Constantin Hays

Clara Lambert

Sophie Brinster

Gilles Lamberet

Laurence Du

Karine Gloux

Alexandra Gruss

Claire Poyart
email: claire.poyart@inserm.fr.

Agnès Fouet

Laurence Du Merle

Agnes Fouet
email: agnes.fouet@inserm.fr

Agnes Fouet

ring formation are dispensable during Enterococcus faecalis systemic infection

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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

INTRODUCTION

Bacterial fatty acids (FAs) are usually synthesized by the type II Fatty Acid Synthesis (FASII) pathway. FAs constitute the two hydrophobic tails which, together with a hydrophilic head, comprise phospholipids (PL) that are essential components of cell membranes. Enterococcus faecalis synthesizes both saturated and unsaturated fatty acids (SFA and UFA, respectively), as do numerous streptococci [START_REF] Brinster | Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens[END_REF][START_REF] Altabe | Isolation and characterization of unsaturated fatty acid auxotrophs of Streptococcus pneumoniae and Streptococcus mutans[END_REF][START_REF] Montgomerie | Fatty acid composition of L-forms of Streptococcus faecalis cultured at different osmolalities[END_REF]. Additionally, E. faecalis and numerous other bacteria encode a cyclopropane fatty acid synthase (Cfa), which catalyzes the transfer of a methyl group from Sadenosyl-L-methionine to the double bond of a lipid chain, thereby forming a cyclopropane ring [START_REF] Grogan | Cyclopropane ring formation in membrane lipids of bacteria[END_REF]. Recent studies uncovered that E. faecalis also synthesizes trans-UFA, further expanding the variety of fatty acids produced by this bacterium [START_REF] Kondakova | A novel synthesis of trans-unsaturated fatty acids by the Gram-positive commensal bacterium Enterococcus faecalis FA2-2[END_REF]. Both cyclopropane and trans fatty acids are more rigid than their non-modified substrates, and contribute to E. faecalis membrane homeostasis in starvation and stress conditions [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF][START_REF] Parsons | Bacterial lipids: metabolism and membrane homeostasis[END_REF][START_REF] Poger | A ring to rule them all: the effect of cyclopropane fatty acids on the fluidity of lipid bilayers[END_REF].

FASII pathway defects prevent bacterial growth in vitro in FA-non-supplemented media; this suggested that FASII enzymes may be essential for bacterial viability and therefore feasible targets for antibiotic design [START_REF] Heath | Lipid biosynthesis as a target for antibacterial agents[END_REF][START_REF] Parsons | Is bacterial fatty acid synthesis a valid target for antibacterial drug discovery?[END_REF][START_REF] Zhang | Inhibiting bacterial fatty acid synthesis[END_REF][START_REF] Heath | Inhibitors of fatty acid synthesis as antimicrobial chemotherapeutics[END_REF][START_REF] Lu | Inhibitors of FabI, an enzyme drug target in the bacterial fatty acid biosynthesis pathway[END_REF][START_REF] Zhu | The two functional enoylacyl carrier protein reductases of Enterococcus faecalis do not mediate triclosan resistance[END_REF][START_REF] Parsons | Staphylococcus aureus fatty acid auxotrophs do not proliferate in mice[END_REF]. However, most bacteria incorporate exogenous FA into their membranes. FA abundance in host compartments and in serum, led us to hypothesize, and then prove that FA incorporation can bypass FASII-targeted antibiotics in the neonatal pathogen Streptococcus agalactiae, as well as other Streptococcaceae [START_REF] Brinster | Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens[END_REF]. Nevertheless, antibiotic bypass may vary according to whether bacteria can survive on exclusively exogenous FAs; for example, Mycobacterium tuberculosis remains sensitive to the anti-FASII isoniazid because of its complex lipid requirement [START_REF] Bhatt | The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development: the mycobacterial FAS-II condensing enzymes[END_REF]. In contrast, Staphylococcus aureus, which synthesizes FAs not produced by humans, can nevertheless bypass FASII inhibitors with host-derived fatty acids [START_REF] Kénanian | Permissive fatty acid incorporation promotes staphylococcal adaptation to FASII antibiotics in host environments[END_REF][START_REF] Gloux | Clinical relevance of type II fatty acid synthesis bypass in Staphylococcus aureus[END_REF][START_REF] Morvan | Environmental fatty acids enable emergence of infectious Staphylococcus aureus resistant to FASII-targeted antimicrobials[END_REF].

