Anti-infective activities of long-chain fatty acids against foodborne pathogens

Abstract Free fatty acids (FFAs) have long been acknowledged for their antimicrobial activity. More recently, long-chain FFAs (>12 carbon atoms) are receiving increased attention for their potent antivirulence activity against pathogenic bacteria. In the gastrointestinal tract, foodborne pathogens encounter a variety of long-chain FFAs derived from the diet, metabolic activities of the gut microbiota, or the host. This review highlights the role of long-chain FFAs as signaling molecules acting to inhibit the infectious potential of important foodborne pathogens, including Salmonella and Listeria monocytogenes. Various long-chain FFAs interact with sensory proteins and transcriptional regulators controlling the expression of infection-relevant genes. Consequently, long-chain FFAs may act to disarm bacterial pathogens of their virulence factors. Understanding how foodborne pathogens sense and respond to long-chain FFAs may enable the design of new anti-infective approaches.


Introduction
T he concern o v er antibiotic r esistance is incr easing, and ne w strategies for combating bacterial infections are needed (Kwon and Po wderly 2021 ). F ree fatty acids (FFAs) have long been known for their antimicrobial activity, and FFAs ar e, ther efor e, consider ed as possible alternatives or complements to classical antibiotics (Desbois andSmith 2010 , Yoon et al. 2018 ).
FFAs consist of a saturated or unsaturated carbon chain, and a terminal carboxy group (Table S1 , Supporting Information). Since the carbon chain is hydrophobic and the carboxyl group is hydr ophilic, FFAs ar e defined as amphipathic molecules holding the ability to drive spontaneous self-assembly (Yoon et al. 2018 ). The biological activity of FFAs depends on the length of the carbon c hain, whic h r anges fr om short ( < 8 carbon atoms) to medium (between 8 and 12 carbon atoms) and long ( > 12 carbon atoms). Furthermore, the number, position, and orientation ( cis vs. trans ) of double bonds determine the structure and functionality of unsaturated FFAs. In general, medium-chain saturated FFAs and longc hain unsatur ated FFAs ar e the most activ e a gainst bacteria, and Gr am-positiv e species appear to be more susceptible to antimicrobial FFAs relative to Gram-negative species (Desbois andSmith 2010 , Yoon et al. 2018 ). For long-c hain unsatur ated FFAs, the antimicr obial activity incr eases with length and degr ee of unsatur ation. Reported minimal inhibitory concentrations (MIC values) of medium-and long-chain FFAs often differ between studies due to variations in experimental conditions (i.e. growth medium, temper atur e, and pH) (Yoon et al. 2018 ). Furthermore, the use of different or ganic solv ents for solubilizing the antimicr obial FFAs likel y affects their concentration-dependent molecular self-assembly (Yoon et al. 2018 ).
In biological systems, FFAs are typically bound to other compounds to form lipids , e .g. triglycerides in dietary fat, or phospholipids in membranes, but they can be released as FFAs by the enzymatic activities of lipases. Since FFAs serve as nutrient sources and are vital for building membranes, bacteria willingly take up FFAs from the environment (Yao and Rock 2017 ). Inside the cytosol, exogenous FFAs are activated in an organism-specific manner and may then be degraded to generate energy or serve as membrane building blocks. In the absence of exogenous FFAs, bacteria must synthesize these building blocks to sustain growth (Radka and Rock 2022 ). The bacterial fatty acid biosynthesis type II system (FASII) performs all steps r equir ed for de novo synthesis of fatty acids, and FASII enzymes ar e, ther efor e, essential for growth in environments devoid of FFAs.
FFAs are used as antimicrobial agents by many organisms to defend themselv es a gainst micr obial pathogens . T he human skin, r espir atory tr act, and gastr ointestinal tr act r epr esent thr ee major en vironments , where bacterial pathogens encounter antimicrobial FFAs produced by the host or resident microbiota (Fischer 2020, Metzler-Zebeli et al. 2021 ). Due to their amphipathic nature, medium-and long-chain FFAs with antimicrobial properties are thought to target and accumulate within the bacterial membrane (Desbois andSmith 2010 , Yoon et al. 2018 ). High doses of antimicrobial FFAs may destabilize the bacterial membrane, leading to increased cell permeability and cell lysis . Furthermore , FFAs ma y interfere with essential biological processes in the membrane, suc h as ener gy pr oduction and n utrient uptak e, in direct or indirect wa ys . Importantly, bacteria ma y sense the presence of FFAs and build up pr otectiv e r esponses a gainst their to xic acti vities (Desbois andSmith 2010 , Kengmo Tchoupa et al. 2022 ). To defend themselv es a gainst FFAs , bacteria ma y pr oduce a mor e pr otectiv e cell surface to pr e v ent FFAs from reaching the cell membrane. Furthermore, some pathogens are known to encode efflux systems acting to export FFAs from the cell, or detoxification factors that inactiv ate antimicr obial FFAs.
Although resistance development potentially could limit the use of FFAs as antimicrobial agents in the future, recent findings indicate that they may be used in alternative ways to combat pathogenic bacteria. At low doses, medium-and long-chain FFAs act as signaling compounds controlling important traits of bacterial pathogens, such as biofilm formation and virulence (Kumar et al. 2020, Mirzaei et al. 2022, Mitchell and Ellermann 2022. Specific FFAs modulate the activity of sensory proteins and transcriptional regulators to suppress the expression of k e y virulence genes r equir ed for establishing a successful infection. Notably, medium-and long-chain FFAs without antimicrobial activity may act to reduce the virulence of a pathogen, suggesting that some FFAs hold potential as anti-infectiv e a gents without affecting the growth and survival of the resident microbiota. This r e vie w will focus on how long-chain FFAs inhibit the growth and/or virulence-potential of foodborne pathogens. Specifically, we wish to address how this knowledge could be used against important foodborne pathogens in the future. To illustrate some of the central findings, we will focus on two major foodborne pathogens, the Gr am-negativ e bacterium Salmonella enterica ser ov ar Typhim urium , and the Gr am-positiv e bacterium Listeria monocytogenes . For detailed information on how long-chain FFAs affect other bacterial pathogens (e.g. Staphylococcus aureus and Pseudomonas aeruginosa ), we refer to recent reviews covering these organisms (Kumar et al. 2020, Kengmo Tchoupa et al. 2022 ).
Importantly, most fatty acids in food are bound to glycerol in the form of trigl ycerides, whic h ar e not activ e a gainst bacteria. T hus , foodborne pathogens are not expected to encounter high le v els of fr eel y av ailable long-c hain FFAs until they r eac h the gastr ointestinal tr act (Chadaideh and Carmody 2021 ), unless they reside in fermented or partially spoiled foods, where lipases produced by other micr oor ganisms (e.g. Pseudomonas spp.) catal yze the release of long-chain FFAs from triglycerides in the foods (Arslan et al. 2011 ). In the intestine, dietary fat is emulsified by the actions of bile salts and FFAs ar e liber ated fr om the trigl ycerides through the enzymatic actions of pancreatic lipase. Longchain FFAs are predominantly incorporated into bile salt-mixed micelles, whic h incr eases the water solubility of the FFAs, thus priming them for uptake (Murota and Storch 2005 ). Then, FFAs are absorbed by the epithelial cells of the small intestine. Dietary fat affects the composition and function of the natural microbial inhabitants of the gut, and the gut microbes contribute to the ov er all fatty acid metabolism in the gastr ointestinal tr act (Agans et al. 2018, Martinez-Guryn et al. 2018, Saika et al. 2019, Chadaideh and Carmody 2021, Mirzaei et al. 2022. The gut microbiota is w ell kno wn to pr oduce short-c hain fatty acids (SCFA), with important roles as anticarcinogenic and anti-inflammatory agents. Mor eov er, SCFAs act to modulate the expression of bacterial virulence factors. For an ov ervie w on how SCFAs influence foodborne pathogens, we refer to a recent review covering this topic (Mirzaei et al. 2022 ).
