Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 79
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Phys Chem Lett ; 15(18): 4823-4827, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38668706

RESUMO

Amphotericin B is a popular antifungal antibiotic, but the exact way it works is still a matter of debate. Here, we used monolayers composed of phosphatidylcholine with ergosterol as a model of fungal lipid membranes to study drug incorporation from the aqueous phase and analyze the molecular reorganization of membranes underlying the biological activity of the antibiotic. The results show that the internalization of antibiotic molecules into membranes occurs only in the presence of ergosterol in the lipid phase. Comparison of images of solid-supported monolayers obtained by atomic force microscopy and lifetime imaging fluorescence microscopy shows the formation of intramembrane clusters of various sizes in the lipid phase, consisting mainly of antibiotic dimers and relatively large membrane pores (∼15 nm in diameter). The results reveal multiple modes of action of amphotericin B, acting simultaneously, each of which adversely affects the structural properties of the lipid membranes and their physiological functionality.


Assuntos
Anfotericina B , Fosfatidilcolinas , Anfotericina B/química , Fosfatidilcolinas/química , Ergosterol/química , Antifúngicos/química , Microscopia de Força Atômica , Antibacterianos/química , Membrana Celular/química , Microscopia de Fluorescência
2.
Int J Mol Sci ; 24(19)2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37834049

RESUMO

Legionella pneumophila is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the L. pneumophila strain Heysham-1, lacking the O-acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with Acanthamoeba castellanii cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to A. castellanii, suggesting that the O-acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose O-acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of L. pneumophila sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.


Assuntos
Legionella pneumophila , Doença dos Legionários , Humanos , Legionella pneumophila/genética , Lipopolissacarídeos/metabolismo , Ramnose/metabolismo , Sorogrupo , Proteínas de Bactérias/metabolismo
3.
Int J Mol Sci ; 24(16)2023 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-37629129

RESUMO

Lutein, zeaxanthin, and meso-zeaxanthin (a steroisomer of zeaxanthin) are macular pigments. They modify the physical properties of the lipid bilayers in a manner similar to cholesterol. It is not clear if these pigments are directly present in the lipid phase of the membranes, or if they form complexes with specific membrane proteins that retain them in high amounts in the correct place in the retina. The high content of macular pigments in the Henle fiber layer indicates that a portion of the lutein and zeaxanthin should not only be bound to the specific proteins but also directly dissolved in the lipid membranes. This high concentration in the prereceptoral region of the retina is effective for blue-light filtration. Understanding the basic mechanisms of these actions is necessary to better understand the carotenoid-membrane interaction and how carotenoids affect membrane physical properties-such as fluidity, polarity, and order-in relation to membrane structure and membrane dynamics. This review focuses on the properties of lutein.


Assuntos
Carotenoides , Luteína , Zeaxantinas , Membranas , Bicamadas Lipídicas
4.
J Phys Chem Lett ; 14(33): 7440-7444, 2023 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-37578906

RESUMO

Here, we address the problem of the antioxidant activity of carotenoids in biomembranes. The activity of lutein and zeaxanthin in the quenching of singlet oxygen generated by photosensitization was monitored in lipid vesicles using a singlet oxygen-sensitive fluorescent probe and with the application of fluorescence lifetime imaging microscopy. The antioxidant activity of xanthophylls was interpreted on the basis of electron paramagnetic resonance oximetry results showing that xanthophylls constitute a barrier to the penetration of molecular oxygen into lipid membranes: to a greater extent in the 13-cis configuration than in all-trans. These results are discussed in relation to the trans-cis photoisomerization of xanthophylls observed in the human retina. It can be concluded that photoisomerization of xanthophylls is a regulatory mechanism that is important for both the modulation of light filtration through the macula and photoprotection by quenching singlet oxygen and creating a barrier to oxygen permeation to membranes.


Assuntos
Antioxidantes , Xantofilas , Humanos , Xantofilas/metabolismo , Oxigênio Singlete , Estresse Oxidativo , Oxigênio , Lipídeos
5.
Int J Mol Sci ; 24(15)2023 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-37569419

