Abstract

Acinetobacter baumannii (Ab) is a clinically important pathogen that has rapidly developed resistance to many antimicrobials. The outer membrane (OM) of A. baumannii (and other Gram-negative pathogens) plays a crucial role in intrinsic antibiotic resistance by acting as a physical barrier that prevents drugs from reaching their targets inside the cell. The Maintenance of Lipid Asymmetry (Mla) transporter has recently emerged as a key player in maintaining the integrity of the OM in all Gram-negative bacteria through the transport of phospholipids (PLs). The importance of the OM in conferring intrinsic resistance to antibiotics and requirement of the Mla transporter in maintaining OM integrity renders this complex an attractive drug target.

The Mla transporter contains six components, including the inner membrane (IM) MlaFEDB complex, periplasmic MlaC, and MlaA which is located in the OM. MlaC is proposed to shuttle PLs between the OM and IM, through interactions with MlaA and MlaD, respectively. Through bioinformatic analysis, we revealed that MlaD from A. baumannii contains a novel mammalian cell entry (MCE) domain, with an additional 47-amino acid region not found in any MlaD orthologs. Upon commencement of this project, the structure and function of the additional region were unknown.

We successfully purified the soluble domain of AbMlaD in its hexamric form and solved the crystal structure of AbMlaD at 2.0 Å. AbMlaD forms a typical 7-stranded β-barrel MCE domain fold with a helical additional region. Although there is less than 35% sequence identity between the MCE domain of MlaD from E. coli and AbMlaD, the two structures are superimposible, with the exeption of the 47-amino acid additional region in AbMlaD.

Through biochemical and biophysical approaches, we investigated the functional role of the additional region in AbMlaD, focusing on its impact on the AbMlaD-AbMlaC interaction. Our findings indicate that the additional region is essential for stability and oligomerisation of AbMlaD. It also influences the interaction between AbMlaD and AbMlaC, as well as the efficiency of PL transfer. Moreover, A. baumannii with a chromosomal deletion of the mlaD gene showed a defect in growth in the presence of SDS, suggesting a role for AbMlaD in the maintenance of the OM integrity. In trans complementation of A. baumannii ΔmlaD with AbmlaD lacking the additional region restored the ability of the deletion mutant strain to grow on SDS agar, showing that the additional region is not directly responsible for maintaining OM integrity.

In additional to their role in PL transport and membrane homeostasis, Mla transporter components have been linked to the virulence of various pathogens. We used an in vivo mouse model of infection to investigate the contribution of MlaD to the pathogenicity of A. baumannii. Our results revealed that the absence of mlaD renders A. baumannii susceptible to various environmental stressors, including antibiotics and detergents. Moreover, A. baumannii ΔmlaD displayed reduced growth under static conditions, diminished biofilm formation, reduced adhesion to and invasion of airway epithelial cells, and reduced virulence in mice. The impaired OM of A. baumannii ΔmlaD, along with its reduced growth under static conditions, could be responsible for the reduced survival and growth of the bacterium within the mice, ultimately contributing to its reduced virulence.

This study increases our understanding of the structure and function of the Mla transporter, as well as its role in A. baumannii pathogenesis. The insights gained through this work pave the way for inhibition studies to ascertain the likelihood of being able to inhibit or modulate the Mla transporter with a small molecule drug.