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1.
Molecules ; 28(12)2023 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-37375242

RESUMO

Amphotericin B is a popular antifungal antibiotic, and despite decades of pharmacological application, the exact mode of its biological activity is still a matter of debate. Amphotericin B-silver hybrid nanoparticles (AmB-Ag) have been reported to be an extremely effective form of this antibiotic to combat fungi. Here, we analyze the interaction of AmB-Ag with C. albicans cells with the application of molecular spectroscopy and imaging techniques, including Raman scattering and Fluorescence Lifetime Imaging Microscopy. The results lead to the conclusion that among the main molecular mechanisms responsible for the antifungal activity of AmB is the disintegration of the cell membrane, which occurs on a timescale of minutes.


Assuntos
Anfotericina B , Nanopartículas , Anfotericina B/farmacologia , Anfotericina B/química , Antibacterianos/análise , Prata/química , Antifúngicos/química , Membrana Celular/metabolismo , Nanopartículas/química , Candida albicans
2.
J Phys Chem B ; 127(16): 3632-3640, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37071547

RESUMO

Amphotericin B (AmB) is a life-saving and widely used antifungal antibiotic, but its therapeutic applicability is limited due to severe side effects. Here, we report that the formulation of the drug based on a complex with albumin (BSA) is highly effective against Candida albicans at relatively low concentrations, which implies lower toxicity to patients. This was also concluded based on the comparison with antifungal activities of other popular commercial formulations of the drug, such as Fungizone and AmBisome. Several molecular spectroscopy and imaging techniques, e.g., fluorescence lifetime imaging microscopy (FLIM), were applied to understand the phenomenon of enhanced antifungal activity of the AmB-BSA complex. The results show that the drug molecules bound to the protein remain mostly monomeric and are most likely bound in the pocket responsible for the capture of small molecules by this transport protein. The results of molecular imaging of single complex particles indicate that in most cases, the antibiotic-protein stoichiometry is 1:1. All of the analyses of the AmB-BSA system exclude the presence of the antibiotic aggregates potentially toxic to patients. Cell imaging shows that BSA-bound AmB molecules can readily bind to fungal cell membranes, unlike drug molecules present in the aqueous phase, which are effectively retained by the cell wall barrier. The advantages and prospects of pharmacological use of AmB complexed with proteins are discussed.


Assuntos
Anfotericina B , Antifúngicos , Antifúngicos/farmacologia , Antifúngicos/química , Anfotericina B/farmacologia , Anfotericina B/química , Candida albicans , Albuminas , Antibacterianos/farmacologia
3.
Int J Mol Sci ; 24(6)2023 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-36982826

RESUMO

Antimicrobial peptides (AMPs) are short, mainly positively charged, amphipathic molecules. AMPs are important effectors of the immune response in insects with a broad spectrum of antibacterial, antifungal, and antiparasitic activity. In addition to these well-known roles, AMPs exhibit many other, often unobvious, functions in the host. They support insects in the elimination of viral infections. AMPs participate in the regulation of brain-controlled processes, e.g., sleep and non-associative learning. By influencing neuronal health, communication, and activity, they can affect the functioning of the insect nervous system. Expansion of the AMP repertoire and loss of their specificity is connected with the aging process and lifespan of insects. Moreover, AMPs take part in maintaining gut homeostasis, regulating the number of endosymbionts as well as reducing the number of foreign microbiota. In turn, the presence of AMPs in insect venom prevents the spread of infection in social insects, where the prey may be a source of pathogens.


