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1.
PLoS Pathog ; 15(11): e1008163, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31738806

RESUMO

Macrophages mediate the elimination of pathogens by phagocytosis resulting in the activation of specific signaling pathways that lead to the production of cytokines, chemokines and other factors. Borrelia burgdorferi, the causative agent of Lyme disease, causes a wide variety of pro-inflammatory symptoms. The proinflammatory capacity of macrophages is intimately related to the internalization of the spirochete. However, most receptors mediating this process are largely unknown. We have applied a multiomic approach, including the proteomic analysis of B. burgdorferi-containing phagosome-enriched fractions, to identify surface receptors that are involved in the phagocytic capacity of macrophages as well as their inflammatory output. Sucrose gradient protein fractions of human monocyte-derived macrophages exposed to B. burgdorferi contained the phagocytic receptor, CR3/CD14 highlighting the major role played by these proteins in spirochetal phagocytosis. Other proteins identified in these fractions include C-type lectins, scavenger receptors or Siglecs, of which some are directly involved in the interaction with the spirochete. We also identified the Fc gamma receptor pathway, including the binding receptor, CD64, as involved both in the phagocytosis of, and TNF induction in response to B. burgdorferi in the absence of antibodies. The common gamma chain, FcγR, mediates the phagocytosis of the spirochete, likely through Fc receptors and C-type lectins, in a process that involves Syk activation. Overall, these findings highlight the complex array of receptors involved in the phagocytic response of macrophages to B. burgdorferi.

2.
Biochem J ; 476(14): 1995-2016, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320388

RESUMO

Bacterial capsules have evolved to be at the forefront of the cell envelope, making them an essential element of bacterial biology. Efforts to understand the Mycobacterium tuberculosis (Mtb) capsule began more than 60 years ago, but the relatively recent development of mycobacterial genetics combined with improved chemical and immunological tools have revealed a more refined view of capsule molecular composition. A glycogen-like α-glucan is the major constituent of the capsule, with lower amounts of arabinomannan and mannan, proteins and lipids. The major Mtb capsular components mediate interactions with phagocytes that favor bacterial survival. Vaccination approaches targeting the mycobacterial capsule have proven successful in controlling bacterial replication. Although the Mtb capsule is composed of polysaccharides of relatively low complexity, the concept of antigenic variability associated with this structure has been suggested by some studies. Understanding how Mtb shapes its envelope during its life cycle is key to developing anti-infective strategies targeting this structure at the host-pathogen interface.

3.
Adv Exp Med Biol ; 1140: 289-298, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347054

RESUMO

Membrane proteins are of utmost importance in different cellular processes including: cell signaling, substrate transport, homeostasis control, immune surveillance, etc. In addition, they represent between 60% and 70% of the therapeutic targets currently used. Therefore, the identification and characterization of these proteins is crucial in many fields of research. Although proteomics has undergone an extraordinary advance in recent years thanks to the development of mass spectrometry, the methods used for the identification and quantification of soluble proteins generally fail to be used for membrane proteins, mainly due to their hydrophobic character.In this chapter, we revised the different alternatives, modifications and improvements that have been developed over the years with the aim of adapting the methods used in proteomics to the particular study of membrane proteins, thus allowing to increase the number of membrane proteins identified, as well as their coverage.


Assuntos
Espectrometria de Massas , Proteínas de Membrana/análise , Proteômica , Interações Hidrofóbicas e Hidrofílicas
4.
BMC Vet Res ; 15(1): 188, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174546

