L Rhamnosus

Segers and Lebeer Microbial Cell Factories 2014, 13(Suppl 1):S7
http://www.microbialcellfactories.com/content/13/S1/S7
PROCEEDINGS Open Access
Towards a better understanding of Lactobacillus

rhamnosus GG - host interactions
Marijke E Segers1,2, Sarah Lebeer1,2*
From 11th International Symposium on Lactic Acid Bacteria
Egmond aan Zee, the Netherlands. 31 August - 4 September 2014
Abstract
Lactobacillus rhamnosus GG (LGG) is one of the most widely used probiotic strains. Various health effects are well
documented including the prevention and treatment of gastro-intestinal infections and diarrhea, and stimulation of
immune responses that promote vaccination or even prevent certain allergic symptoms. However, not all
intervention studies could show a clinical benefit and even for the same conditions, the results are not univocal.
Clearly, the host phenotype governed by age, genetics and environmental factors such as the endogenous
microbiota, plays a role in whether individuals are responders or non-responders. However, we believe that a
detailed knowledge of the bacterial physiology and the LGG molecules that play a key role in its host-interaction
capacity is crucial for a better understanding of its potential health benefits. Molecules that were yet identified as
important factors governing host interactions include its adhesive pili or fimbriae, its lipoteichoic acid molecules, its
major secreted proteins and its galactose-rich exopolysaccharides, as well as specific DNA motifs. Nevertheless,
future studies are needed to correlate specific health effects to these molecular effectors in LGG, and also in other
probiotic strains.
Introduction influence on the commensal microbiota [2,4]. A second

Lactobacillus rhamnosus GG (LGG), ATCC 53103 was mechanism is the capacity of certain probiotic strains to
originally isolated from fecal samples of a healthy enhance the epithelial barrier function by modulating
human adult by Sherwood Gorbach and Barry Goldwin, signaling pathways, such as nuclear factor-B (NF-kB),
explaining its typical surname letters GG. It was identi- Akt and mitogen- activated protein kinase (MAPK)-
fied as a potential probiotic strain because of its resis- dependent pathways, which lead to for example the
tance to acid and bile, good growth characteristics and induction of mucus [5], or increased tight junction func-
adhesion capacity to the intestinal epithelial layer [1]. tioning [6]. Thirdly, most probiotic strains can also mod-
Ever since, it has been one of the most widely studied ulate host immune responses, exerting strain-specific
probiotic strains, used in a variety of commercially avail- local and systemic effects [7]. Many of the interactions
able probiotic products. The beneficial effects of this between probiotic bacteria and intestinal epithelial and
strain have been studied extensively in clinical trials and immune cells are thought to be mediated by molecular
human intervention studies. structures, known as microbe- associated molecular pat-
Probiotic bacteria are proposed to benefit human terns (MAMPs), which can be recognized through speci-
health mainly by three general mechanisms of action fic pattern recognition receptors (PRRs) such as Toll-like
[2,3]. Firstly, certain probiotics can clearly exclude or receptors (TLRs) [8].
inhibit pathogens, either through direct action or through Even though many experimental in vitro data and
experiments in animal models validate these mechanisms
for probiotic strains in general and for LGG in specific,
* Correspondence: sarah.lebeer@uantwerpen.be
1
University of Antwerp, Department of Bioscience Engineering, Research most published in vivo data in humans pay less attention
Group Environmental Ecology and Applied Microbiology, Groenenborgerlaan to mechanisms of action. Nevertheless, we believe that for
171, B-2020 Antwerp, Belgium an optimized and more tailored application of probiotics,
Full list of author information is available at the end of the article
© 2014 Segers and Lebeer; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
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(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Segers and Lebeer Microbial Cell Factories 2014, 13(Suppl 1):S7 Page 2 of 16
it is imperative to understand the mechanisms of interaction also in vivo in humans. LGG has been shown to be a very
with the host in great detail. LGG is an interesting model good mucus adhering Lactobacillus strain compared to
probiotic strain, because of its wide use, its available genome related strains such as the dairy strain L. rhamnosus Lc705
sequence [9] and the availability of numerous knock-out and other probiotic strains such as L. johnsonii LJ1 and
mutants that allow the study of gene-function relations L. casei Shirota [16] (Figure 1A). In human intervention
[10-15]. In this review we aim to give an overview of the studies, LGG was also reported to persist longer and in
recent advances in the molecular knowledge on LGG-host higher concentrations compared to closely related strains
interactions and try to provide a molecular framework for a such as L. rhamnosus LC705 [9]. Orally administered LGG
better understanding of the health effects of LGG. can be recovered from the feces at least one week after
administration in adults [9,17]. Of note, the colonization
Pili-mediated adhesive capacity of LGG capacity of LGG appears to be significantly better in new-
In vivo and in vitro evidence borns [18], which is related to reduced colonization resis-
One of the widely studied key features of LGG is its strong tance exerted by a less established microbiota of infants
adhesive capacity, which has been documented in vitro but and which is probably a general feature for many probiotic
Figure 1 SpaCBA pili and the molecular mechanisms of adhesion. LGG is very good mucus adhering Lactobacillus strain compared to other
probiotic strains such as L. casei Shirota and L. johnsonii LJ1 and the closely related strain L. rhamnosus Lc705. Radioactively labeled bacteria
were allowed to adhere to isolated human intestinal mucus. The adhesion ratio (%) was determined by comparing radioactivity of bacteria
added to the radioactivity of bound bacteria after washing (A). Data were published before [16]. Presence of SpaCBA pili LGG cells based on a
TEM image of LGG labeled with SpaA antiserum and 10 nm protein A gold particles [26] (B) and on a AFM image of LGG in air [37] (C). The
predicted model of the pili shows a pilus backbone formed by the major subunit SpaA, as shown in the schematic figure. The minor subunit
SpaC is present on the tip and decorates the pilus over the length at ratio 1:2 with SpaA. The Spa B minor pilin serves as a molecular switch for
pilus termination and is bound to the peptidoglycan layer. However, it is suggested that leaky activity of the pilin-specific sortase can include
SpaB decorations on the pilus (D). Adapted from [26]. The SpaC pilin is thought to serve as a major adhesin of LGG. It can interact with other
SpaC molecules, inducing homophilic adhesion, and with intestinal epithelial cells or their extracellular matrix, in heterophilic adhesion. The exact
adhesion sites however remain unkown. Pili can have molecular spring properties which makes them capable to withstand shearing stress.