Enterococcus faecalis is a Gram-positive bacterium that inhabits the gastrointestinal tract of humans and animals [START_REF] Fisher | The ecology, epidemiology and virulence of Enterococcus[END_REF] and has long been associated with food fermentations. E. faecalis has emerged as a serious nosocomial pathogen, and is the second most frequently isolated Grampositive pathogen isolated in hospitals after S. aureus [START_REF] Karchmer | Nosocomial bloodstream infections: organisms, risk factors, and implications[END_REF][START_REF] Wisplinghoff | Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study[END_REF]. E. faecalis is responsible for urinary tract infections, bacteremia and endocarditis [START_REF] Arias | The rise of the Enterococcus: beyond vancomycin resistance[END_REF]. It is intrinsically resistant to many antibiotics, and its acquisition of antibiotic resistance genes has made it a public health concern worldwide [START_REF] Arias | The rise of the Enterococcus: beyond vancomycin resistance[END_REF]. This situation prompts the search for new antibiotic targets, notably within the FASII pathway [START_REF] Feng | Environmental DNAencoded antibiotics fasamycins A and B inhibit FabF in type II fatty acid biosynthesis[END_REF][START_REF] Gerusz | Recent advances in the inhibition of bacterial fatty acid biosynthesis[END_REF].

Whether targeting FASII pathway or cyclopropanation enzymes could lead to treatment of E. faecalis systemic infection is not resolved. E. faecalis was shown to use environmental fatty acids [START_REF] Saito | Incorporation of exogenous fatty acids protects Enterococcus faecalis from membrane-damaging agents[END_REF]. In the presence of 15 % human serum, E. faecalis membrane comprises about 10% vaccenic acid (VA; C18:1D11), an FA not present in serum [START_REF] Saito | Incorporation of exogenous fatty acids protects Enterococcus faecalis from membrane-damaging agents[END_REF]. Thus, the E. faecalis FASII pathway is likely active even in the presence of exogenous FAs. The E. faecalis FASII pathway is encoded by the twelve gene cluster, fabT -accA, whose genes are present in numerous Grampositive species, and a set of three genes, fabO -fabN and the divergent fabI gene, encoding enzymes involved in UFA and SFA synthesis, respectively [START_REF] Zhu | The two functional enoylacyl carrier protein reductases of Enterococcus faecalis do not mediate triclosan resistance[END_REF][START_REF] Eckhardt | Transcriptional regulation of fatty acid biosynthesis in Lactococcus lactis[END_REF]. Finally, cfa encodes the cyclopropanation reaction. In one study, deletion of fabN resulted in E. faecalis UFA auxotrophy [START_REF] Diederich | Deletion of fabN in Enterococcus faecalis results in unsaturated fatty acid auxotrophy and decreased release of inflammatory cytokines[END_REF]. In E. faecalis, expression of FASII genes is controlled by FabT, the transcriptional repressor.

An auxiliary acyl-carrier-protein carrying long chain FAs binds to FabT, and facilitates repression of a consensus sequence that regulates FASII gene expression [START_REF] Zhu | The two functional enoylacyl carrier protein reductases of Enterococcus faecalis do not mediate triclosan resistance[END_REF][START_REF] Jerga | Acyl-Acyl carrier protein regulates transcription of fatty acid biosynthetic genes via the FabT repressor in Streptococcus pneumoniae[END_REF][START_REF] Zhu | Enterococcus faecalis encodes an atypical auxiliary acyl carrier protein required for efficient regulation of fatty acid synthesis by exogenous fatty acids[END_REF].

Although E. faecalis incorporates FA from its environment, its FA synthesis requirement in an FA-rich environment cannot be deduced [START_REF] Harp | Exogenous fatty acids protect Enterococcus faecalis from daptomycin-induced membrane stress independently of the response regulator LiaR[END_REF][START_REF] Kau | Enterococcus faecalis tropism for the kidneys in the urinary tract of C57BL/6J mice[END_REF]. In this study we characterized the repressive effect of serum on expression of FASII and FA-related genes. We also tested whether FASII pathway and/or cyclopropanation functions are possible antibiotic targets in a murine model of E. faecalis systemic infection.

