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STRUCTURAL INSIGHT INTO THE BIOGENESIS OF OUTER MEMBRANE PROTEINS IN PATHOGENIC NEISSERIA

thesis
posted on 2024-04-23, 18:37 authored by Evan M BillingsEvan M Billings

The obligate human pathogen, Neisseria gonorrhoeae (Ngo), has continued to acquire widespread antibiotic resistance. Ngo is the causative agent of the sexually transmitted disease gonorrhea, and can cause additional complications such as endocarditis, septicemia, and infertility if left untreated. The Centers for Disease Control and Prevention (CDC) now recommends a treatment option of a single drug of last resort, ceftriaxone, leaving a need for novel therapeutics against this pathogen.

Like many bacterial pathogens, Ngo is Gram-negative consisting of both an inner membrane (IM) and outer membrane (OM). The transmembrane proteins in the IM have primarily an α-helical fold, while the transmembrane proteins in the OM have a β-barrel fold. These β-barrel outer membrane proteins (OMPs) have essential functions in regulating the homeostasis and nutrient acquisition of the cell, in addition to promoting virulence in pathogenic strains. These OMPs are folded and inserted into the outer membrane by the β-barrel assembly machinery (BAM) complex. In E. coli, BAM consists of five proteins: BamA, an OMP itself, and four lipoproteins, BamB, C, D, and E.

Here we present our work toward the structural characterization of BAM from Ngo (NgBAM) using cryo-EM. Ngo lack a homolog of BamB and may function as a four component complex. To better understand the mechanism for how NgBAM is able to mediate OMP biogenesis despite lacking a component that is critical in E. coli, we determined the cryo-EM structure of NgBAM, which revealed several distinct features including that the barrel domain of BamA being observed in the inward-open conformation. We also investigated NgBAM as a therapeutic target, by studying its interaction with a novel broad spectrum antibiotic darobactin. We first showed darobactin is effective against the laboratory strains of NgoFA19 and ATCC-49226. We also show it is effective against the human isolate WHOX, with a comparable MIC to ceftriaxone. To structurally characterize the mechanism of inhibition by darobactin, we used cryo-EM to determine the structures of NgBAM bound to two darobactin compounds. In these structures, darobactin binding was accompanied by large conformational changes in NgBamA. To further probe the effects of darobactin on the conformational plasticity of NgBAM we performed experiments using double electron-electron resonance spectroscopy, which showed distance changes between the engineered site labels consistent with the conformational changes observed in our structural observation. In addition, narrowing of the peak distributions indicated that darobactin binding was reducing the overall conformational heterogeneity of the complex. Taken together, the work presented here contributes to the understanding of how NgBAM functions in folding and inserting OMPs and provides a foundation for future structure based drug design of darobactin and other potential compounds.

Funding

Characterization of a novel antibiotic against Neisseria gonorrhoeae

American Heart Association

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Purdue University Molecular Biophysics Training Program

National Institute of General Medical Sciences

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Unraveling the mechanism by which the BAM complex mediates OMP biogenesis

National Institute of General Medical Sciences

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History

Degree Type

  • Doctor of Philosophy

Department

  • Biological Sciences

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Nicholas Noinaj

Additional Committee Member 2

John Tesmer

Additional Committee Member 3

Leifu Chang

Additional Committee Member 4

Kenneth Ritchie