RESUMEN
Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength, and stiffness prevent dehydration and mechanical disruption of the biofilm. Bacteria in the genus Gluconacetobacter secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused-ion-beam milling of native bacterial biofilms to image cellulose-synthesizing Gluconacetobacter hansenii and Gluconacetobacter xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several micrometers in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, which we have named the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We found that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line to form higher-order cellulose structures, such as sheets and ribbons.IMPORTANCE This work's relevance for the microbiology community is twofold. It delivers for the first time high-resolution near-native snapshots of Gluconacetobacter spp. (previously Komagataeibacter spp.) in the process of cellulose ribbon synthesis, in their native biofilm environment. It puts forward a noncharacterized cytoskeleton element associated with the side of the cell where the cellulose synthesis occurs. This represents a step forward in the understanding of the cell-guided process of crystalline cellulose synthesis, studied specifically in the Gluconacetobacter genus and still not fully understood. Additionally, our successful attempt to use cryo-focused-ion-beam milling through biofilms to image the cells in their native environment will drive the community to use this tool for the morphological characterization of other studied biofilms.
Asunto(s)
Celulosa/ultraestructura , Citoesqueleto/ultraestructura , Gluconacetobacter/metabolismo , Gluconacetobacter/ultraestructura , Acetobacteraceae/metabolismo , Acetobacteraceae/ultraestructura , Biopelículas , Celulosa/metabolismo , Cristalización , Citoesqueleto/metabolismo , Tomografía con Microscopio Electrónico , Electrones , Escherichia coli/metabolismo , Gluconacetobacter xylinus/metabolismo , Gluconacetobacter xylinus/ultraestructura , MicrofibrillasRESUMEN
During growth and development, the skin expands to cover the growing skeleton and soft tissues by constantly responding to the intrinsic forces of underlying skeletal growth as well as to the extrinsic mechanical forces from body movements and external supports. Mechanical forces can be perceived by two types of skin receptors: (1) cellular mechanoreceptors/mechanosensors, such as the cytoskeleton, cell adhesion molecules and mechanosensitive (MS) ion channels, and (2) sensory nerve fibres that produce the somatic sensation of mechanical force. Skin disorders in which there is an abnormality of collagen [e.g. Ehlers-Danlos syndrome (EDS)] or elastic (e.g. cutis laxa) fibres or a malfunction of cutaneous nerve fibres (e.g. neurofibroma, leprosy and diabetes mellitus) are also characterized to some extent by deficiencies in mechanobiological processes. Recent studies have shown that mechanotransduction is crucial for skin development, especially hemidesmosome maturation, which implies that the pathogenesis of skin disorders such as bullous pemphigoid is related to skin mechanobiology. Similarly, autoimmune diseases, including scleroderma and mixed connective tissue disease, and pathological scarring in the form of keloids and hypertrophic scars would seem to be clearly associated with the mechanobiological dysfunction of the skin. Finally, skin ageing can also be considered as a degenerative process associated with mechanobiological dysfunction. Clinically, a therapeutic strategy involving mechanoreceptors or MS nociceptor inhibition or acceleration together with a reduction or augmentation in the relevant mechanical forces is likely to be successful. The development of novel approaches such as these will allow the treatment of a broad range of cutaneous diseases.
Asunto(s)
Dermis/fisiopatología , Epidermis/fisiopatología , Enfermedades de la Piel/fisiopatología , Animales , Enfermedades Autoinmunes/metabolismo , Moléculas de Adhesión Celular/metabolismo , Citoesqueleto/metabolismo , Dermis/metabolismo , Epidermis/metabolismo , Regulación de la Expresión Génica , Humanos , Células Receptoras Sensoriales/metabolismo , Envejecimiento de la Piel , Enfermedades de la Piel/metabolismoRESUMEN
This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Viabilidad Microbiana , Mycobacterium leprae/patogenicidad , Adhesinas Bacterianas/análisis , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Citoesqueleto/metabolismo , Células Epiteliales/ultraestructura , Humanos , Lepra/microbiología , Lepra/patología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Fagocitosis , Proteoma/análisis , Alveolos Pulmonares/microbiología , Alveolos Pulmonares/patología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The predilection of Mycobacterium leprae (ML) for Schwann cells (SCs) leads to peripheral neuropathy, a major concern in leprosy. Highly infected SCs in lepromatous leprosy nerves show a foamy, lipid-laden appearance; but the origin and nature of these lipids, as well as their role in leprosy, have remained unclear. The data presented show that ML has a pronounced effect on host-cell lipid homeostasis through regulation of lipid droplet (lipid bodies, LD) biogenesis and intracellular distribution. Electron microscopy and immunohistochemical analysis of lepromatous leprosy nerves for adipose differentiation-related protein expression, a classical LD marker, revealed accumulating LDs in close association to ML in infected SCs. The capacity of ML to induce LD formation was confirmed in in vitro studies with human SCs. Moreover, via confocal and live-cell analysis, it was found that LDs are promptly recruited to bacterial phagosomes and that this process depends on cytoskeletal reorganization and PI3K signalling. ML-induced LD biogenesis and recruitment were found to be independent of TLR2 bacterial sensing. Notably, LD recruitment impairment by cytoskeleton drugs decreased intracellular bacterial survival. Altogether, our data revealed SC lipid accumulation in ML-containing phagosomes, which may represent a fundamental aspect of bacterial pathogenesis in the nerve.