Moreover, the SpaC pilin decorated over the pilus length provide a molecular zipper mechanism that can facilitate a close interaction between
the host and the bacteria or bacteria with each other (E). Adapted from [35].
strains. LGG can adhere to intestinal mucus [9,19] and Structural properties of the SpaCBA pili
persist in the descending colon [20]. Colonic biopsies even The LGG pilus contains three distinct pilin monomers
suggest that LGG colonization continues for longer than which are covalently linked in a sortase-dependent man-
indicated by fecal recovery [21]. LGG could also be recov- ner: the major pilin SpaA and the minor pilins SpaB
ered from the tonsils [22], vagina [23] and oral cavity [24] and SpaC (Figure 1.D). Mass spectrometry revealed a
after probiotic therapy, but it does not seem as efficient as SpaA/SpaC/SpaB ratio of 5:2:1 [26]. As observed in
other Lactobacillus strains in colonizing these niches. other Gram-positive pili, the major pilin SpaA exerts
LGG seems to show a preferential tissue tropism for the merely a structural function forming the pilus backbone,
intestinal mucus layer, although these findings could be while the accessory pilins play a functional role [33,34].
biased, as only limited studies have investigated the adher- The larger minor pilin SpaC, located on the tip of the
ence and colonization at other body sites. pilus and covering the pilus length [26], is thought to
play a pivotal role in adhesion to mucus. This was
Pili as key intestinal adhesins shown using whole bacteria with a isogenic spaCBA
A major breakthrough in our understanding of the mutation [9], competitive blocking experiments with
excellent adherence capacity of LGG was the discovery SpaC antiserum [9], experiments with recombinant
of fimbria-like appendages [25], later named as pili [9]. SpaC in a mucus binding assay [9] and single molecule
Pili or fimbriae are long and thin proteinaceous protru- atomic force microscopy (AFM) [35]. The smaller minor
sions of the cell surface present on specific Gram- pilin SpaB is thought to act as a molecular switch
positive and Gram-negative bacteria. A genomic com- responsible for pilus termination and initiation of pepti-
parison with the closely related strain LC705 resulted in doglycan binding by the housekeeping sortase [26].
identification of the spaCBA gene cluster involved in the Another interesting feature of pili expression is the
biosynthesis of the LGG-specific SpaCBA pili, and the presence of numerous insertion sequence (IS) elements
confirmation by Western blot and immunogold trans- flanking the spaCBA operon in the LGG genome. It was
mission electron microscopy of structures of ca. 1 µm even suggested that the iso-IS30 element actually
that are around 5 nm thin (Figure 1B) [9]. Subsequently, enhances pili expression in LGG, while not in L. casei
we found by mutational analysis of several predicted strains [30]. It is however also possible that spontaneous
adhesins that the SpaCBA pili play a key role in adhe- removal of the IS element would stop expression of pili
sion to mucus, the Caco-2 intestinal epithelial cell line in LGG, although Douillard et al. [30] showed that LGG
and promote biofilm formation [14]. Of note, the LGG could be recovered from various commercial products
genome also encodes another pili gene cluster, spaDEF, with a very limited amount of genomic changes (2-3
but these pili do not seem to be expressed, at least not SNPs) and was still capable of producing pili, indicating
under the tested conditions [9,14,26]. Interestingly, the its robustness under fermentation conditions. Neverthe-
pili of Bifidobacterium breve UCC2003 appear to be less, others showed in a comparative genome analysis of
only expressed in the murine gastro-intestinal tract and three LGG dairy product isolates that in two of these
not in laboratory conditions [27], highlighting that con- strains, major stretches of genomic DNA were deleted,
dition-dependent expression of the pili cannot be ruled including the spaCBA operon [36]. Moreover, LGG pili
out. However, LGG remains piliated with SpaCBA pili are susceptible to shearing stress. Our AFM study
under different stress conditions such as bile salts and showed that bacterial cells subjected to 8000 × g centri-
low pH [28]. Recently pili were also found in various fugal forces are completely devoid of pili [37]. As pili
other lactobacilli and lactococci [29-31]. Remarkably, seem to play an important role for the probiotic func-
comparative genome and functional analysis of L. rham- tion of LGG, it is important for industrial production of
nosus species showed that functional SpaCBA pili are LGG that detrimental shearing stresses are avoided. The
significantly more prevalent in human isolates than in presence of pili should be a key question for future
dairy isolates. Moreover, the pili are most prevalent in intervention studies with LGG.
intestinal isolates, while none of the strains that origi- Based on single molecule AFM, we could also suggest
nated from the oral and vaginal cavities were shown to that the LGG pili have two important nanoscale proper-
have functional pili [28]. Intriguingly, recent gut metage- ties mediating interactions with the host (Figure 1.E).
nomic studies also showed that pili genes form impor- Firstly, pili appear to function as nanosprings capable of
tant examples of highly abundant functions that could withstanding forces such as shearing stress. Secondly,
be identified, even if mainly expressed by some low- pili can function as a mechanical zipper [35]. As SpaC is
abundance microbes such as Echerichia coli, further distributed not only at the tip, but also over the pilus
supporting a key role for mucus-binding pili in the length [26], it is thought that a zipper-like mechanism
intestinal niche [32]. can facilitate a close interaction with the host after the
first interaction is initiated by a SpaC subunit, enabling Moreover, LPS and LTA interact with different PRRs.
