Growth of the lactic acid bacterium Streptococcus thermophilus in milk depends on its capacity to hydrolyze proteins of this medium through its surface proteolytic activity. Thus, strains exhibiting the cell envelope proteinase (CEP) PrtS are able to grow in milk at high cellular density. Due to its LPNTG motif, which is possibly the substrate of the sortase A (SrtA), PrtS is anchored to the cell wall in most S. thermophilus strains. Conversely, a soluble extracellular PrtS activity has been reported in the strain 4F44. It corresponds, in fact, to a certain proportion of PrtS that is not anchored to the cell wall but rather is released in the growth medium. The main difference between PrtS of strain 4F44 (PrtS4F44) and other PrtS concerns the absence of a 32-residue imperfect duplication in the prodomain of the CEP, postulated as being required for the maturation and correct subsequent anchoring of PrtS. In fact, both mature (without the prodomain at the N-terminal extremity) and immature (with the prodomain) forms are found in the soluble PrtS4F44 form along with an intact LPNTG at their C-terminal extremity. Investigations we present in this work show that (i) the imperfect duplication is not implied in PrtS maturation; (ii) the maturase PrtM is irrelevant in PrtS maturation which is probably automaturated; and (iii) SrtA allows for the PrtS anchoring in S. thermophilus but the SrtA of strain 4F44 (SrtA4F44) displays an altered activity.

Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic
acid bacterium widely used in dairy industry, requires further documentation of its physiological
status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo
technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under
simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library
were designed to positively select activated genes. Second, recombinant clones were introduced
into complementary models mimicking the human gut, the Netherlands Organization for Applied
Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine,
the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human
feces as a colon model. All inserts of activated clones displayed a promoter activity that differed
from one digestive condition to another. Our results also showed that S. thermophilus adapted its
metabolism to stressful conditions found in the gastric and colonic competitive environment and
modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated
in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses,
a first stage in the identification of gut resistance markers and a key step in probiotic selection.

The mucus, mainly composed of the glycoproteins mucins, is a rheological substance that covers the intestinal epithelium and acts as a protective barrier against a variety of harmful molecules, microbial infection and varying lumen environment conditions. Alterations in the composition or structure of the mucus could lead to various diseases such as inflammatory bowel disease or colorectal cancer. Recent studies revealed that an exogenous intake of probiotic bacteria or other dietary components (such as bioactive peptides and probiotics) derived from food influence mucus layer properties as well as modulate gene expression and secretion of mucins. Therefore, the use of such components for designing new functional ingredients and then foods, could constitute a novel approach to preserve the properties of mucus. After presenting some aspects of the mucus and mucins in the gastrointestinal tract as well as mucus role in the gut health, this review will address role of dietary ingredients in improving mucus/mucin production and provides new suggestions for further investigations of how dietary ingredients/probiotics based functional foods can be developed to maintain or improve the gut health.

The acquisition of prtS by Streptococcus thermophilus strains allowed hydrolysis
of caseins into peptides and then to increase their growth in milk. This leads to faster
milk acidification, which is important in dairy industry. However, some strains harboring
the same allele of prtS present different acidification rates, which could be explained by a
difference in the regulation of prtS expression.We chose two strains with the same allele
of prtS (including the same promoter region): one, PB302, is with high acidification rate
while the other, PB18O, is without. They exhibited similar growth in M17, but not in
milk, where PB302 showed better growth. The expression of prtS and activity of PrtS
were lower in PB18O, in the two media tested.We demonstrated that other genes known
to be involved in carbon and nitrogen metabolism were overexpressed in PB302.
Interestingly, these genes were overexpressed in milk compared to M17. Nearly all these
genes possessed a putative CodY-box in their promoter region. Taken together, difference
of gene expression detected in PB302 between milk (low-peptide medium) and
M17 (rich-peptide medium) and presence of a putative CodY-box is a feature of the
transcriptional pattern of CodY-regulated genes. Altogether, our results propose that
acquisition of prtS is not enough in certain strains to achieve rapid milk acidification.
High transcriptional level of dtpT, amiF, ilvC, ilvB, bcaT, livJ, ackA, codY, and prtS in fast
acidifying strain suggests that this transcriptional pattern could be required for fast milk
acidification in Streptococcus thermophilus.