RESUMO

Legionella gormanii is a fastidious, Gram-negative bacterium known to be the etiological agent of atypical community-acquired pneumonia. The human cathelicidin LL-37 exhibits a dose-dependent bactericidal effect on L. gormanii. The LL-37 peptide at the concentration of 10 µM causes the bacteria to become viable but not cultured. The antibacterial activity of the peptide is attributed to its effective binding to the bacterial membrane, as demonstrated by the fluorescence lifetime imaging microscopy. In this study, to mimic the L. gormanii membranes and their response to the antimicrobial peptide, Langmuir monolayers were used with the addition of the LL-37 peptide to the subphase of the Langmuir trough to represent the extracellular fluid. The properties of the model membranes (Langmuir monolayers) formed by phospholipids (PL) isolated from the L. gormanii bacteria cultured on the non-supplemented (PL-choline) and choline-supplemented (PL+choline) medium were determined, along with the effect of the LL-37 peptide on the intermolecular interactions, packing, and ordering under the monolayer compression. Penetration tests at the constant surface pressure were carried out to investigate the mechanism of the LL-37 peptide action on the model membranes. The peptide binds to the anionic bacterial membranes preferentially, due to its positive charge. Upon binding, the LL-37 peptide can penetrate into the hydrophobic tails of phospholipids, destabilizing membrane integrity. The above process can entail membrane disruption and ultimately cell death. The ability to evoke such a great membrane destabilization is dependent on the share of electrostatic, hydrogen bonding and Lifshitz-van der Waals LL-37-PL interactions. Thus, the LL-37 peptide action depends on the changes in the lipid membrane composition caused by the utilization of exogenous choline by the L. gormanii.


Assuntos
Legionella , Humanos , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/metabolismo , Catelicidinas/farmacologia , Colina/farmacologia , Fosfolipídeos/farmacologia
6.
Int J Mol Sci ; 24(13)2023 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-37445880

RESUMO

Zeaxanthin and lutein are xanthophyll pigments present in the human retina and particularly concentrated in its center referred to as the yellow spot (macula lutea). The fact that zeaxanthin, including its isomer meso-zeaxanthin, is concentrated in the central part of the retina, in contrast to lutein also present in the peripheral regions, raises questions about the possible physiological significance of such a heterogeneous distribution of macular xanthophylls. Here, we attempt to address this problem using resonance Raman spectroscopy and confocal imaging, with different laser lines selected to effectively distinguish the spectral contribution of lutein and zeaxanthin. Additionally, fluorescence lifetime imaging microscopy (FLIM) is used to solve the problem of xanthophyll localization in the axon membranes. The obtained results allow us to conclude that one of the key advantages of a particularly high concentration of zeaxanthin in the central part of the retina is the high efficiency of this pigment in the dynamic filtration of light with excessive intensity, potentially harmful for the photoreceptors.


Assuntos
Luteína , Macula Lutea , Humanos , Luteína/química , Zeaxantinas , beta Caroteno , Retina/química , Xantofilas/análise , Macula Lutea/química
7.
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
8.
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
9.
Plant J ; 115(1): 7-17, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36994646

RESUMO

The safe and smooth functioning of photosynthesis in plants is ensured by the operation of numerous regulatory mechanisms that adjust the density of excitation resulting from photon absorption to the capabilities of the photosynthetic apparatus. Such mechanisms include the movement of chloroplasts inside cells and the quenching of electronic excitations in the pigment-protein complexes. Here, we address the problem of a possible cause-and-effect relationship between these two mechanisms. Both the light-induced chloroplast movements and quenching of chlorophyll excitations were analyzed simultaneously with the application of fluorescence lifetime imaging microscopy of Arabidopsis thaliana leaves, wild-type and impaired in chloroplast movements or photoprotective excitation quenching. The results show that both regulatory mechanisms operate over a relatively wide range of light intensities. By contrast, impaired chloroplast translocations have no effect on photoprotection at the molecular level, indicating the direction of information flow in the coupling of these two regulatory mechanisms: from the photosynthetic apparatus to the cellular level. The results show also that the presence of the xanthophyll zeaxanthin is necessary and sufficient for the full development of photoprotective quenching of excessive chlorophyll excitations in plants.


Assuntos
Arabidopsis , Cloroplastos , Cloroplastos/metabolismo , Fotossíntese , Clorofila/metabolismo , Xantofilas/metabolismo
10.
Int J Mol Sci ; 23(11)2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35682729

RESUMO

The effect of the chemical structure of selected phenolic acids on the molecular organization of gliadins was investigated with the application of Fourier Transform Infrared (FTIR) technique, steady-state, and time-resolved fluorescence spectroscopy. Hydroxybenzoic (4-hydroxybenzoic, protocatechuic, vanillic, and syringic) and hydroxycinnamic (coumaric, caffeic, ferulic, sinapic) acids have been used as gliadins modifiers. The results indicated that hydroxybenzoic acids due to their smaller size incorporate into spaces between two polypeptide chains and form a hydrogen bond with them leading to aggregation. Additionally, syringic acids could incorporate into hydrophobic pockets of protein. Whereas hydroxycinnamic acids, due to their higher stiffness and larger size, separated polypeptide chains leading to gliadin disaggregation. These acids did not incorporate into hydrophobic pockets.


