Modulatory Role of Polyphenols in the Molecular Adaptation of Lactobacilli to the Host Gut

Abstract

Plant polyphenols are a class of chemically diverse molecules that contribute, among many other health benefits, to sustain an enhanced host gut microecological niche. It is thought that the modulation of the gut microbiota by these metabolites is a crucial process that contributes to maintain gut homeostasis. Polyphenols are known to shift the gut microbiota composition inhibiting opportunistic pathogens and exerting a prebiotic-like effect on beneficial gut microbes, being Lactobacillus spp. one of the enriched taxa. In this chapter we describe how different polyphenol classes target the relative abundance and growth of Lactobacillus spp. Although lactobacilli can positively respond to polyphenols, mechanistic insights into how polyphenols stimulate these gut microbes is generally limited. However in recent years it has been revealed that some polyphenols can modulate molecular functions implicated in the adaptation of lactobacilli to the gut environment. In addition, some polyphenols can modulate the expression of molecular functions that are engaged in the crosstalk between lactobacilli and intestinal host cells. These developments can provide molecular-based scientific support for polyphenol-mediated improvement of effector capacities of Lactobacillus associated with beneficial effects on host-physiology.

1. Introduction

Lactobacilli belong to the lactic acid bacteria (LAB), which are Gram-positive organisms with a low G + C content that belong to the phylum of the Firmicutes. The genus Lactobacillus encompasses well-studied and very diverse species. Recently, the genus Lactobacillus has been reclassified into 25 genera, including an emended description of the genus Lactobacillus and 23 novel genera. This reclassification allows lactobacilli to be grouped into clades with shared ecological and metabolic properties [1]. Many lactobacilli are industrially relevant microbes that are widely used in a large variety of industrial food fermentations where they rapidly acidify the raw material to critically contribute in the preservation of the final food product. In addition, lactobacilli have the capacity to lend these products other characteristics such as texture and flavor.

Lactobacilli also have the potential to modulate the human physiology and health and have been recognized as potential health beneficial microorganisms in the human gastrointestinal tract. The host diet markedly impacts the adaptation responses of lactobacilli to the gut environment [2].

Among the dietary components, the role of polyphenols to determine the composition of the gut microbiota has been addressed. Polyphenols are chemically diverse molecules that are primarily divided into flavonoid and non-flavonoid classes. According to animal and in vitro studies that evidenced antioxidant, radical-scavenging, and antimutagenic properties, long-term consumption of different polyphenols has been correlated with chronic disease prevention [3]. The gut microbiota composition is an essential factor to extract these polyphenol benefits and counteract metabolic diseases. The potential of polyphenols to influence the composition of the gut microbiota arise in part from its antimicrobial potential, which can result in significant and long-term modification of host gut microbiomes. As will be seen below, Lactobacillus spp. is one of the bacterial populations usually boosted by the administration of polyphenols as they are more tolerant to these metabolites than other bacterial groups [4]. Given lactobacilli have been traditionally recognized as potential health-promoting microbes in the human gastrointestinal tract, considerable information has been accumulated on the Lactobacillus-polyphenol interaction. Besides the positive influence of a number of polyphenols on the growth of Lactobacillus spp. in the host gut, in recent years it has been revealed that polyphenols can modulate molecular functions that are required for adaptation to the gut environment. Interestingly, it has been also observed that some polyphenols can modulate the expression of molecular functions that are engaged in the communication between lactobacilli and intestinal host cells, which in part underlie the host-health effects derived from the administration of these plant metabolites.

2. Modulation of the relative abundance of host gut Lactobacillus spp. by polyphenols

Many polyphenols have antimicrobial potential and as dietary constituents, may induce transformation changes in our gut microbiota. These changes are crucial for the functional efficacy in the gastrointestinal (GI)-tract of a particular microorganism as it depends in part on its (relative) numerical abundance and viability. Therefore, the effects of different structural classes of polyphenols on the relative abundance and viability of Lactobacillus spp. (Table 1) are important for the potential health benefits associated with these microorganisms. However, although the polyphenols can improve the relative abundance of Lactobacillus spp. and impact their activity, these should not be necessarily considered as prebiotic effects. Only if the benefit of the polyphenol arises from the selective utilization by members of the gut microbiota, it could be regarded as prebiotic.

Table 1.

Polyphenol-mediated modulation of in vivo and in vitro relative abundance and viability of Lactobacillus spp.

2.1 Anthocyanins

The anthocyanins are the most abundant polyphenols in a wide variety of blue, red and purple-colored fruits such as berries, red plums or pomegranates. Representative compounds of this family are cyanidin, delphinidin, pelargonidin and malvidin. The intact (glycosylated) dietary anthocyanins generally escape from the the upper gastrointestinal tract and undergo biotransformation in the colon by the action of the enzymatic action of β-glucosidase activity, which can arise from brush border enzymes and/or from gut microbial β-glucosidase activity. Several Lactobacillus strains from species such as Lactiplantibacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus or Lacticaseibacillus casei are involved in anthocyanin degradation [28, 34]. Correlation between the bulk β-glucosidase activity of gut Lactobacillus spp. and degradation of anthocyanins (pure malvidin-3-glucoside and delphinidin-3-glucoside) has been reported and suggested to be strain dependent [28, 34]. However, up to date no specific glycoside hydrolase enzymes from Lactobacillus displaying β-glucosidase activity towards anthocyanins, have been yet described.

