologia , Células A549 , Células Epiteliais Alveolares/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Carga Bacteriana , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose Pulmonar/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , beta-Defensinas/metabolismo , CatelicidinasRESUMO
Tuberculosis kills more than 1 million people every year, and its control depends on the effective mechanisms of innate immunity, with or without induction of adaptive immune response. We investigated the interaction of type II alveolar epithelial cells (AEC-II) infected by Mycobacterium tuberculosis with dendritic cells (DCs). We hypothesized that the microenvironment generated by this interaction is critical for the early innate response against mycobacteria. We found that AEC-II infected by M. tuberculosis induced DC maturation, which was negatively regulated by HIF-1α-inducible NOS2 axis, and switched DC metabolism from an early and short peak of glycolysis to a low energetic status. However, the infection of DCs by M. tuberculosis up-regulated NOS2 expression and inhibited AEC-II-induced DC maturation. Our study demonstrated, for the first time, that HIF-1α-NOS2 axis plays a negative role in the maturation of DCs during M. tuberculosis infection. Such modulation might be useful for the exploitation of molecular targets to develop new therapeutic strategies against tuberculosis.
Assuntos
Células Epiteliais Alveolares/imunologia , Células Dendríticas/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Mycobacterium tuberculosis/imunologia , Óxido Nítrico Sintase Tipo II/imunologia , Tuberculose Pulmonar/imunologia , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/patologia , Animais , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/microbiologiaRESUMO
The cell wall is one of the most important structures of pathogenic fungi, enabling initial interaction with the host and consequent modulation of immunological responses. Over the years, some researchers have shown that cell wall components of Histoplasma capsulatum vary among fungal isolates, and one of the major differences is the presence or absence of α-(1,3)-glucan, classifying wild-type fungi as chemotypes II or I, respectively. The present work shows that an isolate of H. capsulatum chemotype I induced lower levels of interleukin (IL)-8 secretion by the lung epithelial cell line A549, when compared to chemotype II yeasts. Thus, we expected that the absence of α-glucan in spontaneous variant yeasts, which were isolated from chemotype II cultures, would modify IL-8 secretion by A549 cells, but surprisingly, these fungi promoted similar levels of IL-8 secretion as their wild-type counterpart. Furthermore, when using a specific inhibitor for Syk activation, we observed that this inhibitor reduced IL-8 levels in A549 cell cultures infected with wild type chemotype I fungi. This inhibitor failed to reduce this cytokine levels in A549 cell cultures infected with chemotype II and their spontaneous variant yeasts, which also do not present α-glucan on their surface. The importance of SFKs and PKC δ in this event was also analyzed. Our results show that different isolates of H. capsulatum modulate distinct cell signaling pathways to promote cytokine secretion in host epithelial cells, emphasizing the existence of various mechanisms for Histoplasma pathogenicity.
Assuntos
Células Epiteliais Alveolares/metabolismo , Histoplasma/metabolismo , Interleucina-8/metabolismo , Células A549 , Células Epiteliais Alveolares/microbiologia , Parede Celular/metabolismo , Glucanos/metabolismo , Histoplasma/isolamento & purificação , Interações Hospedeiro-Patógeno , Humanos , Pulmão/patologia , Proteína Quinase C-delta/metabolismo , Transdução de Sinais , Especificidade da Espécie , Quinase Syk/metabolismo , Quinases da Família src/metabolismoRESUMO
Smoking increases susceptibility to becoming infected with and developing tuberculosis. Among the components of cigarette smoke, nicotine has been identified as the main immunomodulatory molecule; however, its effect on the innate immune system is unknown. In the present study, the effect of nicotine on molecules of the innate immune system was evaluated. Lung epithelial cells and macrophages were infected with Mycobacterium tuberculosis (Mtb) and/or treated with nicotine. The results show that nicotine alone decreases the expression of the Toll-like receptors (TLR)-2, TLR-4 and NOD-2 in all three cell types, as well as the production of the SP-D surfactant protein in type II pneumocytes. Moreover, it was observed that nicotine decreases the production of interleukin (IL)-6 and C-C chemokine ligand (CCL)5 during Mtb infection in epithelial cells (EpCs), whereas in macrophages derived from human monocytes (MDMs) there is a decrease in IL-8, IL-6, tumor necrosis factor (TNF)-α, IL-10, CCL2, C-X-C chemokine ligand (CXCL)9 and CXCL10 only during infection with Mtb. Although modulation of the expression of cytokines and chemokines appears to be partially mediated by the nicotinic acetylcholine receptor α7, blocking this receptor found no effect on the expression of receptors and SP-D. In summary, it was found that nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis.