RESUMO

BACKGROUND: Both bovine tuberculosis (TB) and paratuberculosis (PTB) are serious and widespread bacterial infections affecting many domestic and wild animal species. However, current vaccines do not confer complete protection and cause interference with other diagnostics tests, including bovine TB. Therefore, the development of "Differentiating Infected from Vaccinated Animals" (DIVA) tests are a pressing need. In this study, we have tested the feasibility of mycobacterial extracellular vesicles (EVs) as potential source of biomarkers to discriminate between Mycobacterium bovis infected, Mycobacterium avium subsp. paratuberculosis (MAP) infected and MAP-vaccinated cows. We have, initially, characterized vesicle production in the two most medically relevant species of mycobacteria for livestock, MAP and M. bovis, for being responsible for tuberculosis (TB) and paratuberculosis (PTB). RESULTS: Our results indicate that these two species produce EVs with different kinetics, morphology and size distribution. Analysis of the immunogenicity of both type of EVs showed some cross reactivity with sera from PTB+ and TB+ cows, suggesting a limited diagnostic capacity for both EVs. Conversely, we noticed that Mycobacterium tuberculosis (Mtb) EVs showed some differential reactivity between sera from MAP-vaccinated or PTB+ cows from TB+ ones. Mass spectrometry analysis (MS) identified a 19-kDa EV-associated lipoprotein as the main source of the differential reactivity. CONCLUSIONS: LpqH could be a good plasma biomarker with capacity to distinguish PTB+ or MAP-vaccinated cows from cows infected with TB.


Assuntos
Doenças dos Bovinos/microbiologia , Vesículas Extracelulares/química , Lipoproteínas/análise , Mycobacterium tuberculosis/química , Paratuberculose/diagnóstico , Tuberculose Bovina/diagnóstico , Animais , Vacinas Bacterianas , Biomarcadores/sangue , Bovinos , Reações Cruzadas , Mycobacterium avium subsp. paratuberculosis/química , Mycobacterium bovis/química , Vacinação/veterinária
5.
J Biol Chem ; 294(27): 10471-10489, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31118223

RESUMO

Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins, such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-associated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans We observed that melanin is assembled into the cryptococcal cell wall in spherical structures ∼200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres ∼30 nm in diameter, termed fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans' melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.

6.
mSphere ; 4(2)2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30918063

RESUMO

Changes in serum glycans discriminate between disease statuses in cancer. A similar connection has not been established in the context of infectious diseases such as tuberculosis (TB). The inflammation arising from infection by Mycobacterium tuberculosis may affect host protein glycosylation, thereby providing information about disease status in TB. A mouse model of infection was used to study glycoprotein N-glycosylation in serum. Following digestion of serum glycoproteins with peptide-N-glycosidase F (PNGase F), released glycans were permethylated and analyzed by multidimensional mass spectrometry (MS). Conditions included naive or Mycobacterium bovis BCG-vaccinated animals, which were either uninfected or infected with M. tuberculosis MS results were validated by lectin blotting. We found that both glycoprotein fucosylation and sialylation were particularly sensitive to M. tuberculosis infection. We observed that M. tuberculosis infection elevates serum IgM levels and induces changes in glycosylation that could inform about the disease.IMPORTANCE We demonstrate that M. tuberculosis infection influenced host protein glycosylation in a mouse model. The mechanism by which infection modifies glycans in serum proteins is not understood. Investigation of the regulation of such modifications by M. tuberculosis opens a new field that could lead to the discovery of novel biomarkers. Validation of such findings in human samples will reveal the clinical relevance of these findings.


Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina M/química , Polissacarídeos/sangue , Tuberculose/imunologia , Animais , Vacina BCG/imunologia , Modelos Animais de Doenças , Feminino , Glicoproteínas/sangue , Glicoproteínas/química , Glicosilação , Imunoglobulina M/sangue , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Tuberculose/sangue , Vacinação
7.
J Biol Chem ; 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504226

RESUMO

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis.  Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins.  Using immunogold electron microscopy we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