Based on several studies using models of in vitro colonic fermentation or in vivo animal fed models with plant extracts rich in anthocyanins or a mix of anthocyanins, it has been hypothesized that anthocyanins (malvidin-3-glucoside or a mix of anthocyanins glucosides) alter the gut microbiota and increase the prevalence of beneficial bacteria, including Lactobacillus spp. (Table 1) [26, 27]. However, cyanidin-3-glucosides from mulberry have shown neutral effects on the growth of L. plantarum or L. acidophilus pure cultures (Table 1) [28].

New metabolites can arise from Lactobacillus anthocyanin metabolism including gallic acid (GA), which was proposed as a critical metabolite to change the structure of the gut microbiota and to prompt the increased abundance of Lactobacillus spp. [26]. However, the potential effects of GA in boosting Lactobacillus cannot be considered specific of anthocyanins as this acid can be found as free acid in the diet or as a product of the metabolism of other polyphenols.

2.2 Flavan-3-ols

Similarly to other polyphenols, the flavan-3-ols can influence the distribution and abundance of microbial populations in the digestive tract but apparently conflicting results have been reported. Some studies have evidenced that the administration of flavanol-rich foods, such as peach peel extracts (mainly epicatechin 3-O-glucoside), alter the gut microbiota and boost the Lactobacillus population (Table 1) [5]. However, the dietary suplementation of other flavanol-rich fruit, camu-camu (rich in galloylated flavanols such as ellagitannins (ETs) and ellagic acid) reduced the abundance of Lactobacillus spp. (Table 1) [6].

Furthermore green tea, which is abundant in flavanols, including the monomers catechin or epicatechin (EC) and the oligomers epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG) (rich in EGCG (48%), can promote the growth of beneficial bacteria, including Bifidobacterium and Lactobacillus, and inhibit pathogenic bacteria, such as Clostridium [7]. In apparent contrast, growth stimulation of Lactobacillus spp. was not observed during the in vitro fermentation of EGCG, the most abundant catechin in green tea, by human gut microbiota (Table 1) [8].

2.2.1 Monomeric flavan-3-ols

Regarding the different impact of monomeric flavan-3-ols on gut lactobacilli it has been reported that a L. plantarum [9] and a L. casei strain [35] are able to cleave the heterocyclic ring of monomeric flavan-3-ols. The formed end-products were more effective antioxidant agents than their flavan-3-ols precursors, albeit these metabolic conversions, which seem to be strain-dependent, did not alter the growth characteristics of the Lactobacillus spp. involved (Table 1). In fact, it has been suggested that most Lactobacillus plantarum strains do not present the ability to metabolize catechins [36]. Interestingly, it has been reported that stimulation of Lactobacillus growth by some monomeric flavanols can be uncoupled from flavanol degradation [10]. Exposure of L. plantarum to catechin (which was found intact at the end of the fermentation) prompted quicker and more efficient sugar utilization and stimulated malic acid fermentation to target an improved growth. This effect was suggested to be caused by catechin-induced alteration of the biophysical properties of the membrane which would positively affect the efficacy of sugar and organic acid transporters.

2.2.2 Polymeric flavan-3-ols

The flavan-3-ols may form polymers with high molecular weights, being condensed tanins (or proanthocyanidins) or hydrolysable tannins (mainly gallotannins and ellagitannins (ETs) the most reported. These flavan-3-ols polymers exert a marked antimicrobial activity against gram-negative bacteria, including opportunistic pathogens. Lactobacillus are inherently more resistant to these polyphenols [4] and usually show an increased relative abundance among the gut microbiota in presence of foods with high content in these polymers suh as red wine [37] or mango (Table 1) [11]. However, in pure cultures the effect of hydrolysable tannins such as tannic acid on final viable Lactobacillus cells is nearly neutral albeit a longer lag phase and cellular injuries were observed respect to the controls (Table 1) [38].

The effects of ETs (another kind of hydrolysable tannins) have been mainly studied by using pomegranate (POM) extracts. These ETs-rich extracts (mainly ellagic acid and punicalagin) have shown its capacity to reshape the gut microbiota and stimulate the relative abundance of Lactobacillus spp. during the fermentation of fecal slurries [39] or when administered in rats (Table 1) [13]. However when pure cultures of Lactobacillus spp. were tested against individual ETs, neutral or very weak inhibitory effects were observed (Table 1) [14]. Recently it has been reported that POM extracts did not support the growth of Lactobacillus strains in the absence of an additional sugar source [15]. Cell growth experiments using POM extracts showed a positive effect for L. acidophilus (increased cell numbers) whereas L. plantarum and Lacticaseibacillus rhamnosus grew to the same extent as the controls, showing that POM extracts triggers species-specific responses by probiotic strains (Table 1). Although these studies highlight a rising interest in using POM as a prebiotic compound, further investigation is required to identify the POM constituents that result in the phenotypes observed. Further, the extent of metabolic degradation of ETs seems to differ among strains as the lack of punicalagin degradation by the probiotic strains used in this study is controversial with a recent study where different Lactobacillus probiotic spp. were able to metabolize punicalagin into ellagic acid and urolithin [40]. No urolithin formation was detected in these studies but it was not unexpcted as lactobacilli are able to metabolize urolithins [40].

