extfieldsofstudy: Medicine Biology

text:
The Biochemistry of Sensing: Enteric Pathogens Regulate Type III Secretion in Response to Environmental and Host Cues

ABSTRACT Enteric pathogens employ sophisticated strategies to colonize and infect mammalian hosts. Gram-negative bacteria, such as Escherichia coli, Salmonella, and Campylobacter jejuni, are among the leading causes of gastrointestinal tract infections worldwide. The virulence strategies of many of these Gram-negative pathogens rely on type III secretion systems (T3SSs), which are macromolecular syringes that translocate bacterial effector proteins directly into the host cytosol. However, synthesis of T3SS proteins comes at a cost to the bacterium in terms of growth rate and fitness, both in the environment and within the host. Therefore, expression of the T3SS must be tightly regulated to occur at the appropriate time and place during infection. Enteric pathogens have thus evolved regulatory mechanisms to control expression of their T3SSs in response to specific environmental and host cues. These regulatory cascades integrate multiple physical and chemical signals through complex transcriptional networks. Although the power of bacterial genetics has allowed elucidation of many of these networks, the biochemical interactions between signal and sensor that initiate the signaling cascade are often poorly understood. Here, we review the physical and chemical signals that Gram-negative enteric pathogens use to regulate T3SS expression during infection. We highlight the recent structural and functional studies that have elucidated the biochemical properties governing both the interaction between sensor and signal and the mechanisms of signal transduction from sensor to downstream transcriptional networks.

mental habitats and use diverse domestic and wild animals as reservoirs (6). The oral sources of infection by these pathogenic bacteria vary as widely as their environmental niches. Salmonella and Campylobacter infections are usually acquired from undercooked chicken and eggs, while GI tract infections caused by Yersinia species are usually acquired from undercooked pork and vegetables (3,(6)(7)(8). Many Vibrio parahaemolyticus infections are caused by contaminated raw or undercooked seafood, and Vibrio cholerae infections are typically caused by contaminated drinking water (9,10). Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) infections, as well as Shigella infections, are linked to consumption of contaminated food and water (11). Interestingly, these pathogens colonize different parts of the GI tract. For example, V. cholerae and EPEC prefer the small intestine, whereas Campylobacter jejuni and EHEC infect the cecum and colon, respectively (12,13).
Toxin delivery systems are key to the environmental survival and persistence of enteric pathogens, as well as to their mechanisms of pathogenesis within human hosts (14). An important toxin delivery system used by many Gram-negative pathogens is the type III secretion system (T3SS). The T3SS is a macromolecular injectisome that translocates bacterial toxins, termed effectors, directly from the bacterial cytoplasm into the host cytosol (15). The expression of the T3SS apparatus and effector proteins must be tightly regulated to coincide with both the appropriate host cell environment and correct stage and location of infection (16). To achieve this coordination, bacteria have evolved mechanisms to sense different physical and chemical host signals and integrate the sensing of these signals into the regulatory control of pathogens via the T3SS apparatus and effector expression. This review focuses on the signals and sensors mediating T3SS expression during gastrointestinal tract infections.

TEMPERATURE
For bacteria to survive and thrive in diverse environments, they must be able to sense and respond to changes in temperature (17). For intestinal pathogens, temperature change is a primary indicator for successful invasion of a mammalian host (18). Therefore, temperature-sensing mechanisms are almost ubiquitously integrated into the regulatory circuits governing virulence gene expression of human intestinal pathogens (18). Many temperature-sensing mechanisms exist in bacteria. These include temperature-induced structural changes in intrinsically bent or supercoiled DNA, thermosensing regulatory proteins, and RNA thermometers (RNATs) (18)(19)(20). RNATs are RNA sequences present in the 5=-untranslated region (UTR) of certain mRNAs that adopt ribosomal binding site-occluding secondary structures at restrictive temperatures but not at permissive temperatures (21). The intestinal pathogens Yersinia pseudotuberculosis and Yersinia enterocolitica utilize these temperature-sensing strategies to efficiently regulate the expression of virulence factors, including the T3SS, within mammalian hosts (22). In pathogenic Yersinia species, the T3SS genes are encoded on the pYV virulence plasmid. Like the majority of the pYV-encoded virulence genes, the T3SS genes are not expressed at temperatures below 30°C and are most highly expressed at 37°C (23). The expression of the T3SS-encoding ysc genes (yscA to yscL), as well as of the yop effector genes (yopE, yopH, yopK, and yopO), is activated by the AraC-type regulator LcrF, which responds to both temperature and host cell contact (24)(25)(26). The lcrF gene is in an operon with yscW, and their transcription is controlled by a promoter sequence upstream of yscW (Fig. 1A).
In Y. pseudotuberculosis, temperature regulation of the ysc/yop regulon is achieved by the combined action of temperature-dependent proteolysis of the transcriptional repressor YmoA and an RNAT within the lcrF 5=-UTR (20). At moderate temperatures, similar to what a bacterium might experience outside a mammalian host (25°C), expression of the yscW-lcrF operon is repressed by two distinct mechanisms (Fig. 1A). First, the transcriptional repressor YmoA, which can form a homodimer or a heterodimer with the nucleoid-associated protein H-NS, binds to sequences downstream of the transcription initiation site and partially represses transcription of the yscW-lcrF operon (Fig. 1A) (20,27). Second, any transcribed lcrF mRNA is not translated because of a two-stem-loop structure that forms in the 5=-UTR at moderate temperatures. This RNA structure masks the Shine-Dalgarno sequence, thereby preventing the binding of the 30S ribosomal subunit, ribosome assembly, and translation of the lcrF mRNA ( Fig. 1A) (20). Without the transcriptional activator LcrF, the expression of the genes encoding the T3SS apparatus and effectors is not induced (Fig. 1A) (23,28). At higher temperatures, such as those that would be encountered within mammalian hosts (37°C), both types of repression of lcrF expression are relieved. Transcriptional repression of the yscW-lcrF operon is lifted by the rapid, temperature-dependent degradation of YmoA by both ClpXP and Lon proteases at 37°C (Fig. 1B). Several mechanisms for the temperature-dependent degradation of YmoA have been proposed. These include increased expression or activity of ClpXP or Lon proteases at 37°C, conformational changes in YmoA that make it a better substrate for ClpXP or Lon at higher temperatures, or direct modification and targeting of YmoA for degradation by an accessory protein at 37°C. Another possible mechanism is that Lon and ClpXP have differing affinities for YmoA homodimers and YmoA-H-NS heterodimers, and the higher-affinity species may dominate at elevated temperatures (29). Translational repression of the lcrF mRNA is also relieved at 37°C, because the inhibitory two-stem-loop structure melts at this temperature, thereby revealing the sequestered Shine-Dalgarno sequence and allowing translation of LcrF (Fig. 1B) (20). LcrF then induces production of the T3SS by binding to specific TTTaGYcTtTat (highly conserved nucleotides are in capital letters) DNA motifs in the promoter regions of many T3SS genes of the ysc/yop regulon and activating their expression (Fig. 1B) (30,31).

HORMONES
The locus for enterocyte effacement (LEE)-encoded T3SS of EHEC, which is essential for formation of the characteristic attaching and effacing (AE) lesions, is regulated by two host hormones, epinephrine (Epi) and norepinephrine (NE), and by an autoinducer (AI-3) that is produced by the gut microbiota. Epi and NE are at the core of stress response signaling in humans and are recognized by G-protein-coupled receptors in mammalian cells (32). However, bacteria lack homologues of these mammalian adren-ergic receptors and have instead evolved sensor histidine kinases (HKs) to recognize these host signals. These chemical signals are received and transmitted by two HKs, QseC and QseE (33)(34)(35). Why EHEC and other enteric pathogens have developed mechanisms to sense Epi and NE remains unclear, but given the important functions neurotransmitters play in gut homeostasis, it is thought that sensing these molecules may allow the pathogen to assess the fitness of the host and modulate its virulence program accordingly (36). In fact, it has been shown that a qseC qseE double mutant is attenuated in an EHEC infant rabbit infection model (36). The importance of qseC and qseE in virulence has also been demonstrated in Citrobacter rodentium, a murine pathogen that harbors a LEE-encoded T3SS (36). C. rodentium LEE expression and gut colonization are both diminished in Dbh Ϫ/Ϫ mice, because they do not produce Epi and NE (36).
For EHEC, AI-3, Epi, and NE are first sensed by the membrane-bound HK QseC, resulting in autophosphorylation and transfer of a phosphate to its cognate response regulator (RR), QseB. Phosphorylated QseB (P-QseB) then activates the transcription of the qseB-qseC operon as well as the flagellar motility operon flhD-flhC ( Fig. 2A) (32,34). QseC can also phosphorylate the noncognate RRs QseF and KdpE (32,37). P-KdpE directly activates transcription of ler, which encodes the master transcriptional activator of all LEE genes (37). QseC autophosphorylation in response to AI-3, Epi, or NE also activates the expression of the sensor HK QseE, which can itself sense Epi, sulfate, and phosphate ( Fig. 2A). Both QseC and QseE can phosphorylate the RR QseF, thereby regulating the expression of the non-LEE-encoded effector T3SS EspFu, which is directly involved in AE lesion formation. QseC also indirectly induces expression of the phage-encoded Shiga toxin (stxAB) through the QseF RR by activating the SOS response. During the SOS response, expression of recA is upregulated and, through its coprotease activity, RecA cleaves the cl repressor of the stxAB genes, allowing for their expression ( Fig. 2A) (32,38).
The structural basis by which QseC or QseE senses AI-3, Epi, or NE remains undefined. Although the crystal structure of the cytoplasmic domain of QseC is available, it does not include the periplasmic sensor region. Recently, Parker et al. used software to generate a predicted structure of the periplasmic domain of QseC (39). The authors identified 8 conserved residues in the periplasmic domain that may mediate ligand To reach the intestinal epithelium, EHEC produces mucinases that obliterate the mucosal layer and create a gluconeogenic environment. B. thetaiotaomicron then switches to gluconeogenic metabolism and secretes large amounts of succinate, which EHEC senses through the transcriptional regulator Cra. Sensing of succinate by Cra induces T3SS expression and AE lesion formation. The gluconeogenic environment also triggers EHEC's stringent response, during which the alarmone ppGpp is synthesized. ppGpp directly binds RNA polymerase and modulates its activity, such that LEE expression is upregulated.
binding. These residues reside within a potential binding pocket formed by a series of ␤-sheets and proximal ␣-helices. However, mutation of these residues did not abrogate the ability of QseC to sense Epi and NE (39). Future structural studies of QseC and its ligands, NE and Epi, are required to completely understand how these signals are sensed.