Asunto(s)
Metabolismo de los Lípidos , Mycobacterium leprae/patogenicidad , Fagosomas/microbiología , Células de Schwann/microbiología , Células Cultivadas , Citoplasma/química , Citoplasma/ultraestructura , Citoesqueleto/metabolismo , Humanos , Inmunohistoquímica , Proteínas de la Membrana/análisis , Viabilidad Microbiana , Microscopía , Mycobacterium leprae/metabolismo , Perilipina-2 , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
A hallmark of LL is the accumulation of Virchow's foamy macrophages. However, the origin and nature of these lipids, as well as their function and contribution to leprosy disease, remain unclear. We herein show that macrophages present in LL dermal lesions are highly positive for ADRP, suggesting that their foamy aspect is at least in part derived from LD (also known as lipid bodies) accumulation induced during ML infection. Indeed, the capacity of ML to induce LD formation was confirmed in vivo via an experimental model of mouse pleurisy and in in vitro studies with human peripheral monocytes and murine peritoneal macrophages. Furthermore, infected cells were shown to propagate LD induction to uninfected, neighboring cells by generating a paracrine signal, for which TLR2 and TLR6 were demonstrated to be essential. However, TLR2 and TLR6 deletions affected LD formation in bacterium-bearing cells only partially, suggesting the involvement of alternative receptors of the innate immune response besides TLR2/6 for ML recognition by macrophages. Finally, a direct correlation between LD formation and PGE(2) production was observed, indicating that ML-induced LDs constitute intracellular sites for eicosanoid synthesis and that foamy cells may be critical regulators in subverting the immune response in leprosy.
Asunto(s)
Eicosanoides/biosíntesis , Lepra/metabolismo , Lepra/microbiología , Metabolismo de los Lípidos , Mycobacterium leprae/patogenicidad , Orgánulos/metabolismo , Receptores Toll-Like/metabolismo , Animales , Biopsia , Medios de Cultivo Condicionados/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Dinoprostona/biosíntesis , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Leucocitos Mononucleares/patología , Metabolismo de los Lípidos/efectos de los fármacos , Activación de Macrófagos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Mycobacterium leprae/efectos de los fármacos , Orgánulos/microbiología , Comunicación Paracrina/efectos de los fármacos , Perilipina-2 , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Piel/microbiología , Piel/patología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 6/metabolismoRESUMEN
OBJECTIVE: To study the phosphorylation state of neurofilament (NF) proteins and activity of KSPXK kinase in the sciatic nerves of Swiss white (S/W) mice inoculated in the hind foot pads with M. leprae. DESIGN: Test group includes S/W mice inoculated in the foot pads with freshly harvested human derived (viable) M. leprae. Control groups were constituted by (1) Age matched un-inoculated mice, (2) Mice similarly inoculated with M. smegmatis and (3) heat killed M. leprae. Phosphorylation state of NF was studied using Western blot analysis and phosphor-specific NF antibody (SMI 31; Sternberger Monoclonals, Inc.). The KSPXK kinase activity was assayed by using KSPXK fusion protein in a radiometric method using gamma(22)P ATP. RESULTS: Several fold increase in M. leprae numbers was seen in viable M. leprae group while M. smegmatis failed to show any fold increase in the foot pads of S/W mice. Western immunoblot analysis of cytoskeletal preparation from sciatic nerves of uninoculated mice and mice inoculated with M. smegmatis showed immunoreactivity to SMI 31 antibody and protein bands corresponding to both NF-H and NF-M at all the time points from 4-20 months post inoculation. In case of viable M. leprae; SMI 31 reactive protein bands were seen at 4 months but not at any of the later intervals, i.e., between 6-20 months. With heat killed M. leprae transient loss of immunoreactivity to SMI 31 was seen. Decrease in KSPXK kinase activity was recorded in sets inoculated with viable and heat killed M. leprae, and corroborated with loss of immunoreactivity seen in WBs reacted with SMI 31 antibody. CONCLUSIONS: Alterations in the sciatic nerve NF cytoskeleton was seen following inoculation in the hind foot pad with both viable and heat killed M. leprae. The hypophosphorylation of NF observed in this study corroborates with the earlier observations in human leprous nerves.
