(en)The present invention relates to a recombinant vector for expressing and secreting, by a propionibacterium , at least one eukaryotic peptide or protein of interest, comprising at least:
under the control of at least one suitable promoter, at least one nucleic acid sequence encoding a propionibacterial signal peptide and, at least one nucleic acid sequence encoding said eukaryotic peptide or protein of interest;

wherein said at least one nucleic acid sequence encoding a propionibacterial signal peptide is translationally fused to said at least one nucleic acid sequence encoding said eukaryotic peptide or protein of interest.


The invention further relates to the uses of such a vector in the pharmaceutical field or for the large-scale production of peptides or proteins of interest.

1.ApplicationNumber: US-201213679344-A
1.PublishNumber: US-2014154213-A1
2.Date Publish: 20140605
3.Inventor: JAN GWENAEL
DIMANCHE-BOITREL MARIE-THÉRÉSE
FALENTIN HÉLÉNE
HALOUZE CHARLES

4.Inventor Harmonized: JAN GWENAEL(FR)
DIMANCHE-BOITREL MARIE-THÉRÉSE(FR)
FALENTIN HÉLÉNE(FR)
HALOUZE CHARLES(FR)

5.Country: US
6.Claims:
(en)The present invention relates to a recombinant vector for expressing and secreting, by a propionibacterium , at least one eukaryotic peptide or protein of interest, comprising at least:
under the control of at least one suitable promoter, at least one nucleic acid sequence encoding a propionibacterial signal peptide and, at least one nucleic acid sequence encoding said eukaryotic peptide or protein of interest;

wherein said at least one nucleic acid sequence encoding a propionibacterial signal peptide is translationally fused to said at least one nucleic acid sequence encoding said eukaryotic peptide or protein of interest.


The invention further relates to the uses of such a vector in the pharmaceutical field or for the large-scale production of peptides or proteins of interest.

7.Description:
(en)The present invention relates to the field of genetic engineering applicable in particular in the pharmaceuticals, chemicals, agri-foods and cosmetics industries, etc.
More precisely, the present invention relates to a recombinant vector for expressing and secreting, by a propionibacterium , one or more amino acid sequences of interest, wherein said vector comprises at least:
under the control of at least one suitable promoter, at least one nucleotide sequence coding for a propionibacteria signal peptide and, in translational fusion with said nucleotide sequence, one or more nucleotide sequences coding for said amino acid sequence or sequences of interest.

In other words, the invention relates to a recombinant vector, particularly for expressing and secreting, by a propionibacterium , at least one eukaryotic peptide or protein of interest, comprising at least:
under the control of at least one suitable promoter, at least one nucleic acid sequence encoding a propionibacterial signal peptide and, at least one nucleic acid sequence encoding said eukaryotic peptide or protein of interest;

wherein said at least one nucleic acid sequence encoding a propionibacterial signal peptide is translationally fused to said at least one nucleic acid sequence encoding said peptide or protein of interest.


The invention also relates to the uses of such a vector in the pharmaceutical field or for the large-scale production of peptides or proteins whose activity is of interest in industries as diverse as pharmaceuticals, chemicals, agri-foods, cosmetics, etc.
There are today a large number of methods and means for the large-scale production of peptides and/or proteins. With traditional chemical synthesis, which is hardly suitable for large-scale production of proteins comprising several dozen amino acids, the generally preferred method is synthesis in vivo, that is, in living biological systems. Many commercial enterprises in France and elsewhere have made a principal business of said production, and thus propose technologies based on genetic engineering and biotechnology to provide industrial quantities of peptides and/or proteins, preferably active and purified. Depending on needs, it is now possible to have recourse to various living systems such as bacteria (e.g., Escherichia coli ), yeasts (e.g., Saccharomyces cerevisiae, Pichia pastoris ), insect cells (baculovirus, Sf9, Sf21, etc.), plant cells and mammalian cells (e.g., CHO, HEK, COS, etc.).
Nevertheless, there still does not exist, at present, a “perfect” living system, that is to say, a system that has universal application (in particular to express any protein and to produce any quantity) and is at the same time simple to make use of, powerful, reliable and affordable.
The present invention aims precisely at mitigating these shortcomings while proposing for the first time the use of propionibacteria as living systems for producing and secreting recombinant peptides and proteins, especially from eukaryotic origin.
Propionibacteria (PB) and, more particularly, dairy propionibacteria (DPB) (notably Propionibacterium freudenreichii ) have a particular metabolism which rests on the anaerobic conversion of sugars or lactic acid into short-chain fatty acids (SCFA), such as propionate, acetate and valerate SCFA. These bacteria are used principally as ripening starter for cooked, pressed cheeses. Until now, few scientific teams or research and development laboratories have been interested in these bacteria. Several probiotic applications are known for said bacteria (for example, the Propiofidus® formulation marketed by Laboratoires Standa, France). They are indeed able to modulate the complex ecosystem of the colon in terms of microbial flora (Bougle et al., 1999) and enzymatic activities (Zarate et al., 2000).
The Inventors have recently cloned and sequenced the genome of the probiotic anaerobic firmicute bacterium Propionibacterium freudenreichii (Falentin et al., 2010, Plos One; Genbank accession No. FN806773). This bacterium has in particular cytotoxic properties with respect to colon cancer cells (Jan et al., 2002; Lan et al., 2007). Among other notable properties, it adheres to colonic epithelial cells and has no toxic effect on healthy cells (Lan et al., 2008).
Although they grow rather slowly, propionibacteria have the considerable advantage of being naturally able to secrete peptides and proteins into the extracellular medium, thus facilitating the recovery of said peptides and proteins without denaturation and without deterioration of the producing cells which can thus be advantageously recycled. These bacteria are very robust and adapt to particular media (such as media containing milk or milk derivatives, e.g., whey, and media containing molasses) that are possibly hostile to the growth and development of other living systems (presence in the medium of lactic acid, salt, etc.), and have good tolerance with respect to variations, changes or disturbances of the environmental conditions likely to occur during large-scale culture operations. Moreover, they can be described as “natural antifungals” because they naturally produce metabolites (for example, propionate) that inhibit the development of contaminating fungi. Furthermore, they are able to produce recombinant proteins of significant size (for example, proteins of more than 500 amino acids) and can even produce several different proteins simultaneously (for example, more than a dozen different proteins).
Propionibacteria are thus completely suitable to serve as living “factories” for the large-scale production of recombinant peptides and proteins of interest, in particular in the context of in vitro or ex vivo processes or applications.
But their utility does not stop there. Indeed, in mammals, including humans, propionibacteria are naturally able to target the intestine where their survival time can reach roughly two weeks, compared to that of lactic bacteria in particular which, although they are natural hosts of this ecosystem, is only two or three days on average. Thus, propionibacteria can also be used as tool for specific addressing or targeting in vivo for colon delivery of peptides and/or proteins of interest, in particular of therapeutic interest.
Some major advantages of propionibacteria over various other bacteria proposed so far for use in anti-tumoral therapy (e.g., in WO 01/25397 in the name of Vion Pharmaceuticals, Inc. and in WO 2009/111177 in the name of Mount Sinai School of Medicine of New York University) are that propionibacteria per se are an efficient and specific anti-tumoral agent that can safely be used in mammals, in particular in humans. This inherent property of propionibacteria can thus be further enhanced upon using propionibacteria to deliver therapeutic agents, such as drugs, to eradicate tumor cells while at the same time preventing damage to normal cells. To do so, propionibacteria do not need to be genetically attenuated or enhanced by genomic mutations as it is the case for other bacteria such as Clostridium, Salmonella, Listeria , and the like. propionibacteria eventually are a “super” anti-tumoral agent thanks to both their intrinsic anti-tumoral properties and their ability to efficiently, specifically and safely deliver other anti-tumoral drugs in order to kill cancer cells.
Thus, the present invention relates to a recombinant vector for expressing and secreting, by propionibacteria, one or more eukaryotic amino acid sequences of interest, comprising at least:
under the control of at least one suitable promoter, at least one nucleotide sequence coding for a propionibacteria signal peptide and, in translational fusion with said nucleotide sequence, one or more nucleotide sequences coding for said eukaryotic amino acid sequence or sequences of interest.