MATERIALS AND METHODS

Bacterial strains, media and culture conditions. E. faecalis V583 vancomycin resistant strain was isolated from a bloodstream infection and sequenced. It was used for the in silico analysis. VE14089, a plasmid-cured derivative of the V583 strain, was the wild type (WT) reference strain used for the construction of mutant and back to the wild type (BWT) strains [START_REF] Paulsen | Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis[END_REF][START_REF] Rigottier-Gois | Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors[END_REF]. Two mutants were constructed, EfΔFASII and EfΔcfa corresponding to the in-frame deletion of nine genes from fabK to accA (7863 bp) and of cfa (1176 bp), respectively; the isogenic BWT strains were obtained, BWT-FASII and BWT-cfa, respectively. Escherichia coli TG1 (Invitrogen) was used for cloning experiments. E. faecalis was cultured in Todd-Hewitt (TH) broth or agar (Difco Laboratories, Detroit, MI) or in RPMI + (RPMI-1640; Life technologies, Gibco) supplemented with amino acids 1X (Gibco®, Life Technologies), vitamins 1X (Sigma Aldrich), glucose 1% and Hepes 0.1mM (Gibco®, Life Technologies). E. coli was cultured in Trypticase-Soy (TS) medium (Difco Laboratories, Detroit, MI). When needed, antibiotics were used at the following concentrations: for E. coli, ticarcillin, 100 µg/ml, and erythromycin, 160 µg/ml; for E. faecalis, erythromycin, 50 μg/ml. Cerulenin (Sigma) was added at 8 μg/ml final concentration.

In silico analysis. The AGTTTGATAATCAAATT sequence was used to seek potential FabT binding sites in the regulatory regions or within the FASII genes [START_REF] Eckhardt | Transcriptional regulation of fatty acid biosynthesis in Lactococcus lactis[END_REF].

General DNA techniques. E. coli plasmid DNA was prepared by rapid alkaline lysis using the QIAprep Spin Miniprep kit (Qiagen). Genomic DNA from E. faecalis was prepared using the DNeasy Blood and Tissue Kit (Qiagen) with pretreatment for Gram-positive bacteria as recommended by the manufacturer. PCR was carried out with the AmpliTaq Gold polymerase as described by the manufacturer (Applied Biosystems). The molecular weight standard used in agarose gels is the 1kb-plus DNA ladder (Life Technologies). Amplification products were purified on QIAquick PCR purification kit (Qiagen). PCR products and plasmids used were sequenced with an ABI 310 automated DNA sequencer, using the ABI PRISM dyes terminator cycle sequencing kit (Applied Biosystem). To determine the genetic and transcriptional organization of VE14089 FASII genes, primer pairs were designed to flank neighboring FASII genes. Primers are listed in Table S1.

Strain construction. The primers used for the construction of the in-frame EfΔFASII and EfΔcfa mutant strains are listed in Table S1. Primers used are based on the published genome sequence of E. faecalis V583 (GenBank accession number NC004668.1) [START_REF] Paulsen | Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis[END_REF]. The ΔFASII deletion was constructed by using splicing-by-overlap extension PCR as described [START_REF] Biswas | High-efficiency gene inactivation and replacement system for Gram-positive bacteria[END_REF]. The corresponding PCR fragment was cloned into the thermosensitive shuttle plasmid pG1. The Δcfa (ef0203, NCBI reference sequence NC_004668.1) deletion was constructed using In-Fusion PCR Cloning Kit (Clontech), cloning the two PCR-amplified fragments into pG1 previously linearized in a one-step reaction following manufacturer's recommendations. The resulting intermediate plasmids, pG1ΔFASII and pG1Δcfa, were checked by sequencing the inserts. Electroporation of VE14089 and allelic exchange were performed as described with minor modifications [START_REF] Biswas | High-efficiency gene inactivation and replacement system for Gram-positive bacteria[END_REF][START_REF] Cruz-Rodz | High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation[END_REF]. For the EfΔFASII construction Tween 80 0.1% (Sigma), a rich source of FA containing 70% oleic acid (OA; C18:1 D9), was added in all steps of the experiment after the electroporation. The in-frame deletion mutants and BWT strains were confirmed by PCR and sequence analysis.

Growth curves. E. faecalis strains were inoculated into RPMI + supplemented with Tween 80 (0.1%) and cultured overnight at 37°C. Overnight cultures were washed twice in PBS and subcultured (1:100) in RPMI + and in RPMI + supplemented with 40% human serum. Growth was determined by measuring absorbance at 600 nm (OD600). Experiments were repeated three times.

RNA extraction and RT-PCR.

RNA was extracted from bacteria by the phenol chloroform technique as described (37). RNA (2-5µg) was treated with DNAse I (Promega) according to manufacturer's recommendations. RNA was quantified by absorbance at 260 and 280 nm. RNA purity and integrity were controlled and RNA was stored at -80°C until use.

Reverse-transcription was carried out using the Superscript II kit (Invitrogen) and Random Hexamer Primers (Fermentas), according to manufacturers' instructions. PCRs were carried out on cDNA, as well as on gDNA and RNA as positive and negative controls, using primer pairs flanking neighboring FASII genes (Table S1). PCR Amplicons were examined on a 1% agarose gel.

