Stress Adaptation Responses of a Listeria monocytogenes 1/2a Strain via Proteome Profiling

Listeria monocytogenes is a foodborne pathogen that is ubiquitous and largely distributed in food manufacturing environments. It is responsible for listeriosis, a disease that can lead to significant morbidity and fatality in immunocompromised patients, pregnant women, and newborns. Few reports have been published about proteome adaptation when L. monocytogenes is cultivated in stress conditions. In this study, we applied one-dimensional electrophoresis and 2D-PAGE combined with tandem mass spectrometry to evaluate proteome profiling in the following conditions: mild acid, low temperature, and high NaCl concentration. The total proteome was analyzed, also considering the case of normal growth-supporting conditions. A total of 1,160 proteins were identified and those related to pathogenesis and stress response pathways were analyzed. The proteins involved in the expression of virulent pathways when L. monocytogenes ST7 strain was grown under different stress conditions were described. Certain proteins, particularly those involved in the pathogenesis pathway, such as Listeriolysin regulatory protein and Internalin A, were only found when the strain was grown under specific stress conditions. Studying how L. monocytogenes adapts to stress can help to control its growth in food, reducing the risk for consumers.


Introduction
Listeria monocytogenes is considered as one of the most severe foodborne pathogens, which is responsible for listeriosis, a systemic illness due to the ingestion of contaminated food, such as vegetables, raw meat, milk, and ready-to-eat foods. The clinical manifestations vary from self-limited gastroenteritis with fever, diarrhea, nausea, and vomiting to invasive infections characterized by bacteremia, encephalitis, and fetal loss [1]. Listeriosis has a significant morbidity and a high fatality rate, around 20-30% [2], mainly in immunocompromised individuals, such as cancer patients or those with autoimmune diseases [3], elderly, pregnant women, and newborns [4].
The incidence of listeriosis outbreaks has increased in recent years and several events have been reported in different continents. In the European Union (EU), listeriosis is the fifth most commonly reported zoonosis in humans, with a notification rate of 0.49 cases per 100,000 population in 2021, 14% higher than the rate of 0.43 in 2020. According to the latest available report from EFSA and ECDC [5], a total of 2,183 reported cases of human invasive listeriosis (923 hospitalized and 196 deaths) were observed in the EU, with meat products from bovines or pigs, fruits and vegetables, and sheep's milk cheeses accounting for the highest values (from 2 to 5%). L.M.ST7 (PFGE ApaI 0246 AscI 0356) was grown in brain heart infusion (BHI) broth (Oxoid Thermo Fisher Scientific, Rodano, Italy) and modified BHI broth (pH 5.5, NaCl 7%) at 37 • C and 12 • C according to D'Onofrio et al. [25] in different conditions: C1 (temperature 37 • C, pH 7.0, NaCl 0.5%); C2 (temperature 37 • C, pH 5.5, NaCl 7%); C3 (temperature 12 • C, pH 7.0, NaCl 0.5%); and C4 (temperature 12 • C, pH 5.5, NaCl 7%).
Bacterial cells were collected at the late log phase (optical density at 600 nm). After centrifugation (Eppendorf 5424, Eppendorf, Hamburg, Germany) (5600× g at 4 • C for 10 min), the cell pellets were washed three times with 10 mL phosphate-buffered saline solution. All experiments were performed in triplicate.