In other words, the invention relates to a recombinant vector, particularly for expressing and secreting, by a propionibacterium , at least one eukaryotic peptide or protein of interest, comprising at least:
under the control of at least one suitable promoter, at least one nucleic acid sequence encoding a propionibacterial signal peptide and, at least one nucleic acid sequence encoding said eukaryotic peptide or protein of interest;

wherein said at least one nucleic acid sequence encoding a propionibacterial signal peptide is translationally fused to said at least one nucleic acid sequence encoding said peptide or protein of interest.


Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one skilled in the relevant art.
For convenience, the meaning of certain terms and phrases employed in the specification, examples and claims are provided.
A vector can be a cloning or an expression vector. The vectors according to the invention can be viral vectors such as bacteriophages, or non-viral, such as plasmid vectors. A plasmid vector is a non-viral DNA molecule hosted by a cell, distinct from the natural chromosomal DNA of said host cell and capable of autonomous replication. The choice of plasmid vector and, more particularly, the origin of replication it carries thus depend on the host cell. According to the type of host cell, several copies of a plasmid vector and/or several different plasmid vectors can be hosted simultaneously. A plasmid vector according to the invention can possibly be carried by (or “integrated in” or “inserted in”) the chromosome of the host cell.
A “recombinant vector” is a vector obtained by traditional molecular biology and genetic engineering techniques, in which one or more exogenous nucleotide sequences have been inserted (or cloned).
The recombinant vector according to the present invention, for expressing and secreting, by a propionibacterium , at least one eukaryotic peptide or protein of interest allows the expression and secretion of said peptide or protein by a propionibacterium . Indeed, it carries a nucleotide sequence which is transcribed and then translated in the host cell, to produce the peptide or protein of interest. It is thus an “expression” or “production” or “synthesis” vector for said peptide or protein of interest.
The peptide or protein of interest expressed via said recombinant vector according to the invention is transported towards the outside of the host cell. The terms “secretion,” “transport towards the outside of the host cell,” “export” and “externalization” are equivalent herein and mean that the peptide or protein of interest is expressed and then is exposed to the extracellular medium. Subject to this exposure, it can possibly remain anchored to the membrane of the host cell. However, preferably, the peptide or protein of interest expressed by the host cell is “released” (or “delivered” or “sorted out”) into the extracellular medium.
The “extracellular medium” is the medium surrounding the host cell, in particular a propionibacterium . It is understood herein that the expressions “extracellular medium,” “exterior medium,” “external medium,” “surrounding medium” and “environment” are synonymous. In some embodiments of the present invention, the propionibacterium is grown in vitro or ex vivo (use of the propionibacterium as a “factory” to produce the peptide and/or protein of interest). In this case, the extracellular medium is the culture medium. It can also be the supernatant of the culture after separation of the biomass (for example, after cell pellets are centrifuged and separated). In other embodiments, the propionibacterium hosting the recombinant vector is administered to a mammal, in particular a human, for in situ expression and delivery of the peptide and/or protein of interest (use of the propionibacterium hosting said vector as a specific targeting tool for colon delivery of said peptide and/or protein of interest). In this case, the extracellular medium is the ecosystem of the mammal, more particularly its intestine, and even more particularly its colon.
In the context of the present invention, an “amino acid sequence” is a sequence of a peptide or a protein, as well the one of fragments, analogs, derivatives and combinations of peptides and proteins.
A protein or peptide “fragment” is a smaller peptide or protein, having an amino acid sequence included in the one of the initial peptide or protein. A protein “fragment” could be a peptide, for example.
“Analog” refers to any modified version of an initial compound, in this case a protein or a peptide, wherein said modified version is natural or synthetic, and wherein one or more atoms, such as carbon, hydrogen or oxygen atoms, or heteroatoms such as nitrogen, sulfur or halogen, have been added or removed from the structure of the initial compound, so as to obtain a new molecular compound.
A “derivative” in the context of the invention is any compound that has a resemblance or a structural motif in common with a reference compound (in this case a protein or a peptide). This definition further includes, on the one hand, compounds that, alone or with other compounds, can be precursors or intermediate products in the synthesis of a reference compound, via one or more chemical reactions, and, on the other hand, compounds that can be formed from said reference compound, alone or with other compounds, via one or more chemical reactions. Thus, the term “derivative” covers at least protein and/or peptide hydrolysates, in particular tryptic hydrolysates, hydrolysate fractions and mixtures of hydrolysates and/or hydrolysate fractions. This definition also covers peptidomimetics or pseudopeptides, which are small molecules that mimic the bioactive properties of a reference peptide (Patch et al., 2002).
Moreover, the terms “analog” and “derivative” of a peptide or protein cover, for example, a peptide or a protein that is glycosylated or phosphorylated or has undergone any grafting of a chemical group.