Assuntos
Anti-Infecciosos , Peptídeos Antimicrobianos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Insetos , Anti-Infecciosos/farmacologia , Antibacterianos
4.
Pharmaceuticals (Basel) ; 14(9)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34577538

RESUMO

The interaction of positively charged polymers (polycations) with a biological membrane is considered to be the cause of the frequently observed toxicity of these macromolecules. If it is possible to obtain polymers with a predominantly negative effect on bacterial and fungal cells, such systems would have great potential in the treatment of infectious diseases, especially now when reports indicate the growing risk of fungal co-infections in COVID-19 patients. We describe in this article cationic derivatives of natural beta-glucan polymers obtained by reacting the polysaccharide isolated from Saccharomyces boulardii (SB) and Cetraria islandica (CI) with glycidyl trimethyl ammonium chloride (GTMAC). Two synthesis strategies were applied to optimize the product yield. Fungal diseases particularly affect low-income countries, hence the emphasis on the simplicity of the synthesis of such drugs so they can be produced without outside help. The three structures obtained showed selective anti-mycotic properties (against, i.e., Scopulariopsis brevicaulis, Aspergillus brasiliensis, and Fusarium solani), and their toxicity established using fibroblast 3T3-L1 cell line was negligible in a wide range of concentrations. For one of the polymers (SB derivative), using in vivo model of Aspergillus brasiliensis infection in Galleria mellonella insect model, we confirmed the promising results obtained in the preliminary study.

5.
Molecules ; 26(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34443685

RESUMO

Recognition of pathogen-associated molecular patterns (PAMPs) by appropriate pattern recognition receptors (PRRs) is a key step in activating the host immune response. The role of a fungal PAMP is attributed to ß-1,3-glucan. The role of α-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of α-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as ß-1,3-glucan recognition proteins, bound to A. niger α-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among α-1,3-glucan-interacting proteins. In response to α-1,3-glucan, a 4.5-fold and 3-fold increase in the gene expression of antifungal peptides galiomicin and gallerimycin was demonstrated, respectively. The significant increase in the level of five defense peptides, including galiomicin, corresponded well with the highest antifungal activity in hemolymph. Our results indicate that A. niger α-1,3-glucan is recognized by the insect immune system, and immune response is triggered by this cell wall component. Thus, the role of a fungal PAMP for α-1,3-glucan can be postulated.


Assuntos
Aspergillus/química , Glucanos/metabolismo , Interações Hospedeiro-Patógeno , Mariposas/microbiologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Corpo Adiposo/efeitos dos fármacos , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hemolinfa/metabolismo , Imunização , Larva , Mariposas/efeitos dos fármacos , Mariposas/genética , Ligação Proteica/efeitos dos fármacos , Análise de Sobrevida
6.
Pathog Dis ; 78(9)2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33232457

RESUMO

Alpha-1,3-glucan, in addition to ß-1,3-glucan, is an important polysaccharide component of fungal cell walls. It is reported for many fungal species, including human pathogenic genera: Aspergillus, Blastomyces, Coccidioides, Cryptococcus, Histoplasma and Pneumocystis, plant pathogens, e.g. Magnaporthe oryzae and entomopathogens, e.g. Metarhizium acridum. In human and plant pathogenic fungi, α-1,3-glucan is considered as a shield for the ß-1,3-glucan layer preventing recognition of the pathogen by the host. However, its role in induction of immune response is not clear. In the present study, the cellular immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan was investigated for the first time. The changes detected in the total hemocyte count (THC) and differential hemocyte count (DHC), formation of hemocyte aggregates and changes in apolipophorin III localization indicated activation of G. mellonella cellular mechanisms in response to immunization with A. niger α-1,3-glucan. Our results, which have clearly demonstrated the response of the insect immune system to this fungal cell wall component, will help in understanding the α-1,3-glucan role in immune response against fungal pathogens not only in insects but also in mammals, including humans.


