Unravelling the Molecular Mechanisms Underlying the Protective Effect of Lactate on the High-Pressure Resistance of Listeria monocytogenes

Formulations with lactate as an antimicrobial and high-pressure processing (HPP) as a lethal treatment are combined strategies used to control L. monocytogenes in cooked meat products. Previous studies have shown that when HPP is applied in products with lactate, the inactivation of L. monocytogenes is lower than that without lactate. The purpose of the present work was to identify the molecular mechanisms underlying the piezo-protection effect of lactate. Two L. monocytogenes strains (CTC1034 and EGDe) were independently inoculated in a cooked ham model medium without and with 2.8% potassium lactate. Samples were pressurized at 400 MPa for 10 min at 10 °C. Samples were subjected to RNA extraction, and a shotgun transcriptome sequencing was performed. The short exposure of L. monocytogenes cells to lactate through its inoculation in a cooked ham model with lactate 1h before HPP promoted a shift in the pathogen’s central metabolism, favoring the metabolism of propanediol and ethanolamine together with the synthesis of the B12 cofactor. Moreover, the results suggest an activated methyl cycle that would promote modifications in membrane properties resulting in an enhanced resistance of the pathogen to HPP. This study provides insights on the mechanisms developed by L. monocytogenes in response to lactate and/or HPP and sheds light on the understanding of the piezo-protective effect of lactate.


Introduction
Listeria monocytogenes is a facultative anaerobic Gram-positive pathogen that can cause listeriosis, with several outbreaks being associated with ready-to-eat (RTE) products. The risk assessments developed so far indicate that within the RTE meat products, cooked meat products have to be considered of high risk due to the exposure to recontamination with L. monocytogenes during the preparation of convenient formats (i.e., sliced/diced and packaged) and due to the potential of L. monocytogenes to grow during the refrigerated storage thanks to its psychrotrophic nature [1].
Differences in food safety microbiological criteria regarding L. monocytogenes are found between countries, setting from a maximum of 100 CFU/g of L. monocytogenes during the shelf-life of the product in EU [2] to the zero-tolerance policy (not detected in 25 g) in USA [3]. In this regard, control measures can be implemented by food manufacturers to comply with the legislation by minimizing the prevalence of the pathogen as well as by limiting its growth in contaminated products.
Among all the available control strategies, high pressure processing (HPP) is an emergent non-thermal technology widely applied in the meat industry. HPP is often used phase. After incubation, cultures were preserved frozen at −80 • C supplemented with 20% of glycerol until used [15].

Preparation of the Samples and HPP
For each biological replicate, cultures of L. monocytogenes strains CTC1034 and EGDe were thawed at ambient temperature and centrifuged at 8240× g for 7 min at 12 • C. Supernatants were discarded and cell pellets were resuspended in the same volume of CHMM without or with 2.8% of lactate. Cultures were distributed in 4 × 10 cm PA/PE pouches (oxygen permeability of 50 cm 3 /m 2 /24 h and a low water vapor permeability of 2.8 g/m 2 /24 h; Sistemvac, Estudi Graf S.A., Girona, Spain), which were closed by thermosealing. Cultures were kept for 1 h at 10 • C to allow the adaptation of L. monocytogenes cells in CHMM medium without and with 2.8% of lactate. Half of the samples were subsequently pressurized at 400 MPa for 10 min using an industrial HPP equipment (Wave 6000; Hiperbaric, Burgos, Spain). The come-up time was 2.50 min and the pressure release time was almost immediate (<2 s). The pressurization fluid was water and the initial temperature was set at 10 • C. After pressurization, samples were kept for 30 min at 10 • C before L. monocytogenes enumeration and RNA extraction. Non-pressurized samples were kept at 10 • C until analysis together with the HPP samples.

L. monocytogenes Enumeration and Data Analysis
For each treatment and biological replicate, L. monocytogenes concentration was determined by plate colony count method from the appropriate tenfold serial dilution prepared in 0.1% Bacto Peptone (Difco Laboratories, Detroit, MI, USA) with 0.85% NaCl. Samples were spread on CHROMagar TM Listeria (CHROMagar, Paris, France) and incubated at 37 • C for 48 h according to the manufacturer instructions. Chromogenic media for L. monocytogenes are known to be able to recover high pressure injured L. monocytogenes [16,17]. In any case, plates were further checked after additional 24-48 h to make sure that sub-lethally injured cells had time to recover and form colonies and, thus, minimize the overestimation of the lethal effect of HPP [10,11]. L. monocytogenes counts were Log transformed, and the inactivation value in terms of Log reduction was calculated by subtracting from the counts found in non-pressurized cultures (Log N 0 ) those of the pressurized cultures (Log N), i.e., LogN 0 − LogN = Log N 0 /N, both in the control and 2.8%-lactate lots.