other SpaC subunits along the tip, but also other cell The specific PRRs for LTA were shown to consist of
surface adhesins on the LGG surface to adhere step by TLR2 in a heterodimer with TLR6 and co-receptors
step, resulting in a more stable interaction. For instance, CD14 and CD36 [42]. The structure of LTA of LGG
two other LPxTG-like surface adhesins were found in was shown by NMR to consist of a glycerolphosphate
LGG: MabA as modulator of biofilm formation and backbone with D-alanyl esters (ca. 70%) as unique
adhesion [13] and the mucus-binding factor MBF [38]. detectable substituents and an average chain length
between n = 30 and n = 76. The glycolipid moiety con-
Possible immunomodulatory role of the SpaCBA pili tains 2 fatty acid chains with an average length of C14,
As key adhesive components, the immunomodulatory with one double bound per fatty acid [12,43]. Analysis
capacity of the pili is also of high interest, although cur- of structure-activity relations showed the importance of
rent data are very limited. In a first study, focusing on the lipid chains of LTA in LGG in interaction with
the nonpiliated spaCBA mutant of LGG, we could show TLR2-6 and the induction of NF-B signaling [43].
a ca. twofold increased induction of interleukin-8 (IL-8) To investigate the in situ role of LTA in live bacteria,
and other pro-inflammatory markers in intestinal a mutant of LGG that showed a modified LTA structure
epithelial Caco-2 cells compared to wild-type, while a lacking D-alanine residues and an altered glycolipid
mutant lacking exopolysaccharide (EPS) and showing an anchor, was created by mutating the dltD gene. This
increased exposure of SpaCBA pili resulted in ca. two- gene encodes a membrane protein that facilitates the
fold less IL-8 mRNA induction [14]. In a more recent ligation of the D-alanyl carrier protein with D-alanine
study, Lactococcus lactis was genetically engineered to [12]. This mutant showed a strongly reduced interaction
express the LGG SpaCBA pili. These constructs with TLR2-6 and a lower induction of IL-8 mRNA in
appeared to promote interaction with TLR2 and expres- the Caco-2 cell line compared to wild type, further sub-
sion of IL-8 [39]. Moreover, in a comparison between stantiating a key role for LGG LTA in promoting more
LGG and other commercial probiotic L. rhamnosus and pro- inflammatory responses [43]. Furthermore, the
L. casei strains, it was shown that certain closely related dltD mutant significantly improved DSS- induced colitis
strains lacking pili expression have a reduced interaction in treated mice compared to buffer control, while the
with TLR2 compared to LGG [28]. As part of our wild type strain showed actually detrimental effects in
ongoing research, we are investigating whether the pili that model [44]. Interestingly, similar effects were
have a direct effect as MAMP interacting with PRRs, observed with LTA mutants in other lactobacilli [45].
such as TLR2, like has been shown for the type I pilus Complete removal of LTA in Lactobacillus acidophilus
of Streptococcus pneumonia [40], or whether they mainly NCFM [46] or a shift from mainly D-alanine to glucosyl
act indirect by mediating a close interaction with the substitutions in Lactobacillus plantarum NCIMB8826
host cells and other LGG exposing surface-bound [47] also resulted in strains capable of more efficiently
ligands. Recent data suggest that it is not specifically the alleviating inflammation. This shows that LTA is not an
presence or absence of SpaCBA pili that mediate LGG-specific effector molecule, but that it is an impor-
immune status. Rather, the pili appear to play an indir- tant molecule in understanding Lactobacillus-host inter-
ect modulating role by promoting close interactions actions and is a crucial factor to take into account when
with host cells such as epithelial cells, probably by the investigating anti-inflammatory effects.
zipper-like mechanism discussed above, so that other
effector molecules can exert their immune modulating Secreted proteins as probiotic effectors
activities [14]. It will be very interesting to substantiate In 2002, Yan and Polk showed that LGG promotes the
this indirect immunomodulatory role in human volun- survival of intestinal epithelial cells (IECs) by preventing
teers, e.g. by intervention studies with spontaneous pili cytokine-induced apoptosis through blocking of p38
mutants of LGG. MAP kinase. They found that the survival-promoting
effect was also present in other probiotic strains such as
Lipoteichoic acid (LTA) as key immune effector L. acidophilus ATCC393 and L. casei ATCC4356, but
of LGG the strongest in LGG [48]. The supernatant of LGG was
One of the first effector molecules of LGG that were shown to prevent apoptosis in IECs [49] and induce
studied are its lipoteichoic acid (LTA) molecules, heat shock proteins [50]. Consequently, two proteins
because LTA is considered as the Gram-positive equiva- from the LGG supernatans were found to cause the
lent of Gram-negative LPS in stimulating strong antiapoptic effect [49]. These proteins p75 (~75 kDa)
immune responses. However, the bio-active concentra- and p40 (~40 kDa) were later renamed as Major
tion of LTA is typically in de micromolar range, while Secreted Protein Msp1 and Msp2 respectively, because
LPS is active in nanomolar concentrations [41]. of the discrepancy in molecular weight [15]. Each of the
purified proteins was shown to activate the Akt signal- because of its essential role in LGG. Immunofluores-
ing peptide, inhibit cytokine-induced IEC apoptosis and cence analysis suggests a possible role in early stage cell
reduce tumor necrosis factor (TNF)- induced epithelial septum formation [15].
damage. The proteins also promoted cell growth in Because of their action as PG hydrolases, it also
human and mouse colon IECs and cultured mouse remains to be studied whether Msp1 and Msp2 could
colon explants [51]. Moreover, they were shown to pro- have immunomodulatory functions by release of PG frag-
tect the intestinal epithelial barrier function from hydro- ments. Intriguingly, a recent study showed that PG might
gen peroxide-induced damage through blocking of MAP be a central mediator for the beneficial effect of certain
kinases [52]. probiotic lactobacilli, such as Lactobacillus salivarius
A subsequent in vivo mice study proved the efficacy of Ls33 in inflammatory bowel disease with NOD-2 as a key
recombinant Msp2/p40 delivered in a pectin/zein hydrogel receptor [58]. Although the PG of most lactobacilli share
bead for the prevention and treatment of DSS- induced the same basic structure, the PG molecules of this Lacto-
intestinal injury and acute colitis and for ameliorating bacillus strain was shown to contain an additional muro-
colon epithelial cell apoptosis and chronic inflammation in peptide, MurNAc-l-Ala-g-d-isoGln-l-Lys, which was
oxazolone-induced colitis [53]. Moreover, Msp2/p40 was suggested to be the NOD2 ligand displaying anti-inflam-
shown to cause phosphorylation of the epidermal growth matory properties [59]. The PG structure of LGG was
factor receptor (EGF-R) leading to activation of Akt. The previously determined when analyzing the PG hydrolyz-
importance of EGF-R in the mechanism was confirmed in ing activity of Msp1/p75 [15]. It is at present not known
the in vivo tests [53]. Nevertheless, the exact PRR could whether LGG PG contains such strong NOD2 ligands
not be identified. The activation of EGF-R is thought to be and how this NOD2 interaction could be influenced by
indirect as Msp2/p40 was shown to stimulate the catalytic its collection of PG hydrolases.