Protein Extraction and Immunoblotting
Proteins were extracted from the sample using CelLytic B Cell Lysis Reagent and Cel-Lytic IB Inclusion Body Solubilization Reagent (Sigma-Aldrich, Milan, Italy). Subsequently, proteins were precipitated by TCA protocol and solubilized with 0.05% SDS, 1M NaOH. The protein concentration was determined by Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Mass Spectrometry (MS) and Data Analysis
The proteins were resolved by SDS-PAGE and made visible by Coomassie stain from a 4-12% NuPAGE ® precast gel (Thermo Fisher Scientific). Briefly, the gel lanes were cut [27] and reduced/alkylated by 10 mM dithiothreitol (DTT) and 55 mM iodoacetamide (IAA), respectively. The proteins were trypsin digested at 37 • C o/n. Peptides were desalinated and concentrated using StageTip C18, which was applied before peptides nLC-MS/MS analysis [28]. Peptide mixtures were dried under vacuum and solubilized in 5% formic acid. The samples were analyzed by a quadrupole Orbitrap Q-Exactive HF mass spectrometer associated with an UHPLC Easy-nLC 1200 (Thermo Fisher Scientific) combined with a 25 cm-C18 column (75 µm inner diameter) (Dr. Maisch Gmbh, Ammerbuch, Germany). In order to separate peptides, a linear gradient was applied: over 23 min from 95% solvent A consisting of 2% ACN (0.1% formic acid) to 50% solvent B by 80% ACN (0.1% formic acid), and over 2 min from 50% to 100% with a flow rate of 0.25 µL/min with a single run time of 33 min. MS data were obtained in positive mode by a DDA (data-dependent acquisition) top 15 method. Using Orbitrap Q-Exactive HF with 60,000 resolution, automatic gain control (AGC) target 1e6, IT 120 ms, survey full scan MS spectra were acquired. For higherenergy collisional dissociation (HCD) spectra, the resolution was set to 15,000, AGC target 1e5, IT 120 ms, normalized collision energy 28%, isolation width of 3.0 m/z, and a dynamic exclusion of 5 s.
The nLC-MS/MS method applied and data analysis were carried out according to D'Onofrio et al. [25].

Gene Ontology Analysis
Gene ontology (GO) analysis and classification were performed using ShinyGO v0.76 software [29]. The gene sets identified for each growth condition were used to perform the enrichment analysis. The enriched pathways were identified, and genes were grouped by functional categories. Furthermore, the proteins identified by nLC-ESI-MS/MS analysis were analyzed and summarized by Venn diagram.

STRING Analysis
The open-source STRING software (Search Tool for the Retrieval of Interacting Genes/Proteins) version 11.05 was used to conduct the in-silico analysis. The proteins related to pathogenesis and stress response pathways given by GO analysis were selected in order to identify the protein-protein interactions. A network image was obtained for visualization purposes.

Gene
Enolase (Eno) and DNA protection during starvation (Dps) proteins were identified for all experimental conditions. Eno plays a crucial role in the degradation of carbohydrates via glycolysis, and it catalyzes the conversion of 2-phosphoglycerate into phosphoenolpyruvate. Furthermore, Eno binds plasminogen during host infection when it is expressed on the cell wall. This reaction allows L. monocytogenes to develop surface-associated proteolytic activity, which can contribute to the invasion of tissues and virulence [22]. Eno has also been implicated in the regulation of gene expression of L. monocytogenes. It seems that Eno binds specific DNA sequences and acts as a transcription factor, regulating the expression of genes involved in a variety of cellular processes, such as general stress response and pathogenesis. In L. monocytogenes, Eno has been shown to bind to the promoter region of the hly gene, which encodes the hemolysin protein. By binding to this region, Eno can modulate the encoding of hly protein [30].
On the other side, Dps protects DNA from oxidative damage [31] and has a role both in resistance to heat and cold shocks and in the virulence pathway, modulating listeriolysin O (LLO) production and its stability. LLO is an essential secreted pore-forming protein that induces vacuole lysis during L. monocytogenes internalization [32,33]. Dps was involved in other biological processes. For example, it is shown to play a role in regulating iron homeostasis in L. monocytogenes. Specifically, Dps binds iron ions and regulates their intracellular concentration, including several cellular pathways, such as respiration, metabolism, and stress responses [34]. Moreover, Dps has been implicated in the response of L. monocytogenes to various environmental stressors, such as osmotic stress and pH changes. In response to these stressors, Dps could undergo conformational changes that modulate its DNA-binding properties and its ability to protect DNA from oxidative damage [35]. Additionally, it has been suggested to have a role in the formation and maintenance of biofilms [36]. D-alanine-D-alanyl carrier protein ligase (DltA) was identified in all stress conditions; this protein is involved in the lipoteichoic acid (LTA) biosynthesis to preserve cation homeostasis and assimilation, through action on cell wall net charge [37].
