Metabolic Shift of an Isogenic Strain of Enterococcus Faecalis 14, Deficient

International Journal of Molecular Sciences

Article Metabolic Shift of an Isogenic Strain of Enterococcus faecalis 14, Deﬁcient in Its Own Bacteriocin Synthesis, as Revealed by a Transcriptomic Analysis

Rabia Ladjouzi , Anca Lucau-Danila and Djamel Drider*

UMR Transfrontalière BioEcoAgro N◦ 1158, Univ. Lille, INRAE, Univ. Liège, UPJV, YNCREA, Univ. Artois, Univ. Littoral Côte d’Opale, ICV—Institut Charles Viollette, F-59000 Lille, France; [email protected] (R.L.); [email protected] (A.L.-D.) * Correspondence: [email protected]

Received: 6 May 2020; Accepted: 29 June 2020; Published: 30 June 2020

Abstract: The production of antimicrobial molecules often involves complex biological pathways. This study aimed at understanding the metabolic and physiological networks of enterocin EntDD14-associated function, in the bacteriocinogenic strain, Enterococcus faecalis 14. A global and comparative transcriptomic study was carried out on E. faecalis 14 and its isogenic mutant ∆bac, inactivated in genes coding for EntDD14. The in vitro ability to form biofilm on polystyrene plates was assessed by the crystal violet method, while the cytotoxicity on human colorectal adenocarcinoma Caco-2 cells was determined by the Cell Counting Kit-8. Transcriptomic data revealed that 71 genes were differentially expressed in both strains. As expected, genes coding for EntDD14 were downregulated in the ∆bac mutant, whereas the other 69 genes were upregulated. Upregulated genes were associated with phage-related chromosomal islands, biofilm formation capability, resistance to environmental stresses, and metabolic reprogramming. Interestingly, the ∆bac mutant showed an improved bacterial growth, a high capacity to form biofilm on inanimate surfaces and a very weak cytotoxicity level. These multiple metabolic rearrangements delineate a new line of defense to counterbalance the loss of EntDD14.

Keywords: biological cost; bacterial physiology; leaderless two-peptides; enterocin EntDD14; bioﬁlm; antimicrobial peptide; microarray analysis

1. Introduction Bacteriocins are proteinaceous ribosomally synthesized antimicrobial peptides that kill or inhibit the growth of undesired bacteria. They have either a narrow spectrum, acting on phylogenetically closely related bacteria [1], or a broad spectrum, acting on phylogenetically distant bacteria [1,2]. Bacteriocins are produced by both Gram-negative and Gram-positive bacteria [3], and to a lesser extent by Archea [4]. Currently there is no unique and universally adopted scheme of bacteriocin classification. Cotter et al. [5] subdivided bacteriocins into two main classes. Class I contains bacteriocins that undergo significant post-translational modifications and class II contains unmodified peptides that only undergo slight modification such as the formation of disulfide bridges or circularization. Due to their cationic nature, and the anionic property the bacterial cell surface, they can form pores in bacterial cell membrane, resulting in dissipation of the proton motive force and depletion of intracellular ATP [6,7]. Bacteriocins can also utilize a set of docking molecules such as lipid II, maltose ABC-transporters, Zn-dependent metallopeptidase, undecaprenyl pyrophosphate phosphatase. For a review, see Cotter et al. [5]. The production of antagonistic substances, such as bacteriocins, is a key (and ancient) mechanism of defense that has been conserved throughout evolution. There have been a large number of bacteriocins

Int. J. Mol. Sci. 2020, 21, 4653; doi:10.3390/ijms21134653 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 4653 2 of 13 isolated from nature. Production of bacteriocins occurs in Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria according to Drissi et al. [8], who carried out a large genome mining project. Bacteriocins, mainly those produced by lactic acid bacteria, designated as LAB-bacteriocins, have potential applications as food preservatives being a potentially safer alternative to the chemicals used for food preservation [9]. Furthermore, research on bacteriocins has spurred an increasing interest in their other multifaceted biological functions, as reviewed by Drider et al. [10], and Chikindas et al. [11]. They are steadily reported as therapeutic agents to fight malevolent pathogens and drug-resistant bacteria [12,13]. Bacteriocin-encoding DNAs are usually organized into operon clusters on the chromosome or any other DNA genetic element such as plasmids [14]. Their export outside of the producing cell is carried out by two main mechanisms: the ABC-transporter and the sec-dependent pathways [15]. Enterocin DD14 (EntDD14) is a leaderless two peptide bacteriocin produced by Enterococcus faecalis 14, a strain isolated from a meconium sample obtained from the Victor Provo Hospital (Roubaix, France) [16]. The strong antibacterial activity of EntDD14 was mainly noticeable against a range of Gram-positive bacteria, including Staphylococcus aureus, Listeria monocytogenes, E. faecalis, Bacillus subtilis, and Clostridium perfringens [16,17]. The chromosomal genetic organization of the EntDD14 cluster has recently been reported and the isogenic mutant ∆bac, inactivated in genes coding for EntDD14 was constructed [18]. The production of bacteriocins is complex and influenced by different environmental factors, such as media composition, pH, or temperature [19], allowing bacteriocinogenic strains to thrive under environmental conditions. Nevertheless, no study so far been reported on the capacity of any bacteriocinogenic strain to reorganize and adapt its metabolism following site-directed inactivation of DNA coding for its bacteriocin. Thus, we establish here through a microarray study, a first snapshot highlighting the physiological and metabolic capabilities of the bacteriocinogenic E. faecalis 14 strain to reorganize its cell machinery following the loss of its normal mechanism of defense.