activity of the metalloproteinase ADAM-17, which subse-
quently releases heparin-bound EGF in IECs, activating Exopolysaccharides (EPS) as modulating
EGF-R [54]. adaptation factors
As mentioned before, Msp1/p75 and Msp2/p40 are An extracellular polysaccharide (EPS) layer is commonly
not unique for LGG. For instance, the homologous pro- found in lactobacilli. Of interest, EPS molecules show a
teins of L. casei BL23 can also cause EGF-R phosphory- large structural diversity [60], so that they are clearly
lation [55]. However, there exists some heterogeneity strain-specific molecules. The LGG cell surface appears
between the homologues. Analysis of Msp1/p75 and to contain two major types of polysaccharides: long
Msp2/p40 gene sequence showed homology with cell galactose-rich polysaccharides and shorter glucose/man-
wall hydrolases in LGG [51], but also in L. casei [55]. nose-rich polysaccharides [61]. We could yet identify the
Msp1 was characterized in LGG as a D-glutamyl-L-lysyl operon responsible for galactose-rich EPS synthesis [62].
endopeptidase with an important role in daughter cell A knock-out mutant of the welE gene, encoding the
separation [15]. Intriguingly, we could also show that priming glycosyltransferase, is completely devoid of the
the Msp1 protein is glycosylated in LGG with Con-A long galactose-rich EPS but shows a higher concentration
reactive residues [56], which could explain the apparent of short glucose-rich EPS [62]. This mutant shows an
discrepancy previously seen between the predicted mole- increased adhesion to Caco-2 IECs, linked to increased
cular weight and the molecular weight shown on Wes- exposure of the SpaCBA pili [14,62]. In addition, this
tern blot [51]. The serine-rich glycosylation site was not type of galactose-rich EPS appears to be an important
found in homologues of Msp1 in several L. casei strains, adaptation factor for LGG as the welE mutant shows a
which suggest a species-specific glycosylation. The gly- reduced in vivo survival in the murine gastrointestinal
cosylation does not impede enzyme activity and was tract (GIT) [63]. More specifically, the long galactose-
suggested not to interfere with activation of Akt, rich EPS were shown to protect the cell against comple-
although a the glycan chain appears to have a modulat- ment-mediated lysis and cathelicidins, specific cationic
ing role as shield. The glycosylation seems to play a role antimicrobial peptides [63]. It thus seems important that
in increasing protein stability and protein binding to the this type of EPS production is balanced between optimal
cell wall [56]. Interestingly, the ConA reactive S-layer protection and optimal adhesion.
protein SlpA of L. acidophilus is suggested to be recog- The role of EPS in the interaction between LGG and
nized by DC-SIGN [57], although this remains to be the host remains largely unclear. Results obtained in our
further substantiated. The Msp2 protein, on the other lab indicate that isolated galactose-rich EPS are not
hand, appears not to be glycosylated. The exact function principal inducers of cytokines in the Caco-2 intestinal
of LGG Msp2 remains unclear since its hydrolytic pepti- epithelial cell line [14]. On the other hand, isolated EPS
doglycan (PG) degrading activity is limited and an msp2 from LGG appears to counteract the cytotoxicity of
knock-out mutant could not be constructed, possibly Bacillus cereus on Caco-2 cells and of streptolysin-O on
rabbit erythrocytes [64]. However, as low concentrations participate as a recent study showed that the antimicro-
of the major secreted proteins have an anti-apoptotic bial effect appears not to be dependent on lactic acid
effect in IECs [51], it remains to be ruled out that pro- concentration alone [68]. Seven heat-stable peptides
tein contaminants in the purified EPS are not interfering with antibacterial activity against enteroaggregative
with the results. In addition, the role of the other glu- E. coli strain EAEC 042, Salmonella Typhi, and Staphy-
cose-rich type of EPS from LGG remains to be substan- lococcus aureus were identified in LGG culture medium
tiated. A recent network based in silico analysis of the [78]. Unfortunately, these peptides have not yet been
glycosyltranferase genes of LGG could identify the puta- identified, to the best of our knowledge. Of note, the
tive gene cluster involved in their biosynthesis [65], genome sequence of LGG encodes several bacteriocin-
which opens perspectives for functional analyses with related genes [9]. Despite several attempts, we and
mutants. others were not yet able to shown bacteriocin produc-
Apart from EPS, other genes and molecules play a role tion under laboratory conditions and coculture with
as factors that promote the adaptation of LGG to the possible inducing strains, although a bacteriocin locus
human host and gastro-intestinal tract in particular. For was found to be induced in the murine gastro-intestinal
instance, an elegant study combining transcriptomics tract after R-IVET (Sarah Lebeer et al., in preparation).
and proteomics in LGG identified putative adaptation Bacterial cell-cell communication through quorum sen-
factors involved in the bile stress response of LGG. sing (QS) might also interfere with pathogen infection as
Among the identified functions were general stress strains present in the gut microbiota are thought to com-
responses as well as cell envelope-related functions, municate to coordinate adaptive processes such as com-
including pathways affecting fatty acid composition, cell petition and cooperation for nutrients and adhesion sites
surface charge, and thickness of the EPS layer [66]. Our [2]. LGG was reported to produce autoinducer-2 (AI-2),
recent recombinase-based in vivo expression technology which is suggested to be an important interspecies QS
(R-IVET) experiment, similar as in L. plantarum molecules, produced by both Gram-positive and Gram-
WCFS1 [67], also indicated a remarkable metabolic flex- negative bacteria [79]. However, the role of QS in patho-
ibility of LGG in the murine gastro-intestinal tract gen exclusion is difficult to investigate since the AI-2
(Sarah Lebeer et al., in preparation). synthase LuxS also interferes with the cell metabolism.
