N-dodecanoyl-homoserine lactone influences the levels of thiol and proteins related to oxidation-reduction process in Salmonella

Quorum sensing is a cell-cell communication mechanism mediated by chemical signals that leads to differential gene expression in response to high population density. Salmonella is unable to synthesize the autoinducer-1 (AI-1), N-acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. This study aimed to evaluate the fatty acid and protein profiles of Salmonella enterica serovar Enteritidis PT4 578 throughout time of cultivation in the presence of AHL. The presence of N-dodecanoyl-homoserine lactone (C12-HSL) altered the fatty acid and protein profiles of Salmonella cultivated during 4, 6, 7, 12 and 36 h in anaerobic condition. The profiles of Salmonella Enteritidis at logarithmic phase of growth (4 h of cultivation), in the presence of C12-HSL, were similar to those of cells at late stationary phase (36 h). In addition, there was less variation in both protein and fatty acid profiles along growth, suggesting that this quorum sensing signal anticipated a stationary phase response. The presence of C12-HSL increased the abundance of thiol related proteins such as Tpx, Q7CR42, Q8ZP25, YfgD, AhpC, NfsB, YdhD and TrxA, as well as the levels of free cellular thiol after 6 h of cultivation, suggesting that these cells have greater potential to resist oxidative stress. Additionally, the LuxS protein which synthesizes the AI-2 signaling molecule was differentially abundant in the presence of C12-HSL. The NfsB protein had its abundance increased in the presence of C12-HSL at all evaluated times, which is a suggestion that the cells may be susceptible to the action of nitrofurans or that AHLs present some toxicity. Overall, the presence of C12-HSL altered important pathways related to oxidative stress and stationary phase response in Salmonella.

The cell-cell communication mechanism mediated by AI-1 in the Proteobacteria phylum is composed of a pair of proteins named LuxI (acyl homoserine lactone synthase) and LuxR (transcriptional factor) and its homologous proteins. In contrast, some Proteobacteria belonging to the Enterobacteriaceae family, such as Salmonella spp. and Escherichia coli, do not synthesize AHL since they lack a LuxI homologue [9,10,22]. However, a homologue of LuxR, known as SdiA, is present in these bacteria and allows the detection of AHLs synthesized by other microorganisms such as Aeromonas hydrophila and Yersinia enterocolitica, leading to gene regulation [23,24]. These AIs are able to enter and leave the cell by diffusion or through efflux pumps depending on the type of AHL [1,[25][26][27]. The AHL molecules bind to the Nterminal domain of the SdiA protein, altering the binding affinity of its C-terminal domain to the DNA and, consequently, regulating the expression of target genes [2,[28][29][30][31].
It has been shown that AHLs regulate the expression of the rck operon (resistant to complement killing), which codes for pefI, srgD, srgA, srgB, rck and srgC genes, and it is found in plasmids influencing virulence of Salmonella Typhimurium [9,10,32,33]. Campos-Galvão et al.
A global analysis was carried out on the influence of AHL on the abundance of proteins and the levels of extracellular organic acids of Salmonella Enteritidis [35]. It was shown that the abundance of proteins involved in translation (PheT), transport (PtsI), metabolic processes (TalB, PmgI, Eno and PykF) and response to stress (HtpG and Adi) increased while the abundance of other proteins related to translation (RplB, RplE, RpsB and Tsf), transport (OmpA, OmpC and OmpD) and metabolic processes (GapA) decreased at 7 h of cultivation in the presence of AI-1. Additionally, there were changes in the formate consumption. It was hypothesized that these observed changes are correlated with entry into the stationary phase of growth. In other organisms, the effect of AHLs in anticipating the stationary phase responses was confirmed by global analysis, such as the transcriptome of Pseudomonas aeruginosa [36] and Burkholderia thailandensis [37], as well as the metabolomes of Burkholderia glumae, Burkholderia pseudomallei and B. thailandensis [38].
Besides the global changes in intracellular metabolites described above, other phenotypes are altered when Salmonella is grown in the presence of AIs. For instance, Nesse et al. [39] showed that invasion of HEp-2 cells by Salmonella Typhimurium was increased in the presence of N-hexanoyl homoserine lactone (C6-HSL) and N-octanoyl homoserine lactone (C8-HSL) at 37˚C. The addition of C8-HSL in cells of S. enterica serovar Typhi containing plasmid pRST98, which harbors the virulence gene rck, increased adhesion to HeLa cells after 1 h at 37˚C in the presence of 5% CO 2 gas [40]. Biofilm formation by Salmonella Enteritidis PT4 578 in polystyrene surface was positively regulated by C12-HSL after 36 h in anaerobiosis, even though no growth changes were observed in planktonic cells [34,41]. Similarly, Bai and Rai [42] showed that biofilm formation on polystyrene was increased by Salmonella Typhimurium when cultivated with N-butyryl homoserine lactone (C4-HSL) e C6-HSL.
On the other hand, a cell free supernatant (CFS) rich in AHLs, AI-2 and other unknown compounds of Y. enterocolitica and Serratia proteamaculans altered growth of different phagetypes of Salmonella Enteritidis and Salmonella Typhimurium under aerobiosis [43]. Similarly, the CFS of P. aeruginosa containing AHLs and different metabolites decreased growth of nine serovars of S. enterica [44]. Conversely, the CFS of Hafnia alvei containing AHLs, as well as the addition of synthetic N-3-oxo-hexanoyl homoserine lactone (3-oxo-C6-HSL) to the growth medium in aerobiosis did not influence biofilm formation by Salmonella Typhimurium [45]. It is noteworthy that in these studies, the CFS of different bacteria contained metabolites other than AHLs which might have interfered in the detection of the AIs subtle effects in the cells.
According to Atkinson and Williams [29], the absence of an AHL synthase in Salmonella and E. coli can be related to ecological aspects, since the bacterium would avoid the information transfer to other microorganisms present in the medium and there would be no energy expenditure with the synthesis of these signaling molecules. However, these bacteria would be favored by the environmental information provided by others through the AHLs detection. Thus, what remains uncertain is the real advantage for Salmonella to use cell-cell communication mediated by AI-1. According to Di Cagno et al. [46], the global analyses of the proteome and transcriptome should help elucidate the influences of chemical signals on the cellular physiology. However, most studies on AHL signaling in Salmonella were targeted at specific genes, as previously mentioned [9,10,32,34], while overall changes in Salmonella physiology in the presence of AHLs is still largely unknown.
The aim of this work was to evaluate the altered physiological aspects of Salmonella, such as the cells fatty acid and protein profiles during growth in the presence of AHL. This is the first work that carefully examines the AHL effect throughout growth in anaerobic conditions and the first to show that levels of thiol and proteins related to the oxidation-reduction stress response are altered by a quorum sensing signal in Salmonella, suggesting that these cells may have a greater potential to resist oxidative stress.
bottles that were sealed with butyl rubber stoppers and then autoclaved (anaerobic TSB). Before each experiment, cells were cultivated in 20 mL of anaerobic TSB for 24 h at 37˚C. Then, 1.0 mL was transferred into 10 mL of anaerobic TSB and incubated at 37˚C. After 4 h of incubation, exponentially growing cells were harvested by centrifugation at 5000 g at 4˚C for 10 min (Sorvall, USA), washed with 0.85% saline, and the pellet resuspended in 0.85% saline. The inoculum was standardized to 0.1 of optical density at 600 nm (OD 600nm ) (approximately 10 7 CFU/mL) using a spectrophotometer (Thermo Fisher Scientific, Finland).

