The production of mycobacterial antigens by homogeneous culture in a fermentor

Nyabenda, J; Bautens, Erwin; Borremans, Martine; Verhofstadt, Roger; Weckx, Maurice; Marchal, André

doi:10.1016/0092-1157(88)90013-3

Cited by 4 publications

(2 citation statements)

References 9 publications

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“…This study allowed the effect of oxygen limitation on the transcriptome of M. tuberculosis to be investigated at a constant growth rate and independently of any other environmental parameters (3). Mycobacterium bovis bacillus Calmette-Guerin (BCG) has also been cultivated successfully in a bioreactor (16,30,42). These studies investigated mycobacterial growth at one specific growth rate.…”

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Compiling a Molecular Inventory for Mycobacterium bovis BCG at Two Growth Rates: Evidence for Growth Rate-Mediated Regulation of Ribosome Biosynthesis and Lipid Metabolism

et al. 2005

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An experimental system of Mycobacterium tuberculosis growth in a carbon-limited chemostat has been established by the use of Mycobacterium bovis BCG as a model organism. For this model, carbon-limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 h (D ‫؍‬ 0.03 h ؊1 ) was adopted to represent cells during the acute phase of infection, whereas a lower dilution rate equivalent to a doubling time of 69 h (D ‫؍‬ 0.01 h ؊1 ) was used to model mycobacterial persistence. This chemostat model allowed the specific response of the mycobacterial cell to carbon limitation at different growth rates to be elucidated. The macromolecular (RNA, DNA, carbohydrate, and lipid) and elemental (C, H, and N) compositions of the biomass were determined for steady-state cultures, revealing that carbohydrates and lipids comprised more than half of the dry mass of the BCG cell, with only a quarter of the dry weight consisting of protein and RNA. Consistent with studies of other bacteria, the specific growth rate impacts on the macromolecular content of BCG and the proportions of lipid, RNA, and protein increased significantly with the growth rate. The correlation of RNA content with the growth rate indicates that ribosome production in carbon-limited M. bovis BCG cells is subject to growth rate-dependent control. The results also clearly show that the proportion of lipids in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. Finally, this study demonstrates the utility of the chemostat model of mycobacterial growth for functional genomic, physiology, and systems biology studies.With three million people dying from tuberculosis (TB) annually, Mycobacterium tuberculosis remains a formidable pathogen. Tuberculosis ranks among the top 10 causes of global mortality and morbidity and is the leading cause of infectious disease (66). The ability of M. tuberculosis to adapt to and survive harsh environmental conditions in order to establish and maintain long-term infections within its human host is fundamental to this organism's success. Modification of the mycobacterial cell in response to changes in the environment is crucial to this adaptive process, but detailed information about how M. tuberculosis changes its macromolecular composition in response to its environment and growth rate is lacking.The genome sequence of M. tuberculosis has been available since 1998 (9). Although information obtained from the genome sequence provided new and valuable insights into the biology of the tubercle bacillus, the genome itself provides few clues regarding how the pathogen responds to its environment by changing its cellular composition. One of the principal tasks of postgenomic biological studies of M. tuberculosis is to understand how the genome orchestrates the structure and dynamics of the cell in response to changes in the environment. This task requires an integrat...

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Compiling a Molecular Inventory for Mycobacterium bovis BCG at Two Growth Rates: Evidence for Growth Rate-Mediated Regulation of Ribosome Biosynthesis and Lipid Metabolism

et al. 2005

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“…To circumvent these difficulties, we chose to investigate the growth of Map in a bioreactor to increase the biomass, standardize the culture conditions and control potential contaminations. Previous studies using bioreactors have described the standardization of M. bovis BCG cultures for vaccine production [19,20]. Growth of Map in a bioreactor was compared to conventional culture flasks inoculated in parallel with the same Map pre-culture at an OD 600nm of 0.06.…”

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Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis

et al. 2013

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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.

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