Nucleic Acid Extraction and Sequencing
DNA of the samples prepared according to Section 2.3 was extracted from L. monocytogenes strain CTC1034 by using 1 mL of an overnight culture of BHI centrifuged at 14,000× g for 10 min. The pellet was then used for DNA extraction according to the protocol described in Cocolin et al. [18]. DNA was quantified using the QUBIT DS-HS kit (Thermo Fisher Scientific, Milan, Italy) and it was standardized at 50 ng/µL. Whole genome sequencing (WGS) was performed using NEBNext ® library prep Kit according to the manufacturers' instructions in paired-end (2 × 150 bp) on a NextSeq 550 Illumina system by the Novagene Company (Cambridge, United Kingdom).
For the transcriptomic analysis, L. monocytogenes cultures of CTC1034 and EGDe strains were centrifuged at 10,416× g for 5 min at 10 • C and pellets corresponding to 3.6 mL of culture were resuspended with 125 µL of RNAlater solution (Invitrogen, Thermo Fisher Scientific, Barcelona, Spain,) and kept at −80 • C. Total RNA was extracted from the pellets using the RNeasy PowerMicrobiome Kit (QIAGEN, Hilden, Germany) following the manufacturers' instructions, and residual DNA was removed with TURBO DNase (Invitrogen, Thermo Fisher Scientific, Milan, Italy) according to the manufacturers' instructions. RNA concentrations were quantified by using a Nanodrop Instrument (Spectrophotometer ND-1000, Thermo Fisher Scientific, Milan, Italy). The RNA integrity was verified by agarose gel electrophoresis. The RNA sequencing library preparation and cDNA synthesis were performed using the NEBNext Ultra RNA Library Prep Kit according to the manufacturers' instructions at Genewiz Inc. (Leipzig, Germany). The transcriptome was studied for all the samples from the experiment and sequencing was carried out on a NextSeq 550 Sequencer yielding 150 bp paired-end reads.
In order to investigate the molecular background that could explain the observed differences in the inactivation between the two L. monocytogenes strains as well as the piezoprotective effect of lactate, a transcriptomic approach was implemented. Total RNA was extracted, sequenced, and compared between L. monocytogenes cultures shortly exposed to (i) CHMM (control without HPP), (ii) CHMM supplemented with lactate (without HPP), (iii) CHMM and subjected to HPP, and (iv) CHMM supplemented with lactate and subjected to HPP.
Raw reads were quality filtered by SolexaQA++ software and PRINSEQ (Phred score < 20, <60 bp). Reads were aligned against the respective build database by using Bowtie2 in end-to-end, sensitive mode according to the strain used. The number of reads mapped to each gene (.sam files) were then used for KEGG functional analysis using MEGAN6 software [24]. Data normalization and determination of differentially abundant KEGG genes, among the studied conditions (lactate and HPP, alone, or in combination) or strains, were conducted using the Bioconductor DESeq2 package [25] in the statistical environment R [26] with default parameters. The statistical significance (p-values) was adjusted for multiple testing using the Benjamini-Hochberg procedure, which assesses the false discovery rate (FDR) by using the DESeq2 package.
Gene set enrichment for pathway analysis was then performed on KEGG orthologs table imported in the GAGE Bioconductor package [27] to identify biological pathways overrepresented or underrepresented between sample without lactate and without HPP treatment against the other combination.

Availability of Data and Material
WGS and Metatranscriptomic raw sequence reads were deposited at the Sequence Read Archive of the National Center for Biotechnology Information (Bioproject accession number: PRJNA692371 and PRJNA692360, for L. monocytogenes CTC1034 and EGDe, respectively).