Asunto(s)
Lepra/inmunología , Mycobacterium leprae , Proteínas de Neurofilamentos/metabolismo , Enfermedades del Sistema Nervioso Periférico/patología , Nervio Ciático/enzimología , Animales , Western Blotting , Citoesqueleto/metabolismo , Citoesqueleto/patología , Lepra/patología , Ratones , Mycobacterium leprae/crecimiento & desarrollo , Fosforilación , Fosfotransferasas/metabolismo , Proteínas Quinasas/metabolismo , Nervio Ciático/metabolismo , Nervio Ciático/ultraestructuraRESUMEN
Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.
Asunto(s)
Lepra/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/patología , Atrofia , Axones/patología , Western Blotting , Citoesqueleto/metabolismo , Citoesqueleto/patología , Enfermedades Desmielinizantes/patología , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunohistoquímica , Fibras Nerviosas/patología , Neuronas/ultraestructura , Adhesión en Parafina , FosforilaciónRESUMEN
Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.
Asunto(s)
Humanos , Atrofia , Axones , Citoesqueleto , Enfermedad de Alzheimer , Enfermedades Desmielinizantes , Electroforesis en Gel de Poliacrilamida , Esclerosis Amiotrófica Lateral , Fibras Nerviosas , Fosforilación , Lepra , Inmunohistoquímica , Adhesión en Parafina , Neuronas , Proteínas de Neurofilamentos , Western BlottingRESUMEN
The interaction between mycobacterial phenolic glycolipids (PGLs) and phagocytes was studied. Human neutrophils were allowed to interact with each of four purified mycobacterial PGLs and the neutrophil production of reactive oxygen metabolites was followed kinetically by luminol-/isoluminol-amplified chemiluminescence. The PGLs from Mycobacterium tuberculosis and Mycobacterium kansasii, respectively, were shown to stimulate the production of oxygen metabolites, while PGLs from Mycobacterium marinum and Mycobacterium bovis BCG, respectively, were unable to induce an oxidative response. Periodate treatment of the M. tuberculosis PGL decreased the production of oxygen radicals, showing the importance of the PGL carbohydrate moiety for the interaction. The activation, however, could not be inhibited by rhamnose or fucose, indicating a complex interaction which probably involves more than one saccharide unit. This is in line with the fact that the activating PGLs from M. tuberculosis and M. kansasii contain tri- and tetrasaccharides, respectively, while the nonactivating PGLs from M. marinum and M. bovis BCG each contain a monosaccharide. The complement receptor 3 (CR3) has earlier been shown to be of importance for the phagocyte binding of mycobacteria, but did not appear to be involved in the activation of neutrophils by PGLs. The subcellular localization of the reactive oxygen metabolites formed was related to the way in which the glycolipids were presented to the cells.