The terms “nucleotide sequence” or a “nucleic acid sequence” according to the invention refer to the sequence of a nucleic acid molecule, DNA and RNA, wherein the former can be genomic DNA, plasmid DNA, recombinant DNA or complementary DNA (cDNA), for example, and the latter can be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA). Preferably, the nucleotide or nucleic acid sequences of the invention are sequences of a DNA molecule.
For the sake of convenience, when the invention refers to “a” vector, “a” protein or “a” nucleotide or amino acid sequence, etc., it is understood that “a” or “the” also covers the use of several vectors, proteins, sequences, etc.
The recombinant vector for expressing and secreting, by a propionibacterium , at least one eukaryotic peptide or protein of interest according to the present invention comprises at least one suitable promoter for the expression of said peptide and/or protein of interest in a propionibacterium . Particularly, said suitable promoter is a promoter for bacterial RNA polymerase, in particular for propionibacterial RNA polymerase. Said suitable promoter can be a constitutive and/or inducible promoter well known by one skilled in the art. Preferably, said promoter is inducible. The promoter can be developmentally regulated, inducible or tissue specific, preferably inducible in the digestive tract. Preferably, said promoter is a promoter of propionic origin, in particular of P. freudenreichii origin, such as the promoter of protein PF963 of P. freudenreichii , used by the Inventors in the examples below. More preferably, said promoter is a strong propionic promoter. As noted by the Inventors, it could be advantageous to use a promoter ensuring “basic” constitutive expression which could be strengthened by induced expression under particular conditions (for example, conditions of digestive stress). In one embodiment, said suitable promoter is the promoter of a gene encoding a surface layer protein of a Propionibacterium freudenreichii.
Unexpectedly, the Inventors have shown that said promoters of genes encoding a surface layer protein of a Propionibacterium freudenreichii in combination with signal peptides of genes encoding a surface layer protein of a Propionibacterium freudenreichii allow the secretion into the extracellular medium of a peptide and/or a protein of interest by a propionibacterium at a level up to 100 times greater than that obtained with other promoters and peptide signals.
These surface layer proteins have notably been described in the article of Lortal et al. (1993).
In particular, said suitable promoter is the promoter of a gene encoding a surface layer protein of a Propionibacterium freudenreichii , said surface layer protein being selected from the group consisting of the surface layer protein A, the surface layer protein B, the surface layer protein C, the surface layer protein D, the surface layer protein E and the surface layer protein F, in particular of the surface layer protein A.
In particular, said suitable promoter is the promoter of the gene encoding the surface layer protein A of P. freudenreichii CIRM BIA 118 strain.
The sequences of suitable promoters according to the present invention can also be the sequences having at least 80%, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with the sequence of the promoter of a gene encoding a surface layer protein of a Propionibacterium freudenreichii over the entire length of said sequence and corresponding to a promoter.
Advantageously, the vector according to the invention will contain at least two suitable promoters such as defined above which, even more advantageously, will be situated preferably in a series (but not necessarily coupled to each other), in order to increase the expression level of the peptide and/or protein of interest (“expression booster” effect).
The recombinant vector according to the present invention comprises at least one nucleotide sequence encoding a propionibacterial signal peptide for the secretion, in particular into the extracellular medium, of a peptide and/or protein of interest. In other words, the recombinant vector of the invention comprises at least one nucleic acid sequence encoding a propionibacterial signal peptide, said sequence being translationally fused to at least one nucleic acid sequence encoding a peptide or protein of interest, allowing the secretion, in particular into the extracellular medium, of said peptide or protein, particularly by a propionibacterium . In particular, said propionibacterial signal peptide is a propionibacterial secretion signal peptide, allowing the secretion, in particular into the extracellular medium, of said peptide or protein of interest, particularly by a propionibacterium . Preferably, a signal peptide of a propionibacterium selected from dairy propionibacterium and so-called “cutaneous” propionibacterium (CPB) can be used. Among other examples, the following dairy propionibacterium can be cited: Propionibacterium freudenreichii, P. jensenii, P. thoenii P. acidipropionici and P. microaerophilum . The following cutaneous propionibacterium can also be cited: P. acnes, P. granulosum, P. avidum and P. propionicum . Even more preferably, a signal peptide of a propionibacterium selected from the following species can be used: Propionibacterium freudenreichii , more particularly the subspecies P. freudenreichii subsp. freudenreichii and P. freudenreichii subsp. shermanii , and Propionibacterium acnes , wherein said propionibacterium is preferentially P. freudenreichii subsp. shermanii . More preferentially, a nucleotide sequence coding for a signal peptide can be selected from the following sequences:
sequences SEQ ID NO: 1 to 18 (see table I below); complementary sequences of same; sequences at least 80% similar to same or to complementary sequences of same; and sequences at least 80% hybridizable in strict conditions with same or with complementary sequences of same.

