Post on 06-Mar-2023
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: borja.sanchez@uvigo.es
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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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)
FEMS Microbiol Lett && (2014) 1–11 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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)
FEMS Microbiol Lett && (2014) 1–11ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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
FEMS Microbiol Lett && (2014) 1–11 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
Extracellular effectors of probiotic/gut bacteria 5
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-
FEMS Microbiol Lett && (2014) 1–11ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
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.
FEMS Microbiol Lett && (2014) 1–11 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved
Extracellular effectors of probiotic/gut bacteria 7
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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