2.3 Flavonols

The effects of representative members of flavonols such as quercetin, kaempferol or myricetin on the gut microbiota composition have been studied and significant shifts have been reported. Reduction of Lactobacillus populations has been observed upon quercetin supplementation (Table 1) [16]. It has been also found that myricetin is able to provoke remarkable reductions in Lactobacillus [17, 19], Lactobacillus intestinalis [17] and Limosilactobacillus spp. (Table 1) [19]. In contrast to quercetin and myricetin, the administration of kaempferol led to an increase in the relative abundance of Lactobacillus (and other beneficial gut microbes such as Akkermansia or Bacteroides) in the fecal samples of mice that were fed a high-fat diet (Table 1) [18].

Some Lactobacillus spp., such as L. acidophilus, were reported to utilize quercetin [41]. Experiments with pure cultures have shown that L. plantarum is unable to metabolize this flavonol albeit exposure to quercetin accelerates L. plantarum growth as a result of earlier sugar uptake and lactic acid production in a pH and concentration-dependent process (Table 1) [20].

Besides in its aglycone forms, flavonols can be found in planta as flavonol-glycosides. The effects of these glycosides on the viability and growth of Lactobacillus spp. have been also reported. Pure rutin or isoquercitrin increased the abundance of Lactobacillus among gut microbiota (Table 1) [21]. However, no effect on the growth of L. acidophilus pure cultures were observed in presence of rutin [22]. Several α-rhamnosidases from Lactobacillus spp. are involved in rutin utilization (Table 1) [42]. However, it has been proposed that since flavonoid absorption mainly occurs in the small intestine, the conversion by Lactobacillus spp. in the colon will occur too late in the digestive system to be of direct benefit [43]. Nevertheless it must be taken into account that Lactobacillus spp. can be predominant in the small intestine (depending on the inter-individual variation) [44], the significance of which for the bioavailability and effects of flavonoids and other polyphenols mainly absorbed in the small intestine, deserves further research.

2.4 Flavanones and flavonones

Flavanones, which are found mainly in citrus fruits (lemons, oranges) and berries, reach the colon intact. Among flavanones, hesperidin and naringin are well studied glycosylated compounds which can undergo microbial biotransformation into the aglycones hesperetin and naringenin. Strains of Levilactobacillus brevis, Lacticaseibacillus paracasei and L. acidophilus [45] have been reported to hydrolyze hesperidin glycosides showing the implication of Lactobacillus spp. in the deglycosylation of flavanones in the gut. Furthermore, an increase in the relative abundance of Lactobacillus spp. has been observed in hesperidin-feed rats (Table 1) [23]. In addition the flavanone glucosides hesperetin-7-O-glucoside and prunin (naringenin 7-O-beta-D-glucoside), also increased the relative abundance of Lactobacillus in the gut microbiota (Table 1) [18]. Among the gut microbial enzymes involved in the hydrolysis of these flavanones, α-rhamnosidases capable to hydrolyze naringin have been identified in Lactobacillus acidophilus [20]. Apart from the enrichment of Lactobacillus spp. in hesperidin-fed rats [23], data obtained on the re-shaping of the gut microbiota induced by flavanones is scarce but suggests that stimulation of Lactobacillus spp. by these polyphenols, at least with hesperidin, depends on various factors. Thus, the ability to hydrolyze flavanones seems to be strain-dependent, can be limited by the structure of the glycoside and can be subjected to catabolite repression [46].

Regarding the flavonones, few studies have addressed the effects of these phenolics on Lactobacillus but it has been found that apigenin does not alter the growth of L. rhamnosus pure cultures (Table 1) [25]. However, treatment of rats with luteolin leads to Lactobacillus as one of the dominant genera in the context of ulcerative colitis (Table 1) [24].

According to these data some flavanone and flavonones have the potential to increase the relative abundance of some Lactobacillus spp. in the gut. However, the underlying mechanisms supporting this advantage are currently unknown.

2.5 Phenolic acids

Phenolics acids, mainly hydroxycinnamic (HCAs) and hydroxybenzoic (HBAs) acids, might occur in the diet esterified with polysaccharides in the lignocellulosic biomass or as free acids (for example in cacao and coffee). In addition, some phenolic acids such as GA can be produced in the gut as end products of the metabolism of distinct classes of more complex polyphenols, including anthocyanins or tannic acid.

The HCAs, such as p-coumaric acid (p-CA), can exert strong inhibitory effects against several Gram-positive and Gram-negative bacteria [47] and therefore may provoke gut community modifications and contribute to define the distribution and abundance of microbial populations in the digestive tract.