SUGARS AND NUTRITIONAL STRESS
While inside the host gastrointestinal tract, enteric pathogens must not only adapt to the nutritional conditions of the gut but also use these nutritional cues to direct the appropriate expression of virulence factors. Pathogenic bacteria that colonize the colon (e.g., EHEC) do not have access to simple dietary sugars because these sugars are absorbed in the small intestine (40). These pathogens must compete with a specialized microbiota that resides in the lumen and outer mucous layer of the intestine. Many members of the microbiota can use the abundant undigested plant polysaccharides and host glycans found in the colon (40). However, neither commensal nor pathogenic E. coli, such as EHEC, can use polysaccharides. EHEC avoids competition with commensal E. coli for monosaccharides and disaccharides by invading the mucosal layer and adhering to enterocytes of the intestinal epithelium (41). To achieve this, EHEC must sense and appropriately respond to a variety of environmental signals.
A prominent member of the luminal microbiota, Bacteroides thetaiotaomicron, produces multiple fucosidases that cleave fucose from the host glycans comprising the mucosal layer (42). Initially, EHEC senses the high concentration of fucose through the HK and RR pair FusKR (Fig. 2B). Activation of the FusKR signaling cascade by fucose represses transcription of several genes, including Ler, the master regulator of the LEE-encoded T3SS (43). The downregulation of the T3SS and other LEE-encoded virulence factors, which are unnecessary for EHEC's survival in the lumen, is a crucial energy-saving measure that helps EHEC outcompete commensal E. coli. During the second stage of its virulence strategy, EHEC senses other B. thetaiotaomicron metabolites (e.g., succinate) and ramps up expression of mucinase, which clears the mucous layer of the intestine and opens a path to the intestinal epithelium (Fig. 2B). Without the mucous layer as a primary carbon source, B. thetaiotaomicron switches to a gluconeogenic metabolism and secretes large amounts of succinate. The high levels of succinate are sensed by EHEC via an unknown mechanism and induce the transcriptional regulator Cra, which in turn activates expression of the T3SS (44).
The gluconeogenic, nutrient-poor environment also stimulates EHEC's stringent response (Fig. 2B). The stringent response is a universal bacterial transcriptional regulatory mechanism that involves synthesis of the alarmone ppGpp by the RelA and SpoT enzymes during times of nutrient deprivation (45). ppGpp, along with the starvationinduced small protein DksA, directly binds to and modulates the activity of RNA polymerase. This activates or represses transcription at specific promoters, resulting in upregulation of LEE expression in EHEC (Fig. 2B) (46). The double activation of LEE expression by both Cra and ppGpp in response to the nutrient-poor conditions at the intestinal epithelium ensures that the LEE-encoded T3SS is expressed at the appropriate stage of infection.

CATIONIC ANTIMICROBIAL PEPTIDES
Cationic antimicrobial peptides (CAMPs) are produced by the host as part of the immune response to bacterial, viral, protozoan, or fungal infections (47). Due to their net positive charge and amphipathicity, CAMPs can interact with and permeate phospholipid membranes. Many bacterial pathogens, such as Salmonella enterica serovar Typhimurium (48), several streptococcal species (49), Neisseria meningitidis (50), and Pseudomonas aeruginosa (51), have evolved mechanisms of CAMP resistance. S. Typhimurium achieves this by sensing the CAMPs and modifying the lipopolysaccharides (LPSs) that decorate its cell surface. In S. Typhimurium, the genes involved in LPS modification are regulated by the PhoPQ two-component system (TCS) (48,52). This TCS is composed of PhoQ, a membrane-spanning HK, and PhoP, a DNA-binding RR. In addition, the PhoPQ TCS also controls the expression of Salmonella pathogenicity islands 1 (SPI-1) and SPI-2, each of which encodes a T3SS (Fig. 3A) (53). PhoQ can function as both an HK and a phosphatase and thereby modulate the phosphorylation status of PhoP in response to environmental cues, such as CAMPs, divalent cations, and acidic pH (54,55). Phosphorylated PhoP induces expression of SPI-2 T3SS genes and represses the expression of most SPI-1 T3SS genes (Fig. 3A) (56,57). The SPI-1 T3SS is used for the initial invasion of nonphagocytic cells, and the SPI-2 T3SS is essential for maintaining the Salmonella-containing vacuole (SCV) and survival within phagocytic cells (58)(59)(60).
Crystallographic studies have demonstrated that divalent cations bind to an acidic patch created by the PhoQ-DcuS-CitA (PDC) sensor fold within the periplasmic domain (PD) (Fig. 3B) (61). In vitro studies using recombinant truncations of PhoQ reconstituted in lipid vesicles have shown that PhoQ can directly sense CAMPs through its PD (55). Binding of divalent cations to this acidic patch represses PhoQ HK activity (61,62). The acidic patch is also important for CAMP recognition, suggesting that CAMPs and divalent cations compete for binding to this region (55). In vivo, divalent cations are thought to be coordinated between the PhoQ PD and the phosphate groups of the outer leaflet of the inner membrane (IM) (Fig. 3A and B) (61). The divalent cations bridge the negative charges of the PD acidic patch and the IM phosphates, thus tethering the PhoQ PD to the membrane. Based on nuclear magnetic resonance studies, the metalbound, and therefore membrane-tethered, PhoQ PD is believed to be limited in structural flexibility and locked in a state that inactivates the HK domain (Fig. 3A) (55). However, when Salmonella is exposed to host CAMPs, the positively charged peptides compete for binding to the PhoQ acidic patch and displace bound divalent cations. This displacement is thought to release the PhoQ sensor domain from its membrane-locked state and induce a conformational change that promotes PhoQ HK activity (Fig. 3A) (55,61).
The exact nature of the conformational change induced by CAMPs, as well as the mechanism of PhoQ HK activation, remains unclear. In a recent study, however, multistate Bayesian modeling of disulfide cross-linking data and existing structures of PhoQ homologous domains predicted that PhoQ transitions between two major states. These states are defined by the insertion or displacement of the PD acidic patch in the IM (Fig. 3A) (63). The authors found that insertion or removal of the PD acidic patch within the membrane was coupled with scissoring transitions, or diagonally opposing displacements, in the PD sensor domain, transmembrane (TM) helical bundles, HAMP, and DHp domains. These changes in conformation are predicted to alter HK domain activity ( Fig. 3A and B), but further studies are needed to confirm these changes and to elucidate how they mechanistically alter PhoQ's HK activity.