2. Results

2.1. Gene Expression Analysis in the E. faecalis 14 ∆bac Mutant Previous sequencing of the E. faecalis 14 genome showed that the EntDD14 encoding genes are chromosomally located [20]. Recently, we constructed a mutant unable to produce EntDD14 by deleting the ddA and ddB genes encoding this bacteriocin [18]. To explain the particular behavior of the ∆bac mutant strain and the impact of the bacteriocin EntDD14 on the global regulation and gene expression in E. faecalis 14, we performed a comparative transcriptomic analysis of RNA isolated from the ∆bac mutant strain versus the wild-type (WT), after 6 h of growth in GM17 medium under semi-aerobic conditions. Notably under these conditions, the bacteriocin EntDD14 is produced both in wild-type and in the ∆bac-Comp complemented strain but not in the ∆bac mutant (Figure1). The results revealed a total of 71 genes that were diﬀerentially expressed in the ∆bac strain (Figure2 and Supplementary Table S1). Of note, only the ddA and ddB structural genes were as expected downregulated in the mutant ∆bac strain (Figure2, lines 1–2), whereas the 69 other remaining genes were upregulated. The functional assembly indicated that in the absence of a functional bacteriocin gene, numerous other genes were upregulated, enabling most likely a substitutionary induced defensive option (Figure2). Int. J. Mol. Sci. 2020, 21, 4653 3 of 13

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Figure 1. AntimicrobialFigure 1. Antimicrobial activity activity of supernatants of supernatants of ofE. E. faecalis faecalis 1414 wild wild-type,-type, its isogenic its isogenic ∆bac mutant∆bac mutant and and ∆bac-Comp complemented strains against L. innocua at 6 h of growth on GM17 medium. ∆bac: E. ∆bac-Comp complemented strains against L. innocua at 6 h of growth on GM17 medium. ∆bac: E. faecalis faecalis 14 mutant deleted in ddA and ddB bacteriocin structural genes. If present, the inhibition zone 14 mutant deletedindicated in theddA susceptibilityand ddB bacteriocinof the bacterialstructural lawn (L. innocua genes.) to the If present,produced EntDD14 the inhibition bacteriocin. zone indicated the susceptibilityThe data of are the representative bacterial lawn of at least (L. three innocua independent) to the experiments. produced EntDD14 bacteriocin. The data are representativeInt. J. Mol. of Sci. at 20 least20, 21, x three FOR PEER independent REVIEW experiments. 4 of 14 An important group of genes with phage-related functions was found to be upregulated,

including genesΔb ac1coding Δbac1 forΔb aac1 metallo -β-lactamase superfamily domain protein in the prophage, phage vs WT Holliday junctionvs WT resolvase,vs WT phage capsid proteins or a bacteriophage transcriptional regulator 251_PI442896112 1 Bacteriocin ddA Genes involved in belonging250_PI442896112 to the Cro/CI family2 Bacteriocin (FigureddB 2, lines 3–7). In the same group werevirulence embedded mechanism genes 1305_PI442896112 3 Metallo-beta-lactamase superfamily domain protein in prophage involved1877_PI442896112 in the transposition or4 DNAPhage Holliday replicationjunction resolvase and repair (Figure 2, lines 3–15). Genes playing an 1327_PI442896112 5 Phage minor capsid protein important1857_PI442896112 role in the cell division,6 Phage cell protein adhesion, biofilm formation, or competence mechanisms were 1886_PI442896112 7 Transcriptional regulator, Cro/CI family also766_PI442896112 found to be upregulated in8 E.Transposase faecalis Δbac mutant strain (Figure 2, lines 16Genes–23). related Another to phage, group 584_PI442896112 9 DNA ligase including2681_PI442896112 genes implied in the 10resistanceDNA mismatch repairto antibioticsprotein MutS (transcriptional regulator,DNA TetRreplication family), and repairin the 47_PI442896112 11 DNA recombination and repair protein RecF outer309_PI442896112 envelope biosynthesis (OxaA,12 Single-stranded NucleosideDNA-binding protein-diphosphate-sugar epimerase), in the response to 1878_PI442896112 13 Helicase loader DnaI general1315_PI442896112 stress (DegP/HtrA, Gls2414 Replicase familydomain generalprotein stress protein, apo-citrate lyase phosphoribosyl- 1879_PI442896112 15 DnaD domain protein dephospho841_PI442896112 -CoA transferase), or16 moreCell division specifically protein FtsL to oxidative stress survival (YkgE, Npx), were also 2256_PI442896112 17 Cell surface protein precursor upregulated2799_PI442896112 in the E. faecalis Δbac mutant strain, probably related to environmental resistance of this 18 Cell surface protein Genes involved in cell mutant1126_PI442896112 (Figure 2, lines 24–31).19 InFibronectin the same/fibrinogen group,-binding protein several transporters acting as efflux pumps were 278_PI442896112 20 Surface exclusion protein Sea1/PrgA division, adhesion and found280_PI442896112 markedly to be upregulated21 Aggregation (Figuresubstance 2, Asa1/linesPrgB 32–35). An important set of genes involved in the 2074_PI442896112 22 Late competence protein ComEA, DNA receptor competence transcription,1620_PI442896112 translation, and23 variousComF operon metabolicprotein C pathways such as glycolysis, tagatose pathway, 1397_PI442896112 24 Transcriptional regulator, TetR family glycerol2031_PI442896112 and arginine catabolism25 Inner (Figuremembrane 2, protein linestranslocase 36–component57) were YidC, OxaAnoticedprotein as being upregulated and are 1122_PI442896112 26 Nucleoside-diphosphate-sugar epimerases likely2561_PI442896112 related to the shift to a new27 Serine defensive protease, DegP strategy./HtrA Finally, a group of 14 genes (Figure 2, lines 58– 111_PI442896112 28 General stress protein, Gls24 family Genes involved in 71)28_PI442896112 were found to be upregulated29 Apo in-citrate E. lyasefaecalis phosphoribosyl Δbac-dephospho mutant-CoA straintransferase but their roles are resistanceunknown. 989_PI442896112 30 Predicted L-lactate dehydrogenase, Fe-S oxidoreductase subunit YkgE 1090_PI442896112 31 NADH peroxidase Npx 72_PI442896112 32 ABC transporter, permease protein 8_PI443061160 33 ABC transporter, ATP-binding protein 1226_PI442896112 34 Mg(2+) transport ATPase, P-type 985_PI442896112 35 D-serine/D-alanine/glycine transporter 779_PI442896112 36 Response regulator receiver: transcriptional regulatory protein 202_PI442896112 37 SSU ribosomal protein S8p (S15Ae) 182_PI442896112 38 Translation elongation factor G 1257_PI442896112 39 Tripeptide aminopeptidase 2498_PI442896112 40 Ribokinase 1547_PI442896112 41 Adenine phosphoribosyltransferase 868_PI442896112 42 PTS system, cellobiose-specific IIA component 867_PI442896112 43 PTS system, cellobiose-specific IIB component 1775_PI442896112 44 Enolase Genes involved in 1393_PI442896112 45 NAD-dependent glyceraldehyde-3-phosphate dehydrogenase metabolic pathways 464_PI442896112 46 Beta-propeller domains of methanol dehydrogenase type 135_PI442896112 47 Arginine deiminase 580_PI442896112 48 Tagatose-6-phosphate kinase / 1-phosphofructokinase 2268_PI442896112 49 5'-methylthioadenosine nucleosidase / S-adenosylhomocysteine nucleosidase 2379_PI442896112 50 Nicotinate-nucleotide adenylyltransferase 1860_PI442896112 51 Glycerate kinase 2070_PI442896112 52 2-dehydropantoate 2-reductase 1944_PI442896112 53 Flavodoxin 2211_PI442896112 54 Heat shock protein 60 family co-chaperone GroES 2488_PI442896112 55 Ubiquinol-cytochrome C reductase iron-sulfur subunit 1361_PI442896112 56 V-type ATP synthase subunit G 1229_PI442896112 57 Dihydrolipoamide acetyltransferase 1435_PI442896112 58 DegV family protein 463_PI442896112 59 LemA family protein 549_PI442896112 60 Hypothetical protein SAV1839 277_PI442896112 61 FIG00630374: hypothetical protein 292_PI442896112 62 FIG00632142: hypothetical protein 2697_PI442896112 63 FIG00628641: hypothetical protein 1293_PI442896112 64 FIG00631897: hypothetical protein Genes with unknown 1955_PI442896112 65 FIG00627620: hypothetical protein function 675_PI442896112 66 FIG00631180: hypothetical protein 295_PI442896112 67 FIG00630159: hypothetical protein 2468_PI442896112 68 FIG00628307: hypothetical protein 127_PI442896112 69 FIG00630781: hypothetical protein 1608_PI442896112 70 Hypothetical protein, homolog of fig|393130.3.peg.2627 109_PI442896112 71 Conserved hypothetical protein -3.0 0.0 3.0 Figure 2. DiﬀFigureerentially 2. Differentially expressed expresse genesd genes (DEGs) (DEGs) in in the theE. E. faecalis faecalis 1414 Δ∆bacbac mutantmutant strain. strain. DEGs with DEGs with fold change (FC) >fold2.0 change and < (FC0.5) of>2.0 individual and <0.5 of ∆ individualbac (1–3) Δb vs.ac (1 mean–3) vs. ofmean the of wild-type the wild-type (WT) (WT) were were represented. represented. The putative functional groups are indicated on the right of the figure. The putative functional groups are indicated on the right of the ﬁgure. 2.2. Impact of the Deletion of the Structural Genes Coding for the Bacteriocin on the Bacterial Growth and An importantUltrastructure group of genes with phage-related functions was found to be upregulated, including genes coding forAs a metallo-revealed byβ this-lactamase transcriptomic superfamily analysis, a set domain of genes involved protein in in metabolism, the prophage, cell division, phage Holliday junction resolvase,transport phage of molecule capsids, and proteinsresistance to or oxid a bacteriophageative stress were over transcriptional-expressed in the ∆b regulatorac mutant as belonging to compared to WT. Presumably, these multiple adaptations have developed to counterbalance the absence of bacteriocin EntDD14 synthesis, and could impact on bacterial physiology and growth. To verify this hypothesis, we performed growth kinetics in 96-well microplate in GM17 medium at 37 °C for 14 h. As expected, the ∆bac mutant showed better bacterial growth than the WT (Figure 3). The

