Lung infections with Mycobacterium abscessus, a species of multidrug-resistant nontuberculous mycobacteria, are emerging as an important global threat to individuals with cystic fibrosis (CF), in whom M. abscessus accelerates inflammatory lung damage, leading to increased morbidity and mortality. Previously, M. abscessus was thought to be independently acquired by susceptible individuals from the environment. However, using whole-genome analysis of a global collection of clinical isolates, we show that the majority of M. abscessus infections are acquired through transmission, potentially via fomites and aerosols, of recently emerged dominant circulating clones that have spread globally. We demonstrate that these clones are associated with worse clinical outcomes, show increased virulence in cell-based and mouse infection models, and thus represent an urgent international infection challenge.

Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium

Crystal Structures of Membrane Transporter MmpL3, an Anti-TB Drug Target.

The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which, in part, render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. Although the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. Mycobacterial membrane protein Large 3 (MmpL3), an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several antimycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism of action of BM212 as a potent MmpL3 inhibitor, and use it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future antituberculosis drugs.

https://www.pnas.org/content/pnas/114/30/7993.full.pdf

MmpL3 is the flippase for mycolic acids in mycobacteria

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is a challenging infection which is becoming increasingly prevalent, particularly in the elderly, for reasons which are unknown. While underlying lung disease is a well-established risk factor for NTM-PD, it may also occur in apparently healthy individuals. No single common genetic or immunological defect has been identified in this group, and it is likely that multiple pathways contribute towards host susceptibility to NTM-PD which further interact with environmental and microbiological factors leading to the development of disease. The diagnosis of NTM-PD relies on the integration of clinical, radiological and microbiological results. The clinical course of NTM-PD is heterogeneous, with some patients remaining stable without the need for treatment and others developing refractory disease associated with considerable mortality and morbidity. Treatment regimens are based on the identity of the isolated species, drug sensitivity testing (for some agents) and the severity of disease. Multiple antibiotics are typically required for prolonged periods of time and treatment is frequently poorly tolerated. Surgery may be beneficial in selected cases. In some circumstances cure may not be attainable and there is a pressing need for better regimens to treat refractory and drug-resistant NTM-PD. This review summarises current knowledge on the epidemiology, aetiology and diagnosis of NTM-PD and discusses the treatment of two of the most clinically significant species, the M. avium and M. abscessus complexes, with a focus on refractory disease and novel therapies.

https://erj.ersjournals.com/content/erj/54/1/1900250.full.pdf

Non-tuberculous mycobacterial pulmonary disease

Mycobacterial genomes contain large sets of loci encoding membrane proteins that belong to a family of multidrug resistance pumps designated Resistance-Nodulation-Cell Division (RND) permeases. Mycobacterial membrane protein Large (MmpL) transporters represent a subclass of RND transporters known to participate in the export of lipid components across the cell envelope. These surface-exposed lipids with unusual structures play key roles in the physiology of mycobacteria and/or can act as virulence factors and immunomodulators. Defining the substrate specificity of MmpLs and their mechanisms of regulation helps understanding how mycobacteria elaborate their complex cell wall. This review describes the diversity of MmpL proteins in mycobacteria, emphasising their high abundance in a few opportunistic rapid-growing mycobacteria. It reports the conservation of mmpL loci between Mycobacterium tuberculosis and non-tuberculous mycobacteria, useful in predicting the role of MmpLs with unknown functions. Paradoxically, whereas MmpLs participate in drug resistance mechanisms, they represent also attractive pharmacological targets, opening the way for exciting translational applications. The most recent advances regarding structural/functional information are also provided to explain the molecular basis underlying the proton-motive force driven lipid transport. Overall, this review emphasises the Janus-face nature of MmpLs at the crossroads between antibiotic resistance mechanisms and exquisite vulnerability to drugs.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/mmi.13675

The diverse family of MmpL transporters in mycobacteria: from regulation to antimicrobial developments.

The MmpL family of proteins translocates complex (glyco)lipids and siderophores across the cell envelope of mycobacteria and closely related Corynebacteriaceae and plays important roles in the biogenesis of the outer membrane of these organisms. Despite their significance in the physiology and virulence of Mycobacterium tuberculosis, and from the perspective of developing novel antituberculosis agents, little is known about their structure and mechanism of translocation. In this study, the essential mycobacterial mycolic acid transporter, MmpL3, and its orthologue in Corynebacterium glutamicum, CmpL1, were investigated as prototypical MmpL proteins to gain insight into the transmembrane topology, tertiary and quaternary structures, and functional regions of this transporter family. The combined genetic, biochemical, and biophysical studies indicate that MmpL3 and CmpL1 are structurally similar to Gram-negative resistance-nodulation and division efflux pumps. They harbor 12 transmembrane segments interrupted by two large soluble periplasmic domains and function as homotrimers to export long-chain (C22-C90) mycolic acids, possibly in their acetylated form, esterified to trehalose. The mapping of a number of functional residues within the middle region of the transmembrane domain of MmpL3 shows a striking overlap with mutations associated with resistance to MmpL3 inhibitors. The results suggest that structurally diverse inhibitors of MmpL3 all target the proton translocation path of the transporter and that multiresistance to these inhibitors is enabled by conformational changes in MmpL3.

/pdf/structure-function-profile-of-mmpl3-the-essential-mycolic-7ijqsh08mo.pdf

Structure–Function Profile of MmpL3, the Essential Mycolic Acid Transporter from Mycobacterium tuberculosis

Nontuberculous mycobacterial (NTM) pulmonary infections are emerging as a global health problem and pose a threat to susceptible individuals with structural or functional lung conditions such as cystic fibrosis, chronic obstructive pulmonary disease and bronchiectasis. Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) species account for 70-95% of the pulmonary NTM infections worldwide. Treatment options for these pathogens are limited, involve lengthy multidrug regimens of 12-18 months with parenteral and oral drugs, and their outcome is often suboptimal. Development of new drugs and improved regimens to treat NTM infections are thus greatly needed. In the last 2 years, the screening of compound libraries against M. abscessus in culture has led to the discovery of a number of different chemotypes that target MmpL3, an essential inner membrane transporter involved in the export of the building blocks of the outer membrane of all mycobacteria known as the mycolic acids. This perspective reflects on the therapeutic potential of MmpL3 in Mycobacterium tuberculosis and NTM and the possible reasons underlying the outstanding promiscuity of this target. It further analyzes the physiological and structural factors that may account for the apparent looser structure-activity relationship of some of these compound series against M. tuberculosis compared to NTM.

/pdf/mmpl3-as-a-target-for-the-treatment-of-drug-resistant-2o7h0ujflc.pdf

Amira Yazidi

Papers

Structure–Function Profile of MmpL3, the Essential Mycolic Acid Transporter from Mycobacterium tuberculosis

MmpL3 as a Target for the Treatment of Drug-Resistant Nontuberculous Mycobacterial Infections.

A “Drug Sweeping” State of the TriABC Triclosan Efflux Pump from Pseudomonas aeruginosa