extracellular molecular effectors mediating probiotic attributes

M IN I R E V I EW

Extracellular molecular effectors mediating probiotic attributes

Lorena Ruiz1, Arancha Hevia2, David Bernardo3, Abelardo Margolles2 & Borja S�anchez4

1Department of Microbiology, University College Cork, Cork, Ireland; 2Department of Microbiology and Biochemistry of Dairy Products, Instituto

de Productos L�acteos de Asturias – Consejo Superior de Investigaciones Cient�ıficas (IPLA-CSIC), Asturias, Spain; 3Antigen Presentation Research

Group, Imperial College London, Harrow, UK; and 4Nutrition and Bromatology Group, Department of Analytical and Food Chemistry, Food

Science and Technology Faculty, University of Vigo, Ourense, Spain

Correspondence: Borja S�anchez, Nutrition

and Bromatology Group, Department of

Analytical and Food Chemistry, Food Science

and Technology Faculty, University of Vigo –

Ourense Campus, E-32004 Ourense, Spain.

Tel./fax: 98 838 73 23;

e-mail: [email protected]

Received 17 June 2014; revised 4 August

2014; accepted 11 August 2014.

DOI: 10.1111/1574-6968.12576

Editor: Hermann Heipieper

Keywords

probiotics; gut bacteria; extracellular

molecules; bioactive compounds.

Abstract

Interest in probiotic bacteria, in the context of health and disease, is increasing

and gathering scientific evidence, as is reflected by their growing utilization in

food and pharma industry. As a consequence, many research effort over the

past few years has been dedicated to discern the molecular mechanisms respon-

sible for their purported attributes. Remarkably, whereas the traditional probi-

otic concept assumes that bacteria must be alive during their administration to

exert health-promoting effects, evidence is being accumulated that supports

defined bacterial secreted molecules and/or isolated surface components medi-

ating attributed cross talk dialogue between the host and the probiotic cells.

Indeed, administration of the isolated bacterial-derived metabolites or mole-

cules may be sufficient to promote the desired effects and may represent a

promising safer alternative in inflammatory disorders. Here, we summarize the

current knowledge of molecular effectors of probiotic bacteria that have been

involved in mediating their effects.

Introduction

The interaction between bacteria and our gut mucosa is a

continuous and bidirectional process in which a mutual-

istic relationship is established (Hooper et al., 2012). Gut

microorganisms perform important physiological func-

tions through their metabolism, while being controlled by

the host immune system to avoid potential threats by

pathogens (Littman & Pamer, 2013). Nevertheless, the

host/microbiota interaction is reciprocal as commensals

can also modulate gut homeostasis, notably during the

early stage of life, either by altering the balance of the

microbial communities, or even by shaping the outcome

of immune responses. Commensals also play a role in

triggering some gut disorders, such as inflammatory

diseases (Kamada et al., 2013).

According to the definition of the Food and Agricul-

ture Organization of the United Nations, ‘probiotics are

live microorganisms which when administered in adequate

amounts confer a health benefit on the host’, with most of

the probiotics currently used in human nutrition belong-

ing to the Bifidobacterium and Lactobacillus genus.

However, many gut bacteria are proposed to be also

probiotics, with their implementation in functional foods

remaining a technological challenge for the future.

Among the health benefits of some probiotic strains/gut

bacteria that have been proved to date, the improvement

or maintenance of gastrointestinal tract (GIT) homeosta-

sis through the balance of microbial composition, patho-

gen inhibition, enhancement of the epithelial barrier, and

immunomodulation are worth mentioning. In this sense,

anti-inflammatory effects exerted by certain probiotic

strains in the framework of inflammatory bowel disease

(IBD) and other chronic inflammatory and metabolic dis-

orders are particularly promising (Martin et al., 2013;

Whelan & Quigley, 2013).

During the last few years, several scientific studies have

reported on the immunomodulatory activity of bacterial

supernatants on relevant immune cell types, including

dendritic cells (DCs) (Bermudez-Brito et al., 2012a). Cell-

and spore-free bacteria supernatants contain probiotic/gut

bacteria-derived molecules that, from a molecular point

of view, may justify the physiological effects observed on

the host. For instance, cell-free culture supernatants from

members of the genus Bifidobacterium and Lactobacillus

can down-regulate pro-inflammatory signaling pathways

FEMS Microbiol Lett && (2014) 1–11 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

