Klevorn, T.; Brown, C.; Hardy, C. D.; Cuthbert, B. D.; Spencer, A.; Jinich, A.; Chan, L.; Mendoza, J.; de Miranda, R.; Kim, H.; Schnappinger, D.; Rhee, K.; Goulding, C.; Ehrt, S.

The intrinsic drug resistance of Mycobacterium tuberculosis (Mtb) is a major barrier to effective tuberculosis (TB) treatment and is largely attributed to its complex, impermeable cell envelope. We identified a periplasmic protein complex comprising FecB and Rv3035 that is essential for maintaining cell envelope integrity and mediating intrinsic multidrug resistance in Mtb. FecB interacts directly with Rv3035, forming a stable heterodimer that also associates with the cell envelope biosynthesis proteins AftB and Rv0227c. We report the structures of Rv3035 alone and in complex with FecB, and identify critical residues required for complex formation and function. Co-essentiality analysis and genetic interaction experiments further supported a functional link between FecB, Rv3035 and AftB, the enzyme responsible for the last step in the biosynthesis of the cell envelope components arabinogalactan and lipoarabinomannan. Mtb mutants of fecB, rv3035 and aftB accumulated trehalose monomycolates in the outer membrane, consistent with a perturbation of membrane assembly and/or maintenance. Importantly, we show that FecB is required for Mtb virulence in mice, underscoring its physiological relevance. Altogether, our findings uncover a previously uncharacterized periplasmic protein complex that supports cell envelope integrity and intrinsic drug resistance in Mtb, highlighting FecB and Rv3035 as promising targets for therapeutic intervention.