Long-chain fatty acids produced by the host and intestinal bacteria play important roles in the regulation of human health and diseases. Conjugated linoleic acid (CLA), which is an isomer of linoleic acid (C18:2) with conjugated double bonds, is produced by enzymatic activities of the gut microbiota (O'Shea et al. 2012, Salsinha et al. 2018 (Table S1 , Supporting Information). CLA has gained significant attention due to its wide range of potential health benefits, including anticarcinogenic, antiobese, antidiabetic, and anti-inflammatory effects (Koba andYanagita 2014 , Saika et al. 2019 ). Notabl y, CLA also exerts anti-infectiv e activities against some enteric pathogens. Recent studies suggest that unknown members of the gut microbiota produce a cis -2 unsatur ated long-c hain FFA, whic h belongs to a famil y of diffusible signal factors (DSF)   (Table S1 , Supporting Information). Inter estingl y, specific DSFs hav e been found to inhibit the production of virulence factors in Salmonella and Vibrio cholerae . In line with this, se v er al unsatur ated long-c hain FFAs pr esent in bile, such as palmitoleic acid (C16:1) and oleic acid (C18:1), are known to affect the virulence-potential of enteric pathogens (Table S1 , Supporting Information) (Chatterjee et al. 2007, Golube v a et al. 2016. Altogether, foodborne pathogens are expected to encounter a v ariety of biologicall y activ e long-c hain fatty acids deriv ed fr om the diet, host, and gut micr obiota in the gastr ointestinal tr act. Although the concentrations and bioavailability of long-chain FFAs in the human gut remain unknown, recent studies in pigs revealed that the fatty acids profiles vary along the gastrointestinal tract, with long-chain FFAs found at higher levels in the stomach, cecum, and colon, r elativ e to the jejunum and ileum of the small intestine (Metzler-Zebeli et al. 2021 , Mitchell andEllermann 2022 ). T hus , foodborne pathogens ma y use long-chain FFAs as signaling molecules to establish a successful infection at the right time and place during intestinal passage. In the following sections, examples of how Salmonella , L. monocytogenes , and related bacteria sense and respond to long-chain FFAs will be presented.

Long-chain FFAs inhibit the activity of Ar aC-famil y regula tors in Salmonella and rela ted Gr am-nega ti v e pathogens
The Gr am-negativ e pathogen S. Typhim urium is the causativ e agent of both acute gastroenteritis and systemic diseases (Menard et al. 2022 ). Salmonella infections are often associated with ingestion of contaminated food pr oducts, suc h as eggs and c hic ken, or consumption of water contaminated with feces from infected hosts (Velge et al. 2012 ). At the initial stages of infection, Salmonella invades the intestinal epithelial cells using a type III section system (T3SS) encoded by the Salmonella pathogenicity island-1 (SPI-1) (Patel and Galan 2005 ). The needle-like T3SS a ppar atus injects bacterial effector proteins into the cytosol of the infected host cells, leading to r earr angements of the actin cytoskeleton and activation of inflammatory pathwa ys . A second Salmonella pathogenicity island, SPI-2, encodes another T3SS system, whic h is r equir ed for intr acellular gr owth and surviv al of this pathogen (Jennings et al. 2017 ). The SPI-2 T3SS translocates a broad range of effector proteins with diverse biochemical functions from Salmonella -containing vacuoles into the host cells.
Medium-length satur ated FFAs, suc h as ca prylic acid (C8:0) and lauric acid (C12:0) (Table S1 , Supporting Information), show gr owth-inhibitory activity a gainst Salmonella species, wher eas the antimicrobial activity of long-chain FFAs is limited against these Gr am-negativ e bacteria (P etscho w et al. 1998, Skriv anov a et al. 2004, Van Immerseel et al. 2004, Shin et al. 2007 ). Although S. Typhim urium gr o ws w ell in the pr esence of high le v els of long-c hain FFAs, they do not leave this pathogen unaffected. Specific longchain FFAs act as signaling molecules that modulate expression of virulence genes in S. Typhimurium by affecting the activities of k e y virulence regulators of SPI-1, SPI-2, and other virulencer ele v ant genes (Viar engo et al. 2013, Golube v a et al. 2016 ). In the following, the effect of long-chain FFAs on AraC-like transcriptional regulators controlling SPI-1 will be addressed.
The genes encoding T3SS from SPI-1 are controlled by the transcriptional regulator HilA (Bajaj et al. 1995 , Eichelberg andGalan 1999 ). This regulator is positively controlled by three other regulators, HilD, HilC, and RtsA; together, these regulators form a feed-forw ar d loop controlling the expression of SPI-1 T3SS (Ellermeier et al. 2005 ) (Fig. 1 ). Inter estingl y, exposur e to satur ated or unsatur ated long-c hain FFAs down-r egulates tr anscription of hilA , leading to decreased expression of SPI-1 genes (Golube v a et al. 2016 ). Specifically, oleic acid (C18:1), myristic acid (C14:0) (Table S1 , Supporting Information), and palmitic acid (C16:0) exert an inhibitory effect on hilA tr anscription. Salmonella Typhim urium can take up and use long-chain FFAs as an energy source, but degradation of these FFAs is not r equir ed for r epr ession of virulence genes (Golube v a et al. 2016 ). Indeed, mouse infection experiments with mutant strains defective in the uptake and/or metabolism of long-chain FFAs sho w ed that Salmonella primarily uses long-chain FFAs as signaling compounds to dictate the expression of T3SS at the right time and place. Regarding the mechanism underlying the anti virulence acti vity of long-c hain unsatur ated FFAs, experiments with oleic acid (C18:1) r e v ealed that this FFA acts dir ectl y on HilD to pr e v ent the r egulator fr om binding to DNA (Golube v a et al. 2016 ) (Fig. 1 ). Consequentl y, tr anscription of hilA is suppr essed in response to long-chain unsaturated FFAs, leading to reduced expression of T3SS from SPI-1.
HilD belongs to the Ar aC-famil y of proteins, and other members of this protein family have been shown to respond directly to long-chain FFAs . T he best studied example is the virulence regulator ToxT in V. cholerae . To xT acti v ates tr anscription of ctxAB and tcpA encoding c holer a toxin and the toxin-coregulated pilus, whic h ar e r equir ed for V. c holerae to cause disease (Peterson and Gellings 2018 ). The To xT regulator acti vates transcription by binding as a monomer or dimer to specific to x-bo xes located in the promoter regions of To xT-acti vated genes (Withey and DiRita 2006 ). Initiall y, experiments wer e conducted with crude bile to identify Figure 1. Long-chain FFAs inhibit the expression of SPI-1 virulence genes in Salmonella . The regulatory proteins HilD, HilC, and RtsA form a feed-forw ar d loop controlling the virulence regulator HilA, which activ ates expr ession of SPI-1 genes. Long-c hain FFAs, suc h as oleic acid (C18:1) and the DSF compound c2-HDA (Table S1 , Supporting Information), interfere with the regulatory activity of HilD. Furthermore, c2-HDA promotes Lon-mediated degradation of HilD and inhibits the regulatory activities of HilC and RtsA.