Fatty Acid Profile of L. monocytogenes
For the strain CTC1034 the fatty acid profile was analyzed to confirm potential changes in the membrane composition due to exposure to lactate and/or HPP. For this, samples of L. monocytogenes CTC1034 were centrifuged at 10,416× g for 6 min at 10 • C. Supernatant was discarded and pellets were resuspended in 1 mL of purified water. Cells were disrupted with 0.5 g of glass beads in a mixer mill (Mixer Mill MM200, Retsch, Llanera, Spain) for 5 min at 30 Hz, centrifuged and supernatant was discarded. Pellets were frozen at −20 • C for 2 h before being freeze dried (Lyomicron LM-181004, Coolvacuum, Granollers, Spain). Methyl esters of fatty acids (FAME) were obtained by methylation described by Castro-Gómez et al. [28], using tritridecanoine as an internal standard. FAME analysis was carried out on an Autosystem chromatograph (Perkin Elmer, Beaconsfield, UK) fitted with a VF-23ms, fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 µm film thickness, Varian, Middelburg, The Netherlands) and FID, according to Calvo et al. [29]. The statistical difference of the results among conditions was assessed through the MANOVA test.

Inactivation of L. monocytogenes by HPP
Inactivation of L. monocytogenes strains CTC1034 and EGDe submitted to HPP at 400 MPa for 10 min in the CHMM resembling the composition of a cooked ham, with or without potassium lactate, is shown in Figure 1. The results show that the application of HPP in a medium without lactate inactivated CTC1034 and EGDe strains by an average reduction of 1.17 ± 0.20 and 2.96 ± 0.43 Log units, respectively. Thus, the strain CTC1034 was significantly (p < 0.05) more resistant to HPP than EGDe. In the presence of lactate in the CHMM, HPP resulted in a lower inactivation of the strains, recording 0.44 ± 0.04 and 2.36 ± 0.22 Log reduction for CTC1034 and EGDe, respectively. In particular, for the CTC1034 strain, the lethal effect of HPP was lower (p < 0.05) in the presence of lactate, corroborating the piezo-protective effect of this antimicrobial on L. monocytogenes inactivation as previously shown for this and other strains inoculated in different types of meat products [9,10,30,31]. UK) fitted with a VF-23ms, fused silica capillary column (30 m × 0.25 mm i.d. × 0 film thickness, Varian, Middelburg, The Netherlands) and FID, according to Calv [29]. The statistical difference of the results among conditions was assessed throu MANOVA test.

Inactivation of L. monocytogenes by HPP
Inactivation of L. monocytogenes strains CTC1034 and EGDe submitted to HPP MPa for 10 min in the CHMM resembling the composition of a cooked ham, with o out potassium lactate, is shown in Figure 1. The results show that the application in a medium without lactate inactivated CTC1034 and EGDe strains by an average tion of 1.17 ± 0.20 and 2.96 ± 0.43 Log units, respectively. Thus, the strain CTC10 significantly (p < 0.05) more resistant to HPP than EGDe. In the presence of lactate CHMM, HPP resulted in a lower inactivation of the strains, recording 0.44 ± 0.04 an ± 0.22 Log reduction for CTC1034 and EGDe, respectively. In particular, for the CT strain, the lethal effect of HPP was lower (p < 0.05) in the presence of lactate, corrobo the piezo-protective effect of this antimicrobial on L. monocytogenes inactivation as ously shown for this and other strains inoculated in different types of meat pr [9,10,30,31].  The comparison of L. monocytogenes genomes of CTC1034 and EGDe strains s the presence of 2967 core genes including 394 genes encoding hypothetical protein 77 genes were absent or present in one L. monocytogenes strain compared to the ot genes being found in CTC1034 but not in EGDe and 42 being found in EGDe but CTC1034. Most of the 35 genes found in CTC1034, but not in EGDe, were related t scription factors, while the major fraction of genes found in EGDe were involved tein export and transcription factors. As transcription factors regulate gene expres greater abundance in the CTC1034 could be related to the major resistance to HPP this strain has shown [32]. The comparison of L. monocytogenes genomes of CTC1034 and EGDe strains showed the presence of 2967 core genes including 394 genes encoding hypothetical proteins. Only 77 genes were absent or present in one L. monocytogenes strain compared to the other, 35 genes being found in CTC1034 but not in EGDe and 42 being found in EGDe but not in CTC1034. Most of the 35 genes found in CTC1034, but not in EGDe, were related to transcription factors, while the major fraction of genes found in EGDe were involved in protein export and transcription factors. As transcription factors regulate gene expression, a greater abundance in the CTC1034 could be related to the major resistance to HPP stress this strain has shown [32].

