Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) is an exclusively human pathogen that proliferates within phagosomes of host phagocytes. Host lipids are believed to provide the major carbon and energy sources for Mtb, with only limited availability of carbohydrates. There is an apparent paradox because five putative carbohydrate uptake permeases are present in Mtb, but there are essentially no host carbohydrates inside phagosomes. Nevertheless, carbohydrate transporters have been implicated in Mtb pathogenesis, suggesting that acquisition of host sugars is important during some stages of infection. Here we show, however, that the LpqY-SugA-SugB-SugC ATP-binding cassette transporter is highly specific for uptake of the disaccharide trehalose, a sugar not present in mammals, thus refuting a role in nutrient acquisition from the host. Trehalose release is known to occur as a byproduct of the biosynthesis of the mycolic acid cell envelope by Mtb's antigen 85 complex. The antigen 85 complex constitutes a group of extracellular mycolyl transferases, which transfer the lipid moiety of the glycolipid trehalose monomycolate (TMM) to arabinogalactan or another molecule of TMM, yielding trehalose dimycolate. These reactions also lead to the concomitant extracellular release of the trehalose moiety of TMM. We found that the LpqY-SugA-SugB-SugC ATP-binding cassette transporter is a recycling system mediating the retrograde transport of released trehalose. Perturbations in trehalose recycling strongly impaired virulence of Mtb. This study reveals an unexpected accessory component involved in the formation of the mycolic acid cell envelope in mycobacteria and provides a previously unknown role for sugar transporters in bacterial pathogenesis.

Fig. 1.

The LpqY-SugA-SugB-SugC ABC transporter mediates trehalose uptake in mycobacteria. (A) Organization of the lpqY-sugA-sugB-sugC locus in M. smegmatis and Mtb. The triangles indicate the transposon insertion sites in trehalose-resistant M. smegmatis ΔglgE suppressor mutants. (B) M1P accumulation from endogenous trehalose in transposon-induced M. smegmatis suppressor mutants revealed by TLC analysis. Cells were cultivated in absence of exogenous trehalose.::Tn, transposon insertion. (C) Growth of M. smegmatis transporter mutants on defined carbon sources after 48 h of incubation. Data represent means of triplicates ± SD. (D) Growth kinetics of Mtb transporter mutants on defined carbon sources. Data represent means of triplicates ± SD. Comp., complemented mutant strains. Data in B to D are representative of at least two independent experiments.

Fig. 2.

The LpqY-SugA-SugB-SugC ABC transporter is a high-affinity trehalose-specific importer. (A) Trehalose uptake rates in Mtb mutant strains. HI, cells heat-inactivated by incubation at 95 °C for 15 min before addition of the radiolabeled trehalose. Comp., complemented mutant strains. (B) Dependence of trehalose uptake rate on substrate concentration. (C) Trehalose uptake activity in Mtb wild-type does not require induction by the substrate. Wild-type cells were preincubated for 24 h in presence of 2 mM trehalose before uptake experiments. (D) Determination of the substrate specificity in trehalose uptake competition assays in presence of 1 mM of the indicated unlabeled competing sugars. (E) Comparison of the uptake rates for trehalose and maltose in Mtb. Data represent means of triplicates ± SD and are representative of two independent experiments.

Fig. 3.

Trehalose acquisition by the LpqY-SugA-SugB-SugC ABC transporter is essential for virulence of Mtb in mice. (A) Survival of SCID mice after low-dose aerosol infections with Mtb trehalose transporter mutants. n = 10 mice per group. (B) Growth kinetics of Mtb trehalose transporter mutants in lungs and spleens of C57BL/6 mice after low-dose aerosol infections. Values represent means of triplicates ± SD. Comp., complemented mutant strains.

Fig. 4.

The LpqY-SugA-SugB-SugC ABC transporter is a trehalose recycling system. (A) Growth kinetics of Mtb strains on 40 mM glycerol as sole carbon source. (B) Trehalose accumulation in cell-free culture supernatants of Mtb strains during growth on glycerol as sole carbon source, as indicated in A. Trehalose was determined using an enzymatic quantification assay. Data in A and B represent means of triplicates ± SD and are representative of two independent experiments. (C) Verification of trehalose secretion by Mtb transporter mutants, as revealed by GC/MS analyses. Samples were taken from 26-d-old cultures, as shown in A and B. The peak at 14.7 min corresponded to that of an authentic trehalose standard and showed an identical mass spectrum (Fig. S3). Comp., complemented mutant strains.

Fig. 5.

Model of trehalose recycling as an accessory component in mycolic acid processing. AG, arabinogalactan layer; Ag85, antigen 85 complex; CM, cytoplasmic membrane; PG, peptidoglycan layer; TMM, trehalose monomycolate; TDM, trehalose dimycolate.