Assuntos
Células Epiteliais Alveolares/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Nicotina/farmacologia , Tuberculose Pulmonar/imunologia , Células A549 , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/patologia , Citocinas/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Macrófagos/microbiologia , Macrófagos/patologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Tuberculose Pulmonar/patologiaRESUMO
Fungi that belong to the genus Paracoccidioides are the etiologic agents of paracoccidioidomycosis, a human systemic mycosis, which occurs in Latin America. Epithelial cell is one of the first cells that interact with these fungi and responds by secreting inflammatory mediators such as cytokines. In the present study, we demonstrate that yeasts of different isolates of Paracoccidioides brasiliensis (Pb18 and Pb03) and Paracoccidioides lutzii (Pb01) distinctly promoted interleukin (IL)-8 secretion by the lung epithelial cell line A549. Depending on the isolate, this cytokine release may rely on the epithelial cell interaction with fungal secreted components or direct contact with the pathogen. In addition, adhesion of yeasts to the pulmonary epithelial cells was also different among Paracoccidioides isolates, and the highest percentage of A549 cells with adhered fungi was observed with P. lutzii. All Paracoccidioides isolates induced an expression increase of α3 and α5 integrins in A549 cells and, using small interfering RNA, we observed that the integrin silencing promoted a reduction of P. lutzii adhesion, which suggests the involvement of integrins in this event. Together, these results indicate that host epithelial cell response may depend on the isolate of Paracoccidioides.
Assuntos
Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/microbiologia , Interleucina-8/biossíntese , Paracoccidioides/fisiologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , Células A549 , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Inativação Gênica , Humanos , Integrinas/genéticaRESUMO
The Paracoccidioides brasiliensis strain downregulated the expression of adhesin Pb14-3-3 (Pb14-3-3 aRNA) was evaluated in a murine model of paracoccidioidomycosis (PCM). Pb14-3-3 aRNA displays attenuated virulence and triggered the formation of fewer granulomas by lowering the fungal burden in the lungs. Additionally, the Pb14-3-3 aRNA showed more elongated yeast cells and less ability to induce pneumocytes apoptosis in vitro. Our results show that 14-3-3 is an important virulence factor in P. brasiliensis-induced pulmonary infection.
Assuntos
Proteínas 14-3-3/genética , Proteínas Fúngicas/genética , Paracoccidioides/genética , Paracoccidioides/patogenicidade , Fatores de Virulência/genética , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/patologia , Animais , Apoptose/genética , Modelos Animais de Doenças , Expressão Gênica , Pulmão/citologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioidomicose/microbiologiaRESUMO
Tuberculosis is a disease caused by Mycobacterium tuberculosis (Mtb). Innate immunity is the first line of defense against Mtb and malfunctions in any of its components are associated with the susceptibility to the disease. Epithelial products such as host defense peptides (HDPs) are the first molecules produced to counteract the infection. Although a wide variety of HDPs are produced by epithelial cells only a few of them have been studied during Mtb infection. Here, we assessed the expression and production of the HDPs psoriasin, secreted phospholipases A2 (sPLA2-IIA) and Ribonuclease (RNase) 7 in airway epithelial cells (NCI-H292), type II pneumocytes (A549 cells) and monocyte-derived macrophages from human peripheral blood mononuclear cells and from the human cell line THP1 after Mtb in vitro infection. Results show that psoriasin and sPLA2-IIA were not induced by Mtb in any of the evaluated cells, while RNase 7 was overexpressed in infected airway epithelial cells. Intracellular analysis by flow cytometry demonstrated that the highest levels of RNase 7 were observed 6 h post-infection and the induction was dependent on direct interaction between airway epithelial cells and Mtb. In addition, analysis by electron microscopy showed that RNase 7 was capable of attaching to the cell wall of intracellular mycobacteria. Our studies suggest that the induction of RNase 7 in response to Mtb could have a role in anti-mycobacterial immunity, which needs to be studied as an innate immune mechanism.
Assuntos
Células Epiteliais Alveolares/microbiologia , Fosfolipases A2 do Grupo II/metabolismo , Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/metabolismo , Ribonucleases/metabolismo , Proteína A7 Ligante de Cálcio S100/metabolismo , Células A549 , Células Epiteliais Alveolares/imunologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Monócitos/imunologia , Monócitos/microbiologiaRESUMO
BACKGROUND: Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection. RESULTS: CRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h. CONCLUSION: M. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by avoiding phagosome maturation. Once recovered from infected macrophages, CRM0019 remains infective and displays greater intracellular growth in A549 cells compared to the ATCC 19977 strain. This evasion strategy in alveolar epithelial cells may contribute to the long survival of the CRM0019 strain in the host and thus to the inefficacy of in vivo treatment.
