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1.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32886672

RESUMEN

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Asunto(s)
Anticuerpos/química , Disulfuros/aislamiento & purificación , Dominios de Inmunoglobulinas , Fragmentos de Péptidos/aislamiento & purificación , Dominios y Motivos de Interacción de Proteínas , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Afinidad de Anticuerpos , Formación de Anticuerpos , Especificidad de Anticuerpos , Antígenos/genética , Antígenos/inmunología , Linfocitos B/fisiología , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/inmunología , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Disulfuros/química , Disulfuros/inmunología , Mapeo Epitopo/métodos , Humanos , Inmunización , Dominios de Inmunoglobulinas/genética , Modelos Moleculares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Dominios y Motivos de Interacción de Proteínas/genética
2.
FEBS J ; 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-38982771

RESUMEN

Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.

3.
Front Immunol ; 15: 1384467, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38605965

RESUMEN

Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Methods: Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Results: Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Discussion: Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.


Asunto(s)
Anticuerpos Biespecíficos , Animales , Bovinos , Humanos , Anticuerpos Biespecíficos/química , Péptidos , Proteínas de la Nucleocápside
4.
Methods Mol Biol ; 2681: 83-97, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37405644

RESUMEN

Phage display is an in vitro technique used in the discovery of monoclonal antibodies that has been used successfully in the discovery of both camelid VHH and shark variable new antigen receptor domains (VNAR). Bovines also contain a unique "ultralong CDRH3" with a conserved structural motif, comprising a knob domain and ß-stalk. When removed from the antibody scaffold, either the entire ultralong CDRH3 or the knob domain alone, is typically capable of binding an antigen, to produce antibody fragments that are smaller than both VHH and VNAR. By extracting immune material from bovine animals and specifically amplifying knob domain DNA sequences by PCR, knob domain sequences can be cloned into a phagemid vector producing knob domain phage libraries. Target-specific knob domains can be enriched by panning the libraries against an antigen of interest. Phage display of knob domains exploits the link between phage genotype and phenotype and could prove to be a high throughput method to discover target-specific knob domains, helping to explore the pharmacological properties of this unique antibody fragment.


Asunto(s)
Bacteriófagos , Técnicas de Visualización de Superficie Celular , Animales , Bovinos , Antígenos , Anticuerpos Monoclonales/genética , Receptores de Antígenos/genética , Bacteriófagos/genética , Biblioteca de Péptidos
5.
Front Immunol ; 14: 1216967, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37483614

RESUMEN

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (KD 40 nM-5.5 µM) and inhibit downstream IL-13 signalling (IC50 0.2-53.8 µM). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13Rα1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4Rα. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful 19F fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these 19F containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.


Asunto(s)
Interleucina-13 , Anticuerpos de Dominio Único , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/metabolismo , Citocinas
6.
MAbs ; 15(1): 2289681, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38084840

RESUMEN

Gremlin-1, a high-affinity antagonist of bone morphogenetic proteins (BMP)-2, -4, and -7, is implicated in tumor initiation and progression. Increased gremlin-1 expression, and therefore suppressed BMP signaling, correlates with poor prognosis in a range of cancer types. A lack of published work using therapeutic modalities has precluded the testing of the hypothesis that blocking the gremlin-1/BMP interaction will provide benefits to patients. To address this shortfall, we developed ginisortamab (UCB6114), a first-in-class clinical anti-human gremlin-1 antibody, currently in clinical development for the treatment of cancer, along with its murine analog antibody Ab7326 mouse immunoglobulin G1 (mIgG1). Surface plasmon resonance assays revealed that ginisortamab and Ab7326 mIgG1 had similar affinities for human and mouse gremlin-1, with mean equilibrium dissociation constants of 87 pM and 61 pM, respectively. The gremlin-1/Ab7326 antigen-binding fragment (Fab) crystal structure revealed a gremlin-1 dimer with a Fab molecule bound to each monomer that blocked BMP binding. In cell culture experiments, ginisortamab fully blocked the activity of recombinant human gremlin-1, and restored BMP signaling pathways in human colorectal cancer (CRC) cell lines. Furthermore, in a human CRC - fibroblast co-culture system where gremlin-1 is produced by the fibroblasts, ginisortamab restored BMP signaling in both the CRC cells and fibroblasts, demonstrating its activity in a relevant human tumor microenvironment model. The safety and efficacy of ginisortamab are currently being evaluated in a Phase 1/2 clinical trial in patients with advanced solid tumors (NCT04393298).


