Acinetobacter baumannii is a clinically important pathogen that has rapidly developed resistance to many antimicrobials. The membrane envelope of A. baumannii is uniquely robust and can resist mechanical desiccation, antimicrobials, and is even able to cope with the complete loss of lipopolysaccharide. Bacterial membrane homeostasis systems, such as the maintenance of lipid asymmetry system (Mla), are important for membrane remodelling in response to attacks from the immune system or antimicrobials. A. baumannii MlaD is a component of the Mla system that contains a mammalian cell entry domain (MCE). Several MCE domains have recently been shown to be important components in membrane remodelling systems. Here we characterise the structure of the MlaD protein from A. baumannii.
Bioinformatics approaches were used to identify the mlaD gene in A. baumannii and compare the amino acid sequence identity to other MlaD bacterial orthologs. The amino acid sequence of AbMlaD was also compared to all known MCE domains. Recombinant AbMlaD was expressed, purified and crystallised, and structurally characterised using spectrometry methods and X-ray crystallography.
Our results show that AbMlaD has a 47 amino acid region not found in any MlaD orthologs. In addition, this unique region is highly conserved in the Acinetobacter genus. The unique region is located in the MCE domain and is not found in any other MCE domain containing proteins. Purified AbMlaD was shown to be folded, and formed a hexamer. Finally, we present the purification and crystallisation conditions of AbMlaD that resulted in diffraction to 2.5 Å.
This work provides important insights into the unique components of A. baumannii membrane homeostasis. Additionally, we show that AbMlaD contains a novel MCE domain, the biological role of which is the subject of ongoing investigation.