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1.
Cell ; 149(3): 684-92, 2012 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-22541437

RESUMEN

Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of the matrix in Bacillus subtilis. Here we report that B. subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both factors formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis, Escherichia coli, and Staphylococcus aureus.


Asunto(s)
Bacillus subtilis/fisiología , Biopelículas , Polisacáridos Bacterianos/metabolismo , Espermidina/análogos & derivados , Aminoácidos/biosíntesis , Aminoácidos/metabolismo , Bacillus subtilis/genética , Escherichia coli/fisiología , Mutación , Poliaminas/metabolismo , Espermidina/biosíntesis , Espermidina/metabolismo , Staphylococcus aureus/fisiología
2.
Proc Natl Acad Sci U S A ; 121(40): e2403842121, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-39264745

RESUMEN

Modern molecular microbiology elucidates the organizational principles of bacterial biofilms via detailed examination of the interplay between signaling and gene regulation. A complementary biophysical approach studies the mesoscopic dependencies at the cellular and multicellular levels with a distinct focus on intercellular forces and mechanical properties of whole biofilms. Here, motivated by recent advances in biofilm research and in other, seemingly unrelated fields of biology and physics, we propose a perspective that links the biofilm, a dynamic multicellular organism, with the physical processes occurring in the extracellular milieu. Using Bacillus subtilis as an illustrative model organism, we specifically demonstrate how such a rationale explains biofilm architecture, differentiation, communication, and stress responses such as desiccation tolerance, metabolism, and physiology across multiple scales-from matrix proteins and polysaccharides to macroscopic wrinkles and water-filled channels.


Asunto(s)
Bacillus subtilis , Biopelículas , Biopelículas/crecimiento & desarrollo , Bacillus subtilis/fisiología , Bacillus subtilis/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética
3.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-34983851

RESUMEN

The viscoelectric effect concerns the increase in viscosity of a polar liquid in an electric field due to its interaction with the dipolar molecules and was first determined for polar organic liquids more than 80 y ago. For the case of water, however, the most common polar liquid, direct measurement of the viscoelectric effect is challenging and has not to date been carried out, despite its importance in a wide range of electrokinetic and flow effects. In consequence, estimates of its magnitude for water vary by more than three orders of magnitude. Here, we measure the viscoelectric effect in water directly using a surface force balance by measuring the dynamic approach of two molecularly smooth surfaces with a controlled, uniform electric field between them across highly purified water. As the water is squeezed out of the gap between the approaching surfaces, viscous damping dominates the approach dynamics; this is modulated by the viscoelectric effect under the uniform transverse electric field across the water, enabling its magnitude to be directly determined as a function of the field. We measured a value for this magnitude, which differs by one and by two orders of magnitude, respectively, from its highest and lowest previously estimated values.

4.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-35042817

RESUMEN

Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix components, spores, and water. By simultaneously following the X-ray fluorescence signal of biofilms and isolated matrix components, we discovered that the ECM preferentially binds calcium ions over other metal ions, specifically, zinc, manganese, and iron. These ions, remaining free to flow below macroscopic wrinkles that act as water channels, eventually accumulate and may possibly lead to sporulation. The possible link between ECM properties, regulation of metal ion distribution, and sporulation across whole, intact biofilms unravels the importance of molecular-level heterogeneity in shaping biofilm physiology and development.


Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Biopelículas/crecimiento & desarrollo , Proteínas Amiloidogénicas/metabolismo , Proteínas Bacterianas/metabolismo , Matriz Extracelular/fisiología , Iones/metabolismo , Dispersión de Radiación , Dispersión del Ángulo Pequeño , Rayos X
5.
J Bacteriol ; 206(2): e0004923, 2024 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-38289062

RESUMEN

The international workshop "Interdisciplinary life of microbes: from single cells to multicellular aggregates," following a virtual preassembly in November 2021, was held in person in Dresden, from 9 to 13 November 2022. It attracted not only prominent experts in biofilm research but also researchers from broadly neighboring disciplines, such as medicine, chemistry, and theoretical and experimental biophysics, both eukaryotic and prokaryotic. Focused brainstorming sessions were the special feature of the event and are at the heart of this commentary.