The antimicrobial activity of HCAs is mainly exerted against gram negative bacteria. Gram positive bacteria are generally less sensible to these acids which show no antimicrobial activity against several Lactobacillus spp. [33]. Thus, intake of dietary HCAs may credit those bacteria able to resist these phenolic acids to persist and colonize the gastrointestinal (GI)-tract of mammals. In fact, phenolic acids (p-CA, ferulic acid and GA) bound to rice bran fiber act as prebiotics for Lactobacillus spp. when released from the fibers (Table 1) [32]. The higher tolerance of Lactobacillus spp. to HCAs is based on an array of diverse mechanisms developed to counter this stress. These mechanisms include the activation of general stress pathways, induction of efflux pumps, differential regulation of genes and proteins involved in the biosynthesis of macromolecular components of the cell envelope as well as a marked response to cope with the oxidative stress imposed by these acids [48].

Besides these mechanisms, lactobacilli are able to metabolize free hydroxycinnamic acids by decarboxylation and/or reduction pathways [49, 50].

Decarboxylation of HCAs give rise to less toxic vinyl derivatives compounds. The reduction of hydroxycinnamic acids into penylpropionic acids can add a growth advantage to certain strictly heteroferementative Lactobacillus spp. under anaerobic conditions. Thus, by using HCAs as external electron acceptors these heterofermentative lactobacilli regenerate NADH in the reduction reaction to gain additional ATP [51]. This mechanism constitutes a true prebiotic effect that could explain the preference of reduction over the decarboxylation pathway in these species.

Regarding the hydroxybenzoic acids one of the most studied is GA. GA is able to stimulate the growth and increase the relative population of Lactobacillus spp. in fecal slurries (Table 1) [26]. These stimulations fit well with DNA microarray experiments and physiological analyses, which established that the metabolism of GA by L. plantarum provides a source of chemiosmotic energy that generates ATP [52].

2.6 Stilbenes

Supplementation with resveratrol (RSV), a stilbene-type compound, can increase the relative abundance of Lactobacillus spp. together with other beneficial microorganisms such as Bifidobacterium and Akkermansia spp. (Table 1) [29]. Contrarily to members of the Enterobacteriaceae family such as E. coli, Lactobacillus spp. can tolerate RSV and increase their growth in the presence of this stilbene [53]. A transcriptomic study has revealed that L. plantarum induces DNA repair mechanisms and a response that decrease the load of copper and H₂S, two compounds promoting oxidative DNA damage. In the same vein, chaperone induction and marked changes in the expression of genes coding for some MFS efflux systems and ABC transporters indicates that export of RSV plays a key role in the tolerance to this stilbene [54].

2.7 Lignans

Lignans, another class of polyphenols, can also shift the composition of the gut microbiota. Among the lignans, syringaresinol increased the abundance of Lactobacillus spp. among the gut microbiota (Table 1) [30]. In the same vein, oleuropein (OLE) a secoiridoid present in olive leafs, olives and extra-virgin olive oil (EVOO) has been recently shown to enrich the Lactobacillus spp. population during in vitro fecal fermentation of olive leaf extracts or EVOO (Table 1) [31]. Lactobacillus spp. are producers of β-glucosidases able to hydrolyze lignans by first removing the sugar moiety from the glycosylated forms. Lactobacillus tolerates high concentrations of OLE [55]. In spite that OLE can be degraded by Lactobacillus spp. to elenoic acid and hydroxytyrosol (HXT), no growth advantage has been associated to the consumption of this polyphenol. Furthermore, L. plantarum downregulated the expression of genes encoding functions associated to rapid growth upon exposure to OLE [56]. In addition, HXT is the sole compound found to be bactericidal, also to L. plantarum, at low concentration. In line with this observation genes involved in L. plantarum cell proliferation were also downregulated in presence of HXT [57]. These studies indicate that lignans present in olive products or flaxseeds may impact positively on the population of lactobacilli by clearing other taxa due to the bactericidal or bacteriostatic effect on susceptible taxa.

Most of these studies described above agree on the fact that exposure to polyphenols increase the relative abundance of Lactobacillus spp. among the gut microbiota. However, when polyphenols were tested against pure cultures of Lactobacillus spp., neutral or slightly inhibitory effects were observed. These findings suggest that the polyphenols examined might reduce the microbial competition in the gut by exerting inhibitory effects on susceptible bacteria. Therefore, the bloom of Lactobacillus spp., which are more tolerant than other taxa to polyphenols, could result from an indirect stimulation rather than a direct effect. However this could not be the sole mechanism underlying the positive impact of polyphenols in the relative abundance of Lactobacillus spp. in the gut.

Since the host diet markedly impact the different responses displayed by some lactobacilli to the gut environment [2], it was reasonable to ask whether dietary polyphenols could contribute to modulate functions involved in the adaptive response of these microorganisms to the gut environment. Studies based on transcriptomic or proteomic approaches have provided molecular details on how individual polyphenols can modulate the expression of molecular traits from Lactobacillus spp. reportedly involved in the adaptive response to host environments.

3. Polyphenol-mediated modulation of Lactobacillus molecular tools involved in the adaptation to the gut habitat

The gut is a harsh environment where lactobacilli undergo different stress conditions and these microorganisms have to adapt by coordinating the expression of genes to improve stress tolerance.

