Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis

Abstract

BackgroundParatuberculosis 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.FindingsFor 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.ConclusionsAn 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.

Novel Feature of Mycobacterium avium subsp. paratuberculosis, Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin

“…Investigation of the T-cell responses in animals infected by strains of each type thus requires establishment of the exact methylation patterns of the HBHA produced by each lineage and the availability of mapC-HBHA and mapS-HBHA in their correctly methylated forms. We have observed recently that lysine residues 168 and 153 of native mapC-HBHA are methylated (24), but the precise methylation patterns and other translational modifications remain to be determined.…”

“…Until now, the HBHA molecules reported for the subspecies M. avium subsp. paratuberculosis belonged to type C (23,24). The variability between mapC-HBHA and mapS-HBHA is restricted to the C-terminal domain of the protein.…”

“…Recombinant HBHA proteins produced in E. coli were analyzed by heparin-Sepharose chromatography. As described previously (24), mapC-HBHA bound weakly to heparin-Sepharose and was eluted at 200 mM NaCl in a NaCl gradient from 0 to 1 M (Fig. 5A).…”

“…Alignment of HBHA protein sequences reveals that the large N-terminal domains are highly conserved, while the C-terminal domains are heterogeneous between mycobacterial species (24,33). To identify the hbhA genes in M. avium subsp.…”

“…One of the bestcharacterized mycobacterial adhesins is the heparin-binding hemagglutinin (HBHA), initially identified in Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin (BCG) (19,20). However, HBHA is also present in many other mycobacteria, both pathogenic and nonpathogenic (21)(22)(23)(24).…”