Assuntos
Apolipoproteínas/imunologia , Aspergilose/imunologia , Glucanos/imunologia , Hemócitos/imunologia , Imunidade Celular , Mariposas , Animais , Apolipoproteínas/metabolismo , Aspergillus niger/imunologia , Aspergillus niger/metabolismo , Parede Celular/química , Modelos Animais de Doenças , Glucanos/metabolismo , Hemócitos/microbiologia , Interações entre Hospedeiro e Microrganismos , Larva/imunologia , Larva/microbiologia , Mariposas/imunologia , Mariposas/microbiologia
7.
Int J Mol Sci ; 21(6)2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32168818

RESUMO

Anionic antimicrobial peptides constitute an integral component of animal innate immunity, however the mechanisms of their antifungal activity are still poorly understood. The action of a unique Galleria mellonella anionic peptide 2 (AP2) against fungal pathogen Candida albicans was examined using different microscopic techniques and Fourier transform infrared (FTIR) spectroscopy. Although the exposure to AP2 decreased the survival rate of C. albicans cells, the viability of protoplasts was not affected, suggesting an important role of the fungal cell wall in the peptide action. Atomic force microscopy showed that the AP2-treated cells became decorated with numerous small clods and exhibited increased adhesion forces. Intensified lomasome formation, vacuolization, and partial distortion of the cell wall was also observed. FTIR spectroscopy suggested AP2 interactions with the cell surface proteins, leading to destabilization of protein secondary structures. Regardless of the anionic character of the whole AP2 molecule, bioinformatics analyses revealed the presence of amphipathic α-helices with exposed positively charged lysine residues. High content of the α-helical structure was confirmed after deconvolution of the IR absorption spectrum and during circular dichroism measurements. Our results indicated that the antimicrobial properties of G. mellonella AP2 rely on the same general characteristics found in cationic defense peptides.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mariposas/química , Peptídeos/farmacologia , Animais , Proteínas de Bactérias/metabolismo , Candida albicans/ultraestrutura , Parede Celular/efeitos dos fármacos , Proteínas de Membrana/química , Viabilidade Microbiana/efeitos dos fármacos , Microscopia de Força Atômica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier
8.
J Invertebr Pathol ; 171: 107341, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32057750

RESUMO

Phenoloxidase (PO) is a key enzyme in the melanization process involved in elimination of pathogens in insects. The PO system is rapidly activated in response to pathogen recognition. Inhibition of PO activity can be a way to avoid immune response and increase infection effectiveness. In this study, the effects of inoculation of Galleria mellonella larvae with Aspergillus niger α-1,3-glucan and conidia on PO activity in hemolymph are analyzed in comparison with the effects of ß-1,3/1,6-glucan inoculation. Our results indicate that α-1,3-glucan, a fungal cell wall polysaccharide, can play a role of a virulence factor involved in inhibition of the insect PO system.


Assuntos
Aspergillus niger/fisiologia , Glucanos/fisiologia , Proteínas de Insetos/antagonistas & inibidores , Monofenol Mono-Oxigenase/antagonistas & inibidores , Mariposas/microbiologia , Fatores de Virulência/fisiologia , Animais , Larva/crescimento & desenvolvimento , Larva/microbiologia , Mariposas/crescimento & desenvolvimento , Esporos Fúngicos/fisiologia
9.
Amino Acids ; 51(2): 175-191, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30167962

RESUMO

Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Cecropinas/química , Cecropinas/farmacologia , Escherichia coli/efeitos dos fármacos , Proteínas de Insetos/química , Proteínas de Insetos/farmacologia , Mariposas/química , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Aderência Bacteriana/fisiologia , Cecropinas/isolamento & purificação , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hemolinfa/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Lipopolissacarídeos/metabolismo , Fluidez de Membrana/fisiologia , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Periplasma/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier
10.
J Insect Physiol ; 105: 18-27, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29289504