Assuntos
Gliadina , Hidroxibenzoatos , Ácidos Cumáricos
11.
Cancers (Basel) ; 13(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072003

RESUMO

AIM: The anti-glioma effect of lensoside Aß alone and in combination with sorafenib (pro-survival Raf kinase inhibitor) was evaluated for the first time in terms of programmed cell death induction in anaplastic astrocytoma and glioblastoma multiforme cell lines as an experimental model. Apoptosis, autophagy, and necrosis were identified microscopically (fluorescence and scanning microscopes) and confirmed by flow cytometry (mitochondrial membrane potential MMP and cell death). The expression of apoptotic (caspase 3) and autophagic markers (beclin 1) as well as Raf kinase were estimated by immunoblotting. The FTIR method was used to determine the interaction of the studied drugs with lipid and protein groups within cells, while the modes of drug action within the cells were assessed with the FLIM technique. RESULTS: Lensoside Aß itself does not exhibit anti-glioma activity but significantly enhances the anti-cancer potential of sorafenib, initiating mainly apoptosis of up to 90% of cells. It was correlated with an increased level of active caspase 3, a reduced MMP value, and a lower level of Raf kinase. The interaction with membrane structures led to morphological changes typical of programmed death. CONCLUSIONS: Our results indicate that lensoside Aß plays an important role as an adjuvant in chemotherapy with sorafenib and may be a potential candidate in anti-glioma combination therapy.

12.
J Phys Chem B ; 125(23): 6090-6102, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34038114

RESUMO

The functioning of the human eye in the extreme range of light intensity requires a combination of the high sensitivity of photoreceptors with their photostability. Here, we identify a regulatory mechanism based on dynamic modulation of light absorption by xanthophylls in the retina, realized by reorientation of pigment molecules induced by trans-cis photoisomerization. We explore this photochemically switchable system using chromatographic analysis coupled with microimaging based on fluorescence lifetime and Raman scattering, showing it at work in both isolated human retina and model lipid membranes. The molecular mechanism underlying xanthophyll reorientation is explained in terms of hydrophobic mismatch using molecular dynamics simulations. Overall, we show that xanthophylls in the human retina act as "molecular blinds", opening and closing on a submillisecond timescale to dynamically control the intensity of light reaching the photoreceptors, thus enabling vision at a very low light intensity and protecting the retina from photodegradation when suddenly exposed to strong light.


Assuntos
Retina , Protetores Solares , Humanos , Luteína , Análise Espectral Raman , Xantofilas
13.
Plant J ; 107(2): 418-433, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33914375

RESUMO

Safe operation of photosynthesis is vital to plants and is ensured by the activity of processes protecting chloroplasts against photo-damage. The harmless dissipation of excess excitation energy is considered to be the primary photoprotective mechanism and is most effective in the combined presence of PsbS protein and zeaxanthin, a xanthophyll accumulated in strong light as a result of the xanthophyll cycle. Here we address the problem of specific molecular mechanisms underlying the synergistic effect of zeaxanthin and PsbS. The experiments were conducted with Arabidopsis thaliana, using wild-type plants, mutants lacking PsbS (npq4), and mutants affected in the xanthophyll cycle (npq1), with the application of molecular spectroscopy and imaging techniques. The results lead to the conclusion that PsbS interferes with the formation of densely packed aggregates of thylakoid membrane proteins, thus allowing easy exchange and incorporation of xanthophyll cycle pigments into such structures. It was found that xanthophylls trapped within supramolecular structures, most likely in the interfacial protein region, determine their photophysical properties. The structures formed in the presence of violaxanthin are characterized by minimized dissipation of excitation energy. In contrast, the structures formed in the presence of zeaxanthin show enhanced excitation quenching, thus protecting the system against photo-damage.