FATTY ACIDS
Fatty acids (FAs) in the intestinal tract are generated through the combined action of the host metabolism and the gut flora. Long-chain fatty acids (LCFAs) enter the small intestine through the diet and via fat metabolism (64). The resident microbiota in the large intestine produces abundant quantities of short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate (reviewed in reference 65). Thus, host and bacterial metabolism result in different LCFA and SCFA concentrations along the GI tract. In addition to their metabolic roles, LCFAs and SCFAs act as cues for bacteria to sense their location in the GI tract and modulate the expression of their virulence factors.
Individual FAs have differential effects on S. Typhimurium virulence. For example, acetate induces invasion genes, while propionate, butyrate, and LCFAs reduce invasiveness (66)(67)(68). Regulation of S. Typhimurium virulence by FAs is dependent on the multilayered regulatory network that controls the expression of the invasive SPI-1 T3SS (Fig. 4). The BarA/SirA TCS, which is composed of the hybrid HK BarA and the Minireview ® DNA-binding RR SirA (also known as UvrY), is at the top of this network. The BarA/SirA TCS is activated by various SCFAs through different mechanisms (Fig. 4). Acetate activates SirA via the metabolite acetyl-phosphate, which phosphorylates SirA directly (66). This sensing mechanism bypasses BarA, as it depends on the conversion of acetate to acetyl-phosphate by metabolic enzymes (66). While the role of BarA in SCFA sensing for S. Typhimurium is not clear, work on its homologue in E. coli demonstrated that BarA senses formate (69), a signal that induces SPI-1 in S. Typhimurium (68). In addition, deletion of acetyl-phosphate synthesis genes in E. coli still results in low-level activation of a SirA-dependent operon in a BarA-dependent manner when acetate is present, suggesting that BarA also senses acetate (69). Once phosphorylated, the SirA RR activates the expression of CsrB and CsrC, two regulatory RNAs that repress the RNA-binding protein CsrA (Fig. 4) (70, 71). CsrB and CsrC sequester CsrA, preventing it from binding to the hilD mRNA Shine-Dalgarno sequence. The free hilD mRNA can then be translated.
LCFA and propionate negatively affect SPI-1 T3SS expression by modulating HilD activity via different mechanisms (Fig. 4) (67, 72). LCFAs bind to HilD and reduce its affinity for DNA, thus reducing transcription from HilD-dependent promoters (72). HilD controls its own expression, as well as that of the regulators hilC and rtsA, resulting in a feed-forward loop that further amplifies LCFA suppression of SPI-1 expression (Fig. 4) (3). The effect of propionate is less direct, as its metabolite, propionyl coenzyme A (CoA), destabilizes HilD and thereby limits its activity (Fig. 4) (67). Golubeva et al. (72) proposed that absorption along the small intestine results in a low concentration of LCFAs in the distal ileum, which would relieve HilD repression and allow the expression of the SPI-1 activator hilA at this site. Since the propionate concentration is high in the colon and cecum, this molecule has been proposed to serve as a signal for S. Typhimurium that it has left the optimal site, the ileum, for infection (67). The repressive effects of certain FAs in Salmonella virulence and their antimicrobial activities have resulted in their use as supplements in animal feed (73). In addition to their direct effect on bacterial pathogens, SCFAs also influence host physiology in diverse ways, including alteration of immune function (74). Thus, the effects of SCFAs on gut pathogens such as Salmonella are probably due to a combination of their inhibitory effect on virulence, their antimicrobial properties, and the regulation of the host immune response.

IRON
Along with their eukaryotic hosts, most bacterial species require access to free iron for survival and replication (75,76). Due to the toxicity of free ferric Fe 3ϩ and host iron-sequestering mechanisms, the concentration of free iron is low in most host tissues (76). However, in the lumen of the small intestine, which is the primary site of iron absorption, free iron concentrations are high (77). S. Typhimurium uses the iron concentration to sense when it has entered the intestinal lumen (78). Many of the genes that are repressed by high Fe 2ϩ concentrations are also repressed when S. Typhimurium is confined to the intestinal lumen (79). Iron concentration has been found to directly affect the expression of the S. Typhimurium SPI-1 T3SS. The activation of SPI-1 expression in response to high iron concentrations depends on the ferric uptake regulator Fur (78). Both metal chelation by 2,2-dipryridyl and deletion of fur have been shown to greatly reduce the transcription of the SPI-1 master regulator hilA under otherwise-SPI-1-inducing conditions (78). One mechanism by which Fur modulates hilA is by repressing expression of the gene encoding the global regulator H-NS (80). Under iron-limiting conditions, such as outside the host or within host macrophages, H-NS directly binds and represses transcription from the hilA promoter (Fig. 5A). Under iron-replete conditions, such as in the intestinal lumen, iron-bound Fur (Fur · Fe 2ϩ ) binds to a regulatory site, termed the Fur box, in the hns promoter region and represses hns expression. By this mechanism, Fur · Fe 2ϩ indirectly activates HilA and downstream SPI-1 expression (Fig. 5B) (81). In addition to acting through H-NS, Fur · Fe 2ϩ also directly activates expression of hilD, a gene encoding the major transcriptional activator for hilA (82). Fur · Fe 2ϩ binds to a Fur box upstream of the hilD promoter (positions Ϫ191 to Ϫ163) and activates the expression of hilD, which in turn activates the expression of hilA (Fig. 5B) (82). In most bacterial species, Fur · Fe 2ϩ binds in the Ϫ35 to ϩ12 region of target promoters and represses transcription through steric hindrance (83). However, in Salmonella, Fur · Fe 2ϩ binds farther upstream in the hilD promoter region, thereby possibly activating hilD expression by directly recruiting RNA polymerase (RNAP) or by altering the conformation of the promoter DNA to facilitate RNAP loading (82).
Fur is a major regulator of gene expression that is conserved in several bacterial species. It controls the expression of iron homeostasis genes as well as diverse iron-sensitive cellular processes, including respiration, DNA synthesis, and redox stress resistance (84,85). In E. coli as well as many other bacterial species, Fur binds the Fur box in an Fe 2ϩ -dependent manner (86). The structural basis for the activation of Fur as a transcription factor by metal ions has not been specifically studied in S. Typhimurium, but structural studies performed on Fur homologues have begun to elucidate this mechanism. The Fur domain structure consists of an N-terminal DNA-binding domain (DBD) and a C-terminal dimerization domain linked by a disordered hinge region (Fig. 5C). A recent study of Magnetospirillum gryphiswaldense MSR-1 Fur, which solved high-resolution structures of apo, holo, and operator-bound forms of MSR-1 Fur, demonstrated that metal ion binding induces a caliper-like rotation and movement of the DBDs, orienting them for efficient DNA binding and stabilizing the structure (Fig. 5C) (87). Since no completely apo structures of any additional Fur homologues have been published, it is not known if a similar conformational change is induced by metal ion binding in Fur proteins from other species. However, all published holo structures of Fur (i.e., for E. coli, P. aeruginosa, and V. cholerae) adopt the same V-shaped conformation with the dimerization domains at the nexus and DNA-binding domains at the periphery (88).

BILE SALTS
Bile salts are a component of biliary secretions that are released into the small intestine to solubilize fats. They also have antimicrobial properties against bacteria that inhabit or transit the gastrointestinal tract (89). Successful enteric pathogens have developed ways to tolerate bile salt-induced stress and to use these compounds as markers of intestinal location. The marine bacterium Vibrio parahaemolyticus infects humans through the consumption of contaminated raw or undercooked seafood, causing acute gastroenteritis. V. parahaemolyticus enterotoxicity is largely mediated by a pathogenicity island (Vp-PAI) which encodes T3SS2, one of two T3SSs in this bacterium. T3SS2 expression is induced by bile salts via signaling, which includes the IM proteins VtrA, VtrB, and VtrC (Fig. 6A) (90)(91)(92). VtrA and VtrB are transcription factors that are embedded in the IM by a single transmembrane ␣-helix (Fig. 6A). VtrA also contains a C-terminal periplasmic domain that interacts with the periplasmic domain of VtrC, which is also anchored to the inner membrane. VtrA and VtrC form a 1:1 complex in which eight ␤-strands from VtrC and a single ␤-strand from VtrA form a ␤-barrel (Fig. 6B). Binding of bile salts to the hydrophobic interior of the VtrA/C dimer displaces a loop that covers the ␤-barrel in the apo structure (Fig. 6B, inset). This heterodimeric receptor transmits a signal upon bile salt binding that activates the DNA-binding domain of VtrA to induce transcription from the vtrB promoter (Fig. 6A) (92). VtrB then promotes transcription of T3SS2-related genes (90,91). Interestingly, the topologies of the VtrA and VtrB transcription factors tether them to the IM, which may serve to localize T3SS gene products to the membrane site, where they will be assembled and exported. The V. parahaemolyticus bile-sensing system is conserved in V. cholerae strains that contain a pathogenicity island similar to Vp-PAI. In V. cholerae AM-19226, which lacks toxin-coregulated pilus and cholera toxin genes but contains a Vp-PAI-like island, T3SS gene expression depends on the VtrA and VtrB homologues VttR A and VttR B (93). The encoded VtrC homologue is also likely to be part of this regulatory mechanism (93-95). The inner membrane proteins ToxR and ToxS also play roles in the regulation of T3SS2 expression in V. parahaemolyticus. ToxR and ToxS have been linked to bile salt sensing in V. cholerae; however, the sensing mechanism remains unclear (96,97). ToxR has the same domain arrangement as VtrA, with a cytoplasmic DBD, a transmembrane helix, and a periplasmic domain (Fig. 6B). Like VtrC, ToxS is composed of a transmembrane helix and a periplasmic domain. Despite having similar architectures, the periplasmic domains of V. parahaemolyticus ToxR and ToxS have less than 25% sequence similarity to VtrA and VtrC, respectively. Recent work identified ToxR in a genetic screen for factors contributing to colonization of the mammalian intestine and showed that ToxR is necessary for vtrB expression in V. parahaemolyticus (98). This suggests that ToxR works with VtrA and VtrC to regulate T3SS2 expression via VtrB. Work with V. cholerae AM-19226 supported a similar role for ToxR in the regulation of this strain's T3SS (99). ToxR and ToxS also interact through their periplasmic domains (100). Furthermore, Miggett et al. (100) demonstrated that bile salts destabilize the ToxR periplasmic domain, which promotes its interaction with ToxS (100). Additional studies are needed to determine if bile salts have any direct effect on ToxR's partner, ToxS.