Real-time quantitative PCRs. Overnight VE14089 RPMI + cultures were then subcultured (1:100) in RPMI + with or without addition of 40% human serum to mid-log phase (OD600 = 0.3).

RNAs were extracted and cDNA samples were diluted to 50 ng/μl. qPCR was performed using the LightCycler (Roche) and the SYBR Green PCR kits (Applied Biosystems) using primers listed in Table S1. Each assay was performed in duplicate, with two independently prepared total RNA samples. The relative quantification in gene expression was determined by the 2 -ΔΔCt method, using rpoB as the reference gene [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Fatty acid analysis.

Strains were inoculated into RPMI + supplemented with Tween 80 (0.1%) and grown overnight at 37°C. Cells were washed three times in RPMI + , diluted 100-fold in fresh RPMI + cultures with or without 40% human serum. Cells were harvested after 14 h of growth (stationary phase) in static conditions at 37 °C, and washed once in NaCl 0.9% containing 0.01% Triton X-100 and twice in NaCl 0.9%. Cell pellets were stored at -20°C until analysis. The FA composition of serum used for experiments was also determined. Whole cell esterified fatty acid determinations were done on an AutoSystem XL Gas Chromatograph (Perkin-Elmer) equipped with a DB-Wax (Agilent, XXX; for all analyses except those shown in Fig. 3D) or ZB-Wax capillary column (30 m x 0.25 mm x 0.25mm; Phenomenex, France; for Fig. 3D) as described [START_REF] Brinster | Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens[END_REF][START_REF] Kénanian | Permissive fatty acid incorporation promotes staphylococcal adaptation to FASII antibiotics in host environments[END_REF]. Bacterial fatty acid composition of each sample was analyzed in three independent experiments. Intravenous infection of mice. All in vivo infections in this study were performed with 6week-old female BALB/c mice (Charles River, L'Arbresle cedex, France). Bacterial suspensions used for intravenous (i.v.) infection were prepared as follows. Overnight broth cultures of mutant and isogenic BWT strains, grown in TH supplemented with 0.1 % Tween 80, were diluted 1:100 into 50 ml of TH-Tween 80 medium and grown until OD600 = 0.6. The bacterial suspensions were washed twice in NaCl 0.9% and resuspended in NaCl 0.9% to obtain from 6x10 8 to 8x10 8 colony forming units (CFU)/ml final concentration in 500 µl for intravenous (iv) injections. Mice were injected with a 28-gauge, 0.5-in. needle in the tail vein.

Dissemination assay and organ analysis. Randomized groups of 9-21 mice or of 6 mice, BWT-FASII and EfΔFASII, and BWT-cfa and and EfΔcfa, respectively, were infected and mice were sacrificed by cervical dislocation at days 1, 7 and 14 or day 7, post infection (pi); blood, liver, spleen and kidneys were harvested. All organs were weighed prior to further experimentation.

Liver, spleen and kidneys were homogenized in 0.9% NaCl. Serial dilutions were immediately plated on TH agar plates containing 0.1 % Tween80 (TH-Tween agar). CFU were enumerated after 24 h incubation at 37°C.

RESULTS and DISCUSSION

FASII pathway genetic organization and regulation of expression. In E. faecalis, a total of 15 genes belonging to the FASII pathway were previously identified [START_REF] Zhu | The two functional enoylacyl carrier protein reductases of Enterococcus faecalis do not mediate triclosan resistance[END_REF][START_REF] Eckhardt | Transcriptional regulation of fatty acid biosynthesis in Lactococcus lactis[END_REF][START_REF] Lu | Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity[END_REF]. However, a 16 th gene, ef1773 (NCBI reference sequence for the protein, WP_010706667.1), named fabG, is present in the annotated V583 genome. This gene is conserved among E. faecalis isolates. As two genes were assigned the same annotation we suggest renaming ef2881, which belongs to the 12 gene cluster, as fabGa, and ef1773, fabGb. FabGa and FabGb proteins share 32% identity and 65% similarity. Physiological and enzymatic properties of the two FabG homologues remain unknown and we hypothesized that they may catalyze the same 3-oxoacyl-ACP reduction step but with different substrate preferences or conditions of expression. In silico analysis using the E. faecalis FabT consensus sequence indicated the presence of a putative FabT binding site, AATTTGAAACAAAACG (palindromic nucleotides in bold), 10 bp downstream of the fabGb translation start codon (Fig. 1A) [START_REF] Lu | Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity[END_REF]. This further suggests that FabGb is involved in FA synthesis.