Asunto(s)
Antígenos Bacterianos/inmunología , Glucolípidos/inmunología , Activación Neutrófila , Metabolismo de los Hidratos de Carbono , Carbohidratos/fisiología , Proteínas Inactivadoras de Complemento/farmacología , Gránulos Citoplasmáticos/metabolismo , Citoesqueleto/fisiología , Inducción Enzimática/inmunología , Humanos , Antígeno de Macrófago-1/metabolismo , Monosacáridos/farmacología , Mycobacterium bovis/química , Mycobacterium bovis/inmunología , Mycobacterium kansasii/química , Mycobacterium kansasii/inmunología , Mycobacterium marinum/química , Mycobacterium marinum/inmunología , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/inmunología , NADPH Oxidasas/biosíntesis , Neutrófilos/enzimologíaRESUMEN
Dystroglycan connects the extracellular matrix and cytoskeleton. Key findings in the past year indicate that dystroglycan interacts with a wider repertoire of extracellular ligands than originally appreciated, that dystroglycan plays a critical role in organizing extracellular matrix molecules on the cell surface and in basement membranes, and that at least two human pathogens utilize dystroglycan to gain access to host cells. Together, these advances begin to help elucidate important biological roles for dystroglycan in development and disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas del Citoesqueleto/fisiología , Glicoproteínas de Membrana/fisiología , Animales , Adhesión Bacteriana , Moléculas de Adhesión Celular/fisiología , Proteínas del Citoesqueleto/química , Citoesqueleto/metabolismo , Distroglicanos , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Proteína Adaptadora GRB2 , Humanos , Laminina/metabolismo , Ligandos , Glicoproteínas de Membrana/química , Proteínas Musculares/fisiología , Distrofias Musculares/metabolismo , Mycobacterium leprae/metabolismo , Unión Proteica , Proteínas Tirosina Quinasas/fisiología , Proteínas/fisiología , Dominios Homologos srcRESUMEN
A rare spindle-cell pseudotumor caused by Mycobacterium avium-intracellulare (MAI) that mimics a mesenchymal tumor, was recently reported (7,14). We report on three such pseudotumors in patients with the acquired immunodeficiency syndrome (AIDS), two involving lymph nodes and one involving the bone marrow. In the course of investigating the first-encountered example of this tumor for evidence of smooth-muscle origin of the spindle cells, it was noted that these cells stained positively for desmin by immunoperoxidase techniques (IPX), as did a variety of other cytoskeleton filaments of all sizes. Electron microscopic examination of one of these lesions revealed spindle cells containing lysosomes and large numbers of microorganisms compatible with MAI but no filaments or organelles suggestive of smooth-muscle cells. Further studies revealed that the typical lesions produced by MAI in patients with AIDS, namely aggregates of histiocytes or individual histiocytes laden with organisms, rather than the expansile spindle-cell pseudotumor, also strain strongly for cytoskeleton filaments, as do M. tuberculosis and Mycobacterium leprae. Awareness of the existence of this unusual manifestation of MAI infection in AIDS patients and its desmin positivity can avoid misdiagnosis of a primary or metastatic smooth-muscle neoplasm. The cell of origin appears to be the histiocyte.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Citoesqueleto , Complejo Mycobacterium avium/aislamiento & purificación , Infección por Mycobacterium avium-intracellulare/patología , Adulto , Citoesqueleto/ultraestructura , Diagnóstico Diferencial , Humanos , Técnicas para Inmunoenzimas , Masculino , Músculo Liso , Infección por Mycobacterium avium-intracellulare/diagnóstico , Infección por Mycobacterium avium-intracellulare/microbiología , Neoplasias de Tejido Muscular/diagnóstico , Coloración y EtiquetadoRESUMEN
Certain structures which indicate probable involvement of a filamentous phase in life cycle of M. leprae have been noted in preserved skin biopsy suspensions from lepromatous leprosy cases. These include (i) filaments with empty or pink round spaces within them (ii) conidia-like structures and (iii) membranes with acid fast bacilli. These structures were rare in the fresh material.
Asunto(s)
Citoesqueleto/fisiología , Filamentos Intermedios/fisiología , Mycobacterium leprae/crecimiento & desarrollo , Ciclo Celular , División Celular , Frío , Humanos , Uniones Intercelulares , Membranas/citología , Membranas/fisiología , Mycobacterium leprae/fisiologíaRESUMEN
Sera from 34 patients with lepromatous leprosy were screened for the presence of autoantibodies by indirect immunofluorescence using two epithelial cell lines, PTK2 and HEp2, as substrates. Indirect immunofluorescence staining of both substrates with the serum of a patient with lepromatous leprosy revealed a cytoplasmic intermediate filament staining pattern. After exposure of PTK2 cells to colchicine, the filaments collapsed into thick perinuclear coils, confirming the presence of intermediate filament reactivity. Immunofluorescence of rat fibroblasts with the same serum also revealed an intermediate filamentous staining pattern. Human keratinocytes exposed to the patient's serum revealed a diffuse cytoplasmic staining pattern. Our study suggests the presence of autoantibodies to cytoskeletal intermediate filaments or to molecules associated with vimentin and possibly keratin subunit proteins in the serum of a patient with lepromatous leprosy.