Whole Transcriptome Analysis
For the transcriptomic analysis involving both L. monocytogenes strains, a total of 152.43 Gbp of clean reads were obtained. For each sample, approximately 6.62 Gbp of reads  Table S1). The KEGG analysis assigned 864 genes to 24 KEGG pathways.
Results from the statistical analysis of the KEGG genes obtained with the transcriptomic analysis revealed that the number of differentially expressed genes (DEGs) found in the pairwise comparisons between all the condition combinations studied (effect of lactate, effect of HPP and effect of both factors) was strain-dependent ( Figure 2; Supplementary Tables S2-S10).

Whole Transcriptome Analysis
For the transcriptomic analysis involving both L. monocytogenes strains, a total of 152.43 Gbp of clean reads were obtained. For each sample, approximately 6.62 Gbp of reads were found (Supplementary Table S1). The KEGG analysis assigned 864 genes to 24 KEGG pathways.
Results from the statistical analysis of the KEGG genes obtained with the transcriptomic analysis revealed that the number of differentially expressed genes (DEGs) found in the pairwise comparisons between all the condition combinations studied (effect of lactate, effect of HPP and effect of both factors) was strain-dependent ( Figure 2; Supplementary Tables S2-S10). due to the exposure of cells to lactate, the application of the HPP (400 MPa for 10 min) and the application of both stresses compared to control conditions (exposed to CHMM without lactate).
In this framework, the stress induced by the exposure of L. monocytogenes cultures to CHMM with lactate compared to those exposed to CHMM without the antimicrobial resulted in a different response depending on L. monocytogenes strain. While the presence of lactate in the CHMM resulted in 104 DEGs in CTC1034, no DEGs were found in EGDe ( Figure 2; Supplementary Table S3). A similar pattern was obtained when analyzing the effect of the application of both stresses, lactate and HPP, on L. monocytogenes compared to control conditions, resulting in 286 DEGs for the CTC1034 and only 1 DEGs for the EGDe strain ( Figure 2; Supplementary Tables S6 and S10). Therefore, these results suggest that the response to stress is highly dependent on the particularities of the L. monocytogenes strain. In the study of the transcriptional response of two L. monocytogenes strains due to exposure to organic acids (lactate and diacetate) reported by Stasiewicz et al. [33], large differences on the number of transcribed genes were found and only a minor fraction of the differentially transcribed genes were shared between the two strains.
Additionally, it was interesting to observe that DEGs found for EDGe in the pairwise comparison of pressurized samples with and without the presence of lactate (Supplementary Table S8) were the same or involved in the same metabolic pathways as those DEGs found in non-pressurized cultures of CTC1034 in response to lactate stress (Supplementary Table S3). The different pairwise comparisons between the stressing conditions involving lactate also support this hypothesis (Supplementary Tables S4, S5, S8 and S9). These results would lead to the hypothesis that both L. monocytogenes strains employ similar molecular mechanisms in response to the lactate stress, although they seem to be activated in a different magnitude and/or time frame.
On the other hand, the application of the HPP resulted in 386 and 120 DEGs for the CTC1034 and EGDe strains, respectively, when compared to control conditions, i.e., L. monocytogenes cultures exposed to CHMM without lactate (Figure 2; Supplementary Tables S2 and S7). due to the exposure of cells to lactate, the application of the HPP (400 MPa for 10 min) and the application of both stresses compared to control conditions (exposed to CHMM without lactate).
In this framework, the stress induced by the exposure of L. monocytogenes cultures to CHMM with lactate compared to those exposed to CHMM without the antimicrobial resulted in a different response depending on L. monocytogenes strain. While the presence of lactate in the CHMM resulted in 104 DEGs in CTC1034, no DEGs were found in EGDe ( Figure 2; Supplementary Table S3). A similar pattern was obtained when analyzing the effect of the application of both stresses, lactate and HPP, on L. monocytogenes compared to control conditions, resulting in 286 DEGs for the CTC1034 and only 1 DEGs for the EGDe strain ( Figure 2; Supplementary Tables S6 and S10). Therefore, these results suggest that the response to stress is highly dependent on the particularities of the L. monocytogenes strain. In the study of the transcriptional response of two L. monocytogenes strains due to exposure to organic acids (lactate and diacetate) reported by Stasiewicz et al. [33], large differences on the number of transcribed genes were found and only a minor fraction of the differentially transcribed genes were shared between the two strains.