Asunto(s)
Bacterias , Biopelículas , Humanos , Bacterias/genética , Eucariontes/genética
6.
Soft Matter ; 20(5): 1047-1060, 2024 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-38205608

RESUMEN

Biofilms are multicellular heterogeneous bacterial communities characterized by social-like division of labor, and remarkable robustness with respect to external stresses. Increasingly often an analogy between biofilms and arguably more complex eukaryotic tissues is being drawn. One illustrative example of where this analogy can be practically useful is the process of wound healing. While it has been extensively studied in eukaryotic tissues, the mechanism of wound healing in biofilms is virtually unexplored. Combining experiments in Bacillus subtilis bacteria, a model organism for biofilm formation, and a lattice-based theoretical model of biofilm growth, we studied how biofilms recover after macroscopic damage. We suggest that nutrient gradients and the abundance of proliferating cells are key factors augmenting wound closure. Accordingly, in the model, cell quiescence, nutrient fluxes, and biomass represented by cells and self-secreted extracellular matrix are necessary to qualitatively recapitulate the experimental results for damage repair. One of the surprising experimental findings is that residual cells, persisting in a damaged area after removal of a part of the biofilm, prominently affect the healing process. Taken together, our results outline the important roles of nutrient gradients and residual cells on biomass regrowth on macroscopic scales of the whole biofilm. The proposed combined experiment-simulation framework opens the way to further investigate the possible relation between wound healing, cell signaling and cell phenotype alternation in the local microenvironment of the wound.


Asunto(s)
Bacillus , Bacterias , Biopelículas , Transporte Biológico , Cicatrización de Heridas
7.
Proc Natl Acad Sci U S A ; 116(45): 22478-22484, 2019 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-31636220

RESUMEN

The term amyloid defines a group of proteins that aggregate into plaques or fibers. Amyloid fibers gained their fame mostly due to their relation with neurodegenerative diseases in humans. However, secreted by lower organisms, such as bacteria and fungi, amyloid fibers play a functional role: for example, when they serve as cement in the extracellular matrix of biofilms. Originating either in humans or in microorganisms, the sequence of amyloid proteins is decorated with hexapeptides with high propensity to form fibers, known as steric zippers. We have found that steric zippers form globular structures on route to making fibers and exhibit a characteristic force-distance (F-D) fingerprint when pulled with an atomic force microscope (AFM) tip. Particularly, the F-D pulling curves showed force plateau steps, suggesting that the globular structures were composed of chains that were unwound like a yarn ball. Force plateau analysis showed that the F-D characteristic parameters were sequence sensitive, representing differences in the packing of the hexapeptides within the globules. These unprecedented findings show that steric zippers exhibit a characteristic nanomechanical signature in solution in addition to previously observed characteristic crystallographic structure. Getting to the fundamental interactions that govern the unzipping of full-length amyloid fibers may initiate the development of antiamyloid methods that target the physical in addition to the structural properties of steric zippers.


Asunto(s)
Amiloide/química , Fenómenos Biomecánicos , Microscopía de Fuerza Atómica , Oligopéptidos/química
8.
Soft Matter ; 16(26): 6180-6190, 2020 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-32567645