3.1 Molecular chaperones

The molecular chaperones are involved in the adaptation of Lactobacillus spp. to the gut habitat. In fact, when L. plantarum WCFS1 (a model strain) is submitted to a simulator mimicking the oro-gastric–intestinal (OGI) conditions [58] this bacterium upregulates a set of proteases (clpB, clpE, clpP, fstH), chaperones (groEL, dnaK), and heat-shock proteins (hsp1, hsp2, hsp3). Studies that addressed the transcriptomic response of L. plantarum to different polyphenols (PPCs) have shown that most of these genes were also induced by p-CA [59], RSV [54], OLE [56] or HXT [57] exposure, implying common resistance mechanisms. Therefore, these polyphenols can modulate the expression of these molecular functions and facilitate the adaptation to the host gut.

3.2 Cell wall modifications

Lactobacilli are challenged to maintain the integrity of its cell envelope in the duodenum mainly due to the high osmolarity, acidic conditions and contact with bile salts. Lactobacilli respond accordingly by inducing a relatively high number of genes involved in functions related to the biosynyhesis of the cell envelope [60].

Some polyphenols are also able to injure the cell wall of Lactobacillus [38] and transcriptomic and proteomic studies have revealed polyphenol-induced modulation of genes and proteins involved in the biosynthesis of macromolecular components of the cell envelope [48]. Therefore, modulation of genes that play roles in proper functioning of cell integrity and that are involved in the biosynthesis of cell envelope constituents are common to polyphenols and GI-tract-induced stress.

An example is the peptidoglycan (PG). Genes and proteins involved in the biosynthesis of PG precursors, including LdhD (D-lactate dehydrogenase) and DapF (diaminopimelate (DAP) epimerase) are induced by L. plantarum [12] or L. hilgardii [61] upon tannic acid exposure and by L. acidophilus NCFM in presence of rutin [22]. Rutin also induced the L. acidophilus NCFM GlmU, an enzyme involved in the biosynthesis of the PG precursor UDP-GlcNAc. The L. plantarum D-alanyl-D-alanine dipeptidase (aad) and D-hydroxyisocaproate dehydrogenase (hicD2), two enzymes known to modify the biosynthesis of PG precursors were transcriptionally induced by p-CA [59]. In addition, L. plantarum downregulated the expression of peptidoglycan hydrolases in presence of p-CA. Altogether these expression profiles indicate that Lactobacillus spp. can modify the PG structure, reduce the PG turnover and represses PG lysis as a strategy to counter the injuries of polyphenols on the cell wall and maintain cell integrity.

3.3 Cell membrane modifications

Membrane modifications in its fatty-acid (FA) or phopholipid composition, are crucial strategies for bacterial adaptability to environmental stress including the GI-tract [62]. L. plantarum decreased the expression of the FA biosynthesis (fab) locus in presence of p-CA [59] or OLE. In the same vein this bacterium downregulated the expression of cyclopropane-fatty-acyl-phospholipid synthase (Cfa2) in the presence of TA [12] or OLE [56] also indicative of membrane modifications in its FA composition.

These expression profiles are aimed to strengthen the cell envelope and/or maintain its membrane integrity under polyphenol pressure and may therefore have cross-protective consequences to lactobacilli under GI-tract stress.

3.4 High osmolarity

Passage through the duodenum imposes high osmolarity conditions to lactobacilli. Besides cell wall and cell membrane modifications, L. plantarum differentially regulates the expression of transporters of compatible solutes to counter this stress, including glycine-betaine/carnitine/choline [63]. The expression of these transporters is downregulated in presence of p-CA [59] (a phenolic compound that turns leaky the membrane of diverse bacteria) and HXT [57], thus potentially allowing the amassing of these osmoprotectants in the extracelular space and provide stabilization of the external membrane, as suggested previously for trehalose [64].

3.5 Bile stress

Apart of the above mentioned cell wall and cell membrane adaptations, Lactobacillus also possesses other mechanisms involved in bile tolerance and bile-responsive genes that have been previously identified [63]. According to transcriptomic studies, the exposure of Lactobacillus to some polyphenols also lead to differential expression of bile-responsive genes in ways to enhance bile resistance denoting common resistance mechanisms for bile and polyphenols. Among these genes, L. plantarum induces F₀F₁-ATPase in presence of HXT which might compensate for the proton motive force (PMF) dissipation caused by bile salts. Multidrug transporters play an important role for bile tolerance in Lactobacillus spp. [65]. In this regard, p-CA [59] and RSV [54] markedly induced L. plantarum genes encoding an efflux pump induced in the small intestine and proposed to be involved in the extrusion of bile salts [66]. L. plantarum also overexpress a multidrug transporter potentially able to use deoxycholate as a substrate (lp_3368 gene) upon exposure to RSV [54], which is located in the vicinity of bsh3, a bile salt hydrolase-encoding gene.

GA also induces genes involved in GlcNAc utilization. This amino sugar is present in the human intestinal mucus glycoproteins and serves as a carbon source to Lactobacillus, particularly under bile stress [65]. The increased capacity to use GlcNAc permits adaptation to this specific nutritional environment and could be important to lactobacilli to ensure their residence time and survival in the GIT where nutrients are not in constant supply.