Int. J. Mol. Sci. 2020, 21, 4653 4 of 13 the Cro/CI family (Figure2, lines 3–7). In the same group were embedded genes involved in the transposition or DNA replication and repair (Figure2, lines 3–15). Genes playing an important role in the cell division, cell adhesion, biofilm formation, or competence mechanisms were also found to be upregulated in E. faecalis ∆bac mutant strain (Figure2, lines 16–23). Another group including genes implied in the resistance to antibiotics (transcriptional regulator, TetR family), in the outer envelope biosynthesis (OxaA, Nucleoside-diphosphate-sugar epimerase), in the response to general stress (DegP/HtrA, Gls24 family general stress protein, apo-citrate lyase phosphoribosyl-dephospho-CoA transferase), or more specifically to oxidative stress survival (YkgE, Npx), were also upregulated in the E. faecalis ∆bac mutant strain, probably related to environmental resistance of this mutant (Figure2, lines 24–31). In the same group, several transporters acting as efflux pumps were found markedly to be upregulated (Figure2, lines 32–35). An important set of genes involved in the transcription, translation, and various metabolic pathways such as glycolysis, tagatose pathway, glycerol and arginine catabolism (Figure2, lines 36–57) were noticed as being upregulated and are likely related to the shift to a new defensive strategy. Finally, a group of 14 genes (Figure2, lines 58–71) were found to be upregulated in E. faecalis ∆bac mutant strain but their roles are unknown.

2.2. Impact of the Deletion of the Structural Genes Coding for the Bacteriocin on the Bacterial Growth and Ultrastructure As revealed by this transcriptomic analysis, a set of genes involved in metabolism, cell division, transport of molecules, and resistance to oxidative stress were over-expressed in the ∆bac mutant as compared to WT. Presumably, these multiple adaptations have developed to counterbalance the absence of bacteriocin EntDD14 synthesis, and could impact on bacterial physiology and growth. To verify this hypothesis, we performed growth kinetics in 96-well microplate in GM17 medium at 37 ◦C for 14 h. As expected, the ∆bac mutant showed better bacterial growth than the WT (Figure3). Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 14 The mutant reached a maximum optical density OD600nm of 0.71 which was significantly higher than that of themutant WT (OD reached 0.52). a maximum The growth optical ratesdensity of OD the600 nm both of 0.71 strains which werewas significantly significantly higher di thanfferent that (p < 0.05). Remarkably,of the the WT∆ bac (ODstrain 0.52). The was growth able torates recover of the both the WTstrains phenotype were significantly when different transformed (p < 0.05). by a pAT18 plasmid containingRemarkably, the the ddA∆bac andstrainddB was genesable to whichrecover the encode WT phenotype a functional when bacteriocintransformed by EntDD14 a pAT18 as shown plasmid containing the ddA and ddB genes which encode a functional bacteriocin EntDD14 as shown by the ∆bac-Comp kinetic. Notably, no significant differences were observed between the growth rates by the ∆bac-Comp kinetic. Notably, no significant differences were observed between the growth rates of the ∆bac-Compof the ∆baccomplemented-Comp complemented strain strain and and that that of the the WT WT (p (=p 0.931)= 0.931),, arguing arguing therefore, therefore, a role for a role for EntDD14 inEntDD14 the global in the bacterialglobal bacterial growth. growth.