Genetic organization of the 12 gene cluster was initially defined in silico on the basis of an upstream FabT consensus binding sequence [START_REF] Eckhardt | Transcriptional regulation of fatty acid biosynthesis in Lactococcus lactis[END_REF]. Depending on the species, an organization in two or three operons was suggested [START_REF] Zhu | The two functional enoylacyl carrier protein reductases of Enterococcus faecalis do not mediate triclosan resistance[END_REF][START_REF] Eckhardt | Transcriptional regulation of fatty acid biosynthesis in Lactococcus lactis[END_REF][START_REF] Lu | Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity[END_REF]. In E. faecalis, FabT consensus binding sequences are present upstream of fabT and fabK (Fig. 1A), suggesting a two-operon organization. Also, the UFA synthesis genes, fabO and fabN, may constitute another operon [START_REF] Zhu | The two functional enoylacyl carrier protein reductases of Enterococcus faecalis do not mediate triclosan resistance[END_REF][START_REF] Eckhardt | Transcriptional regulation of fatty acid biosynthesis in Lactococcus lactis[END_REF][START_REF] Lu | Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity[END_REF]. This organization was tested by overlapping RT-PCR mapping (Fig. 1A,1B). Results show that the 12-gene cluster is organized as a single operon, which ends before ef2874. A transcription reinitiation site may be linked to the FabT consensus binding sequence upstream of fabK. Furthermore, respectively intense and faint bands were obtained for fabO-fabN and fabN-pyrD-1 (= ef0285), suggesting that fabO and fabN constitute an operon and that there may be some read-through downstream of fabN.

Altogether our data show that E. faecalis FASII genes are organized in two operons and two monocistronic transcriptional units (Fig. 1A,1B).

The FASII pathway can be strongly repressed by exogenous fatty acids, such that phospholipid synthesis relies on exogenous FA substrates [START_REF] Brinster | Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens[END_REF][START_REF] Yao | Exogenous fatty acid metabolism in bacteria[END_REF]. Human serum is rich in lipids comprising long acyl-chain fatty acids such as 18:1 (15%) and 16:0 (25%), which can act as FabT corepressors (29). To our knowledge, the regulation of E. faecalis FASII gene expression elicited by addition of FAs has not been studied. To address this question, and after checking that there was no growth difference in the presence or absence of added human serum (data not shown), we used qRT-PCR to analyze the effect of 40% human serum on E. faecalis FASII gene expression (Fig. 1A,1C). Serum addition resulted in decreased expression of all FASII genes varying from 2.5-to 14-fold depending on the gene. Interestingly, a putative FabT binding site was found close to the most strongly repressed genes, fabGb, fabO and fabK. This suggests that a FabT-controlled transcription initiation site or a transcription repression site exists upstream of these genes.

Altogether, our data show that the 12 gene operon (from fabT to accA), as defined above, is strongly repressed in the presence of a physiological concentration of serum.

E. faecalis FASII pathway and cyclopropanation are dispensable for growth in the

presence of serum. Supplementation with serum or unsaturated FA overcomes growth inhibition mediated by cerulenin, a FASII inhibitor [START_REF] Brinster | Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens[END_REF][START_REF] Kénanian | Permissive fatty acid incorporation promotes staphylococcal adaptation to FASII antibiotics in host environments[END_REF][START_REF] Saito | Enterococcus faecalis responds to individual exogenous fatty acids independently of their degree of saturation or chain length[END_REF]. We confirmed these results with the WT strain VE14089 (data not shown). The consequences of a non-functional FASII pathway were then examined by constructing EfΔFASII, in which 9 genes of the 12-gene operon, from fabK to accA (ef2883 to ef2875) were deleted. The genes that were removed encode products implicated in the initiation and elongation modules (Fig. 1A). Growth of the WT, EfΔFASII mutant and back-tothe-wild-type (BWT-FASII) (see Materials and Methods) strains was first compared in RPMI + (Fig. 2A). The WT and the BWT-FASII strains behaved similarly. In contrast, EfΔFASII growth was essentially arrested in RPMI + ; slight observed growth is likely due to residual Tween 80 used in precultures. These results confirm that an active FASII pathway is required for growth in FAfree medium. Addition of 40% human serum to growth medium led to equivalent growth of all strains (Fig. 2A), indicating that serum is sufficient to fully restore growth of the EfΔFASII mutant strain. Altogether, these results indicate that the exogenous FAs present in serum are necessary and sufficient to restore growth when E. faecalis FASII pathway is abolished, either via the addition of a FASII inhibitor (1) or by deletion of FASII genes.