Bile has also been shown to induce oxidative stress, as indicated by the bile-mediated induction of glutathione reductase and genes from the metC-cysK operon are induced by bile [63]. These genes are also induced in L. plantarum to respond to the oxidative stress imposed by OLE [56], HXT [57] or p-CA [59]. In addition, Lactobacillus activates other different mechanisms to counter oxidative stress in the presence of some polyphenols [48], including the induction of enzymes inactivating toxic oxygen radicals such as NADH-peroxidase, catalase and pyruvate oxidase, which can cross-protect these bacteria against the oxidative stress imposed by bile or in the mucosal surface of the colon. Of note, Lactobacillus overexpresses methionine-sulfoxide reductases (Msr) to counter the loss of biological activity of proteins caused by oxidation of methionine to methionine sulfoxide under the oxidative stress imposed by phenolic compounds such as p-CA [59], HXT [31] or OLE [30]. Due to the crucial importance of the msr genes to reverse the oxidative damage on Lactobacillus proteins induced by the NO produced by epithelial cells, the induction of msr genes by these polyphenols would provide an ecological advantage for maintenance of lactobacilli in gut ecosystems.

Since many resistance mechanisms to oxidative stress are common to bile and polyphenol exposure, these compounds may have cross-protective consequences to Lactobacillus.

3.6 Key genes for survival and persistence in the host gut

Some polyphenols are able to modulate the expression of specific genes that have been shown to play a crucial role for the survival and persistence of L. plantarum in the host. Among these, the lp_2940 gene was strongly induced by L. plantarum in presence of tannic acid [38] or GA [52]). In addition to lp_2940, tannic acid induced copA (also crucial for the survival and persistence of L. plantarum in the digestive tract) as well as argG, ram2 which were markedly induced in the GI-tracts of humans and mouse [66]. Tannic acid was also able to inactivate the penicillin-binding PBP2A protein, a biomarker negatively related with GI survival [67]. These studies show that certain polyphenols can prime the adaptation to the GI-tract by modulating not only the (gene) functions required to face the different type of stress imposed in the gut, but also of specific biomarkers that are known to be crucial for the persistence and survival in the GI-tract.

As can be seen, substantial overlaps exist in the expression patterns of genes involved in the adaptation of lactobacilli to their transit through the GIT and exposure to polyphenols (Figure 1). This capacity may be of utility to improve fitness of lactobacilli in this niche, more in view that gut robustness of individual strains may depend on differential gene expression levels rather than on the presence or absence of conserved genes [68].

Figure 1.

Schematic representation of overlapping between the expression patterns of molecular attributes involved in the adaptive responses to polyphenols and the host gut. For more information on the specific actors involved (genes and proteins), see text.

4. Role of polyphenols in the modulation of Lactobacillus cell surface factors implicated in host-Lactobacillus interactions

4.1 Adherence: Polyphenol-mediated regulation of proteins engaged in adhesion

Adhesion to intestinal mucosa is considered to be preconditions for temporary colonization, stimulation of the immune system, and resistance to intestinal pathogens [60].

Moonlighting proteins are proteins that might have two or more unrelated functions depending on their cellular context. Analysis of differential surface proteomes of the probiotic L. acidophilus NCFM strain revealed that ferulic acid, resveratrol, tannic acid, and caffeic acid varied the abundance of moonlighting proteins engaged in adhesion [69]. The abundance of some of these moonlighting proteins including elongation factor EF-P, pyruvate kinase, and elongation factor EF-Tu correlated well with binding capabilities to HT-29 cells triggered by RSV or caffeic acid (Figure 2). L. acidophilus NCFM also overexpresses EF-P and pyruvate kinase (as well as GAPDH and EF-G) in the presence of rutin [22], however the same positive correlation between these moonlighting proteins, rutin and L. acidophilus adhesion, has to be yet experimentally validated.

Figure 2.

Schematic representation of the different mechanisms engaged in the Lactobacillus-host crosstalk that are modulated by polyphenols. CPS, capsular polysaccharide. MAMP, microorganism-associated molecular patterns. WTA, wall-teichoic acid. LTA, lipoteiochoic acid. QS-systems, quorum sensing-systems. pts19, N-acetyl-galactosamine/glucosamine:PTS system components. Msa, mannose-specific adhesion factor. EF, elongation factor. PK, pyruvate kinase. TLR, Toll-like receptor.

Another Lactobacillus protein that can moonlight and is known to be implicated in adhesion to host cells is the elongation factor EF-GreA which as markedly induced by tannic acid [12]. In the same vein some molecular chaperones displaying moonlighting functionality and implicated in adhesion to host cells [70, 71, 72], such as the GroEL is markedly induced by p-CA [59], HXT [57] or OLE [56] whereas some small heat-shock proteins (HSP1 and HSP3) are induced by p-CA [59]. However, albeit these chaperones might participate in the binding of Lactobacillus johnsonii or L. plantarum WCFS1 to host cells, the positive correlation between their role as surface-translocated adhesins, induction by polyphenols and Lactobacillus adhesion, has to be yet experimentally validated (Figure 2).

