Transcription of genes involved in sulfolipid and polyacyltrehalose biosynthesis of Mycobacterium tuberculosis in experimental latent tuberculosis infection

Abstract

The Influence of trehalose-based glycolipids in the virulence of Mycobacterium tuberculosis (Mtb) is recognised; however, the actual role of these cell-wall glycolipids in latent infection is unknown. As an initial approach, we determined by two-dimensional thin-layer chromatography the sulfolipid (SL) and diacyltrehalose/polyacyltrehalose (DAT/PAT) profile of the cell wall of hypoxic Mtb. Then, qRT-PCR was extensively conducted to determine the transcription profile of genes involved in the biosynthesis of these glycolipids in non-replicating persistent 1 (NRP1) and anaerobiosis (NRP2) models of hypoxia (Wayne model), and murine models of chronic and progressive pulmonary tuberculosis. A diminished content of SL and increased amounts of glycolipids with chromatographic profile similar to DAT were detected in Mtb grown in the NRP2 stage. A striking decrease in the transcription of mmpL8 and mmpL10 transporter genes and increased transcription of the pks (polyketidesynthase) genes involved in SL and DAT biosynthesis were detected in both the NRP2 stage and the murine model of chronic infection. All genes were found to be up-regulated in the progressive disease. These results suggest that SL production is diminished during latent infection and the DAT/PAT precursors can be accumulated inside tubercle bacilli and are possibly used in reactivation processes.

Figure 1. Structure of SL-I and DAT/PAT of M. tuberculosis.

The trehalose core is sulphated in position 2′ and esterified with palmitic, multimethyl-branched phtioceranic and hydroxyphtioceranic acids in positions 2, 4, 6 and 6′ for SL-I biosynthesis; it is also esterified with stearic and multimethyl-branched mycolipenic acids in positions 2, 2′, 3′, 4 and 6′ for PAT, or with mycosanoic acids for DAT biosynthesis. The enzymatic actions of Pks: polyketidesynthase, PapA: acyltransferases and MmpL: mycobacterial membrane protein-large polyketide transporters are shown by dashed lines and arrows.

Figure 2. Trehalose-derived glycolipid profile and neutral red staining of M. tuberculosis grown in NRP2 stage.

2D-TLC analysis of Mtb H37Rv grown under oxygen-replete (A and C) and NRP2 (B and D) conditions. Crude extracts were resolved using the two-solvent system: chloroform:methanol:water (60∶12:1, v/v) for the first direction and chloroform:methanol:water (75∶11:1, v/v) for the second direction. For resolving polar glycolipids (PGs) accumulated in the inoculation point, the elusion time in C and D was prolonged for 30 min after the resolving system reached the edge of the TLC plate. The neutral red staining observed for M. tuberculosis H37Rv cultured in aerobic (E) and NRP2 stages (F) is shown. TDM, trehalose dimycolate; SL, sulfolipid; DAT, diacyltrehalose; PGs, polar glycolipids.

Figure 3. In vitro transcription of genes in M. tuberculosis H37Rv during the NRP1 and NRP2 stages.

The relative quantification is expressed as the ratio of NRP1 and NRP2/transcription of exponential-oxygenated phase. The presented data have statistically significant differences compared to exponential phase values (P<0.05).

Figure 4. In vivo transcription of genes in M. tuberculosis H37Rv during long-lasting TB infection in mice.

The relative quantification is expressed as the ratio of infection at months 1, 3, 5 and reactivation/transcription at day 28 of progressive infection TB. The presented data have statistically significant differences compared to transcription values at day 28 of progressive infection TB (P<0.05).

Figure 5. In vivo transcription of genes of M. tuberculosis H37Rv during progressive tuberculosis in mice.

The relative quantification is expressed as the ratio of infection at days 1 and 120/transcription at day 28 of progressive infection TB. The presented data have statistically significant differences compared to transcription values at day 28 of progressive infection TB (P<0.05).

Figure 6. Model showing accumulation of DAT and SL/PAT precursors in latent M. tuberculosis.

The diminished transcription of mmpL8 and mmpL10 might avoid the complete biosynthesis of trehalose-based glycolipids and the subsequent accumulation of precursors as polar glycolipids and DAT in the plasma membrane (See discussion for details). Plasma membrane (PM); external side of cell wall (ESCW).