CONCLUSIONS
T3SSs are essential to many bacterial pathogens for successful host colonization and invasion. Because their production can reduce bacterial growth rates under certain conditions (101) and can trigger the host's immune response (102), the timing of their expression must be tightly controlled. It is not surprising, then, that T3SS regulatory pathways are often multileveled and complex. Enteric pathogens have devised strategies to sense signals specifically related to the mammalian gut, and these strategies include molecules involved in host nutrition (iron, fatty acids), metabolism (bile salts), homeostasis (hormones), and microbiota by-products (fucose, succinate). These sensing mechanisms have been integrated into the regulatory networks controlling virulence and colonization factors, which often include T3SSs. As seen in the examples presented here, multiple signals often feed into a single regulatory cascade which has evolved complex feedback mechanisms that allow the bacterium to fine-tune T3SS expression in the signal-rich environment of the mammalian GI tract (Fig. 7).
Bacteria use T3SSs in different ways to promote their survival and proliferation within a host. Pathogens that use T3SSs to maintain their extracellular life cycle, like EHEC, inject effectors essential for establishing a strong attachment to intestinal epithelial cells (13). Other pathogens (e.g., S. flexneri and V. parahaemolyticus) use their T3SSs both extracellularly, to promote attachment and invasion, and intracellularly, to enhance their proliferation, facilitate vacuolar escape, and mediate cell-to-cell spread (103)(104)(105)(106). Salmonella has two T3SSs that are used at different stages of infection and are often oppositely regulated by related signals to accommodate the complex wiring of downstream transcriptional regulatory networks.
There is still much to be learned about the signals and sensors that control T3SS expression in enteric bacterial pathogens. For example, it has been postulated that contact between a bacterium and its host cell plays a role in T3SS regulation. Recent work with EHEC suggested that the outer membrane lipoprotein NlpE may regulate T3SS expression through a surface-sensing mechanism involving the Cpx TCS (107,108). However, NlpE is only required for activation of the LEE-encoded T3SS in response to attachment to abiotic surfaces and undifferentiated Caco-2 cells (107). It is possible that other lipoproteins, such as YafY, NlpA, Pal, or OsmB, sense adhesion to other cell types. However, further studies are needed to determine if these other lipoproteins are involved in the regulation of the T3SS in response to surface sensing (109). The mechanism by which the sensor domains NlpE and other outer membrane lipoproteins may sense hydrophobic surfaces is poorly understood and awaits further study. We anticipate that future work will further elucidate how biochemical signals from both the host and the environment are sensed by enteric pathogens to regulate T3S and other mechanisms of virulence. Elucidation of these sensing mechanisms could provide new potential therapeutic targets for the improved management of GI tract infections by enteric bacteria.

ACKNOWLEDGMENTS
We thank the members of the Orth lab for thoughtful discussion and revision of the manuscript.
This work was funded by Welch Foundation grant I-1561 and the Once Upon a Time. . . Foundation. N.J.D. was funded by NIH T32AI070116-10. K.O. is a Burroughs Wellcome Investigator in Pathogenesis of Infectious Disease, a Beckman Young Investigator, and a WW. Caruth, Jr., Biomedical Scholar and is an Earl A. Forsythe Chair in Biomedical Science.
The funders had no role in creating this work or in the decision to submit it for publication.


extfieldsofstudy:
Medicine
Materials Science

s2fieldsofstudy: Biology Psychology

text:
Epigenetic Modification of OXTR is Associated with Openness to Experience

Oxytocin is a neuropeptide known to influence social and cognitive processing across several mammalian species. There currently exists a mixed and controversial pattern of evidence that oxytocin pathway genes confer individual differences in social cognition and personality in humans. Inconsistencies across studies may in part be explained by the presence of intermediary, epigenetic, variables that exist between genotype and phenotype. This study was designed to investigate the association between epigenetic modification of the Oxytocin Receptor Gene (OXTR), via DNA methylation, and Big-5 personality traits. Genetic data were collected via saliva samples and analyzed to quantify DNA methylation within the promoter region of OXTR. The results indicate that Openness to Experience is associated with OXTR DNA methylation, while controlling for the remaining Big-5 personality dimensions (Neuroticism, Extraversion, Agreeableness, and Conscientiousness) and sex and age. This finding provides additional support for models associating oxytocin with individual differences in personality and identity in humans.

In spite of the growth of the field of behavioral genetics, established links between personality traits and specific genes are currently tenuous, at best. Indeed, several recent important meta-analyses show that many published studies linking single-nucleotide polymorphisms (SNPs) with personality traits fail to replicate (De Moor et al., 2012;Montag & Reuter, 2014;Rietveld et al., 2014). One reason underlying the presence of inconsistencies across gene × personality studies may be that many studies have yet to consider epigenetic factors impacting the way genes function and are ultimately expressed (Kumsta & Heinrichs, 2013). Genes do not directly impact psychological phenomena: they initiate biochemical and physiological mechanisms. Furthermore, identical SNPs that occur in different people typically do not function in identical ways, and may result in a variety of phenotypes being expressed. Thus, investigating the association between SNPs and individual differences in human traits is limited because several intermediary factors that exist between genotype and phenotype are not typically considered. One novel way to elucidate the way genes are associated with phenotypes, such as with personality, is to use an epigenetic approach.
Epigenetics is the study of how gene functioning is altered by exogenous and endogenous factors. One epigenetic process that influences the expression of genes is DNA methylation, which occurs when a methyl group forms a covalent attachment with the 5' carbon of cytosine in the context of a cytosine phosphodiester guanine (CpG) dinucleotide, commonly called a CpG site. DNA methylation regulates the expression of genes by influencing the recruitment and binding of regulatory protein to DNA. Typically, an increase in DNA methylation is associated with a decrease in expression of that gene. Although several studies show that stable genetic variants, such as SNPs, confer tendencies toward specific personality types, very little is currently known regarding the association between epigenetic modification of genes and personality.
The oxytocin (OT) system is one biological substrate that may be associated with individual differences in human behavior and social cognition. OT is a neuropeptide, synthetized within the hypothalamus in the brain. Some evidence indicates that greater endogenous OT is associated with higher trait novelty-seeking temperament and OT administration leads to increased holistic processing, more flexible thinking, more original ideas, and better creative problem solving (De Dreu et al., 2014). In addition, Cardoso, Ellenbogen, and Linnen (2012) demonstrated that OT administration is associated with changes in self-report personality. Following OT administration, people tend to self-report higher extraversion and Openness to Experience. These findings have motivated the search for genes within the OT system that confer individual differences in personality traits and social cognition.
One OT pathway gene linked to human traits and social cognition is the Oxytocin Receptor Gene (OXTR), located on chromosome 3p25, which codes for a G-protein-coupled receptor that mediates the effects of OT on postsynaptic activity. Some research shows that SNPs of OXTR confer individual differences in cognition, sociability, and novelty-seeking (Melchers, Montag, Markett, & Reuter, 2013;Montag et al., 2017). In spite of mounting data, there also exist evidence that OXTR SNPs fail to explain a significant portion of individual differences in human behavior and traits (Apicella et al., 2010;Bakermans-Kranenburg & Van IJzendoorn, 2014). One reason underlying the presence of inconsistences across studies may be the fact that many investigations have not taken into account epigenetic modification of OXTR.
In this study, we sought to investigate the association between epigenetic modification of OXTR, via DNA methylation, and selfreported, Big-5, personality traits. Genetic data were collected from saliva samples and analyzed to target and quantify DNA methylation in the MT2 region (Kusui et al., 2001) within the promoter region of OXTR. A recent study demonstrated that methylation of DNA extracted from saliva is more similar to the methylation patterns observed in brain tissue than DNA extracted from blood (Smith et al., 2015). The promotor region of OXTR contains many CpG sites. However, there currently exists consistent and converging evidence that epigenetic modification of CpG site -934 is associated with psychological functioning (Bell et al., 2015;Kader, Ghai, & Maharaj, 2018;Rubin et al., 2016), and individual differences in brain structure and function (Jack, Connelly, & Morris, 2012;Puglia, Lillard, Morris, & Connelly, 2015), and that increased DNA methylation in the MT2 region, which includes this site, associates with lower expression levels (Kusui et al., 2001). This evidence guided our decision to target DNA methylation at CpG site -934. However, we could not isolate the -934 from the -924 CpG sites based on a limitation of the mass spectrometry technology employed for this assay.
In this study, we explored the association between in Big-5 personality traits and epigenetic modification of OXTR. On the basis of existing evidence that OT administration is associated with increased holistic processing, more flexible thinking, more original ideas and better creative problem solving (De Dreu et al., 2014), and changes in self-reported Openness to Experience (Cardoso, Ellenbogen, & Linnen, 2012), we predicted that DNA methylation at promoter region of OXTR would be associated individual differences in Openness to Experience. Furthermore, there currently exists a mixed pattern of results linking the OXTR gene with sociability (Bakermans-Kranenburg & Van IJzendoorn, 2014). We therefore explored the associated between epigenetic modification of OXTR and prosocial personality traits, Extraversion and Agreeableness. To test these predictions, we conducted a multiple regression analysis with all Big-5 personality traits entered simultaneously as predictor variables and OXTR DNA methylation entered as the criterion variable (controlling for age and sex).