The msa gene from L. plantarum encodes a mannose-specific adhesion factor which improves adhesion to mannose-containing intestinal cells and is important for the health-promoting effects of probiotics (Figure 2) [73]. This gene is induced by L. plantarum in presence of RSV [54], therefore exposure to RSV may help improving the adhesion capacity and probiotics effects of this bacterium.

4.2 Inmunomodulation

The host microbiome markedly influences the host phenotype and has essential effects on physiological host homeostasis including metabolic function, immunoregulation and barrier maintenance [74]. This is achieved by the crosstalk between the microbiota and host cells, i.e., the intestinal epithelial cells (IEC) and immune cells located in the host intestine. The interaction of Lactobacillus with the intestinal cells occurs via recognition of certain Lactobacillus components named microorganism-associated molecular patterns (MAMPs) (host-commensal interactions) through host innate immune receptors (pattern-recognition receptors (PRRs), including cell membrane toll-like receptors (TLRs). This interaction induces the production of innate effector molecules and signaling pathways that result in Lactobacillus probiotic effects [60].

As shown above the intake of flavonoids is able to modulate the composition of the gut microbiota. Although the modification of the relative abundance of the Lactobacillus and other beneficial bacteria can be at the earliest level involved in modifying the host-bacteria crosstalk, much less is known on how polyphenols can influence the expression and types of MAMPs, which is one of the major factors influencing TLR activation.

The cell envelope is the main source of Lactobacillus MAMPs that interact with host receptors inducing signaling pathways that result in probiotic effects [60]. Since the biosynthesis of these MAMPs (peptidoglycan, teichoic acids, polysaccharides, and also lipids, lipoproteins or proteins) is subjected to regulation by polyphenols, exposure to these metabolites is expected to modify their structures and consequently the recognition by host receptors. Hence, polyphenols emerge as new potential modulators of the cell surface properties and signaling ability of Lactobacillus spp. (Figure 2).

The link between the variable biochemistry of MAMPs and the inmunomodulatory capacity of Lactobacillus, has been previously observed. For example, variation in the PG composition of L. acidophilus NCFM respect to the PG of Ligilactobacillus salivarius Ls33 hinders the activation of host defense mechanisms that the Ls33 strain is able to activate to protect against chemically induced colitis [75]. The capacity of polyphenols to modify the biosynthesis of PG may therefore influence the inmunomodulatory capacity of Lactobacillus albeit the understanding of the role played by these PG modifications by polyphenols is still to be developed.

Polyphenols can also modify the biosynthesis of the capsular polysaccharides (CPSs) from lactobacilli. In this case, the decreased expression of many of the cps genes encompassed within the four cps gene clusters of the L. plantarum WCFS1 genome is a core and common transcriptome response of this bacterium to p-CA [59], RSV [54], and OLE (Figure 2) [56]. Also, L. plantarum WCFS1 exposed to tannic acid did not detectably accumulate CPS over the cell outer surface [38].

Since CPSs shield adhesion proteins and MAMPs interacting with PRRs of dendritic cells, it can prevent the crosstalk and alter the innate and adaptive immunity. In fact, deletion mutants in the four cps gene clusters of L. plantarum WCFS1 alter its immunomodulatory capacities by increasing the exposure of bacterial MAMPs to their host receptors to induce signaling cascades [76]. In the same vein, CPS downregulation has been found to be required for optimal adherence of L. rhamnosus GG to intestinal epithelial cells [77]. In view of this, decreased expression of cps genes driven by the exposure to some polyphenols is likely to optimize the immunomodulatory and adhesion capacities of Lactobacillus (Figure 2).

Other cell wall constituents that are directly related to the inmunodulatory capacity of Lactobacillus spp. and which severely impact its capacity to communicate with their hosts are the teichoic acids (TAs) [78], i.e., cell wall teichoic acids (WTAs) and lipoteichoic acids (LTAs). Variable backbone alditol composition of the WTA affects recognition by host receptors and bacterial adhesion. The expression of genes and proteins implicated in the biosynthesis or modification of L. plantarum TAs is responsive to the presence of some polyphenols (Figure 2). Therefore, exposure to these polyphenols is expected to modify the TA backbone and hence the signaling ability of L. plantarum and its capacity to communicate with their hosts. Specific examples of genes or proteins involved in WTA biosynthesis modulated by polyphenols are TagE6 (poly(glycerol-phosphate) α-glycosyltransferase), an enzyme involved in glucosyl substitution of poly(ribitol-5-P) WTA [78], which was induced after exposure to tannic acid [12] and transcriptionally overexpressed in response to OLE [56] or HXT [57]. In the same vein, induction of genes of the tarIJKL locus is a genetic marker of WTA backbone alditol switching in L. plantarum [78], which originates an alternative WTA variant to the wild-type glycerol-containing backbone.