Preparation of HSL solution
The autoinducer-1, N-dodecanoyl-DL-homoserine lactone (C12-HSL; PubChem CID: 11565426; Fluka, Switzerland) was suspended in acetonitrile (PubChem CID: 6342; Merck, Germany) at a concentration of 10 mM and further diluted to a working solution of 10 μM in acetonitrile. Control experiment was performed using acetonitrile with final concentration in the media less than 1% (v/v) to avoid interference in the growth and response of Salmonella to C12-HSL [9].

Saponification, methylation and extraction of fatty acids.
An aliquot of 10 mL of the standardized inoculum was added into anaerobic bottles containing 90 mL of anaerobic TSB supplemented with 50 nM of C12-HSL or the equivalent volume of acetonitrile as control and then, incubated at 37˚C. After 4, 6, 7, 12 and 36 h of incubation, OD 600nm was determined. Concomitantly, 10 mL of the culture was centrifuged at 5000 g at 4˚C for 15 min (Sorvall, USA). The cells in the pellet were resuspended in 1 mL of sterilized distilled water, and once again, centrifuged at 5000 g at 4˚C for 10 min. The pellet was transferred to glass tubes free from fatty acids and afterwards, the fatty acids were saponified, methylated, and extracted by the procedure of the Sherlock 1 Analysis Manual (version 6.2; MIDI, USA) [48].

Analysis and identification of fatty acids.
The cellular fatty acid composition was determined by 7890A gas chromatograph equipped with flame ionization detector (Agilent Technologies, USA). Afterwards, the fatty acids were identified and quantified in the Sherlock 1 Microbial Identification System software by the procedure of the Sherlock 1 Analysis Manual (version 6.2; MIDI, USA) using the Calibration Standard 1 (Microbial ID Part # 1200-A) containing the straight chain C9:0 to C20:0 fatty acid methyl esters [48].

Statistical analysis.
Experiments were carried out in three biological replicates. The values of the triplicates were used for Principal Component Analysis (PCA) using RStudio software (version 1.0.143; USA). All data were subjected to analysis of variance (ANOVA) followed by Tukey's test using the Statistical Analysis System and Genetics Software 1 [49]. A p-value < 0.05 was considered to be statistically significant.