components responsible for repression of virulence genes in V. cholerae (Chatterjee et al. 2007 ). The expression of cholera toxin was found to be efficiently repressed by the fatty acid-containing component of bile, and five individual long-chain FFAs were isolated from this fraction for further analyses . T he long-chain unsaturated FFAs arachidonic acid (C20:4) (Table S1 , Supporting Information), linoleic acid (C18:2), and oleic acid (C18:1) were found to inhibit c holer a toxin pr oduction, wher eas the long-chain saturated FFAs palmitic acid (C16:0) and stearic acid (C18:0) had no effect. Notabl y, high le v els of the thr ee unsatur ated long-c hain FFAs had a growth-inhibitory effect as well on V. cholerae (Chatterjee et al. 2007 ). In later studies, structur al anal yses of ToxT revealed that palmitoleic acid (C16:1) was tightly bound to a ToxT monomer (Lowden et al. 2010 ). Palmitoleic acid (C16:1), oleic acid (C18:1), and linoleic acid (C18:2) were found to inhibit transcription of tcp and ctxAB , and they pr e v ented ToxT fr om binding to the pr omoter r egions of virulence genes (Lowden et al. 2010 , Plecha and. In contrast, the saturated FFA palmitic (C16:0) had no effect on the regulatory and DNA-binding activity of ToxT (Lowden et al. 2010 ). Further structural and mutational studies supported a model where FFA-free ToxT adopts an active conformation that can dimerize and bind DNA, whereas binding of unsatur ated long-c hain FFAs to ToxT results in an inactive conformation that precludes dimerization and binding to DNA (Childers Since linoleic acid (C18:2) is a potent inhibitor of To xT acti vity, the antivirulent effect of commercially acquired CLA was investigated in V. cholerae . CLA was found to inhibit the production of c holer a toxin and toxin-coregulated pilus in vitro , and it reduced the DNA-binding activity of T oxT . Interestingl y, administr ation of CLA reduced the production of cholera toxin in a rabbit ileal loop model for c holer a disease. Furthermor e, c holer a toxin-induced fluid accumulation was reduced upon administration of CLA in vivo . These findings suggest that CLA could be used as a ther a peutic a gainst c holer a.
Enter ohemorrha gic Esc hericia coli (EHEC) is known to cause sev er e diarrhea upon consumption of contaminated foods (Khalil et al. 2018 ). Gut colonization by EHEC relies on the production of a variety of adhesive pili, and their expression is controlled by the Ar aC famil y member Rns (Munson 2013 ). Structur al studies on Rns r e v ealed the pr esence of the medium-c hain satur ated FFA decanoic acid (C10:0) (Table S1 , Supporting Information) bound in a pocket within Rns (Midgett et al. 2021 ). Importantl y, exposur e to decanoic acid (C10:0) inhibits Rns-dependent expression of pili in EHEC. The structur al mec hanism by whic h Rns is regulated by decanoic acid (C10:0) remains to be clarified, but this study supports that medium-chain saturated FFAs also act to control virulence gene expression in enteric pathogens by binding dir ectl y to an Ar aC famil y member (Fig. 2 ). Inter estingl y, it should be noted that the medium-chain saturated FFA caprylic acid (C8:0) exerts dual effects on Salmonella . As mentioned abo ve , caprylic acid (C8:0) sho ws gro wth-inhibitory activity against Salmonella , but at subinhibitory concentrations, this fatty acid acts to reduce transcription of hilA (Van Immerseel et al. 2004 ). The mechanism underlying the inhibitory effect of caprylic acid (C8:0) on hilA expression remains to be uncovered. Clearly, future studies should address whether medium-c hain satur ated FFAs affect the activity of the thr ee Ar aC-like r egulators contr olling the expr ession of hilA .
Recent findings further substantiate a role for long-chain unsaturated FFAs in controlling the activity of regulators of the AraC family in enteric bacteria. A search for chemicals acting to inhibit invasion gene expression in S . Typhimurium resulted in the identification of fatty acids belonging to the DSF-class of quorum sensing signaling molecules (Bosire et al. 2020 ). DSF molecules ar e cis -2 unsatur ated fatty acids pr oduced by Gr am-negativ e bac-teria, and they are known to control important biological processes in plant pathogens, including biofilm formation and virulence (Kumar et al. 2020. The DSF compound cis -2-hexadecenoic acid (c2-HDA) has a chain length of 16 carbons with a single cis unsaturation at the second carbon (Table S1 , Supporting Information). Inter estingl y, c2-HDA acts to inhibit the expression of SPI-1 virulence genes hilA and sopB in S . Typhimurium and the c holer a toxin-encoding genes ctxAB in V. cholerae (Bosire et al. 2020 ). Indeed, c2-HDA is a more efficient inhibitor of virulence gene expression in S . Typhimurium than the unsaturated longchain FFA oleic acid (C18:1) from bile. In Salmonella , HilD is the primary target for c2-HDA activity, and the DSF-compound pr e v ents HilD from binding to DNA. Notably, c2-HDA also acts to destabilize the HilD protein via Lon protease activity (Bosire et al. 2020, Chowdhury et al. 2021b (Fig. 1 ). In comparison, oleic acid (C18:1) inhibits the DNA-binding activity of HilD, but this FFA does not affect the stability of HilD. Exposure to c2-HDA, as well as oleic acid (C18:1), reduces invasion of epithelial cells, suggesting that long-c hain unsatur ated FFAs could act to pr e v ent Salmonella fr om causing intestinal infections.
So far, no dietary sources for cis -2-unsaturated fatty acids are known, but a recent study suggests that Salmonella and other intestinal pathogens may well encounter DSF compounds in the gut (Chowdhury et al. 2021b ). Mor e specificall y, c2-HDA could be extracted from the large intestinal content of mice, showing that this signaling compound indeed is present in the mammalian intestine. Structural and mutational studies identified differ ent r esidues of HilD r equir ed for r epr ession by v arious types of long-chain FFAs. For c2-HDA, the carboxylate head group as well as the cis -2 unsaturation are important for recognition by HilD. Notabl y, c2-HDA r eadil y outcompetes other intestinal long-chain fatty acids for HilD-mediated r epr ession of inv asion, suggesting that Salmonella uses this specific signal over other fatty acids to contr ol inv asion in the intestinal environment (Chowdhury et al. 2021b ). Intriguingl y, r ecent findings show that the AraC-like virulence regulators HilC and RtsA respond to the presence of c2-HDA as w ell (Cho wdhury et al. 2021a ) (Fig. 1 ). As mentioned abo ve , the HilC and RtsA r egulators, together with HilD, ensur e complete activation of SPI-1 by activating transcription of each other as well as hilA . Indeed, c2-HDA can bind dir ectl y to all thr ee r egulators, perturbing their interaction with hilA promoter DNA, but in contrast to HilD, the stability of HilC and RtsA is not affected by c2-HDA (Chowdhury et al. 2021a , b ). Since HilC and RtsA are not degraded, HilD pr otein le v els ar e r a pidl y r estor ed when c2-HDA signaling ends, leading to induction of SPI-1 expression. HilC and RtsA are gener all y less r esponsiv e to c2-HDA compared to HilD, enabling Salmonella to regain virulence more quickly after removal of c2-HDA (Chowdhury et al. 2021a ).
Altogether, these findings demonstrate that c2-HDA and other long-chain FFAs act as signaling molecules to modulate bacterial virulence by direct binding to AraC-like transcription activators (Fig. 2 ). The FFAs encountered by foodborne pathogens during passage of the intestinal tract may be derived from food sour ces, b y activities of the host or the resident microbiota. Ho w ever, the specific origin of the most potent fatty acid inhibitor of Salmonella virulence, c2-HDA, is not yet known. DSF compounds, including c2-HDA, ar e most likel y pr oduced by Gr am-negativ e species of the intestinal microbiota, serving as one of the many signals sensed by Salmonella and other enteric pathogens during intestinal passage . Ultimately, these signaling molecules determine where and when Salmonella chooses to initiate the infection program encoded from SPI-1. Intriguingly, a recent study found that r ecombinant pr oduction of c2-HDA by E. coli in the gut of c hic kens could inhibit Salmonella invasion and animal carriage (Rather et al. 2023 ). Further investigations are required to evaluate the practical use of in situ production of c2-HDA to reduce the carriage of Salmonella in production animals (Rather et al. 2023 ).