Asunto(s)
Neoplasias , Transducción de Señal , Humanos , Animales , Ratones , Línea Celular , Neoplasias/tratamiento farmacológico , Microambiente Tumoral
7.
Front Immunol ; 14: 1170357, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37251411

RESUMEN

Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering. Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.


Asunto(s)
Anticuerpos Biespecíficos , Albúmina Sérica , Animales , Bovinos , Humanos , Albúmina Sérica/metabolismo , Fragmentos Fab de Inmunoglobulinas , Epítopos , Técnicas de Visualización de Superficie Celular
8.
MAbs ; 13(1): 1980942, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34850665

RESUMEN

Therapeutic antibodies must have "drug-like" properties. These include high affinity and specificity for the intended target, biological activity, and additional characteristics now known as "developability properties": long-term stability and resistance to aggregation when in solution, thermodynamic stability to prevent unfolding, high expression yields to facilitate manufacturing, low self-interaction, among others. Sequence-based liabilities may affect one or more of these characteristics. Improving the stability and developability of a lead antibody is typically achieved by modifying its sequence, a time-consuming process that often results in reduced affinity. Here we present a new antibody library format that yields high-affinity binders with drug-like developability properties directly from initial selections, reducing the need for further engineering or affinity maturation. The innovative semi-synthetic design involves grafting natural complementarity-determining regions (CDRs) from human antibodies into scaffolds based on well-behaved clinical antibodies. HCDR3s were amplified directly from B cells, while the remaining CDRs, from which all sequence liabilities had been purged, were replicated from a large next-generation sequencing dataset. By combining two in vitro display techniques, phage and yeast display, we were able to routinely recover a large number of unique, highly developable antibodies against clinically relevant targets with affinities in the subnanomolar to low nanomolar range. We anticipate that the designs and approaches presented here will accelerate the drug development process by reducing the failure rate of leads due to poor antibody affinities and developability.Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CQA: critical quality attribute; ELISA: enzyme-linked immunoassay; FACS: fluorescence-activated cell sorting; Fv: fragment variable; GM-CSF: granulocyte-macrophage colony-stimulating factor; HCDR3: heavy chain CDR3; IFN2a: interferon α-2; IL6: interleukin-6; MACS: magnetic-activated cell sorting; NGS: next generation sequencing; PCR: polymerase chain reaction; SEC: size-exclusion chromatography; SPR: surface plasmon resonance; TGFß-R2: transforming growth factor ß-R2; VH: variable heavy; VK: variable kappa; VL: variable light; Vl: variable lambda.


Asunto(s)
Anticuerpos Monoclonales , Regiones Determinantes de Complementariedad , Anticuerpos Monoclonales/química , Afinidad de Anticuerpos , Linfocitos B , Regiones Determinantes de Complementariedad/química , Biblioteca de Genes , Humanos , Biblioteca de Péptidos
9.
Nat Commun ; 12(1): 583, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33495445

RESUMEN

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Complejos Multiproteicos/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos Monoclonales/farmacología , Células Cultivadas , Cristalografía por Rayos X , Epítopos/química , Epítopos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Complejos Multiproteicos/química , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/química , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Necrosis Tumoral alfa/química
10.
ACS Synth Biol ; 9(10): 2828-2839, 2020 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-32927940