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https://www.researchgate.net/publication/236337970_Role_of_gut_microbiota_in_immunity_and_inflammatory_disease?el=1_x_8&enrichId=rgreq-634b65b9-601f-4180-984e-8bf9e7c4ac0b&enrichSource=Y292ZXJQYWdlOzI2NDcxMDIwODtBUzoxNDIwMDExMjMxMDY4MTZAMTQxMDg2NzExNDA5OA==

https://www.researchgate.net/publication/225275596_Interactions_Between_the_Microbiota_and_the_Immune_System?el=1_x_8&enrichId=rgreq-634b65b9-601f-4180-984e-8bf9e7c4ac0b&enrichSource=Y292ZXJQYWdlOzI2NDcxMDIwODtBUzoxNDIwMDExMjMxMDY4MTZAMTQxMDg2NzExNDA5OA==

in immune cells (Bermudez-Brito et al., 2012b). There-

fore, the identification and usefulness of probiotic-pro-

duced bioactive molecules – rather than the bacteria

themselves – have been proposed for the development of

a new generation of functional foods (Tsilingiri & Rescig-

no, 2013).

Microbiota-secreted compounds also have deleterious

effects on the host, such as metalloproteases secreted by

bacteria, which are associated with their invasive capacity

in the colon of patients with IBD (Steck et al., 2011).

Some protease-secreting bacteria have the capacity to

degrade gliadin in the duodenum, the protein that in

patients with celiac disease fails to establish the mecha-

nisms of immune tolerance, therefore suggesting a link

with the disease (Bernardo et al., 2009). The role of bac-

teria extracellular metabolites in the human gut has tradi-

tionally been described in the context of disease. Bacterial

metabolites have not usually been studied in the context

of providing beneficial effects on the host. For instance,

extracellular vesicles secreted by the gut microbiota have

been shown to have a therapeutic effect on an IBD mouse

model (Kang et al., 2013). Compounds secreted to the

media by Faecalibacterium prausnitzii, populations of

which are decreased in the context of IBD, had a positive

effect in strengthening epithelial paracellular permeability,

thereby decreasing the severity of the dextran sodium sul-

fate (DSS) induced colitis in mice (Carlsson et al., 2013).

Likewise, among the factors mediating the cross talk

between commensals and the host, bacterial extracellular

constituents are expected to be central (Lebeer et al.,

2010). The following sections describe current knowledge

on the benefits attributed to extracellular proteins, metab-

olites, and other surface associated or compounds

secreted by probiotic or gut bacteria.

Extracellular proteins and encryptedbioactive peptides

Among the huge variety of metabolites described as effec-

tors of probiotic/gut bacteria, extracellular proteins, and

small peptides secreted by them are frequently cited

(S�anchez et al., 2010). Extracellular or surface-associated

proteins are translocate through the cytoplasmic mem-

brane, being the molecular systems analogous in Gram-

positive and Gram-negative bacteria (further information

can be retrieved in Saier, 2006 and Schneewind & Missia-

kas, 2012). Some of them have been shown to have a

beneficial effect on the human GIT after being released to

the lumen, where they can interact directly with mucosal

cells, activating surface receptors/downstream signaling

pathways that lead to different cytokine secretion and

gene expression profiles (Tsilingiri & Rescigno, 2013). For

instance, serpin protein secreted by Bifidobacterium lon-

gum efficiently inhibits both pancreatic and neutrophil

elastase, the latter being secreted in acute inflammation

episodes (Ivanov et al., 2006). The interaction of extracel-

lular proteins secreted by bacteria with DCs is worth

noting. In the gut, DCs maintain the mechanisms of

immune tolerance against commensal bacteria and food

antigens. To that end, DCs have a regulatory profile

which is acquired once they have entered the GIT mucosa

milieu following modulation by intestinally derived fac-

tors, including TGF-b and retinoic acid (Mann et al.,

2012), and also bacterial proteins (Bernardo et al., 2012).