8.
J Biol Chem ; 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30385508

RESUMO

Natural brown-black eumelanin pigments confer structural coloration in animals and potently block ionizing radiation and antifungal drugs. These functions also make them attractive for bioinspired materials design, including coating materials for drug-delivery vehicles, strengthening agents for adhesive hydrogel materials, and free-radical scavengers for soil remediation. Nonetheless, the molecular determinants of the melanin 'developmental road traveled' and the resulting architectural features have remained uncertain because of the insoluble, heterogeneous, and amorphous characteristics of these complex polymeric assemblies. Here, we used 2D solid-state NMR, EPR, and dynamic nuclear polarization (DNP) spectroscopic techniques, assisted in some instances by the use of isotopically enriched precursors, to address several open questions regarding the molecular structures and associated functions of eumelanin. Our findings uncovered (1) that the identity of the available catecholamine precursor alters the structure of melanin pigments produced either in Cryptococcus neoformans fungal cells or under cell-free conditions; (2) that the identity of the available precursor alters the scaffold organization and membrane lipid content of melanized fungal cells; (3) that the fungal cells are melanized preferentially by an L-dopa precursor; (4) and that the macromolecular carbon- and nitrogen-based architecture of cell-free L-dopa and fungal dopamine melanins includes indole, pyrrole, indolequinone, and open-chain building blocks that develop depending on reaction time. In conclusion, the availability of catecholamine precursors plays an important role in eumelanin development by affecting the efficacy of pigment formation, the melanin molecular structure, and its underlying scaffold in fungal systems.

9.
Curr Opin Immunol ; 53: 30-37, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29656063

RESUMO

A more effective vaccine to control tuberculosis (TB), a major global public health problem, is urgently needed. Current vaccine candidates focus predominantly on eliciting cell-mediated immunity but other arms of the immune system also contribute to protection against TB. We review here recent studies that enhance our current knowledge of antibody-mediated functions against Mycobacterium tuberculosis. These findings, which contribute to the increasing evidence that antibodies have a protective role against TB, include demonstrations that firstly distinct human antibody Fc glycosylation patterns, found in latent M. tuberculosis infection but not in active TB, influence the efficacy of the host to control M. tuberculosis infection, secondly antibody isotype influences human antibody functions, and thirdly that antibodies targeting M. tuberculosis surface antigens are protective. We discuss these findings in the context of TB vaccine development and highlight the need for further research on antibody-mediated immunity in M. tuberculosis infection.

10.
Emerg Microbes Infect ; 7(1): 19, 2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29511161

RESUMO

Macrophages are cells of the innate immune system with the ability to phagocytose and induce a global pattern of responses that depend on several signaling pathways. We have determined the biosignature of murine bone marrow-derived macrophages and human blood monocytes using transcriptomic and proteomic approaches. We identified a common pattern of genes that are transcriptionally regulated and overall indicate that the response to B. burgdorferi involves the interaction of spirochetal antigens with several inflammatory pathways corresponding to primary (triggered by pattern-recognition receptors) and secondary (induced by proinflammatory cytokines) responses. We also show that the Toll-like receptor family member CD180 is downregulated by the stimulation of macrophages, but not monocytes, with the spirochete. Silencing Cd180 results in increased phagocytosis while tempering the production of the proinflammatory cytokine TNF. Cd180-silenced cells produce increased levels of Itgam and surface CD11b, suggesting that the regulation of CD180 by the spirochete initiates a cascade that increases CR3-mediated phagocytosis of the bacterium while repressing the consequent inflammatory response.


Assuntos
Antígenos CD/imunologia , Borrelia burgdorferi/fisiologia , Doença de Lyme/genética , Macrófagos/imunologia , Animais , Antígenos CD/genética , Borrelia burgdorferi/genética , Citocinas/genética , Citocinas/imunologia , Humanos , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Macrófagos/química , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/química , Monócitos/imunologia , Monócitos/microbiologia , Fagocitose , Proteômica , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-30687646