RESUMEN

Synthetic biology and metabolic engineering offer potentially green and attractive routes to the production of high value compounds. The provision of high-quality parts and pathways is crucial in enabling the biosynthesis of chemicals using synthetic biology. While a number of regulatory parts that provide control at the transcriptional and translational level have been developed, relatively few exist at the protein level. Single domain antibodies (sdAb) such as camelid heavy chain variable fragments (VHH) possess binding characteristics which could be exploited for their development and use as novel parts for regulating metabolic pathways at the protein level in microbial cell factories. Here, a platform for the use of VHH as tools in Escherichia coli is developed and subsequently used to modulate linalool production in E. coli. The coproduction of a Design of Experiments (DoE) optimized pBbE8k His6-VHHCyDisCo system alongside a heterologous linalool production pathway facilitated the identification of anti-bLinS VHH that functioned as modulators of bLinS. This resulted in altered product profiles and significant variation in the titers of linalool, geraniol, nerolidol, and indole obtained. The ability to alter the production levels of high value terpenoids, such as linalool, in a tunable manner at the protein level could represent a significant step forward for the development of improved microbial cell factories. This study serves as a proof of principle indicating that VHH can be used to modulate enzyme activity in engineered pathways within E. coli. Given their almost limitless binding potential, we posit that single domain antibodies could emerge as powerful regulatory parts in synthetic biology applications.


Asunto(s)
Monoterpenos Acíclicos/metabolismo , Proteínas Bacterianas/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroliasas/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Ingeniería Metabólica/métodos , Anticuerpos de Dominio Único/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reactores Biológicos , Camélidos del Nuevo Mundo/inmunología , Codón , Activación Enzimática/inmunología , Hidroliasas/genética , Hidroliasas/metabolismo , Streptomyces/enzimología , Streptomyces/genética , Biología Sintética/métodos
11.
FEMS Microbiol Lett ; 284(2): 237-46, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18507683

RESUMEN

Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Biopelículas , Células Epiteliales/microbiología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Mucosa Intestinal/microbiología , Adhesinas de Escherichia coli , Animales , Adhesión Bacteriana , Células Cultivadas , Escherichia coli/metabolismo , Femenino , Ratones , Ratones Endogámicos BALB C , Factores de Tiempo
12.
Front Immunol ; 9: 1698, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30083160

RESUMEN

Every human possesses millions of distinct antibodies. It is now possible to analyze this diversity via next-generation sequencing of immunoglobulin genes (Ig-seq). This technique produces large volume sequence snapshots of B-cell receptors that are indicative of the antibody repertoire. In this paper, we enrich these large-scale sequence datasets with structural information. Enriching a sequence with its structural data allows better approximation of many vital features, such as its binding site and specificity. Here, we describe the structural annotation of antibodies pipeline that maps the outputs of large Ig-seq experiments to known antibody structures. We demonstrate the viability of our protocol on five separate Ig-seq datasets covering ca. 35 m unique amino acid sequences from ca. 600 individuals. Despite the great theoretical diversity of antibodies, we find that the majority of sequences coming from such studies can be reliably mapped to an existing structure.

13.
Biochim Biophys Acta ; 1713(2): 92-112, 2005 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-15993836

RESUMEN

Recent genomic analyses of the two sequenced strains F. nucleatum subsp. nucleatum ATCC 25586 and F. nucleatum subsp. vincentii ATCC 49256 suggested that the major protein secretion systems were absent. However, such a paucity of protein secretion systems is incongruous with F. nucleatum pathogenesis. Moreover, the presence of one or more such systems has been described for every other Gram-negative organism sequenced to date. In this investigation, the question of protein secretion in F. nucleatum was revisited. In the current study, the absence in F. nucleatum of a twin-arginine translocation system (TC #2.A.64.), a Type III secretion system (TC #3.A.6.), a Type IV secretion system (TC #3.A.7.) and a chaperone/usher pathway (TC #1.B.11.) was confirmed. However, contrary to previous findings, our investigations indicated that a Type I protein secretion system was also absent from F. nucleatum. In contrast, members of the holin family (TC #1.E) and the machinery required for a Type 4 piliation/fimbriation system (TC #3.A.15.2.) were identified using a variety of bioinformatic tools. Furthermore, a complete range of proteins resembling members of the Type V secretion pathway, i.e., the Type Va (autotransporter; TC #1.B.12.), Type Vb (two-partner secretion system; TC #1.B.20.) and Type Vc (YadA-like trimeric autotransporter; TC #1.B.42.), was found. This work provides new insight into the protein secretion and virulence mechanisms of F. nucleatum.