In silico analysis of the genome of Streptococcus thermophilus LMD-9 revealed that this strain has a potential new peptide/nickel ABC transporter. We named this system OTS for Oligopeptide Transporter of S. thermophilus. It is composed of a peptide/nickel binding protein OtsA, two permeases OtsB and OtsC and a double ATPase OtsD. This system was presumably acquired by horizontal transfer from Actinobacteria or distant species like Lactococcus raffinolactis or Enterococcus asini may be via an intermediate like Lactococcus lactis or its ancestor. RT-PCR experiments proved that OTS gene cluster is transcribed and that at least the otsB, otsC, and otsD genes constitute an operon. A mutant LMD-9?ots, partially deleted for the otsA and otsB genes was constructed. Growth of LMD-9 and LMD-9?ots strains was monitored in the presence of different nitrogen sources and in the presence of urea and nickel. Results revealed that OTS is not implicated in nickel transport, but constitutes a new characterized transporter of peptides of small size, possibly di- and tripeptides in S. thermophilus. 

Streptococcus thermophilus (ST) is a lactic acid
bacterium widely used in dairy industry and displays several
properties which could be beneficial for host. The objective of
this study was to investigate, in vitro, the implication of
sortase A (SrtA) and sortase-dependent proteins (SDPs) in
the adhesion of ST LMD-9 strain to intestinal epithelial cells
(IECs) and resistance to bile salt mixture (BSM;
taurocholoate, deoxycholate, and cholate). The effect of mutations
in prtS (protease), mucBP (MUCin-Binding Protein),
and srtA genes in ST LMD-9 in these mechanisms were examined.
The HT29-MTX, HT29-CL.16E, and Caco-2 TC7
cell lines were used. HT29-MTX and HT29-CL.16E cells
express different mucins found in the gastro intestinal tract;
whereas, Caco-2 TC7 express cell surface proteins found in
the small intestine. All mutants showed different adhesion
profiles depending on cell lines. The mutation in genes srtA
and mucBP leads to a significant decrease in LMD-9 adhesion
capacity to Caco-2 TC7 cells. A mutation in mucBP gene has
also shown a significant decrease inLMD-9 adhesion capacity
to HT29-CL.16E cells. However, no difference was observed
using HT29-MTX cells. Furthermore, ST LMD-9 and srtA
mutant were resistant to BSM up to 3 mM. Contrariwise, no
viable bacteria were detected for prtS and mucBP mutants at
this concentration. Two conclusions could be drawn. First,
SDPs could be involved in the LMD-9 adhesion depending
on the cell lines indicating the importance of eukaryotic-cell
surface components in adherence. Second, SDPs could contribute
to resistance to bile salts probably by maintaining the
cell membrane integrity.

Streptococcus thermophilus is the second most used bacterium in dairy industry. It is daily consumed by millions of people through the worldwide consumption of yogurts, cheeses and fermented milks. S. thermophilus presents many features that make it a good candidate for the production of heterologous proteins. First, its ability to be naturally transformable allows obtaining swiftly and easily recombinant strains using various genetic tools available. Second, its Generally Recognised As Safe status and its ability to produce beneficial molecules or to liberate bioactive peptides from milk proteins open up the way for the development of new functional foods to maintain health and well-being of consumers. Finally, its ability to survive the intestinal passage and to be metabolically active in gastrointestinal tract allows considering S. thermophilus as a potential tool for delivering various biological molecules to the gastrointestinal tract. The aim of this review is therefore to take stock of various genetic tools which can be employed in S. thermophilus to produce heterologous proteins and to highlight the advantages and future trends of use of this bacterium as a heterologous expression host.

AIMS:

To construct and validate the recombinase-based in vivo expression technology (R-IVET) tool in Streptococcus thermophilus (ST).