RESUMO

The capacity of Mycobacterium tuberculosis (Mtb) to sense, respond and adapt to a variable and hostile environment within the host makes it one of the most successful human pathogens. During different stages of infection, Mtb is surrounded by a plethora of lipid molecules and current evidence points out the relevance of fatty acids during the infectious process. In this study, we have compared the transcriptional response of Mtb to hypoxia in cultures supplemented with a mix of even long-chain fatty acids or dextrose as main carbon sources. Using RNA sequencing, we have identified differential expressed genes in early and late hypoxia, defined according to the in vitro Wayne and Hayes model, and compared the results with the exponential phase of growth in both carbon sources. We show that the number of genes over-expressed in the lipid medium was quite low in both, early and late hypoxia, relative to conditions including dextrose, with the exception of transcripts of stable and non-coding RNAs, which were more expressed in the fatty acid medium. We found that sigB and sigE were over-expressed in the early phase of hypoxia, confirming their pivotal role in early adaptation to low oxygen concentration independently of the carbon source. A drastic contrast was found with the transcriptional regulatory factors at early hypoxia. Only 2 transcriptional factors were over-expressed in early hypoxia in the lipid medium compared to 37 that were over-expressed in the dextrose medium. Instead of Rv0081, known to be the central regulator of hypoxia in dextrose, Rv2745c (ClgR), seems to play a main role in hypoxia in the fatty acid medium. The low level of genes associated to the stress-response during their adaptation to hypoxia in fatty acids, suggests that this lipid environment makes hypoxia a less stressful condition for the tubercle bacilli. Taken all together, these results indicate that the presence of lipid molecules shapes the metabolic response of Mtb to an adaptive state for different stresses within the host, including hypoxia. This fact could explain the success of Mtb to establish long-term survival during latent infection.


Assuntos
Anaerobiose , Exposição Ambiental , Ácidos Graxos/metabolismo , Mycobacterium tuberculosis/fisiologia , Estresse Fisiológico , Adaptação Fisiológica , Carbono/metabolismo , Meios de Cultura/química , Perfilação da Expressão Gênica , Glucose/metabolismo , Mycobacterium tuberculosis/genética , Análise de Sequência de RNA
12.
Int Rev Immunol ; 36(6): 315-337, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-28933579

RESUMO

Modulation of the immune system has been widely targeted for the treatment of several immune-related diseases, such as autoimmune disorders and cancer, due to its crucial role in these pathologies. Current available therapies focus mainly on symptomatic treatment and are often associated with undesirable secondary effects. For several years, remission of disease and subsequently recovery of immune homeostasis has been a major goal for immunotherapy. Most current immunotherapeutic strategies are aimed to inhibit or potentiate directly the adaptive immune response by modulating antibody production and B cell memory, as well as the effector potential and memory of T cells. Although these immunomodulatory approaches have shown some success in the clinic with promising therapeutic potential, they have some limitations related to their effectiveness in disease models and clinical trials, as well as elevated costs. In the recent years, a renewed interest has emerged on targeting innate immune cells for immunotherapy, due to their high plasticity and ability to exert a potent and extremely rapid response, which can influence the outcome of the adaptive immune response. In this review, we discuss the immunomodulatory potential of several innate immune cells, as well as they use for immunotherapy, especially in autoimmunity and cancer.


Assuntos
Doenças Autoimunes/terapia , Células Dendríticas/imunologia , Imunidade Inata , Imunoterapia Adotiva/métodos , Células T Matadoras Naturais/imunologia , Neoplasias/terapia , Animais , Doenças Autoimunes/imunologia , Plasticidade Celular , Humanos , Imunidade , Células T Matadoras Naturais/transplante , Neoplasias/imunologia
14.
PLoS Pathog ; 13(3): e1006250, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28278283

RESUMO

Currently there are a dozen or so of new vaccine candidates in clinical trials for prevention of tuberculosis (TB) and each formulation attempts to elicit protection by enhancement of cell-mediated immunity (CMI). In contrast, most approved vaccines against other bacterial pathogens are believed to mediate protection by eliciting antibody responses. However, it has been difficult to apply this formula to TB because of the difficulty in reliably eliciting protective antibodies. Here, we developed capsular polysaccharide conjugates by linking mycobacterial capsular arabinomannan (AM) to either Mtb Ag85b or B. anthracis protective antigen (PA). Further, we studied their immunogenicity by ELISA and AM glycan microarrays and protection efficacy in mice. Immunization with either Abg85b-AM or PA-AM conjugates elicited an AM-specific antibody response in mice. AM binding antibodies stimulated transcriptional changes in Mtb. Sera from AM conjugate immunized mice reacted against a broad spectrum of AM structural variants and specifically recognized arabinan fragments. Conjugate vaccine immunized mice infected with Mtb had lower bacterial numbers in lungs and spleen, and lived longer than control mice. These findings provide additional evidence that humoral immunity can contribute to protection against Mtb.