Table I below presents the nucleotide (NT) sequences mentioned above, as well as the corresponding amino acid (AA) sequences, identified following sequencing by the Inventors of the P. freudenreichii genome.







TABLE I



Protein

SEQ ID NO






identified
Putative signal peptide
NT
AA



PF# 1058
MSKTLSRIASVASVAALAGSITVIAGQNASA-DS
1
19


Atgtcaaagacactctctcggatcgcatccgtcgcttcggttg




Ccgcgctcgccggcagcatcaccgtcatcgccgggcagaa




cgcgtccgccgacagc





PF# 1328
MKNGLKTLLIGGVGIATLAVGGVGTAIA-DN
2
20


Gtgaagaacggtctcaagaccctgctcattggtggagtcgg




Catcgcgacccttgcggtcggcggcgtcggaactgccatcg




cagacaat





PF# 1347
MRSTTTKAFAGVAVLALALAGCGSNSGSSTKSA-DS
3
21


Atgcgatccaccacgacgaaggcgtttgccggtgtcgctgtgctggcgct




Ggcgcttgctggctgcggctcgaattcgggctcgtccaccaagtcggccg




acagc





PF# 146
MLTRKRVVAAGAAATLSLTAFAGLQPASA-AT
4
22


Atgctcactcgcaagagagtggttgcagcgggagctgccgcc




Accctgtccctcacggcgtttgccgggttgcagcccgccagcg




ccgccacc





PF# 1885
MGFRVGRRPLIGAVLAGSMATLVGCSTSGSGSGA-SS
5
23


Atgggattcagggttggccgtcgtcccctcatcggggcagttctcgccgggtc




Gatggcaacactcgtgggctgttccacctcgggtagcggcagtggagcctc




cagc





PF# 190
MQALQGRRRSRRVMAAAVAALTAMTVLPSQLNAVA-AP
6
24


Atgcaggccctccaaggaaggcgccggtcacgacgggtgatggcggccgc




Ggtagcagccctcaccgccatgaccgtgctgccctcccagctcaacgccgttg




ctgcaccc





PF# 2074
MSTGRMKFIKLAVPVIVACCLTPMAALA-DV
7
25


Atgtccactggccgcatgaagttcatcaagctggcagttcctg




tcatcgttgcctgctgcttgacgccaatggctgccttagctgatgtg





PF# 241
MAMRARHGVVRLGLVCLTALAVFGTANVSGQVAVMA-
8
26


EG




Atggcgatgagggcacgtcacggcgtcgtccggcttggtctggtctgtctcaccgc




attggcggtcttcggcacggcaaatgtgtcgggtcaggttgcggtgatggctgagggc





PF# 2732
MNQALSTMRLKIGDSTKRIRIFFVVMAVAITLLA-GR
9
27


Ttgaaccaggccctgtcgacgatgcgcctgaagatcggcgactccacc




Aagcgcatccggatcttcttcgtcgtgatggccgtggcgatcaccctgctc




gcgggacgg





PF# 279
MRRRTTIAALAAVLSFSPLAAQA-AP
10
28


Atgcgacgtcgcaccacgattgcagccctcgctg




Ctgtcttgagtttcagtcccctggccgcccaggccg




caccc





PF# 2818
MPSHAVRETRANKLRRFLRPTVAQGVLGIAFCLVAAVGV
11
29


VQI-RS




Atgcctagtcatgcggtgcgggagacgcgggcgaacaagttgcgccggttcctgcggcc




c




Accgttgcccagggcgtgctcggtatcgcgttctgcctcgtggccgccgtcggcgtggtg




ca




gatccgctcc





PF# 2932
MSRIQLPRLSRIAIAAAASAALIGTSFIAPATAFA-AP
12
30


Atgtcacggattcaactcccccggctgagccggattgcgatcgcagc




Agcagcttccgctgccctgatcggcaccagcttcatcgccccggcca




cggcctttgccgcgccg





PF# 3042
MKRRTLLGTLGIMGLSVPLAACS-SK
13
31


Ctagccgaccttctcggccttgctggccaggtcgtc




ggggatcgtgacccccatcagggaggcggccttctcatt





PF# 3412
MVTGGNDMPSKRITTWPGISALSALIAGMLLAPLPVAA-
14
32


DG Ttagttgttggggacgaggagggagtggagttcgatgacgtcgagggtgggtgc




Ggtggcggggcgggtgatggtttcggtgccggtgtgtccttgggcggtccaggtg




atggtccaggt





PF# 3427
MAMVMASLAMFGASRASA-AD
15
33


Tcagccagttggtgccggccttggcgtcgg




cggcgtgggatacacgcggaactgggcgcc





PF# 527
MFISRFRRAAAVGLAAVTALSATACSGSSSSSSSSA-SS
16
34


Atgttcatttcgcgcttccgtcgtgcggctgcggtcggcctggccgc




Cgtcaccgcattgtccgccactgcctgtagcggttcctcgtcgtcgtc




cagctcatccgcgagctcg





PF# 876
MKSATRRPLTRWIVAFGVVLVLVIAGSVGLHASG-AL
17
35


Atgaagtccgcgacgcgacgcccgctgacgcgctggattgtcgccttcg




Gggtggtgttggtgctggtcatcgccgggtcggtggggctgcatgcctccg




gtgccctg





PF# 963
MNPFVKTARVAITSTLVAGSLATASLVFAPLAQA-DY
18
36


Gtgaatcccttcgtcaagacggcgcgcgtggctatcacctcgacgc




Tggtggcaggctcgctcgccactgccagcctcgtgtttgcaccactt




gcacaggccgattac













































































































Even more preferably, a nucleotide sequence coding for a signal peptide can be selected from the following sequences:
sequence SEQ ID NO: 18 encoding the signal protein of protein PF963 of P. freudenreichii (table I above); the complementary sequence of same; sequences at least 80% similar to same or to the complementary sequence of same; and sequences at least 80% hybridizable in strict conditions with same or with the complementary sequence of same.

A nucleotide sequence that is “complementary” to a reference nucleotide sequence refers herein to any DNA whose nucleotides are complementary to those of the reference sequence, and whose orientation is reversed (the complementary sequence is thus an antiparallel sequence). Two “complementary” nucleotide sequences are thus such that each base of one is paired with the complementary base of the other, with the orientation of the two sequences being reversed. The complementary bases are A and T (or A and U in the case of RNA), and C and G.
A nucleotide sequence that is “similar” or “homologous” to a reference nucleotide sequence refers herein to a nucleotide sequence with a percent identity with the reference nucleotide sequence of at least roughly 80%, preferably at least roughly 85%, more preferably at least roughly 90%, even more preferably at least roughly 95%, even more preferably at least roughly 98%, wherein said percentage is purely statistical and the differences between the two nucleotide sequences can be distributed randomly over their entire length. Similar sequences can thus include variations related to mutations in the reference sequence, wherein said mutations correspond in particular to truncations, substitutions, deletions and/or additions of at least one nucleotide. Similar sequences can also include variations related to degeneration of the genetic code.
