Deciphering the growth behaviour of Mycobacterium africanum

Abstract

Background: Human tuberculosis (TB) in West Africa is not only caused by M. tuberculosis but also by bacteria of the two lineages of M. africanum. For instance, in The Gambia, 40% of TB is due to infections with M. africanum West African 2. This bacterial lineage is associated with HIV infection, reduced ESAT-6 immunogenicity and slower progression to active disease. Although these characteristics suggest an attenuated phenotype of M. africanum, no underlying mechanism has been described. From the first descriptions of M. africanum in the literature in 1969, the time to a positive culture of M. africanum on solid medium was known to be longer than the time to a positive culture of M. tuberculosis. However, the delayed growth of M. africanum, which may correlate with the less virulent phenotype in the human host, has not previously been studied in detail.

Methodology/principal findings: We compared the growth rates of M. tuberculosis and M. africanum isolates from The Gambia in two liquid culture systems. M. africanum grows significantly slower than M. tuberculosis, not only when grown directly from sputa, but also in growth experiments under defined laboratory conditions. We also sequenced four M. africanum isolates and compared their whole genomes with the published M. tuberculosis H37Rv genome. M. africanum strains have several non-synonymous SNPs or frameshift mutations in genes that were previously associated with growth-attenuation. M. africanum strains also have a higher mutation frequency in genes crucial for transport of sulphur, ions and lipids/fatty acids across the cell membrane into the bacterial cell. Surprisingly, 5 of 7 operons, recently described as essential for intracellular survival of H37Rv in the host macrophage, showed at least one non-synonymously mutated gene in M. africanum.

Conclusions/significance: The altered growth behaviour of M. africanum might indicate a different survival strategy within host cells.

Figure 1. Culture methods and spoligotyping results from isolates obtained from sputum samples of 552 patients in the study.

Figure 2. Frequency distributions for the Time-to-Positivity of mycobacterial cultures in two liquid culture systems.

All samples were incubated for 42 days. Upper panels: results for the Bactec 9000, Lower panels: Bactec MGIT 960, solid bars: M. tuberculosis, open bars: M. africanum.

Figure 3. In vitro growth curves from standardized inocula.

M. tuberculosis (solid line) and M. africanum (dashed line) were grown from standardized inocula in Bactec MGIT 960 and measured growth units (GU) are plotted versus time [days].

Figure 4. Schematic overview of molecular transport mechanisms present in M. africanum.

Genetic information of 132 genes encoding various families of membrane transporters was compared between the sequenced M. africanum strains and M. tuberculosis H37Rv. The analysis comprised both, transport mechanisms specific to nutrients and macromolecules (upper and lower figure). Genes that are identical to the wildtype M. tuberculosis H37Rv gene homologue are displayed with a solid border line and white background. Genes, with non-synonymous mutation are displayed in blue with dashed lines. Genes with a frameshift mutation, are displayed with striped background.

Figure 5. Status of putative M. africanum operons essential for intracellular survival.

Seven operons were previously defined as essential for growth within the macrophage . Genes that are identical to the wildtype M. tuberculosis H37Rv gene homologue are displayed with a solid border line and white background. Genes, with a non-synonymous mutation are displayed in blue with dashed lines. Genes with a frameshift mutation, are displayed with striped background.