PPE51 mediates uptake of trehalose across the mycomembrane of Mycobacterium tuberculosis

Mohammed Rizwan Babu Sait,Jessica A Stewart,Benjamin M Swarts,Marc Jacobsen,Thomas R Ioerger,Rainer Kalscheuer,Hendrik Koliwer-Brandl

doi:10.1038/s41598-022-06109-7

Mohammed Rizwan Babu Sait, Jessica A Stewart + Show 5 more

Open Access

https://doi.org/10.1038/s41598-022-06109-7

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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.

Highlights

The disaccharide trehalose is essential for viability of Mycobacterium tuberculosis, which synthesizes trehalose de novo but can utilize exogenous trehalose
The mycolic acids are long chain α-branched β-hydroxy fatty acids that are either covalently bound to arabinogalactan, which in turn is linked to peptidoglycan, or esterified to sugars such as trehalose to give rise to the glycolipids, trehalose monomycolates (TMM) or trehalose dimycolates (TDM)[3]
TMM is synthesized in the cytoplasm by 6-O-mycoloylation of trehalose catalysed by Pks[13] and is transported to the mycomembrane through the mycobacterial membrane protein large 3 (MmpL3) transporter[10]

Summary

Introduction

The disaccharide trehalose is essential for viability of Mycobacterium tuberculosis, which synthesizes trehalose de novo but can utilize exogenous trehalose. We harnessed the growth inhibitory properties of 6-azido trehalose (6-TreAz), which has antimycobacterial activity at high concentration[14], for the isolation of spontaneously resistant mutants to identify genes potentially involved in trehalose uptake and its metabolism This led to the identification of a member of the proline-proline-glutamate (PPE) family proteins. The growth inhibition of wild-type strain H37Rv by 6-TreAz likely results from the depletion of TDM in the mycomembrane as Mtb could not conjugate an additional mycolic acid to TMM because the 6′ position is occupied by the azide group, as already hypothesized previously for Mycobacterium smegmatis[23]. Since TreS has been shown to be fully dispensable for growth of Mtb under nutrient-proficient in vitro culture conditions[8,25,26], it is unlikely that potential inhibition of TreS contributes relevantly to the growth inhibitory effect of 6-TreAz as observed in our study

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