Significant under expression of the DosR regulon in M. tuberculosis complex lineage 6 in sputum

Abstract

Mycobacterium africanum lineage (L) 6 is an important pathogen in West Africa, causing up to 40% of pulmonary tuberculosis (TB). The biology underlying the clinical differences between M. africanum and M. tuberculosis sensu stricto remains poorly understood. We performed ex vivo expression of 2179 genes of the most geographically dispersed cause of human TB, M. tuberculosis L4 and the geographically restricted, M. africanum L6 directly from sputa of 11 HIV-negative TB patients from The Gambia who had not started treatment. The DosR regulon was the most significantly decreased category in L6 relative to L4. Further, we identified nonsynonymous mutations in major DosR regulon genes of 44 L6 genomes of TB patients from The Gambia and Ghana. Using Lebek's test, we assessed differences in oxygen requirements for growth. L4 grew only at the aerobic surface while L6 grew throughout the medium. In the host, the DosR regulon is critical for M. tuberculosis in adaptation to oxygen limitation. However, M. africanum L6 appears to have adapted to growth under hypoxic conditions or to different biological niches. The observed under expression of DosR in L6 fits with the genomic changes in DosR genes, microaerobic growth and the association with extrapulmonary disease.

Keywords: DosR; Gene expression; Hypoxia; Mycobacterium africanum; Mycobacterium tuberculosis; Sputum.

Fig. 1

Volcano plot of all genes tested visualizing fold change in expression and statistical significance. Dark blue dots indicate genes with no significant difference in expression between M. africanum L6 and M. tuberculosis L4. Mid-dark blue shows genes with unadjusted p value less than 0.05 and log 2 Fold change between −1 and 1. Light blue describes genes with unadjusted p value less than 0.05 and log 2 Fold change greater than (−)1. DosR genes significantly under expressed in M. africanum L6 relative to M. tuberculosis L4 following Bonferroni correction are shown in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Median qPCR cycle threshold values of the 48 dormancy regulon genes of M. africanum L6 and M. tuberculosis L4. Genes with significantly lower expression in M. africanum L6 relative to M. tuberculosis L4 are shown in green (same genes as the green ones in Fig. 1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Single Nucleotide Polymorphisms detected in 44 M. africanum L6 strains from Gambia and Ghana relative to H37Rv in under expressed genes. Blue and yellow indicate a SNP in Gambian and Ghanaian isolates respectively and white indicates wildtype. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Lebek test for oxygen preference. Test for oxygen preference in Lebek medium for the reference strain H37Rv (L4), a clinical L4 strain and 2 clinical M. africanum strains (L6). From left to right, tube 1, Negative control; tube 2, M. tuberculosis H37Rv; tube 3, L4 M. tuberculosis clinical strain (991508); tube 4, L6 M. africanum clinical strain (112287); tube 5, L6 M. africanum clinical strain (133158), showing diffused growth by M. africanum L6 compared to surface growth by M. tuberculosis.