Figure 3. E. faecalis E. faecalis bac FigureGrowth 3. Growth curves curves of of E. faecalisstrains strains in in G GM17M17 medium. medium. E. faecalis 14 WT, ∆b14ac mutant WT, ∆ and themutant and the ∆bac complemented∆bac complemented strain. strain. The The verticalvertical bars bars represent represent the thestandard standard deviations. deviations. The data The are the data are the means of threemeans independentof three independent experiments. experiments. The The asterisk asterisk (*) (*) indicates indicates that thatthe growth the growth rate is significantly rate is signiﬁcantly diﬀerent fromdifferent that from of thethat WTof the strain WT strain using using the the Student Student tt test.test.

To strengthen the above-presented results, we verified whether any changes in the bacterial cell size, cell shape or other morphological structures (filaments or mucus) can be observed for the ∆bac mutant, as opposed to the WT. Such differences might explain the differences in the observed OD, as cell size can directly influence absorbance values. For this reason, exponential phase cultures, after 6 h of growth on GM17 medium under semi-aerobic conditions, were observed by transmission electron microscopy (TEM). Several micrographs of E. faecalis 14 and the ∆bac mutant were compared but no significant differences were noticed in the cell ultrastructure (Figure 4). Both strains exhibited cells of similar size and morphology of the bacterial cell wall with a predominant diplococcal organization. It was noted that both strains displayed colonies of the same appearance and average size when cultured on GM17 agar.

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To strengthen the above-presented results, we verified whether any changes in the bacterial cell size, cell shape or other morphological structures (filaments or mucus) can be observed for the ∆bac mutant, as opposed to the WT. Such differences might explain the differences in the observed OD, as cell size can directly influence absorbance values. For this reason, exponential phase cultures, after 6 h of growth on GM17 medium under semi-aerobic conditions, were observed by transmission electron microscopy (TEM). Several micrographs of E. faecalis 14 and the ∆bac mutant were compared but no significant differences were noticed in the cell ultrastructure (Figure4). Both strains exhibited cells of similar size and morphology of the bacterial cell wall with a predominant diplococcal organization. It was noted that both strains displayed colonies of the same appearance and average size when Int.cultured J. Mol. Sci. on 20 GM1720, 21, agar.x FOR PEER REVIEW 6 of 14

Figure 4. TransmissionTransmission electron micrographs of E. faecalis 14 and its ∆b∆bacac mutant strain.strain. No significant significant differencesdifferences in the size or organizationorganization ofof bacterialbacterial cellscells werewere observed.observed. 2.3. Absence of EntDD14 and the Biofilm Production Ability of the ∆bac Mutant Strain 2.3. Absence of EntDD14 and the Biofilm Production Ability of the Δbac Mutant Strain Among the genes over-expressed in the ∆bac mutant, two groups were related to biofilm formation Among the genes over-expressed in the ∆bac mutant, two groups were related to biofilm and maintenance. Therefore, we assessed the ability of the ∆bac mutant and WT strains to form biofilm formation and maintenance. Therefore, we assessed the ability of the ∆bac mutant and WT strains to using crystal violet method on polyester plates. A deeper analysis revealed a better adhesion score for form biofilm using crystal violet method on polyester plates. A deeper analysis revealed a better the ∆bac mutant compared to the WT (Figure5). There were significantly higher absorbance values adhesion score for the ∆bac mutant compared to the WT (Figure 5). There were significantly higher recorded for the ∆bac mutant (OD630nm = 1.241) compared to WT (OD630 nm = 0.909) with a p value of absorbance values recorded for the ∆bac mutant (OD630 nm = 1.241) compared to WT (OD630nm = 0.909) p = 0.011. Nevertheless, following the recommendations of Stepanovićet al. [21] we considered both with a p value of p = 0.011. Nevertheless, following the recommendations of Stepanović et al. [21] we strains to be strongly adherent, since the recorded absorbance values were 4 times greater than that of considered both strains to be strongly adherent, since the recorded absorbance values were 4 times the control (OD630nm = 0.09). These results confirmed those obtained by transcriptomic analyses and greater than that of the control (OD630 nm = 0.09). These results confirmed those obtained by showed that a mutant deleted in the genes coding for bacteriocin increased its adhesion capacity and transcriptomic analyses and showed that a mutant deleted in the genes coding for bacteriocin consequently its biofilm formation. increased its adhesion capacity and consequently its biofilm formation. 2.4. Absence of Bacteriocin Reduced the Cytotoxicity of E. faecalis 14 We previously showed that neither E. faecalis 14 nor its enterocin EntDD14 were cytotoxic to the intestinal porcine epithelial cell line IPEC-1 [17]. Here, we tested the cytotoxicity of E. faecalis 14 against Caco-2 cells. As expected, E. faecalis 14 exert only a slight cytotoxic effect on these eukaryotic cells, since 89% survival was observed after 24 h of contact (Figure6). Interestingly, in the absence of EntDD14 bacteriocin the viability of Caco-2 cells increased after 24 h incubation, as shown by a very

Figure 5. Adhesion of E. faecalis 14 and its ∆bac mutant strain to polystyrene plates as determined by

OD630 nm measurements. The absorbance values are the means of three independent experiments. Sterile GM17 was used as control. The error bars represent the standard deviations. Columns with

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Figure 4. Transmission electron micrographs of E. faecalis 14 and its ∆bac mutant strain. No significant differences in the size or organization of bacterial cells were observed.

