The Middle Fragment of Helicobacter pylori CagA Induces Actin Rearrangement and Triggers Its Own Uptake into Gastric Epithelial Cells

Cytotoxin-associated gene product A (CagA) is a major virulence factor secreted by Helicobacter pylori. CagA activity in the gastric epithelium is associated with higher risk of gastric cancer development. Bacterial type IV secretion system (T4SS)-mediated translocation of CagA into the cytosol of human epithelial cells occurs via a poorly understood mechanism that requires CagA interaction with the host membrane lipid phosphatidylserine (PS) and host cell receptor integrin α5β1. Here we have characterized the isolated recombinant middle fragment of CagA (CagA-M) that contains the positively-charged PS-binding region (aa 613–636) and a putative β1 integrin binding site, but lacks the EPIYA region, secretion signal peptide and the CagA multimerization motif. We show that CagA-M, when immobilized on latex beads, is capable of binding to, and triggering its own uptake into, gastric epithelial cells in the absence of infection with cagA-positive H. pylori. Using site-directed mutagenesis, fluorescent and electron microscopy, and highly-specific inhibitors, we demonstrate that the cell-binding and endocytosis-like internalization of CagA-M are dependent on (1) binding to PS; (2) β1 integrin activity; and (3) actin dynamics. Interaction of CagA-M with the host cells is accompanied by the development of long filopodia-like protrusions (macrospikes). This novel morphology is different from the hummingbird phenotype induced by the translocation of full-length CagA. The determinants within CagA-M and within the host that are important for endocytosis-like internalization into host cells are very similar to those observed for T4SS-mediated internalization of full-length CagA, suggesting that the latter may involve an endocytic pathway

Structural and Functional Characterization of the Helicobacter pylori protein CagA

Helicobacter pylori is a pathogenic bacteria which infects the gastric epithelium and induces adenocarcinoma. CagA is a major virulence factor of H. pylori, which is translocated into the cytosol of gastric epithelium, mainly by the aid of type IV secretion system. The main objectives of this thesis are the structural and functional analysis of CagA middle fragments using methods such as crystallization and X-ray diffraction, circular dichroism, multi-angle light scattering, and microscopy techniques. Results showed that CagA middle domain is folded and monomeric. It induces the development of a novel macrospike phenotype and triggers its uptake into the gastric epithelium

The middle fragment of <i>Helicobacter pylori </i>CagA induces Actin rearrangement and triggers its own uptake into gastric epithelial cells

Cytotoxin-associated gene product A (CagA) is a major virulence factor secreted by Helicobacter pylori. CagA activity in the gastric epithelium is associated with higher risk of gastric cancer development. Bacterial type IV secretion system (T4SS)-mediated translocation of CagA into the cytosol of human epithelial cells occurs via a poorly understood mechanism that requires CagA interaction with the host membrane lipid phosphatidylserine (PS) and host cell receptor integrin α5β1. Here we have characterized the isolated recombinant middle fragment of CagA (CagA-M) that contains the positively-charged PS-binding region (aa 613–636) and a putative β1 integrin binding site, but lacks the EPIYA region, secretion signal peptide and the CagA multimerization motif. We show that CagA-M, when immobilized on latex beads, is capable of binding to, and triggering its own uptake into, gastric epithelial cells in the absence of infection with cagA-positive H. pylori. Using site-directed mutagenesis, fluorescent and electron microscopy, and highly-specific inhibitors, we demonstrate that the cell-binding and endocytosis-like internalization of CagA-M are dependent on (1) binding to PS; (2) β1 integrin activity; and (3) actin dynamics. Interaction of CagA-M with the host cells is accompanied by the development of long filopodia-like protrusions (macrospikes). This novel morphology is different from the hummingbird phenotype induced by the translocation of full-length CagA. The determinants within CagA-M and within the host that are important for endocytosis-like internalization into host cells are very similar to those observed for T4SS-mediated internalization of full-length CagA, suggesting that the latter may involve an endocytic pathway

The Cytotoxin-Associated Gene A (CagA) of Helicobacter pylori: the Paradigm of an Oncogenic Virulence Factor

Helicobacter pylori is a microaerophilic, spiral-shaped and gram-negative microorganism that produces various virulence factors such as CagA, VacA, urease, and host cells adhesins, which in a synchronous concert, allow H. pylori to colonize and infect the host gastric epithelium. H. pylori infection is associated with some severe side effects in human, such as gastritis, peptic ulcer, non-Hodgkin’s lymphoma and adenocarcinoma. CagA is the most notorious virulence factor of H. pylori. It is known as the first bacterial oncoprotein. The gene encoding CagA is localized on the cag pathogenicity island (cagPAI), a 40kbp DNA segment which also carries genes for the type four secretion system (T4SS) of H. pylori. The interaction of CagA with intracellular partner proteins leads to some irreversible alteration of host cells by increasing cell size, elevating motility, phenomena known as the “hummingbird phenotype”. CagA also disrupts the epithelium apical junctions and thereby destroys the normal epithelial architecture. A tyrosine phosphorylation site, named EPIYA motif, helps CagA to bind to cytosolic proteins in a phosphorylation-dependent manner. CagA is also interacts with host proteins in a phosphorylation-independent fashion, which altogether will assist to develop adenocarcinoma in infected cells. This review summarizes the core data on the structure and function of CagA and its role in conferring the main pathophysiologic effects of H. pylori infection as well as suggesting a therapeutic option for treatment of H. pylori infection based on CagA virulence