Assuntos
Mananas/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/imunologia , Vacinas Conjugadas/imunologia , Aciltransferases/imunologia , Transferência Adotiva , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Imunidade Humoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Mycobacterium tuberculosis/imunologia , Análise de Sequência com Séries de Oligonucleotídeos
15.
J Infect Dis ; 214(3): 426-37, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27234419

RESUMO

BACKGROUND: Bacillus Calmette-Guerin (BCG) vaccine is widely used for the prevention of tuberculosis, despite limited efficacy. Most immunological studies of BCG or Mycobacterium tuberculosis strains grow bacteria in the presence of detergent, which also strips the mycobacterial capsule. The impact of the capsule on vaccine efficacy has not been explored. METHODS: We tested the influence of detergent in cultures of BCG and M. tuberculosis strains on the outcome of vaccination experiments on mice and transcriptional responses on M. tuberculosis RESULTS: Vaccination of mice with encapsulated BCG promoted a more potent immune response relative to vaccination with unencapsulated BCG, including higher polysaccharide-specific capsule antibody titers, higher interferon γ and interleukin 17 splenic responses, and more multifunctional CD4(+) T cells. These differences correlated with variability in the bacterial burden in lung and spleen of mice infected with encapsulated or unencapsulated M. tuberculosis The combination of vaccination and challenge with encapsulated strains resulted in the greatest protection efficacy. The transcriptome of encapsulated M. tuberculosis was similar to that of starvation, hypoxia, stationary phase, or nonreplicating persistence. CONCLUSIONS: The presence of detergent in growth media and a capsule on BCG were associated with differences in the outcome of vaccination, implying that these are important variables in immunological studies.


Assuntos
Vacina BCG/imunologia , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Meios de Cultura/química , Mycobacterium bovis/imunologia , Mycobacterium bovis/metabolismo , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/sangue , Linfócitos T CD4-Positivos/imunologia , Feminino , Perfilação da Expressão Gênica , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos Endogâmicos C57BL , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo
16.
J Infect Dis ; 214(2): 300-10, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27056953

RESUMO

BACKGROUND: The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans. METHODS: Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages. RESULTS: Immunoglobulin G (IgG) responses to AM increased significantly 4-8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera. CONCLUSIONS: Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans.


Assuntos
Anticorpos Antibacterianos/imunologia , Mananas/imunologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Proteínas Opsonizantes/imunologia , Fagocitose , Adulto , Anticorpos Antibacterianos/metabolismo , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Mananas/metabolismo , Análise em Microsséries , Viabilidade Microbiana , Mycobacterium tuberculosis/fisiologia , Proteínas Opsonizantes/metabolismo , Ligação Proteica
17.
Appl Microbiol Biotechnol ; 100(9): 3887-92, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27020292

RESUMO

The release of cellular factors by means of extracellular vesicles (EVs) is conserved in archaea, bacteria, and eukaryotes. EVs are released by growing bacteria as part of their interaction with their environment and, for pathogenic bacteria, constitute an important component of their interactions with the host. While EVs released by gram-negative bacteria have been extensively studied, the vesicles released by thick cell wall microorganisms like mycobacteria were recognized only recently and are less well understood. Nonetheless, studies of mycobacterial EVs have already suggested roles in pathogenesis, opening exciting new avenues of research aimed at understanding their biogenesis and potential use in antitubercular strategies. In this minireview, we discuss the discovery of mycobacterial vesicles, the current understanding of their nature, content, regulation, and possible functions, as well as their potential therapeutic applications.