Participants
In order to estimate an appropriate sample size for this study, we carried out an effect size estimate based on existing evidence linking DNA methylation of OXTR with individual differences in behavior. We pooled effect sizes from three recently published studies (Kim, Kim, Kim, & Treasure, 2014;Rubin et al., 2016;Ziegler et al., 2015). The average reported effect size for associations between OXTR DNA methylation and behavior was .34. We then conducted a power analysis, using π = 0.80, to determine an appropriately sized sample. On the basis of these reported effect sizes, we estimated that a sample size of greater than N = 66 would be sufficient. However, it is also clear that this analysis did not account for many other factors such as publication bias. We acknowledge that this is a single study, and that any potential results obtained here, warrant replication.
We recruited 128, healthy, fluent English-speaking (71 females, 57 males; mean age = 21.32 years, SD = 3.46 years) adults from the surrounding community to participate in genetic and behavioral testing. All participants were screened for neurological and psychiatric conditions, via self-report. All participants provided written informed consent as detailed in the Declaration of Helsinki, and the University of Georgia Institutional Review Board approved all procedures within this study.
Lastly, we confirmed the reliability of the OXTR methylation assay by using Epitect unmethylated (0%) and methylated (100%) DNA samples (Qiagen) as positive controls (Figure 1). For each participant, percent OXTR DNA methylation values were retained and used as the criterion variable in subsequent statistical analyses.

Personality assessment
Each participant completed the NEO Personality Inventory-3. The NEO Personality Inventory-3 covers each of the Big-5 personality traits (Neuroticism, Extraversion, Openness to Experience, Agreeableness, and Conscientiousness). Personality data were scored to represent T-values, with the population mean defined as T = 50 and 1 SD of T = 10. An examination of the internal consistency for all items included for each trait dimension showed high internal consistency (Table 1).

Statistical analyses
We undertook a statistical approach designed to test for the significance of associations between OXTR DNA methylation and Big-5 personality traits (Neuroticism, Extraversion, Openness to Experience, Agreeableness, and Conscientiousness). Statistical analyses were initiated by performing a multiple regression analysis, with each of the Big-5 personality dimensions entered as predictor variables and OXTR DNA methylation values entered as the criterion variable, while controlling for sex and age. Thus, this analysis tests for OXTR × personality trait associations, while controlling for the variance of all other Big-5 personality dimensions, as well as sex and age. We used a p = .05 significance threshold for this analysis. Next, we performed a confirmatory analysis using a standard, correction for multiple comparisons, approach. In this analysis, we tested for gene × personality trait associations independently, using a standard Bonferroni (.05/5 comparisons: α = .01) statistical threshold, while controlling for sex and age.

Results
Across our sample, mean OXTR DNA methylation at CpG -934/-924 was 0.580 (SD = 0.053). We tested for associations between personality and OXTR DNA methylation (Table 1). Openness to Experience was significantly associated with OXTR DNA methylation, while controlling for the remaining Big-5 personality dimensions (Neuroticism, Extraversion, Agreeableness, and Conscientiousness) and sex and age (standardized β = −.232, t = 2.72, p = .007). People that exhibit reduced OXTR DNA methylation (presumably associated with higher OXTR expression) tend to score higher on Openness to Experience. No other Big-5 personality trait was found to be significantly associated with OXTR DNA methylation. Next, we performed a confirmatory analysis, for gene × personality trait associations independently, using a standard Bonferroni (.05/5 comparisons: α = .01) statistical threshold, while controlling for sex and age. The results of this analysis were consistent with the results derived from the multiple regression analysis: reduced OXTR DNA methylation is associated with higher Openness to Experience, r = −.22, p = .007. Lastly, no other Big-5 personality trait was found to be significantly associated with OXTR DNA methylation using the standard, Bonferroni corrected, approach. Lastly, we performed an exploratory analysis associating OXTR DNA methylation with facet level Openness to Experience. The results of this analysis indicated that reduced OXTR DNA methylation was associated with higher Actions (O4: r = −.60, p = .037), Ideas (O5: r = −23, p = .006), and Values (O6: r = −.17, p = .028) but not Fantasy, Esthetics, or Feelings.

Discussion
In this study, we observed an association between epigenetic modification of OXTR and trait Openness to Experience. People that exhibit reduced OXTR DNA methylation (presumably associated with higher OXTR expression) tend to score higher on Openness to Experience. This finding is consistent with evidence that individual differences in endogenous OT is associated with trait novelty-seeking (De Dreu et al., 2014) and that OT administration is associated with increased self-reported Openness to Experience (Cardoso, Ellenbogen, & Linnen, 2012). This finding provides additional support for models associating OT with individual differences in personality and identity in humans.
Openness to Experience is a Big-5 personality trait that represents the affinity toward variety and the tendency toward thinking in a flexible and fluid way (McCrae & Costa, 1997). Behavioral research shows that high Openness to Experience is associated with intelligence (Ashton, Lee, Vernon, & Jang, 2000;Harris, 2004), social and political attitudes (Van Hiel, Kossowska, & Mervielde, 2000), and hypnotizability (Glisky, Tataryn, Tobias, Kihlstrom, & McConkey, 1991). People scoring higher on Openness to Experience tend to be more open to different cultures and lifestyles and lower on right-wing authoritarianism (McCrae, 1996). Interestingly, recent empirical research shows that OT administration influences interpersonal and political trust (Merolla, Burnett, Pyle, Ahmadi, & Zak, 2013) as well as ethnocentrism (De Dreu, Greer, Van Kleef, Shalvi, & Handgraaf, 2011). It is important to note, however, that De Dreu et al. (2011) demonstrated that OT administration was associated with greater ethnocentrism, while Openness to Experience is typically associated with lower ethnocentrism. There also exists evidence that OT is associated with hypnotizability. Following OT administration, people tend to be more easily hypnotized (Bryant, Hung, Guastella, & Mitchell, 2012) and OXTR SNPs are associated with individual differences in hypnotizability (Bryant, Hung, Dobson-Stone, & Schofield, 2013). Combined, these findings indicate that epigenetic modification of OXTR influences the relationship between OT and a variety of cognitive styles in humans.
There exists prior evidence that individual differences in Openness to Experience can be explained by genetics. Bergeman et al. (1993) performed an adoption/twin study and demonstrated that for Openness to Experience, genetic influence was substantial and that there was little evidence of shared rearing environment. Peciña et al. (2013) demonstrated that a commonly occurring genetic variant within the dopamine system (DRD2) is associated with trait Openness to Experience. Interestingly, the OT and dopamine systems have been shown to interact with one another to influence the way many social and cognitive tasks are performed (Baskerville & Douglas, 2010;Love et al., 2012;Rosenfeld, Lieberman, & Jarskog, 2011). Together, these findings strengthen existing models that link genetic predispositions to personality phenotypes. Based on prior evidence that OT administration is associated with increased self-reported Openness to Experience and Extraversion (Cardoso, Ellenbogen, & Linnen, 2012), we predicted that epigenetic modification would also be associated with individual differences in trait Extraversion. The results of this study did not support this hypothesis. These findings indicate that the way OT administration affects self-perceptions of Extraversion is different than the way epigenetic modification of OXTR affects selfperceptions of Extraversion. This is consistent with prior evidence indicating OT administration affect brain function differently according OXTR genotype (Feng et al., 2015;Marsh et al., 2012). In addition, there is also evidence that effects of OT administration vary according to sex and age (Ebner et al., 2016). Future studies are required that combine OT administration, measurement of DNA methylation, and personality and behavioral measures.
In this study, we characterized epigenetic modification of OXTR via DNA methylation values derived from saliva samples. Despite its association with personality, methylation patterns observed in saliva may not reflect DNA methylation patterns in the brain specifically. Some studies, however, indicate that DNA derived from the saliva may be a good proxy for methylation within brain tissue (Smith et al., 2015;Smith et al., 2014) and may reflect epigenetic programming that is not tissue-specific. It is currently unclear how exogenous and endogenous factors may impact DNA methylation of the OXTR gene. Changes in DNA methylation occur in response to early childhood experience (Essex et al., 2013), neuronal activity (Guo et al., 2011), and social stress (Unternaehrer et al., 2012). It will be important for future epigenetic studies on OXTR to include variables representative of individual life experience and psychological stress.
The current results do not provide an indication of directionality. Specifically, it is currently unclear if highly "open" people are exposed to factors that reduce OXTR methylation. Alternatively, it is currently unclear if reduced OXTR methylation leads to being more "open." There is evidence that both Openness to Experience and DNA methylation are dynamic. For example, out of the Big-5 personality traits, Openness to Experience tends to be the most dynamic (Bleidorn, Kandler, Riemann, Angleitner, & Spinath, 2009), and tends to reduce with age (Gregory, Nettelbeck, & Wilson, 2010). In addition, mindfulness-based training (van den Hurk et al., 2011), as well as psilocybin intake (MacLean, Johnson, & Griffiths, 2011) are associated with increased Openness to Experience. DNA methylation is also subject to change in response to experience (Essex et al., 2013), and according to age (Horvath, 2013). Unternaehrer et al. (2012) showed that OXTR methylation increases following acute psychosocial stress (Trier social stress test). Together these studies indicate that the relationship between OXTR methylation and trait Openness to Experience is dynamic and likely bidirectional. In addition, these findings highlight the need for future studies to incorporate longitudinal approaches to investigate epigenetics and personality.
There exist several important limitations of this study that warrant consideration. First and foremost, the sample size in this study is modest and the current finding requires replication. This study is also limited in terms of the number of predictor and outcome variables. We targeted CpG -934/-924 based on extant data demonstrating this site to be associated with psychological and brain-based metrics (Bell et al., 2015;Jack, Connelly, & Morris, 2012;Kader, Ghai, & Maharaj, 2018;Puglia et al., 2015;Rubin et al., 2016). However, this study fails to examine the association between other CpG sites within the promoter region of OXTR or other genes with the OT system and personality.
In addition, other genes, such as within the serotonin or dopamine systems, may also be associated with trait Openness to Experience, but were not considered here. Lastly, it will be important for future epigenetic studies of personality to take into account measures of gene expression as well a life experience.
In conclusion, this study presents new evidence that epigenetic modification of OXTR confers individual differences in personality. Reduced DNA methylation of OXTR is associated with higher trait Openness to Experience. This finding sheds new light onto an important psychological question: "what factors influence human identity?"