2.3. Absence of EntDD14 and the Biofilm Production Ability of the Δbac Mutant Strain Among the genes over-expressed in the ∆bac mutant, two groups were related to biofilm formation and maintenance. Therefore, we assessed the ability of the ∆bac mutant and WT strains to form biofilm using crystal violet method on polyester plates. A deeper analysis revealed a better adhesion score for the ∆bac mutant compared to the WT (Figure 5). There were significantly higher absorbance values recorded for the ∆bac mutant (OD630 nm = 1.241) compared to WT (OD630nm = 0.909) Int.with J. Mol.a p value Sci. 2020 o,f21 p, = 4653 0.011. Nevertheless, following the recommendations of Stepanović et al. [21]6 of we 13 considered both strains to be strongly adherent, since the recorded absorbance values were 4 times greater than that of the control (OD630 nm = 0.09). These results confirmed those obtained by weak cytotoxicity level of the ∆bac mutant on Caco-2 cells (viability of 98.8%, Figure5). The di fference transcriptomic analyses and showed that a mutant deleted in the genes coding for bacteriocin between the ∆bac mutant and WT was significant but with a p value close to the 0.05 cut-off value increased its adhesion capacity and consequently its biofilm formation. (p = 0.045).

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 14

different letters are significantly different using one-way ANOVA with Tukey test and Student t test for pairwise comparisons. (*), p < 0.05; (**), p < 0.01.

2.4. Absence of Bacteriocin Reduced the Cytotoxicity of E. faecalis 14 We previously showed that neither E. faecalis 14 nor its enterocin EntDD14 were cytotoxic to the intestinal porcine epithelial cell line IPEC-1 [17]. Here, we tested the cytotoxicity of E. faecalis 14 against Caco-2 cells. As expected, E. faecalis 14 exert only a slight cytotoxic effect on these eukaryotic

cells,Figure since 89% 5. Adhesion survival of wasE. faecalis observed14 and after its 24∆bac h ofmutant contact strain (Figure to polystyrene 6). Interestingly, plates as in determined the absence of EntDD14 bacteriocin the viability of Caco-2 cells increased after 24 h incubation, as shown by a very byFigure OD630nm 5. Adhesionmeasurements. of E. faecalis The 14 absorbance and its ∆b valuesac mutant are thestrain means to polystyrene of three independent plates as determined experiments. by weak cytotoxicity level of the ∆bac mutant on Caco-2 cells (viability of 98.8%, Figure 5). The difference SterileOD630 nm GM17 measurements. was used as The control. absorbance The error values bars are represent the means the standard of three deviations. independent Columns experiments. with betweendiSterilefferent the GM17 letters∆bac wasmutant are used significantly and as control. WT di wasff erentThe significant error using bars one-way butrepresent with ANOVA athe p value withstandard Tukey close deviations. test to the and 0.05 Student Columns cutt-offtest with value for (p = 0.045).pairwise comparisons. (*), p < 0.05; (**), p < 0.01.

Figure 6. Cytotoxicity of E. faecalis 14 and its ∆bac mutant on Caco-2 cells (24 h). Data are means of Figure 6. Cytotoxicity of E. faecalis 14 and its ∆bac mutant on Caco-2 cells (24 h). Data are means of three independent experiments. Error bars represent standard deviations. Columns with different three independent experiments. Error bars represent standard deviations. Columns with different letters are significantly different using one-way ANOVA with Tukey test and the Student t test for letters are significantly different using one-way ANOVA with Tukey test and the Student t test for pairwise comparisons. (*), p < 0.05; (**), p < 0.01; not significant (n.s.), p > 0.05. pairwise comparisons. (*), p < 0.05; (**), p < 0.01; not significant (n.s.), p > 0.05. 3. Discussion 3. Discussion Bacteriocins are known for their effectiveness in fighting and eradicating microbial pathogens includingBacteriocins bacteria, are and known to a lesser for their extent effectiveness fungi and virusesin fighting [22– 24and]. Initially,eradicating the bacteriocinmicrobial pathogens synthesis andincluding its impact bacteria, on a specificand to a bacteriocin lesser extent gene fungi was and studied viruses at the [22 transcriptional–24]. Initially, the level, bacteriocin and in very synthesis limited casesand its at impact the post-transcriptional on a specific bacteriocin levels. Togene the was best studied of our knowledge,at the transcriptional there are level, no data and reporting in very thelimited impact cases of bacteriocin at the post- ontranscriptional the whole bacterial levels. transcriptome.To the best of Thus, our knowledge, there is a need there to are understand no data howreporting a bacteriocinogenic the impact of strainbacteriocin or any on probiotic the whole strain bacterial would behavetranscriptome when it. Th is naturallyus, there oris purposelya need to deprivedunderstand of how the ability a bacteri toocinogenic produce bacteriocin. strain or any In probiotic fact, this wouldstrain w happenould behave following when a i spontaneoust is naturally mutationor purposely of one deprived or more of genes the ability involved to produce in the biosynthesis bacteriocin. ofInthe fact, bacteriocin. this would To happen gain new following insights a spontaneous mutation of one or more genes involved in the biosynthesis of the bacteriocin. To gain new insights into this important question we conducted a comparative transcriptomic study using the total RNA isolated from the bacteriocinogenic E. faecalis 14 and the ∆bac mutant strain, after 6 h of growth. The ∆bac mutant, previously engineered to delete its structural genes encoding the peptides A and B of the EntDD14 bacteriocin, was unable to produce this bacteriocin [18]. Overall, the loss of EntDD14 synthesis caused metabolic and physiological changes in E. faecalis 14 to result in what is probably an alternative mechanism of defense. Thus, genes involved in mobile genetic element activity and biofilm formation were upregulated in the ∆bac mutant, as well as genes involved in resistance to environmental stresses and metabolism, thereby proving the ability of this strain to adapt and thrive under the experimental conditions tested here. These transcriptomics data were consolidated by the assays of adhesion to polystyrene plates, and the assessment of cytotoxicity

