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Probiotics are live microorganisms that provide health benefits to the host when ingested in adequate amounts. The strains most frequently used as probiotics include lactic acid bacteria and bifidobacteria. Probiotics have demonstrated significant potential as therapeutic options for a variety of diseases, but the mechanisms responsible for these effects have not been fully elucidated yet. Several important mechanisms underlying the antagonistic effects of probiotics on various microorganisms include the following: modification of the gut microbiota, competitive adherence to the mucosa and epithelium, strengthening of the gut epithelial barrier and modulation of the immune system to convey an advantage to the host. Accumulating evidence demonstrates that probiotics communicate with the host by pattern recognition receptors, such as toll-like receptors and nucleotide-binding oligomerization domain-containing protein-like receptors, which modulate key signaling pathways, such as nuclear factor-ĸB and mitogen-activated protein kinase, to enhance or suppress activation and influence downstream pathways. This recognition is crucial for eliciting measured antimicrobial responses with minimal inflammatory tissue damage. A clear understanding of these mechanisms will allow for appropriate probiotic strain selection for specific applications and may uncover novel probiotic functions. The goal of this systematic review was to explore probiotic modes of action focusing on how gut microbes influence the host.
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Systematic Review
Ann Nutr Metab 2012;61:160–174
DOI: 10.1159/000342079
Probiotic Mechanisms of Action
Miriam Bermudez-Brito Julio Plaza-Díaz Sergio Muñoz-Quezada
Carolina Gómez-Llorente Angel Gil
Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology ‘José Mataix’,
Biomedical Research Center, University of Granada, Armilla , Spain
damage. A clear understanding of these mechanisms will al-
low for appropriate probiotic strain selection for specific ap-
plications and may uncover novel probiotic functions. The
goal of this systematic review was to explore probiotic
modes of action focusing on how gut microbes influence the
host. Copyright © 2012 S. Karger AG, Bas el
Introduction
According to the Food and Agriculture Organization
of the United Nations and the World Health Organiza-
tion
[1] , probiotics are live microorganisms that confer a
hea lth benef it to the host when administered in adequate
amounts. In particular, strains belonging to Bifidobacte-
rium and Lactobacillus , which are the predominant and
subdominant groups of the gastrointestinal microbiota,
respectively
[2] , are the most widely used probiotic bacte-
ria and are included in many functional foods and di-
etary supplements
[3–5] . Saccharomyces boulardii yeast
has also been shown to have health benefits
[6] . After a
long history of safe use of probiotics in fermented dairy
products and an increased recognition of their beneficial
effects on human health
[7] , the food industry has be-
come increasingly interested in these types of microor-
ganisms. Often the criteria for the selection of probiotics
include the tolerance to gastrointestinal conditions (gas-
Key Words
Antimicrobial responses Bifidobacteria Lactic acid
bacteria Lactobacilli Probiotic mechanism of action
Probiotics
A b s t r a c t
Probiotics are live microorganisms that provide health ben-
efits to the host when ingested in adequate amounts. The
strains most frequently us ed as p robiotics include lactic acid
bacteria and bifidobacteria. Probiotics have demonstrated
significant potential as therapeutic options for a variety of
diseases, but the mechanisms responsible for these effects
have not been fully elucidated yet. Several important mech-
anisms underlying the antagonistic effects of probiotics on
various microorganisms include the following: modification
of the gut microbiota, competitive adherence to the mucosa
and epithelium, strengthening of the gut epithelial barrier
and modulation of the immune system to convey an advan-
tage to the host. Accumulating evidence demonstrates that
probiotics communicate with the host by pattern recogni-
tion receptors, such as toll-like receptors and nucleotide-
binding oligomerization domain-containing protein-like re-
ceptors, which modulate key signaling pathways, such as
nuclear factor- B and mitogen-activated protein kinase, to
enhance or suppress activation and influence downstream
pathways. This recognition is crucial for eliciting measured
antimicrobial responses with minimal inflammatory tissue
Recei ved: July 19, 2012
Accepted: July 20, 2012
Published online: O ctober 2, 2012
Prof. A ngel Gil
Institute of Nutr ition and Food Technolog y ‘José Mataix’ (I NyTA)
Biomedical Research Center, University of Granada
Avenid a del Conocimiento s/n, ES–18100 Ar milla (Spain)
E-Mail agil @ ugr.es
© 2012 S. Ka rger AG, Basel
0250– 6807/12/0612–0160$38.00/0
Accessible online at:
www.karger.com/anm
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tric acid and bile), ability to adhere to the gastrointestinal
mucosa and competitive exclusion of pathogens [8, 9].
The mechanisms underlying the beneficial effects of pro-
biotics are largely unknown but are likely to be multifac-
torial. Several mechanisms related to the antagonistic ef-
fects of probiotics on various microorganisms include the
following mechanisms: secretion of antimicrobial sub-
stances, competitive adherence to the mucosa and epithe-
lium, strengt hening of the gut epithelia l barrier and mod-
ulation of the immune system
[10 ] .
The results of evidence-based analyses from human
studies and animal models have shown the clinical po-
tential of probiotics against many diseases
[11] . Probiotics
have been reported to suppress diarrhea
[1 2] , alleviate
lactose intolerance
[13 ] and postoperative complications
[14 ] , exhibit antimicrobial [1 5] and anti-colorectal cancer
activities
[16 , 17] , reduce irritable bowel symptoms [1 8]
and prevent inflammatory bowel disease
[19 ] . However,
generalizations concerning the potential health benefits
of probiotics should not be made because probiotic effects
tend to be strain specific. Thus, the health benefit attrib-
uted to one strain is not necessarily applicable to another
strain even within one species
[20] .