A few examples that belong to these types of molecular

factors are the cell wall-associated proteins p40 and p75

from Lactobacillus casei ssp. rhamnosus GG, the S-layer

protein from L. acidophilus, or the STp peptide encoded

in the secreted protein D1 from L. plantarum (Konstanti-

nov et al., 2008; Seth et al., 2008; Yan et al., 2011; Ber-

nardo et al., 2012; Al-Hassi et al., 2013). In L. casei BL23,

the presence of two proteins associated with the bacterial

surface, and which can also be secreted, was reported

(B€auerl et al., 2010). Indeed, such proteins resulted in

being homologous of p40 and p75 in L. rhammnosus GG,

which are known to prevent cytokine-induced apoptosis

in intestinal epithelial cells and decreasing susceptibility

to DSS-induced colon epithelial injury in mice (Yan

et al., 2007). In the work of B€auerl et al., it is shown how

p75 is particularly involved in epithelial cell separation,

and that both p75 and p40 stimulate epidermal growth

factor receptor phosphorylation ex vivo in mice. Both

proteins bound extracellular matrix proteins such as

mucin, fibronectin, and fibrinogen, which suggests a

potential role in the gut colonization and persistence of

L. casei. Supporting this, both proteins bound to the epi-

thelial cell lines T84 and Caco-2 in vitro. In addition,

L. casei synthesize a surface-associated protease (lactoce-

pin), which is able to hydrolyze the pro-inflammatory

lymphocyte chemoattractant (IP-10), secreted by epithe-

lial cells (von Schillde et al., 2012). Other Lactobacillus

species secrete extracellular proteins with an ability to

bind glycosylated proteins (i.e. mucin and proteins pres-

ent on the surface of epithelial cell lines), such as the chi-

tin-binding protein from L. plantarum (S�anchez et al.,

2011).

Other extracellular proteins mediating probiotic effects

are S-layer proteins (Table 1). Some Lactobacillus strains

are surrounded by a surface layer, the S-layer, made of

protein subunits packed into a paracrystalline hexago-

nal or tetragonal monolayer. S-layer-containing lactobacil-

li are L. acidophilus, L. gasseri, L. johnsonii, L. brevis,

L. helveticus, and L. crispatus, as well as about L. kefir,

L. parakefir, L. amylovorus, L. sobrius, and L. mucosae

(Hyn€onen & Palva, 2013). These proteins are usually

small (40–60 kDa) and highly basic with highly stable

FEMS Microbiol Lett && (2014) 1–11ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

2 L. Ruiz et al.

Table 1. List of the molecules supporting probiotic attributes, included the respective producer microorganism and the observed effects

Molecule Microorganism Observed effect

Scientific

evidence Reference

Extracellular and secreted proteins

Serpin B. longum Inhibit pancreatic and neutrophil elastases In vitro Ivanov et al. (2006)

p40 and p75 L. rhamnosus GG

L. casei BL23

Prevent cytokine-induced apoptosis in

intestinal epithelial cells and decrease

susceptibility to DSS-induced colon

epithelial injury in mice

Stimulate epidermal growth factor

receptor phosphorylation ex vivo in mice

In vitro and

in vivo

Yan et al. (2007) and

B€auerl et al. (2010)

Bind extracellular protein matrix In vitro B€auerl et al. (2010)

Lactocepin L. casei Hydrolyze pro-inflammatory chemokine IP-10 In vivo von Schillde et al. (2012)

slpA L. acidophilus NCFM Adhesion to Caco-2 cells In vitro Konstantinov et al. (2008)

Flagellin E. coli Nissle 1917 Induce release of beta-defensin-2 in epithelial

cells through NF-kD and AP-1-dependent

pathways

In vitro Schlee et al. (2007)

Fimbriae Bifidobacterium sp. Gut colonization factors, promote production

of cytokines

In vitro and

in vivo

O’Connell Motherway

et al. (2011), Turroni

et al. (2013) and

Lebeer et al. (2012a, b)

STp from protein D1 L. plantarum Partially restore DC phenotype in ulcerative

colitis

In vitro Bernardo et al. (2012)

and Al-Hassi et al.

(2013)

Exopolysaccharides

Polysaccharide A Bacteroides fragilis Inhibit production of pro-inflammatory

interleukin-17 and favor the production

of interleukin-10-producing CD4+ T cells

In vivo Mazmanian et al. (2008)

Exopolysaccharide

fractions

B. breve UCC2003 Reduce Citrobacter rodentium infection in a

mice model, attributed to attenuated

production of pro-inflammatory cytokines

In vitro and

in vivo

Fanning et al. (2012)

Exopolysaccharide

fractions

B. adolescentis Responsible of immunomodulatory

properties of the strain

In vitro Hosono et al. (1997)

B. longum Promote growth of macrophages, IL-10

production

Inhibits TNF-a secretion

In vitro Wu et al. (2010)

Antimicrobial bacteriostatic effects In vitro Wu et al. (2010)

Reduction of infection-related cytotoxic

effects on enterocytes

In vitro Ruas-Madiedo et al. (2010)

Bacillus sp.,

B. animalis

and Lactobacillus sp.