Extraction and quantification of proteins.
An aliquot of 10 mL of the standardized inoculum was added into anaerobic bottles containing 90 mL of anaerobic TSB supplemented with 50 nM of C12-HSL or the equivalent volume of acetonitrile as control and then, incubated at 37˚C. After 4, 6, 7, 12 and 36 h of incubation, OD 600nm was determined and 10 mL of the culture was centrifuged at 5000 g at 4˚C for 15 min (Sorvall, USA). The cells in the pellet were resuspended in 1 mL of sterilized distilled water, transferred to 1.5 mL microtubes and once again centrifuged at 9500 g at 4˚C for 30 min (Brikmann Instruments, Germany). The pellet was resuspended in 1 mL of Tris-HCl 50 mM, pH 8.0 added of 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT). Next, the mixture was kept in ice for 1 min, then 1 min in ultrasound bath (154 W, 37 KHz; Unique, Brazil) and this cycle was repeated five times. Posteriorly, five cycles of 1 min in ice, 1 min in vortex and 8 min in ultrasound bath were performed. The mixture was centrifuged at 9500 g at 4˚C for 30 min, the supernatant containing the intracellular proteins was transferred to 1.5 mL microtube and stored at −20˚C. The pellet was resuspended in 100 μL of ammonium bicarbonate 50 mM and 1 mL of 2:1 trifluoroethanol:chloroform (TFE:CHCl 3 ) was added. Then, the mixture was subjected again to five cycles on ice and ultrasound bath and five cycles on ice, vortex and ultrasound bath as previously described. The mixture was centrifuged at 9500 g at 4˚C for 4 min to obtain three phases. The upper phase, composed by proteins soluble in TFE, was transferred to 1.5 mL microtubes and was dried in SpeedVac (Genevac, England). The supernatant with intracellular proteins was transferred to microtube containing the proteins soluble in TFE. The protein extract was precipitated with 10% (w/v) trichloroacetic acid (TCA) and kept on ice for 30 min and after that, the material was centrifuged at 9500 g at 4˚C for 10 min. The supernatant was discarded and the precipitate washed three times with cold acetone. After evaporation of the residual acetone at room temperature, the precipitate was resuspended in 400 μL of ammonium bicarbonate 50 mM. Proteins were quantified using Coomassie blue dye [50] and the protein extracts were stored at -20˚C.

In-solution protein digestion.
The trypsin digestion of proteins in solution was performed according to Villen and Gygi [51], with modifications. An aliquot of the extract containing 10 μg of protein was transferred to 1.5 mL microtube and the final volume was adjusted to 150 μL with ammonium bicarbonate 50 mM and 150 μL of urea 8 M were added. The disulfide bonds of the proteins were reduced by 5 mM DTT for 25 min at 56˚C and the protein mixture was cooled to room temperature. Next, the alkylation was carried out by adding 14 mM iodoacetamide and followed by incubation for 30 min at room temperature in the dark. The free iodoacetamide was quenched by adding 5 mM DTT with further incubated for 15 min at room temperature in the dark. The urea concentration in the protein mixture was reduced to 1.6 M by diluting it in 1:5 in ammonium bicarbonate 50 mM with the addition of 1 mM calcium chloride. An aliquot containing 20 ng of trypsin (Sequencing grade modified trypsin; Promega, USA) in ammonium bicarbonate 50 mM was added to a final ratio of 1:50 of trypsin:protein and it was incubated for 16 h at 37˚C. After the incubation, the solution was cooled to room temperature and the enzymatic reaction was quenched with 1% (v/v) trifluoroacetic acid (TFA) until pH 2.0. After centrifugation at 2500 g for 10 min at room temperature, the supernatant containing the peptides was collected.
2.5.3. Peptide desalting. The supernatant containing the peptides was desalted according to Rappsilber et al. [52], with modifications. Two membranes of octadecyl (C18; 3M EMPORE, USA) were packed in each stage tip to load 10 μg of digested proteins. The stage tip was conditioned with 100 μL of 100% (v/v) methanol and was equilibrated with 100 μL 0.1% (v/v) of formic acid. After that, the supernatant containing the peptides was loaded two times onto the stage tip and this was washed 10 times with 100 μL of 0.1% (v/v) of formic acid. The peptides were eluted with 200 μL of 80% (v/v) acetonitrile containing 0.1% (v/v) of formic acid. In each previous step, the stage tip was centrifuged at 400 g for 2 min (Eppendorf, Germany). The eluate was dried in SpeedVac (Thermo Fisher Scientific, Finland), resuspended in 22.5 μL of 0.1% (v/v) formic acid and stored at -20˚C.

Mass spectrometric analysis.
The solution containing the peptides was centrifuged at 9000 g for 5 min and 15 μL of the supernatant was transferred to vials. An aliquot of 4.5 μL of peptides, equivalent to 2 μg of protein, was separated by C18 (100 mm x 100 mm) RP-nanoUPLC (nanoAcquity; Waters, USA) coupled to a Q-Tof Premier mass spectrometer (Waters, USA) with nanoelectrospray source at a flow rate of 0.6 μL.min -1 . The gradient was 2-90% (v/v) acetonitrile in 0.1% (v/v) formic acid over 45 min. The nanoelectrospray voltage was set to 3.0 kV, a cone voltage of 50 V and the source temperature was 80˚C. The instrument was operated in the 'top three' mode, in which one MS spectrum is acquired followed by MS/ MS of the top three most-intense peaks detected. After MS/MS fragmentation, the ion was placed on exclusion list for 60 s.