The role of cyclopropanation on E. faecalis growth in the presence of exogenous FAs remained an open question. E. faecalis produces cyclopropane FAs, which are produced from both endogenous and exogenous substrates, presumably to mediate membrane fluidity adjustment by modifying UFA [START_REF] Poger | A ring to rule them all: the effect of cyclopropane fatty acids on the fluidity of lipid bilayers[END_REF]. A cfa in-frame deletion mutant was constructed to assess the role of cyclopropane FA synthesis in E. faecalis growth in medium without or with host serum. The mutant strain grew like the WT and BWT-cfa strains in both media, indicating that the presence or absence of cyclic FA does not impact E. faecalis growth in vitro (Fig 2B).

E. faecalis WT and Ef

DFASII strains show identical FA composition upon growth in serum. Strong repression of E. faecalis FASII genes in the presence of human serum correlates with incorporation of FAs, as previously observed in the same culture condition [START_REF] Saito | Incorporation of exogenous fatty acids protects Enterococcus faecalis from membrane-damaging agents[END_REF]. We determined FA compositions of WT VE14089 and BWT derivative (BWT-FASII) grown in the absence or presence of 40% serum (Table S2, S3, Fig. 3A). BWT-FASII profiles were similar to those of the WT; the slight differences probably arose from growth variations that could alter the efficiency of conversion between the UFA and cyclo forms. As previously described, FA of cells grown in FA-free medium (RPMI + ) comprised mainly palmitic acid (16:0; PA) (40%), vaccenic acid (18:1Δ 11 ; VA) (20%) and cyclopropanated-VA form (C19Δ 11 ) (20%) (Fig. 3A). In contrast, membrane FA composition of bacteria grown in serum resembled profiles of serum alone (Table S3, Fig. 3A) [START_REF] Zhu | The two functional enoylacyl carrier protein reductases of Enterococcus faecalis do not mediate triclosan resistance[END_REF][START_REF] Saito | Incorporation of exogenous fatty acids protects Enterococcus faecalis from membrane-damaging agents[END_REF][START_REF] Harp | Exogenous fatty acids protect Enterococcus faecalis from daptomycin-induced membrane stress independently of the response regulator LiaR[END_REF]. Thus, the proportion of endogenously produced 18:1Δ 11 dropped to 1.7%, and that of serum-provided oleic acid (18:1Δ 9 ; OA) rose to 13.5%. These latter percentages are different from previously reported ones [START_REF] Saito | Incorporation of exogenous fatty acids protects Enterococcus faecalis from membrane-damaging agents[END_REF][START_REF] Harp | Exogenous fatty acids protect Enterococcus faecalis from daptomycin-induced membrane stress independently of the response regulator LiaR[END_REF]; this could be due to the use here of 40% rather than 15% serum. Thus, VE14089, like other E. faecalis isolates, incorporates serum-supplied exogeneous FA [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. Another notable change in the presence of serum was a decrease in the amount of C19Δ 11 , as reported [START_REF] Saito | Incorporation of exogenous fatty acids protects Enterococcus faecalis from membrane-damaging agents[END_REF], while cyclopropanated-OA (C19Δ 9 ), which is not found in serum, rose to 15%, indicating that E. faecalis CFA synthase cyclizes both 18:1Δ 9 and 18:1Δ 11 . This indicates that E. faecalis CFA synthase is active on different substrates which is in agreement with the fact that bacterial CFAs display lax requirements for positioning of the cis double bond, except for cis double-bond positions at FA extremities, or trans double bonds, which are generally not converted [START_REF] Grogan | Cyclopropane ring formation in membrane lipids of bacteria[END_REF].

We also established the FA content of the EfΔFASII mutant strain grown in 40% human serum (Fig. 3B). The FA profiles of the EfΔFASII and BWT-FASII strains were similar (Fig. 3B).

Both strains similarly incorporated serum FAs and generated C19Δ 9 . These data demonstrate that FASII activity has no impact on membrane composition in the presence of human serum.

