RESUME Avec près de huit millions de personnes atteintes et deux millions de décès chaque année, ... more RESUME Avec près de huit millions de personnes atteintes et deux millions de décès chaque année, la tuberculose reste un problème majeur de santé à l'échelle mon-diale. La faible protection conférée par le seul vaccin disponible, le Bacille de Calmette et Guérin (BCG), et l'émergence de souches multi-résistantes aux antibio-tiques ainsi que l'avènement du SIDA, sont les trois causes principales qui ont contribué à l'augmentation de l'incidence de la tuberculose lors de la dernière décennie. L'organisation mondiale de la santé (OMS) estime qu'entre les années 2000 et 2020, près d'un mil-liard de personnes seront nouvellement infectées par Mycobactérium tuberculosis (Mtb), l'agent étiologique de la tuberculose, 200 millions d'entre elles développe-ront la maladie et parmi ceux-ci 35 millions mourront si aucune amélioration n'est apportée dans le contrôle de cette infection. Une telle amélioration nécessite un accroissement de nos connaissanc...
Early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) are c... more Early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) are complex proteins secreted by Mycobacterium tuberculosis that play a major role in the pathogenesis of tuberculosis. However, studies focusing on the biological functions of ESAT-6 led to discordant results and the role of ESAT-6 remains controversial. In the present study, we aim to address a potential explanation for this discrepancy and to highlight the physiological impact of two conformational states of ESAT-6. Analysis of a recombinant form of ESAT-6 by native gel electrophoresis, size exclusion chromatography and CD spectroscopy revealed that ESAT-6 forms dimers/multimers with higher molecular weight, which disappeared under the action of the detergent amidosulfobetaine-14 (ASB), giving rise to another conformational state of the protein. NMR has further indicated that ASB-treated versus nontreated ESAT-6 adopted distinct structural forms but with no well defined tertiary structure. However, protein-protein docking analysis favored a dimeric state of ESAT-6. Interestingly, the two preparations presented opposing effects on mycobacterial infectivity, as well as macrophage survival, interferon-γ secretion and membrane pore formation. Thereafter, we generated a recombinant form of the physiological heterodimer ESAT-6/CFP-10 that ASB was also able to dissociate and which showed functions similar to those of ESAT-6 dimers/multimers. Our data suggest that, in the absence of CFP-10, the hydrophobic regions of the ESAT-6 can form dimers/multimers, mimicking the ESAT-6/CFP-10 heterodimer, whereas their dissociation generates a protein presenting entirely different activities. Overall, the present study clarifies the intriguing divergences between reports that could be attributed to the ESAT-6 oligomeric state and sheds light on its importance for a better comprehension of the physiopathology of tuberculosis.
Taguchi's methods were used for the design of an experimental strategy aimed at optimizing ce... more Taguchi's methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or beta 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the alpha subunit CD11b. Anti beta 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these...
With nearly eight million new cases each year and two million deaths, tuberculosis (TB) remains a... more With nearly eight million new cases each year and two million deaths, tuberculosis (TB) remains a major health problem worldwide. The limited protection afforded by the only available vaccine, Bacille Calmette-Guerin BCG, and the emergence of multi-resistant strains to antibiotics along with the advent of AIDS, are three main causes that contributed to the increase of TB incidence during the last decade. The World Health Organization (WHO) estimates between 2000 and 2020, nearly one billion people will be newly infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, and 200 millions of them will develop the disease, of which 35 million will die if there is no improvement in controlling infection. Such improvement requires an increase in our knowledge of the fundamental biology of this very complex disease and in particular a better understanding and characterization of the types of interactions between mycobacteria and the immune system. The alveolar mac...
In response to cell activation signals, integrins switch from a low to a high affinity state. Phy... more In response to cell activation signals, integrins switch from a low to a high affinity state. Physiologic ligands bind to integrins through a von Willebrand Factor A-type domain. Crystallographic studies revealed two conformations of this domain, "closed" and "open." The latter crystallizes in complex with a pseudoligand or ligand, suggesting that it represents the high affinity state; data linking structure and activity are lacking however. In this communication, we expressed stable low and high affinity forms of integrin CD11b A-domain and determined their binding isotherms and crystal structures. The low affinity form, generated by deleting an N-terminal extension extrinsic to the domain, did not bind to physiologic ligands, and crystallized in the closed conformation. The high affinity form was generated by either deleting or substituting an invariable C-terminal Ile(316), wedged into a hydrophobic socket in the closed form, but displaced from it in the open ...