Antioxidants and free-radicals scavenging In vitro Kodali & Sen (2008),

Xu et al. (2011) and

Zhang et al. (2013)

Teichoic acids and other cell wall components

High-molecular

mass components

of the cell wall

L. casei Shirota Decrease lipopolysaccharide-induced IL-6

production in macrophages

In vitro Yasuda et al. (2008)

Polysaccharide-

peptidoglycan

L. casei Shirota Decrease IL-6 production in lipopolysaccharide-

stimulated mononuclear cells and macrophage

cell lines

In vitro Matsumoto et al. (2005)

Teichoic acids Lactobacillus sp. Mediate anti-inflammatory properties – induce

IL-10 production via TLR2 recognition in

macrophages

In vitro Kaji et al. (2010)

Modified lipoteichoic

acids

L. rhamnosus GG Improved colitis in murine model correlated to

decreased TLR and pro-inflammatory cytokine

secretion

In vivo Claes et al. (2010)

L. plantarum Increased secretion of IL-10 and decreased

secretion IL-12

In vitro and

in vivo

Grangette et al. (2005)

LTA fraction L. sakei Decrease matrix metalloproteinase-1 in skin

epithelial cells after UV treatment

In vitro You et al. (2013)

(continued)


Extracellular effectors of probiotic/gut bacteria 3

tertiary structures. S-layer proteins of lactobacilli have

commonly been suggested to be involved in the adherence

process, although not all lactobacilli have an S-layer. It has

been reported that the slpA mutant of L. acidophilus

NCFM was severely affected in its capacity to adhere to

Caco-2 cells, although it is likely that other surface-associ-

ated proteins are no longer targeting the surface following

the removal of the S-layer (Konstantinov et al., 2008).

Interestingly, the cell morphology of this slpA mutant was

significantly altered (small, curved bacilli), indicating

an additional role for SlpA in cell shape determination

(Lebeer et al., 2008).

Surface-associated proteins, such as the motility pro-

teins of fimbria and flagella, have also been shown to

mediate certain probiotic traits. In probiotic/gut bacteria,

flagellin is not only used as a propulsion mechanism, but

it is also responsible for adhesion to mucosal cells

through binding to specific targets such as mucin or

fibronectin (S�anchez et al., 2009; Troge et al., 2012). In

addition, flagellin monomers shed to the bacterial sur-

roundings can interact directly with epithelial cells, trig-

gering downstream responses. For instance, flagellin from

the probiotic Escherichia coli Nissle 1917 induced the

release of b-defensin-2 in epithelial cells through NF-KB-

and AP-1-dependent pathways, two well-known signaling

pathways in eukaryotic cells (Schlee et al., 2007). Regard-

ing Bifidobacterium and Lactobacillus species, genome

sequencing has revealed the presence of fimbrial appendi-

ces, which have the peculiarity of only being produced

when the bacteria grow within the gut or on the surface

of agar plates (Kankainen et al., 2009; O’Connell Mother-

way et al., 2011; Turroni et al., 2013). In addition to their

role in adhesion to the gut mucus, fimbrial subunits are

able to interact with epithelial and immune cells, promot-

ing the production of the pro-inflammatory cytokines

interleukin (IL)-8 and tumor necrosis factor-alpha

(TNF)-a, respectively (Lebeer et al., 2012a; Turroni et al.,

2013).

Serine-rich proteins from certain microorganisms have

been related to binding to eukaryotic components, such

as the serine-rich fragment from the SrpA protein of

Staphylococcus aureus, which mediates platelet-aggregation

(Siboo et al., 2005), or the pneumococcal serine-rich

repeat protein (Sanchez et al., 2010). In this sense, the

presence of encrypted peptides in larger extracellular pro-

teins that may be released by the action of intestinal pro-

teases interacting with the mucosal cells, and with a high

proportion of serine and threonine amino acids, has

recently been discovered. In L. plantarum, a serine/threo-

nine rich protein (homologous to gi|28270057 from

L. plantarum WCFS1) has been shown to promote cell

aggregation (Hevia et al., 2013). A peptide of 6.8 kDa

encoded within such protein, named STp, had regulatory

effects on the human GIT through its interaction with

human intestinal DCs, whose immune function was

prone toward a tolerogenic response (Bernardo et al.,

2012; Al-Hassi et al., 2013). These kinds of peptides rep-

resent a new way of understanding the probiotic/gut bac-

teria/host interaction and may be the basis for the

development of new functional ingredients targeting IBD

or other chronic inflammatory gastrointestinal disorders.