One of the important functions of DltA is its role in resistance to cationic antimicrobial peptides (CAMPs). CAMPs are small cationic peptides that are produced by various organisms as part of their innate immune system. They can disrupt bacterial cell membranes and are an important defense mechanism against bacterial infections. DltA plays a crucial role in L. monocytogenes resistance to CAMPs by modifying the net charge of the cell wall. In particular, DltA catalyzes the transfer of D-alanine-D-alanine dipeptides to the carrier protein DltC, which then transfers the D-alanine-D-alanine to LTA. This modification reduces the net negative charge of the cell wall and makes it less susceptible to CAMPs. Moreover, DltA has been implicated in the regulation of virulence in L. monocytogenes. Specifically, DltA has been also implicated in regulating the expression of virulence genes by modulating the activity of the transcriptional regulator "Listeriolysin positive regulatory protein" (PrfA) [38].
PrfA, encoded by prfA gene, was found only in acidic and osmotic stress conditions; this is a very interesting finding, as this protein has been pointed out in the literature as a "master regulator of virulence" [16]. The lack of PrfA greatly attenuates the virulence of L. monocytogenes, in both vertebrates and invertebrates, as well as in cell culture infection models [39][40][41]. This protein is also known as a positive regulator of listeriolysin, 1-phosphatidylinositol phosphodiesterase (PI-PLC), and several pathogenic factors [42]. Moreover, prfA gene seems to be related to the adaptative response to acid stress of L. monocytogenes. The production of this protein only in the presence of both osmotic and acidic stress conditions confirms the link between this protein and stress-resistant cells, similarly to what was recently reported for oxidative stress resistance [43]. On this basis, L. monocytogenes strains able to survive/grow in foods with low pH and high salt concentration could be potentially considered as more virulent. Therefore, more attention should be paid to strains isolated in these types of food (e.g., dried meat products or long-aged cheeses, as well as foods containing acidic preservatives), in which the pathogen is often exposed to severe osmotic and acid stresses, even at the same time. The presence of food preservatives in the gastrointestinal tract, particularly organic and inorganic acids, is an important hurdle for L. monocytogenes. In fact, organic acid dissociation in the cell cytoplasm implies acidification and proton/anion influx, which inhibits both transport of catabolites and ATP synthesis, leading to cell membrane, nucleic acid, and protein damages. To face acidic stress, L. monocytogenes genome acts by a wide spectra of gene expression changes, such as rB, CtsR, and PrfA involved in DNA repair, fatty acid biosynthesis related to cell wall modification, and oxidative stress defense [44].
Motility gene repressor (MogR) and Internalin A (InlA) proteins were identified in low temperature conditions, C3 and C4, respectively. The former induces transcriptional repression of flagellar motility genes at 37 • C and during infection [45], whereas the latter acts as mediator of L. monocytogenes entrance into host intestinal epithelial cells, binding the cadherin-1 receptor (E-cadherin, CDH1) in human subjects. This could explain the potential role of storage of foods at a low temperature in favoring the production of these proteins highly involved in the pathogenesis of listeriosis. Recently, the joint effects of low temperature and osmotic stress were evaluated in relation to the capacity of L. monocytogenes to invade human intestinal CACO-2 cells, and the results were strain-dependent [46]. In our study, these proteins associated with cellular invasion during infection were identified only under stressful conditions, namely at a low temperature, in both the presence (C4) and absence (C3) of osmotic stress, and thus shown to be mostly related to cold stress rather than to high salt concentration.
The list of all proteins identified for each condition and GO enrichment analysis results are available as Supplementary Material. As for the genes grouped under the "stress response" category, listed here below, damage control and DNA-repair-associated genes were expressed for all conditions, shown to be not strictly related to the presence of stressful environmental conditions.
In addition, Ssb1, RadA, and RecA proteins have been found to be involved in various other biological processes in L. monocytogenes. Ssb1, for example, influenced stress response and virulence; it was upregulated in response to heat shock and oxidative stress, and its expression was required for the full virulence of L. monocytogenes in a mouse model of infection. Additionally, Ssb1 has been implicated in cell wall biosynthesis and antibiotic resistance. On the other hand, RadA has been found to be important for the survival of such bacteria under DNA-damaging conditions [48]. Specifically, RadA was required for the repair of DNA double-stranded breaks, which can be caused by exposure to ionizing radiation or other DNA-damaging agents. Moreover, RadA was shown to be important for the formation and maintenance of L. monocytogenes biofilms. Finally, RecA protein has been implicated in the regulation of bacterial virulence. Specifically, RecA regulated the expression of virulence genes by modulating transcriptional regulator PrfA activity. Additionally, RecA has been implicated in the regulation of stress, including responses to heat shock and DNA damage [49].