In the present study, we sought to conduct a system-
atic review on the mechanisms of action of probiotic
strains. Using the following equation: ‘epithelial barrier’
[All Fields] OR ‘antimicrobial substances’[All Fields] OR
‘bacteriocins’[All Fields] OR ‘BIF’[All Fields] OR ‘adhe-
sion’[All Fields] OR ‘competitive exclusion’[All Fields]
OR ‘defensins’[All Fields] OR ‘mucins’[All Fields] OR
‘bacterial adhesins’ [All Fields] OR ‘antifungals’[All
Fields] OR ‘intestinal microbiota’[All Fields] OR ‘fatty
acids’[All Fields] OR ‘mechanisms’[All Fields] OR
‘TLR2’[All Fields] OR ‘TLR4’[All Fields] OR ‘TLR9’[All
Fields] OR ‘toll-like receptor’[All Fields] OR ‘NOD1’[All
Fields] OR ‘NOD2’
[All Fields] OR ‘inflammasome’[All
Fields] OR ‘NLRP3’ [All Fields]
AND ‘probiotics’[MeSH],
we ha ve se le ct ed 165 re le va nt art ic le s o f 1 ,731 a rti cl es pu b-
lished until June 25, 2012, from the PubMed and SCO-
PUS databases.
Mechanisms of Action of Probiotics
Major probiotic mechanisms of action include en-
hancement of the epithelial barrier, increased adhesion to
intestinal mucosa, and concomitant inhibition of patho-
gen adhesion, competitive exclusion of pathogenic mi-
croorganisms, production of anti-microorganism sub-
stances and modulation of the immune system ( fig.1 ).
Enhancement of the Epithelial Barrier
The intestinal epithelium is in permanent contact with
luminal contents and the variable, dynamic enteric flora.
The intestinal barrier is a major defense mechanism used
to maintain epithelial integrity and to protect the organ-
ism from the environment. Defenses of the intestinal bar-
rier consist of the mucous layer, antimicrobial peptides,
secretory IgA and the epithelial junction adhesion com-
plex
[21] . Once this barrier function is disrupted, bacte-
rial and food antigens can reach the submucosa and can
induce inflammatory responses, which may result in in-
testinal disorders, such as inflammatory bowel disease
[22–24] . Consumption of non-pathogenic bacteria can
contribute to intestinal barrier function, and probiotic
bacteria have been extensively studied for their involve-
ment in the maintenance of this barrier. However, the
mechanisms by which probiotics enhance intestinal bar-
rier function are not fully understood.
Several studies have indicated that enhancing the ex-
pression of genes involved in tight junction signaling is a
possible mechanism to reinforce intestinal barrier integ-
rity
[25] . For instance, lactobacilli modulate the regula-
tion of several genes encoding adherence junction pro-
teins, such as E-cadherin and -catenin, in a T84 cell bar-
rier model. Moreover, incubation of intestinal cells with
lactobacilli differentially influences the phosphorylation
of adherence junction proteins and the abund ance of pro-
tein kinase C (PKC) isoforms, such as PKC , thereby pos-
itively modulating epithelial barrier function
[26] .
Recent dat a have indicated that probiotics may initiate
repair of the barrier function after damage. Escherichia
coli Nissle 1917 (EcN1917) not only prevents the disrup-
tion of the mucosal barrier by enteropathogenic E. coli ,
but it even restores mucosal integrity in T84 and Caco-2
cells. This effect is mediated by the enhanced expression
and redistribution of tight junction proteins of the zonu-
la occludens (ZO-2) and PKC resulting in the reconstruc-
tion of the tight junction complex
[27, 28] . Similarly, Lac-
tobacillus casei DN-114001
[29] and VSL3 (a mixture of
pre- and probiotics)
[30] are capable of sustaining the in-
testinal barrier function by similar mechanisms. A recent
paper has reported that VSL3 protects the epithelial bar-
rier and increases tight junction protein expression in
vivo and in vitro by activating the p38 and extracellular
regulated kinase signaling pathways
[31] .
A link between altered levels of pro-inflammatory cy-
tokines and intestinal permeability has been described in
a number of intestinal diseases
[32] . Using probiotics, the
pr event ion of c yt ok ine-i nduced epi th elia l dam age, which
is characteristic of inflammatory bowel disease
[24] , may
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also contribute to the reinforcement of the mucosal bar-
rier. Two isolated and purified peptides secreted by Lac-
tobacillus rhamnosus GG (LGG), which are designated
p40 and p75, have recently been demonstrated to prevent
cytokine-induced cell apoptosis by activating the anti-
apoptotic protein kinase B (PKB/Akt) in a phosphatidyl
inositol-3 -kinase-dependent pathway and by inhibiting
the pro-apoptotic p38/mitogen-activated protein kinase
(MAPK)
[33, 34] . The evidence that p40 and p75 are re-
sponsible for the observed effects is derived from the ob-
servation that the anti-apoptotic function is abolished
when p40- and p75-specific antibodies are added in vitro
to murine and human epithelial cells or to colon explants
derived from mice
[34] . Other low-molecular-weight
(LMW) peptides secreted from LGG induce expression of
heat shock proteins and activate MAPKs
[35] .
Mucin glycoproteins (mucins) are major macromolec-
ular constituents of epithelial mucus and have long been
implicated in health and disease. Probiotics may promote
mucous secretion as one mechanism to improve barrier
function and the exclusion of pathogens. Several Lacto-
bacillus species increase mucin expression in human in-
testinal cell lines. However, this protective effect is de-
pendent on Lactobacillus adhesion to the cell monolayer,
which likely does not occur in vivo
[36 , 37] . Conversely,
another group has shown that Lactobacillus acidophilus
A4 cell extract is sufficient to increase MUC2 expression
in HT29 cells independent of attachment
[38] . Addition-
ally, VSL3, which contains some Lactobacillus species, in-
creases the expression of MUC2 , MUC3 and MUC5AC in
HT29 cells
[30] . In vivo studies are less consistent because
only a few have been performed. Mice given VSL3 daily
for 14 days do not exhibit altered mucin expression or
mucous layer thickness
[39] . Conversely, rats given VSL3
at a similar daily dose for 7 days have a 60-fold increase
in MUC2 expression and a concomitant increase in mu-
cin secretion
[40] . Therefore, mucous production may be
increased by probiotics in vivo, but further studies are
needed to make a conclusive statement.
