PPE51 mediates uptake of trehalose across the mycomembrane of Mycobacterium tuberculosis

Abstract

The disaccharide trehalose is essential for viability of Mycobacterium tuberculosis, which synthesizes trehalose de novo but can also utilize exogenous trehalose. The mycobacterial cell wall encompasses two permeability barriers, the cytoplasmic membrane and the outer mycolic acid-containing mycomembrane. The ABC transporter LpqY-SugA-SugB-SugC has previously been demonstrated to mediate the specific uptake of trehalose across the cytoplasmic membrane. However, it is still unclear how the transport of trehalose molecules across the mycomembrane is mediated. In this study, we harnessed the antimycobacterial activity of the analogue 6-azido trehalose to select for spontaneous resistant M. tuberculosis mutants in a merodiploid strain harbouring two LpqY-SugA-SugB-SugC copies. Mutations mediating resistance to 6-azido trehalose mapped to the proline-proline-glutamate (PPE) family member PPE51 (Rv3136), which has recently been shown to be an integral mycomembrane protein involved in uptake of low-molecular weight compounds. A site-specific ppe51 gene deletion mutant of M. tuberculosis was unable to grow on trehalose as the sole carbon source. Furthermore, bioorthogonal labelling of the M. tuberculosis Δppe51 mutant incubated with 6-azido trehalose corroborated the impaired internalization. Taken together, the results indicate that the transport of trehalose and trehalose analogues across the mycomembrane of M. tuberculosis is exclusively mediated by PPE51.

Figure 1

6-Azido trehalose (6-TreAz) inhibits Mtb growth at higher concentrations. (A) Structure of trehalose and 6-TreAz. (B) Growth inhibition of Mtb H37Rv wild-type (WT), the ΔlpqY-sugC mutant and the complemented mutant ΔlpqY-sugC pMV306(Kan)::Rv1235–1238 on Middlebrook 7H10 agar containing 1 mM 6-TreAz or just solvent control (1% DMSO = w/o 6-TreAz). Equal amounts of diluted cell suspensions of each strain were plated onto both types of agar and incubated for 3 weeks at 37 °C.

Figure 2

Isolation of spontaneous 6-TreAz-resistant Mtb mutants. 10 µl aliquots of tenfold-serially diluted cell suspensions of Mtb H37Rv wild-type (WT), the merodiploid strain Mtb H37Rv pMV306::Rv1235–1238, the ΔlpqY-sugC gene deletion mutant, the complemented mutant ΔlpqY-sugC pMV306::Rv1235–1238 and spontaneous 6-TreAz-resistant mutant clones were spotted each onto Middlebrook 7H10 agar containing 1 mM 6-TreAz or just solvent control (1% DMSO = w/o 6-TreAz). Plates were incubated for 3 weeks at 37 °C. For genotypes of mutant clones 1–4 and 6 refer to Table 1.

Figure 3

6-TreAz resistance of the Mtb Δppe51 deletion mutant. 10 µl aliquots of tenfold-serially diluted cell suspensions of Mtb H37Rv wild-type, the ΔlpqY-sugC gene deletion mutant and the Δppe51 gene deletion mutant were spotted onto Middlebrook 7H10 agar containing 1 mM 6-TreAz and incubated for 3 weeks at 37 °C. The Δppe51 gene deletion mutant is unable to utilize glucose as will be demonstrated in Fig. 4 and therefore exhibits a growth defect on the used solid medium that contains glycerol and glucose as main carbon sources.

Figure 4

Growth of the Mtb Δppe51 deletion mutant in minimal medium with glucose or trehalose as the sole carbon source. Cells of Mtb H37Rv wild-type (WT) (red bars), the Δppe51 mutant (green bars) and the complemented mutant Δppe51 pMV361::ppe51 (blue bars) were cultivated in minimal medium containing increasing concentrations (0–5 mM) of trehalose (A) or glucose (B), respectively. Growth was determined using the resazurin microplate assay and normalized to cells of Mtb H37Rv wild-type grown in Middlebrook 7H9 complete medium (= 100% growth) or medium only control (= 0% growth). Values are means of triplicates ± SD. Growth defects of the Δppe51 mutant observed at 5 mM concentration of both sugars compared to wild-type or the complemented mutant was statistically significant (p < 0.005 as determined using unpaired t test).

Figure 5

Growth of the Mtb Δppe51 deletion mutant in 7H9 limited medium with glycerol or trehalose as the sole carbon source. Cells of Mtb H37Rv wild-type (WT), the Δppe51 mutant and the complemented mutant Δppe51 pMV361::ppe51 were cultivated in 7H9 limited medium containing 0.5% (v/v) glycerol (blue bars), 10 mM trehalose (red bars) or no additional carbon source (brown bars). Strains grown in 7H9 complete medium served as positive controls (green bars). Growth was determined using the resazurin microplate assay and normalized to cells of Mtb H37Rv wild-type grown in 7H9 complete medium (= 100% growth) or medium only control (= 0% growth). Values are means of two independent experiments each performed in triplicates ± SD. Growth differences between the indicated groups were statistically significant (***p < 0.005 as determined using unpaired t test).

Figure 6

Metabolic labelling of Mtb strains employing click chemistry proves PPE51-dependent uptake of 6-TreAz. Mtb strains were incubated with 100 µM 6-TreAz for 3 days and subsequently conjugated with AF488 DIBO alkyne. Mean fluorescence intensity (MFI) was measured for 50,000 cells from each sample in each experiment. Data shown is the mean of three independent experiments ± SD. Differences in MFI between the indicated groups were statistically significant (***p < 0.005 as determined using unpaired t test). The high background in the controls that were not incubated with 6-TreAz is presumably due to non-specific association of the secondary DIBO-fluorophore.

Figure 7

Simplified scheme summarizing transport and metabolism of trehalose and trehalose mycolates in Mtb. Exogenous trehalose is translocated across the mycomembrane via the PPE51 transporter. The LpqY-SugABC transporter allows the uptake of trehalose through the cytoplasmic membrane and recycles free trehalose that is released during synthesis of trehalose dimycolates (TDM) from trehalose monomycolates (TMM) by the mycoloyl transferases of the Antigen 85 complex (Ag85). The Ag85 also catalyzes the synthesis of arabinogalactan-mycolates from TMM, which is not depicted here for the sake of simplicity. Intracellularly, mycolates are conjugated to trehalose to yield TMM involving the enzymes Pks13 and CmrA, whereas the MmpL3 transporter mediates the secretion of TMM. IM inner membrane, AG arabinogalactan.