METHODS AND RESULTS:

The R-IVET system we constructed in the LMD-9 strain includes the plasmid pULNcreB allowing transcriptional fusion with the gene of the site-specific recombinase Cre and the chromosomal cassette containing a spectinomycin resistance gene flanked by two loxP sites. When tested in M17 medium, promoters of the genes encoding the protease PrtS, the heat-shock protein Hsp16 and of the lactose operon triggered deletion of the cassette, indicating promoter activity in these conditions. The lactose operon promoter was also found to be activated during the transit in the murine gastrointestinal tract.

CONCLUSIONS:

The R-IVET system developed in ST is relatively stable, functional, very sensitive and can be used to assay activity of promoters, which are specifically active in in vivo conditions.

SIGNIFICANCE AND IMPACT OF THE STUDY:

This first adaptation of R-IVET to ST provides a highly valuable tool allowing an exploration of the physiological state of ST in the GIT of mammals, fermentation processes or dairy products.

Milk proteins contain numerous potential bioactive peptides, which may be released by digestive proteases or by the proteolytic system of lactic acid bacteria during food processing. The capacity of Streptococcus thermophilus to generate peptides, especially bioactive peptides, from bovine caseins was investigated. Strains expressing various levels of the Cell Envelope Proteinase, PrtS, were incubated either with αs1-, αs2- or β-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the β-casein was preferentially hydrolyzed first, followed by αs2-casein and then αs1-casein. Numbers and types of peptides released were strain-dependent. Hydrolysis appeared to be linked with the accessibility of different casein regions by protease. Analysis of bonds hydrolyzed in the region 1-23 of αs1-casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from β-casein, 5 from αs2-casein and 2 from αs1-casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors.

Lactobacillus helveticus is a lactic acid bacterium very used in fermented milks and cheese. The rapid growth of L. helveticus in milk is supported by an efficient cell envelope proteinase (CEP) activity, due to subtilisin-like serine proteases. These enzymes play also crucial roles in texture and flavor formation in dairy products as well as in generating in situ bioactive peptides. In L. helveticus, several genes encoding putative CEPs were detected and characterized by a large intraspecific diversity; little is known about regulation of expression of CEP-encoding genes. Anchored at the bacterial surface, CEPs are large-sized enzymes (>150kDa) hydrolyzing β- and α(s1)-casein as well. Substrate cleavages occur after almost all types of amino acids residues, but mass spectrometry analysis revealed L. helveticus strains with specific profiles of substrate hydrolysis, which could explain identification of strains associated with interesting technological properties. In this review, the most recent data regarding CEP-encoding genes, CEP activities toward caseins and L. helveticus strain diversity are discussed.

Proteolytic and acidifying properties of Streptococcus thermophilus strains isolated from yoghurt or cheeses were evaluated. Among 30 strains tested, 12 exhibited cell envelope-associated proteinase activity (PrtS+), three displayed a slight PrtS activity (PrtS+/−) and 15 were PrtS−, despite the presence of the corresponding gene (prtS) in eight of them. Sequencing of the prtS gene in four PrtS− and one PrtS+ strains revealed that the absence of PrtS activity in the PrtS− strain probably results from an alteration of the prtS regulation. The strains displaying the highest acidifying capacities were all PrtS+. All but one PrtS+ strains were phylogenetically close, as shown by the sequencing of their rDNA internal transcribed spacer (ITS) 16S-23S. More specifically, the high proteolytic and acidifying capacities are associated with the presence of a type II-ITS.