(6)
F i g . 1 . Major mechan isms of action of pro-
biotics.
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Increased Adhesion to Intestinal Mucosa
Adhesion to intestina l mucosa is regarded as a prereq-
uisite for colonization and is important for the interac-
tion between probiotic strains and the host [41–43] . Ad-
hesion of probiotics to the intestinal mucosa is also im-
portant for modulation of the immune system
[43, 44]
and antagonism against pathogens
[45] .
Thus, adhesion has been one of the main selection cri-
teria for new probiotic strains
[41, 46–48] and has been
related to certain beneficial effects of probiotics
[49] . Lac-
tic acid bacteria (LABs) display various surface determi-
nants that are involved in their interaction with intestinal
epithelial cells (IECs) and mucus. IECs secrete mucin,
which is a complex glycoprotein mixture that is the prin-
cipal component of mucous, thereby preventing the ad-
hesion of pathogenic bacteria
[47, 50] . Additionally, lip-
ids, free proteins, immunoglobulins and salts are present
in mucous gel
[51] . This specific interaction has indicated
a possible association between the surface proteins of
probiotic bacteria and the competitive exclusion of patho-
gens from the mucus
[52–54] . As mentioned above, sev-
eral Lactobacillus proteins have been shown to promote
mucous adhesion
[54] , and bacteria display surface ad-
hesins that mediate attachment to the mucous layer
[55] .
This process is mainly mediated by proteins, although
saccharide moieties and lipoteichoic acids have also been
implicated
[56] . The most studied example of mucus-tar-
geting bacterial adhesins is MUB (mucus-binding pro-
tein) produced by Lactobacillus reuteri
[55, 57] . The pro-
teins playing a role in the mucous adhesion phenotype of
lactobacilli are mainly secreted and surface-associated
proteins, which are either anchored to the membrane
through a lipid moiety or embedded in the cell wall
[58
61]
. The involvement of surface proteins in the interac-
tion with human plasminogen or enterocytes has been
reported in Bifidobacterium animalis subsp. lactis and Bi-
fidobacterium bifidum , respectively. Under certain cir-
cumstances, these proteins may play a role in facilitating
the colonization of the human gut through degradation
of the extracellular matrix of cells or by facilitating close
contact with the epithelium
[62– 66] . MapA (mucous ad-
hesion-promoting protein) has been reported to mediate
the binding of L. reuteri and L. fermentum to mucus
[52] .
Probiotics, such as L. plantarum , have been reported to
induce MUC2 and MUC3 mucins and to inhibit the ad-
herence of enteropathogenic E. coli . These observations
indicate that enhanced mucous layers and glycocalyx
overlying the intestinal epithelium as well as the occupa-
tion of microbial binding sites by Lactobacillus spp. pro-
vide protection against invasion by pathogens
[45, 67, 68] .
Collado et al.
[69] evaluated the adhesion of Bifidobacte-
rium longum and Bifidobacterium catenulatum strains to
human intestinal mucus and compared the results to
those of control experiments that were run with the orig-
inal acid-sensitive strains. They reported that in half of
the 4 studied cases, the acid-resistant derivative shows a
greater ability to adhere to human intestinal mucus than
the original strain. The ability of bifidobacteria to inhib-
it pathogen adhesion to mucus is not generally improved
by the acquisition of acid resistance. Overall, the induc-
tion of acid resistance in bifidobacteria may be a strate-
gy
for selecting strains with enhanced stability and im-
proved surface properties that favor their potential func-
tionality as probiotics against specific pathogens.
The mixture of probiotics and VSL3 has been reported
to increase the synthesis of cell surface mucins and to
modulate mucin gene expression in a manner dependent
on the adhesion of bacterial cells to the intestinal epithe-
lium
[40] .
Probiotics also cause qualitative alterations in intesti-
nal mucins that prevent pathogen binding
[68] . The bac-
terial component involved in the adhesion of the LB and
BG2FO4 L. acidophilus strains is protease resistant and is
associated with the bacterial surface
[70 –72] . Interesting-
ly, the bacterial component is also degraded into an anti-
microbial peptide, which lends anti-pathogenic proper-
ties to the host and provides an example of how large
surface proteins may exhibit evolutionarily beneficial
pleiotropic effects
[73] .
Probiotic strains can also induce the release of defen-
sins from epithelial cells. These small peptides/proteins
are active against bacteria, fungi and viruses. Moreover,
these small peptides/proteins stabilize the gut barrier
function
[74] . Observations have indicated that in re-
sponse to attack by pathogenic bacteria, the host engages
its first line of chemical defense by increasing the produc-
tion of antimicrobial proteins (AMPs), such as - and -
defensins, cathelicidins, C-type lectins and ribonucleases
[75 –80] . Many AMPs are enzymes that kill bacteria by
carrying out an enzymatic attack on cell wall structures
and/or non-enzymatic disruption of the bacterial mem-
brane. Enzymes expressed by Paneth cells attack the bac-
terial membranes. Lysozyme hydrolyzes the glycosidic
linkage of wall peptidoglycan
[81] and phospholipase A
2
bacterial membrane phospholipids
[82] . Defensins com-
prise a major family of membrane-disrupting peptides in
vertebrates. The interaction is non-specific and mainly
by binding to anionic phospholipid groups of the mem-
brane surface through electrostatic interactions. This in-
teraction creates defensin pores in the bacterial mem-
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brane that disrupt membrane integrity and promote lysis
of microorganisms
[83] . Cathelicidins are usually cation-
ic, -helical peptides that bind to bacterial membranes
through electrostatic interactions and, like the defensins,
induce membrane disruption [84] .