In particular, sequences “at least roughly X % similar” to a reference nucleotide sequence coding for a PB signal peptide refer to variants of this sequence, wherein said variants have, over their entire length, at least roughly X % of bases identical to those of the reference sequence. The identical bases can be consecutive in their entirety or only in part. The variants thus envisaged can be the same length as the reference nucleotide sequence, or a different length, as it acts as a signal peptide in a PB. Indeed, those persons skilled in the art know that the expression products of nucleotide sequences with a certain level of similarity (at least roughly X %) can nevertheless, taking into account degeneration of the genetic code on the one hand, and the preferential use of certain codons according to the host organisms (bacteria, yeasts, etc.) on the other, fulfill the same function. These expression products can themselves be identical or similar. Here, these expression products are functional signal peptides in PB.
The definition above can be extended to amino acids sequences, or peptide/protein sequences, at least roughly X % similar to a reference amino acid sequence. This case includes protein variants with, over their entire length, amino acids at least roughly X % similar to those of the reference sequence. Here again, the similar amino acids can be consecutive in their entirety or only in part. Peptide/protein variants can be the same length or a different length, given that, preferably, the biological function of the reference amino acid sequence is conserved.
The expression “similar amino acids” refers herein to amino acids with the same side-chain reactivity. Thus, polarity and comparable ionization properties are used by persons skilled in the art to define groups of similar amino acids. For example, it is useful to categorize aliphatic amino acids, namely glycine, alanine, valine, leucine and isoleucine, within the same group. Similarly, dicarboxylic amino acids, aspartic acid and glutamic acid are similar. Also, serine and threonine belong to the same group in that they both carry an esterifiable alcohol group. Additionally, lysine, arginine and histidine can be cited as similar basic amino acids, etc.
In all the definitions above, “X” equals 80. In particular, “X” equals 85, preferably 90, more preferably 95 and even more preferably 98.
A sequence that is “hybridizable in strict conditions” with a reference nucleotide sequence refers herein to a nucleotide sequence capable of hybridizing under temperature and ionic strength conditions suitable to maintain hybridization between two complementary fragments of DNA. The “strict hybridization conditions” are consistent with the classic definition known to those persons skilled in the art (Sambrook and Russell, 2001). “Strict hybridization conditions” are, for example, conditions that enable the specific hybridization of two single-stranded nucleotide sequences after at least one washing step as described below. The hybridization step can in particular be carried out at roughly 65° C. for 12 h in a solution comprising 6× SSC, 0.5% SDS, 5×Denhardt's solution and 100 μg nonspecific DNA (salmon sperm DNA, for example), or in any another solution of equivalent ionic strength. The following step, comprising at least one washing, is carried out, for example, at roughly 65° C. in a solution comprising at most 0.2×SSC and at most 0.1% SDS, or in any another solution of equivalent ionic strength. The parameters defining the hybridization conditions depend on the temperature (Tm) at which 50% of the paired strands separate. For sequences of more than 30 bases, the temperature (Tm) is calculated according to the formula: Tm=81.5+0.41*[% G+C]+16.6* Log(cation concentration)−0.63*[% formamide]−(600/number of bases). For sequences of less than 30 bases, the temperature (Tm) is defined by the following relationship: Tm=4*(number of G+C)+2*(number of A+T). The hybridization conditions can thus be adapted by those persons skilled in the art according to the size of the sequences used, their GC content and other parameters, as indicated in particular in the protocols described in Sambrook and Russell (2001).
In particular, “reference nucleotide sequences” and “reference amino acid sequences” from P. freudenreichii can be obtained from its genomic sequence that has been recently made available by the Inventors (Falentin et al., 2010, Plos One; Genbank accession No. FN806773).
Preferably, a signal peptide will be selected from:
sequences SEQ ID NO 19 to 36 (table I above) and 45 to 57 (see table II below); sequences at least 80% similar to same; and analogs and derivatives of same.

