Mycobacterium bovis BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism

Abstract

Mycobacterium bovis BCG is the only accepted vaccine for the prevention of tuberculosis (TB) in humans.BCG is a live vaccine, and induction of immunity to TB requires productive infection of the host by BCG. However, BCG is not a satisfactory vaccine, because it fails to protect against pulmonary TB in adults. In this study, we found that BCG strains cannot utilize many naturally occurring amino acids as the sole nitrogen source for growth. This defect is caused, at least partially, by the lack of functional metabolic enzymes. All BCG strains are unable to catabolize L-alanine or D-alanine due to a frameshift mutation in the L-alanine dehydrogenase gene (ald). Some BCG strains, such as BCG-Pasteur and BCG-Frappier, cannot catabolize L-serine, apparently due to inadequate expression of L-serine deaminase (sdaA). We also found that undegraded alanine and serine inhibit the growth of BCG through blockage of glutamine synthetase. These results suggest that BCG strains are limited in nitrogen metabolic capacity and predict defects that may restrict multiplication and persistence of the live vaccine within the host.Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis isolated in 1921, is currently administered to over 100 million newborns per year as the only available vaccine against tuberculosis (TB) (13). BCG is only efficacious as a live vaccine; killed BCG does not provide protection in animal models, and killed Mycobacterium tuberculosis in human trials provided only weak and transient protection. In clinical trials of BCG vaccination, the observed efficacy has ranged from no protection to 80% fewer cases of TB (11, 13). A better understanding of this variable efficacy should help guide BCG programs and suggest avenues for the development of novel anti-TB vaccines.Several hypotheses have been generated to explain the vaccine trial data (3). The most prominent is that exposure to environmental mycobacteria partially sensitizes the host against mycobacteria and thereby provides heterologous immunity that obscures the potential benefits of BCG vaccination (14, 15). To support this, a recent study showed that the multiplication of the Danish strain of BCG was inhibited in animals previously sensitized with environmental mycobacteria. Consequently BCG vaccination elicited only a transient immune response and afforded no protection against a subsequent TB challenge (8). A second hypothesis involves differences in the vaccine strains used in clinical trials (9). After their introduction in 1921, BCG vaccines were maintained by in vitro passage in a variety of vaccine laboratories for 4 to 5 decades, resulting in phenotypic and genetic differences between BCG strains (6, 27). The capability of each of these BCG vaccines to protect against TB is unknown, because most clinical trials were performed with vaccine strains that have not been preserved. Notably, among genetic changes in BCG strains after 1921, one observes deletions in regulatory genes (which are postulated to govern the capacity to survi...

Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin

“…Cells were then washed thrice with iron-free Sauton's media and resuspended in 100 ml of media with or without 50 mg transferrin at 4°C or 37°C for 6 h. Finally, cells were washed and fluorescence data from 10,000 cells per sample were acquired, using a Guava Soft Express Pro Flowcytometer. To confirm the sensitivity of the assay, M.tb H37Ra cells were pre-incubated with calcein as described above followed by incubation with Sauton's media 60 containing ferric ammonium citrate at 4°C or 37°C or media without iron. Data are plotted as mean fluorescence intensity (MFI)±s.e.m.…”

Contribution of L‐Alanine Dehydrogenase to In Vivo Persistence and Protective Efficacy of the BCG Vaccine

“…This suggests the Ald protein may be a virulence determinant expressed by M. tuberculosis during the latent state of infection. In addition, the inability of BCG to utilise L-alanine inhibits BCG growth in axenic culture, and this has raised the intriguing possibility that loss of Ald function may limit vaccine persistence in vivo and hence influence protective efficacy (6). In this report we have restored Ald activity to BCG and determined the effect on virulence and protective efficacy of the vaccine against aerosol M. tuberculosis infection.…”

“…Protective efficacy of BCG:Ald against aerosol M. tuberculosis infection: The addition to BCG of M. tuberculosis of specific and shared antigens improves protective efficacy (11,16). Ald is a highly abundant secreted protein of M. tuberculosis that is absent from BCG, and the inability to catabolise alanine has been postulated to limit BCG efficacy (6). We therefore determined if addition of Ald to BCG improved its protective effect.…”

“…The loss of enzyme activity has been attributed to mutation in the BCG coding sequence (6). Analysis of BCG Pasteur genome sequence (http:// www.sanger.ac.uk/Projects/M_bovis/) revealed a point mutation at position 266 in the BCG sequence, which results in early termination of Ald synthesis (Fig.…”

Immunoproteomic Identification of Human T Cell Antigens of Mycobacterium tuberculosis That Differentiate Healthy Contacts from Tuberculosis Patients

“…The novel proteins identified in this study can be classified into five groups: (i) the metabolic enzymes Acn (13), GgtB (40), Pgi (41), Tal, and FabG4; (ii) proteins with a reduced presence or that are absent in BCG such as Ald (42) and Rv1558 (43) The presence of metabolic enzymes has been previously reported in the CF or extracellular milieu of M. tuberculosis and various pathogens (57). The extracellular localization of these proteins may be due to autolysis given the prolonged culture time (4 weeks) of M. tuberculosis.…”