Traffic Control: Investigating the lipoprotein trafficking stress response in Escherichia coli and Acinetobacter baumannii Restricted; Files & ToC

Abstract

Antibiotic resistant infections are one of the leading global public health threats, as treatment of infections is becoming increasingly more difficult. Gram-negative pathogens particularly present formidable difficulties due to the impermeability of their protective outer membrane (OM) barrier. The OM is built by several essential and highly conserved molecular machines that rely on lipoprotein trafficking (Lol), as each requires an essential lipoprotein component. Therefore, targeting the Lol pathway offers a promising avenue to compromise OM integrity, enabling the use of existing antibiotics that struggle to penetrate the OM of Gram-negative pathogens. Lipoproteins are extracted from the inner membrane by LolCDE, transported across the periplasm by LolA chaperone, and inserted into the OM by LolB. In Escherichia coli, the Cpx two-component system monitors lipoprotein trafficking. Lipoprotein NlpE was previously identified as a trafficking sensor for Cpx, however it had remained unclear how NlpE signaled. This work employs a multifaceted approach, combining genetics, biochemistry, and AlphaFold2 complex modeling, to elucidate the molecular details of how NlpE triggers the Cpx response through an interaction with the CpxA histidine kinase. The investigation of lipoprotein trafficking extends to another Gram-negative bacteria, Acinetobacter baumannii, which encodes a LoLF hybrid protein instead of LolC/E. AlphaFold multimer modeling is used to explore A. baumannii LolA:LolF and LolA:LolB interactions, comparing them to E. coli models and solved structures. These insights reveal potential important functional differences in the Lol pathway between organisms. Additionally, A. baumannii lacks a Cpx homolog system or clear stress response that oversees lipoprotein trafficking. In this final study, a CRISPRi strategy is employed to induce lipoprotein trafficking defects in A. baumannii. RNA-seq identifies several candidate stress response systems upregulated under CRISPRi-induced stress. Following further investigation, the BfmRS system is revealed as an important system for maintaining the OM via a different mechanism from E. coli, not involving lipoprotein NlpE. Overall, this work reveals the mechanisms of lipoprotein trafficking monitoring in two Gram-negative organisms, advancing the field’s understanding that the preservation of this essential and conserved cellular mechanism (Lol) may not be similar across species. These differences could ultimately be exploited for species-specific antibiotic development.

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About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Microbiology
Keyword	stress response Acinetobacter baumannii lipoprotein Escherichia coli Gram-negative bacteria bacteriology outer membrane
Committee Chair / Thesis Advisor	Marcin Grabowicz, Emory University
Committee Members	Philip Rather, Emory University Christine Dunham, Emory University William Shafer, Emory University David Weiss, Emory University

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