Type II Fatty Acid Synthesis Pathway and Cyclopropane Ring Formation Are Dispensable during Enterococcus faecalis Systemic Infection

Abstract

Enterococcus faecalis , a multi-antibiotic-resistant Gram-positive bacterium, has emerged as a serious nosocomial pathogen. Here, we used a genetic approach to characterize the strategies used by E. faecalis to fulfill its requirements for endogenous fatty acid (FA) synthesis in vitro and in vivo . The FA synthesis (FASII) pathway is encoded by two operons and two monocistronic genes. Expression of all these genes is repressed by exogenous FAs, which are incorporated in the E. faecalis membrane and modify its composition. Deletion of nine genes of the 12-gene operon abolished growth in a FA-free medium. Addition of serum, which is lipid-rich, restored growth. Interestingly, the E. faecalis membrane contains cyclic fatty acids that modify membrane properties, but are unavailable in host serum. The cfa gene that encodes the cyclopropanation process, is located in a locus independent of the FASII genes. Its deletion did not alter growth under the conditions tested, but yielded bacteria devoid of cyclic FAs. No differences were observed between mice infected with wild-type, or FASII or cyclopropanation mutant strains, in terms of bacterial loads in blood, liver, spleen or kidneys. We conclude that in E. faecalis , neither FASII nor cyclopropanation enzymes are suitable antibiotic targets. Importance Membrane lipid homeostasis is crucial for bacterial physiology, adaptation, and virulence. Fatty acids are constituents of the phospholipids that are essential membrane components. Most bacteria incorporate exogenous fatty acids into their membranes. Enterococcus faecalis has emerged as a serious nosocomial pathogen, which is responsible for urinary tract infections, bacteremia and endocarditis, and is intrinsically resistant to numerous antibiotics. E. faecalis synthesizes saturated and unsaturated fatty acids, but also cyclic fatty acids that are not found in the human host. We characterized mutant strains deficient in fatty acid synthesis and modification using genetic, biochemical, and in vivo approaches. We conclude that neither the fatty acid synthesis pathway nor the cyclopropanation enzyme are suitable targets for E. faecalis antibiotic development.

The Enterococcus faecalis FabT Transcription Factor Regulates Fatty Acid Biosynthesis in Response to Exogeneous Fatty Acids

“…The sequences tested are indicated by the red stars whereas the fabT gene is referred by the blue arrow. Note that the first fours genes were previously shown to be cotranscribed ( Zhu et al, 2019 ) and a recent report showed that this is the case for the entire eleven gene operon ( Hays et al, 2021 ). (B) The nucleotide sequences of FabT-binding sites of the promoters of fab -related genes above.…”

“…However, FabT failed to bind hypothesized promoter regions of the fabK or acpB genes, neither of which play significant roles in E. faecalis fatty acid biosynthesis ( Bi et al, 2014 ; Zhu et al, 2019 ). The fabK gene is coexpressed with fabT , fabH , and acpA genes encoded early in the fab gene operon ( Figure 3A ) but is inefficiently translated in vivo ( Bi et al, 2014 ) whereas the role of the AcpB protein is incorporation of exogeneous fatty ( Bi et al, 2014 ; Zhu et al, 2019 ), Note also that the fabK gene has been shown to be cotranscribed with the upstream and downstream genes ( Zhu et al, 2019 ) and others have recently shown that all the genes of the FabT operon are cotranscribed ( Hays et al, 2021 ). Hence, the Streptococcus pneumoniae fabK promoter ( Jerga and Rock, 2009 ) is not present in E. faecalis.…”

“…In E. faecalis most of the fatty acid synthesis genes are transcribed as a single mRNA species encoding 12 genes ( Zhu et al, 2019 ; Hays et al, 2021 ) ( Figure 3A ). The exceptions are the two genes ( fabO and fabN ) required for synthesis of the unsaturated acids and fabI , the gene encoding the essential enoyl-ACP reductase.…”

“…It is a facultative anaerobe that inhabits the gastrointestinal tract of humans and other animals ( Fisher and Phillips, 2009 ; Zhu et al, 2019 ). Indeed, E. faecalis can use either de novo synthesized fatty acids or exogenous fatty acids for membrane phospholipid synthesis ( Zhu et al, 2013 ; Zhu et al, 2019 ; Hays et al, 2021 ) ( Figure 1A ). Exogenous fatty acids enter the cells probably by diffusion and are activated by the Fak system which produces acyl-phosphates.…”

FabT, a Bacterial Transcriptional Repressor That Limits Futile Fatty Acid Biosynthesis

“…The main Acyl Carrier Protein (ACP) encoded by acpA is always present on the main locus, in an operon comprising three or more genes, the first of which is the MarR family regulator FabT (Fig. 1B) (12)(13)(14). A second transcript starting with acpA may indicate the existence of a second operon, or of a stabilized part of a longer mRNA (12,15).…”