Identification and quantification of proteins.
The spectra were acquired using software MassLynx (version 4.1; Waters) and the .raw data files were converted to a peak list format .mgf without summing the scans by the Mascot Distiller software (version 2.3.2.0; Matrix Science, United Kingdom) and searched against the knowledgebase UniProtKB using the Mascot software (version 2.4.0; Matrix Science, United Kingdom). For the search, the following parameters were considered: taxonomy Salmonella and all entries (separately), monoisotopic mass, trypsin, allowing up to one missed cleavage site, peptide charge +2, +3 and +4, fixed modification for carbamidomethylation of cysteine residues and variable modification for oxidation of methionine residues, peptide and MS/MS tolerance equal to 0.1 Da [53][54][55], ESI-QUA-TOF for instrument. The proteins identifications by Mascot software were validated and the proteins were quantified by Scaffold software (version 4.7.2; Proteome Software, USA). For the protein validation, peptide and protein identifications were accepted if they could be established at higher than 90% probability as specified by the Peptide Prophet algorithm [56] and by the Protein Prophet algorithm [57], respectively. In addition, the proteins should contain at least one identified peptide. The false discovery rates (FDR) for identification of proteins and peptides using decoy method by Scaffold software should to be less than or equal to 1% (FDR 1%) [54,58,59]. For the protein quantitation, the quantitative value of total ion current (TIC) of each protein was normalized by sum total of TIC. For the unidentified proteins, a TIC value of 0.05 was adopted (cut off of method sensibility).
2.5.6. Statistical analysis. Experiments were carried out in three biological replicates. The logarithms of normalized TIC values of the triplicates were used for PCA as well as the logarithm of normalized mean of TIC values of the triplicates of each protein which were used to construct the heatmap and dendrogram using RStudio software. The statistical difference of normalized TIC values among the samples were calculated by T-test with correction for multiple hypotheses by FDR using RStudio software. The fold changed was calculated as the ratio of the normalized mean of TIC values of the treatment with C12-HSL by the control and the result was shown as logarithm in base two (Log 2 FC). The proteins with p-value less than 0.05 (p-value < 0.05) or negative logarithm of p-value more than 1.301 (-Log 10 p > 1.301) and fold changed less than 0.667-fold or more than 1.500-fold or Log 2 FC less than -0.585 or more than 0.585 (Log 2 FC < -0.585 or > 0.585) were considered differentially abundant proteins [60]. Moreover, when the protein was not detected in one of the treatments, the p-value was not considered and the fold-changed was calculated by adopting a TIC value of 0.05 (cut off of method sensibility) for the sample in which the protein was not identified, being this protein also considered differentially abundant.
2.5.7. Bioinformatics analysis. The Gene Ontology (GO) annotations of the process and function of proteins were acquired with the tool QuickGO, implemented by the European Bioinformatics Institute (http://www.ebi.ac.uk/QuickGO/). Then, the Protein-Protein Interaction (PPI) network was generated for some proteins of Salmonella Typhimurium LT2 using the STRING database version 10.5 (http://string-db.org/, [61,62]). The confidence levels of the PPI were considered in relation to the average local clustering coefficient: interactions at low confidence or better (coefficient ! 0.150), interactions at medium confidence or better (coefficient ! 0.400), interactions at high confidence or better (coefficient ! 0.700) and interactions at highest confidence (coefficient ! 0.900), available at http://string-db.org/ [61,62].

Quantification of free cellular thiol
2.6.1. Extraction of free cellular thiol. The pellet obtained as described in item 2.5.1. was resuspended in 250 μL of sterilized distilled water. Next, the mixture was kept on ice for 1 min, 1 min mixed by vortex for 1 min and treated with ultrasound (400 W, 20 KHz; Sonics & Materials Inc., USA) for 30 s; and this cycle was repeated five times. The mixture was then centrifuged at 9500 g at 4˚C for 15 min and the supernatant containing the free cellular thiol was used immediately.
2.6.2. Quantification of free cellular thiol. The quantification of free cellular thiol was performed according to Ellman [63] and Riddles et al. [64], with modifications. For each 25 μL of the supernatant or standard, 5 μL of 0.4% (w/v) 5,5 0 -dithiobis(2-nitrobenzoic acid) (DTNB or Ellman's reagent; Sigma, USA) in buffer sodium phosphate 0.1 M pH 8.0 containing 1mM EDTA and 250 μL of the buffer sodium phosphate were added in microplate. The microplate was incubated at room temperature for 15 min and the absorbance at 412 nm measured by using a spectrophotometer (Thermo Fisher Scientific, Finland). The free cellular thiol was quantified by using cysteine hydrochloride monohydrate (Sigma, USA) as standard at concentrations from 0.0 to 1.5 mM. The obtained equation was as follows: absorbance = (0.9421 x concentration) + 0.0432, with R 2 = 0.9936. Next, the quantification of free cellular thiol was normalized by OD 600nm .

Statistical analysis.
Experiments were carried out in four biological replicates. The statistical analysis was performed according to item 2.4.3, except that the PCA was not performed.