Assuntos
Células Epiteliais Alveolares/microbiologia , Proliferação de Células , Interações Hospedeiro-Patógeno/fisiologia , Viabilidade Microbiana , Mycobacterium abscessus/fisiologia , Mycobacterium abscessus/patogenicidade , Células A549 , Animais , Contagem de Colônia Microbiana , Humanos , Evasão da Resposta Imune , Lisossomos/metabolismo , Macrófagos/microbiologia , Camundongos , Fagossomos/microbiologia , Células RAW 264.7RESUMO
Apoptosis is considered an escape mechanism from the host immune system for the fungus Paracoccidioides spp, and it serves as a vehicle for entry into macrophages without stimulating microbicidal activities. Recently, gp43 of P. brasiliensis was demonstrated to be involved in this process. Therefore, as a new therapeutic alternative, it is very important to study compounds that could reduce the modulation of the induction of apoptosis caused by this fungus. Decyl gallate (G14) is a known antifungal compound, and we decided to investigate its anti-apoptotic properties. Our results demonstrate that G14 was effective against apoptosis induced by gp43, as observed in epithelial cells, and led to a reduction in DNA damage, Bak down-regulation and Bcl-2 up-regulation. Together, these data show that G14 presents promising anti-apoptotic activity.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Glicoproteínas/fisiologia , Paracoccidioides/fisiologia , Células A549 , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/patologia , Antígenos de Fungos/metabolismo , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Genes bcl-2/genética , Humanos , Paracoccidioidomicose/fisiopatologia , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
Paracoccidioides spp., which are temperature-dependent dimorphic fungi, are responsible for the most prevalent human systemic mycosis in Latin America, the paracoccidioidomycosis. The aim of this study was to characterise the involvement of elongation factor Tu (EF-Tu) in Paracoccidioides brasiliensis-host interaction. Adhesive properties were examined using recombinant PbEF-Tu proteins and the respective polyclonal anti-rPbEF-Tu antibody. Immunogold analysis demonstrated the surface location of EF-Tu in P. brasiliensis. Moreover, PbEF-Tu was found to bind to fibronectin and plasminogen by enzyme-linked immunosorbent assay, and it was determined that the binding to plasminogen is at least partly dependent on lysine residues and ionic interactions. To verify the participation of EF-Tu in the interaction of P. brasiliensis with pneumocytes, we blocked the respective protein with an anti-rPbEF-Tu antibody and evaluated the consequences on the interaction index by flow cytometry. During the interaction, we observed a decrease of 2- and 3-fold at 8 and 24 h, respectively, suggesting the contribution of EF-Tu in fungal adhesion/invasion.
Assuntos
Interações Hospedeiro-Patógeno , Paracoccidioides/enzimologia , Fator Tu de Elongação de Peptídeos/metabolismo , Fatores de Virulência/metabolismo , Células Epiteliais Alveolares/microbiologia , Adesão Celular , Linhagem Celular , Fibronectinas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Paracoccidioides/fisiologia , Plasminogênio/metabolismo , Ligação ProteicaRESUMO
High glucose concentration in the airway surface liquid (ASL) is an important feature of diabetes that predisposes to respiratory infections. We investigated the role of alveolar epithelial SGLT1 activity on ASL glucose concentration and bacterial proliferation. Non-diabetic and diabetic rats were intranasally treated with saline, isoproterenol (to increase SGLT1 activity) or phlorizin (to decrease SGLT1 activity); 2 hours later, glucose concentration and bacterial proliferation (methicillin-resistant Sthaphylococcus aureus, MRSA and Pseudomonas aeruginosa, P. aeruginosa) were analyzed in bronchoalveolar lavage (BAL); and alveolar SGLT1 was analyzed by immunohistochemistry. BAL glucose concentration and bacterial proliferation increased in diabetic animals: isoproterenol stimulated SGLT1 migration to luminal membrane, and reduced (50%) the BAL glucose concentration; whereas phlorizin increased the BAL glucose concentration (100%). These regulations were accompanied by parallel changes of in vitro MRSA and P. aeruginosa proliferation in BAL (r = 0.9651 and r = 0.9613, respectively, Pearson correlation). The same regulations were observed in in vivo P. aeruginosa proliferation. In summary, the results indicate a relationship among SGLT1 activity, ASL glucose concentration and pulmonary bacterial proliferation. Besides, the study highlights that, in situations of pulmonary infection risk, such as in diabetic subjects, increased SGLT1 activity may prevent bacterial proliferation whereas decreased SGLT1 activity can exacerbate it.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Pulmão/metabolismo , Pneumonia Bacteriana/metabolismo , Infecções por Pseudomonas/metabolismo , Transportador 1 de Glucose-Sódio/genética , Infecções Estafilocócicas/metabolismo , Aloxano , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/microbiologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/microbiologia , Broncodilatadores/farmacologia , Contagem de Colônia Microbiana , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Regulação da Expressão Gênica , Isoproterenol/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Florizina/farmacologia , Pneumonia Bacteriana/complicações , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/patologia , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Ratos , Ratos Wistar , Transportador 1 de Glucose-Sódio/agonistas , Transportador 1 de Glucose-Sódio/antagonistas & inibidores , Transportador 1 de Glucose-Sódio/metabolismo , Infecções Estafilocócicas/complicações , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/patologiaRESUMO
Both CCL20 and human ß-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1ß, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 µg/ml) was markedly higher than that against E. coli (1.5 µg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site.
