Comparative transcriptional study of the putative mannose donor biosynthesis genes in virulent Mycobacterium tuberculosis and attenuated Mycobacterium bovis BCG strains

Abstract

Mycobacterium tuberculosis contains mannosylated cell wall components which are important in macrophage recognition and response. The building block for the mannosyl constituents of these components is GDP-mannose, which is synthesized through a series of enzymes involved in the mannose donor biosynthesis pathway. Nothing is known about the expression levels of the genes encoding these enzymes during the course of infection. To generate transcriptional profiles for the mannose donor biosynthesis genes from virulent M. tuberculosis and attenuated Mycobacterium bovis BCG, bacteria were grown in broth culture and within human macrophages. Our results with broth-grown bacteria show that there are differences in expression of the selected genes between M. tuberculosis and BCG, with increased expression of manC in M. tuberculosis and manA in BCG during stationary-phase growth. Results for M. tuberculosis extracted from within macrophages show that whiB2 is highly expressed and manB and manC are moderately expressed during infection. Rv3256c, Rv3258c, and ppm1 have high expression levels early and decreased expression as the infection progresses. Results with BCG show that, as in M. tuberculosis, whiB2 is highly expressed throughout infection, whereas there is either low expression or little change in expression of the remaining genes studied. Overall, our results show that there is differential regulation of expression of several genes in the mannose donor biosynthesis pathway of M. tuberculosis and BCG grown in broth and within macrophages, raising the possibility that the level of mannose donors may vary during the course of infection and thereby impact the biosynthesis of mannose-containing cell wall molecules.

Fig. 1.

Putative mannose donor biosynthesis pathway for the building of two key mannose donor molecules, GDP-mannose and polyprenyl monophosphate mannose (PPM), which serve as substrates for mannosyltransferases. The inset shows the genomic location and arrangement of genes encoding key enzymes, manA, manB, and manC, in the pathway as well as the other genes of interest with unknown functions, Rv3253c, Rv3256c, and Rv3258c, used for the transcriptional expression study.

Fig. 2.

Transcriptional profile of putative mannose donor biosynthesis genes of M. tuberculosis (A) and BCG (B) grown in broth medium. cDNA was made from RNA extracted from both strains at their different growth phases and subjected to real-time PCR for determining the amount of transcripts of each gene as a measure of its expression. Expression of target genes is relative to that of the housekeeping gene, rpoB, and is plotted against the OD₆₀₀ of the culture corresponding to the time points (in days) used for generating growth curves. Data are means ± standard deviations of values from triplicate wells. Graphs were plotted from representative experiments (n = 3).

Fig. 3.

Transcriptional profiles of putative mannose donor biosynthesis genes in M. tuberculosis and BCG from infected MDMs. Single-cell suspensions of M. tuberculosis grown on 7H11 agar plates were used to infect MDM monolayers at an MOI of 5:1. GTC lysis buffer was used to extract RNA at 24, 48, 72, and 120 h. cDNA was synthesized from RNA and subjected to real-time PCR to determine the amount of transcripts of each gene as a measure of its expression. (A) Transcriptional profile of the single-cell suspension prior to infection. (B and C) Transcriptional profiles of M. tuberculosis and BCG harvested from infected macrophages. Data are means ± standard deviations of values from triplicate wells. Graphs were plotted from representative experiments (n = 5).