Indeed, a luxS knock-out mutant of LGG was shown to
Secreted antimicrobials produced by LGG have numerous pleiotropic effects, which could not be
Several in vitro studies have shown the efficacy of LGG complemented by exogenous addition of synthetic AI-2
against the viability, adherence or infection of GIT patho- molecules [10]. It remains to be investigated whether
gens. Indeed, LGG was shown to reduce the viability of AI-2 or other QS systems play a role as a probiotic
Salmonella enterica subsp. enterica serovar Typhimur- mechanism for LGG. For instance, McCormick and col-
ium [68-72], Shigella sonei [73], and Pseudomonas, leagues could nicely show that cyclic dipeptides of strain
Staphylococcus and Streptococcus strains [70] in vitro. In Lactobacillus reuteri RC-14 quench agr-mediated expres-
vivo mice experiments confirmed that LGG pretreatment sion of toxic shock syndrome toxin- 1 in staphylococci
reduces S. Typhimurium infection parameters [74]. How- [80], highlighting that QS could play a role in antipatho-
ever, there is some controversy concerning the results genic mechanisms of probiotics.
obtained with S. Typhimurium, as not all studies could
detect a reduction in viability [74,75] or reduced adher- Unmethylated CpG-rich DNA motifs as
ence to human intestinal mucus [75,76]. Unfortunately, intracellular MAMPs
to our knowledge no human trials have been carried out Other important bacterial MAMPs are derived from
focusing on Salmonella specifically. bacterial DNA and become only available after cell lysis.
There have been a number of studies trying to identify Bacterial DNA can be distinguished from eukaryotic
the antibacterial compounds, mostly focusing on DNA in frequency of unmethylated cytosine-guanine
S. Typhimurium. LGG grown in de Man-Rogosa-Sharpe dinucleotides (CpG) motifs. These CpG motifs are rela-
(MRS), a medium recommended for lactobacilli, was tively widespread in viral and bacterial DNA, but are
shown to inhibit growth of S. Typhimurium SL1344 in a not common in mammalian DNA. CpG motifs and syn-
pH-dependent manner [76]. Interestingly, lactic acid was thetic unmethylated CpG oligonucleotide mimics
identified as the main antimicrobial compound in differ- (ODN) are generally recognized by TLR9 and can
ent conditions [69,71,72], which is clearly not a specific induce a strong T-helper-1 (T H -1) like inflammatory
factor for LGG. As lactic acid permeabilizes the Gram- response [81]. Targeting TLR9 with CpG or ODN has
negative outer membrane, it might facilitate antibacterial been a strategy for a number of clinical trials studying
action of other compounds, such as organic acids or the effect on cancer treatment, allergy and infection dis-
bacteriocins [77]. These compounds might indeed eases, reviewed in [82]. It is important to note that
TLR9 function in the intestinal epithelial layer is proteomics and metabolomics show great potential, espe-
thought to be polarized as IECs respond differently to cially if methods are integrated and combined with net-
apical or basolateral exposure to CpG. As basolateral work biology approaches [90]. For instance, a gene
TLR9 activation signals activation of the NF-B path- expression analysis of the small bowel mucosa from
way, apical TLR9 stimulation seems to prevent NF-B patients treated with LGG compared with placebo treat-
activation. This mechanism is thought to play an impor- ment, showed that LGG affected genes involved in
tant role in epithelial homeostasis [83]. immune response and inflammation, apoptosis, cell-cell
A bioinformatic analysis of the frequency of CpG signaling, cell growth and cell differentiation, cell adhe-
motifs in the genomes of gut commensals demonstrated sion and signaling. It should be noted that these analyses
a correlation with genomic GC content [84]. Indeed, in were done at a rather late time point, i.e. in biopsy sam-
vitro treatment of polarized IEC layers showed that ples of patients consuming LGG during one month
DNA form different probiotic strains have differential (1.2 × 10 10 colony forming units, CFU, daily) [91]. A
effects on NF-B activation [85] and in vivo studies more recent in vivo transcriptome analysis compared the
using a mouse model showed differential effects on mucosal responses towards LGG (1.68 × 1010) with two
immune proliferation activity [86]. The genome of LGG, other commercially available lactobacilli (i.e. L. acidophi-
but also of other lactobacilli such as L. plantarum lus Lafti-L10, L. casei CRL-431) in a placebo-controlled
WCFS1, appears to have a higher frequency of the opti- randomized double-blind cross-over design in which the
mal motif for interaction with TLR9, i.e. GTCGTT, than volunteers consumed all three probiotic preparations and
could be expected by their genomic GC content [84]. a placebo control in a randomized order with each time a
Moreover, a potent ODN, TTTCGTTT named ID35, 2-week wash-out period. Interestingly, even after only
was identified in the LGG genome [86]. The effect of 6 h, the mucosal response to LGG was also mainly char-
chromosomal DNA of LGG was also tested in polarized acterized by the induction of TH1 development via the
IECs, where it was shown to diminish TNF-induced NF- IFN-STAT4 (signal transducer and activator of transcrip-
B activation and reduction of trans- epithelial resistance tion 4) axis and affected pathways include cellular growth
thus protecting the epithelial layer [87]. The chromoso- and proliferation pathways, wound healing, angiogenesis,
mal DNA of LGG and derived ODNs were also shown to interferon mediated responses, calcium signaling and ion
be strong in vivo inducers of murine B cell proliferation homeostasis [92]. These pathways contain signatures of
and were able to stimulate TH1 immunity in murine sple- the previously documented activity of Msp1/p75 and
nocyte cells [86]. ID35 isolated form LGG genome even Msp2/p40 to promote cell proliferation and epithelial
seems to be beneficial in allergy prevention in an ovalbu- integrity [51,53,54], but clearly other factors play a role.