Exopolysaccharides

Production of exopolysaccharide layers is a widespread trait

within the microbial world, including gut microorganisms

and common probiotic bacteria such as Lactobacillus and

Bifidobacterium strains (Ismail & Nampoothiri, 2010;

Leivers et al., 2011). The functional role of these exter-

nal polysaccharide coats has mainly been studied in

pathogens, where the polymers have been attributed a

role in protecting bacterial cells against environmental

stress factors, such as dehydratation or acid conditions,

bacteriophages, immune evasion, or even biofilm forma-

tion or tissue adhesion (Ruas-Madiedo & de los Reyes-

Gavil�an, 2005; Hidalgo-Cantabrana et al., 2013). The

Table 1. Continued

Molecule Microorganism Observed effect

Scientific

evidence Reference

Bacterial metabolites

CLA – Mediates suppression of lipopolysaccharide

-induced IL-12 in dendritic cells and promotes

IL-10 production

In vitro Loscher et al. (2005)

and Reynolds et al.

(2009)

VSL#3 probiotic mix Macrophage immunomodulation mediating

colitis amelioration

In vivo Bassaganya-Riera et al.

(2012)

Acetate B. longum NCC2705 Protection against E. coli EC157:O7 induced-

tissue damage

In vivo Fukuda et al. (2011)

Acetate and Lactate B. breve and L. casei Modulate host-epithelial genes involved in

cell-cycle regulation and cell differentiation

In vitro Matsuki et al. (2013)

Bacteriocins L. salivarius UCC118 Reduce Listeria infection in a mice model In vivo Corr et al. (2007)

Thuricin CD Bacillus thuringensis Combat Clostridium difficile infection In vitro Rea et al. (2010)


4 L. Ruiz et al.

latest research is shedding light on some beneficial

properties that might also be directly attributed to the

exopolysaccharide coat of probiotic/gut bacteria, either

directly modulating host responses, or mediating modifi-

cations in the gut microbial population.

Perhaps the best known example is polysaccharide A

from Bacteroides fragilis, which has been shown to inhibit

the production of pro-inflammatory interleukin-17 and

favoring the function of interleukin-10-producing CD4+

T cells (Mazmanian et al., 2008). Exopolysaccharide coats

have been shown to exert a key role for probiotic intesti-

nal colonization, which has been attributed, on one hand,

to a better tolerance to gastrointestinal conditions and,

on the other hand, to immune evasion contribution

(Mozzi et al., 2009; Lebeer et al., 2011; Fanning et al.,

2012). Administration of B. breve UCC2003 in a mice

model reduced the infection promoted by Citrobacter

rodentium as compared to the non-exopolysaccharide-

derivative strain, which was associated with an attenuated

production of proinflammatory cytokines (Fanning et al.,

2012). Whereas these effects might be attributed to an

exopolysaccharide coating effect of otherwise exposed

antigenic compounds, the fact is that isolated exopolysac-

charide fractions from different probiotic/gut bacteria

have themselves also demonstrated immunomodulatory

properties.

Immunomodulatory properties of a B. adolescentis

strain had earlier been attributed to a water soluble frac-

tion of its exopolysaccharide (Hosono et al., 1997). As

then, several in vitro and in vivo analyses confirmed a

direct effect of the exopolysaccharide fraction produced

from different probiotic/gut strains on immunological

responses. The exopolysaccharide fraction produced by a

B. longum strain promoted in vitro growth of macrophag-

es, stimulated IL-10 production and inhibited TNF-asecretion. Notably, these effects were opposite to the effect

exerted by the lipopolysaccharide from E. coli, which is an

example of an immunogenic carbohydrate polymer pro-

duced by Gram-negative pathogenic strains. It is also

worth remarking that pretreatment with the bifidobacterial

exopolysaccharide prevented the lipopolysaccharide effect

on macrophages, including inhibition of macrophage

growth and TNF-a secretion (Wu et al., 2010). Although

the molecular mechanisms underlying these responses

remain for the most unclear, these results suggest that pro-

biotic-derived exopolysaccharide might somehow protect

against pathogen infection, or even against other immune-

related disorders (Ciszek-Lenda et al., 2011; Fanning

et al., 2012). Nagai et al. (2011) showed that the acidic

fraction of a L. bulgaricus exopolysaccharide, when

administered in vivo, promoted a better recovery rate

following an influenza virus infection. This effect has

been correlated to an augmentation of NK cell activa-

tion, which has been proposed as taking place upon exo-

polysaccharide -mediated immune stimulation through

Peyer’s patch cells in the intestinal mucosa.