Additionally, it was interesting to observe that DEGs found for EDGe in the pairwise comparison of pressurized samples with and without the presence of lactate (Supplementary Table S8) were the same or involved in the same metabolic pathways as those DEGs found in non-pressurized cultures of CTC1034 in response to lactate stress (Supplementary Table S3). The different pairwise comparisons between the stressing conditions involving lactate also support this hypothesis (Supplementary Tables S4, S5, S8 and S9). These results would lead to the hypothesis that both L. monocytogenes strains employ similar molecular mechanisms in response to the lactate stress, although they seem to be activated in a different magnitude and/or time frame.
On the other hand, the application of the HPP resulted in 386 and 120 DEGs for the CTC1034 and EGDe strains, respectively, when compared to control conditions, i.e., L. monocytogenes cultures exposed to CHMM without lactate (Figure 2; Supplementary Tables S2 and S7).
The pathway enrichment analysis (performed by GAGE) of the KEGG genes of CTC1034 strains showed an enrichment of several pathways in CHMM subjected to HPP (with and without lactate) compared with the control CHMM (without HPP nor lactate), including Flagellar assembly (ko02040), Fructose and mannose metabolism (ko00051), Phosphotransferase system (ko02060), Biosynthesis of amino acids (ko01230) and Pheny-lalanine, and tyrosine and tryptophan biosynthesis (ko00400). Moreover, an enrichment of the flagellar assembly (ko02040) and a reduction in glycolysis/gluconeogenesis (ko00010) in CHMM supplemented with lactate without HPP was observed when compared with CHMM. Regarding EGDe, an enrichment in cysteine and methionine metabolism (ko00270), peptidoglycan biosynthesis (ko00550), fatty acid metabolism (ko01212), biosynthesis of amino acids (ko01230) and citrate cycle (ko00020), and a downregulation of the flagellar assembly (ko02040) and phosphotransferase system (PTS) (ko02060) were observed in CHMM subjected to HPP if compared with non-pressurized CHMM (data not shown).

Effect of Lactate Exposure on L. monocytogenes
Some studies support that in order to counteract the intracellular osmotic pressure caused by an increased amount of lactate, bacteria (i) reduce intracellular pools of anions and (ii) shift the flux in the central carbon metabolism [34]. The results from the present transcriptomic analysis reveal that L. monocytogenes could use both strategies to overcome the stress suffered by its exposure to lactate. Regarding the possible effect of lactate on the central carbon metabolism of the pathogen, the results of the present study show that genes involved in the pentose phosphate pathway coupled with oxidative reactions to produce reducing equivalents (rpiB, tktA, tktB, G6PD) were upregulated. Additionally, a downshift was observed in the conversion of pyruvate to acetyl-CoA and ethanol, as indicated by the downregulation of genes such as pdhC, plfD, and adhE. In line with the output of the pathway enrichment analysis described above, these transcriptomic results suggest that in presence of lactate, L. monocytogenes redistributed its metabolic carbon flux from the glycolytic pathway to oxidative reactions producing reducing equivalents (Figure 3).
The enrichment of flagellar assembly pathways and in detail of flagellar genes (FlhA, FlhF, FliC, FliE, FliF, FliG, FliH, FliI, FliR, FliP, FlgB, FlgC, FlgD, FlgE, FlgG, FlgK, and FlgL) found in the presence of lactate (Supplementary Tables S3 and S8) could indicate that the electrochemical potential of protons across the cytoplasmic membrane could also contribute to fuel the flagellar motor of the pathogen [43] and/or that the unfavorable environment faced by L. monocytogenes would promote the pathogen to elicit the chemotactic response and to move to a more favorable environment [44].
The activation of all the strategies to counteract the osmotic pressure and membrane potential changes due to lactate would result in less efficient pathways for ATP production and in a higher energy expenditure, leading to the limitation of growth in the presence of lactate [45][46][47]. A decrease of metabolic energy generation due to the increase in external lactate concentration was described in Streptococcus cremoris [48].
