# The role of cardiolipin in the biogenesis of the Gram-negative bacterial cell envelope

> **NIH NIH R01** · UNIVERSITY OF GEORGIA · 2023 · $677,076

## Abstract

Abstract
The increasing rise in antibiotic resistance and the diminished discovery of new antimicrobials threatens global
healthcare. Of particular concern are Gram-negative pathogens, as these organisms are intrinsically resistant
to multiple classes of antibiotics and the discovery of novel drugs targeting these bacteria has remained
challenging. The innate resistance of these organisms is provided primarily by their outer membrane (OM), a
defining feature of Gram negatives that encapsulates their peptidoglycan layer. Unlike the inner membrane
(IM) that is composed solely of glycerophospholipids (GPLs), the OM is asymmetrical with GPLs found in the
inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane organization
affords protection from large polar molecules, as well as lipophilic compounds, creating an impervious barrier.
Since the OM is essential, pathways required for its assembly are key targets for antimicrobial design.
Currently, there are no antibiotics that directly target OM biogenesis in clinical use and first attempts have
proven difficult. Thus, it remains critical to investigate cell envelope biology for future and current antimicrobial
design.
Recently, we discovered a connection between the GPL cardiolipin (CL) and the synthesis and transport of
LPS. E. coli harbors three distinct enzymes that synthesize CL, yet CL is not required for cell viability and is the
least abundant of the three major GPLs in Gram negatives. We found LpxM, the enzyme that adds the last acyl
chain to the lipid anchor of LPS, to be critical for viability in the absence of clsA. Suppressors of clsA and lpxM
synthetic lethality were identified in msbA, a gene that encodes the essential, homodimeric ABC transporter
that “flips” LPS across the IM. Multiple pieces of genetic and biochemical data supported a model in which CL
enhances MsbA activity driving LPS transport. Also, we observed that single mutants lacking either ClsA, the
primary CL synthase, or LpxM have reduced LPS levels. This suggests the cell can “sense” defects in LPS
transport at the cytoplasmic face of the IM and slow LPS synthesis to balance OM lipid content. In the current
application we will define (i) the functional role of CL in MsbA-dependent LPS transport, (ii) characterize
specific MsbA-CL interactions and determine how they impact MsbA activity, (iii) determine if ClsA and MsbA
are co-localized in the bacterial cell envelope, and (iv) determine how defects in LPS transport results in
feedback inhibition of LPS synthesis. Completion of these Aims will provide novel insights into cell envelope
biogenesis and promote the development of novel therapeutics targeting Gram-negative pathogens.

## Key facts

- **NIH application ID:** 10731444
- **Project number:** 1R01AI174416-01A1
- **Recipient organization:** UNIVERSITY OF GEORGIA
- **Principal Investigator:** Michael Stephen Trent
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $677,076
- **Award type:** 1
- **Project period:** 2023-06-01 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10731444

## Citation

> US National Institutes of Health, RePORTER application 10731444, The role of cardiolipin in the biogenesis of the Gram-negative bacterial cell envelope (1R01AI174416-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10731444. Licensed CC0.

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