Assuntos
Proteínas de Arabidopsis/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Zeaxantinas/metabolismo , Arabidopsis/metabolismo , Clorofila/metabolismo , Metabolismo Energético , Luz , Microscopia de Fluorescência , Folhas de Planta/metabolismo , Análise Espectral Raman , Tilacoides/metabolismo , Tilacoides/efeitos da radiação , Tilacoides/ultraestrutura
14.
Nanoscale ; 13(6): 3686-3697, 2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33543744

RESUMO

Amphotericin B (AmB), one of the most powerful but also toxic drugs used to treat systemic mycoses, is believed to selectively permeabilize fungal cell membranes to ions in a sterol-dependent manner. Unfortunately, the structure of the biologically active AmB channels has long eluded researchers, obstructing the design of safer alternatives. Here, we investigate the structural and thermodynamic aspects of channel formation, stability, and selective ion conduction. We combine fluorescence lifetime imaging and molecular simulations to trace the process of channel assembly until the formation of stable, roughly octameric double-length channels (DLCs). This stoichiometry is confirmed by matching the predicted channel conductances with the past results of patch-clamp measurements. We then use free energy calculations to explain the effect of sterols on DLC stability and discuss the observed cation selectivity in structural terms, addressing several long-standing controversies in the context of their physiological relevance. Simulations of ion permeation indicate that only solvated ions pass through DLCs, revealing surprising solvation patterns in the channel lumen. We conclude our investigation by inspecting the role of the tail hydroxyl in the assembly of functional channels, pointing at possible origins of the cholesterol-ergosterol selectivity.


Assuntos
Anfotericina B , Bicamadas Lipídicas , Anfotericina B/farmacologia , Membrana Celular , Colesterol , Ergosterol
15.
Int J Mol Sci ; 21(16)2020 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823647

RESUMO

The growth of Legionella dumoffii can be inhibited by Galleria mellonella apolipophorin III (apoLp-III) which is an insect homologue of human apolipoprotein E., and choline-cultured L. dumoffii cells are considerably more susceptible to apoLp-III than bacteria grown without choline supplementation. In the present study, the interactions of apoLp-III with intact L. dumoffii cells cultured without and with exogenous choline were analyzed to explain the basis of this difference. Fluorescently labeled apoLp-III (FITC-apoLp-III) bound more efficiently to choline-grown L. dumoffii, as revealed by laser scanning confocal microscopy. The cell envelope of these bacteria was penetrated more deeply by FITC-apoLp-III, as demonstrated by fluorescence lifetime imaging microscopy analyses. The increased susceptibility of the choline-cultured L. dumoffii to apoLp-III was also accompanied by alterations in the cell surface topography and nanomechanical properties. A detailed analysis of the interaction of apoLp-III with components of the L. dumoffii cells was carried out using both purified lipopolysaccharide (LPS) and liposomes composed of L. dumoffii phospholipids and LPS. A single micelle of L. dumoffii LPS was formed from 12 to 29 monomeric LPS molecules and one L. dumoffii LPS micelle bound two molecules of apoLp-III. ApoLp-III exhibited the strongest interactions with liposomes with incorporated LPS formed of phospholipids isolated from bacteria cultured on exogenous choline. These results indicated that the differences in the phospholipid content in the cell membrane, especially PC, and LPS affected the interactions of apoLp-III with bacterial cells and suggested that these differences contributed to the increased susceptibility of the choline-cultured L. dumoffii to G. mellonella apoLp-III.


Assuntos
Apolipoproteínas/farmacologia , Colina/farmacologia , Suplementos Nutricionais , Legionella/efeitos dos fármacos , Mariposas/microbiologia , Animais , Membrana Celular/efeitos dos fármacos , Ácidos Graxos/análise , Fluorescência , Corantes Fluorescentes/metabolismo , Legionella/ultraestrutura , Lipopolissacarídeos/farmacologia , Lipossomos , Microscopia de Força Atômica , Açúcares/análise
16.
J Phys Chem Lett ; 11(9): 3242-3248, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32271019

RESUMO

Photosystem II (PSII) converts light into chemical energy powering almost all life on Earth. The primary photovoltaic reaction in the PSII reaction center requires energy corresponding to 680 nm, which is significantly higher than in the case of the low-energy states in the antenna complexes involved in the harvesting of excitations driving PSII. Here we show that despite seemingly insufficient energy, the low-energy excited states can power PSII because of the activity of the thermally driven up-conversion. We demonstrate the operation of this mechanism both in intact leaves and in isolated pigment-protein complex LHCII. A mechanism is proposed, according to which the effective utilization of thermal energy in the photosynthetic apparatus is possible owing to the formation of LHCII supramolecular structures, leading to the coupled energy levels corresponding to approximately 680 and 700 nm, capable of exchanging excitation energy through the spontaneous relaxation and the thermal up-conversion.


