Characterisation of MlaD from Acinetobacter baumannii

Abstract

Acinetobacter baumannii (Ab) is a clinically important pathogen that has rapidly developed resistance to many antibiotics. The outer membrane of A. baumannii (and other Gram-negative pathogens) plays a crucial role in intrinsic antibiotic resistance. The Maintenance of Lipid Asymmetry (Mla) transporter is essential for maintaining the integrity of the outer membrane in all Gram-negative bacteria through the transport of phospholipids. The Mla transporter contains six components, including the inner membrane MlaFEDB complex, periplasmic MlaC, and MlaA which is embedded in the inner leaflet of the outer membrane. MlaC is proposed to shuttle phospholipids between the outer and inner membranes through interactions with MlaA and MlaD, respectively. Bioinformatics analysis revealed that AbMlaD contains a novel mammalian cell entry (Mce) domain with a unique 47 amino acid (aa) additional region, which is highly conserved within Acinetobacter species, but not found in any other MlaD orthologs. This current study investigated the structure and function of AbMlaD, including that of the 47-aa additional region.

The crystal structure of the soluble domain of AbMlaD, solved at 2 Angstrom resolution, revealed a conserved seven-stranded beta-barrel fold connected by loops, with the 47-aa additional region forming a helix-turn-helix structure between the fourth and fifth beta-strands. Similar to EcMlaD, six AbMlaD monomers are assembled into a homo-hexameric ring structure, and the 47-aa additional region (of each monomer) is found on the exterior of the hexameric ring, protruding from the inner membrane into the periplasm. This study employed a combination of in silico, biochemical, and biophysical analyses to investigate the functional role of the AbMlaD 47- aa additional region. A thermal shift assay, a tryptophan quenching assay, and thin layer chromatography were used to assess the interaction between AbMlaC and AbMlaD or an AbMlaD derivative lacking the 47-aa additional region, as well as the efficiency of phospholipid transfer. In vitro results demonstrated that the 47-aa additional region strengthens the interaction between AbMlaD and AbMlaC, promotes phospholipid transfer from AbMlaC to AbMlaD, and stabilises the AbMlaD hexamer. However, the additional region was not required for maintaining outer membrane integrity or for forming a complex with other Mla inner membrane components.

In addition to their role in phospholipid transport and membrane homeostasis, Mla transporter components have been linked to the virulence of various pathogens. This study investigated the role of MlaD in the pathogenicity and survival of the multidrug-resistant A. baumannii strain BAA-1605. A markerless deletion method was used to construct a BAA-1605 mlaD deletion mutant, which was characterised phenotypically and complemented to validate the direct involvement of MlaD (and the 47-aa additional region) in the observed phenotypes. Deletion of mlaD led to a compromised outer membrane, a growth defect under static conditions, decreased biofilm formation, decreased ability to kill eukaryotic cells, and attenuated virulence in a murine model of A. baumannii infection. The 47-aa additional region of AbMlaD did not play a role in these pathogenetic traits.

This study increases our understanding of the structure and function of MlaD, and by extension, the Mla transporter, as well as their 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, and in doing so, reduce A. baumannii virulence.