HSL alters the fatty acid profile of Salmonella throughout time
Analysis of the fatty acid profile of Salmonella cells, both in the absence and presence of C12-HSL, showed alterations in the composition at different time points. The largest change appears to be at the 4 h time point with four fatty acids presenting altered abundance at this time of cultivation. Of these fatty acids, 17:0 cyclo ɷ7c and 19:0 cyclo ɷ8c had their abundance decreased in the presence of C12-HSL, while two unresolved mixtures of 16:1 ɷ6c/16:1 ɷ7c and 18:1 ɷ6c/18:1 ɷ7c had their abundance increased (Table 1 and S1 Table).
The concentration of the fatty acids 19:00 and 18:1 ɷ7c 11-methyl, as well as, an unresolved mixture of the 18:0 anteiso/18:2 ɷ6,9c did not change throughout time of cultivation in the presence of C12-HSL, but changed in the control (Table 1). Additionally, after 6 h of cultivation, there was no difference in the concentration of the fatty acids 17:0 cyclo ɷ7c, 19:0 cyclo ɷ8c, 17:1 ɷ7c and 18:1 2OH, as well as, an unresolved mixture of the 19:1 ɷ6c/19:1 ɷ7c/19:0 cyclo in the treatment with C12-HSL (Table 1). Interestingly, the maintenance of the cyclopropane fatty acids 17:0 cyclo ɷ7c and 19:0 cyclo ɷ8c suggests that the cells are prepared for a possible stress condition. This hypothesis is strengthened by the fact that the cyclopropane fatty acids 17:0 cyclo and 19:0 cyclo are formed by transmethylation of cis monounsaturated fatty acids 16:1 ɷ7c and 18:1 ɷ7c, respectively, when the cell enters into stationary phase [65]. The level of cyclopropane fatty acids increases during the stationary phase due to the increased expression of the synthase that produces this fatty acid and is mediated by sigma S factor (σ S ) [66,67]. According to several studies, this modification may help reduce the impact of environmental stresses such as starvation, oxidation, acid and heavy metal addition, organic compound toxicity, increased temperature and pressure because it increases the stability and fluidity of the membrane, and at the same time reduces its permeability against toxic compounds [66][67][68][69][70][71][72][73]. An example of the effect of fatty acid modifications on bacterial cell membrane related to a stress condition was presented by Guckert et al. [68] who observed an increased proportion of cyclopropane fatty acids during nutrient deprivation in Vibrio cholerae.
The PCA was used in order to evaluate the variations among triplicates and to the understanding of the global difference of levels and types of fatty acids among samples stimulated by C12-HSL (Fig 1). The distance of the dots in the PCA figure (Fig 1) is proportional to the difference between the fatty acid composition of the experimental groups. Based on this information, there is a high dispersion between the global profile of fatty acids identified in the absence and presence of C12-HSL, which is represented by the distance between control and treatment at the same time. However, the graphic shows that the distance between control and treatment is higher at the 4 h and 6 h time points, with the dispersion tending to reduce over time as both groups tend to cluster more closely. On the other hand, within the treatment with C12-HSL, the identified fatty acids tended to be less dispersed throughout growth. Thus, the fatty acid profile of the cells in the logarithmic (4 h) and stationary (36 h) phases of growth were more similar to each other in the presence of AI-1 than in its absence.
The data also indicate that the fatty acid composition of Salmonella cells growing with C12-HSL for 4 h is similar to cells cultured in the absence of this autoinducer for 7 h (Fig 1). At this time of cultivation (7 h), the culture is already in the stationary phase of growth and the maintenance of the cyclopropane fatty acids suggest that the cells are prepared to support stressful conditions, as previously discussed. These results suggest that AHL signaling in Salmonella induces a profile of fatty acids which is typical to the stationary phase of growth, even in logarithmic phase of growth when the cell density was relatively low. According to Schuster et al. [74], this early preparation of the cells has a high fitness cost, but leads to more resistance should a stress condition arrive.