More preferably, said signal peptide will be selected from:
sequence SEQ ID NO 36 corresponding to the signal peptide of protein PF963 of P. freudenreichii (table I above); sequences at least 80% similar to same; and analogs and derivatives of same.

Table II below presents said amino acid sequences, identified by the Inventors from the genomic sequence of P. acnes , accessible from databases (strain P. acnes KPA171202; accession number: NCBI: NC — 006085; GenBank: AEO17283).









TABLE II



Protein





sequence

SEQ

Homologous

accession
Putative sequence of
ID
Associated
protein in P .

number
the signal peptide
NO
function

freudenreichii




PPA2239
MSKVVASAIA
45

PF1328


GALSTLSAGG





LTMVQA






PPA1840
mrkaivtpva vlavlvmalt
46

PF1347


gcgqknqsgg






PPA1786
mastprrrwa wvlllvvasl
47

PF1885


vivgvyrka






PPA2198
mssmkg1s1v latsfmlsfs
48

PF2074


pgssfa






PPA0721
mehrygasqv sgsaprrgrg
49

PF241



PPA2198
mssmkglslv latsfmlsfs
50

PF3412


pgssfas






PPA0257
mphsdqptsk rvmsaprrrm
51

PF876


pgwvpvtvgi avvvivvvav





ivsslrs






AAA51650
mfgtpsrrtf ltasalsama
52
hyaluronidase



laasptvtda





ia






CAA67627
mkinarfavm aasvavlmaa
53
triacylglycerol



apiaqa

lipase




AAT83976
mypvhlplrn esefsfrahn
54
lipoprotein



hggtvpsrlt rrsvlatgav





alpmtaaaca






AAT83859
mrhmrplial slaglmtlsa
55
peptide-binding



cgedvaa

protein




AAT83771
mnrtlkvaav gaiailclaa
56
secreted sugar-



csdpgsdsaq s

binding protein




AAT83059
mekssfaaan mtimsepttp
57
secreted protease



tsqa




























































In one embodiment, said nucleic acid sequence encoding a propionibacterial signal peptide can be selected from the group consisting of:
the sequences SEQ ID NO: 1 to 18, particularly the sequence SEQ ID NO: 18; the nucleic acid sequences encoding the signal peptides of sequences SEQ ID NO: 45 to 57; and the sequences having at least 80%, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with one of said sequences over the entire length of said sequence and corresponding to a peptide signal, in particular to a peptide signal allowing the secretion, in particular into the extracellular medium, of said peptide and/or protein of interest by a propionibacterium.

The percentage of identity to which reference is made in the presentation of the invention are determined on the basis of global alignment of sequences to be compared, that is to say, on an alignment of sequences over their entire length, using for example the algorithm of Needlman and Wunsch 1970. This sequence comparison can be done for example using the needle software by using the parameter “Gap open” equal to 10.0, the parameter “Gap Extend” equal to 0.5, and a matrix “BLOSUM 62”. Software such as needle is available on the website ebi.ac.uk worldwide, under the name “needle”.
Advantageously, said nucleic acid sequence encoding a propionibacterial signal peptide can be selected from the group consisting of:
the sequence of the signal peptide of a gene encoding a surface layer protein of a Propionibacterium freudenreichii; the sequences having at least 80% more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with said sequence over the entire length of said sequence and corresponding to a peptide signal, in particular to a peptide signal allowing the secretion, in particular into the extracellular medium, of said peptide and/or protein of interest by a propionibacterium.

Said surface layer protein can be selected from the group consisting of the surface layer protein A, the surface layer protein B, the surface layer protein C, the surface layer protein D, the surface layer protein E and the surface layer protein F of a Propionibacterium freudenreichii , in particular the surface layer protein A.
Unexpectedly, the Inventors have demonstrated that signal peptides of genes encoding a surface layer protein of a Propionibacterium freudenreichii allow the secretion into the extracellular medium of a peptide and/or a protein of interest by a propionibacterium . These results are really surprising since surface layer proteins are, as indicated by their name, described as proteins anchored in the surface of Propionibacterium freudenreichii (Lortal et al. 1993).
More surprisingly, the Inventors have shown that said signal peptides in combination with promoters of genes encoding a surface layer protein of a Propionibacterium freudenreichii allow the secretion into the extracellular medium of a peptide and/or a protein of interest by a propionibacterium at a level up to 100 times greater than that obtained with other signal peptides.
In one embodiment, the recombinant vector according to the invention for expressing and secreting, by a propionibacterium , at least one eukaryotic peptide or protein of interest, comprising at least:
under the control of at least one suitable promoter, at least one nucleic acid sequence encoding a propionibacterial signal peptide and, at least one nucleic acid sequence encoding said peptide or protein of interest;

wherein said at least one nucleic acid sequence encoding a propionibacterial signal peptide is translationally fused to said at least one nucleic acid sequence encoding said peptide or protein of interest; and

wherein said promoter and said propionibacterial signal peptide are the promoter and the propionibacterial signal peptide of at least one gene encoding a surface layer protein of a Propionibacterium freudenreichii , in particular said surface layer protein being selected from the group consisting of the surface layer protein A, the surface layer protein B, the surface layer protein C, the surface layer protein D, the surface layer protein E and the surface layer protein F more particularly the surface layer protein A.