min-sensitized mouse model, by inducing the T H 1- The same method was also used to investigate whether
response and suppressing ovalbumine-specific IgE pro- humans respond differently to different growth stages of
duction [88]. Moreover, a study using peripheral blood L. plantarum WCFS1 [93]. They indeed observed clear
mononuclear cells (PBMCs) from allergic patients differences in the transcriptional response to exponen-
showed that LGG as well as its genomic DNA can modu- tially growing or stationary phase bacteria, and
late the T H 1/T H 2 response to specific allergens dose- between viable and heat-killed stationary bacteria. It
specifically. More than 50% of the effect of LGG could be will be very interesting to use the same analyses to
explained by the effect of the genomic DNA, as stated by investigate the transcriptional responses towards LGG
the authors [89]. wild type and spontaneous non-GMO food-grade
mutants, such as spontaneous pili mutants, to explore
Profiling of host responses against LGG their relative contribution to the human host response.
The above mentioned LGG molecules and their corre- Alternatively, experiments with dedicated isogenic
sponding mutants are studied one-by- one but it should mutants such as of Msp1/p75 [15] could be designed
be highlighted that in situ the host interaction towards for analyses in animal models, since Lin et al. [94]
LGG will be an integrated sum of different interactions. have also nicely shown by transcriptomics that LGG
A combination of all MAMP-PRR interactions decides also upregulates cytoprotective gene expression and
how the immune system is triggered, while also various MAPK-related expression in the developing murine
metabolites such as lactic acid can be envisaged to modu- intestine. In addition, ex vivo and in vitro models such
late host responses. Therefore, molecular profiling of the as the porcine small intestinal epithelial cell line
host responses upon LGG application can reveal impor- (IPEC-J2) appear to be good models for the study of
tant novel insights (Figure 2), especially if time course innate immune responses to probiotics [95].
studies are included. Until now, these host responses Nevertheless, and probably most importantly, the
towards LGG have been mainly characterized by tran- abovementioned duodenal transcriptome studies of
scriptomics methods, but also other approaches such as Kleerebezem and coworkers showed a remarkable large
Figure 2 Molecular interactions of LGG with intestinal epithelial cells. LTA as a MAMP interacts with TLR2-6, activating NF- signaling [43].
Secreted protein Msp2/p40 induces release of HB-EGF that causes phosphorylation of EGF-R, activating downstream protein kinase C (PKC) and
phosphoinositide 3-kinase (PI3K) -Akt signaling [51,53,54]. A recent human duodenal transcriptome study indicates that JUN and STAT4
transcription factors play a central role in downstream signaling after consumption of LGG, leading to mainly TH1 cytokine production and
activating pathways involved in cellular growth and proliferation, wound healing, angiogenesis, interferon-mediated responses, calcium signaling
and ion homeostasis [92]. Adapted from [96]
distance of the transcriptome profiles between the molecular “bandwidth of human health” [96]. Clearly,
human participants. As all participants were healthy, this complicates the selection of the most appropriate
the large inter-person variation indicates that mucosal biomarkers to monitor probiotic intervention in
tissues have multiple mucosal solutions to accomplish human study subjects and the possibility for stratifica-
healthy homeostasis, which is suggested to be a tion of responders and non-responders.
Clinical benefits of LGG irritable bowel syndrome (IBS) patients. However, it is

In the next paragraphs, we tried to summarize some important to mention that the clinical effects were sig-
well documented clinical benefits and explain some of nificant but moderate [107], which is not unexpected if
the findings with the molecular framework we provided you consider the large subject heterogeneity in IBS
above, although this association should be taken with patients.
caution. In other conditions, LGG effects were better stratified.
For instance, oral supplementation with LGG (6 × 109
Promotion of gastro-intestinal health in children and CFU with human milk) has been shown to prevent
adults enteric colonization by Candida species in preterm neo-
As mentioned before, LGG has been shown to colonize nates in a randomized study [108], although the under-
the gut of newborns significantly better than adults [18]. lying mechanisms need to be explored. Two pilot
Interestingly, prenatal supplementation with LGG studies also showed promising results for LGG treat-
(1.8×10 10 CFU in capsules, daily from 36 th week of ment (respectively 1010 CFU in skim milk and 1.2 × 109
gestation) has been reported to change the composition CFU in lyophilized powder daily) of recurrent Clostri-
of the neonates microbiota, promoting a beneficial pro- dium difficile induced colitis in children [109,110], but
file dominated by bifidobacteria [97,98], although the this should be repeated in larger trials. Furthermore,
overall microbial diversity did not seem to have changed application of a commercial yoghurt with LGG to renal
[99]. Others showed that postnatal application of LGG patients during 8 weeks has been shown to succeed in
(109 CFU daily, lyophilized powder mixed with breast clearing vancomycin-resistant enterococci in all patients
milk) appears to affect neonatal intestinal colonization in an double-blind, randomized, placebo-controlled trial
patterns causing a higher species diversity compared to [111]. A larger single-blind, randomized, placebo-con-
placebo [100], although the analysis was not done at trolled trial focused on children (3 × 109 CFU daily, dis-
detailed level. How exactly LGG could promote the solved in water or milk) and could show a significant
colonization of bifidobacteria in vivo remains to be difference between the treated and the control groups
further explored. only after three weeks [112]. The mechanism of clearing
Given its excellent intestinal mucus adherence capaci- of this vancomycin-resistant enterococci remains to be
ties, LGG has also been often selected as candidate pro- explored, but the SpaCBA pili of LGG share some
biotic for the prevention and treatment of gastro- sequence identity (30-40%) with the pili of Enterococcus
intestinal infections and diarrhea, although the efficacy faecalis and Enterococcus faecium [9]. Clearly, experi-
is not uniformly proven. Three subsequent meta-analysis ments with non-GMO pili deficient variants of LGG
studies have discussed the use of LGG for the treatment would be highly interesting to study their role in the
of acute diarrhea in children [101-103]. Overall, the cur- gastro-intestinal and pathogen exclusion effects of LGG.