The microbial adhesion process of LAB also includes
passive forces, electrostatic interactions, hydrophobic in-
teractions, steric forces, lipoteichoic acids and specific
structures, such as external appendages covered by lec-
tins. A wide variety of molecules mediating the adhesion
of pathogenic bacteria has been characterized. However,
the understanding of the factors that mediate adhesion
for Lactobacillus is extremely limited
[85–87] . Further
studies are needed for the identification and analysis of
the functional significa nce of various components of mu-
cous layers as well as the complex interactions of mucous
layers, microbiota (including probiotics) and epithelial
cells with underlying innate and adaptive immune sys-
tems
[68] .
Competitive Exclusion of Pathogenic Microorganisms
In a report addressing the total exclusion of Salmo-
nella typhimurium from maggots of blowflies published
in 1969, Greenberg
[88] first used the ‘competitive exclu-
sion’ term for the scenario in which one species of bacte-
ria more vigorously competes for receptor sites in the in-
testinal tract than another species. The mechanisms used
by one species of bacteria to exclude or reduce the growth
of another species are varied, including the following
mechanisms: creation of a hostile microecology, elimina-
tion of available bacterial receptor sites, production and
secretion of antimicrobial substances and selective me-
tabolites, and competitive depletion of essential nutrients
[89] .
Specific adhesiveness properties due to the interaction
between surface proteins and mucins may inhibit the col-
onization of pathogenic bacteria and are a result of an-
tagonistic activity by some strains of probiotics against
adhesion of gastrointestinal pathogens
[90] . Lactobacilli
and bifidobacteria have been shown to inhibit a broad
range of pathogens, including E. coli , Salmonella , Helico-
bacter pylori, Listeria monocytogenes and Rotavirus
[91–
97]
. Exclusion is the result of different mechanisms and
properties of probiotics to inhibit pathogen adhesion, in-
cluding the production of substances and the stimulation
of IECs. Competitive exclusion by intestinal bacteria is
based on a bacterium-to-bacterium interaction mediated
by competition for available nutrients and for mucosal
adhesion sites. To gain a competitive advantage, bacteria
can also modify their environment to make it less suitable
for their competitors. The production of antimicrobial
substances, such as lactic and acetic acid, is one example
of this type of environmental modification
[98] . Some
lactobacilli and bifidobacteria share carbohydrate-bind-
ing specificities with some enteropathogens
[99, 100] ,
which makes it possible for the strains to compete with
specific pathogens for the receptor sites on host cells
[10 1] .
In general, probiotic strains are able to inhibit the attach-
ment of pat hogenic bacteria by mea ns of steric hind rance
at enterocyte pathogen receptors
[10 2] .
The effect of probiotic bacteria on the competitive ex-
clusion of pathogens has been demonstrated using hu-
man mucosal material in vitro
[45, 103] as well as chicken
[10 4] and pig mucosal material in vivo [1 05] . Hirano et al.
[45] s ho w ed t h at L. rhamnosus, a strong ly adhering strai n,
is capable of inhibiting the internalization of EHEC (en-
terohemorrhagic E. coli ) in a human intestinal cell line.
Production of Antimicrobial Substances
One of the proposed mechanisms involved in the
he alth benef it s aff orded by pro bioti cs inclu des t he forma-
tion of LMW compounds ( ! 1,000 Da), such as organic
acids, and the production of antibacterial substances
termed bacteriocins ( 1 1,000 Da).
Organic acids, in particular acetic acid and lactic acid,
have a strong inhibitory effect against Gram-negative
bacteria, and they have been considered the main antimi-
crobial compounds responsible for the inhibitory activity
of probiotics against pathogens
[10 6 –1 08 ] . The undissoci-
ated form of the organic acid enters the bacterial cell and
dissociates inside its cytoplasm. The eventual lowering of
the intracellular pH or the intracellular accumulation of
the ionized form of the organic acid can lead to the death
of the pathogen
[109, 110] .
Many LAB produce antibacterial peptides, including
bacteriocins and small AMPs. Bacteriocins produced by
Gram-positive bacteria (usually LAB, including lactacin
B from L. acidophilus , plantaricin from L. plantarum and
nisin from Lactococcus lactis ) have a narrow activity
spectrum and act only against closely related bacteria, but
some bacteriocins are also active against food-borne
pathogens
[111] . The common mechanisms of bacterio-
cin-mediated killing include the destruction of target
cells by pore formation and/or inhibition of cell wall syn-
thesis
[11 2] . For example, nisin forms a complex with the
ultimate cell wall precursor, lipid II, thereby inhibiting
cell wall biosynthesis of mainly spore-forming bacilli.
Subsequently, the complex aggregates and incorporates
peptides to form a pore in the bacterial membrane
[11 3] .
Several studies have revealed that bacteriocin production
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confers producing strains with a competitive advantage
within complex microbial environments as a conse-
quence of their associated antimicrobial activity. Bacte-
riocin production may enable the establishment and in-
crease the prevalence of producing strains as well as en-
able the direct inhibition of pathogen growth within the
gastrointestinal tract
[114 ] .
Some specific antibacterial compounds have been de-
scribed for several Bifidobacterium strains, and a unique
bacteriocin, bifidocin B, which is produced by B. bifidum
NCFB 1454 and is active towards Gram-positive bacteria,
has been described as well
[108, 115] . Liévin et al. [116 ]
described a strong killing activity of two Bifidobacterium
strains against several pathogenic bacteria, including Sal-
monella enterica ser. typhimurium SL1344 and E. coli
C1845. This activity has been attributed to the produc-
tion of a potential LMW lipophilic molecule
[11 7] . In ad-
dition, an LMW protein termed BIF, which is produced
by B. longum BL1928, is the only compound character-
ized thus far that is active against Gram-negative bacteria
[100, 118, 119] . This protein has no direct inhibitory or
killing effect, but it inhibits the binding of E. coli to hu-
man epithelial cell lines.
Intestinal bacteria also produce a diverse array of
health-promoting fatty acids. Indeed, certain strains of
intestinal bifidobacteria and lactobacilli have been shown
to produce conjugated linoleic acid (CLA), a potent anti-
carcinogenic agent
[114, 120] . An anti-obesity effect of
CLA-producing L. plantarum has been observed in diet-
induced obesity in mice
[1 21] . Recently, the ability to
modulate the fatty acid composition of the liver and adi-
pose tissue of the host upon oral administration of CLA-
producing bifidobacteria and lactobacilli has been dem-
onstrated in a murine model
[114 ] .