Assuntos
Vesículas Extracelulares/metabolismo , Mycobacterium/fisiologia , Fatores de Virulência/metabolismo
18.
Antimicrob Agents Chemother ; 60(3): 1646-55, 2015 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-26711773

RESUMO

The importance of Alternaria species fungi to human health ranges from their role as etiological agents of serious infections with poor prognoses in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria, which was used as a model organism of the genus, and was designed to unravel melanin production in response to antifungals. After we characterized the pigment produced by A. infectoria, we studied the dynamics of 1,8-dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in cell walls in response to nikkomycin Z, caspofungin, and itraconazole but not in response to fluconazole or amphotericin B. These results indicate that A. infectoria activates DHN-melanin synthesis in response to certain antifungal drugs, possibly as a protective mechanism against these drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin and enhanced morphological changes (increased hyphal balloon size), characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs, and its susceptibility is influenced by melanization, suggesting the therapeutic potential of drug combinations that affect melanin synthesis.


Assuntos
Alternaria/efeitos dos fármacos , Antifúngicos/farmacologia , Parede Celular/efeitos dos fármacos , Melaninas/biossíntese , Aminoglicosídeos/farmacologia , Anfotericina B/farmacologia , Caspofungina , Equinocandinas/farmacologia , Fluconazol/farmacologia , Itraconazol/farmacologia , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Naftóis , Pirróis/farmacologia , Quinolinas/farmacologia
19.
Nat Rev Microbiol ; 13(10): 620-30, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26324094

RESUMO

Extracellular vesicles (EVs) are produced by all domains of life. In Gram-negative bacteria, EVs are produced by the pinching off of the outer membrane; however, how EVs escape the thick cell walls of Gram-positive bacteria, mycobacteria and fungi is still unknown. Nonetheless, EVs have been described in a variety of cell-walled organisms, including Staphylococcus aureus, Mycobacterium tuberculosis and Cryptococcus neoformans. These EVs contain varied cargo, including nucleic acids, toxins, lipoproteins and enzymes, and have important roles in microbial physiology and pathogenesis. In this Review, we describe the current status of vesiculogenesis research in thick-walled microorganisms and discuss the cargo and functions associated with EVs in these species.


Assuntos
Vesículas Extracelulares/fisiologia , Fungos/fisiologia , Bactérias Gram-Positivas/fisiologia , Mycobacterium/fisiologia , Fungos/patogenicidade , Bactérias Gram-Positivas/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Mycobacterium/patogenicidade
20.
J Agric Food Chem ; 63(33): 7326-32, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26244793

RESUMO

Many of the most widely consumed edible mushrooms are pigmented, and these have been associated with some beneficial health effects. Nevertheless, the majority of the reported compounds associated with these desirable properties are non-pigmented. We have previously reported that melanin pigment from the edible mushroom Auricularia auricula can protect mice against ionizing radiation, although no physicochemical characterization was reported. Consequently, in this study we have characterized commercial A. auricula mushroom preparations for melanin content and carried out structural characterization of isolated insoluble melanin materials using a panel of sophisticated spectroscopic and physical/imaging techniques. Our results show that approximately 10% of the dry mass of A. auricula is melanin and that the pigment has physicochemical properties consistent with those of eumelanins, including hosting a stable free radical population. Electron microscopy studies show that melanin is associated with the mushroom cell wall in a manner similar to that of melanin from the model fungus C. neoformans. Elemental analysis of melanin indicated C, H, and N ratios consistent with 5,6-dihydroxyindole-2-carboxylic acid/5,6-dihydroxyindole and 1,8-dihydroxynaphthalene eumelanin. Validation of the identity of the isolated product as melanin was achieved by EPR analysis. A. auricula melanin manifested structural differences, relative to the C. neoformans melanin, with regard to the variable proportions of alkyl chains or oxygenated carbons. Given the necessity for new oral and inexpensive radioprotective materials coupled with the commercial availability of A. auricula mushrooms, this product may represent an excellent source of edible melanin.


Assuntos
Basidiomycota/química , Melaninas/química , Parede Celular/química , Cryptococcus neoformans/química , Espectroscopia de Ressonância de Spin Eletrônica , Indóis/química , Espectroscopia de Ressonância Magnética , Melaninas/análise , Melaninas/isolamento & purificação , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Naftóis/química , Pós/química
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