In addition to the up/downregulation of molecular mechanisms involved in restoring osmotic pressure and membrane potential, it is worth to highlight that in the presence of lactate, L. monocytogenes specifically upregulated genes involved in the methionine synthesis (Figure 4), in particular a higher expression of the methyltransferases mmuM in CTC1034 (Supplementary Table S3) and MetE in pressurized EGDe (Supplementary Table S8) was found. Both enzymes are responsible for converting homocysteine to methionine, thus suggesting that in the presence of lactate L. monocytogenes promoted the oxidation of homocysteine to methionine, avoiding the accumulation of the toxic metabolite homocysteine and increasing the amount of intracellular methionine. In accordance with this, genes associated with the sulfur metabolism (metC, metX, cysE or cysO) involved in the methionine synthesis were also found to be upregulated by the exposure of L. monocytogenes to lactate (Figure 4; Supplementary Tables S3 and S8). In previous studies dealing with the transcriptome analysis of L. monocytogenes cells exposed to lactate, the upregulation of the methionine biosynthesis was not reported [33,49]. However, in those experiments L. monocytogenes was exposed to lactate for a much longer time, i.e., 8 h at 7 • C and 48 h at 15 • C, than the exposure time used in the present study (<2 h at 10 • C). It can be hypothesized that the upregulation of the methionine synthesis would only occur in the early exposure of the pathogen to lactate as a first step of the overall mechanism to overcome the stress suffered by the presence of lactate. In addition to the time-related factor, other potential reasons leading to different results include the pathogen strains, the concentration and the type of salt (sodium vs. potassium), and the incubation temperature or the matrix composition (culture medium) used for the experiment.
Among all the multiple factors that can determine the expression of genes involved in the methionine synthesis, the observed upregulation of this metabolic pathway by L. monocytogenes in the presence of lactate could be relevant in relation to the piezo-resistance mechanisms since another organic acid such as acetate has been shown to specifically inhibit the synthesis of methionine in Escherichia coli, favoring the accumulation of the toxic compound homocysteine and consequently limiting or even inhibiting the growth of the pathogen [50]. Moreover, Roe et al. [50] reported that the addition of methionine in the medium containing acetate restores E. coli growth to 80% of that observed in medium without acetate, indicating that the inhibition of the methionine biosynthesis is one of the main factors responsible for the growth depletion of E. coli cultured in the presence of acetate. Supporting these results, Pinhal et al. [51] reported that the uncoupling effect of acetate or the perturbation of the anion composition of the cell played only a limited role (20%) in the E. coli growth depletion, suggesting that other molecular mechanisms, such as the inhibition of the methionine synthesis, could have a more prominent role on the bacterial growth-inhibitory effect.
Methionine can be converted to S-adenosyl-L-methionine (SAM), which represents a methyl group donor for many fundamental cellular processes, such as cellular signaling and epigenetic regulations that promote cellular anabolism and proliferation in bacteria and yeasts [52,53]. Specifically, SAM is involved in the methylation of proteins, RNAs, biotin, polyamines, and lipids [53,54]. In the present study, the metK gene responsible for the conversion of methionine to SAM was found to be upregulated in the L. monocytogenes CTC1034 strain when it was exposed to lactate, suggesting a higher production of SAM. Moreover, an increased intracellular concentration of methionine was also reported to contribute to the antioxidant defense in bacteria [55], although its role in the piezo-protection remains unknown. Among all the multiple factors that can determine the expression of genes involved in the methionine synthesis, the observed upregulation of this metabolic pathway by L. monocytogenes in the presence of lactate could be relevant in relation to the piezo-resistance mechanisms since another organic acid such as acetate has been shown to specifically inhibit the synthesis of methionine in Escherichia coli, favoring the accumulation of the toxic compound homocysteine and consequently limiting or even inhibiting the growth of the pathogen [50]. Moreover, Roe et al. [50] reported that the addition of methionine in the medium containing acetate restores E. coli growth to 80% of that observed in medium without acetate, indicating that the inhibition of the methionine biosynthesis is one of the main factors responsible for the growth depletion of E. coli cultured in the presence of acetate. Supporting these results, Pinhal et al. [51] reported that the uncoupling effect of acetate or the perturbation of the anion composition of the cell played only a limited role (20%) in the E. coli growth depletion, suggesting that other molecular mechanisms, such as the inhibition of the methionine synthesis, could have a more prominent role on the bacterial growth-inhibitory effect.