extfieldsofstudy: Biology Medicine

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Crosstalk Between Peroxisome Proliferator-Activated Receptor Gamma and the Canonical WNT/β-Catenin Pathway in Chronic Inflammation and Oxidative Stress During Carcinogenesis

Inflammation and oxidative stress are common and co-substantial pathological processes accompanying, promoting, and even initiating numerous cancers. The canonical WNT/β-catenin pathway and peroxisome proliferator-activated receptor gamma (PPARγ) generally work in opposition. If one of them is upregulated, the other one is downregulated and vice versa. WNT/β-catenin signaling is upregulated in inflammatory processes and oxidative stress and in many cancers, although there are some exceptions for cancers. The opposite is observed with PPARγ, which is generally downregulated during inflammation and oxidative stress and in many cancers. This helps to explain in part the opposite and unidirectional profile of the canonical WNT/β-catenin signaling and PPARγ in these three frequent and morbid processes that potentiate each other and create a vicious circle. Many intracellular pathways commonly involved downstream will help maintain and amplify inflammation, oxidative stress, and cancer. Thus, many WNT/β-catenin target genes such as c-Myc, cyclin D1, and HIF-1α are involved in the development of cancers. Nuclear factor-kappaB (NFκB) can activate many inflammatory factors such as TNF-α, TGF-β, interleukin-6 (IL-6), IL-8, MMP, vascular endothelial growth factor, COX2, Bcl2, and inducible nitric oxide synthase. These factors are often associated with cancerous processes and may even promote them. Reactive oxygen species (ROS), generated by cellular alterations, stimulate the production of inflammatory factors such as NFκB, signal transducer and activator transcription, activator protein-1, and HIF-α. NFκB inhibits glycogen synthase kinase-3β (GSK-3β) and therefore activates the canonical WNT pathway. ROS activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling in many cancers. PI3K/Akt also inhibits GSK-3β. Many gene mutations of the canonical WNT/β-catenin pathway giving rise to cancers have been reported (CTNNB1, AXIN, APC). Conversely, a significant reduction in the expression of PPARγ has been observed in many cancers. Moreover, PPARγ agonists promote cell cycle arrest, cell differentiation, and apoptosis and reduce inflammation, angiogenesis, oxidative stress, cell proliferation, invasion, and cell migration. All these complex and opposing interactions between the canonical WNT/β-catenin pathway and PPARγ appear to be fairly common in inflammation, oxidative stress, and cancers.

Inflammation and oxidative stress are common and co-substantial pathological processes accompanying, promoting, and even initiating numerous cancers. The canonical WNT/β-catenin pathway and peroxisome proliferator-activated receptor gamma (PPARγ) generally work in opposition. If one of them is upregulated, the other one is downregulated and vice versa. WNT/β-catenin signaling is upregulated in inflammatory processes and oxidative stress and in many cancers, although there are some exceptions for cancers. The opposite is observed with PPARγ, which is generally downregulated during inflammation and oxidative stress and in many cancers. This helps to explain in part the opposite and unidirectional profile of the canonical WNT/β-catenin signaling and PPARγ in these three frequent and morbid processes that potentiate each other and create a vicious circle. Many intracellular pathways commonly involved downstream will help maintain and amplify inflammation, oxidative stress, and cancer. Thus, many WNT/β-catenin target genes such as c-Myc, cyclin D1, and HIF-1α are involved in the development of cancers. Nuclear factor-kappaB (NFκB) can activate many inflammatory factors such as TNF-α, TGF-β, interleukin-6 (IL-6), IL-8, MMP, vascular endothelial growth factor, COX2, Bcl2, and inducible nitric oxide synthase. These factors are often associated with cancerous processes and may even promote them. Reactive oxygen species (ROS), generated by cellular alterations, stimulate the production of inflammatory factors such as NFκB, signal transducer and activator transcription, activator protein-1, and HIF-α. NFκB inhibits glycogen synthase kinase-3β (GSK-3β) and therefore activates the canonical WNT pathway. ROS activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/ Akt) signaling in many cancers. PI3K/Akt also inhibits GSK-3β. Many gene mutations of the canonical WNT/β-catenin pathway giving rise to cancers have been reported (CTNNB1, AXIN, APC). Conversely, a significant reduction in the expression of PPARγ has been observed in many cancers. Moreover, PPARγ agonists promote cell cycle arrest, cell differentiation, and apoptosis and reduce inflammation, angiogenesis, iNTRODUCTiON Cancer is a complex process that can be defined in term of three steps: initiation, promotion, and progression (1). Several chemical, physical, and biological factors may induce chronic inflammation, thereby increasing the risk of cancers (2). This link between cancer and inflammation has been reported in experimental and epidemiological studies (3,4) and demonstrated through the efficacy of anti-inflammatory therapies in cancer (5). Chronic inflammation is responsible for various steps involved in carcinogenesis, such as promotion, survival, cellular transformation, invasion, proliferation, angiogenesis, and metastasis (6,7).
Oxidative stress also operates at these stages by promoting DNA damages and genes mutations (8). In recent years, several studies have shown that the link between inflammation and cancer can involve oxidative stress through reactive oxygen species (ROS) production. Tumor promoters have the capacity to recruit inflammatory factors and then stimulate ROS production (9,10). Oncogenic transformation is promoted by oxidative stress that acts as a DNA-damaging effector (11). ROS generation, together with oxidative stress, stimulates several signaling pathways that contribute to cancer development by regulating proliferation, invasion, angiogenesis, and metastasis (12).
The canonical WNT/β-catenin pathway regulates several signaling pathways involved in development and tissue homeostasis. This pathway is modulated from transcription level regulations to post-transcriptional modifications. An aberrant WNT/βcatenin pathway is observed in cancers (13,14). This results in stimulating the expression of numerous WNT target genes involved in tumor development, such as c-Myc, cyclin D1, and HIF-1α (15), the production of ROS (16), and the activation of chronic inflammation (17).