The Cytotoxin-Associated Gene A (CagA) of Helicobacter pylori: the Paradigm of an Oncogenic Virulence Factor

Helicobacter pylori is a microaerophilic, spiral-shaped and gram-negative microorganism that produces various virulence factors such as CagA, VacA, urease, and host cells adhesins, which in a synchronous concert, allow H. pylori to colonize and infect the host gastric epithelium. H. pylori infection is associated with some severe side effects in human, such as gastritis, peptic ulcer, non-Hodgkin’s lymphoma and adenocarcinoma. CagA is the most notorious virulence factor of H. pylori. It is known as the first bacterial oncoprotein. The gene encoding CagA is localized on the cag pathogenicity island (cagPAI), a 40kbp DNA segment which also carries genes for the type four secretion system (T4SS) of H. pylori. The interaction of CagA with intracellular partner proteins leads to some irreversible alteration of host cells by increasing cell size, elevating motility, phenomena known as the “hummingbird phenotype”. CagA also disrupts the epithelium apical junctions and thereby destroys the normal epithelial architecture. A tyrosine phosphorylation site, named EPIYA motif, helps CagA to bind to cytosolic proteins in a phosphorylation-dependent manner. CagA is also interacts with host proteins in a phosphorylation-independent fashion, which altogether will assist to develop adenocarcinoma in infected cells. This review summarizes the core data on the structure and function of CagA and its role in conferring the main pathophysiologic effects of H. pylori infection as well as suggesting a therapeutic option for treatment of H. pylori infection based on CagA virulence

Цитотоксин-ассоциированный ген А (CagA) Helicobacter pylori: парадигма онкогенного фактора вирулентности

Helicobacter pylori is a microaerophilic, spiral-shaped and gram-negative microorganism that produces various virulence factors such as CagA, VacA, urease, and host cells adhesins, which in a synchronous concert, allow H. pylori to colonize and infect the host gastric epithelium. H. pylori infection is associated with some severe side effects in human, such as gastritis, peptic ulcer, non-Hodgkin’s lymphoma and adenocarcinoma. CagA is the most notorious virulence factor of H. pylori. It is known as the first bacterial oncoprotein. The gene encoding CagA is localized on the cag pathogenicity island (cagPAI), a 40kbp DNA segment which also carries genes for the type four secretion system (T4SS) of H. pylori. The interaction of CagA with intracellular partner proteins leads to some irreversible alteration of host cells by increasing cell size, elevating motility, phenomena known as the “hummingbird phenotype”. CagA also disrupts the epithelium apical junctions and thereby destroys the normal epithelial architecture. A tyrosine phosphorylation site, named EPIYA motif, helps CagA to bind to cytosolic proteins in a phosphorylation-dependent manner. CagA is also interacts with host proteins in a phosphorylation-independent fashion, which altogether will assist to develop adenocarcinoma in infected cells. This review summarizes the core data on the structure and function of CagA and its role in conferring the main pathophysiologic effects of H. pylori infection as well as suggesting a therapeutic option for treatment of H. pylori infection based on CagA virulenceHelicobacter pylori – это микроаэрофильная, спиралевидная, грамотрицательная бактерия, которая производит различные факторы вирулентности, такие как CagA, VacA, уреаза, а также адгезины, которые обеспечивают адгезию к клетке-хозяину. Синхронизированное взаимодействие факторов вирулентности позволяет H. pylori колонизировать и инфицировать эпителий желудка хозяина. Инфицирование организма человека H. pylori вызывает ряд побочных эффектов, таких как гастрит, язвенная болезнь желудка и двенадцатиперстной кишки, неходжкинская лимфома и аденокарцинома. CagA является наиболее печально известным фактором вирулентности H. pylori и признан первым бактериальным онкогеном. Он расположен на островке патогенности cag (cagPAI) – сегменте ДНК размером 40 т.п.н., который также содержит гены системы секреции четвертого типа (T4SS) H. pylori. Взаимодействие CagA с внутриклеточными белками-партнерами приводит к некоторым необратимым изменениям в клетках хозяина (увеличение их размера, повышение подвижности клеток), а также возникновению в клетках феномена, известного под названием «фенотип колибри». CagA также разрушает соединения в апикальном полюсе эпителиальных клеток, и тем самым разрушает нормальную архитектуру эпителия. Сайт фосфорилирования тирозина, называемый EPIYA мотивы, помогает CagA связываться с цитозольными белками фосфорилированно-зависимым образом. Также CagA может взаимодействовать с белками хозяина фосфорилированно-независимым способом, что в совокупности способствует развитию аденокарциномы в инфицированных клетках. В данном обзоре обобщены основные данные о структуре и функциях CagA, его роли в развитии основных патофизиологических эффектов в результате инфицирования H. pylori, а также о терапевтическом варианте лечения инфекции, вызываемой H. pylori, содержащей CagA фактор вирулентност