Int. J. Mol. Sci. 2020, 21, 4653 7 of 13 into this important question we conducted a comparative transcriptomic study using the total RNA isolated from the bacteriocinogenic E. faecalis 14 and the ∆bac mutant strain, after 6 h of growth. The ∆bac mutant, previously engineered to delete its structural genes encoding the peptides A and B of the EntDD14 bacteriocin, was unable to produce this bacteriocin [18]. Overall, the loss of EntDD14 synthesis caused metabolic and physiological changes in E. faecalis 14 to result in what is probably an alternative mechanism of defense. Thus, genes involved in mobile genetic element activity and bioﬁlm formation were upregulated in the ∆bac mutant, as well as genes involved in resistance to environmental stresses and metabolism, thereby proving the ability of this strain to adapt and thrive under the experimental conditions tested here. These transcriptomics data were consolidated by the assays of adhesion to polystyrene plates, and the assessment of cytotoxicity to Caco-2 cells. However, deeper analyses using additional experiments such as a qRT-PCR or Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 14 transcriptional fusions are needed to strengthen these conclusions. The productionto Caco-2 cells. of Ho bacteriocinswever, deeper might analyses provide using aadditional competitive experiments advantage such as to a a qRT producer-PCR or in certain ecologicaltranscriptional niches [25]. fusions This would are needed explain to strengthen the high these frequencies conclusions. of bacteriocin-producing enterococci The production of bacteriocins might provide a competitive advantage to a producer in certain in the humanecological intestinal niches [25] tract. This [26 would]. So, explain in the the absence high frequencies of this of key bacteriocin element,-producing the bacterium enterococci is able to reorganizein its the metabolism, human intestin toal provide tract [26]. an So, alternative in the absence mechanism of this key element to maintain, the bacterium its competitiveness is able to in the environmentreorganize (Figure its7 metabolism,). to provide an alternative mechanism to maintain its competitiveness in the environment (Figure 7).

Figure 7. ModelFigure 7. proposing Model prop theosing impact the impact of deletion of deletion of of genes genes encoding EntDD14 EntDD14 bacteriocin bacteriocin in E. faecalis in E. faecalis 14. 14. Transcriptomic data obtained here show that E. faecalis can enhance expression of several genes in order to compensateTranscriptomic for the data loss obtained of the here antibacterial show that E. activity faecalis can attributable enhance expression to EntDD14. of several genes The groupin order of to DEGs compensate involved for the in loss the of phage the antibacterial activity andactivity also attributable in DNA to replication EntDD14. and repair (Figure2, The group of DEGs involved in the phage activity and also in DNA replication and repair (Figure lines 3–15)2, includeslines 3–15) includes genes that genes present that present high high homology homology with with prophage prophage-related-related genes described genes described in in E. faecalis V538E. faecalis [27 V538] and [27] obvious and obvious functional functional similarities similarities with with the the phage phage-related-related chromosomal chromosomal islands islands (PRCIs) that(PRCIs) were that first were identified first identified in Staphylococcus in Staphylococcus aureusaureus [28][28. C].losely Closely related related elements elements with the same with the same genetic organizationgenetic organization were reportedwere reported as wellas well in inE. E. faecalisfaecalis PRCIsPRCIs [28] [,28 and], andmobile mobile genetic geneticelements elementswere were found to act as a reservoir for acquisition and dissemination of drug resistance factors in this species found to act[29] as. aMatos reservoir et al. for, showed acquisition that p andhage- disseminationrelated genes play of drug an important resistance role factors in genetic in this and species [29]. Matos et al.,physiological showed flexibility that phage-related for optimal adaptation genesplay in E. faecalis an important V583 [27]. roleThese in authors genetic indicated and physiologicalthat flexibilityp forrophage optimal induction adaptation in E. faecalis in subpopulationsE. faecalis V583 could favor [27]. their These survival authors and found indicated this mechanism that prophage inductionto in beE. similar faecalis to subpopulations that described for Shewanella could favor oneidensis their, Pseudomonas survival and aeruginosa found and this Streptococcus mechanism to be pneumoniae, especially in biofilms [30–32]. similar to thatBesides described these for putativeShewanella PRCI, other oneidensis genes ,involvedPseudomonas in the cell aeruginosa division,and cell adhesionStreptococcus, biofilm pneumoniae , especiallyformation in biofilms, and [ 30competence–32]. were found to be upregulated in the ∆bac mutant (Figure 2, lines 16–23). BesidesInterestingly, these putative genes with PRCI, similar other functions genes were involved reported in asthe involved cell division, in Enterococcus cell adhesion, biofilm biofilm formation,formation and competence [33]. FtsL is an were essential found protein to for be the upregulated cell division [34] in and the it was∆bac foundmutant to be involved (Figure 2, lines in E. faecalis biofilm formation [35]. Different cell surface proteins and aggregation substances were 16–23). Interestingly,reported to be genesinvolved with in E. similar faecalis biofilm functions maintenance were reported[36]. The late as competence involved protein in Enterococcus ComEA biofilm formationor [33 the]. ComF FtsL operon is an essentialprotein C are protein involved for in thecompetence, cell division a function [34 that] and in Streptococcus it was found mutants to be is involved in E. faecalisrequiredbiofilm for biofilm formation formation [35]. [37] Di. ffImportantly,erent cell our surface results proteins suggest that and the aggregation biofilm generatio substancesn was were reported tostimulated be involved in the in absenceE. faecalis of thebiofilm bacteriocin maintenance active gene [ in36 E.]. Thefaecalis. late The competence biofilm formation protein was ComEA or already described as the basis of antibiotic resistance in E. faecalis [35], and also as a factor in resistance to environmental stress [38]. In this study, we provide a new insight and show that the bacteriocinogenic E. faecalis 14, genetically modified and thus deprived of its bacteriocin production, has increased its biofilm formation ability (Figure 5). Interestingly, this form of cellular organization