Long-chain FFAs affect the activity of the two-component system PhoP/PhoQ in Salmonella
The two-component system PhoP/PhoQ plays a major role in controlling the expression of virulence-related genes in Salmonella , including SPI-1 and SPI-2 genes (Groisman et al. 2021 ). In the presence of an activating signal, the transmembrane sensor PhoQ autophosphorylates, after which the phosphoryl group is transferred to the cytoplasmic response regulator PhoP. Phosphorylated PhoP exerts its role as transcriptional regulator by binding to specific sequences in the promoter regions of PhoP/PhoQ regulated genes (Zwir et al. 2012 ). In the absence of an activating signal, PhoQ dephosphorylates PhoP, which attenuates its transcriptional regulator activity. Among the PhoQ activating conditions are Mg 2 + limitation, specific cationic antimicrobial peptides, low pH, and high osmolarity (Garcia Vescovi et al. 1996, Bader et al. 2005, Prost et al. 2007, Yuan et al. 2017 (Fig. 3 ). Intriguingly, a screen for molecules from plant extracts that would modulate PhoP/PhoQ activity led to the identification of unsaturated longchain FFAs as inhibitory input signals (Viarengo et al. 2013 ). More specificall y, unsatur ated long-c hain FFAs of the C16 and C18 series were found to reduce the expression of genes activated by PhoP, wher eas the satur ated long-c hain FFAs palmitic acid (C16:0) and stearic acid (C18:0) had no effect on PhoP-dependent activation of transcription. The inhibitory effect does not r el y on cellular uptake of fatty acids or a functional metabolic β-o xidati ve pathway, demonstrating that exogeneous unsaturated long-chain FFAs are sensed directly by the two-component system (Viarengo et al. 2013 ) (Fig. 3 ). Indeed, C18:2 FFAs were shown to inhibit the autokinase activity of PhoQ by a mechanism that involves a conformational change in the periplasmic sensor domain (Carabajal et al. 2020 ). Accordingl y, PhoP-dependent r egulation of gene expression is repressed upon exposure to these unsaturated longchain FFAs.
CLA, corresponding to conjugated C18:2, inhibits PhoQ autokinase activity as well, and the effect of oral administration of CLA on Salmonella -induced colitis in mice was, ther efor e, tested (Carabajal et al. 2020 ). Notably, the animals were pretreated with Figure 3. Long-c hain unsatur ated FFAs inhibit the PhoP/PhoQ two-component system in Salmonella . The histidine kinase PhoQ autophosphorylates in the presence of an activating signal, such as Mg 2 + limitation, cationic antimicrobial peptides (CAMPs), low pH, and high osmolarity. The phosphoryl group is transferred to the response r egulator PhoP, whic h acts as a tr anscription r egulator of virulence genes. Long-c hain unsatur ated FFAs, suc h as palmitoleic acid (C16:1) and linoleic acid (C18:2) (Table S1 , Supporting Information), interfere with the autokinase activity of PhoQ, thereby preventing phosphorylation of the cytoplasmic response regulator PhoP. Consequentl y, PhoP-dependent r egulation of virulence genes is r epr essed. OM: outer membrane. IM: inner membrane. stre ptom ycin to provide a more robust model for Salmonellainduced colitis. In this model, phoP primarily contributes to the later stages of infection, where Salmonella reaches the spleen (Carabajal et al. 2020 ). Curiously, mice treated with CLA turned out to be more susceptible to Salmonella infection compared to untr eated contr ols, most likel y r eflecting the complex interplay between bacterial and host responses when exposed to dietary CLA supplementation.

Long-chain FFAs control the activity of FadR in Salmonella and related Gram-negati v e pathogens
In Gr am-negativ e enteric pathogens, the metabolism of exogenous long-chain FFAs has been linked to infection via the regulatory actions of the GntR/TetR family regulator, FadR (Pifer et al. 2018, Ellermann et al. 2021. When long-chain FFAs are taken up from the environment, they are esterified in the cytoplasm into long-chain acyl coenzyme A (CoA) thioesters (Fujita et al. 2007 ). Long-chain acyl-CoAs serve as substrates for membrane biosynthesis or ener gy pr oduction, and they are directly sensed by the F adR regulator. F adR binds the CoA moiety of the activated fatty acids, resulting in a decreased affinity for DNA (van Aalten et al. 2000 ). In its unbound form, FadR binds to DNA and activates the fab genes encoding the fatty acid biosynthesis pathway, whereas the fatty acid degradation machinery, encoded by the fad genes, is under negative control by FadR (Fujita et al. 2007 ). During the intestinal phase of infection, the regulatory activities of FadR contribute to gut colonization of Salmonella (Hoshino et al. 2022 ). More specificall y, FadR affects the expr ession of genes involv ed in fla gellar motility, and proper flagellar motility is required for colonization of the gut. Notably, fatty acid metabolism by β-oxidation seems to be dispensable during colonization of the gut, supporting that long-chain FFAs are primarily used by Salmonella as a signaling molecule, rather than an energy source in the gut (Hoshino et al. 2022 ). Ho w e v er, during the systemic phase of infection, fatty acid metabolism appears to be required for tissue colonization (Reens et al. 2019 ).
FadR was r ecentl y shown to play an important role in virulence gene expression in EHEC (Pifer et al. 2018, Ellermann et al. 2021. FadR is an activator of genes belonging to the locus of enterocyte effacement (LEE) pathogenicity island encoding a type 3 secretion system (T3SS). The T3SS injects effector molecules into colonic e pithelial cells, where the y promote cytosk eletal r earr angements known as the attaching and effacing (A/E) lesions c har acteristic for EHEC disease. Upon exposure to long-chain fatty acids, including palmitic acid (C16:0) and ar ac hidonic acid (C20:4), the FFAs are taken up by EHEC and converted into acyl-CoA deri vati ves. Next, FadR-dependent regulation of LEE is modulated by direct binding of the long-chain acyl-CoAs to FadR, leading to decreased binding of FadR to its DNA motifs and, consequently, inhibition of EHEC virulence. Indeed, treatment of EHEC with ar ac hidonic acid (C20:4) pr e v ents this pathogen from forming the characteristic A/E lesions on epithelial cells in a FadR-dependent manner (Ellermann et al. 2021 ). Inter

Dietary supplementation of omega-3 fatty acids affects the course of Salmonella infection
Dietary fish oil rich in omega-3 long-chain polyunsaturated fatty acids is well known for its various health beneficial effects, and omega-3 fatty acids ar e widel y r ecommended for tr eating c hr onic inflammatory diseases , heart diseases , type 2 diabetes and autoimmune diseases, amongst others (Husson et al. 2016 ). More specificall y, long-c hain pol yunsatur ated FFAs serv e as pr ecursors for eicosanoids, which play important roles as signaling molecules in diverse biological processes, including immunomodulatory roles (Sheppe and Edelmann 2021 ). In the past, mouse infection studies demonstrated that a diet rich in fish oil for 4 weeks decr eases surviv al after per or al c hallenge with S. Typhim urium (Chang et al. 1992 ). In line with this, the bacterial counts in the liver and spleen were higher in mice fed on fish oil diet compared to mice fed on a low-fat diet, following intr a peritoneal infection to mimic a systemic infection (Chang et al. 1992 ). In contr ast, a mor e r ecent study r eported a pr otectiv e effect of the longc hain pol yunsatur ated omega-3 fatty acids eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) against Salmonella infection (Liu et al. 2020 ). Mice fed with an omega-3 enriched diet for 7 days before infection with S. Typhimurium demonstrated an incr eased surviv al r elativ e to mice fed with an omega-6 enric hed diet or a regular diet. Salmonella was found to be phagocytized mor e efficientl y by macr opha ges in the pr esence of the omega-3 FFAs, and the intestinal production of SCFAs with antibacterial and anti-inflammatory properties was significantly increased. In line with this, the composition of the intestinal microbiota was changed in a direction that likely promotes host resistance tow ar ds Salmonella infection. Notabl y, gr owth of Salmonella is not inhibited by the omega-3 FFAs, and virulence gene expression is unaffected in the presence of omega-3 FFAs (Liu et al. 2020 ). These findings suggest that the long-c hain pol yunsatur ated omega-3 FFAs pr otects a gainst Salmonella infections by inducing beneficial changes to gut microbiota and host immune responses.
