Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

Structural and functional characterisation of MlaD from Acinetobacter baumannii (#140)

Jing Zhang 1 , Jiansi Long 1 , Xiaojun Yuan 1 , Fei-ju Li 2 , Simon Wiliams 1 , Colin Jackson 3 , Gaetan Burgio 2 , Matthew Johnson 4 , Denisse Leyton 1
  1. Research School of Biology, Australian National University, Acton, ACT, Australia
  2. Department of Immunology and Infectious Disease, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia
  3. Research School of Chemistry, Australian National University, Canberra, -, Australia
  4. Research School of Biology, Australian National University, Canberra, ACT, Australia

Acinetobacter baumannii is a clinically important pathogen with a robust membrane resistant to mechanical desiccation and antimicrobials. The maintenance of lipid asymmetry (Mla) transporter plays an important role to maintain the membrane homeostasis. The Mla transporter contains six components: the inner membrane (IM) MlaFEDB complex, the periplasmic MlaC, and the outer membrane MlaA. MlaC is proposed to shuttle PLs between the OM and IM, through interactions with MlaA and MlaD, respectively.

Bioinformatic analysis indicated that AbMlaD has a 47 amino acid region not found in any MlaD orthologs. This unique region is highly conserved in the Acinetobacter genus. Here, we characterised the structure and function of MlaD from A. baumannii. We solved the structure of the AbMlaD ortholog at 2.0 Å. AbMlaD forms a typical 7-stranded beta-barrel MCE domain fold, and the additional region, located between strands 4 and 5, was shown to form a helical domain. Through biochemical and biophysical methods, we found that this region is crucial for AbMlaD stability, oligomerization, and efficient phospholipid (PL) transfer to AbMlaC. A. baumannii lacking the mlaD gene displayed growth defects in the presence of SDS, suggesting a role for AbMlaD in outer membrane integrity. Complementation with AbMlaD lacking the additional region restored SDS resistance, indicating that this region isn't directly responsible for outer membrane integrity.

In addition to PLs transport and membrane stability, MlaD plays a role in pathogen virulence; A. baumannii lacking mlaD gene is more susceptible to antibiotics and detergents and exhibits reduced growth under static conditions and diminished biofilm formation. In an in vivo mouse infection model, the absence of mlaD rendered the bacteria less virulent. These effects may arise from compromised OM integrity and reduced growth under static conditions in A. baumannii lacking the mlaD gene, collectively contributing to 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.