RESUMEN

Biofilms are aggregates of cells that form on surfaces or at the air-water interface. Cells in a biofilm are encased in a self-secreted extracellular matrix (ECM) that provides them with mechanical stability and protects them from antibiotic treatment. From a soft matter perspective, biofilms are regarded as colloidal hydrogels, with the cells playing the role of colloids and the ECM compared with a cross-linked hydrogel. Here, we examined whole biofilms of the soil bacterium Bacillus subtilis utilizing methods that are commonly used to characterize hydrogels in order to evaluate the uptake of water and the water properties in the biofilms. Specifically, we studied wild-type as well ECM mutants, lacking the protein TasA and the exopolysaccharide (EPS). We characterized the morphology and mesh size of biofilms using electron microscopy, studied the state of water in the biofilms using differential scanning calorimetry, and finally, we tested the biofilms' swelling properties. Our study revealed that Bacillus subtilis biofilms resemble cross-linked hydrogels in their morphology and swelling properties. Strikingly, we discovered that all the water in biofilms was bound water and there was no free water in the biofilms. Water binding was mostly related with the presence of solutes and much less so with the major ECM components, the protein TasA and the polysaccharide EPS. This study sheds light on water uptake and water binding in biofilms and it is therefore important for the understanding of solute transport and enzymatic function inside biofilms.


Asunto(s)
Bacillus subtilis , Hidrogeles , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Biopelículas , Agua
9.
Phys Chem Chem Phys ; 22(40): 23286-23294, 2020 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-33033811

RESUMEN

Functional amyloid proteins are self-secreted by microbial cells that aggregate into extracellular networks and provide microbial colonies with mechanical stability and resistance to antibiotic treatment. In order to understand the formation mechanism of functional amyloid networks, their aggregation has been studied in vitro under different physical conditions, such as temperature, salt concentration, and pH. Typical aggregates' morphologies include fibers or plaques, the latter resembling amyloid aggregates in neurodegenerated brains. Here, we studied the pH-reduction-induced aggregation of TasA, an extracellular functional amyloid appearing as fibers in biofilms of the soil bacterium, Bacillus subtilis. We used turbidity and zeta potential measurements, electron microscopy, atomic force microscopy, and static light scattering measurements, to characterize the aggregates of TasA and to compare them with colloidal aggregates. We further studied the aggregation of TasA in the presence of negatively charged nanoparticles and showed that nanoparticles co-aggregated with TasA, and that the co-aggregation was hindered sterically. Based on these studies, we concluded that, similarly to colloidal aggregation, TasA aggregation occurs due to surface potential modulations and that the aggregation is followed by a rearrangement process. Shedding light on the aggregation mechanism of TasA, our results can be used for the design of TasA aggregation inhibitors and promoters.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Concentración de Iones de Hidrógeno , Modelos Moleculares , Agregación Patológica de Proteínas
10.
Chembiochem ; 20(3): 355-359, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30371005

RESUMEN

Biofilms are aggregates of microbial cells that form on surfaces and at interfaces, and are encased in an extracellular matrix. In biofilms made by the soil bacterium Bacillus subtilis, the protein TapA mediates the assembly of the functional amyloid protein TasA into extracellular fibers, and it anchors these fibers to the cell surface. We used circular dichroism and NMR spectroscopy to show that, unlike the structured TasA, TapA is disordered. In addition, TapA is composed of two weakly interacting domains: a disordered C-terminal domain and a more structured N-terminal domain. These two domains also exhibited different structural changes in response to changes in external conditions, such as increased temperatures and the presence of lipid vesicles. Although the two TapA domains weakly interacted in solution, their cooperative interaction with lipid vesicles prevented disruption of the vesicles. These findings therefore suggest that the two-domain composition of TapA is important in its interaction with single or multiple partners in the extracellular matrix in biofilms.


Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas de la Matriz Extracelular/química , Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular
11.
Biochemistry ; 57(35): 5230-5238, 2018 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-29565118

RESUMEN

Biofilms are aggregates of cells that form surface-associated communities. The cells in biofilms are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins, and sometimes nucleic acids. Some extracellular matrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiate their constructive function from disease-related amyloid fibers. Recent functional amyloid assembly studies have neglected their interaction with membranes, despite their native formation in a cellular environment. Here, we use TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein and ask whether the bacterial functional amyloid interacts with membranes. Using biochemical, spectroscopic, and microscopic tools, we show that TasA interacts distinctively with bacterial model membranes and that this interaction mutually influences the morphology and structure of the protein and the membranes. At the protein level, fibers of similar structure and morphology are formed in the absence of membranes and in the presence of eukaryotic model membranes. However, in the presence of bacterial model membranes, TasA forms disordered aggregates with a different ß sheet signature. At the membrane level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform more considerably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial more than eukaryotic model membranes and that this leads to membrane disruption and to reshaping the TasA fiber formation pathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of antibiofilm drugs to the protein-membrane interface.