RESUMO

A lipid-binding protein apolipophorin III (apoLp-III), an exchangeable component of lipophorin particles, is involved in lipid transport and immune response in insects. In Galleria mellonella, apoLp-III binding to high-density lipophorins and formation of low-density lipophorin complexes upon immune challenge was reported. However, an unanswered question remains whether apoLp-III could form different complexes in a pathogen-dependent manner. Here we report on pathogen- and time-dependent alterations in the level of apoLp-III free and lipophorin-bound form that occur in the hemolymph and hemocytes shortly after immunization of G. mellonella larvae with different pathogens, i.e. Gram-negative bacterium Escherichia coli, Gram-positive bacterium Micrococcus luteus, yeast-like fungus Candida albicans, and filamentous fungus Fusarium oxysporum. These changes were accompanied by differently persistent re-localization of apoLp-III in the hemocytes. The apoLp-III-interacting proteins were recovered from immune hemolymph by affinity chromatography on a Sepharose bed with immobilized anti-apoLp-III antibodies. ApoLp-I, apoLp-II, hexamerin, and arylphorin were identified as main components that bound to apoLp-III; the N-terminal amino acid sequences of G. mellonella apoLp-I and apoLp-II were determined for the first time. In the recovered complexes, the pathogen-dependent differences in the content of individual apolipophorins were detected. Apolipophorins may thus be postulated as signaling molecules responding in an immunogen-dependent manner in early steps of G. mellonella immune response.


Assuntos
Apolipoproteínas/metabolismo , Mariposas/imunologia , Animais , Hemócitos/metabolismo , Hemolinfa/metabolismo , Proteínas de Insetos/análise , Proteínas de Insetos/metabolismo , Mariposas/metabolismo
11.
Postepy Biochem ; 63(4): 269-276, 2017.
Artigo em Polonês | MEDLINE | ID: mdl-29374428

RESUMO

Proteolytic enzymes and their inhibitors are crucial in host-pathogen interaction. Metalloproteases secreted by pathogenic microbes play an important role in destroying not only host tissues but also their immune proteins. Metalloproteinase inhibitors, in contrast, may serve as effective therapeutic agents, which is especially important because of the increasing number of microorganisms resistant to known antibiotics. The role of metalloproteases produced by the bacterium Pseudomonas aeruginosa in the colonization of the host organism is described. Attention has also been paid to the role of inhibitors of these enzymes in defense responses and underlined their potential role in inhibiting the development of infection.


Assuntos
Antibacterianos/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Infecções por Pseudomonas/prevenção & controle , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Antibacterianos/uso terapêutico , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Proteólise/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia
12.
Postepy Biochem ; 63(4): 315-325, 2017.
Artigo em Polonês | MEDLINE | ID: mdl-29374432

RESUMO

Phenoloxidases are oxidoreducting enzymes whose main function is the oxidation of phenols. The term phenoloxidase is often used interchangeably to describe three different enzymes: tyrosinase (EC 1.14.18.1), catechol oxidase, and laccase. Of these, only tyrosinase has two activities: (1) oxygenase activity to hydroxylate monophenols to ortho-diphenols and (2) oxidase activity responsible for further oxidation of ortho-diphenols to ortho-quinones. Tyrosinase is a key enzyme involved in the melanogenesis process, resulting in the formation of black-brown eumelanin and yellow-red feomelanin. In addition to the pigmentary role, human melanin protects against harmful ultraviolet radiation, while in invertebrate animals melanin is involved in the process of cuticle hardening, wound healing, clot formation, maintenance of intestinal homeostasis and defense reactions. In invertebrates, the tyrosinase is synthesized as a proenzyme that is activated by a serine proteases' cascade known as the phenoloxidase system. This system is considered as one of the innate immunity mechanisms.


Assuntos
Monofenol Mono-Oxigenase/metabolismo , Animais , Humanos , Indóis/metabolismo , Melaninas/metabolismo , Melaninas/efeitos da radiação , Serina Proteases/metabolismo , Raios Ultravioleta
13.
Microbiol Res ; 193: 121-131, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27825480

RESUMO

The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and ß-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation.