The tarIJK and the tarK genes are transcriptionally induced in response to OLE [56] and HXT [57], respectively, suggesting that WTA backbone alditol switching occurs in response to these olive polyphenols. Furthermore, downregulation of tagD2, which CDP-activates glycerol-P, was downregulated in presence of HXT, further supporting WTA backbone alditol switching. In addition, downregulation of the gtca1gene, which encodes an LTA glycosylation protein, was downregulated in L. plantarum cells exposed to HXT suggesting changes in the decoration of LTAs. Lactobacillus strains that produce alternative LTA variants can alter cytokine induction capacity thus increasing anti-inflammatory immune modulation [79, 80].

The potential of main olive polyphenols (HXT, OLE) to modify the signaling ability of L. plantarum and the crosstalk with their hosts, is further supported by the OLE-based down-regulation of other documented immunomodulators from L. plantarum, including components of the N-acetyl-galactosamine/glucosamine:PTS system and components of the plantaricin and LamBDCA quorum-sensing systems [81, 82]. In line with these observations, recent studies have shown that EVOO consumption modifies the gut microbiota being lactobacilli one of the promoted taxa [31]. Furthermore, HXT can reverse obsesity and insuline resistance and the crosstalk events involved, i.e. enhanced expression of inflammatory factors (TNF-α, IL-1β, IL-6) and lipid accumulation in liver, caused obesity, and aggravated IR via the JNK/IRS (Ser 307) pathway in HFD mice [83]. More interestingly is that these beneficial effects of HXT were transferable through fecal microbiota transplantation indicating a crucial role of the microbiota (and probably the modification of the signaling ability by polyphenols) in the observed effects.

5. Conclusions

Prebiotic-like effects on host gut Lactobacillus spp. have been usually observed upon the administration of different structural classes of polyphenols during in vivo or in vitro studies. However, when polyphenols were tested against pure cultures of Lactobacillus spp., neutral or slightly inhibitory effects were observed. These findings underline the role of polyphenols as antimicrobials able to reduce the microbial competition in the gut by inhibiting susceptible opportunistic/pathogenic bacteria. Since Lactobacillus spp. own mechanisms to overcome these inhibitory effects, the increase in the relative abundance could result from an indirect stimulation rather than a direct effect. However, polyphenols can also provide direct growth advantages to Lactobacillus in the gut. Up to the best of our knowledge only two selective mechanisms linked to the utilization of polyphenols that can promote the growth of Lactobacillus spp. have been elucidated. One is linked to the use of HCAs as external electron acceptors to gain energy by some strictly heterofermentative lactobacilli and the other is associated to the metabolism of GA as a source of chemiosmotic energy to Lactobacillus plantarum. In addition to their role as antimicrobials, it has been shown that polyphenols can contribute to modulate bacterial functions involved in the adaptive response of Lactobacillus to the gut environment.

Some polyphenols activate different Lactobacillus specialized mechanisms involved in countering the oxidative stress that Lactobacillus have to deal with in the gut. These mechanisms include the induction of enzymes inactivating toxic oxygen radicals or that implicated in countering the loss of biological activity of proteins and lipids caused by oxidation.

In the same vein polyphenols are able to coordinate the gene expression of Lactobacillus in ways that improve the stress tolerance to different conditions imposed by the passage through the gut, mainly high osmolarity, acidic conditions and contact with bile salts. Thus, molecular chaperones known to be involved in the adaptation of Lactobacillus to the gut habitat are markedly induced by polyphenols. Polyphenols are also able to coordinate the expression of a significant number of genes to reinforce the cell wall and maintain the membrane integrity. Furthermore some polyphenols induce the expression of genes such as multidrug transporters that are involved in bile tolerance. Notably, some polyphenols were shown to induce the expression of specific genes, such as lp_2940, that have a proven key role for the survival and persistence of L. plantarum in the host gut.

These findings reveal the capacity of polyphenols to modulate the expression patterns of Lactobacillus genes involved in the adaptation to the gut, in ways that improve its fitness in this niche.

Omics approaches have also shown that polyphenols play a marked modulatory role in the expression of Lactobacillus cell envelope components that participate in the communication between lactobacilli and intestinal host cells. These components, including peptidoglycan, teichoic acids and capsular polysaccharides, are expected to modify their structures upon exposure to these polyphenols and consequently their recognition by host receptors. In addition, exposure of Lactobacillus to specific polyphenols regulates the expression of molecular cell wall components with documented inmunomodulatory capacity. All of this reveals that exposure to some polyphenols, particularly to the main olive polyphenols hydroytyrosol or oleuropein, can modify the signaling ability and crosstalk capacity of Lactobacillus. The consequences of these modifications are an increased exposure of certain Lactobacillus inmunomodulators to their host receptors which induces the production of innate effector molecules and signaling pathways. This in turn result in Lactobacillus probiotic effects, including a diminished induction of pro-inflammatory cytokines and therefore an increased protection against intestinal inflammation.

The finding that specific polyphenols can modulate molecular functions from lactobacilli implicated in the survival and persistence in the host gut and Lactobacillus effector molecules that participate in proposed health-promoting interactions with the host gut provides molecular science-based criteria for polyphenol-mediated improvement of effector capacities of Lactobacillus associated with beneficial effects on host physiology.

Acknowledgments

We acknowledge partial support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).