Asunto(s)
Amiloide/química , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biopelículas , Membrana Celular/química , Membranas Artificiales , Sustitución de Aminoácidos , Amiloide/metabolismo , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Fluidez de la Membrana , Multimerización de Proteína
12.
J Biol Chem ; 288(24): 17559-68, 2013 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-23632024

RESUMEN

Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some ß-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.


Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/química , Biopelículas , Proteínas de la Matriz Extracelular/química , Adsorción , Silicatos de Aluminio/química , Amiloide/química , Amiloide/aislamiento & purificación , Amiloide/ultraestructura , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Proteínas de la Matriz Extracelular/aislamiento & purificación , Proteínas de la Matriz Extracelular/ultraestructura , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Luz , Microscopía de Fuerza Atómica , Tamaño de la Partícula , Multimerización de Proteína , Dispersión de Radiación , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie
13.
Biomaterials ; 309: 122578, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38692146

RESUMEN

Biofilm research has grown exponentially over the last decades, arguably due to their contribution to hospital acquired infections when they form on foreign body surfaces such as catheters and implants. Yet, translation of the knowledge acquired in the laboratory to the clinic has been slow and/or often it is not attempted by research teams to walk the talk of what is defined as 'bench to bedside'. We therefore reviewed the biofilm literature to better understand this gap. Our search revealed substantial development with respect to adapting surfaces and media used in models to mimic the clinical settings, however many of the in vitro models were too simplistic, often discounting the composition and properties of the host microenvironment and overlooking the biofilm-implant-host interactions. Failure to capture the physiological growth conditions of biofilms in vivo results in major differences between lab-grown- and clinically-relevant biofilms, particularly with respect to phenotypic profiles, virulence, and antimicrobial resistance, and they essentially impede bench-to-bedside translatability. In this review, we describe the complexity of the biological processes at the biofilm-implant-host interfaces, discuss the prerequisite for the development and characterization of biofilm models that better mimic the clinical scenario, and propose an interdisciplinary outlook of how to bioengineer biofilms in vitro by converging tissue engineering concepts and tools.


Asunto(s)
Bioingeniería , Biopelículas , Prótesis e Implantes , Biopelículas/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Humanos , Prótesis e Implantes/microbiología , Bioingeniería/métodos , Animales , Modelos Biológicos , Infecciones Relacionadas con Prótesis/microbiología , Microambiente Celular
14.
Mol Microbiol ; 86(2): 426-36, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22882172

RESUMEN

Many bacteria organize themselves into structurally complex communities known as biofilms in which the cells are held together by an extracellular matrix. In general, the amount of extracellular matrix is related to the robustness of the biofilm. Yet, the specific signals that regulate the synthesis of matrix remain poorly understood. Here we show that the matrix itself can be a cue that regulates the expression of the genes involved in matrix synthesis in Bacillus subtilis. The presence of the exopolysaccharide component of the matrix causes an increase in osmotic pressure that leads to an inhibition of matrix gene expression. We further show that non-specific changes in osmotic pressure also inhibit matrix gene expression and do so by activating the histidine kinase KinD. KinD, in turn, directs the phosphorylation of the master regulatory protein Spo0A, which at high levels represses matrix gene expression. Sensing a physical cue such as osmotic pressure, in addition to chemical cues, could be a strategy to non-specifically co-ordinate the behaviour of cells in communities composed of many different species.