Int. J. Mol. Sci. 2020, 21, 4653 8 of 13 the ComF operon protein C are involved in competence, a function that in Streptococcus mutants is required for biofilm formation [37]. Importantly, our results suggest that the biofilm generation was stimulated in the absence of the bacteriocin active gene in E. faecalis. The biofilm formation was already described as the basis of antibiotic resistance in E. faecalis [35], and also as a factor in resistance to environmental stress [38]. In this study, we provide a new insight and show that the bacteriocinogenic E. faecalis 14, genetically modified and thus deprived of its bacteriocin production, has increased its biofilm formation ability (Figure5). Interestingly, this form of cellular organization can enhance the bacterial persistence and resistance to environmental stress [39–41], which correlates with our own observations (Figure2, lines 24–35). Moreover, the involvement of enzymes such as NADH peroxidase Npx, lactate dehydrogenase as well as the general stress protein Gls24, in the response to oxidative stress, has already been demonstrated in E. faecalis [42–44]. Of note, the expression of genes coding for these enzymes in the ∆bac mutant indicates a better ability to maintain its redox balance compared to WT. Such adaptation enabled the strain to withstand different environmental stresses, and tolerance to antibiotics, which is correlated with the resistance to oxidative stress [45–47]. The lack of bacteriocin and enhancement of other putative virulence mechanisms caused this strain to undergo metabolic reorganization. In this respect, different mechanisms have been described in bacteria to explain the relationship between biofilm formation and the metabolic reprogramming [41,48,49]. This would probably explain the elevated in vitro growth rate observed in the ∆bac mutant, which is accompanied by overexpression of genes involved in the glycolysis, tagatose, glycerol, and arginine pathways (Figure2, lines 36–57). Furthermore, the tests on Caco-2 cells revealed a very weak cytotoxicity for the ∆bac mutant which would therefore presumably have little impact on intestinal cells. Nevertheless, this finding does show indirectly that bacteriocin EntDD14 can produce a very low level of cytotoxicity. A previous study showed no reduction in cell viability at concentrations of 50 µg/mL and 100 µg/mL of pure EntDD14 after 4 h of contact with the intestinal epithelial cell line IPEC-1. However, a decrease of 9.6% to 20% in the IPEC-1 cell viability was observed after 24 h of contact in a dose-dependent manner [17]. Collectively these results revealed alternative compensatory mechanisms present in the ∆bac mutant which counterbalance the loss of EntDD14 synthesis and could help the bacterium to maintain its competitiveness in complex environments such as the intestinal tract. This work will open new prospects for studying and better understanding the bacteriocin networks and mainly its role in bacterial pathogenicity.

4. Materials and Methods

4.1. Bacterial Strains and Growth Conditions E. faecalis 14 [16] and the recently constructed E. faecalis 14 ∆bac mutant and E. faecalis 14 ∆bac-Comp [18] were used in this study. The ∆bac mutant deleted in ddA and ddB genes was constructed by allelic exchange using a method based on the conditional replication of the pLT06 vector [50], while the complemented strain was constructed with a DNA fragment containing the entire ddA, ddB and the promoter region cloned in the Gram-positive replicative plasmid pAT18 [51]. Cultures were grown on M17 medium supplemented with 0.5% (w/v) of glucose (GM17) at 37 ◦C under semi-aerobic conditions. Growth kinetics were determined in 96-well microplates by measuring the optical density at 600 nm (OD600) using a SpectraMax i3 spectrophotometer (Molecular Devices, San Jose, CA, USA). The wells were equally inoculated, and the plates were read every 30 min for 12 h. The bacterial master cultures were stored at 80 C in GM17 broth supplemented with 50% (v/v) glycerol. − ◦ 4.2. Antibacterial Activity Assays The screening of antimicrobial activity of WT Ent. faecalis 14, its isogenic ∆bac mutant and the complemented ∆bac-Comp strains against L. innocua ATCC 33090 [52] was performed using well-diﬀusion method [17]. Brieﬂy, Brain Heart Infusion (BHI) plates (1% agar) were inoculated with L. Int. J. Mol. Sci. 2020, 21, 4653 9 of 13 innocua strain and were allowed to dry. Then, 50 µL of culture supernatant of the tested strain was added to the well and incubated overnight at 37 ◦C. The radius of the inhibition zone was measured from the edge of the well to the edge of the inhibition halo.

4.3. RNA Isolation and Microarrays Analysis For the microarray analysis, three distinct cultures of E. faecalis 14 ∆bac were used (∆bac1, ∆bac2 and ∆bac3) which were compared with E. faecalis 14 (WT1, WT2 and WT3), after 6 h of growth in GM17 medium. All cultures were harvested at the same cell concentration of ~2.108 CFU/mL. Analyses were performed with total RNA isolated by using NucleoSpinTM RNA Plus columns (Macherey-Nagel, Hoerdt, France). RNA quality was determined by Nanodrop and the absorbance ratios A260/280 and A260/230 were found to be between 2.0 and 2.2. RNA quality was also examined with a Bioanalyzer 2100 (Agilent, Les Ulis, France) and a minimal RNA integrity number (RIN) of 0.8 was required for all samples. Agilent G2509F E. faecalis 14 custom oligo-based DNA microarray (8 15 K) containing × spots of 60-mer oligonucleotide probes (in 5 replicates) were used to study the gene expression. RNA amplification, staining, hybridization, and washing were conducted according to the manufacturer’s instructions. Slides were scanned at 5 µm/pixel resolution using the GenePix 4000B scanner (Molecular Devices Corporation, Sunnyvale, CA, USA). Images were used for grid alignment and expression data digitization using GenePix Pro 6.0 software. Expression data were normalized by Quantile algorithm. To ascertain the quality of normalized data, filtering of data was mandatory for flagged signals. Expression data of the 3 wild-type samples were filtered for p value < 0.05 and the average was calculated for each gene. A fold change (FC) value was calculated using ∆bac individual samples and the mean of WT. Differentially expressed genes (DEGs) were selected for a FC threshold >2.0 or <0.5 and transformed in log2 FC. As results of biological repetitions were significantly similar, no qRT-PCR validation was performed. Functional annotation of DEGs was based on NCBI GenBank and related-genes physiological processes were assigned with NCBI, AmiGO 2 Gene Ontology and UniProt. KEGG pathway analysis was also used to identify relevant biological pathways of selected genes. All the microarray data have been submitted to the NCBI GEO archive for functional genomics data with the accession number GSE149873.