rent data suggest that LGG can reduce the duration of
diarrhea with 1.05 days, particularly in children from Possible effects at other body niches
geographical Europe, treated with a high dose of LGG Although many intervention studies with LGG are tar-
(≥10 10 CFU/day). LGG (10 9 CFU daily in fermented geting the GIT, it is also interesting to investigate extra-
milk product) was also shown to reduce the risk of intestinal effects of LGG. For instance, LGG has been
acquiring nosocomial gastrointestinal infections when shown to reduce oral counts of Streptococcus mutans, a
administered daily in hospitalized children [104]. Under- bacterium correlated with caries formation, respectively
nourished Peruvian children showed a lower incidence in yogurt, milk and lozenges [113-115]. Especially long-
of diarrhea when treated with LGG (>1010 CFU daily, term consumption of LGG containing milk (5-10 × 105
lyophilized powder mixed with liquid cherry gelatin). CFU) appears to be able to reduce caries development
Effects were the largest in non-breastfed children [105]. in children [114]. Of note, there was no effect of short-
However, long-term consumption of milk containing term consumption of LGG (4×10 8 CFU daily) on the
LGG (108 CFU daily) in children attending day care cen- acidogenicity of plaque nor on caries formation in
ters in Finland could not show an effect on the inci- adults, although it should be noted that LGG was admi-
dence of gastro-intestinal symptoms [106]. It is nistered in a tablet, which might not be the best formu-
important to note that the unsuccessful trial tested a lation [116]. Importantly, LGG appears not to ferment
100-fold lower daily concentration, although other fac- sucrose to a significant level [9], indicating that it is
tors such as probiotic formulation and study subject itself not cariogenic, a property which is sometimes
heterogeneity could of course also play a role. A recent attributed to lactobacilli due to lactic acid production.
meta-analysis concluded that LGG treatment can also Others have investigated the effect of LGG consump-
reduce pain frequency and intensity in children with tion on respiratory health. For instance, Hojsak and col-
abdominal pain-related disorders, particularly among leagues [104] showed that fermented milk containing
LGG was efficient in reducing the risk on respiratory trials also reported that treatment (5×109 CFU daily in
tract infections (RTIs) that lasted longer than three days milk) was efficient in IgE-sensitized infants, but not in
in hospitalized children. Also, preterm infants treated non-IgE-sensitized infants [127,128]. This is probably a
daily with 109 CFU LGG in capsules starting within one good example that patient stratification is important to
week after birth, appear to have significantly lower inci- identify potential responders, but more research is
dence of RTIs and rhinovirus-induced episodes in the necessary to determine the effect of LGG in IgE-sensi-
first 2 months [117]. Furthermore, capsulated LGG (109 tized infants.
CFU) was shown to protect hospitalized patients against Related to food allergy, it was reported that adminis-
ventilator-associated pneumonia, mainly when caused by tration of capsulated LGG (5×109 CFU) in infants with
Gram-negative pathogens like Pseudomonas aeruginosa cow’s milk allergy augments IFN-g production in stimu-
[118]. Moreover, in cystic fibrosis patients colonized lated PBMCs, thus possibly providing beneficial T H 1
with P. aeruginosa, long-term LGG treatment (6×10 9 immunomodulatory signals [128]. Indeed, infants
CFU daily, in oral rehydration solution) significantly acquire more oral tolerance when hydrolyzed casein for-
decreased the incidence of pulmonary exacerbations and mula was administered in combination with LGG (107
increased body weight [119]. Unfortunately, this study CFU/ 100 mL) than with the formula alone [129]. In
did not evaluate P. aeruginosa colonization status after milk-hypersensitive adults, LGG (2.6×108 CFU daily in
LGG treatment. Clearly this area requires further milk) has been shown to reduce the immunoinflamma-
research, because probably a combination of LGG’s anti- tory response by reducing the expression of specific
pathogenic and immune modulating capacities deter- receptors such as the complement receptors CR1 and
mines its potential in RTIs. CR3 [130].
LGG as a vaccine adjuvant
Immunomodulatory applications of LGG Another perhaps more elegant way to investigate the
Allergic diseases immunomodulatory effects of LGG is by studying its
The potential immunomodulatory effects of LGG that capacity to ameliorate humoral responses to vaccines
have yet received most attention include its widely dis- when applied as an adjuvant. One study showed that the
cussed effects against allergic disease. In a study pub- immunogenicity of an oral rotavirus vaccine was signifi-
lished in The Lancet, Kalliomäki and colleagues cantly ameliorated when mixed with 5×10 10 CFU of
[120-122] showed that the combination of prenatal LGG [131]. LGG in milk (1010 CFU daily, 1 week before
maternal (2-4 weeks) and postnatal pediatric (6 months) vaccination, 4 weeks after) was also shown to increase
LGG treatment (1010 CFU daily, capsules or in water) in the poliovirus neutralizing antibody titer with a fourfold
families with a history of atopic disease, significantly increase in poliovirus-specific IgA in adults receiving an
lowered the risk of eczema at the age of 2, 4 and 7. oral vaccine against polio 1, 2 and 3 [132]. Moreover,
However, allergic rhinitis and asthma tended to be more LGG treatment (10 10 CFU in a capsule, daily, 28 days
common in the LGG treated group and no significant starting at vaccination) increased protection rates after
differences were found in incidence of cow milk allergy. an oral life attenuated influenza vaccine. The effect was
Moreover, Kopp and colleagues [123] could not repeat viral strain-dependent as antibody titers against H1N1
the beneficial results against eczema using a similar pro- and B strains were low for placebo and LGG-treated
tocol and concentration. The reason for these different groups. For the H3N2 strain, LGG increased protection
outcomes is unknown, however it is thought that the significantly [133]. However, there was no influence on
different genetic background of the tested populations the effect of an oral S. Typhi Ty21a oral vaccine (4×1010
(Finnish versus German) might play a role. Also, the CFU daily, 7 days) [134]. In addition, a recent study
German trial had more infants with older siblings, even showed that maternal supplementation with LGG
which could be a potential cofounder [120,123]. In addi- (1.8 × 10 10 CFU daily) from 36 weeks gestation until
tion, it seems that different probiotic products have delivery reduces vaccine-specific immune responses for
been used for these studies, so that also differences in tetanus, Haemophilus influenzae type b (Hib) and pneu-
probiotic formulation, and for instance pili presence, mococcal conjugate (PCV7) vaccines in infants at high
cannot be ruled out. risk of developing allergic disease [135], indicating that
Atopic dermatitis in children could not be treated by the timing of administration is important if one desires
LGG in three independent trials, using a daily concen- an adjuvant effect and that LGG might not always be
tration of 5×109 CFU/100 ml formula [124], 5×109 CFU the best choice for these purposes. Moreover, van Baar-
in milk [125] or 1010 CFU in milk [126]. However, in len et al. [92,93] showed other lactobacilli such as L.