Asunto(s)
Fimbrias Bacterianas/metabolismo , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/patogenicidad , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Fenómenos Fisiológicos Bacterianos , Secuencia de Bases , Biopelículas , Biología Computacional/métodos , Fusobacterium/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Unión Proteica , Conformación Proteica , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Programas Informáticos , Virulencia
14.
Biochim Biophys Acta ; 1745(2): 223-53, 2005 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-15950297

RESUMEN

Consistent information about protein secretion in Gram-positive bacteria is essentially restricted to the model organism Bacillus subtilis. Among genome-sequenced clostridia, Clostridium acetobutylicum has been the most extensively studied from a physiological point of view and is the organism for which the largest variety of molecular biology tools have been developed. Following in silico analyses, both secreted proteins and protein secretion systems were identified. The Tat (Twin arginine translocation; TC #2.A.64) pathway and ABC (ATP binding cassette) protein exporters (TC #3.A.1.) could not be identified, but the Sec (secretion) pathway (TC #3.A.5) appears to be used prevalently. Similarly, a flagella export apparatus (FEA; TC #3.A.6.), holins (TC #1.E.), and an ESAT-6/WXG100 (early secreted antigen target of 6 kDa/proteins with a WXG motif of approximately 100 residues) secretion system were identified. Here, we report for the first time the identification of a fimbrilin protein exporter (FPE; TC #3.A.14) and a Tad (tight adherence) export apparatus in C. acetobutylicum. This investigation highlights the potential use of this saprophytic bacterium in biotechnological and biomedical applications as well as a model organism for studying protein secretion in pathogenic Gram-positive bacteria.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clostridium acetobutylicum/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Celulosomas/fisiología , Colina/metabolismo , Clostridium acetobutylicum/enzimología , Clostridium acetobutylicum/fisiología , Proteínas Fimbrias/fisiología , Datos de Secuencia Molecular , Unión Proteica/fisiología , Señales de Clasificación de Proteína/genética , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Ricina/genética , Ricina/metabolismo
15.
FEMS Microbiol Lett ; 263(1): 10-20, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16958845

RESUMEN

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of food and water-borne E. coli-mediated human diarrhoea worldwide. The incidence in developing countries is estimated at 650 million cases per year, resulting in 800 000 deaths, primarily in children under the age of five. ETEC is also the most common cause of diarrhoea among travellers, including the military, from industrialized nations to less developed countries. In addition, ETEC is a major pathogen of animals, being responsible for scours in cattle and neonatal and postweaning diarrhoea in pigs and resulting in significant financial losses. Studies on the pathogenesis of ETEC infections have concentrated on the plasmid-encoded heat-stable and heat-labile enterotoxins and on the plasmid-encoded antigenically variable colonization factors. Relatively little work has been carried out on chromosomally encoded virulence factors. Here, we review the known virulence factors of ETEC and highlight the future for combating this major disease.


Asunto(s)
Cromosomas Bacterianos , Infecciones por Escherichia coli/microbiología , Escherichia coli/patogenicidad , Factores de Virulencia/genética , Factores de Virulencia/toxicidad , Animales , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/toxicidad , Humanos
16.
FEMS Microbiol Lett ; 264(1): 22-30, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17020545