CANONiCAL wNT/β-CATeNiN PATHwAY
The WNT pathway is involved in numerous pathways that control tissue homeostasis and embryogenesis development. WNT ligands belong to the family of genes observed in humans, Xenopus, mice, drosophilia, and Zebrafish (31). Dysregulation of the canonical WNT pathway activity has been reported in several disorders and cancers (32-34).
Canonical WNT signaling is characterized by the interaction between WNT ligand and specific targets resulting in cytosolic β-catenin accumulation and then its nuclear translocation (Figure 1). The nuclear activation of β-catenin results in the stimulation of downstream factors (35). During the "off state" of the WNT/β-catenin pathway, WNT ligands do not bind specific receptors. Cytosolic β-catenin is maintained at a minimal level through the activation of the β-catenin destruction complex, formed by the combination of AXIN (a cytoplasmic protein regulating G-protein signaling), glycogen synthase kinase-3β (GSK-3β, a serine-theronine kinase), adenomatous polyposis coli (APC, a tumor suppressor gene), and casein kinase 1 (CK-1, a serine/threonine-selective enzyme) (36). CK-1 and GSK-3β target β-catenin by phosphorylating the serine and threonine residues located in the amino acid terminus (37-39). CK-1 phosphorylates an N-terminus of β-catenin and GSK-3β phosphorylates a threonine 41 (Th41), Ser33, and Ser37 sites of β-catenin (35, 40). These phosphorylations result in recruiting APC in the destruction complex. APC modulates the degradation of the cytosolic β-catenin into the proteasome through its tumor suppressor properties (36, 41).
The "on state" is characterized by modified WNT/β-catenin signaling. WNT ligands bind Frizzled (FZD, a family of G protein-coupled receptors) and low-density lipoprotein receptor-related protein 5 (LRP 5/6) co-receptors (42). Then, Disheveled (DSH, a phosphoprotein) forms a complex with FZD, which results in the phosphorylation of LRP5/6 by GSK-3β and then the recruitment of the β-catenin destruction complex. DSH phosphorylates LRP6 (43), which inhibits GSK-3β activity leading to the stabilization and then the cytosolic accumulation of β-catenin. Accumulation of β-catenin leads to its nuclear translocation and then β-catenin binds the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors (44, 45). The nuclear complex formed by β-catenin and TCF/LEF activates several WNT target genes, such as c-Myc (a transcription factor) and cyclin D1 (a protein belonging to the highly conserved cyclin family encoded by the CCND1 gene) (37, 38). The WNT target genes are involved in several processes, such as cell division, proliferation, invasion, and stem cell maintenance (46). Furthermore, β-catenin accumulation is involved in cancer phenotype maintenance (47-49).

Crosstalk Between PPARγ and wNT/β-Catenin Signaling in Cancers
Even if, the molecular mechanisms by which TZDs regulate differentiation and stemness programs have been well studied in adipocytes and normal cells, in cancer cells, they still remain unclear (32). In normal cells, PPARγ suppresses tumorigenesis and WNT signaling by targeting phosphorylated β-catenin at the proteasome by a process involving its catenin-binding domain within PPARγ. In contrast, oncogenic β-catenin resists proteasomal degradation by inhibiting PPARγ activity, which requires its TCF/LEF-binding domain (62). In adipocytes, PPARγ increases differentiation and inhibits proliferation by affecting the WNT/β-catenin pathway. PPARγ interacts with GSK3-β to induce the differentiation factor C/EBPα and this leads to the production of adiponectin (101,102). PPARγ activation reduces β-catenin at both the mRNA and protein levels to promote differentiation (103). In human metastatic prostate cancer LnCaP cells, PPARγ inhibits the WNT pathway by targeting phosphorylated β-catenin at the proteasome (62, 104). In gastric and colon cancer cells, PPARγ decreases β-catenin expression, subcellular localization, and downstream effectors, resulting in the modulation of several genes, such as telomerase reverse transcriptase, and Sox9, which are involved in cell development, differentiation, and survival processes (105)(106)(107). PPARγ agonists, by inhibiting activation of the WNT/β-catenin pathway, could be used in combination with other drugs such as inhibitors of tyrosine kinases (108), PI3K/AKT (109), and mitogenactivated protein kinase (MAPK) cascades to maximize the antitumor and pro-differentiating effect.

CARCiNOGeNeSiS: ROLe OF CHRONiC iNFLAMMATiON AND OXiDATive STReSS
Cancer progression is promoted by an environment rich in inflammatory factors, DNA damages, and genetic or epigenetic mutations (110).

Chronic inflammation
Several studies have shown that prolonged inflammation leads to DNA damages and tissue injury (111). Chronic inflammation can affect cell homeostasis, metabolism, and genomic regulation, leading to the initiation of tumorigenesis (112). Furthermore, damages induced by chronic inflammation are responsible for the development of malignancy sites (113,114).
The link between inflammation and cancer initiation has been examined in a recent study (115). Inflammation stimulates the activation of cytotoxic mediators, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), which have a major role in DNA damages (116). DNA damage accumulation is responsible for the initiation of carcinogenesis through the enhancement of genomic instabilities (117).
Pathogenic stimuli can stimulate inflammation and then eradicate the normal host defense response (118). Pathogens promote carcinogenesis through the recruitment of infections and the inhibition of immune response leading to chronic inflammation (3). Stomach, intestine, liver, colon, and skin are the main sites of common pathogenic infections that are believed to be related to cancer progression (119,120).
The inflammatory response is regulated by the canonical WNT/β-catenin pathway (111). Moreover, infection pathogens can overexpress the WNT/β-catenin pathway leading to uncontrolled inflammation and then to an increased risk of carcinogenesis (121).

Oxidative Stress
Oxidative stress is characterized by an imbalance between production and elimination of reactive metabolites and free radicals (ROS and RNS) (8,136). ROS generation is caused by cell damages through nitration and oxidation of macromolecules, such as proteins, lipids, DNA, and RNA. The NADPH oxidase (NOX) enzyme enhances ROS through the oxidation of intracellular NADPH to NADP + . Then, the transfer of electrons through the mitochondrial membrane reduces molecular oxygen and produces the superoxide anion as a primary product. ROS production has a major role in several pathways and in changes of intracellular and extracellular environmental conditions (137).
Leukocytes during inflammation are recruited from the damage sites and this leads to an increased uptake of oxygen, which induces the release of ROS and subsequently its accumulation (6,149).
Several redox-regulated transcription factors have a key role in the stimulation of pro-inflammatory mediators, such as NFκB, a signal transducer and activator of transcription (STAT), activator protein-1 (AP-1), and the hypoxia-inducible factors (HIF) (112). The oxidative stress-induced inflammation induces the production of COX2, iNOS, TNF-α, IL-6, and miRNAs (150). A vicious circle operates between inflammation and oxidative stress leading to carcinogenesis (129).
NADPH-oxidase (NOX) is stimulated by inflammation and leads to oxidative stress and alteration of nuclear signaling (151). ROS, activated by NOX, stimulate the canonical WNT/βcatenin pathway through oxidization and inactivation of the nucleoredoxin (a redox-sensitive regulator), resulting in tumor cell proliferation (112). ROS production leads to the activation of c-Myc (152), STAT (153), and PI3K/Akt (154) and the inactivation of PPARγ (155). ROS production activates Akt signaling through the inhibition of the phosphatase and tensin homolog deleted from chromosome (PTEN) (156,157). The Akt pathway is involved in cellular metabolism and the promotion of cell survival (156,157).