RESUME Avec près de huit millions de personnes atteintes et deux millions de décès chaque année, ... more RESUME Avec près de huit millions de personnes atteintes et deux millions de décès chaque année, la tuberculose reste un problème majeur de santé à l'échelle mon-diale. La faible protection conférée par le seul vaccin disponible, le Bacille de Calmette et Guérin (BCG), et l'émergence de souches multi-résistantes aux antibio-tiques ainsi que l'avènement du SIDA, sont les trois causes principales qui ont contribué à l'augmentation de l'incidence de la tuberculose lors de la dernière décennie. L'organisation mondiale de la santé (OMS) estime qu'entre les années 2000 et 2020, près d'un mil-liard de personnes seront nouvellement infectées par Mycobactérium tuberculosis (Mtb), l'agent étiologique de la tuberculose, 200 millions d'entre elles développe-ront la maladie et parmi ceux-ci 35 millions mourront si aucune amélioration n'est apportée dans le contrôle de cette infection. Une telle amélioration nécessite un accroissement de nos connaissanc...
Early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) are c... more Early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP-10) are complex proteins secreted by Mycobacterium tuberculosis that play a major role in the pathogenesis of tuberculosis. However, studies focusing on the biological functions of ESAT-6 led to discordant results and the role of ESAT-6 remains controversial. In the present study, we aim to address a potential explanation for this discrepancy and to highlight the physiological impact of two conformational states of ESAT-6. Analysis of a recombinant form of ESAT-6 by native gel electrophoresis, size exclusion chromatography and CD spectroscopy revealed that ESAT-6 forms dimers/multimers with higher molecular weight, which disappeared under the action of the detergent amidosulfobetaine-14 (ASB), giving rise to another conformational state of the protein. NMR has further indicated that ASB-treated versus nontreated ESAT-6 adopted distinct structural forms but with no well defined tertiary structure. However, protein-protein docking analysis favored a dimeric state of ESAT-6. Interestingly, the two preparations presented opposing effects on mycobacterial infectivity, as well as macrophage survival, interferon-γ secretion and membrane pore formation. Thereafter, we generated a recombinant form of the physiological heterodimer ESAT-6/CFP-10 that ASB was also able to dissociate and which showed functions similar to those of ESAT-6 dimers/multimers. Our data suggest that, in the absence of CFP-10, the hydrophobic regions of the ESAT-6 can form dimers/multimers, mimicking the ESAT-6/CFP-10 heterodimer, whereas their dissociation generates a protein presenting entirely different activities. Overall, the present study clarifies the intriguing divergences between reports that could be attributed to the ESAT-6 oligomeric state and sheds light on its importance for a better comprehension of the physiopathology of tuberculosis.
Taguchi's methods were used for the design of an experimental strategy aimed at optimizing ce... more Taguchi's methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or beta 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the alpha subunit CD11b. Anti beta 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these...
With nearly eight million new cases each year and two million deaths, tuberculosis (TB) remains a... more With nearly eight million new cases each year and two million deaths, tuberculosis (TB) remains a major health problem worldwide. The limited protection afforded by the only available vaccine, Bacille Calmette-Guerin BCG, and the emergence of multi-resistant strains to antibiotics along with the advent of AIDS, are three main causes that contributed to the increase of TB incidence during the last decade. The World Health Organization (WHO) estimates between 2000 and 2020, nearly one billion people will be newly infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, and 200 millions of them will develop the disease, of which 35 million will die if there is no improvement in controlling infection. Such improvement requires an increase in our knowledge of the fundamental biology of this very complex disease and in particular a better understanding and characterization of the types of interactions between mycobacteria and the immune system. The alveolar mac...
In response to cell activation signals, integrins switch from a low to a high affinity state. Phy... more In response to cell activation signals, integrins switch from a low to a high affinity state. Physiologic ligands bind to integrins through a von Willebrand Factor A-type domain. Crystallographic studies revealed two conformations of this domain, "closed" and "open." The latter crystallizes in complex with a pseudoligand or ligand, suggesting that it represents the high affinity state; data linking structure and activity are lacking however. In this communication, we expressed stable low and high affinity forms of integrin CD11b A-domain and determined their binding isotherms and crystal structures. The low affinity form, generated by deleting an N-terminal extension extrinsic to the domain, did not bind to physiologic ligands, and crystallized in the closed conformation. The high affinity form was generated by either deleting or substituting an invariable C-terminal Ile(316), wedged into a hydrophobic socket in the closed form, but displaced from it in the open ...
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Papers by Makram Essafi