Differences in the experimental setup and methodological analyses (e.g. 4 weeks of feeding on a fish oil diet vs. 7 days on a diet enriched in omega-3 FFAs) may well explain the discrepancies observ ed between r esults of the mouse infection studies (Chang et al. 1992, Liu et al. 2020. Clearl y, mor e r esearc h on long-c hain pol yunsatur ated FFAs and eicosanoid functions ar e r equir ed to fully understand their roles in the course of bacterial infections (Sheppe and Edelmann 2021 ).

Long-chain FFAs exert antimicrobial activity against L. monocytogenes and related intestinal Gram-positi v e bacteria
Listeria monocytogenes can be found in a variety of food-processing environments and food products, and infections by this Grampositiv e pathogen ar e commonl y associated with the consumption of ready-to-eat foods (Kallipolitis et al. 2020, Koopmans et al. 2023 ). Listeria monocytogenes is well known for its ability to grow and survive under a wide range of stressful environmental conditions, including stress factors encountered along the food chain, such as acidic pH, osmotic stress, and low temperatures (Bucur et al. 2018 ). After consumption of contaminated foods, L. monocytogenes enters the gastrointestinal tract leading to gastroenteritis in otherwise healthy individuals. Listeria monocytogenes can cross the intestinal barrier and cause life-threatening conditions such as meningitis and encephalitis in susceptible individuals, or stillbirth and spontaneous abortion in pregnant women (Fr eita g et al. 2009, Koopmans et al. 2023. Listeria monocytogenes has been found in dairy foods and several studies in the past focused on elucidating the antilisterial effect of fatty acids typical for milk (Wang and Johnson 1992, Petrone et al. 1998, Sprong et al. 2001. They found that mediumc hain satur ated FFAs, suc h as lauric acid (C12:0), ar e most activ e a gainst L. monocytogenes , together with long-c hain unsaturated FFAs of the C18 series. Listeria monocytogenes is gener all y sensitive to w ar ds mono-and pol yunsatur ated long-c hain FFAs, including the omega-3 FFA eicosapentaenoic acid (C20:5) (Shin et al. 2007, Sternkopf Lillebaek et al. 2017, Chen et al. 2021 ). The specific mechanisms by which medium-and long-chain FFAs act against L. monocytogenes is presently unclear, but electron microscopic analyses of FFA-treated cells revealed a filamentous appearance and slightly irregular cell surface, supporting that the membrane is the primary target for antimicrobial medium-and long-chain FFAs (Wang and Johnson 1992 ).
Not sur prisingl y, bacteria ar e ca pable of de v eloping r esistance against the antimicrobial activity of FF As. Indeed, FF A-tolerant str ains wer e r eadil y isolated by serial passa ge of L. monocytogenes in broth with increasing concentrations of antimicrobial mediumand long-chain FFAs (Thomasen et al. 2022a , b ). Genome sequencing of FFA-tolerant isolates revealed mutations in genes involved in glycosylation of wall teichoic acids (WTAs) (Thomasen et al. 2022a ). Lack of N -acetylglucosamine (GlcNAc) glycosylation on WTAs allo ws gro wth in the presence of high le v els of lauric acid (C12:0), palmitoleic acid (C16:1), and eicosapentaenoic acid (C20:5) (Thomasen et al. 2022a ). Furthermore, absence of GlcNAc slightly increase the sensitivity to w ar d the antimicrobial peptide CRAMP, and WTA glycosylations are known to confer decreased susceptibility to w ar d gentamicin and ampicillin (Meireles et al. 2020 ). The FFA-tolerant isolates are characterized by a more hydrophilic cell surface and reduced binding of FFAs to the bacterial surface (Thomasen et al. 2022a ). Most likel y, these m utants ar e better pr otected fr om antimicr obial FF As, because the FF As ar e r epulsed from their surface (Fig. 4 ). A different subset of FFA-tolerant isolates was found to carry mutations in the ccpA gene encoding the catabolite control protein A, CcpA, a major transcriptional regulator of carbon catabolite r epr ession in L. monocytogenes (Thomasen et al. 2022b ). Inactiv ation of ccpA pr omotes gr owth in the pr esence of lauric acid (C12:0) and palmitic acid (C16:1) and results in a mor e hydr ophilic cell surface. Notabl y, m utations conferring tolerance to w ar d antimicrobial medium-and long-chain FFAs also support growth of L. monocytogenes at low pH (Thomasen et al. 2022a , b ).
Gr am-positiv e bacteria of the gut microbiota are affected by long-chain FFAs as well. For species of the genus Lactobacillus , growth in synthetic media is stimulated by the addition of Tween 80, which is rich in oleic acid (C18:1); furthermore, this growth supplement enhances survival of some Lactobacilli under acidic conditions, most likely due to the incorporation of exogenous long-chain FFAs in the bacterial membrane (Corcoran et al. 2007 ). Inter estingl y, some beneficial Lactobacillus species are inhibited by long-c hain unsatur ated FFAs linoleic acid (C18:2), oleic acid (C18:1), and α-linolenic acid (C18:3) (Table S1 , Supporting Information) (Di Rienzi et al. 2018 ), whic h ar e commonl y found in vegetable oil from soybeans (Jiang et al. 1998, Kankaanpaa et al. 2001, Jenkins and Courtney 2003. The consumption of soybean oil in the Western world has increased dramatically during the past century. During the same period, the pr e v alence of beneficial Lactobacilli has declined in Western gut microbiomes (Walter et al. 2011 ). The beneficial Gr am-positiv e bacteria Lactobacillus reuteri and Lactobacillus johnsonii still exist in people who consume a Western diet, suggesting that they are protected from antimicrobial FFAs in the gut, or that they become resistant to these compounds (Di Rienzi et al. 2018 ). In vitro evolution experiments demonstrated that L. reuteri and L. johnsonii become resistant to the toxic effects of linoleic acid (C18:2) through mutations in genes related to fatty acid metabolism, cell wall and membrane, and resistance to acid stress (Di Rienzi et al. 2018 ). The Lactobacilli persisted through both chronic and acute exposures to linoleic acid (C18:2) in the mouse gut. Inter estingl y, FFA-r esistant L. reuteri isolates wer e r etrie v ed fr om the small intestine of mice given a highfat diet, supporting that c hr onic exposur e to a diet high in linoleic acid (C18:2) promotes resistance in the resident population of L. reuteri (Di Rienzi et al. 2018 ). Altogether, Lactobacilli seem to be pr otected fr om the toxic effects of antimicrobial long-chain FFAs in the gut, and they are capable of developing resistance to w ar ds these compounds. Since FFA-tolerant mutants were readily isolated in vitro for L. monocytogenes (described above), it is tempting to speculate that intestinal pathogens may de v elop r esistance tow ar ds antimicrobial long-chain FFAs in the gut as well.