Assuntos
Antifúngicos/farmacologia , Apoptose , Candida albicans/efeitos dos fármacos , Lepidópteros/enzimologia , Muramidase/farmacologia , Animais , Candida albicans/fisiologia , Viabilidade Microbiana/efeitos dos fármacos
14.
Peptides ; 68: 105-12, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25579437

RESUMO

Apolipophorin III (apoLp-III), a lipid-binding protein and an insect homolog of human apolipoprotein E, plays an important role in lipid transport and immune response in insects. In the present study, we have demonstrated a correlation in time between changes in the apoLp-III abundance occurring in the hemolymph, hemocytes, and fat body after immunization of Galleria mellonella larvae with Gram-negative bacteria Escherichia coli, Gram-positive bacteria Micrococcus luteus, yeast Candida albicans, and a filamentous fungus Fusarium oxysporum. Using two-dimensional electrophoresis (IEF/SDS-PAGE) and immunoblotting with anti-apoLp-III antibodies, the profile of apoLp-III forms in G. mellonella larvae challenged with the bacteria and fungi has been analyzed. Besides the major apoLp-III protein (pI=6.5), one and three additional apoLp-III forms differing in the pI value have been detected, respectively, in the hemolymph, hemocytes, and fat body of non-immunized insects. Also, evidence has been provided that particular apoLp-III-derived polypeptides appear after the immune challenge and are present mainly in the hemolymph and hemocytes. The time of their appearance and persistence in the hemolymph was dependent on the pathogen used. At least two of the apoLp-III forms detected in hemolymph bound to the microbial cell surface. The increasing number of hemolymph apoLp-III polypeptides and differences in their profiles observed in time after the challenge with different immunogens confirmed the important role of apoLp-III in discriminating between pathogens by the insect defense system and in antibacterial as well as antifungal immune response.


Assuntos
Apolipoproteínas/sangue , Proteínas de Insetos/sangue , Mariposas/metabolismo , Animais , Candida albicans/imunologia , Cumarínicos/imunologia , Escherichia coli/imunologia , Corpo Adiposo/metabolismo , Hemolinfa/metabolismo , Imunidade Inata , Larva/imunologia , Larva/metabolismo , Larva/microbiologia , Micrococcus luteus/imunologia , Mariposas/imunologia , Mariposas/microbiologia , Especificidade de Órgãos , Isoformas de Proteínas/sangue
15.
Peptides ; 53: 194-201, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24472857

RESUMO

The lysozymes are well known antimicrobial polypeptides exhibiting antibacterial and antifungal activities. Their antibacterial potential is related to muramidase activity and non-enzymatic activity resembling the mode of action of cationic defense peptides. However, the mechanisms responsible for fungistatic and/or fungicidal activity of lysozyme are still not clear. In the present study, the anti-Candida albicans activity of Galleria mellonella lysozyme and anionic peptide 2 (AP2), defense factors constitutively present in the hemolymph, was examined. The lysozyme inhibited C. albicans growth in a dose-dependent manner. The decrease in the C. albicans survival rate caused by the lysozyme was accompanied by a considerable reduction of the fungus metabolic activity, as revealed by LIVE/DEAD staining. In contrast, although AP2 reduced C. albicans metabolic activity, it did not influence its survival rate. Our results suggest fungicidal action of G. mellonella lysozyme and fungistatic activity of AP2 toward C. albicans cells. In the presence of AP2, the anti-C. albicans activity of G. mellonella lysozyme increased. Moreover, when the fungus was incubated with both defense factors, true hyphae were observed besides pseudohyphae and yeast-like C. albicans cells. Atomic force microscopy analysis of the cells exposed to the lysozyme and/or AP2 revealed alterations in the cell surface topography and properties in comparison with the control cells. The results indicate synergistic action of G. mellonella AP2 and lysozyme toward C. albicans. The presence of both factors in the hemolymph of naive larvae suggests their important role in the early stages of immune response against fungi in G. mellonella.