Probiotic-derived exopolysaccharide has also demon-

strated an in vitro capability to inhibit biofilm formation

of enterohemorrhagic E. coli (Kim et al., 2009) and to

even exert antimicrobial bacteriostatic effects on certain

common pathogenic bacteria (Wu et al., 2010). Likewise,

in vitro analyses have revealed that lactobacilli or

bifidobacteria-derived exopolysaccharide might reduce

infection-related cytotoxic effects on enterocytes, the

particular effect being strain and dose dependent (Ruas-

Madiedo et al., 2010). In this sense, exopolysaccharide

fractions isolated from Bacillus sp., B. animalis and sev-

eral Lactobacillus strains have demonstrated antioxidant

and free radical scavenging activity in vitro (Kodali &

Sen, 2008 Xu et al., 2011; Zhang et al., 2013) which

might be, at least in part, responsible for the observed

attenuation of cytotoxic effects on enterocytes.

Finally, in vitro studies with purified exopolysaccharide

fractions isolated from bifidobacterial and lactobacilli

strains have demonstrated their ability to modulate the

intestinal microbiota, promoting the growth of certain

relevant bacterial groups, thus suggesting that exopolysac-

charide polymers might act as fermentable substrates for

selected populations of the gut microbiota and could be

considered as prebiotic substrates (Bello et al., 2001;

Salazar et al., 2009).

Teichoic acids (TA) and other cell wallcomponents

Certain immunomodulatory properties of probiotic/gut

bacteria may be performed by some components from

the cell wall. Lactobacillus casei strain Shirota (LcS)

improves inflammatory conditions both in vitro and

in vivo by decreasing lipopolysaccharide-induced IL-6

production in macrophages (Yasuda et al., 2008). A KO

LcS strain, lacking a gene cluster encoding for high-molec-

ular-mass components from the cell wall, was unable to

elicit the anti-inflammatory properties of the WT strain

(Yasuda et al., 2008). Similarly, polysaccharide–peptidogly-can complex derived from LcS cell wall also decreased

the release of IL-6 in vitro in both lipopolysaccharide-

stimulated lamina propria mononuclear cells and macro-

phage cell lines from mice, as well as in peripheral blood

mononuclear cells from human patients with ulcerative

colitis, a form of IBD (Matsumoto et al., 2005).

Among the components of the cell wall performing

immunomodulatory properties, the relevance of TA and li-

poteichoic acids (LTA) cannot be disregarded (Hernandez-

Mendoza et al., 2009). TA are molecules which provide

rigidity to the cell wall of Gram-positive bacteria and have



pro-inflammatory effects via Toll-like receptor 2 (TLR2)

(Matsuguchi et al., 2003). Recent evidence suggests that TA

can also mediate directly some anti-inflammatory proper-

ties of lactic acid bacteria, because they were revealed as

essential, but not sufficient factors, to induce IL-10 produc-

tion via TLR2 recognition in murine macrophages exposed

to different Lactobacillus strains (Kaji et al., 2010). How-

ever, and given that TA are usually important pro-inflam-

matory molecules of Gram-positive bacteria, their mutants

often show a high therapeutic performance in experimental

murine models of colitis (Lebeer et al., 2012b). While

L. rhamnosus GG exacerbated the severity of colitis murine

models compared with untreated mice, modification of

LTA on their surface improved the condition of the mice at

the time that correlated with decreased TLR and pro-

inflammatory cytokine expression in the colon (Claes et al.,

2010). A modified TA of L. plantarum increased secretion

of anti-inflammatory IL-10 and decreased pro-inflamma-

tory IL-12 in both human and murine peripheral blood

mononuclear cells. In vivo, the strain producing the modi-

fied TA was more protective than the WT in a 2,4,6-trini-

trobenzene sulfonic acid (TNBS)-induced murine colitis

model (Grangette et al., 2005). An L. acidophilus strain,

deficient in LTA synthesis, increased the tolerogenic prop-

erties of intestinal DCs compared to the wild-type strain,

which was mirrored in vivo both by amelioration of

DSS-induced colitis and a larger number of T cells with

regulatory properties (Mohamadzadeh et al., 2011). Some

probiotic strains also have the capacity in murine models

to protect from visceral pain perception, likely in a process

mediated by some components from the cell wall and

TLR-2 signaling (Kamiya et al., 2006; Duncker et al.,

2008). Finally, an LTA isolated from L. sakei was able to

decrease the matrix metalloproteinase-1 (MMP-1) produc-

tion in skin epithelial cells after UV treatment (You et al.,

2013). In summary, cell wall components from probiotic/

gut bacteria can mediate, at least partially, the cross talk

between the commensals and the host.