Long-chain FFAs with antibiofilm activity against L. monocytogenes
Belo w gro wth-inhibitory concentrations, FFAs are known to selectiv el y inhibit or disrupt biofilm formation by various pathogens (Kumar et al. 2020 ). Listeria monocytogenes forms disinfectanttolerant biofilms on a variety of surfaces in food production envir onments. Consequentl y, biofilms ar e of major concern to the food industry since they can lead to contamination of food products (Mazaheri et al. 2021, Osek et al. 2022. A high-throughput screen for antibiofilm agents identified saturated medium-and long-chain FFAs ranging from C9 to C14 as effective inhibitors of L. monocytogenes biofilm de v elopment, while longer satur ated FFAs (C16-C18) had stimulatory effects on biofilm formation (Nguyen et al. 2012 ). The concentrations used for testing antibiofilm activity did not change planktonic cell density, suggesting that saturated medium-and long-chain FFAs act as signaling molecules to modulate biological pathways involved in biofilm formation. Their antibiofilm targets in L. monocytogenes ar e pr esentl y unknown, but in Gr am-negativ e bacteria, satur ated FFAs are known to suppress quorum sensing signaling pathways controlling biofilm formation (Santhakumari et al. 2017, Kumar et al. 2020. Notably, the study by Nguyen et al. ( 2012 ) did not examine the effect of unsaturated medium-and long-chain FFAs on the formation of biofilms by L. monocytogenes . Furthermore, the study focused on single-species L. monocytogenes biofilms, which are consider ed mor e sensitiv e to disinfectants and antimicr obials r elativ e to the mixed-species biofilms commonly found in the food industry (Yuan et al. 2020 ). Uncovering the biofilm-inhibitory properties of saturated and unsaturated FFAs may support the future development of new antibiofilm strategies against L. monocytogenes in single-and/or mixed-species biofilms.

FFAs inhibit the activity of key virulence regulator PrfA in L. monocytogenes
Low le v els of medium-and long-c hain FFAs ar e known to r educe expression of virulence genes essential for the intracellular lifestyle of L. monocytogenes (Sternkopf Lillebaek et al. 2017, Dos Santos et al. 2020. During infection, L. monocytogenes enters and multiplies within a variety of host cells, and the pathogen spreads from cell-to-cell through a mechanism that involves actin polymerization (Fr eita g et al. 2009, Koopmans et al. 2023 (Fig. 5 A). Se v er al virulence factors contribute to the intracellular lifestyle of L. monocytogenes , including a pore-forming toxin, LLO, and ActA, whic h pr omotes actin pol ymerization and cell-to-cell spr ead. The tr anscription r egulator PrfA is essential for the expr ession of genes encoding LLO, ActA, and other k e y virulence factors (Scortti et al. 2007 ). Various signals fr om the envir onment ar e known to affect the le v el or activity of PrfA, such as temper atur e c hanges, the presence of readily utilizable carbohydrates, bacterial-and host-derived glutathione (GSH), or other peptides (Johansson and Fr eita g 2019 , Tiensuu et al. 2019 ) (Fig. 5 B). Curiousl y, exposur e to subinhibitory le v els of medium-c hain and long-c hain FFAs generates an inhibitory signal that pr e v ents PrfA-de pendent acti vation of virulence gene tr anscription (Sternk opf Lillebaek et al. 2017, Dos Santos et al. 2020. The antivirulence activity of medium-and long-chain FFAs seems to rely on the length of the carbon chain and degree of unsaturation (Dos Santos et al. 2020 ). Among the saturated FFAs, lauric acid (C12:0) and myristic acid (C14:0) exert PrfA-inhibitory activity, whereas palmitic acid (C16:0) and stearic acid (C18:0) have no effect on virulence gene expr ession. Exposur e to long-chain unsaturated FFAs, such as eicosapentaenoic acid (C20:5), linoleic acid (C18:2), and γ -linolenic acid (C18:3) (Table S1 , Supporting Information), leads to r epr ession of PrfA-dependent transcription as well (Sternkopf Lillebaek et al. 2017, Dos Santos et al. 2020 (Fig. 5 B). Importantly, the PrfA-inhibitory activity of a specific FFA correlates well with its ability to prevent PrfA from binding to DNA. These findings support a model where long-chain unsaturated FFAs and medium-chain saturated FFAs interfere dir ectl y with the DNA-binding activity of PrfA (Fig. 6 ). Some FFAs act as antivirulent agents only [e.g. myristic acid (C14:0) and oleic acid (18:1)], whereas others exert a dual inhibitory effect: at low (subinhibitory) doses, they show antivirulent activity, whereas at high doses, they pr e v ent bacterial gr owth [e.g. palmitoleic acid (C16:1) and lauric acid (C12:0)]. Importantl y, m utant str ains that grow in the presence of high le v els of antimicr obial medium-and longc hain FFAs r emain susceptible to the PrfA-inhibitory activity of FFAs (Thomasen et al. 2022a , b ). Thus, although L. monocytogenes becomes resistant to the antimicrobial activity of FFAs, it can still be targeted by their antivirulent activity.
Tr anscriptomic anal yses of L. monocytogenes exposed to v arious FFAs confirmed that specific medium-and long-chain FFAs act to down-regulate the expression of PrfA-dependent virulence genes (Chen et al. 2021, Thomasen et al. 2022b ). Exposure to a subinhibitory le v el of lauric acid (C12:0) r epr essed the expr ession of three genes ( plcA , hly , and actA ) belonging to the PrfA regulon, wher eas onl y a single gene, encoding the coc ha per one Gr oES, was significantl y up-r egulated after lauric acid-exposur e (Thomasen et al. 2022b ). Similarl y, tr eatments with MIC le v els of long-c hain unsaturated FFAs led to repression of PrfA-dependent virulence genes (Chen et al. 2021 ). Notably, up to 50% of the coding genes in L. monoc ytogenes w er e differ entiall y expr essed upon exposur e to gr owth-inhibitory le v els of palmitoleic acid (C16:1), linoleic acid (C18:2), and α-linolenic acid (C18:3) (Chen et al. 2021 ). Key genes associated with the response to acid, bile , osmotic , and o xidati ve str ess wer e found to be downr egulated in r esponse to antimicr obial FFAs. In L. monocytogenes , str ess r esponse genes ar e primaril y regulated by the alternative sigma factor Sigma B, which also controls the expression of prfA and other virulence-associated genes contributing to adaptation during the intestinal phase of infection (Tiensuu et al. 2019 ). Importantly, Sigma B is not r equir ed for growth in the presence of the antimicrobial FFAs palmitoleic acid (C16:1) and γ -linoleic acid (C18:3) (Sternkopf Lillebaek et al. 2017 ). Altogether, these findings suggest that exposure to antimicrobial FFAs does not activate the Sigma B-dependent str ess r esponse in L. monocytogenes . In contr ast, antimicr obial FFAs a ppear to downr egulate man y genes involv ed in the ada ptation to str essful conditions encountered in the gastrointestinal tract, including acid, bile, and osmotic stress (Chen et al. 2021 ). Intriguingly, a prfA m utant str ain can gr ow at high concentr ations of medium-and long-chain FFAs, suggesting that unknown regulatory activities of PrfA increases the sensitivity of L. monoc ytogenes to w ar d antimicrobial FFAs (Sternkopf Lillebaek et al. 2017 ). Indeed, in addition to the PrfA-dependent virulence genes described abo ve , PrfA is known to control the expression of multiple genes involved in str ess toler ance and gener al metabolism (Milohanic et al. 2003, Marr et al. 2006, Henderson et al. 2020. Clearly, PrfA plays a centr al r ole in the r esponse of L. monocytogenes a gainst antimicr obial and antivirulent medium-and long-chain FFAs (Sternkopf Lillebaek et al. 2017, Dos Santos et al. 2020.
