Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.

International audienceBACKGROUND: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. FINDINGS: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. CONCLUSIONS: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays

Live Attenuated B. pertussis as a Single-Dose Nasal Vaccine against Whooping Cough

Pertussis is still among the principal causes of death worldwide, and its incidence is increasing even in countries with high vaccine coverage. Although all age groups are susceptible, it is most severe in infants too young to be protected by currently available vaccines. To induce strong protective immunity in neonates, we have developed BPZE1, a live attenuated Bordetella pertussis strain to be given as a single-dose nasal vaccine in early life. BPZE1 was developed by the genetic inactivation or removal of three major toxins. In mice, BPZE1 was highly attenuated, yet able to colonize the respiratory tract and to induce strong protective immunity after a single nasal administration. Protection against B. pertussis was comparable to that induced by two injections of acellular vaccine (aPV) in adult mice, but was significantly better than two administrations of aPV in infant mice. Moreover, BPZE1 protected against Bordetella parapertussis infection, whereas aPV did not. BPZE1 is thus an attractive vaccine candidate to protect against whooping cough by nasal, needle-free administration early in life, possibly at birth

Development and Standardization of a High-Throughput Bordetella pertussis Growth-Inhibition Assay

International audienceBordetella pertussis, the main causative agent of whooping cough, is a reemerging pathogen, and recent vaccine-resistant strain outbreaks and emergence of macrolides-resistant strains in China raised new concerns for control of the disease. New vaccines and potentially new antibiotics are thus needed. B. pertussis is tedious to culture and requires several days of growth to count isolated colonies on agar-based media, making large-scale screening of new anti-B. pertussis compounds or functional evaluation of large sample sizes of immune sera difficult. Here, we developed a scalable, rapid, high-throughput luminescence-based Bordetella growth inhibition assay (BGIA) to quantify surviving bacteria after treatment with anti-B. pertussis compounds. A strong correlation between luminescence and colony-forming units (r 2 = 0.9345, p < 0.0001) was found and the BGIA showed high sensitivity and reproducibility. We demonstrate here that the BGIA can be used to quantify resistance of B. pertussis to antibiotics, sensitivity to complement and to human serum in an easy-to-operate and fast manner. We have optimized the assay and tested the effects of different B. pertussis strains and growth conditions on serum and complement sensitivity. We also uncovered complement-independent antibody-mediated inhibition of B. pertussis growth. The BGIA can thus effectively be implemented for large-scale serum studies to further investigate anti-B. pertussis immune responses at a functional level, as well as for screening of B. pertussis strains for their resistance to antibiotics or complement, and for high-throughput screening of novel anti-B. pertussis compounds

Protection contre les infections hétérologues par la souche vaccinale atténuée Bordetella pertussis BPZE1