these trails there was a consistent but not significant plantarum WCFS1 show a more clear modulating of
effect of LGG in the IgE-sensitized subgroup. Two other the NF-B pathway.
Conclusions similar MAMPS might show different results. Small var-

Is LGG a better probiotic strain than other probiotics on iations in structure (pili, LTA, EPS, etc.), expression
the market? This question is difficult to answer, since level or ratio can also have a large effect on the host
the answer largely depends on the host response that is response. However, it is also apparent that not all
aimed for by the application. As mentioned before, the reported health effects of LGG are univocal. Successful
host response is dependent on the combination of sev- administration appears to depend on the applied dose,
eral bacterial effectors, including MAMPs interacting growth phase, formulation, time of administration, dura-
with PRRs. Even though these effectors might not be tion of treatment, age and genetic background of study
unique for LGG, it seems that the sum of effectors in subjects, among other variables (Figure 3). Nevertheless,
LGG is often beneficial for the host, while strains with one of the clear advantages of LGG is that this probiotic
Figure 3 Pipeline for the design of intervention trials with LGG and related probiotics. In this schema, we have made an overview of
different steps that should ideally been taking when designing novel intervention studies with LGG or related probiotics, taking current
information into account. For more information, the reader is referred to the main text of this manuscript.
is well characterized and so widely used that it has a Competing interests

The authors declare that they have no competing interests.
very good safety track record. LGG has been consumed
in over 40 countries worldwide and is especially popular Acknowledgements
in Finland with a yearly per capita consumption rate of We acknowledge all researchers that help to unravel aspects of the host
interaction of LGG. We especially acknowledge Prof. J. Vanderleyden and Dr.
6L in 2000 [136]. To support the safety of LGG, it was
S. De Keersmaecker (KU Leuven) to initiate the genetic research on LGG
shown that despite increasing LGG consumption in Fin- more than 10 years ago and Prof. W. De Vos (University of Wageningen and
land and Sweden respectively, the rate of Lactobacillus University of Finland) to strongly promote the research on LGG. In addition,
all other previous and current PhD students unravelling LGG’s probiotic
bacteremia remained constant [137,138]. Moreover, the
properties are acknowledged, including Monica Perea Velez, Ingmar Claes,
use of LGG in a wide variety of clinical trials without Mariya Petrova, Prachi Tripathi, Hanne Tytgat, and Cynthia Vargas Garcia. The
serious adverse events confirmed its safety. LGG has current LGG research at the KU Leuven and UAntwerpen is funded by FWO-
Vlaanderen (including KaN 152011N) and by BOF financing KU Leuven (PF
been administered to, among others, low birth weight
financing, spokesman J. Balzarini) and UAntwerpen (DOC PRO and IOF-SBO
infants [139,140], pregnant women [122,123,141], HIV- projects Sarah Lebeer).
infected patients [138] and patients with a mechanical
Declarations
ventilator [118].
This article has been published as part of Microbial Cell Factories Volume 13
Nevertheless, case reports show that probiotic therapy Supplement 1, 2014: Proceedings of the 11th International Symposium on
is to be discouraged in certain groups of patients. In a Lactic Acid Bacteria. The full contents of the supplement are available online
at http://www.microbialcellfactories.com/supplements/13/S1. Publication
review concerning the safety of probiotics, it was con-
charges for this supplement were funded by the Stichting Symposium on
cluded that adverse effects of probiotics were correlated Lactic Acid Bacteria. The articles have undergone the journal’s standard peer
with (i) impaired intestinal barrier function, (ii) immune review process for supplements. EJ is employed by Chr Hansen A/S, is a
shareholder in Chr Hansen A/S; and has applied for patents relating to the
compromised state and (iii) central venous catheter
content of an article which he did not edit but which forms part of this
[142]. Indeed, cases specifically reported for LGG show supplement. MK is an employee of Nizo. The other Supplement Editors
that these risk factors might play a role. For instance, a declare that they have no competing interests.
number of infants treated with LGG for short gut syn-
Authors’ details
drome associated with intestinal friability seemed to 1
University of Antwerp, Department of Bioscience Engineering, Research
manifest sepsis with LGG-like bacteria [143,144] and an Group Environmental Ecology and Applied Microbiology, Groenenborgerlaan
171, B-2020 Antwerp, Belgium. 2KU Leuven, Centre of Microbial and Plant
ulcerative colitis patient was diagnosed with LGG bac-
Genetics, Kasteelpark Arenberg 20, box 2460, B- 3001 Leuven, Belgium.
teremia [145], although these identifications were not
done at a detailed genomic level. Nevertheless, in Published: 29 August 2014
patients with a seriously compromised integrity of the
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Segers and Lebeer Microbial Cell Factories 2014, 13(Suppl 1):S7

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Towards a better understanding of Lactobacillus

Introduction influence on the commensal microbiota [2,4]. A second

Clinical benefits of LGG irritable bowel syndrome (IBS) patients. However, it is

Conclusions similar MAMPS might show different results. Small var-

is well characterized and so widely used that it has a Competing interests

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