Considering the inhibitory effects of specific medium-and long-chain FFAs against L. monocytogenes , they could have a protective effect to w ar ds infections b y this pathogen. Cell-based infection models are commonly used to evaluate the ability of L. monocytogenes to invade host cells, m ultipl y within them, and spread to the neighboring cells . T he Caco-2 enterocyte-like cellline has been used to examine how specific FFAs derived from milk affect the inv asiv eness of L. monocytogenes (Petrone et al. 1998 ). A total of 12 short-, medium-, and long-c hain FFAs wer e tested for their ability to protect Caco-2 fr om inv asion by L. monocytogenes . The FFA le v els used in this experiment did not affect the growth and survival of bacteria and Caco-2 cells, ho w ever, it should be noted that long-chain FFAs are known to upregulate the expression of E-cadherin, which serves as target for the listerial virulence factor InlA (described below) (Jiang et al. 1995 ). After 1 hour of FFA-exposure, the bacterial invasion of FFA-treated cells was reduced by 20-to 500-fold compared with untreated cells (Petrone et al. 1998 ). Among the FFAs tested, the unsatur ated long-c hain FFA linolenic acid (C18:3) had the most potent effect on invasion. In line with this, C18:3 exposure efficiently The internalins InlA and InlB promote bacterial entry into nonphagocytic host cells . T he pore-forming toxin LLO, and the phospholipases PlcA and PlcB, enable bacterial escape from the primary vacuole. Inside the cytosol, the pathogen multiplies and spreads to adjacent cells using the actin assembl y-inducing pr otein ActA. Finall y, LLO, PlcA, and PlcB promote bacterial escape from the secondary vacuole formed upon entry of L. monocytogenes into the neighboring cell. (B) The transcriptional regulator PrfA activates transcription of virulence genes required for the intracellular lifestyle of L. monocytogenes . The le v el or activity of PrfA is affected by v arious signals fr om the envir onment, suc h as high temper atur e, carbohydr ates, and glutathione (GSH). Furthermore, exposure to medium-chain saturated and long-chain unsaturated FFAs prevent PrfA from activating transcription of virulence genes encoding InlA, LLO, ActA, and so on. down-r egulates expr ession of virulence genes in L. monocytogenes , including PrfA-dependent transcription of inlA encoding the surface protein InlA, which is required for invasion of Caco-2 cells (Fig. 5 A) (Sternkopf Lillebaek et al. 2017, Dos Santos et al. 2020. Other FFAs with known PrfA-inhibitory activity, such as lauric acid (C12:0), oleic acid (C18:1), and linoleic acid (C18:2), are potent inhibitors of bacterial invasion as well (Petrone et al. 1998 ). The satur ated long-c hain FFA stearic acid (C18:0) is the least effectiv e in pr e v enting inv asion b y L. monoc ytogenes and this FFA does not affect the expression of PrfA-activated virulence genes, including inlA (Sternkopf Lillebaek et al. 2017, Dos Santos et al. 2020. These findings suggest that FFAs with PrfA-inhibitory activity are interesting candidates for antivirulence compounds acting to pr e v ent L. monocytogenes from invading enterocytes.

Fatty acid biosynthesis plays a role in the virulence of L. monocytogenes
The membrane phospholipids of L. monocytogenes are mainly composed of br anc hed-c hain fatty acids, whic h ar e synthesized by the FASII system (Sauer et al. 2019 ). Listeria monocytogenes encodes putative homologs of the core enzymes for bacterial type II fatty acid synthesis, including the β-ketoac yl-ac yl carrier protein synthase III (FabH), which carries out the first condensation reaction in fatty acid biosynthesis (Singh et al. 2009 ). The FabH enzyme in L. monocytogenes exhibits selectivity for anteiso br anc hed-c hain fatty acid pr ecursors, whic h r esults in a membr ane enric hed in this type of fatty acid. Importantly, a high content of anteiso branchedchain fatty acids increases membrane fluidity, which contributes to L. monocytogenes stress resistance and plays a role in bacterial pathogenesis (Sun and O'Riordan 2010 ). Anteiso br anc hedchain fatty acids enhance the resistance of L. monocytogenes tow ar d macr opha ge killing by pr otecting a gainst the antimicr obial pe ptides and pe ptidogl ycan hydr olases pr oduced in the pha gosome (Sun et al. 2012 ). Furthermore, conditions that support a high content of anteiso br anc hed-c hain fatty acids promote the production of LLO, suggesting that changes in membrane fluidity could alter the activity of PrfA (Sun et al. 2012 ). The molecular mec hanisms underl ying the effect of membr ane fatty acid composition on LLO production remains to be determined.
The eno yl-ac yl carrier pr otein r eductase I (FabI) plays a role in the elongation cycle of fatty acid synthesis (Yao et al. 2016 ). Inhibition of FabI, using a selective FabI inhibitor, prevents endogenous fatty acid synthesis by 80% and lo w ers gro wth of L. monocytogenes in laboratory medium (Yao et al. 2016 ). In the presence of a FabI inhibitor, supplementation with exogenous fatty acids partiall y r estor es normal gr owth in labor atory medium, and the exogenous fatty acids are incorporated into the membrane phospholipids. Importantly, inhibition of FabI during intracellular infection of host cells has detrimental effects on L. monocytogenes , which cannot be reverted by the acquisition of host cell fatty acids (Yao et al. 2016 ). Clearl y, FabI r epr esents an inter esting tar get for drugs acting against intracellular L. monocytogenes .

Exogenous unsa tur a ted long-chain FFAs promote gro wth b y L. monocytogenes a t lo w temper a ture
Listeria monocytogenes is a psyc hr otr ophic bacterium holding the ability to alter the composition of its membrane to sustain optim um membr ane fluidity at low temper atur e (Annous et al. 1997 ). A high content in the membrane of anteiso br anc hed-c hain fatty acids is known to be a critical factor for growth of L. monocytogenes at r efriger ation temper atur es (Annous et al. 1997, Singh et al. 2009 ). When the temper atur e declines, L. monocytogenes r esponds by rising the anteiso-C15:0 fatty acid content in the membrane to maintain optimal membrane fluidity (Annous et al. 1997, Zhu et al. 2005.
Recent studies suggest that exogenous long-chain fatty acids ar e incor por ated in the membrane to pr omote gr o wth at lo w temper atur es (Flegler et al. 2022, Touche et al. 2023. Listeria monocytogenes encodes homologs of the fatty acid kinase system F akA/F akB, which allows the bacterium to utilize exogenous FFA as precursors for membrane building blocks (Yao et al. 2016 , Yao andRock 2017 ). In Gram-positive bacteria, the fatty acid kinase system binds and phosphorylates exogenous FFAs taken up by the cells . T he resulting acyl-phosphates then enters the phospholipid synthesis pathway, and the phospholipids are finally incorporated in the bacterial membrane (Yao and Rock 2017 ). Growth medium supplemented with unsaturated long-chain FFAs induces an increase of the growth rate of L. monocytogenes at 5 • C-6 • C, whereas satur ated long-c hain FFAs inhibit bacterial gr owth or leav e it unaffected at low temper atur es (Flegler et al. 2022, Touche et al. 2023. Incor por ation of unsaturated FFAs decreases the phospholipid melting point temper atur e, whic h allo ws L. monoc ytogenes to compensate for the decrease in membrane fluidity caused b y lo w temper atur es. Notabl y, L. monocytogenes does not encode a functional fatty acid β-oxidation pathway, which means that exogenous long-chain FFAs do not serve as an energy source for L. monocytogenes (Glaser et al. 2001, Sauer et al. 2019. T hus , exogenous unsaturated long-chain FFAs seem to be used exclusively as membrane building blocks to promote the fitness of L. monocytogenes under stressful conditions, such as low temperature.