E. faecalis cyclopropane fatty acid synthase impacts the cellular FA composition.

To characterize the impact of the cfa deletion on E. faecalis membrane FA composition, we compared FA profiles of EfΔcfa, BWT-cfa, and WT strains grown in FA-free medium and in the presence of human serum (Fig. 3C, 3D, Tables S2,S4). The BWT-cfa, profiles were near identical to those of the WT (Tables S2,S4). Compared to the BWT-cfa control strain in RPMI + the EfΔcfa mutant strain was devoid of C19Δ 11 and was enriched in 18:1Δ 11 (Fig. 3C). In serum-supplemented medium, the EfΔcfa strain was devoid of C19Δ 9 and enriched in 18:1Δ 9 (Fig. 3D). These data confirm that the EfΔcfa strain is defective for cyclopropanation of unsaturated fatty acids, mainly 18:1Δ 11 in RPMI + and 18:1Δ 9 in the presence of serum. This implies that, in vivo, the fatty acid composition of cfa defective strains may be different from those of wild-type strains and this could have physiological consequences. E. faecalis septicemic infection is independent of both the FASII pathway and fatty acid cyclopropanation. The WT, EfΔFASII and BWT-FASII strains grew similarly and displayed the same FA composition when grown in the presence of human serum. During infection however, E. faecalis infects organs whose FA contents are variable [START_REF] Cunnane | Lipid and fatty acid composition of organs from copper-deficient mice[END_REF][START_REF] Gentry-Weeks | Intravenous mouse infection model for studying the pathology of Enterococcus faecalis infections[END_REF]. We therefore asked whether exogenous FAs in the host may compensate the loss of the active FASII pathway in vivo.

We first followed the infection process, looking at the physiological consequences of intravenous infection with the BWT-FASII strain. CFU counts and organ weights were examined 1, 7, and 14 days post infection (pi) (Fig. 4A,4B). Throughout the course of the experiment, the CFUs in blood were below the detection threshold (data not shown). The CFU counts in the liver diminished 31-fold, at 14 d pi, with no change in the weight of livers throughout the experiment.

In the spleen, the bacterial levels strongly decreased over a 2-week period, from approximately 10 4 CFU 24 h pi, to 66% spleens being sterile at the end of the experiment. However, d14-spleens weighed twice as much as d1 spleens (Fig. 4B). Finally, kidney weights increased from d7 to d14 (Fig. 4B). This correlated, albeit with a time laps, to the tendency in the CFU counts increase at d7 in BWT-FASII-infected mice (15-fold, respectively) and is in agreement with E. faecalis tropism for the kidneys [START_REF] Harp | Exogenous fatty acids protect Enterococcus faecalis from daptomycin-induced membrane stress independently of the response regulator LiaR[END_REF][START_REF] Kau | Enterococcus faecalis tropism for the kidneys in the urinary tract of C57BL/6J mice[END_REF].

We then assessed the consequences of the FASII gene deletion by comparing the BWT-FASII-and EfΔFASII-infected mice. Bacterial counts in liver, spleen and kidneys, as well as organ weights, were similar for BWT-FASII-and EfΔFASII-infected mice at all-time points (Fig. 4A,4B). This demonstrates that, as in other species, targeting the FASII pathway would be ineffective for the treatment of E. faecalis septicemic infection [START_REF] Brinster | Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens[END_REF]. model. Cfa is reportedly more expressed in stationary phase [START_REF] To | Cyclopropanation of membrane unsaturated fatty acids is not essential to the acid stress response of Lactococcus lactis subsp. cremoris[END_REF]. We reasoned that differences, if any, between EfΔcfa and BWT-cfa would be more pronounced after CFUs reached their highest level in the wild-type strain (here BWT) -infected mice, i.e., at d7. No differences were observed in terms of bacterial loads in the different organs between EfΔcfa-and BWT-cfa-infected mice (Fig. 4C). This indicates that cyclopropanation activity does not confer an advantage to E. faecalis during systemic infection in this model.

In conclusion, our data demonstrate that targeting the FASII pathway or the cyclopropanation process would be ineffective for the treatment of E. faecalis septicemic infection.

  Ethics statement. All animal experiments described in this study were conducted in accordance with guidelines of Paris Descartes University, in compliance with the European animal welfare regulation (http://ec.europa.eu/environment/chemicals/lab_animals/home_en.html) and were approved by the University Paris Descartes animal care and use committee and by the Ministère de l'Education Nationale, l'Enseignement Supérieur et la Recherche (2015032714098562_v1, APAFIS #390).

  Statistical analysis. Statistical analyses were performed using a 2-way ANOVA and a Bonferroni post-test or a Mann-Whitney test (PRISM 8). A p value ≤ 0.05 was considered statistically significant; * p<0.05, ** p<0.01, *** p<0.005, **** p<0.0001. 10

Figure 1

 1 Figure 1 Genetic organization of E. faecalis FASII pathway and impact of exogenous FAs on

Figure 2 .

 2 Figure 2. Impact of FASII pathway mutations on E. faecalis growth. (A) Growth curves of

Figure 3 .

 3 Figure 3. Fatty acid profiles of E. faecalis WT, DFASII, and Dcfa strains grown without and

Figure 4 .

