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ABSTRACT Mycobacterium tuberculosis, the causative agent of the airborne infection tuberculosis (TB), contains 13 mycobacterial membrane protein large (MmpL) transporters that can be divided into two distinct subclasses. These MmpL proteins play important functional roles within the mycobacterium and subsequently are considered attractive drug targets to combat TB infection. Previously, we reported both X-ray and cryo-electron microscopy (cryo-EM) structures of the MmpL3 transporter, providing high-resolution structural information for this subclass of the MmpL proteins. Thus far, there is no structural information available for the other subclass, which includes MmpL5, an inner membrane transporter that plays a critical role in iron hemostasis. Here, we report the first cryo-EM structure of the Mycobacterium smegmatis MmpL5 transporter bound with the meromycolate extension acyl carrier protein M (AcpM) to a resolution of 2.81 Å. Our structural data reveals that MmpL5 and AcpM interact in the cytoplasm to form a complex, and this allows us to propose that MmpL5 may also associate with the mycobactin L (MbtL) protein in a similar fashion to form a heterocomplex important for iron acquisition, which enables the survival and replication of the mycobacterium.IMPORTANCEThe emergence and spread of multidrug-resistant tuberculosis (TB) present enormous challenges to the global public health. The causative agent, Mycobacterium tuberculosis, has now infected more than one-third of the world's population. Here, we report the first structure of the mycobacterial membrane protein large 5 (MmpL5), an essential transporter for iron acquisition, bound with the meromycolate extension acyl carrier protein M (AcpM), indicating a plausible pathway for mycobactin translocation. Our studies will ultimately inform an era in structure-guided drug design to combat TB infection.

Tuberculosis (TB) is one of the major causes of death related to antimicrobial resistance worldwide because of the spread of Mycobacterium tuberculosis multi- and extensively drug resistant (multi-drug resistant (MDR) and extensively drug-resistant (XDR), respectively) clinical isolates. To fight MDR and XDR tuberculosis, three new antitubercular drugs, bedaquiline (BDQ), delamanid, and pretomanid were approved for use in clinical setting. Unfortunately, BDQ quickly acquired two main mechanisms of resistance, consisting in mutations in either atpE gene, encoding the target, or in Rv0678, coding for the repressor of the MmpS5-MmpL5 efflux pump. To better understand the spreading of BDQ resistance in MDR- and XDR-TB, in vitro studies could be a valuable tool. To this aim, in this work an in vitro generation of M. tuberculosis mutants resistant to BDQ was performed starting from two MDR clinical isolates as parental cultures. The two M. tuberculosis MDR clinical isolates were firstly characterized by whole genome sequencing, finding the main mutations responsible for their MDR phenotype. Furthermore, several M. tuberculosis BDQ resistant mutants were isolated by both MDR strains, harboring mutations in both atpE and Rv0678 genes. These BDQ resistant mutants were further characterized by studying their growth rate that could be related to their spreading in clinical settings. Finally, we also constructed a data sheet including the mutations associated with BDQ resistance that could be useful for the early detection of BDQ-resistance in MDR/XDR patients with the purpose of a better management of antibiotic resistance in clinical settings.