There is growing evidence that supplementation with probiotics improves intestinal transit,
induces systemic protective immune responses and presents beneficial effect on stress
and anxiety. Concomitantly, exposure to Polycyclic Aromatic Hydrocarbons (PAH),
especially in juvenile, proves to induce cognitive developmental delay and behavioral
impairments related to anxiety.
This study provides a proof of concept on the use of probiotic beneficial effect to
counteract the neurotoxic effects induced by PAH. It was carried out by using six groups
of 12 Swiss female mice each . Three groups were daily fed with a mixture of probiotics
for 8 weeks whereas the others received the vehicule only. After 1 month of probiotic
supplementation, the 3 groups of each conditions were exposed by oral gavage to a
mixture of 16-PAHs (3 times per week, 0, 20 and 200 µg/kg, 4 weeks). Neurobehavioural
status related to exploration, anxiety and immediate learning were studied during the last
week of PAH exposure. Faeces were collected, -before, -after 4 weeks of probiotic
supplementation and -at the end of PAH exposure to assess the microbiota balance and
the probiotic viability along the gastro-intestinal tract. Preliminary data showed that
probiotics enable a faster growth of mice compared to controls (p<0.05) and reduce the
loss of weight observed in PAH-treated groups. Enumeration of probiotic strains at several
time point, pointed out that the probiotics survive along the gastro-intestinal tract, but PAH
seems to affect their viability as well as this of microbiota at 200 µg/kg of bw. Analysis of
microbiota by 16S ribosomal RNA gene sequencing should confirm this result. In the lightdark
apparatus, supplementation with probiotics partially restore the decrease of the level
of activity observed in mice exposed to PAH 200 µg/kg (p<0.05). Behavioral analyses
currently under evaluation should enable us to understand how PAH-induced
neurotoxicity and if probiotics may prevent their detrimental effects.

Introduction and objectives:

There is growing evidence that supplementation with probiotics improves intestinal transit, induces systemic protective immune responses and presents beneficial effect on stress and anxiety. Concomitantly, exposure to Polycyclic Aromatic Hydrocarbons (PAH), especially in juvenile, proves to induce cognitive developmental delay and behavioral impairments related to anxiety. This study provides a proof of concept on the use of probiotic beneficial effect to counteract the neurotoxic effects induced by PAH.

Material and methods:

We studied six groups of 12 Swiss female mice each. Three groups were daily fed with a mixture of probiotics for 8 weeks whereas the others received the vehicule only. After 1 month of probiotic supplementation, the 3 groups of each conditions were exposed by oral gavage to a mixture of 16-PAHs (3 times per week, 0, 20 and 200 µg/kg, 4 weeks). Neurobehavioural status related to exploration, anxiety and immediate learning were studied during the last week of PAH exposure. Faeces were collected, -before, -after 4 weeks of probiotic supplementation and -at the end of PAH exposure to assess the microbiota balance and the probiotic viability along the gastro-intestinal tract.

Results, discussion, conclusion:

Preliminary data showed that probiotics enable a faster growth of mice compared to controls (p<0.05) and reduce the loss of weight observed in PAH-treated groups. Enumeration of probiotic strains at several time point, pointed out that the probiotics survive along the gastro-intestinal tract, but PAH seems to affect their viability as well as this of microbiota at 200 µg/kg of bw. Analysis of microbiota by 16S ribosomal RNA gene sequencing should confirm this result. In the light-dark apparatus, supplementation with probiotics partially restore the decrease of the level of activity observed in mice exposed to PAH 200 µg/kg (p<0.05). Behavioral analyses currently under evaluation should enable us to understand how PAH-induced neurotoxicity and if probiotics may prevent their detrimental effects.

Streptococcus thermophilus is widely used in the dairy industry to make yogurts and cheeses. In order to define its probiotic potential, we have (i) determined the ability of S. thermophilus LMD-9 to adhere to cell lines (HT29-MTX, HT29-CL.16E and Caco-2) expressing different mucins, (ii) identified genes specifically induced during adhesion to Caco-2 cells using the R-IVET approach (Recombinase-based In Vivo Expression Technology).

We have shown that the adhesion capacity of LMD-9 varies according to the cell line used. It adhered more to the HT29-CL.16E cells, which mainly secrete intestinal mucin MUC2, suggesting that it could colonize the intestines in vivo and have beneficial effects. The role in adhesion of ctsR, srtA, prtS and mucBP genes, two of which encode parietal surface proteins (prtS, mucBP), was also studied. The adhesion pattern of the mutants was different according to the cell lines, showing that the involvement of the proteins CtsR, SrtA, PrtS and MucBP in the adhesion would depend on the properties of the eukaryotic cells (mucus, mucin type ...).