Finally, probiotic bacteria are able to produce so-called
de-conjugated bile acids, which are derivatives of bile
salts. De-conjugated bile acids show a stronger antimi-
crobial activity compared to that of the bile salts synthe-
sized by the host organism. It remains to be elucidated
how probiotics protect themselves from their own bacte-
ricidal metabolites or if they are resistant to de-conjugat-
ed bile acids at all
[1 22] .
It is well known that some strains of probiotics pro-
duce metabolites that inhibit the growth of fungi and oth-
er species of bacteria
[123, 124] . Some researchers have
reported that Lactobacillus can produce antifungal sub-
stances, such as benzoic acid, methylhydantoin, mevalo-
nolactone
[125, 126] and short-chain fatty acids [1 27] .
Magnusson and Schnürer
[1 28 ] discovered that Lacto-
bacillus coryniformis can produce proteinaceous com-
pounds exhibiting antifungal properties, and Rouse et al.
[1 29] characterized the antifungal peptides produced by
LAB. These reports showed that the antifungal culture
has the ability to prevent the growth of molds found in
apple spoilage. Dal Bello et al. [1 30 ] reported the identifi-
cation and chemical characterization of four antifungal
substances produced by L. plantarum FST 1.7, including
lactic acid, phenyllactic acid and two cyclic dipeptides
[cyclo(
L -Leu- L -Pro) and cyclo( L -Phe- L -Pro)]. A study de-
scribed the antifungal culture as having the ability to re-
tard growth of Fusarium culmorum and Fusarium gra-
minearum found on breads. Another such study has re-
ported the production of the antifungal cyclic dipeptides,
cyclo (
L -Phe- L -Pro) and cyclo( L -Phe-traps-4-OH- L -Pro),
by LAB, which inhibit the growth of food- and feed-
borne filamentous fungi and yeasts in a dual-culture agar
plate assay
[13 1] .
Probiotics and the Immune System
It is well known that probiotic bacteria can exert an
immunomodulatory effect. These bacteria have the abil-
ity to interact with epithelial and dendritic cells (DCs)
and with monocy tes/macrophages and lymphocy tes. The
immune system can be divided between the innate and
adaptive systems. The adaptive immune response de-
pends on B and T lymphocytes, which are specific for
particular antigens. In contrast, the innate immune sys-
tem responds to common structures called pathogen-as-
sociated molecular patterns (PAMPs) shared by the vast
majority of pathogens
[13 2] . The primary response to
pathogens is triggered by pattern recognition receptors
(PPRs), which bind PAMPs. The best-studied PPRs are
toll-like receptors (TLRs). In addition, extracellular C-
type lectin receptors (CLRs) and intracellular nucleotide-
binding oligomerization domain-containing protein
(NOD)-like receptors (NLRs) are known to transmit sig-
nals upon interaction with bacteria
[13 3] .
It is well established that the host cells that interact
most extensively with probiotics are IECs. In addition,
probiotics can encounter DCs, which have an important
role in innate and adaptive immunity. Both IECs and
DCs can interact with and respond to gut microorgan-
isms through their PPRs
[132, 133] . Figure 2 shows a sum-
mary of how probiotics may interact and modulate the
immune system
TLRs and Probiotics
TLRs are transmembrane proteins expressed on vari-
ous immune and non-immune cells, such as B cells, nat-
ural killer cells, DCs, macrophages, fibroblasts, epithelial
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cells and endothelial cells. In mammals, the TLR family
includes eleven proteins (TLR1–TLR11). However, there
is a stop codon in the human TLR11 gene that results in
a l ack of produc ti on o f hu ma n TLR11. Acti vat ion of TLR s
occurs af ter binding of the ligand to extracel lular leucine-
rich repeats. In humans, TLR1, TLR 2, TLR4, TLR5, TLR6
and TLR10 are outer membrane associated and primar-
ily respond to bacterial surface-associated PA MPs. TLR3,
TLR7, TLR8 and TLR9 are found on the surface of endo-
somes where they respond primarily to nucleic acid-
based PAMPs from viruses and bacteria
[13 2] . Dimeriza-
tion of TLRs and the highly conserved toll-interleukin-1
(IL-1) receptor (TIR) domains leads to the recruitment of
adaptor molecules, such as myeloid differentiation pri-
mary response protein (MyD88), TIR domain-contain-
ing adaptor protein and TIR domain-containing adapter-
F i g . 2 . Interaction of probiotics with the gut-associated immune
system. ASC = Apoptosis-associated speck-like protein contain-
ing a CARD; B. thetaiotamicron = Bacteroides thetaiotamicron ;
CARD9 = caspase recruitment domain-containing protein 9;
ERK = extracellular regulated kinase; IE-DAP = D-gamma-glu-
tamyl-meso-DAP; IKK = I B kinase; IRAK4 = IL-1 receptor-as-
sociated ki nase 4; JNK = Jun N-termina l kinase; MDP = muramyl
dipeptide; MKK = mitogen-activated kinase kinase; NEMO =
NF- B essential modulator; TAB1/2/3 = TAK binding proteins;
TAK1 = ubiquitin-dependent kinase of MKK and IKK; TBK1 =
serine/threonine-protein kinase 1; TRAF6 = TNF receptor-asso-
ciated factor 6; Ub = ubiquitin.