International journal of precision engineering and manufacturing : IJPEM

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions

Цитотоксин-ассоциированный ген А (CagA) Helicobacter pylori: парадигма онкогенного фактора вирулентности

Helicobacter pylori is a microaerophilic, spiral-shaped and gram-negative microorganism that produces various virulence factors such as CagA, VacA, urease, and host cells adhesins, which in a synchronous concert, allow H. pylori to colonize and infect the host gastric epithelium. H. pylori infection is associated with some severe side effects in human, such as gastritis, peptic ulcer, non-Hodgkin’s lymphoma and adenocarcinoma. CagA is the most notorious virulence factor of H. pylori. It is known as the first bacterial oncoprotein. The gene encoding CagA is localized on the cag pathogenicity island (cagPAI), a 40kbp DNA segment which also carries genes for the type four secretion system (T4SS) of H. pylori. The interaction of CagA with intracellular partner proteins leads to some irreversible alteration of host cells by increasing cell size, elevating motility, phenomena known as the “hummingbird phenotype”. CagA also disrupts the epithelium apical junctions and thereby destroys the normal epithelial architecture. A tyrosine phosphorylation site, named EPIYA motif, helps CagA to bind to cytosolic proteins in a phosphorylation-dependent manner. CagA is also interacts with host proteins in a phosphorylation-independent fashion, which altogether will assist to develop adenocarcinoma in infected cells. This review summarizes the core data on the structure and function of CagA and its role in conferring the main pathophysiologic effects of H. pylori infection as well as suggesting a therapeutic option for treatment of H. pylori infection based on CagA virulenceHelicobacter pylori – это микроаэрофильная, спиралевидная, грамотрицательная бактерия, которая производит различные факторы вирулентности, такие как CagA, VacA, уреаза, а также адгезины, которые обеспечивают адгезию к клетке-хозяину. Синхронизированное взаимодействие факторов вирулентности позволяет H. pylori колонизировать и инфицировать эпителий желудка хозяина. Инфицирование организма человека H. pylori вызывает ряд побочных эффектов, таких как гастрит, язвенная болезнь желудка и двенадцатиперстной кишки, неходжкинская лимфома и аденокарцинома. CagA является наиболее печально известным фактором вирулентности H. pylori и признан первым бактериальным онкогеном. Он расположен на островке патогенности cag (cagPAI) – сегменте ДНК размером 40 т.п.н., который также содержит гены системы секреции четвертого типа (T4SS) H. pylori. Взаимодействие CagA с внутриклеточными белками-партнерами приводит к некоторым необратимым изменениям в клетках хозяина (увеличение их размера, повышение подвижности клеток), а также возникновению в клетках феномена, известного под названием «фенотип колибри». CagA также разрушает соединения в апикальном полюсе эпителиальных клеток, и тем самым разрушает нормальную архитектуру эпителия. Сайт фосфорилирования тирозина, называемый EPIYA мотивы, помогает CagA связываться с цитозольными белками фосфорилированно-зависимым образом. Также CagA может взаимодействовать с белками хозяина фосфорилированно-независимым способом, что в совокупности способствует развитию аденокарциномы в инфицированных клетках. В данном обзоре обобщены основные данные о структуре и функциях CagA, его роли в развитии основных патофизиологических эффектов в результате инфицирования H. pylori, а также о терапевтическом варианте лечения инфекции, вызываемой H. pylori, содержащей CagA фактор вирулентност

Thermal unfolding and refolding transitions of CagA domains monitored by far-UV CD.

<p>The unfolding data is shown with black dots for CagA-N (a), CagA-M (b), CagA-M_c (c) and CagA-R (d). Unfolding was reversible for CagA-M, CagA-M_c and CagA-R; the refolding data is shown with open circles. The insets show the corresponding CD spectra for CagA-M (b), CagA-M_c (c) and CagA-R (d) for the native (solid line), unfolded (85 °C, dashed line) and refolded (dotted line) states. The low signal-to-noise ratio for the CagA-<i>R </i><i>spectrum</i> reflects the fact that the scan for this fragment was performed at lower wavelengths (205 nm rather than 222 nm), where the absorbance is inherently higher and thus the data is collected at a higher dynode voltage.</p