Assuntos
Células Epiteliais Alveolares/metabolismo , Brucella abortus/imunologia , Brucelose/metabolismo , Quimiocina CCL20/biossíntese , beta-Defensinas/biossíntese , Células Epiteliais Alveolares/microbiologia , Antibacterianos/farmacologia , Brucelose/imunologia , Brucelose/microbiologia , Linhagem Celular , Linhagem Celular Tumoral , Quimiocina CCL20/metabolismo , Quimiocina CCL20/farmacologia , Humanos , Imunidade Inata , Lipopolissacarídeos/farmacologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Sistema de Sinalização das MAP Quinases , Testes de Sensibilidade Microbiana , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/microbiologia , Receptor 2 Toll-Like/metabolismo , beta-Defensinas/metabolismo , beta-Defensinas/farmacologiaRESUMO
Streptococcus pneumoniae is a major human pathogen that can survive to stress conditions, such as the acidic environment of inflammatory foci, and tolerates lethal pH through a mechanism known as the acid tolerance response. We previously described that S. pneumoniae activates acidic-stress induced lysis in response to acidified environments, favoring the release of cell wall compounds, DNA and virulence factors. Here, we demonstrate that F(0)F(1)-ATPase is involved in the response to acidic stress. Chemical inhibitors (DCCD, optochin) of this proton pump repressed the ATR induction, but caused an increased ASIL. Confirming these findings, mutants of the subunit c of this enzyme showed the same phenotypes as inhibitors. Importantly, we demonstrated that F(0)F(1)-ATPase and ATR are necessary for the intracellular survival of the pneumococcus in macrophages. Alternatively, a screening of two-component system (TCS) mutants showed that ATR and survival in pneumocytes were controlled in contrasting ways by ComDE and CiaRH, which had been involved in the ASIL mechanism. Briefly, CiaRH was essential for ATR (ComE represses activation) whereas ComE was necessary for ASIL (CiaRH protects against induction). They did not regulate F0F1-ATPase expression, but control LytA expression on the pneumococcal surface. These results suggest that both TCSs and F(0)F(1)-ATPase control a stress response and decide between a survival or a suicide mechanism by independent pathways, either in vitro or in pneumocyte cultures. This biological model contributes to the current knowledge about bacterial response under stress conditions in host tissues, where pathogens need to survive in order to establish infections.
Assuntos
Viabilidade Microbiana , ATPases Translocadoras de Prótons/metabolismo , Transdução de Sinais , Streptococcus pneumoniae/fisiologia , Estresse Fisiológico , Ácidos/toxicidade , Células Epiteliais Alveolares/microbiologia , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Camundongos , ATPases Translocadoras de Prótons/genética , Streptococcus pneumoniae/genéticaRESUMO
The capsule is the most important virulence factor of the fungal pathogen Cryptococcus neoformans. This structure consists of highly hydrated polysaccharides, including glucuronoxylomannan (GXM), and galactoxylomannan (GalXM). It is also composed of mannoproteins (MPs) which corresponds to less than 1% of the capsular weight. Despite MPs being the minority and least studied components, four of these molecules with molecular masses of 115, 98, 88, and 84 kDa were identified and characterized as C. neoformans immunoreactive antigens involved in the pathogenesis, and are potential cryptococcosis vaccine candidates. With the aim to describe the adhesive property of MPs, we cloned and expressed the MP84, a mannoprotein with molecular weight of 84 kDa, on Pichia pastoris yeast, and performed interaction assays of C. neoformans with epithelial lung cells, in the presence or absence of capsule components. Two fungal strains, the wild type, NE-241, and a mutant, CAP67, deficient in GXM production, were used throughout this study. The adhesion assays were completed using epithelial lung cells, A549, and human prostate cancer cells, PC3, as a control. We observed that capsulated wild type (NE-241), and acapsular (CAP67) strains adhered significantly to A549 cells, compared with PC3 cells (p < 0.05). GXM inhibits the NE-241 adhesion, but not the CAP67. In contrast, CAP67 adhesion was only inhibited in the presence of MP84. These results demonstrate the involvement of MP in the adhesion of C. neoformans to epithelial lung cells. We conclude that this interaction possibly involves an adhesion-like interaction between MP on the fungal surface and the complementary receptor molecules on the epithelial cells.