Endonuclease MutS2, DNA mismatch repair MutS, and DNA mismatch repair MutL proteins repair mismatches in DNA. Furthermore, MutS2 seems to have a role in bacterial genetic diversity control, acting as a small molecule with sensor activity [50]. In addition to their roles in DNA repair and maintenance of genetic stability, the endonuclease MutS2, MutS, and MutL proteins have been found to be involved in various other biological processes in L. monocytogenes. MutS2 has been shown to play a role in the regulation of bacterial genetic diversity in such bacteria; specifically, MutS2 had ribonuclease activity and cleaved small non-coding RNAs, leading to changes in gene expression. MutS2 had an important significance for the adaptation of such a pathogen to different environments and for the regulation of its virulence gene expression. Moreover, it seemed to modulate L. monocytogenes full virulence in a mouse model of infection, and its expression was upregulated in response to various stresses, including low pH, salt stress, and exposure to bile salts. MutL has been implicated in the regulation of biofilm formation of the pathogen. Specifically, it was required for robust biofilms formation, and its expression was upregulated in response to nutrient limitation [51].
DEAD-box ATP-dependent RNA helicase (CshB) is involved in heat and cold tolerance, oxidative and alkali stress, and motility [52,53].
RNA helicases are characterized by the specific amino acid sequence D-E-A-D (Asp-Glu-Ala-Asp) in the conserved helicase motif II. They are fundamental for RNA secondary structure resolution under cold stress conditions, allowing transcription and translation events [54]. In addition to its roles in stress tolerance and motility, CshB was involved in the regulation of gene expression. It seemed to interact with a number of different RNA molecules, including small regulatory RNAs and mRNAs encoding virulence factors. By unwinding RNA secondary structures and promoting RNA degradation or stabilizing RNA structures, CshB could influence the levels of these RNAs and thereby modulate gene expression. Furthermore, CshB was required for L. monocytogenes full virulence and its expression was upregulated in response to various stresses, including low pH and oxidative stress [53].
Chaperone DnaJ and DnaK proteins are produced to respond to hyperosmotic and heat shock. They prevent the formation of stress-denatured proteins by means of ATP-dependent interactions, leading to efficient folding [55]. Chaperone protein works with DnaJ and DnaK. It is involved in cell recovery pathways induced by heat and osmotic damage [56,57]. Moreover, ClpB contributes to L. monocytogenes virulence, acting as a chaperone [58]. The clpB gene is related to defective cellular growth when such a microorganism is exposed to NaCl osmotic stress [59,60]. For this reason, chaperone proteins are considered to be of crucial importance to restore protein structures and, consequently, their functions are vitiated by osmotic stress [61]. In addition to their roles in responding to heat and osmotic stress, chaperones DnaJ, DnaK, and ClpB orchestrated L. monocytogenes virulence: DnaJ and DnaK were fundamental for the proper folding and function of the bacterial surface protein InlA [60], while ClpB was fundamental for Actin A (ActA), which is a factor involved in bacterial movement and spreading within host tissues [62].
ATP-dependent helicase/nuclease subunit A and B (ADDA and ADDB) contribute to DNA repair and recombination pathways as UvrABC system proteins (UvrA, UvrB, and UvrC) and RuvA-RuvB complex [50]. In addition, ADDA and ADDB were implicated in the regulation of L. monocytogenes virulence; specifically, their expression was significantly upregulated in L. monocytogenes strains that were more virulent than others.
Moreover, it seemed that the deletion of the genes encoding ADDA and ADDB resulted in decreased virulence in a mouse model of L. monocytogenes infection [62].
The RecF and lexA proteins act in tandem and are involved in the replication of DNA and basal SOS response, both with RecA. Pyrophosphatase (Ppax) plays a role in DNA repair, modulating the intracellular pyrophosphate pool [49]. The stress response category also includes genes involved in oxidoreductase activity, such as peptide methionine sulfoxide reductase (msrB) and thioredoxin reductase (trxB), which contribute to cell redox homeostasis, removing superoxide radicals [63]. Among all the "stress response pathway" proteins, only MsrB and PpaX were specific to certain stress conditions. Specifically, MsrB was expressed only in the presence of both osmotic and acidic stress, while PpaX was found only in response to cold stress. STRING analysis highlighted a network characterized by 28 nodes and 116 edges ( Figure 2). The cluster indicates that the proteins are biologically connected and involved in stress response regulation and pathogenesis [64].