Assuntos
Hemolinfa/química , Mariposas/química , Muramidase/farmacologia , Peptídeos/farmacologia , Animais , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Microscopia de Força Atômica
16.
Biochim Biophys Acta ; 1828(6): 1449-56, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23419829

RESUMO

Insect immune response relies on the humoral and cellular mechanisms of innate immunity. The key factors are the antimicrobial polypeptides that act in concert against invading pathogens. Several such components, e.g. apolipophorin III (apoLp-III), lysozyme, and anionic peptide 2, are present constitutively in the hemolymph of non-challenged Galleria mellonella larvae. In the present study, we demonstrate an evidence for a synergistic action of G. mellonella lysozyme and apoLp-III against Gram-negative bacteria, providing novel insights into the mode of action of these proteins in insect antimicrobial defense. It was found that the muramidase activity of G. mellonella lysozyme considerably increased in the presence of apoLp-III. Moreover, apoLp-III enhanced the permeabilizing activity of lysozyme toward Escherichia coli cells. As shown using non-denaturing PAGE, the proteins did not form intermolecular complexes in vivo and in vitro, indicating that the effect observed was not connected with the intermolecular interactions between the proteins. Analysis of AFM images of E. coli cells exposed to G. mellonella lysozyme and/or apoLp-III revealed evident alterations in the bacterial surface structure accompanied by the changes in their biophysical properties. The bacterial cells demonstrated significant differences in elasticity, reflected by Young's modulus, as well as in adhesive forces and roughness values in comparison to the control ones. The constitutive presence of these two defense molecules in G. mellonella hemolymph and the fact that apoLp-III enhances lysozyme muramidase and perforating activities indicate that they can be regarded as important antibacterial factors acting at the early stage of infection against Gram-negative as well as Gram-positive bacteria.


Assuntos
Antibacterianos/farmacologia , Apolipoproteínas/farmacologia , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Mariposas/enzimologia , Muramidase/farmacologia , Adesividade , Animais , Antibacterianos/isolamento & purificação , Apolipoproteínas/isolamento & purificação , Apolipoproteínas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sinergismo Farmacológico , Módulo de Elasticidade , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hemolinfa/enzimologia , Proteínas de Insetos/isolamento & purificação , Larva/enzimologia , Microscopia de Força Atômica , Mariposas/embriologia , Mariposas/imunologia , Muramidase/isolamento & purificação , Muramidase/metabolismo
17.
J Insect Physiol ; 58(1): 164-77, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22100292

RESUMO

Galleria mellonella apolipophorin III (apoLp-III) has been implicated in the innate immune response against bacterial infections. The protein binds components of bacterial cell wall and inhibits growth of selected Gram-positive and Gram-negative bacteria. Interaction of apoLp-III with fungal ß-1,3-glucan suggests antifungal properties of the protein. In the present study, the effect of apoLp-III on the growth, metabolic activity and cell surface characteristics of selected yeasts and filamentous fungi was investigated using light, confocal and atomic force microscopy. ApoLp-III bound to the cell surface of different yeasts and filamentous fungi as confirmed by immunoblotting with anti-apoLp-III antibodies. Incubation of the fungi in the presence of apoLp-III induced alterations in growth morphology. Candida albicans underwent transition from yeast-like to hyphal growth with formation of true hyphae, whereas Fusarium oxysporum hyphae exhibited decreased metabolic activity, increased vacuolization and appearance of numerous monophialids with microconidia. Atomic force microscopy imaging demonstrated evident alterations in the fungal cell surface after incubation with apoLp-III, suggesting that the protein affected the cell wall components.


Assuntos
Apolipoproteínas/farmacologia , Candida albicans/efeitos dos fármacos , Fusarium/efeitos dos fármacos , Mariposas/imunologia , Animais , Apolipoproteínas/isolamento & purificação , Candida albicans/metabolismo , Fusarium/metabolismo , Hemolinfa/imunologia , Interações Hospedeiro-Patógeno/imunologia , Microscopia de Força Atômica , Mariposas/química , Mariposas/microbiologia
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