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inducing interferon (IFN)- (TRIF), to initiate signaling
activation. The TLR signaling pathway, except for TLR3,
involves the recruitment of MyD88, which activates the
MAPK and nuclear factor (NF)- B signaling pathways
[13 3 –13 5] . TLR3 utilizes the adaptor protein TRIF, lead-
ing to the expression of type 1 IFNs
[13 5] . Furthermore,
TLR-mediated signaling has been shown to control DC
maturation inducing the upreg ulation of various matura-
tion markers, such as CD80, CD83 and CD86, as well as
the CCR7 chemokine receptor. Moreover, commensal
and probiotic microorganisms can create an overall tol-
erant state mediated by the action of TLRs on DCs. It is
clear that TLR9 signaling is essential to mediate the anti-
inflammatory effect of probiotics. However, different
studies have implicated other TLRs, such as TLR3 and
TLR7, in the tolerance induced by commensal and pro-
biotic bacteria. After activation by commensal and pro-
biotic microorganisms, DCs initiate an appropriate re-
sponse, such as the differentiation of Th
0
to T
reg
, which
has an inhibitory effect on Th
1
, Th
2
and Th
17
inflamma-
tory responses.
It is well established t hat probiotics can sup press intes-
tinal inflammation via the downregulation of TLR ex-
pression, secretion of metabolites that may inhibit TNF-
from entering blood mononuclear cells and inhibition
of NF- B signaling in enterocytes
[13 2] .
In this regard, cell wall components of lactobacilli can
potentially signal through binding TLR2 in combination
with TLR6. The diacylated membrane anchors of lipo-
proteins and lipoteichoic acids bind to TLR2 and TLR6,
thereby promoting dimerization and MyD88-mediated
activation of the canonical pathway of NF- B
[13 5] . Stim-
ulation of TLR2 increases the production of cytokines,
and TLR2 activation has an important role in enhancing
transepithelial resistance to invading bacteria
[13 6] .
TLR2 recognizes peptidoglycan, which is the main
component of Gram-positive bacteria, including the Lac-
tobacillus genus. Several studies have demonstrated that
TLR2 is required for some Lactobacillus strains to exert
their immunomodulatory effects. Vinderola et al.
[13 7]
demonstrated that L. casei CRL 431 interacts with epithe-
lial cells through TLR2 and that the interaction between
L. casei and gut-associated immune cells induces an in-
crease in the number of CD-206 and TLR2 receptors,
mainly in the cells involved in the innate immune re-
sponse.
In addition, Shida et al.
[13 8] showed that L. casei in-
duces a high level of IL-12 production in both wild-type
and TLR2-deficient macrophages, and that peptidogly-
can induces low levels of IL-12 production in wild-type
macrophages and even lower levels in TLR2-deficient
macrophages. They also suggested that the intact pepti-
doglycan of lactobacilli actually signals via TLR2 to in-
hibit IL-12 production. Although the recognition by
TLR2 is essential, 12–48% of IL-12 production in TLR2-
deficient macrophages is inhibited by peptidoglycan,
thus suggesting that other TLR2-independent mecha-
nisms may also be involved. Furthermore, it has been
demonstrated that Lactobacillus strains, such as L. rham-
nosus GG (LGG) and L. plantarum BFE 1685, enhance
TLR2 in vitro in experiments using human intestinal
cells, and more recently, L. casei CRL 431 has been shown
to exert a similar effect on healthy mice and mice infect-
ed with S. enterica serovar typhimurium
[139, 140] . For
instance, probiotic administration to healthy mice in-
creases expression of TLR2, TLR4 and TLR9, and it im-
proves the secretion of TNF- , IFN- and IL-10 in Peyer’s
patches
[14 0] .
Similarly, when porcine IECs encounter Lactobacillus
jensenii TL2937, TLR2 may act synergistically and coop-
eratively with one or more PRRs, which may result in a
coordinated sum of signals that induce the upregulation
of several negative regulators of TLRs, including A20,
Bcl-3 and MKP-1
[14 1] .
TLR2 also has an important role in the recognition of
bifidobacteria. Hoarau et al.
[14 2] reported that a fer men -
tation product from Bifidobacterium breve C50 can in-
duce maturation, high IL-10 production and prolonged
survival of DCs via the TLR2 pathway.
Similarly, Zeuthen et al.
[14 3] showed that TLR2/
DCs produce more IL-2 and less IL-10 in response to bi-
fidobacteria, and they concluded that the immuno-in-
hibitory effect of bifidobacteria is dependent on TLR2.
Recently, Kailova et al.
[14 4] reported that oral admin-
istration of B. bifidum OLB 6378 to rats with necrotizing
enterocolitis (NEC) stimulates TLR2 expression in the
ileal epithelium, enhances epithelial expression of COX-2
and increases intestinal production of prostaglandin E
2
.
Indeed, pretreatment of IEC-6 cells with the probiotic
strain stimulates TLR2 and COX-2 expression and blocks
cytokine-induced apoptosis. However, there is no evi-
dence of a clear link between TLR2 activation and the
upregulation of COX-2.
In contrast, it has been shown that the L. reuteri
strains DSM 17938 and ATCC PTA 4659 have a benefi-
cial
effect on preventing NEC in rats. In response to the
probiotic, mRNA expression of IL-6, and expression lev-
els of TNF- , TLR4 and NF- B are significantly down-
regulated, and mRNA levels of IL-10 are significantly
upregulated. Moreover, L. reuteri treatment leads to de-
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creases in intestinal protein levels of TLR4, IL-1 and
TNF- in newborn rats with NEC. Furthermore, L. reu-
teri significantly increases survival rate, reduces both the
incidence and severity of NEC and decreases pro-inf lam-
mator y c y toki ne le ve ls in pa ra l lel w it h i nhi biti on of TL R4
signaling via the NF- B pathway.
Moreover, TLR4 has a significant role in the host
defense against Salmonella infection in vivo . In healthy
mice, L. casei CRL 431 activates this receptor and can be
used as a surveillance mechanism against pathogenic
bacteria
[14 0] . Activation of TLR4 leads to the induction
of pro-inflammatory mediators, an increase in TLR2 ex-
pression, and a reduction in its own expression, which
leads to the recruitment of inflammatory cells and the
initiation of the appropriate responses in the spleen. Col-
lectively, these events allow for the control of bacterial
replication
[140, 146, 147] .