Assuntos
Transferência de Energia , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/metabolismo , Clorofila A/metabolismo , Temperatura Alta , Folhas de Planta/metabolismo , Reciclagem , Espectrometria de Fluorescência
17.
Sci Rep ; 9(1): 17029, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31745151

RESUMO

Amphotericin B is an antibiotic used as the "gold standard" in the treatment of life-threatening fungal infections. Several molecular mechanisms have been proposed to explain exceptionally high effectiveness of amphotericin B in combating fungi. In the present work, we apply fluorescence lifetime imaging microscopy to track, step by step, modes of the toxic activity of amphotericin B towards a clinical strain of Candida albicans. The images recorded reveal that the antibiotic binds to cells in the form of the small aggregates characterized by a relatively short fluorescence lifetime (0.2 ns). Amphotericin B binds preferentially to the cell walls of mature cells but also to the plasma membranes of the daughter cells at the budding stage. The images recorded with the application of a scanning electron microscopy show that the antibiotic interferes with the formation of functional cell walls of such young cells. The results of imaging reveal the formation of the amphotericin B-rich extramembranous structures and also binding of the drug molecules into the cell membranes and penetration into the cells. These two modes of action of amphotericin B are observed in the time scale of minutes.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/metabolismo , Candida albicans/crescimento & desenvolvimento , Candidíase/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Microscopia de Fluorescência/métodos
18.
Front Microbiol ; 10: 2890, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921066

RESUMO

The Legionella pneumophila TF3/1 mutant of the Corby strain, which possesses a point mutation in the active site of the O-acetyltransferase, synthesized the polysaccharide chain with a reduced degree of substitution with O-acetyl groups. The mutant did not produce a high-molecular-weight lipopolysaccharide (LPS) fraction above 12 kDa. The disturbances in LPS synthesis have an effect on the composition of other macromolecules (lipids and proteins), as indicated by differences in the infrared absorption spectra between the L. pneumophila Corby strain and its TF3/1 mutant. The wild-type strain contained less N+-CH3 and C-N groups as well as more CH3 groups than the mutant. The fatty acid composition showed that the wild type strain synthesized more branched acyl residues (a15:0, i16:0, and a17:0), a less unsaturated acid (16:1), and a straight-chain acid (18:0) than the mutant. The mutant synthesized approximately twice more a long-chain fatty acid (20:0) than the wild type. The main differences in the phospholipids between both strains were found in the classes of phosphatidylcholines and phosphatidylglycerols (PG). Substantial differences in the cell surface topography of these bacteria and their nanomechanical properties were shown by atomic force microscopy (AFM). The wild type strain had no undulated surface and produced numerous vesicles. In the case of the mutant type, the vesicles were not numerous, but there were grooves on the cell surface. The average roughness of the cell surface of the mutant was approximately twofold higher than in the wild-type strain. In turn, the wild-type strain exhibited much better adhesive properties than the mutant. The kinetic study of the interaction between the L. pneumophila strains and Acanthamoeba castellanii monitored by Förster resonance energy transfer revealed a pronounced difference, i.e., almost instantaneous and highly efficient binding of the L. pneumophila Corby strain to the amoeba surface, followed by penetration into the amoeba cells. This process was clearly not as efficient in the case of the mutant. The results point to LPS and, in particular, to the length of the polysaccharide fraction as an important L. pneumophila determinant involved in the process of adhesion to the host cell.

19.
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
20.
Sci Rep ; 8(1): 14067, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218099

RESUMO

Amphotericin B is an antibiotic used in pharmacotherapy of life-threatening mycotic infections. Unfortunately, the applicability of this antibiotic is associated with highly toxic side effects. In order to understand molecular mechanisms underlying toxicity of amphotericin B to patients, two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of the drug and imaged with the application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. The results of the cell viability assays confirm high toxicity of amphotericin B towards human cells. The images recorded demonstrate effective binding of amphotericin B to biomembranes. Analysis of the images reveals the operation of a defence mechanism based upon the elimination of molecules of the drug from living cells via formation of small amphotericin B-containing lipid vesicles. The fact that exosomes formed are devoid of cholesterol, as concluded on the basis of the results of Raman analysis, suggests that sequestration of sterols from the lipid phase of biomembranes is not a sole mechanism responsible for the toxic side effects of amphotericin B. Alternatively, the results imply that molecules of the drug present directly within the hydrophobic membrane core disturb the lipid membrane structure and affect their biological functions.


Assuntos
Anfotericina B/toxicidade , Antibacterianos/toxicidade , Antifúngicos/toxicidade , Microscopia de Fluorescência , Sobrevivência Celular/efeitos dos fármacos , Células HT29 , Humanos , Análise Espectral Raman
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...