The production of certain proteins, particularly those involved in pathogenesis, such as listeriolysin regulatory protein and InlA, can be induced by stress conditions as a mechanism of the adaptation and survival of the bacteria. These proteins may help L. monocytogenes to overcome stressors in the environment and continue to grow and cause infection. Furthermore, Manso et al. [65] found that many stress response genes in L. monocytogenes are regulated by SigB, which allows the pathogen to respond to stress by activating the expression of genes involved in stress tolerance and adaptation. Moreover, Sibanda et al. [17] showed that L. monocytogenes can adjust its gene expression to adapt to various stress conditions encountered during foodborne transmission. This mechanism is regulated by the crosstalk between SigB and PrfA. SigB plays a crucial role in the stress response by controlling gene expression that aids the survival of the bacteria in difficult environmental conditions, i.e., shifts in osmotic pressure, temperature, pH, redox potential, and nutrient availability [66].
It seems that Eno binds specific DNA sequences and acts as a transcription factor, regu lating the expression of genes involved in a variety of cellular processes, such as genera stress response and pathogenesis. In L. monocytogenes, Eno has been shown to bind to th promoter region of the hly gene, which encodes the hemolysin protein. By binding to th region, Eno can modulate the encoding of hly protein [30]. On the other side, Dps protects DNA from oxidative damage [31] and has a role bot in resistance to heat and cold shocks and in the virulence pathway, modulating listerioly sin O (LLO) production and its stability. LLO is an essential secreted pore-forming protei that induces vacuole lysis during L. monocytogenes internalization [32,33]. Dps was in volved in other biological processes. For example, it is shown to play a role in regulatin iron homeostasis in L. monocytogenes. Specifically, Dps binds iron ions and regulates the intracellular concentration, including several cellular pathways, such as respiration, me tabolism, and stress responses [34]. Moreover, Dps has been implicated in the response o L. monocytogenes to various environmental stressors, such as osmotic stress and pH changes. In response to these stressors, Dps could undergo conformational changes tha modulate its DNA-binding properties and its ability to protect DNA from oxidative dam age [35]. Additionally, it has been suggested to have a role in the formation and mainte nance of biofilms [36]. D-alanine-D-alanyl carrier protein ligase (DltA) was identified i Figure 2. A network diagram created using STRING v11.05 to show the protein-protein interactions of upregulated proteins in L. monocytogenes after exposure to C2, C3, and C4 environmental stress conditions. Nodes represent proteins, while edges indicate their interactions. The edges are colorcoded to represent different types of interactions, including known (pink and light blue), predicted (green, red, and blue), and other (yellow, black, and grey) interactions.

Conclusions
Elucidating the mechanism of L. monocytogenes stress response implies knowledge about the proteins involved in such pathways and how their expression is regulated. In this respect, the results of this study may be utilized to understand L. monocytogenes metabolism when it is exposed to different stress factors and how these conditions can induce the production of specific proteins involved in the pathogenic pathways. Overall, stress response entailed different pathways, but some proteins seemed to be associated with stress conditions, namely osmotic, acidic, and cold stress. These findings indicate the usefulness of a deep knowledge of the proteomic characteristics of different L. monocytogenes strains, as their response could vary and be linked to the peculiar environmental conditions encountered during food production and storage.
The presence of certain proteins in the proteome, particularly those included in the "pathogenic pathway", is the final proof of the pathogenicity of certain isolates, which cannot be based only on the presence of the codifying gene. The characterization of the other proteins detected in this study by means of bioinformatic tools (i.e., VirulentPred and Vaxijen) is already in progress, with the aim of identifying the potential immunogenic proteins involved in the virulence pathways. Future investigations might involve the development and optimization of a CRISPR-Cas-assisted recombineering system to facilitate bacterial genetic manipulation [67]. Using this system, point mutations, deletions, insertions, and gene replacements can be easily generated on the chromosome or native plasmids in L. monocytogenes. In this way, the pathogenic role of these proteins could be confirmed.