Transcriptomic analysis revealed that the adhesion process did not depend solely on surface proteins, but other metabolic pathways would play an important role. We identified 31 genes specifically induced during adhesion: 4 encoding surface proteins, 8 nutrient transporters, 3 stress response proteins, 2 competency proteins, 1 regulator, 5 hypothetical proteins and 9 others.

The adhesion process is therefore a complex process that depends on both bacterial and eukaryotic surface properties.

The growth in milk of S. thermophilus and Lb. helveticus depends on their proteolytic system, including CEP (Cell Envelope Proteinase) that hydrolyze caseins in peptides. Lb. helveticus and S. thermophilus harbour respectively 0 to 4 CEP (PrtH to PrtH4) and 0 to 1 CEP (PrtS). Better understand their activity and substrate specificity independently will permit to control proteolysis in fermented products according to the strain used. Construction of mutants of Lb. helveticus is not easy, so we chose to express heterologously the prtH gene in a lactic acid bacterium naturally transformable, proteolytic, and growing under the same conditions: S. thermophilus LMD-9. Three mutants were obtained by replacing prtS in LMD-9: i) PrtS- (prtS replaced by ery), ii) PrtH+ (expressing PrtH with its own anchor domain (S-layer) and iii) PrtH+WANS (expressing PrtH with PrtS anchor (LPXTG motif)). In milk, the introduced genes have a similar level of expression to that of prtS (RT-qPCR). A shaving of the cell surface of S. thermophilus and HPLC-MSMS analyses have permit to detect 18 extracellular proteins, for example HtrA and PrtS in LMD-9 and PrtH in PrtH+ and PrtH+WANS mutants. The presence of immature and mature forms of anchored PrtS suggests an autoprocessing maturation while only the immature form of PrtH was detected, suggesting the need for a maturase, as it is the case for other proteases. These results show that S. thermophilus is a good tool to express and anchor to the wall heterologous proteins of interest. Our prospects concern the characterization of the activity and specificity of the different CEP of Lb. helveticus.

Streptococcus thermophilus and Lactobacillus helveticus are lactic acid bacteria (LAB) used in fermented dairy products as co-starters. Their growth depends on the supply of amino acids by a proteolytic system, which contains cell envelope proteinases (CEP) hydrolyzing milk proteins in peptides, peptide transporters and intracellular peptidases hydrolyzing peptides into amino acids. Whereas most LAB possess one CEP, L. helveticus strains can display one to four CEP genes. In L. helveticus CNRZ32, the four PrtH, PrtH2, PrtH3 and PrtH4 are potentially present on the cell?wall and participate to the generation of techno- and bio-functional peptides. To date, activity and specificity of each CEP is still not established although it is an industrial challenge for strain selections. As biochemical and genetical strategies have been ineffective to study each CEP, we decided to express CEP on another LAB surface. Our strategy was to express PrtH on the cell?wall of S. thermophilus LMD­9. This strain was chosen because it grows under the same culture conditions as L. helveticus, expresses a unique CEP, PrtS, and is naturally transformable. Three mutants were obtained by allelic replacement of prtS: i) a negative control PrtS^- with prtS deleted, ii) a PrtH⁺ mutant expressing PrtH, using the lactobacilli S-Layer motif as anchoring domain and iii) a PrtH⁺WANS mutant expressing PrtH fused with PrtS cell-wall spacer and anchor domains. All genes were expressed as shown by RT-qPCR. However, only PrtH⁺WANS proteinase was detected by shaving of the cell-wall proteins and peptide identification by chromatography coupled on line with ESI orbitrap tandem mass spectrometry. These first results show that S. thermophilus is a good tool to secrete and anchor heterologous proteins on the cell wall. Next step is to express the three others L. helveticus proteinases and to determine their catalytic activity on milk proteins.