Assuntos
Células Epiteliais Alveolares/microbiologia , Aderência Bacteriana , Proteínas de Bactérias , Cryptococcus neoformans/fisiologia , Glicoproteínas de Membrana/metabolismo , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The present paper is an overview of the primary events that are associated with the histoplasmosis immune response in the murine model. Valuable data that have been recorded in the scientific literature have contributed to an improved understanding of the clinical course of this systemic mycosis, which is caused by the dimorphic fungus Histoplasma capsulatum. Data must be analyzed carefully, given that misinterpretation could be generated because most of the available information is based on experimental host-parasite interactions that used inappropriate proceedings, i.e., the non-natural route of infection with the parasitic and virulent fungal yeast-phase, which is not the usual infective phase of the etiological agent of this mycosis. Thus, due to their versatility, complexity, and similarities with humans, several murine models have played a fundamental role in exploring the host-parasite interaction during H. capsulatum infection.
Assuntos
Histoplasma/imunologia , Histoplasmose/imunologia , Imunidade Inata , Imunidade Adaptativa , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/microbiologia , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/microbiologia , Parede Celular/química , Parede Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Histoplasma/crescimento & desenvolvimento , Histoplasma/patogenicidade , Histoplasmose/microbiologia , Histoplasmose/patologia , Especificidade de Hospedeiro , Humanos , Camundongos , Especificidade da Espécie , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/microbiologiaRESUMO
Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B. pertussis inside the epithelia. We show here that B. pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B. pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B. pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.
Assuntos
Bordetella pertussis/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/microbiologia , Linhagem Celular , Humanos , Espaço Intracelular/microbiologia , Microdomínios da Membrana/metabolismo , Microtúbulos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Coqueluche/imunologia , Coqueluche/microbiologiaRESUMO
The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.
Assuntos
Células Epiteliais Alveolares/microbiologia , Biofilmes , Histoplasma/patogenicidade , Células Epiteliais Alveolares/metabolismo , Adesão Celular , Linhagem Celular , Histoplasma/metabolismo , Histoplasma/fisiologia , Histoplasmose/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Microscopia Eletrônica de Varredura , Sais de Tetrazólio/metabolismoRESUMO
Transcript profiling is an invaluable strategy to study differential gene expression. Here we describe a detailed protocol for applying a subtractive hybridization technique, representational difference analysis (RDA), as a molecular strategy for the identification of differentially expressed genes in studies of host-fungus interaction. Bioinformatics tools that can be used in the analysis of expressed sequence tags (ESTs) are also detailed.
Assuntos
Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Paracoccidioides/genética , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/imunologia , Células Epiteliais Alveolares/microbiologia , Animais , Biologia Computacional , Interações Hospedeiro-Patógeno , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioides/imunologia , Paracoccidioides/fisiologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/microbiologiaRESUMO
Mycobacterium tuberculosis infection continues to be a major cause of morbidity and mortality throughout the world. The vast complexity of the intracellular pathogen M. tuberculosis and the diverse mechanisms by which it can invade host cells highlight the importance of developing a fully protective vaccine. Our vaccine development strategy consists of including fragments from multiple mycobacterial proteins involved in cell invasion. The aim of this study was to identify high activity binding peptides (HABPs) in the immunogenic protein Rv1980c from M. tuberculosis H37Rv with the ability to inhibit mycobacterial invasion into U937 monocyte-derived macrophages and A549 cells. The presence and transcription of the Rv1980c gene was assessed in members belonging to the M. tuberculosis complex and other nontuberculous mycobacteria by PCR and RT-PCR, respectively. Cell surface localization was confirmed by immuno-electron microscopy. Three peptides binding with high activity to U937 cells and one to A549 cells were identified. HABPs 31100, 31101, and 31107 inhibited invasion of M. tuberculosis into A549 and U937 cells and therefore could be promising candidates for the design of a subunit-based antituberculous vaccine.