Methionine can be converted to S-adenosyl-L-methionine (SAM), which represents a methyl group donor for many fundamental cellular processes, such as cellular signaling and epigenetic regulations that promote cellular anabolism and proliferation in bacteria and yeasts [52,53]. Specifically, SAM is involved in the methylation of proteins, RNAs,

Effect of HPP on L. monocytogenes
The transcriptomic analysis revealed that both L. monocytogenes strains upregulated genes involved in DNA repair mechanisms such as RadA, phrB, uvrB, adaB, and lipid and peptidoglycan biosynthetic pathways (glmS, murF, murG, murC, or fabH), among others (Supplementary Tables S2 and S7), presumably as a consequence of the stress induced by the application of the HPP to L. monocytogenes. In case of flagella assemblage (FlhA, FlhF, FliC, FliE, FliF, FliG, FliH, FliI, FliR, FliP, FlgB, FlgC, FlgD, FlgE, FlgG, FlgK and FlgL) and chemotaxis (MotA, CheA, CheR, CheY, FliG, FliM, and FliN/FliY), an upregulation of genes involved in these pathways was found in CTC1034 (Supplementary Table S2), while a downregulation was observed in EGDe (Supplementary Table S7). These differences could be related to the particularities of each L. monocytogenes strain but also to the higher severity of the HPP injury in the EGDe strain compared to CTC1034, leading to a higher inactivation extent (Figure 1). An important parameter influencing motility of L. monocytogenes is temperature; L. monocytogenes cells are motile at temperatures below 30 • C but not at human body temperature (37 • C) [56]. Additionally, flagella, as cell surface appendices, are considered putative virulence factors. In the current study, the temperature for the experiments could partially explain the upregulation of the flagella genes in CTC1034. In addition to this, we may deduce that these genes would be downregulated when L. monocytogenes is under stress (for example desiccation) [57]. It is therefore puzzling that HPP resulted in an upregulation in CTC1034, and at this point we cannot provide a biological explanation. Nevertheless, this observation is particularly relevant since it suggests that cells of L. monocytogenes surviving the HPP treatment would be prepared to colonize the human body [58]. On the other hand, HPP was found to downregulate genes involved in the septal ring (ftsA, ftsW, ftsQ, mreB). These results were in line with those reported by Bowman et al. [59] regarding the response of L. monocytogenes pressurized at 400-600 MPa for 5 min in tryptone soy yeast extract (TSYE) broth.
As a response to HPP, L. monocytogenes CTC1034 and EGDe upregulated genes involved in the methionine biosynthesis (luxS, mmuM, msrB), suggesting an enhanced methionine production/availability (Supplementary Tables S2 and S8), which also agrees with the enrichment gene analysis for EGDe (see Section 3.2.2). The upregulation of these genes pointed out that, as stated due to the exposure to lactate (Section 3.2.3), the application of HPP would result in a higher generation of SAM in L. monocytogenes, which could affect cellular processes throughout its role in the methyl cycle [60]. These results are in accordance with those reported by Bravim et al. [61], where it was found an upregulation of the sulfur metabolism genes involved in the activation of the methionine biosynthesis when Saccharomyces cerevisiae was submitted to an HPP of 50 MPa for 30 min.
Considering the metabolic pathways in which methionine and SAM are involved, methionine could increase L. monocytogenes resistance to HPP for its role as an endogenous antioxidant in cells [62] and for its involvement in lipid biosynthesis [63]. Since the HPP affects the bacterial membrane properties [64][65][66], the involvement of methionine in lipid biosynthesis could play a role in the HPP resistance ( Figure 4). In this regard, according to the results of the fatty acid profile of L. monocytogenes CTC1034 (Table 1) compared with the control conditions when the pathogen was exposed to lactate and/or HPP stresses, cells tended to increase, although not significantly, the level of total branched-chain fatty acids (BCFAs, specifically iso and/or anteiso conformations of C13, C14, C15, C16, C17). This finding agrees with the fact that in L. monocytogenes BCFAs contribute to membrane fluidity and resistance against environmental stresses [67].