iNTeRACTiONS BeTweeN THe CANONiCAL wNT/β-CATeNiN PATHwAY AND CARCiNOGeNeSiS interactions Between the Canonical wNT/β-Catenin Pathway and inflammation
A positive crosstalk between WNT/β-catenin and NFκB has been reported recently (17). The overexpression of WNT/β-catenin results in the enhancement of IκB-α degradation and then NFκB transactivation (158) (Figure 2). Upregulation of the target gene, CRDBP, by stimulated β-catenin signaling leads to a stabilization of βTrCP mRNA (159). In colon cancer, overexpression of both βTrCP and CRD-BP is associated with the activation of the β-catenin signaling and NFκB, contributing to cell proliferation and metastasis (159,160). In breast cancer, TLR3 stimulation activates β-catenin signaling simultaneously with activation of the NFκB pathway, in a synergistic manner (161). β-catenin and NFκB pathways act in together diffuse large B-cell lymphomas (162). The WNT/β-catenin pathway leads to an increase in COX expression, which then influences the inflammatory response (163). E-cadherin and GSK-3β are decreased in melanoma cells by the stimulated β-catenin signaling (164). Concomitant GSK-3β and E-cadherin inactivation with cytosolic β-catenin accumulation induces NFκB-dependent iNOS expression in hepatic cells (165). The WNT/β-catenin pathway modulates in a positive manner its downstream target TNFRSF19 in colon cancer, which activates the NFκB signaling (166). However, the synergistic effect between β-catenin and NFκB depends on both the TCF/LEF link and the context of the genes or cell types (167).
Nuclear factor-kappaB signaling can regulate WNT/β-catenin signaling through the use of IKKα (170) and RelA (171). IKKα can increase β-catenin signaling, whereas IKKβ downregulates β-catenin signaling (172). IKKα upregulates β-catenin/TCF/LEF activation and then the target gene cyclin D1 (173). GSK3-β and APC are degraded through the activation of IKKα leading to the cytosolic β-catenin accumulation (170). In glioma cells, overexpression of RelA coupled with the knockout of SN50, a NFκB inhibitor, increases the β-catenin nuclear translocation and then enhances β-catenin/TCF/LEF activity (174). The positive crosstalk between the WNT/β-catenin pathway and NFκB pathway participates in the regulation of several pathways involved in cancer development based on inflammation-induced carcinogenesis. This could be explained in part by the synergistic effect observed between β-catenin/TCF4 and NFκB on the overexpression of WNT target genes in colon cancer (171). This positive crosstalk induces several stem cell signature genes, such as Sox9, Ascl2, and Lgr5, leading to tumor growth.
interactions Between the Canonical wNT/β-Catenin Pathway and ROS Reactive oxygen species production activates the PI3K/Akt pathway, which is overactivated in numerous cancers (175). PTEN is a phosphoinositide-3-phosphatase, which downregulates the PI3K/Akt pathway (157). NADPH oxidase and superoxide dismutase oxidize PTEN and inactivate it. Then, the inhibition of PTEN activity by oxidative stress increases the activity of Akt and thus enhances the phosphorylation of GSK-3β by Akt. GSK-3β inactivated by Akt does not inhibit the nuclear β-catenin signaling, resulting in cell proliferation in several cancers. Alkylation of PTEN activates Akt and β-catenin (176). In addition, ROS causes the stabilization of HIF-1α and then the activation of the glycolytic enzymes participating in cell proliferation and angiogenesis (50, 175). The WNT/β-catenin pathway can activate HIF-1α by stimulating the PI3K/Akt pathway (15).
A recent study by Zhang et al. (177) has shown that ROS production activates the WNT/β-catenin pathway, but the mechanism involved remains unclear (177) (Figure 2). Furthermore, carcinogenesis of cells can increase the endogenous level of ROS production (175). Indeed, several oncogenes enhance ROS production, such as Akt (16) and c-Myc (178).

Genetic and epigenetic Regulation of the Canonical wNT/β-Catenin Pathway in Cancers
Several genetic mutations lead to the aberrant activation of the canonical WNT/β-catenin pathway (14). In numerous malignant FiGURe 3 | Peroxisome proliferator-activated receptor gamma (PPARγ) activation can inhibit cancer development through several mechanisms by acting on numerous target genes and pathways, such as the canonical WNT/β-catenin pathway. It also interferes with pro-inflammatory signaling by repressing nuclear factor-kappaB (NFκB) and tumor necrosis factor α (TNF-α).
processes, the regulator genes of CTNNB1, AXIN, and APC have been observed to be mutated (179).
Mutations of CTNNB1, a β-catenin target, have been shown to be involved in the initiation of colon, gastric, ovarian, pancreatic, and prostate cancers, but also in melanoma and medulloblastoma (180,181). APC mutations have been observed in colon cancer and AXIN mutations in hepatocellular carcinoma and medulloblastoma (182,183).
Several studies have shown an interaction between miRNAs and the canonical WNT/β-catenin pathway, such as in osteoblast differentiation and cardiac and bone formation (184-186) (Figure 2). An aberrant expression of miR-374a is coupled with cytosolic β-catenin accumulation in breast cancers (187), and degradation of APC leading to the inactivation of the β-catenin destruction complex and enhancing the transcriptional activity of TCF/LEF (188). Moreover, the canonical WNT/β-catenin pathway controls the activity of several cancer-stem cell (CSC)specific miRNAs. These specific miRNAs play a major role in tumor initiation. Overexpression of the WNT/β-catenin pathway leads to activation of the oncogenic miRNA expression to enhance the self-renewal potential of CSCs, which is involved in the resistance to drug therapy and initiation of new tumor growth (14).
Cancer-stem cells theory is characterized by the fact that cancer cells are derived from certain populations of cells, which possess stem cell properties (189)(190)(191). Several studies have shown that the WNT/β-catenin pathway can regulate stem cells and stem progenitors, plethora system maintenance, and cell self-renewal (37). Moreover, in a recent study, it was observed that the cano nical WNT/β-catenin pathway plays a major role in the regulat ion of the activity of stem self-renewal in numerous cells (192).
The role of miRNAs in the regulation of CSCs is currently being investigated. Nevertheless, it has been observed that several miRNAs, such as miR410, can promote tumor growth, invasion, and migration of NSCLC cells through the activation of the canonical WNT/β-catenin pathway (193). miR-451 expression stimulates the upregulation of the macrophage migration inhibitory factor (MIF) and COX-2 expression to activate WNT/β-catenin pathway in CSCs (194,195).
On the other hand, other miRNAs appear to be tumor suppressors, such as miR-34a. miR-34a directly targets the tumor suppressor p53 and suppresses the expression of several target genes, such as SOX, Nanog, and N-Myc (205,206). Let-7 is considered as a β-catenin negative regulator (207). Future studies will help us to better understand the role of miRNAs in the inhibition or activation of cancer initiation and its development through interaction with the WNT/β-catenin pathway by regulating the epithelial mesenchymal transition in can cer (208,209).

ACTiON OF PPARγ AGONiSTS iN CANCeRS
Several studies have shown a significant reduction of PPARγ expression in cancers such as colon cancer (210,211), gastric cancers (212), follicular thyroid cancer (213), cervical carcinoma (214), and esophageal cancer (215) (Figure 3). Numerous studies have shown that PPARγ has antineoplastic actions on lung,

Proliferation
Several studies have observed an anti-proliferative role played by TZDs (233,265). Moreover, the PPARγ ligand docosahexaenoic acid (DHA) has been shown to have an anti-proliferative role in lung tumor cell cultures (234). In parallel, DHA also downregulates the cell proliferation and angiogenesis processes in breast cancer (231,236). Rosiglitazone reduces the proliferation time of liposarcoma, but troglitazone has a limited effect in prostate, colon, and breast cancers (266)(267)(268).

invasion and Migration
Thiazolidinediones downregulate tumor growth and the migration of tumor cells in colon cancer cells by inducing cell differentiation (264). In the same way, TZDs arrest cell cycle G1 with a decrease in E-cadherin expression (264). To date, few studies have revealed PPARγ agonists to play a positive role in the inhibition of invasion and the migration of cancer cells.
inflammation Peroxisome proliferator-activated receptor gamma agonists, such as DHA and omega-3 fatty acids EPA, are known to induce antiinflammatory activity (252,253). Some natural and synthetic PPARγ agonists can have a chemoprotective role by targeting inflammatory agents (257,271). PPARγ overexpression inhibits the activity of TNF-α and NFκB (257,272) resulting in a reduction of tumor development (273). PPARγ activation seems to act on the tumor environment, especially on inflammation (19).

Angiogenesis
Peroxisome proliferator-activated receptor gamma agonists can modulate angiogenesis in vitro and in vivo models (274). However, some paradoxical effects of PPARγ agonists have also been observed. PPARγ agonists may enhance VEGF in tumor cells (275,276) and may have pro-or anti-angiogenic roles depending on the cell environment (277)(278)(279)(280).

CONCLUSiON
Cancers are readily associated with complex inflammatory phenomena and oxidative stress that may complicate or even initiate them. In cancers, apart from certain exceptions, the canonical WNT/β-catenin signaling is generally upregulated while PPARγ is downregulated. These two major cell pathways work in an opposite manner and this partly explains their unidirectional profile observed in cancers, chronic inflammation, and oxidative stress. This results in an activation of several upstream or downstream pathways involved in carcinogenesis, such as TGF-β, NFκB, TNF-α, TGF-β, IL-6, IL-8, VEGF, iNOS, PI3K/Akt, HIF-1α, and certain target genes such as c-Myc, cyclin D1, COX2, and Bcl2. The use of PPARγ agonists in cancers could reduce both ROS production and chronic inflammation leading to a decrease in the WNT/β-catenin pathway and then an inhibition of carcinogenesis processes (Figure 4). Because of the considerable impact of cancers and inflammatory processes on mortality and morbidity rates worldwide, it is imperative to continue to find new therapeutic pathways by seeking, directly or indirectly, to inhibit the canonical system WN/β-catenin and to activate PPARγ by new agonists free of deleterious effects.

AUTHOR CONTRiBUTiONS
All listed authors have made substantial, direct, intellectual contributions to the study and have given their approval for its submission for publication.