Probiotic-derived metabolites withattributed health benefits: conjugatedlinoleic acid (CLA) and bacteriocins as acase study

Conjugated fatty acids are geometrical and positional iso-

mers of various polyunsaturated fatty acids (PUFA) and

are commonly found in nature, including plant seed oils,

full fat milk, and the fat of ruminants. They have been

reported to mediate a range of metabolic effects on the

host, including anticarcinogenic, antidiabetogenic, and

anti-obesity effects (Coakley et al., 2009). Therefore, there

is an increasing interest in developing suitable strategies

for conjugated fatty acid production, including microbial-

mediated production. In fact, dairy and intestinal bacteria

have been reported to display linoleic acid isomerase

activity that mediates the conjugation of the c9-c12 dou-

ble bond of linoleic acid (C18:2) to yield the production

of c9,t11-C18:2, and t9,t11-C18:2 conjugated isomers.

Hence, microbial ability to conjugate PUFA offers the

possibility to either enrich functional food with CLA, or

to establish a gastrointestinal microbiota capable of pro-

ducing CLA in situ. Indeed, bacterial production of CLA

has been evidenced both ex vivo and in vivo in mice mod-

els, thus supporting the occurrence of CLA production at

intestinal level (Ewaschuk et al., 2006; Rosberg-Cody

et al., 2011). Remarkably, local bacterial production of

CLA has been reported to mediate amelioration of experi-

mental IBD in animal models and Crohn’s disease in

humans. Although the molecular mechanisms behind

CLA-mediated anti-inflammatory effects are not com-

pletely understood, CLA has been demonstrated to medi-

ate suppression of lipopolysaccharide-induced IL-12

production in DCs and promotes IL-10 production and

inhibition of NF-kB activation (Loscher et al., 2005),

therefore providing evidence of the mechanisms underly-

ing its anti-inflammatory effects (Reynolds et al., 2009).

The same effect is achieved with the administration of the

VSL#3 probiotic bacteria, which promoted changes in the

gut microbiota favoring the local production of CLA,

with a concomitant macrophage immunomodulation at

the gut mucosa level (Bassaganya-Riera et al., 2012).

Other proposed metabolites mediating probiotic attri-

butes are short-chain fatty acids (SCFA) and bacteriocins

(Fig 1). Organic SCFA are the result of bacterial metabo-

lism, and the ratio among different SCFA has frequently

been employed as a marker of bacterial population

dynamics in microbial ecosystems. In the distal colon, the

SCFA produced as a result of microbial fermentations

have been reported to exert trophic, regulatory, and

immunomodulatory effects that can be directly due to the

produced SCFA, or be the result of SCFA-mediated

dynamic modulation of other microbial populations, that

is due to cross-feeding (De Vuyst & Leroy, 2011).

Remarkably, a recent work established that the acetate

produced by certain bifidobacterial strains following the

utilization of carbohydrates in the distal colon is the

mediator behind the observed in vivo protection against

E. coli EC157:O7 induced-tissue damage (Fukuda et al.,

2011). In vitro analysis confirmed that acetate induces the

expression of host functions involved in the anti-inflamma-

tory response and prevents reduction in transepithelial

electrical resistance, thus suggesting prevention of bacterial

translocation. Recent results also pointed to a modulation

of host-epithelial genes involved in cell-cycle regulation

and cell differentiation; acetate and lactate produced by

strains of B. breve and L. casei were identified as the molec-


6 L. Ruiz et al.

ular effectors (Matsuki et al., 2013). Bacterial metabolism

within the intestine may promote important changes in the

intestinal ecosystem composition with critical impact on

the host.

Bacteriocins are ribosomal synthesized short peptides

that display antimicrobial activity against a range of

usually closely related bacteria, likely to be present within

the same ecological niche. They have been identified as

being produced by a number of lactobacilli, bifidobacteri-

al, and other gut strains and have been proposed as a

trait conferring on probiotic strains a selective advantage

to colonize the intestinal environment (Dobson et al.,

2012). Notably, bacteriocins produced at intestinal level

have been demonstrated to mediate anti-infective effects.