SAM was reported to be required for the synthesis of phosphatidylcholine from phosphatidylethanolamine [68] and to have a role in transferring a methylene group to mature phospholipids that lead to the formation of cyclopropane fatty acids (CFAs), a major component of the phospholipids of the bacterial membrane bilayers [69]. A higher proportion of CFAs in the membrane bilayer of Escherichia coli has been shown to increase the resistance of the pathogen submitted to HPP of 500 MPa for 5 to 30 min [70]. Since the pressure resistance of E. coli is reported to be related to an altered membrane functionality and with the resistance of this pathogen to oxidative stress [71], it was suggested by Chen et al. [70] that CFAs could contribute to pressure resistance by increasing the resistance of membrane lipids to the oxidative stress derived from the application of the HPP. Therefore, the results of the present study point out that the exposure of L. monocytogenes cells to lactate prior the HPP would upregulate the methionine biosynthesis pathway, thus contributing to enhance the resistance against HPP by changes in the lipidic membrane functionality.
The higher expression of the methionine biosynthesis pathway by L. monocytogenes exposed to lactate and the inhibition of the biosynthesis of this amino acid by acetate reported for E. coli [50] could be the reason why the piezo-protective effect on L. monocytogenes treated at 400 MPa for 10 min was only seen for cooked ham formulated with lactate and not with diacetate [10]. Further studies regarding L. monocytogenes membrane functionality (membrane composition, fluidity, and integrity) as a function of the exposure of lactate and the application of the HPP need to be conducted to experimentally to confirm the role of the membrane properties on the piezo-protective effect exerted by lactate on HPP inactivation of L. monocytogenes. Table 1. Fatty acid profile (mean % ± standard deviation) of L. monocytogenes CTC1034 after exposure of cells to lactate, after the application of the HPP (400 MPa for 10 min), and after the application of both stresses compared to control conditions (exposed to CHMM without lactate).

Fatty Acid Condition
Control Lactate HPP Lactate + HPP membrane and its ability to cope with pressure stress. Further studies regarding the L. monocytogenes membrane functionality (membrane composition, fluidity, and integrity) as a function of the exposure of lactate and the application of the HPP need to be conducted to experimentally confirm the role of the membrane properties on the piezo-protection and piezo-stimulation effect exerted by lactate on HPP inactivation of L. monocytogenes.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biom11050677/s1, Table S1: Number of raw and clean reads from the transcriptomic analysis of both L. monocytogenes strains CTC1034 and EGDe in CHMM without and with lactate and/or without and with HPP of 400 MPa for 10 min, Table S2: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in the L. monocytogenes strain CTC1034 in samples without lactate pressurized and non-pressurized. Positive Log2 fold change indicates genes more abundant in pressurized samples, Table S3: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in the L. monocytogenes CTC1034 strain in non-pressurized samples without and with lactate. Positive Log2 fold change indicates genes more abundant in samples with lactate, Table S4: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in the L. monocytogenes strain CTC1034 in pressurized samples without and with lactate. Negative Log2 fold change indicates genes less abundant in samples with lactate, Table S5: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in the L. monocytogenes strain CTC1034 in samples with lactate non-pressurized and pressurized. Positive Log2 fold change indicates genes more abundant in pressurized samples, Table S6: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in CTC1034 L. monocytogenes strain throughout the comparison of control samples (non-exposed to lactate and nonpressurized) to samples exposed to lactate and pressurized. Positive Log2 fold change indicates genes more abundant in samples exposed to lactate and pressurized, Table S7: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in the L. monocytogenes strain EGDe in samples without lactate pressurized and non-pressurized. Positive Log2 fold change indicates genes more abundant in pressurized samples, Table S8: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in the L. monocytogenes strain EGDe in pressurized samples without and with lactate. Positive Log2 fold change indicates genes more abundant in samples with lactate, Table S9: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in the L. monocytogenes strain EGDe in samples with lactate non-pressurized and pressurized. Positive Log2 fold change indicates genes more abundant in pressurized samples, Table S10: List of KEGG Orthology (KO) genes differentially (FDR < 0.05) expressed in EGDe L. monocytogenes strain throughout the comparison of control samples (non-exposed to lactate and non-pressurized) to samples exposed to lactate and pressurized. Positive Log2 fold change indicates genes more abundant in samples exposed to lactate and pressurized.