Asunto(s)
Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Matriz Extracelular/genética , Regulación Bacteriana de la Expresión Génica , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Matriz Extracelular/metabolismo , Histidina Quinasa , Presión Osmótica , Fosforilación , Proteínas Quinasas , Factores de Transcripción
15.
RSC Med Chem ; 14(5): 983-991, 2023 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-37252093

RESUMEN

Biofilms are surface or interface-associated communities of bacterial cells, embedded in a self-secreted extracellular matrix (ECM). Cells in biofilms are 100-1000 times more resistant to antibiotic treatment relative to planktonic cells due to various reasons, including the ECM acting as a diffusion barrier to antibiotic molecules, the presence of persister cells that divide slowly and are less susceptible to cell-wall targeting drugs, and the activation of efflux pumps in response to antibiotic stress. In this study we tested the effect of two titanium(iv) complexes that have been previously reported as potent and non-toxic anticancer chemotherapeutic agents on Bacillus subtilis cells in culture and in biofilm forming conditions. The Ti(iv) complexes tested, a hexacoordinate diaminobis(phenolato)-bis(alkoxo) complex (phenolaTi) and a bis(isopropoxo) complex of a diaminobis(phenolato) "salan"-type ligand (salanTi), did not affect the growth rate of cells in shaken cultures, however they did affect biofilm formation. Surprisingly, while phenolaTi inhibited biofilm formation, the presence of salanTi induced the formation of more mechanically robust biofilms. Optical microscopy images of biofilm samples in the absence and presence of Ti(iv) complexes suggest that Ti(iv) complexes affect cell-cell and/or cell-matrix adhesion, and that these are interfered with phenolaTi and enhanced by salanTi. Our results highlight the possible effect of Ti(iv) complexes on bacterial biofilms, which is gaining interest in light of the emerging relations between bacteria and cancerous tumors.

16.
ACS Appl Bio Mater ; 2022 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-35977081

RESUMEN

Biofilms are multicellular communities of microbial cells that grow on natural and synthetic surfaces. They have become the major cause for hospital-acquired infections because once they form, they are very difficult to eradicate. Nanotechnology offers means to fight biofilm-associated infections. Here, we report on the synthesis of silver nanoparticles (AgNPs) with the antibacterial ligand epigallocatechin gallate (EGCG) and the formation of a lysozyme protein corona on AgNPs, as shown by UV-vis, dynamic light scattering, and circular dichroism analyses. We further tested the activity of EGCG-AgNPs and their lysozyme bioconjugates on the viability of Bacillus subtilis cells and biofilm formation. Our results showed that, although EGCG-AgNPs presented no antibacterial activity on planktonic B. subtilis cells, they inhibited B. subtilis biofilm formation at concentrations larger than 40 nM, and EGCG-AgNP-lysozyme bioconjugates inhibited biofilms at concentrations above 80 nM. Cytotoxicity assays performed with human cells showed a reverse trend, where EGCG-AgNPs barely affected human cell viability while EGCG-AgNP-lysozyme bioconjugates severely hampered viability. Our results therefore demonstrate that EGCG-AgNPs may be used as noncytotoxic antibiofilm agents.

17.
Nat Commun ; 13(1): 7082, 2022 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-36400765

RESUMEN

Many bacteria in nature exist in multicellular communities termed biofilms, where cells are embedded in an extracellular matrix that provides rigidity to the biofilm and protects cells from chemical and mechanical stresses. In the Gram-positive model bacterium Bacillus subtilis, TasA is the major protein component of the biofilm matrix, where it has been reported to form functional amyloid fibres contributing to biofilm structure and stability. Here, we present electron cryomicroscopy structures of TasA fibres, which show that, rather than forming amyloid fibrils, TasA monomers assemble into fibres through donor-strand exchange, with each subunit donating a ß-strand to complete the fold of the next subunit along the fibre. Combining electron cryotomography, atomic force microscopy, and mutational studies, we show how TasA fibres congregate in three dimensions to form abundant fibre bundles that are essential for B. subtilis biofilm formation. Our study explains the previously observed biochemical properties of TasA and shows how a bacterial extracellular globular protein can assemble from monomers into ß-sheet-rich fibres, and how such fibres assemble into bundles in biofilms.