To highlight this, the capacity of L. salivarius UCC118 to

reduce Listeria infection in a mice model was directly

attributed to the production of two antimicrobial pep-

tides, as a nonproducer derivative failed to confer protec-

tion (Corr et al., 2007). Another example of bacteriocin

with proven anti-infective properties is thuricin CD, pro-

duced by Bacillus thuringensis, that has been proved to be

successful in combating Clostridium difficile in distal

colon models (Rea et al., 2010). Finally, purified bacte-

riocins have also demonstrated anti-infective properties

when administered in vivo to mice models, and therefore,

bacteriocin administration appears to be a promising

Cytoplasm side

PG

Exopolysachharide

Secreted antimicrobial peptides

Serpin

Pro-inflammatory cytokinesM s growth, IL-10, TNF-α

Elastase

↓Inflammation

S-layer

Lactocepin

↑IL-8, TNF-α↑ β-defensin- 2

STp

Prevent cytokine induced apoptosis

p75, p40

Hydrolysepro-inflammatory

cytokines

Modulate DC function (i.e. ↑ IL-10, ↓ IL-12)

CLA isomers

Inte

stin

al lu

men

TA &

LTA

Neutrophil

Pili, Flagella

Cell-wall

SCFAs

Protection against pathogens

Immunomodulatoryproperties

Ephitelial cells

Macrophage

Φ

Fig. 1. Overview of the mechanism of action of molecules supporting probiotic attributes. From left to right, the monomeric subunits from the

pili and flagella, shed from the bacterial surface to the gut environment, interact with immune cells triggering secretion of IL-8, TNF-a, and

b-defensin 2. Serpin, a suicide inhibitor of serine proteases, blocks the action of elastase secreted by neutrophils during acute inflammatory

episodes. Lactocepin, a surface-associated protease of Lactobacillus casei, can hydrolyze certain pro-inflammatory cytokines such as the epithelial-

produced chemokine IP-10. TA and LTA mediate immunomodulatory processes by interacting with specific receptors on the surface of mucosal

cells. Different probiotic secreted effectors such as CLA, encrypted immunomodulatory peptides (STp) or S-layer proteins modulate DC function

by triggering the production of anti-inflammatory cytokines (IL-10) and decreasing the production of pro-inflammatory cytokines (IL-12). p75 and

p40, two surface proteins produced by L. casei and L. rhamnosus, exert protective effects at the epithelial level notably by reinforcing the tight

junctions, conferring protective effects against cytokine-induced apoptosis. Released subunits from exopolysaccharides produced by probiotic

bacteria interact with macrophages (Mφs) promoting their growth, increasing the secretion of anti-inflammatory cytokines (IL-10), and decreasing

the production of pro-inflammatory cytokines (TNF-a). Finally, the action of secreted antimicrobial peptides and SCFAs limits the proliferation of

enteropathogens in the gut.



alternative to prevent gastrointestinal infections (Dobson

et al., 2012). Further research is needed to guarantee the

security and efficiency of their administration.

Future perspectives

The effect of bacterial-derived molecular components or

metabolites that act as effectors of probiotic benefits is

variable and has been proved to be exerted at various

levels. Narrowing down probiotic attributes to defined

molecules that exert health-promoting effects by them-

selves has opened new, promising opportunities for the

development of functional bioactive compounds. This is

of particular importance in cases of severe inflammation,

where epithelial barriers may be compromised and bacte-

rial translocation may occur. Under those circumstances,

administration of defined isolated probiotic molecules,

rather than whole bacterial cells, is expected to be a

promising and safer alternative that may help to tackle

chronic inflammatory disorders. In this regard, further

research is needed to standardize in vivo assays to guar-

antee their safety and efficacy, especially in severe inflam-

mation models. Additional efforts are necessary to

characterize in detail their mechanisms of action to

assure their safety when exploited as bio-active risk-free

alternatives. Finally, the development of strategies for

bio-production and isolation of relevant amounts of

targeted molecules from the producing strains will be

necessary.

Acknowledgements

This research was funded by Grants AGL2010-14952 and

AGL2013-44039-R from the Spanish ‘Plan Nacional de

I+D’ and by the BBSRC Institute Strategic Programme

for Gut Health and Food Safety (Grant BB/J004529/1).

B.S. and A.H. were recipients of a Ram�on y Cajal post-

doctoral contract and a FPI grant, respectively, from the

Spanish Ministry of Economy and Competitiveness.

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