HSL alters the protein profile of Salmonella throughout time
The validation of proteins identifications showed percentages of FDR less than or equal to 0.8% for proteins (FDR 0.8%) and less than or equal to 0.14% for peptides (FDR 0.14%) ( Table 2). These FDR values were lower than those accepted by Liu et al. [58] and Tran et al. [59], which admitted an FDR lower than 1% (FDR < 1.0%) for proteins and peptides of Salmonella Typhimurium.
The protein composition at different time points is shown on Fig 2 and S2 Table. The PCA of the proteomic data showed that the variation among replicates was much lower than the variation among times (Fig 2A). There was a similar dispersion of the protein profile such as   that observed with the fatty acid analysis, with a higher dispersion of protein and fatty acid profiles in the absence of C12-HSL (Fig 2A). The identified proteins in cells treated with C12-HSL tended to be less dispersed throughout growth than in the absence of the quorum sensing molecule (Fig 2A). Cluster analysis by agglomerative hierarchical methods of the proteins at the different time points was prepared generating a heatmap and a dendrogram that separated the proteins and the samples into two major clades, respectively (Fig 2B and 2C, respectively). Based on the color intensity variability in each column of the heatmap, the protein profile of Salmonella in the absence of C12-HSL at the 4 h time point presented more variations and, this was confirmed by the dendrogram (Fig 2C) that discriminates this sample from the others as a single branch in the tree. Interestingly, the PCA, the heatmap and the dendrogram analyses together showed that the addition of C12-HSL at the beginning of Salmonella cultivation resulted in smaller protein profile variations throughout the time of cultivation when compared with the control. Thus, the protein profile of the cells in the logarithmic (4 h) and stationary (36 h) phases of growth were more similar to each other in the presence of AI-1 than in its absence as well as the fatty acid profile previously shown. Based on these similarities, these results suggest that AI-1 can anticipate a stationary phase response. Moreover, these results corroborate our previous observation, in which the differentially abundant proteins and organic acids of Salmonella cultivated for 7 h in the presence of C12-HSL correlated with entry into the stationary phase of growth, mainly in relation to nitrogen and amino acid starvation as well as acid stress [35]. Schuster et al. [36] showed that genes with expression influenced by the growth phase in P. aeruginosa were repressed by quorum sensing during the late logarithmic and stationary phases. Goo et al. [38] showed that quorum sensing anticipates and influences the survival to the stress of the stationary phase of B. glumae, B. pseudomallei and B. thailandensis. The survival of these bacteria requires the activation of cellular enzymes through the quorum sensing mechanism for production of excreted oxalate, which serves to counteract ammonia-mediated alkaline toxicity during stationary phase [38]. In addition, the genes involved in transcription and translation of B. pseudomallei in stationary phase were co-regulated by RpoS protein (RNA polymerase sigma factor) and quorum sensing [75].
The statistical analyses of the identified proteins are shown in Table 3 and S3 Table. The results showed that at 4 and 6 h of incubation in presence of C12-HSL, a higher percentage of differentially abundant proteins was observed in comparison with other times of cultivation (Table 4). In addition, more proteins had their abundance decreased at 4 h in the presence of AHL (50.0%), while an opposite trend was observed at 6 h (54.8%). The greatest number of differentially abundant proteins at the initial times can be due to the addition of AI-1 at the beginning of the cultivation. Growth and cell size are altered by intrinsic and extrinsic factors during the adaptation phase to an environmental condition and, consequently, alter the cellular components resulting in a greater fluctuation in protein abundance at initial times of cultivation [76,77]. In AHL-producing bacteria, such as B. thailandensis and P. aeruginosa, many genes were regulated at the end of the logarithmic and beginning of the stationary phase of growth due to the accumulation of signaling molecules in the medium [36, 37, 78]. Schuster et al. [36] showed that genes involved in carbohydrate utilization or nutrient transport were the most repressed by quorum sensing during the late logarithmic and stationary phases of P. aeruginosa.
The differentially abundant proteins were grouped in order to perform an analysis of enrichment of biological processes based on GO annotations, as shown in Fig 3. The proteins related to translation, transcription, oxidation-reduction, metabolic, protein folding, transport processes as well as unclassified proteins, had their abundance affected by C12-HSL in all the studied time points.       Quorum sensing and redox process in Salmonella An important identified protein is LuxS (S-ribosylhomocysteine lyase), which produces the autoinducer-2 signaling molecule (Table 3 and Fig 3). In our experimental conditions, this protein was identified only in the presence of 50 nM of C12-HSL at 6 h of cultivation, that is, at the end of the logarithmic phase of growth of Salmonella. This result suggests that there  Quorum sensing and redox process in Salmonella could be a cross response between quorum sensing mechanisms mediated by AI-1 and AI-2 in Salmonella depending on the growth phase. Interactions among the different mechanisms of quorum sensing present in P. aeruginosa have been described leading to a hierarchical activation of these mechanisms [79][80][81] and also to the synthesis of inductive and inhibitory molecules [82]. The existence of multiple arrangements between the different mechanisms of quorum sensing might play an important role in processing of environmental cues and thus, dictating necessary and robust collective responses [83]. Additional studies should be performed in Salmonella to confirm this possible connection. Among the identified proteins, a greater number was involved in transcription process showed a variation of their abundance in the presence of C12-HSL (Fig 3). Since transcription is an essential step in gene expression and the transcriptional regulation determines the molecular machinery for developmental plasticity, homeostasis and adaptation [84], a network of interaction between the proteins related to the transcription process and "regulation of transcription, DNA-templated" was generated (Fig 4A). The PPI network showed an average local clustering coefficient of 0.636 and a p-value of <1.06e-10 for enrichment, indicating that the interactions showed at medium confidence or better and, the proteins have more interactions among themselves than what would have been expected for a random set of proteins of similar size. This result also indicates that these proteins are, at least, partially biologically connected as a group [61,62]. The RpoA (DNA-directed RNA polymerase subunit alpha) and Hns (DNA-binding protein H-NS) proteins were the central nodes of two networks that are connected ( Fig 4A). RpoA had its abundance increased at 6 h of cultivation of Salmonella Enteritidis in the presence of C12-HSL (Fig 4B). Soni et al. [85] showed that the abundance of this protein decreased in late logarithmic phase of growth of Salmonella Typhimurium in the presence of AI-2. In addition, these proteins were identified at all the time points evaluated in this study (Fig 4B). Thus, the differential abundance of proteins related to transcription regulation may be responsible for the differences observed in the abundance of other proteins between control and treatment with C12-HSL and throughout the time of cultivation of Salmonella (Figs 3 and 4B).