 4 Figure 4. EfΔFASII and BWT-FASII infection in a mouse septicemic model. (A) CFUs shown

Figure 1

 1 Figure 1 Genetic organization of E. faecalis FASII pathway and impact of exogenous FAs on E. faecalis FASII gene expression. (A) Schematic representation of E. faecalis VE14089 FASII pathway genes. Gene positions with names below are represented with open arrows. Two genes in the locus, pyrD-1 (ef0285), and ef2874, are unrelated to FASII. Above, asterisks indicate putative FabT binding sites; below, transcripts predicted by PCR reactions, straight arrows; transcripts identified by predicted FabT binding sequence and expression levels, dotted arrow. (B) Agarose gel of the RT-PCR amplification products on cDNA using primer pairs from neighboring genes. The order is the same as that in Fig. 1A above; lane: 1, fabI-fabO; 2; fabO-fabN; 3 fabN-ef0285; 4, fabT-fabH; 5, fabH-acpP; 6, acpP-fabK; 7, fabK-fabD; 8, fabD-fabGa; 9, fabGa-fabF; 10, fabF-accB; 11, accB-fabZ; 12, fabZ-accC; 13, accC-accD; 14, accD-accA; 15, accA-ef2874. MW, molecular weight standard. (C) FASII gene expression fold change in E. faecalis grown in RPMI + medium in the absence or the presence of 40% human serum. The means ± SD of three independent experiments are represented.

Figure 2 .

 2 Figure 2. Impact of FASII pathway mutations on E. faecalis growth. (A) Growth curves of VE14089 (WT) (), EfΔFASII (p) and BWT-FASII (¢) strains in RPMI + in the absence (empty symbols) or presence (closed symbols) of 40% human serum. (B) Growth curves of VE14089 (), EfΔcfa (p) and BWT-cfa (¢) strains in RPMI + in the absence (empty symbols) or presence (closed symbols) of 40% human serum. Growth curves shown are representative of at least 3 independent experiments.

Figure 3 .

 3 Figure 3. Fatty acid profiles of E. faecalis WT, DFASII, and Dcfa strains grown without and with serum. E. faecalis was grown in RPMI + or in RPMI + containing 40% human serum to stationary phase. FA composition was compared to assess the roles of medium FA content and genetic background. The means of the percentages of each FA found in E. faecalis, BWT or mutant strains, grown to stationary phase in the presence or absence of 40 % serum of three independent experiments are presented. (see also Table S2, S3, S4). (A) BWT-FASII grown in the absence or presence of serum. (B). BWT-FASII and EfDFASII strains grown in 40% serum. (C) BWT-cfa and EfDcfa strains grown in RPMI + . (D) BWT-cfa and EfDcfa strains grown in 40% human serum. Only major fatty acids (>2% of total in at least one profile) are shown. Green stars, fatty acids derived from serum. Green arrows: cyclopropanation in the BWT strains of unsaturated fatty acids, dark green, produced by E. faecalis (mainly 18:1Δ 11 ) or, light green, incorporated from serum (mainly 18:1Δ 9 ). Columns: dark green BWT-FASII; dark blue, EfDFASII; light green, BWT-cfa; light blue, EfΔcfa; hatched, strains grown in RPMI + ; empty, strains in RPMI + supplemented with 40% human serum.

Figure 4 .

 4 Figure 4. EfΔFASII and BWT-FASII infection in a mouse septicemic model. (A) CFUs shown by scattergraph plots at days 1, 7 and 14 pi of bacteria recovered from BWT-FASII and EfΔFASII infected mice. Nine to 20 mice were intravenously infected with 6.10 8 to 8.10 8 E. faecalis BWT-FASII (closed squares) or EfΔFASII (open squares). (B) Organ weights shown by scattergraph plots at days 1, 7 and 14 pi of 3 to 9 mice infected by EfΔFASII or BWT-FASII. Symbols are as in 'A'. (C) CFUs shown by scattergraph plots at day 7 pi of viable bacteria recovered EfΔcfa and BWT-cfa infected mice. Six mice were intravenously infected with 6-8.10 8 E. faecalis BWT-cfa (closed circles) or EfΔcfa (open circles) strains. Upper panels, liver; middle panels, spleen; lower panels, kidneys. Each symbol represents the number of CFUs per organ of one infected mouse or the weight of one organ. Median ranges are represented. Statistical analysis, A, B, 2-way ANOVA and a Bonferroni post-test; C, Mann-Whitney test. * p<0.05; ** p<0.01, *** p<0.005 **** p<0.0001.

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.

ACKNOWLEDGMENTS

We fondly acknowledge our colleague G. Lamberet, who passed away Dec 23, 2019. We thank A. Tazi for helpful discussions. This work was supported by funding from the Agence Nationale de la Recherche (ANR-13001038), the Fondation pour la recherche Médicale (DBF20161136769) and from INSERM, CNRS and Université de Paris.