Similarly, heat-inactivated LGG and Lactobacillus del-
brueckii subsp. bulgaricus can decrease TLR4 expression
similar to lipopolysaccharide (LPS) after 12 h in human
monocyte-derived DCs. Moreover, LGG downregulates
p38 expression, and L. delbrueckii subsp . bulgaricus re-
duces inhibitor protein B (I B) expression. In addition,
these probiotic strains can modify the immune response
at the post-transcriptional level by modifying miRNA ex-
pression
[14 8] .
Another relevant TLR is TLR9, which recognizes bac-
terial CpG DNA and synthetic unmethylated CpG oligo-
nucleotide mimics (CpG-ODN). Unmethylated DNA
fragments containing CpG motifs that are released from
probiotics in vivo have the potential to mediate anti-in-
flammatory effects through TLR9 signaling at the epithe-
lial surface. It is known that Lactobacillus species differ
in their C+G composition. Thus, the ability of different
species to stimulate TLR9 is likely to be different
[13 5,
149] . TLR9 activation through apical and basolateral sur-
faces activates different intracellular signaling pathways
in polarized epithelial cells. Whereas basolateral TLR9
triggers I B degradation and NF- B pathway activa-
tion, apical TLR9 induces cytoplasmic accumulation of
ubiquitinated I B and inhibition of NF- B activation
[15 0 ] .
Using polarized HT29 and T84 cell monolayers, Gha-
dimi et al.
[15 1] showed that binding of natural com-
mensal-origin DNA to the apical TLR9 initiates an in-
tracellular signaling cascade in a specific manner that
is associated with the attenuation of TNF- -induced
NF- B activation and NF- B-mediated IL-8 expression.
When LGG DNA was apically applied, they showed a de-
tracted TNF- -induced NF- B activation by reduced
I B degradation and p38 MAPK phosphorylation,
thereby indicating that intracellular chemical signals
may coordinately regulate multiple properties of TLR9
expression that are relevant in multicellular functional
responses of TLR9 to bacterial DNA. They also showed
that TLR9 silencing abolishes the inhibitory effect of nat-
ura l c ommens al-o rigi n DNA on TN F- -induced I L-8 se-
cretion.
Similarly, B. breve (NumRes 204), L. rhamnosus
(NumRes 1) and L. casei (DN-114 001) strains induce dif-
ferent cytokine production levels by human and mouse
primary immune cells. It has been demonstrated that the
B. breve strain induces lower levels of the pro-inflamma-
tory cytokine IFN- than L. rhamnosus and L. casei .
Moreover, B. breve and lactobacilli induce cytokines in a
TLR9-dependent manner, and the lower inflammatory
profile of B. breve is due to inhibitory effects of TLR2
[15 2] .
In addition, it has been shown that purified genomic
DNA from L. plantarum (p-gDNA) does not substantial-
ly stimulate pro-inflammatory cytokines. However, p-
gDNA inhibits LPS-induced TNF- production by T HP-
1 cells. Furthermore, p-gDNA reduces the expression of
TLR2, TLR4 and TLR9, which induces the activation of
NF- B through the LPS signaling pathway, leading to the
upregulation of inf lammatory cytokines
[153, 154] . Pre-
treatment of p-gDNA inhibited the phosphorylation of
MAPKs and NF- B, and also inhibited LPS-induced
TNF- production in subsequent LPS stimulation. In
this regard, L. plantarum genomic DNA-mediated inhi-
bition of signaling and TNF- was accompanied by the
suppression of TLR2, TLR4 and TLR9, as well as the in-
duction of IL-1 receptor-associated kinase M (a negative
regulator of TLR)
[15 4 ] .
NLRs and Probiotics
As mentioned before, there is another family of mem-
brane-bound receptors: NLRs. They are located in the cy-
toplasm and are important in tissues where TLRs are ex-
pressed at low levels. The most thoroughly characterized
members are NOD1 and NOD2, but currently more than
20 different NLRs have been identified
[15 5] . Unlike
NOD1, which is ubiquitously expressed, the expression of
NOD2 is restricted to DCs, macrophages, Paneth cells,
intestinal cells, lung cells and oral epithelial cells, and it
is expressed at low levels in T cells. NOD1 can sense pep-
tidoglycan moieties containing meso-diaminopimelic
acid, which are associated with Gram-negative bacteria,
but NOD2 senses muramyl dipeptide motifs, which can
be found in a wide range of bacteria
[15 6] . Upon recogni-
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169
tion of their agonist, both NOD1 and NOD2 self-oligo-
merize to recruit and activate the adaptor protein RICK,
a protein kinase that regulates CD95-mediated apoptosis,
which is essential for the activat ion of NF- B and M APKs,
resulting in the upregulation of transcription and pro-
duction of inflammatory mediators (e.g. cytokines, che-
moattractants, COX-2 and inducible nitric oxide syn-
thase)
[15 7] .
There are a few studies showing the effect of probiotics
on NLR. However, Fernandez et al.
[15 8] recently demon-
strated that the protective capacity of L. salivarius Ls33
correlates with local IL-10 production, which is abolished
in NOD2-deficient mice. Indeed, these authors showed
that the anti-inflammatory effect of Ls33 is mediated via
NOD2.
Another important pathway activated by NLRs in-
volves apoptosis-associated speck-like protein with cas-
pase recruitment to activated caspase 1, an adaptor pro-
tein which is necessary for the cleavage of pro-IL-1 and
pro-IL-18 into their mature and biologically active forms.
NLRs participate in the formation of inflammasomes,
which leads to the activation of caspase-1. There are three
principal inf lammasomes named after the NLR involved
as follows: NOD-like receptor family, pyrin domain con-
taining protein (NLRP) 1, NLRP3 and NLRC4. NLRP3
detects LPS, muramyl dipeptide, bacterial RNA and viral
RNA
[15 7] .