Dietary fat affects the course of L. monocytogenes infection
The effect of different dietary fat on L. monocytogenes infection has been e v aluated using v arious animal infection models (Harrison et al. 2013, Las Heras et al. 2019. Se v er al studies hav e shown that mice fed with fish oil are more susceptible to infection by L. monocytogenes r elativ e to mice fed on a low-fat diet (de P ablo et al. 2000(de P ablo et al. , Puertollano et al. 2004. A diet containing fish oil impairs the clearance of L. monocytogenes from the organs of mice, and survival of the infected animals is significantly reduced. The polyunsatur ated long-c hain fatty acids in fish oil ar e known to hold antiinflammatory and immunomodulatory properties, which may reduce the cellular host defense against systemic infections caused by intracellular pathogens, including L. monocytogenes (Harrison et al. 2013, Husson et al. 2016. When infected with L. monocytogenes , the survival of animals fed diets containing olive oil rich in monounsaturated oleic acid (C18:1), or hydrogenated coconut oil ric h in satur ated lauric acid (C12:0), is higher than the survival of mice fed with fish oil (de Pablo et al. 2000 ). Furthermore, dietary supplementation with CLA for up to 4 weeks does not affect the resistance to L. monocytogenes in mice (Turnock et al. 2001 ). Notably, the mouse experiments were performed by intravenous or intr a peritoneal injection of L. monocytogenes , leaving out the gastr ointestinal sta ge of infection (de P ablo et al. 2000, Turnoc k et al. 2001, Puertollano et al. 2004, Harrison et al. 2013. T hus , the effect of CLA and dietary oils rich in unsaturated long-chain fatty acids should be reassessed using a model that allows evaluation of the gastrointestinal stage of infection by L. monocytogenes . In mice, infection by L. monocytogenes via the intestinal r oute is r elativ el y low due to a poor interaction between the internalin InlA and the m urine r eceptor E-cadherin (Lecuit et al. 1999 ). Notably, mice can be infected perorally using the strain L. monocytogenes EGDe m expressing a modified InlA, which interacts with the murine E-cad receptor (Wollert et al. 2007, Monk et al. 2010. This strain was used in a recent study investigating the influence of a high-fat westernized diet, rich in saturated fat, on host susceptibility to infection by L. monocytogenes via the intestinal route (Las Heras et al. 2019 ). The animals consumed either a high-fat diet, a low-fat diet, or r egular c ho w, for 2 w eeks. Then, the mice were infected perorally with L. monocytogenes , and infection was allowed to pr ogr ess for 3 da ys . Mice fed on westernized high-fat diet were more susceptible to infection compared to lo w-fat-or cho w-fed animals . T he bacterial burden in the spleen, caecum, and lymph nodes was significantly higher in animals fed with high-fat diet compared to chow-fed diet. Furthermore, pr ofound c hanges in the host r esponse and gut micr obiota could be observed both pre-and postinfection. Altogether, this study concludes that consumption of a westernized high-fat diet is a significant factor influencing host resistance to L. monocytogenes infection (Las Heras et al. 2019 ). Notably, the authors did not examine the potential effects of a high-fat westernized diet on listerial expression of virulence factors.
Another study aimed to determine how high intake of milk fat affects intestinal colonization by L. monocytogenes in rats (Sprong et al. 1999 ). After adaptation to either low milk fat or high milk fat diets, rats were infected orally with L. monocytogenes . Inter estingl y, gr eater milk fat consumption inhibits colonization of this pathogen and reduces diarrhea in infected animals. The gastric contents of rats displayed an enhanced antilisterial activity with the amount of fat consumed. When analyzing the composition of the gastric contents, higher concentrations of FFAs wer e observ ed when lar ger amounts of dietary fat were consumed (Sprong et al. 1999 ). The antilisterial activity was primarily observed in the stomach, whereas no killing of L. monocytogenes was found in the contents of the small and large intestine of rats . T hese observations suggest that milk fat-mediated killing of L. monocytogenes most lik ely tak es place in the gastric lumen. Indeed, various FFAs with known antibacterial activity against L. monocytogenes is found at higher concentrations in the stomach of rats fed on high-fat milk, including lauric acid (C12:0). Although no antibacterial activity could be observed, specific medium-and long-chain FFAs with known PrfA-inhibitory activity are present in higher le v els in the small intestine of rats fed on a high-fat milk (Sprong et al. 1999 ). It is , therefore , tempting to speculate that specific FFAs derived from dietary fat could act as antivirulence agents in the small intestine, where L. monocytogenes is expected to initiate its infection.

Concluding remarks
Foodborne pathogens encounter a variety of long-chain FFAs in the gastrointestinal tract, and multiple studies have confirmed their potential as antimicrobial and/or antivirulence agents . T he consumption of dietary fat affects the course of infection, howe v er, for most enteric pathogens the effect of dietary supplementation of specific long-chain FFAs with antimicrobial and/or anti virulent acti vities r emains to be e v aluated. The dietary fat type is expected to affect the complex interplay between the host response, the gut microbiota, and the pathogen at the gastrointestinal stage of infection (Caesar et al. 2015 , Chadaideh andCarmody 2021 ). Furthermore, the dose and timing of fatty acid intake may determine the outcome of pathogen exposure (Husson et al. 2016 ). T hus , mor e studies ar e r equir ed to systematicall y explor e the impact of different types and doses of long-chain FFAs in models that closely simulate the infectious disease in humans caused by foodborne pathogens.
Although natur all y occurring long-c hain FFAs show antivirulence activity in vitro , they may not be the best candidates for antivirulence ther a py. FFAs serv e as substr ates for metabolic activities by the host and resident microbiota in the gastrointestinal tr act, and the concentr ations r equir ed to r educe infection a ppear to be r elativ el y high. Ho w e v er, detailed information on how inhibitory FFAs interact with regulatory proteins may be used as inspiration to develop more potent antivirulence therapeutics. Indeed, structural information on how palmitoleic acid (C16:1) interacts with ToxT allo w ed the design of a new class of ToxT inhibitors that pr e v ents virulence gene expr ession mor e efficientl y than natur all y occurring FFAs (Woodbrey et al. 2017 ). More specifically, the folded conformation of the monounsaturated long-chain FFA found in the crystal structure of ToxT served as inspiration for designing more effective ToxT inhibitors, and further improvements of their inhibitory potential resulted in a second-generation indole deri vati ve with decreased cytotoxicity that protects against V. cholerae colonization in a mouse model (Woodbrey et al. 2018 ). Similar structure-function based approaches may lead to the development of novel anti-infective compounds targeting k e y virulence regulators in other bacterial pathogens.
In the past, the antimicrobial potency of FFAs has been studied very intensively to identify the FFAs with the greatest efficacy against bacterial pathogens. Although foodborne pathogens and members of the gut microbiota may develop resistance to w ar d the antimicrobial actions of FFAs, their role as antivirulence agents should be further investigated. Detailed knowledge on how longc hain FFAs interfer e with bacterial virulence may stimulate the de v elopment of novel anti-infective strategies in the future.