Structural Insights into Outer Membrane Protein Biogenesis in Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae (Ngo), is an obligate human pathogen and the causative agent of the disease gonorrhea. If left untreated, gonorrhea can lead to serious health issues including ectopic pregnancy and infertility. In addition to unsuccessful vaccine development, Ngo has rapidly become resistant to several classes of antibiotics including penicillin's, tetracycline's, and fluoroquinolone's. Recent data published by the Center for Disease Control stated over 500,000 drug resistant Ngo infections occur each year in the United States. Due to this widespread antibiotic resistance, the current and only treatment option is limited to a combination of ceftriaxone and azithromycin. However, resistance to even these drugs has emerged and the disease will likely become untreatable in the near future. This has created a desperate need for the development of novel antibiotics and vaccines in order to fight this disease. Like many bacterial pathogens, Ngo is Gram-negative having both an inner and outer membrane. Outer membrane proteins (OMPs) have been recently identified as a novel class of antibiotic and vaccine targets. The biogenesis of these β-barrel OMPs is mediated by a multi-component protein complex, known as the β-barrel assembly machinery (BAM) complex. Conserved across all Gram-negative bacteria, BAM is required for viability and represents a powerful potential antibiotic or vaccine target. In E. coli, this 200 kDa complex is comprised of five proteins: BamA, an OMP itself, and four lipoproteins, BamB through E. A clear mechanism for how substrate OMPs are folded and inserted into the membrane by BAM is still poorly understood. My research focuses on understanding the mechanism of BAM from Ngo (NgBAM). Previous studies have indicated that Neisseria do not possess a homolog of BamB in their genome and NgBAM may function as a four component complex. I am currently working to characterize NgBAM using a combination of cryo-electron microscopy and in vivo studies. The cloning, expression, and purification of the complex has now been optimized and we recently collected several cryo-EM data sets. Our structural studies have revealed interesting features distinct from E. coli, and we plan to move into in vivo structure guided mutagenesis and knockout studies soon. This work will lay the foundation for characterizing the complex for future vaccine and drug development against gonorrhea.