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

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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.