Levels of thiol and proteins related to the oxidation-reduction process are altered by HSL in Salmonella
The proteins related to the oxidation-reduction process had their abundance affected by the presence of C12-HSL at all times of cultivation (Fig 3). At 4 h of cultivation with this signaling molecule, a greater number of these proteins had their abundance decreased in comparison to the control (Fig 3). Conversely, their abundance was increased at 6 h of cultivation. This might suggest that cells cultivated during this period in the presence of C12-HSL have greater potential to resist oxidative stress than cells cultivated in the absence of this molecule. The proteins related to the oxidative process can be considered crucial to maintenance of the cellular redox balance, as well as to anticipate resistance to a possible oxidative stress due to excessive production of reactive oxygen/nitrogen species (ROS/RNS) [86][87][88].
In other bacteria, quorum sensing has been associated to oxidative stress response. For instance, in P. aeruginosa, the expression of katA (catalase) and sodA (superoxide dismutase) genes and, concomitantly, the activities of the catalase and superoxide dismutase enzymes were up-regulated by quorum sensing [89]. In addition, Garcia-Contreras et al. [90] showed that resistance of P. aeruginosa to oxidative stress caused by the addition of hydrogen peroxide (H 2 O 2 ) was enhanced due to quorum sensing, increasing the production of catalase and NADPH dehydrogenases. In the study herein, the SodC1 (Superoxide dismutase [Cu-Zn] 1) also had its abundance increased at 4 h of incubation (Log 2 FC = 11.567), while SodB  Table 3). The cell-cell communication system via BpsIR (homologous to LuxIR) and N-octanoyl-homoserine lactone (C8-HSL) also increased resistance to oxidative stress of B. pseudomallei, as well as the expression of the dpsA gene (DNAbinding protein from starved cells) [91]. The Dps is a non-specific DNA-binding protein involved in resistance to oxidative stress and it is an abundant protein in stationary phase of growth in E. coli [92,93].
A network of interaction among the proteins of the oxidation-reduction process was generated ( Fig 5A). The PPI network showed an average local clustering coefficient of 0.609 and a pvalue of <1e-16 for enrichment. These results indicate that the interactions showed at medium confidence or better and, the proteins have more interactions among themselves than for a random set of proteins of similar size and also that these proteins are at least partially biologically connected as a group [61,62].
The predicted network is an efficient tool to identify potential interactions between a large number of proteins identified by proteomics providing a biological meaning for the data. Furthermore, the centrality of proteins in this network is generally associated to the importance of this element to the biological process associated to the network [94,95]. Interestingly, the TrxA protein (Thioredoxin 1) stands out as being a central node by interacting with most of the proteins used to generate the network (Fig 5A). In these experiments, this protein had its increased abundance at 7 h of cultivation in the presence of C12-HSL, but its abundance was lower at 12 h (Fig 5B). This protein is known to be essential to activate gene transcription of the pathogenicity island 2 (SPI-2) of Salmonella Typhimurium and consequently, for resistance during mice infection [96][97][98].
Thioredoxin is an oxidoreductase that participates in redox reactions by oxidation of its thiol active-sites which are then reduced by NADPH. It also exerts control over the activity of target proteins via reversible thiol-disulfide exchange reactions by the thioredoxin and glutaredoxin systems [88,[99][100][101][102]. This protein also has a regulatory mechanism independent of thiol redox activity, which thioredoxin interacts with other proteins and forms a functional complex [100]. Thiol, also known as mercaptan or sulfhydryl,-SH side chain of cysteine is susceptible to reactions with ROS or RNS species, giving rise to a range of post-translational oxidative modifications by thiol proteins, including reversible (intra-protein disulfides, interprotein disulfides, S-sulfenation, S-nitrosation, S-thiolation, S-sulfhydration, S-sulfenamidation) and non-reversible hyper-oxidized (S-sulfination, S-sulfonation) redox states. In addition, in some cases it can alter the structure and activity of proteins that contain cysteine residues [87,88,103,104].
From nine proteins that were identified at all cultivation times and used for the generation of the network (Fig 5A), eight are or have some relation to thiol proteins such as: Tpx (Probable thiol peroxidase), Q7CR42 (Putative thiol-alkyl hydroperoxide reductase), Q8ZP25 (Putative thiol-disulfide isomerase and thioredoxin), YfgD (Arsenate reductase), AhpC (Alkyl hydroperoxide reductase subunit C), NfsB (Oxygen-insensitive NAD(P)H nitroreductase), YdhD (Glutaredoxin) and TrxA (Thioredoxin 1) proteins. The thiol proteins: Tpx, Q7CR42, Q8ZP25 and YfgD decreased in abundance at 4 h of cultivation of Salmonella Enteritidis in the presence of the 50 nM C12-HSL (Fig 5B). On the other hand, at 6 h of cultivation, the Tpx, Q7CR42, Q8ZP25, YfgD, AhpC, NfsB and YdhD proteins increased in abundance, as well as Q8ZP25, NfsB and TrxA proteins at 7 h of cultivation (Fig 5B). In addition, more thiol proteins had their abundances altered at 4, 6 and 7 h of culture, which refer to the logarithmic phase up to the early stationary phase of growth compared to the times of 12 and 36 h where the cells were in stationary phase for a long time (Fig 5B). The quantification of free cellular thiol showed a correlation with the abundance of thiol proteins at each time and treatment (Fig 5B and 5C). At 4 h of cultivation, a lower level of thiol was observed in the treatment with the quorum sensing molecule as well as a lower abundance of the thiol proteins in comparison to the control. Subsequently, at 6 and 7 h higher levels of thiol were observed in cells cultivated in presence of C12-HSL (Fig 5C and S4 Table). Then, at 12 and 36 h no differences in the levels of thiol were observed, correlating with the number of differentially abundant thiol proteins. In addition, for the same treatment throughout the time of cultivation of Salmonella, the levels of thiol increased up to 7 h of cultivation in the presence of C12-HSL and decreased after this time (Fig 5C). On the other hand, in the absence of this AHL, the level of thiol varied throughout time without a trend (Fig 5C). These results showed that quorum sensing alters not only the abundance of thiol proteins but also the levels of thiol, suggesting that resistance to possible oxidative stress can be mediated by the signaling molecule. This is the first time that the relationship between thiol proteins and levels of free cellular thiol with quorum sensing is reported.