Asunto(s)
Bacillus subtilis , Arañas , Animales , Bacillus subtilis/metabolismo , Biopelículas , Proteínas Bacterianas/metabolismo , Amiloide/metabolismo , Arañas/metabolismo
18.
Microorganisms ; 9(3)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806534

RESUMEN

Functional amyloid proteins often appear as fibers in extracellular matrices of microbial soft colonies. In contrast to disease-related amyloid structures, they serve a functional goal that benefits the organism that secretes them, which is the reason for the title "functional". Biofilms are a specific example of a microbial community in which functional amyloid fibers play a role. Functional amyloid proteins contribute to the mechanical stability of biofilms and mediate the adhesion of the cells to themselves as well as to surfaces. Recently, it has been shown that functional amyloid proteins also play a regulatory role in biofilm development. TasA is the major proteinaceous fibrilar component of the extracellular matrix of biofilms made of the soil bacterium and Gram-positive Bacillus subtilis. We have previously shown, as later corroborated by others, that in acidic solutions, TasA forms compact aggregates that are composed of tangled fibers. Here, we show that in a neutral pH and above a certain TasA concentration, the fibers of TasA are elongated and straight and that they bundle up in highly concentrated salt solutions. TasA fibers resemble the canonic amyloid morphology; however, these fibers also bear an interesting nm-scale periodicity along the fiber axis. At the molecular level, TasA fibers contain a twisted ß-sheet structure, as indicated by circular dichroism measurements. Our study shows that the morphology of TasA fibers depends on the environmental conditions. Different fibrilar morphologies may be related with different functional roles in biofilms, ranging from granting biofilms with a mechanical support to acting as antibiotic agents.

19.
J Vis Exp ; (147)2019 05 14.
Artículo en Inglés | MEDLINE | ID: mdl-31157787

RESUMEN

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.


Asunto(s)
Biopolímeros/química , Carbonato de Calcio/química , Minerales/química , Cloruro de Calcio/química , Microscopía Electrónica de Rastreo , Solubilidad , Espectrometría Raman , Termodinámica
20.
J Control Release ; 257: 40-50, 2017 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-28017890

RESUMEN

Controlling the interaction of drug delivery systems (DDS) with tissues is critical for the success of therapies. Specifically in cancer, due to the high density of the tumors, tissue penetration of DDS is critical and may be challenging. In previous work we have shown that Solidified Polymer Micelles (SPMs) rapidly internalize into cells and tissues. Using AFM analysis, in the present work we measured differences in rigidity of SPM compared with Wet Polymer Micelles (WPM). We further examined whether the semi-solid form of hydrated SPMs has an effect on the interaction with tumor cells both in mono-layer systems and in multi-layer clusters of cells as spheroids. For that we have performed detailed characterization of SPM compared to WPM, including examinations of particle size, stability, drug release kinetics and cell transcytosis, in melanoma A-375 cells. Cell uptake measurements were done using fluorescent signal analysis, FACS and microscopy imaging, showing enhanced abilities of SPMs to penetrate cells and tissues. A simple physical model is presented that well agrees with the experiments and provides insight about the role of particle rigidity in the engulfment mechanism. We conclude that particle rigidity enhances cellular uptake and tissue penetration and that SPMs have a promising potential as an effective and highly permeable DDS. Our findings can be important in future rational design of DDS for particle adjustment to specific tissues and pathologies.


Asunto(s)
Portadores de Fármacos/metabolismo , Módulo de Elasticidad , Micelas , Polietilenglicoles/metabolismo , Línea Celular Tumoral , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Exocitosis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neoplasias/metabolismo , Tamaño de la Partícula , Polietilenglicoles/química
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