The following two steps are required for the complete
activation of the NLRP3 inflammasome: a priming step
to induce transcription of NLRP3 mRNA and a sequen-
tial step to recognize various PAMPs and danger-associ-
ated molecular patterns by fully expressed NLRP3 itself
[159, 160] . With regard to probiotic mechanisms asso-
ciated with NLRP3, Tohno et al.
[16 1] found that L.
delbrueckii subsp. bulgaricus NIAI B6 and L. gasseri
JCM1131
T
are able to enhance NLRP3 expression in the
GALT of adult and newborn swine. Their results suggest-
ed that immunobiotic Lactobacillus strains directly pro-
mote NLRP3 expression via TLR and NOD-mediated
signaling, resulting in the induction of appropriate
NLRP3 activation in porcine GALT. Furthermore, their
results indicated that NLRP3 expression is upregulated
by TLR2, TLR9, NOD1 and NOD2 agonists in adult and
newborn porcine GALT. It has been suggested that
NLRP3 has an important role in the regulation of human
intestinal inflammation, such as in Crohn’s disease
[16 2] ,
and that dysregulated NLRP3 expression results in the
disruption of immune homeostasis associated with auto-
inflammatory disease in humans
[16 3] . Because the po-
tential expression level of NLRP3 is low in immune cells,
induction of cellular NLRP3 expression itself is a first
step to evoke the appropriate activation of the NLRP3-
mediating signaling pathway in order to respond to dan-
ger-associated molecular patterns and PAMP stimuli
[159, 160, 164, 165] .
Conclusions
Probiotics have considerable potential for preventive
or therapeutic applications in various gastrointestinal
d is orde rs . H owe ve r, it is im po rt ant to no te th at ma ny pr o-
biotic health claims have not yet been substantiated by
experimental evidence. In addition, the efficacy demon-
strated for one given bacterial strain cannot necessarily
be transferred to other probiotic organisms. Moreover,
the mechanisms underlying probiotic action have not yet
been fully elucidated.
This study reviewed the mechanisms of action of pro-
biotics. Several important mechanisms underlying the
antagonistic effects of probiotics on various microorgan-
isms include the following: modification of the gut mi-
crobiota, competitive adherence to the mucosa and epi-
thelium, strengthening of the gut epithelial barrier and
modulation of the immune system to convey an advan-
tage to the host. The recent characterization of the host
families of pattern-recognition molecules, such as TLR
and NOD-like receptors, as well as modulating key sig-
naling pathways, such as NF- B and MAPK, with respect
to their ability to enhance or suppress activation and in-
fluence downstream pathways will shed light onto the
complex interplay of host-microbe interactions. Stimula-
tion of these receptors by commensal bacteria has a cru-
cial role to elicit measured antimicrobial responses with
minimal inflammatory tissue damage.
Future Perspectives
In the present review, we provided an overview of the
mechanisms of action of probiotics. It must be noted that
many reported mechanisms of probiotic action are the
results of in vitro experiments. Considerable effort has
been invested in the development of methods enabling
the in-depth analysis of the molecular mechanisms of
probiotics. The complex and dynamic interactions that
exist between the intestinal epithelium and bacteria on
the luminal side as well as between the epithelium and the
underlying immune system on the basolateral side must
be reconciled in co-culture experiments with probiotics,
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170
DCs and IECs as well as in 3D models. Other models in-
clude tissue explants, bioreactors and organoids. In vitro
models have improved our current knowledge regarding
specific probiotic modes of action. However, a number of
limitations have to be taken into account. For example,
results obtained with different IECs have to be carefully
interpreted because not all cell lines share the same char-
acteris tics. It should a lso be noted that cu lture c onditions
may influence the expression of certain molecular char-
acteristics.
The molecular elucidation of probiotic action in vivo
will help to identify true probiotics and to select the most
suitable ones for the prevention and/or treatment of par-
ticular diseases. It is important to note that results ob-
tai ned in an imal models ca nno t be di rec tl y tra nsfer red to
humans. The physiology of animals differs considerably
from t hat of huma ns, but th is di sadvan tage is outweigh ed
by the possibility of using a nimals with virtually identical
genetic backgrounds, such as human microbiota-associ-
ated animals.
The quest for a better understanding of how probiotics
operate has catalyzed an enormous interest in the mo-
lecular processes underlying host-microbe interactions.
Gaining insight into the mechanisms of probiotic action
may not only help to improve the credibility of the probi-
otic concept but also to foster the development of novel
strategies for the treatment or prevention of gastrointes-
tinal and autoimmune diseases.
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This chapter explores the role of probiotics in adhesion of pathogens. The human intestinal microbiota constitutes a complex microbial ecosystem that plays an important role in health and disease. This microbiota may be divided in commensals and symbiotic and pathogenic microorganisms. A correct individual balance of the microbiota plays a critical role in the maintenance of the health status of the host. The presence of bacterial pathogens alters the intestinal bacterial homeostasis (microbiota composition and activity), leading to either an increased risk of disease or specific diseases. Probiotics are viable beneficial microorganisms with a demonstrated health impact on the host. The assessment of adhesion properties and competitive exclusion of pathogens constitutes an important point in probiotic characterization. Selection of new probiotic strains and combinations counteracts both pathogen challenges against normal healthy microbiota as well as counteracting identified microbiota deviations that may predispose the subjects to later disease. Probiotic strains or combinations that inhibit and displace pathogens may be good candidates for the treatment or prevention of specific diseases caused by known pathogens or microbiota deviations related to disease risk reduction.
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Paneth cells in mouse small intestinal crypts secrete granules rich in microbicidal peptides when exposed to bacteria or bacterial antigens. The dose-dependent secretion occurs within minutes and α-defensins, or cryptdins, account for 70% of the released bactericidal peptide activity. Gram-negative bacteria, Gram-positive bacteria, lipopolysaccharide, lipoteichoic acid, lipid A and muramyl dipeptide elicit cryptdin secretion. Live fungi and protozoa, however, do not stimulate degranulation. Thus intestinal Paneth cells contribute to innate immunity by sensing bacteria and bacterial antigens, and discharge microbicidal peptides at effective concentrations accordingly.