Les infections respiratoires font aujourd hui encore partie des premières causes de morbidité et mortalité à l échelle planétaire. De nombreux pathogènes viraux ou bactériens sont responsables de ces infections. Pour certaines d entre elles aucun vaccin n est disponible, comme c est le cas du virus respiratoire syncytial, et pour d autres les vaccins actuels souffrent d importantes limitations. Ces limitations sont souvent liées à la variation antigénique et l évolution constante des souches circulantes, comme dans le cas du virus de la grippe ou de Streptococcus pneumoniae. Pour d autres, comme la coqueluche, le schéma vaccinal actuel ne permet pas de suffisamment protéger la population la plus vulnérable, càd. les enfants en bas age. La majorité des vaccins actuels sont classiquement administrés par voie parentérale. Or, la porte d entrée des pathogènes respiratoires est la muqueuse nasale. Dès lors une immunisation intranasale semblerait plus indiquée. La vaccination intranasale présente l avantage de cibler à la fois l immunité locale et l immunité systémique. D autre part, elle ne fait pas appel à des procédures invasives et est facile à pratiquer.Dans cette optique, un nouveau vaccin vivant atténué contre la coqueluche a été développé dans le laboratoire d accueil. Ce vaccin, appelé BPZE1, consiste en une souche génétiquement atténuée de Bordetella pertussis, l agent étiologique de la coqueluche. Les études précliniques ont montré l efficacité de cette souche vaccinale après une seule administration nasale. De plus, les premiers essais cliniques chez les adultes ont démontré l innocuité et l immunogénicité de cette souche. Au-delà de l intérêt pour la vaccination contre la coqueluche, la souche BPZE1 présente aussi des propriétés anti-inflammatoires qui protègent contre l asthme et contre la mortalité induite par le virus hautement pathogène de la grippe.Dans la première partie de ma thèse, nous nous sommes intéressés à étudier la protection induite par BPZE1 contre des infections hétérologues. D abord, nous avons montré qu une seule dose de BPZE1 protège contre une infection létale par Bordetella bronchiseptica, une bactérie appartenant au même genre que la souche vaccinale. La protection se traduit par une réduction de l inflammation pulmonaire médiée par les cellules T régulatrices, par une augmentation significative de survie des souris infectées et une diminution de la charge bactérienne. Ensuite, nous avons étudié la protection hétérologue de BPZE1 contre une bactérie non apparentées, S. pneumoniae. Nous avons montré que l administration intranasale de BPZE1 protège contre la mortalité induite par S. pneumoniae. Cette protection est dépendante du facteur de différenciation myéloide 88 (MyD88). Dans la deuxième partie de ma thèse, nous nous sommes intéressés à combiner cet effet anti-inflammatoire, non spécifique de BPZE1 avec l induction d une réponse immunitaire spécifique contre un antigène hétérologue. Ainsi, nous avons étudié la possibilité d utiliser BPZE1 comme plateforme biotechnologique pour la présentation d antigènes protecteurs hétérologues afin de développer des vaccins nasaux multivalents. Trois différents modèles (virus d influenza, virus respiratoire syncytial et S. pneumoniae) ont été développés en exprimant les antigènes les mieux conservés et les plus prometteurs dans la souche BPZE1. L ensemble de ces travaux de recherche ont montré que BPZE1 est un candidat vaccinal prometteur pour exprimer des antigènes hétérologues et protéger contre les infections respiratoires hétérologues.Respiratory infections are still among the leading causes of morbidity and mortality worldwide. Many different bacterial or viral pathogens are responsible for these infections. For some of them no vaccine is available, as is the case for the respiratory syncytial virus, and other current vaccines suffer from significant limitations. These limitations are mainly related to the antigenic variation and the constant evolution of circulating strains, as is the case of influenza virus and Streptococcus pneumoniae. For others, such as pertussis, the current vaccination programs do not result in sufficient protection of the most vulnerable population, i. e. very young infants. Most of the current vaccines are administered parenterally. However, the port of entry of respiratory pathogens is the nasal mucosa. Intranasal vaccination seems therefore more appropriate. Intranasal vaccination has the advantage of targeting both the local and the systemic immune system. In addition, it is non invasive (needle-free) and easy to apply.In this context, a new, live attenuated vaccine against pertussis has been developed in the host laboratory. The vaccine, named BPZE1, is a genetically attenuated Bordetella pertussis strain, the causative agent of whooping cough. Preclinical studies have demonstrated the efficacy of this vaccine strain after a single nasal administration. In addition, the first clinical trials in adults have demonstrated the safety and efficacy of this strain. Beyond pertussis vaccination, BPZE1 has also anti-inflammatory properties that protect against asthma and against mortality induced by highly pathogenic influenza.In the first part of my thesis, we were interested in studying the protection induced by BPZE1 against heterologous infections. Firstly, we showed that a single administration of BPZE1 conferred heterologous protection against a lethal infection with Bordetella bronchiseptica. This protection was evidenced by a reduction of lung inflammation mediated by regulatory T cells, a significant increase in survival of the challenged mice and a significant decrease in bacterial load.Then, we have investigated the heterologous protection of BPZE1 against an unrelated bacterium, S. pneumonia. We found that an intranasal administration of BPZE1 protected against mortality caused by S. pneumoniae. This protection was MyD88 dependent.In the second part of my thesis, we were interested in studying the possibility of using BPZE1 as a biotechnological platform for the presentation of heterologous protective antigens to develop multivalent vaccines. Three different models (influenza virus, respiratory syncytial virus and S. pneumoniae) were developed by expressing the most promising and conserved antigens in BPZE1.In conclusion, our data indicates that BPZE1 is a promising candidate vaccine to express foreign antigens and to protect against heterologous respiratory infections.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

Intrabacterial Lipid Inclusions: overview of an amazing organelle

International audienceIntrabacterial lipid Inclusions (ILI), known as oil/fat bodies or adiposomes, are organelles found in intracellular pathogens, such as mycobacteria. They are composed of neutral lipids, mainly triacylglycerol, surrounded by a phospholipid monolayer containing a wide array of proteins. In the past decade, several groups have fostered the research on elucidating the ILI composition though the combination of a large panel of broad-range techniques, including proteomics, lipidomics or metabolomics, portraying an unexpected level of complexity. Although ILI with their associated proteins are thought to play various roles related to the metabolism and storage of neutral lipids, membrane biosynthesis, reduction of cytotoxicity and signal transduction; their exact role and contribution in virulence, their origin and evolution remain largely unknown. This chapter aims to summarize the current knowledge on the biology of ILI, their occurrence and evolution. It also reports the latest achievements regarding the procedures used to prepare and purify ILI as well as to cutting-edge methods, such as proximity labelling to identify protein partners associated with ILI. The access to newly discovered ILI-associated partners and their possible link with pathogenicity opens the path to a tremendous and fascinating emerging area in the mycobacterial field

Single-Molecule Force Spectroscopy of Mycobacterial Adhesin-Adhesin Interactions▿

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of α-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation

Interaction of the mycobacterial heparin-binding hemagglutinin with actin, as evidenced by single molecule force spectroscopy.

Although Mycobacterium tuberculosis and related species are considered to be typical endosomal pathogens, recent studies have suggested that mycobacteria can be present in the cytoplasm of infected cells and cause cytoskeleton rearrangements, the mechanisms of which remain unknown. Here, we used single-molecule force spectroscopy to demonstrate that the heparin-binding hemagglutinin (HBHA), a surface adhesin from Mycobacterium tuberculosis displaying sequence similarities with actin-binding proteins, is able to bind to actin. Force curves recorded between actin and the coiled-coil, N-terminal domain of HBHA showed a bimodal distribution of binding forces reflecting the detection of single and double HBHA-actin interactions. Force curves obtained between actin and the lysine-rich C-terminal domain of HBHA showed a broader distribution of binding events, suggesting they originate primarily from intermolecular electrostatic bridges between cationic HBHA domains and anionic actin residues. We also explored the dynamics of the HBHA-actin interaction, showing that the binding force and binding frequency increased with the pulling speed and contact time, respectively. Taken together, our data indicate that HBHA is able to specifically bind actin, via both its N-terminal and C-terminal domains, strongly suggesting a role of the HBHA-actin interaction in the pathogenesis of mycobacterial diseases

The mycobacterial heparin-binding hemagglutinin, virulence factor and antigen useful for diagnostics and vaccine development.

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