Affiliation:
1. Department of Chemistry, University of Georgia, Athens, Georgia, USA
2. Department of Microbiology and Immunology, A.T. Still University of Health Sciences, Kirksville, Missouri, USA
3. School of Biological Sciences, Illinois State University, Normal, Illinois, USA
Abstract
ABSTRACT
Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant
Staphylococcus aureus
. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of
S. aureus
or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of
S. aureus
strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance:
mprF
,
yycG
, and
pgsA
. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase (
pdhB
) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of
pdhB
had no effect, complementation of the
pgsA
mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that
pgsA
contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels.
IMPORTANCE
The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with
Staphylococcus aureus
that display reduced susceptibility to β-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of
S. aureus
, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant
S. aureus
, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene,
pgsA
, on membrane fluidity and cell wall thickness in
S. aureus
with high-level daptomycin resistance.
Funder
HHS | NIH | National Institute of Allergy and Infectious Diseases
Publisher
American Society for Microbiology