4.4. Transmission Electron Microscopy

Cultures of E. faecalis 14 and its ∆bac derivative mutant were grown in GM17 at 37 ◦C for 6 h then harvested by centrifugation ( 8000 g, 10 min, 4 C). For TEM, the pellets were fixed with 2.5% (v/v) × ◦ glutaraldehyde solution and 0.1 M (v/v) of Cacodylate buffer (pH 7.4) and prepared as a Formvar film on a 300 square mesh, nickel grid (EMS FF300-Ni). The TEM images were obtained using a JEOL JEM 2100FX TEM instrument (Jeol, Tokyo, Japan) equipped with a GATAN CCD Orius 200D camera (Gatan, Pleasanton, CA, USA) at an acceleration voltage of 200 KV.

4.5. Assessment of Biofilm Formation by Enterococcal Strains on Polystyrene Tissue Culture Plates (TCP) To assess the biofilm formation of E. faecalis 14 and its ∆bac derivative mutant strains to polystyrene plate, a semi quantitative method was used, as previously described [53]. Briefly, 100 µL of culture of each Enterococcus strain (108 CFU/mL), grown in GM17, were added to the wells of sterile 96-well microplates already filled with 100 µL of sterile GM17. The microplates were left for 15 min with gentle agitation before being incubated at 37◦C for 24 h. The cultures were then aspirated and the non-adherent cells were removed by two washes with phosphate-buffered saline (PBS) 10 mM, pH 7.2. Subsequently, 200 µL of 96% ethanol (Sigma–Aldrich, St Louis, MO, USA) were added to each well in order to fix the adherent cells. After 15 min of fixation, the wells were drained, dried and then stained with 0.1% (w/v) crystal violet (Biochem Chemopharma, Quebec, Canada) for 30 min. The stained cells were washed twice with 200 µL of PBS before extracting the dye with 200 µL of 96% ethanol. The number of cells was quantified using a microplate reader (ELX800. BioTek, Winooski, VT, USA) by Int. J. Mol. Sci. 2020, 21, 4653 10 of 13 measuring the absorbance (A) at 630 nm. According to the recommendations of Stepanovićet al. [21], these strains were classified into four categories. Taking Ac as the absorbance of the control (sterile GM17), the following interpretations were applied; A Ac: non-adherent (non-biofilm producer), 2Ac ≤ A > Ac: weakly adherent (weak biofilm producer), 4Ac A > 2Ac: moderately adherent (moderate ≥ ≥ biofilm producer), and strongly adherent (strong biofilm producer): A > 4Ac.

4.6. CCK-8 Cytotoxicity Assay Cytotoxicity of E. faecalis 14 wild-type and its ∆bac derivative mutant strains was assessed in vitro using the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technology, Kumamoto, Japan), for Caco-2 cells [54]. Caco-2 cells were seeded at a density of 6 104 cells/well in 96-well cell culture × plates and preincubated for 7 days at 37 ◦C in the presence of 5% CO2 in Dulbeco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Courtaboeuf, France) containing 4.5 g/L of glucose and supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), 10% of heat-inactivated fetal bovine serum (FBS) and 1% (v/v) non-essential amino acids. All these reagents were provided by PAN-Biotech GmbH (Aidenbach, Germany). Of note; the media were changed three times to maintain optimal conditions for Caco-2 cell growth. Overnight enterococcal cultures were prepared in DMEM without antibiotics and then applied to confluent Caco-2 cell monolayers at a ratio of MOI (Multiplicity of infection) 1:10 (Caco-2/E. faecalis 14 or ∆bac mutant strains). A control test was also performed with non-infected Caco-2 cells. After 24 h of incubation, the medium was removed, and cells were washed twice with PBS and then incubated with 150 µL of DMEM containing gentamicin (50 µg/mL) and 5% of CCK-8 reagent for 2 h at 37 ◦C. The relative viability (%) was then calculated based on absorbance at 450 nm using a microplate reader (Xenius SAFAS, Monaco, France). Results were expressed as percentage of proliferation compared to the viability of untreated cells (control).

4.7. Statistical Analysis Differences in absorbance values among samples were calculated using the Student t test. p values of less than 0.05 were considered to be significant. Data of bacterial growth, biofilm formation, and cytotoxicity experiments were expressed as a mean standard error calculated over three ± independent experiments. Analysis of statistical significance was performed by one-way ANOVA and the post-hoc Tukey Test (p < 0.05).

5. Conclusions This paper reports for the first time a comparative transcriptomics analysis of a bacteriocinogenic strain, namely E. faecalis 14 and its derivative strain, inactivated in genes coding for synthesis of its own bacteriocin EntDD14 ∆bac. The mutant strain ∆bac could express differentially at least 71 genes. Two out of 71 genes were downregulated and 69 out of 71 were upregulated. Downregulated genes were those coding for EntDD14 because these genes had been removed from this strain. Upregulated genes included those coding for proteins involved in cellular defense and virulence, probably as a substitutionary line of defense. These include a group that could be associated with PRCIs, probably involved in physiological flexibility and optimal adaptation. Other upregulated genes were associated with cell division, cell adhesion, and biofilm formation. Genes involved in the environmental resistance were markedly upregulated and a prominent metabolic reprogramming accompanied all these strategies. In addition to these transcriptomic observations, the ∆bac mutant possessed better growth, a high capacity to form biofilm on inert surfaces and a very weak level of cytotoxicity on Caco-2 cells. Collectively, these transcriptomic data suggest a new alternative virulence strategy in case of inability to synthesize bacteriocin.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/13/ 4653/s1, Table S1: Gene expression in Enterococcus faecalis 14 ∆bac mutant strain. Int. J. Mol. Sci. 2020, 21, 4653 11 of 13

Author Contributions: D.D., R.L. and A.L.-D. conceived the ideas, designed the experiments, discussed the data throughout the project and wrote this article. R.L. performed the experiments and A.L.-D. carried out the transcriptomic analysis. D.D., R.L. and A.L.-D. revised and approved the manuscript dissertation. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the CPER/FEDER grant awarded by la Région des Hauts-de-France (2016–2021). Acknowledgments: The authors are grateful to Nacim Barache, Faouzi Chahi and Rezak Mendil for their technical assistance in this work. The authors would like to thank Stephen W. Elson for his critical reading of the manuscript. Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

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