RESUMEN

The plasmid encoded toxin, Pet, is a prototypical member of the serine protease autotransporters of the Enterobacteriaceae. In addition to the passenger and beta-domains typical of autotransporters, in silico predictions indicate that Pet possesses an unusually long N-terminal signal sequence. The signal sequence can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions, which we have termed the extended signal peptide region, demonstrate remarkable conservation. In contrast, the N2, H2 and C regions demonstrate significant variability and are reminiscent of typical Sec-dependent signal sequences. Despite several investigations, the function of the extended signal peptide region remains obscure and surprisingly it has not been proven that the extended signal peptide region is actually synthesized as part of the signal sequence. Here, we demonstrate that the extended signal peptide region is present only in Gram-negative bacterial proteins originating from the classes Beta- and Gammaproteobacteria, and more particularly only in proteins secreted via the Type V secretion pathway: autotransporters, TpsA exoproteins of the two-partner system and trimeric autotransporters. In vitro approaches demonstrate that the DNA region encoding the extended signal peptide region is transcribed and translated.


Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Betaproteobacteria/metabolismo , Gammaproteobacteria/metabolismo , Señales de Clasificación de Proteína/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Secuencia de Bases , Betaproteobacteria/clasificación , Gammaproteobacteria/clasificación , Datos de Secuencia Molecular , Filogenia , Biosíntesis de Proteínas , Transporte de Proteínas/fisiología , Transcripción Genética
17.
Trends Microbiol ; 12(7): 306-9, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15223057

RESUMEN

The term general secretory pathway (GSP) has been usurped and misused in the literature over the past few years. The concept of GSP is discussed from an historical perspective, and the definitions of the general export pathway (GEP), the main terminal branch (MTB) of the GSP, the unified GSP nomenclature and the type II, IV and V secretion pathways are also described to show how they have fuelled the confusion. By putting the record straight and using novel findings within the field of bacterial protein secretion, we hope to bring clarity to this area of science and prevent further promulgation of incorrect terminologies.


Asunto(s)
Proteínas Bacterianas/metabolismo , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/metabolismo , Transporte de Proteínas , Sustancias Macromoleculares , Terminología como Asunto
18.
FEMS Microbiol Lett ; 311(2): 133-9, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20735484

RESUMEN

The plasmid-encoded toxin, Pet, a prototypical member of the serine protease autotransporters of the Enterobacteriaceae, possesses an unusually long signal peptide, which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions correspond to a conserved N-terminal extension previously designated the extended signal peptide region (ESPR), while the N2, H2 and C regions resemble typical Sec-dependent signal sequences and exhibit considerable sequence variability. We have shown previously that the ESPR directs Sec-dependent, post-translational translocation of Pet across the bacterial inner membrane. In this study, we demonstrate that the ESPR is not essential for the secretion or the function of Pet.


Asunto(s)
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Enterotoxinas/química , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Señales de Clasificación de Proteína , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Toxinas Bacterianas/genética , Enterotoxinas/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Eliminación de Secuencia , Serina Endopeptidasas/genética
19.
PLoS One ; 5(1): e8801, 2010 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-20098708

RESUMEN

BACKGROUND: Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. METHODS: In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. CONCLUSION: This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies.


Asunto(s)
Escherichia coli/genética , Genoma Bacteriano , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Secuencia de Bases , Carbono/metabolismo , Cartilla de ADN , Escherichia coli/clasificación , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Interacciones Huésped-Patógeno , Humanos , Hierro/metabolismo , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Filogenia
20.
Nat Rev Microbiol ; 7(3): 206-14, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19182809

RESUMEN

The folding of transmembrane proteins into the outer membrane presents formidable challenges to Gram-negative bacteria. These proteins must migrate from the cytoplasm, through the inner membrane and into the periplasm, before being recognized by the beta-barrel assembly machinery, which mediates efficient insertion of folded beta-barrels into the outer membrane. Recent discoveries of component structures and accessory interactions of this complex are yielding insights into how cells fold membrane proteins. Here, we discuss how these structures illuminate the mechanisms responsible for the biogenesis of outer membrane proteins.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacterias Gramnegativas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Bacterias Gramnegativas/química , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Multimerización de Proteína , Transporte de Proteínas
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