Variations in the abundance of thiol proteins and levels of free cellular thiol due to the growth phase of Salmonella and the presence of acyl homoserine lactone can be related to changes in the structure of the SdiA protein (LuxR homologue) which could alter its ability to bind DNA and, consequently, activate transcription. On the other hand, the thiol proteins and thiol could prevent structural alterations of the SdiA protein caused by ROS/RNS. This rationale is possible because the cysteine residues (C) and their respective positions (C45, C122, C142, and C232) present in the SdiA protein of Salmonella Enteritidis PT4 578 could be susceptible to oxidative stress [105]. The C232 is the main conserved residue among LuxR family proteins [31] and it is involved in the interaction between the Ligand-binding domain (LBD) with DNA-binding domain (DBD) and DBD-DBD [30].
Kafle et al. [106] evaluated all cysteine residues of the LasR protein, a LuxR homologue, of P. aeruginosa to infer their redox sensitivity and to probe the connection between stress response and the activity of that protein. The C79 residue is important for ligand recognition and folding of this domain which further potentiates DNA binding, but it does not seem to be sensitive to oxidative stress when bound to its native ligand. The C201 and C203 residues in the DBD form a disulfide bond when treated with hydrogen peroxide, and this bond seems to disrupt the DNA binding activity of the transcription factor. Mutagenesis of either of these cysteines leads to expression of a protein that no longer binds DNA. Thus, these authors provided a possible mechanism for oxidative stress response by the cysteine residues of the LasR protein in P. aeruginosa and indicated that multiple cysteines within the protein can be useful targets for disabling its activity.
The presence of C12-HSL increased the abundance of thiol proteins, such as oxidoreductases, which can change the structure of this AHL and inactivate it. Some oxidoreductases synthesized by Rhodococcus erythropolis and Bacillus megaterium, as well as by eukaryotic cells, are able to inactivate AHLs by oxidation or reduction of their acyl side chain [107][108][109][110]. In fact, this is one of the known mechanisms of quorum quenching [107].
Finally, the NfsB protein (also named NfnB or NfsI) was the only protein that had its abundance increased at all cultivation times in the presence of C12-HSL compared to the control treatment in Salmonella (Fig 5B, indicated with a black arrow at each time point). This result suggests that Salmonella cultivated in the presence of this quorum sensing molecule can be susceptible to the action of nitrofurans or that C12-HSL has a certain toxicity or mutagenicity activity to the cell. The NfsB protein is a flavin mononucleotide-containing flavoprotein that can use either NADH or NADPH as a source of reducing power in order to reduce the nitro moiety of nitrofurans, yielding biologically inactive end products. This process occurs through a sequence of intermediates, including nitroso and hydroxylamine states, which are assumed to be responsible for toxicity [111,112]. E. coli and Salmonella Typhimurium containing the nfsA and nfsB genes are more sensitive to nitrofurans, which are widely used as antimicrobial agents [113,114]. Carroll et al. [115] also showed that the introduction of plasmids carrying the nfsA and nfsB genes into Salmonella Typhimurium increased sensibility to nitrofuran compounds with mutagenic potential.
Toxicity of AHLs has already been proven in many studies, especially those performed with eukaryotic cells. For instance, Gomi et al. [116] showed that 10 μg/mL of C12-HSL derived from Chromobacterium violaceum induced the production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) by mouse RAW264.7 cells and IL-8 by human THP-1 cells, while C4, C6, C7, C8, C10 and C14-HSL had no effect. The C12-HSL and C12-oxo-HSL also decreased the levels of putrescine in human epidermal cells (HaCat) and, consequently, decreased the rate of cell proliferation [117]. John et al. [118] showed that N-3-oxo-tetradecanoyl-homoserine lactone (3-oxo-C14-HSL) produced by Acinetobacter baumannii had a dosedependent cytotoxic effect on human cervical cancer cells (HeLa), adenocarcinoma human alveolar basal epithelial cells (A549), Dukes' type C colorectal adenocarcinoma cells (HCT15) and Dukes' type B colorectal adenocarcinoma cells (SW480), with induction of apoptosis and reduced viability and proliferation of these cells in the presence of AHL. In addition, these authors showed that 3-oxo-C14-HSL was able to decrease the growth of Staphylococcus aureus.
It is possible that the NfsB protein can be used as a biomarker for the presence of the AI-1 in Salmonella, due to its greater abundance in the presence of this molecule in all the evaluated times. It is noteworthy that nitroreductases homologous to NfsA and NfsB are found in many members of the Enterobacteriaceae family [119].

Conclusions
The fatty acid and protein profiles of Salmonella Enteritidis PT4 578 in logarithmic phase of growth at 4 h of cultivation in the presence of C12-HSL were similar to the profiles of cells in late stationary phase at 36 h, suggesting that quorum sensing signal anticipates a stationary phase response (Fig 6). Overall, the fatty acid and protein profiles varied less along the bacterium growth in the presence of AI-1. In addition, the presence of this signaling molecule increased the abundance of thiol proteins and the levels of free cellular thiol after 6 h of cultivation suggesting that the cells may be prepared for a possible oxidative stress (Fig 6). This increase may lead to modifications in the structure of the AHL or SdiA protein which, consequently, could alter its binding affinities to the DNA. More studies are needed in order to confirm this hypothesis.
There was an increased abundance of LuxS protein in presence of C12-HSL which suggests a cross response between the quorum sensing mechanisms mediated by AI-1 and AI-2 in Salmonella (Fig 6), while the increased abundance of NfsB protein in this condition suggests that the cells can either be susceptible to the action of nitrofurans or this signaling molecule has a certain toxicity or mutagenicity to the cell (Fig 6). Further studies are needed in order to confirm the hypotheses generated in this work.