In one embodiment, said promoter and said propionibacterial signal peptide are the promoter and the propionibacterial signal peptide of the same gene encoding a surface layer protein of a Propionibacterium freudenreichii,
In particular, said nucleic acid sequence encoding a propionibacterial signal peptide is selected from the group consisting of:
the nucleic acid sequences encoding the signal peptides of sequences SEQ ID NO: 59 to 68, in particular of sequence SEQ ID NO: 59; the sequences having at least 80% of identity with one of said sequences over the entire length of said sequence and corresponding to a peptide signal, in particular a peptide signal allowing the secretion, particularly into the extracellular medium, of said peptide and/or protein of interest by a propionibacterium.

Unexpectedly, the inventors have demonstrated that the signal peptide having the sequence set forth in SEQ ID NO: 59 in combination with the promoter of the gene encoding the Surface layer protein A of a Propionibacterium freudenreichii allows the secretion into the extracellular medium of a peptide and/or a protein of interest by different strains of Propionibacterium freudenreichii at a level up to 100 times greater than that obtained with other signal peptides and promoters.





TABLE III



Sequences of the different signal peptides

corresponding to surface layer (slp) genes

identified in different strains of


Propionibacterium freudenreichii









Strain






(CIRM




SEQ

BIA
Gene
Locus


ID

N o )
name
Tag
Product
Signal peptide
NO:



118
slpA
PFCIR
S-layer
MATGAAAAMFVTTFAG
59



M118_
protein
MAPANA




06465
A





118
slpB
PFCIR
S-layer
MSVRKSLTGMALGLALTI
60



M118_
protein
TPLAGAVPASA




02660
B





118
slpD
PFCIR
S-layer
MRRFFSAAIAILLAATLTP
61



M118_
protein
ALNAPMASA




04435
D





121
slpD
PFCIR
S-layer
MRRFFSAAIAILLAATLTP
62



M121_
protein
ALNAPMASA




00945
D





122
slpB
PFCIR
S-layer
MSVRKSLTGMALGLALTI
63



M122_
protein
TPLAGAVPAAA




04010
B





122
slpD
PFCIR
S-layer
MRRFFSAAIAILLAATLTP
64



M122_
protein
ALNAPMASA




11375
D





122
slpE
PFCIR
S-layer
MKTRVRSRKPAAGLAGIA
65



M122_
protein
LFASGLSLMSTVASR




10825
E





125
slpA
PFCIR
S-layer
MATGAAAAMFVTTFAG
66



M125_
protein
MAPANA




05685
A





129
slpA
PFCIR
S-layer
MATGAAAAMFVTTFAG
67



M129_
protein
MAPANA




13370
A





512
slpA
PFCIR
S-layer
MATGAAAAMFVTTFAG
68



M512_
protein
MAPANA




08945
A

































































In one embodiment, the recombinant vector according to the invention comprises the nucleotide sequence SEQ ID NO: 69 translationally fused to said nucleic acid sequence encoding said peptide or protein of interest.
The nucleotide sequence SEQ ID NO: 69 comprises the promoter and the signal peptide of the Surface layer protein A of the Propionibacterium freudenreichii CIRM BIA 118 (CIRM: “Centre International de Ressources Microbiennes”; BIA: “Bactéries d'Intérêt Alimentaire”).
A particularly preferred recombinant vector is obtained by inserting a nucleotide sequence encoding a peptide or protein of interest in vector pFB4 (contained in the recombinant strain Escherichia coli DH5α deposited with the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) on Apr. 15, 2010 and registered under number I-4297).
In the recombinant vector according to the invention, the propionibacterial signal peptide is “translationally fused” with the peptide or protein of interest, an essential condition so that it can be secreted by the host cell. In other words, in the recombinant vector according to the invention said at least one nucleic acid sequence encoding a propionibacterial signal peptide is translationally fused to said at least one nucleic acid sequence encoding said peptide or protein of interest.
It is observed that a signal peptide could be translationally fused with several amino acid sequences in a series, making it possible to express and secrete a chimeric protein, for example. Alternately or additionally, the same vector can possibly carry several translational fusions of a signal peptide and a peptide and/or protein of interest, under the control of one or more promoters. The promoters in question can control the transcription of only one or of several of these translational fusions.
In the context of the present invention, the peptide and/or protein of interest that will be expressed from the recombinant vector of the invention are preferably eukaryotic. In particular, they can be from animal origin, more preferably from mammalian origin. In particular, they can originate from mammals selected from rodents such as mice, rats, rabbits, Chinese pigs, hamsters; canidae (e.g., dogs) and felidae (e.g., cats); domestic livestock, including cows, pigs, goats, sheeps, horses; and humans. Even more preferably, the amino acid sequences of interest are from human origin.
Preferably, at least one eukaryotic peptide and/or protein of interest to be expressed and secreted using the recombinant vector according to the present invention has a chemical mediation activity.
Advantageously, any or all of the eukaryotic peptide and/or protein of interest to be expressed and secreted using the recombinant vector according to the present invention has(ve) a chemical mediation activity.
In other words, at least one of the peptide and/or protein of interest that will be expressed from the recombinant vector according to the present invention has a biological activity of interest, preferably a chemical mediation activity.
The terms and expressions “activity,” “function,” “biological activity,” “biological function,” “bioactivity,” “(biological) activity of interest” and “(biological) function of interest” are equivalent and refer to a biological activity that is of interest, especially for medical purposes, such as an activity of chemical mediation. In particular, the peptide and/or protein of interest that will be expressed from the vector according to the present invention is “functional” or “active” or “bioactive,” that is, it is able to fulfill its natural biological function, which is independent (from a qualitative and/or quantitative point of view) of post-translational modifications not able to be carried out by a propionibacterium.
Any eukaryotic protein whose biological activity is of interest to industry or medicine is thus within the scope of the present invention, insofar as said activity does not depend on post-translational modifications not able to be carried out by a propionibacterium.
The principal utility of the invention is to make it possible to produce an eukaryotic peptide and/or protein of interest. It is described here another utility of the invention that is to recycle industrial organic waste or residual by-products. Thus, for example, whey and molasses, which are produced as unused residues by certain industries, may be recycled as substrates for the culture of recombinant propionibacterium able to synthesize peptide and/or protein of interest.
An eukaryotic peptide or protein, or a fragment or domain thereof, having an activity of “chemical mediation” is a “chemical mediator”, i.e., a peptide or protein naturally secreted by an eukaryotic cell, or a fragment or domain of such a peptide or protein, and that is capable of binding a cell receptor to induce a cellular response. Examples of chemical mediators include neuromediators or neurotransmitters, hormones, growth factors, cytokines, and the like. Chemical mediators also include fusion peptides or proteins capable of binding to a cell receptor.
Preferably, the eukaryotic peptide or protein of interest that can be expressed and secreted from the vector according to the invention, has a chemical mediation activity that is of medical interest such as an activity selected from proapoptotic, anti-inflammatory, immunomodulatory activities, and combinations thereof.
In other words, in one embodiment, said peptide or protein of interest can have a biological activity selected from the group consisting of proapoptotic activity, anti-inflammatory activity and immunomodulatory activity.
The peptide or protein of interest may be selected from cytokines, chemokines, peptide hormones, neurotransmitters, peptides involved in inflammation, satiety, blood pressure, etc. . . . The peptide or protein of interest is preferably a proapoptotic and/or anti-inflammatory peptide or protein, preferably from human origin. In particular, said peptide or protein of interest can be selected from the group consisting of:
a proapoptotic peptide or protein; and an anti-inflammatory peptide or protein.

According to a preferred embodiment, the peptide or protein of interest is a cytokine.
The peptide of interest can be a biologically active fragment or domain of a protein of interest. This means that when the peptide of interest expressed from the vector according to the present invention is a protein fragment or domain, it remains “functional” or “active” or “bioactive,” that is, it is able to fulfill the natural biological function of the corresponding native protein. All definitions provided herein with respect to proteins also apply to biologically active fragments or domains thereof.
In a preferred embodiment, the recombinant vector according to the present invention makes it possible to express and secrete the proapoptotic TRAIL protein (TNF-related apoptosis-inducing ligand, also called TNSF10, TL2, CD253 and Apo-2L), a cytokine of the TNF family. The sequence of the Homo sapiens TRAIL protein can be the sequence set forth in SEQ ID NO: 58 (accession number AAC50332.1). In particular, the amino acid sequence of interest is that of the active C-terminal extracellular domain of TRAIL, preferably the sequence from amino acids 114 to 281 of TRAIL (TRAIL sequence accession number in GenBank: U37518; Uniparc: UPI0000001629).
Thus, in one embodiment, said peptide or protein of interest can be the proapoptotic TRAIL protein or the C-terminal extracellular domain of the TRAIL protein, in particular the Homo sapiens proapoptotic TRAIL protein or the C-terminal extracellular domain of the Homo sapiens TRAIL protein.
In particular, said nucleic acid sequence encoding said peptide or protein of interest can be the sequence from amino acids 114 to 281 of the TRAIL protein sequence set forth in SEQ ID NO: 58.
According to the literature, TRAIL is an antineoplastic agent with strong potential because it induces the death of many tumor cells, independently of p53 and Pgp180 (MDR, multidrug resistance). TRAIL also inhibits the growth of xenografted colon tumors in nude mice (Ashkenazi et al., 1999). Quite interestingly, TRAIL has little cytotoxic effect on most normal tissues (Ashkenazi et al., 1999), including human colon epithelium (Sträter et al., 2002).
Other teams very recently expressed the active C-terminal extracellular domain of TRAIL in bacteria such as Salmonella typhimurium (Ganai et al., 2009), Bifidobacterium longum (Hu et al., 2009) and E. coli (Zhang et al., 2010). In this work, salmonellas, bifidobacteria and coliform bacteria are proposed as systemic TRAIL delivery vectors in cancer models.
However, propionibacteria have major advantages compared to other bacteria such as salmonellas, bifidobacteria and coliform bacteria.
First, dairy propionibacteria enable local delivery because they target colon epithelial cells and have an active fermentative metabolism in the human colon (Hervé et al., 2007), which enables site-specific delivery of TRAIL.
Second, dairy propionibacteria themselves have proapoptotic properties. It was recently shown in vitro that dairy propionibacteria, by the SCFA resulting from their fermentative metabolism, induce the apoptosis of two human colon adenocarcinoma cell lines (Caco2 and HT29) (Jan et al., 2002). This effect is directly related to the release of propionate by these bacteria and to their action on cancer cell mitochondria. It has also been shown that at extracellular pH (pH e ) between 6 and 7.5, SCFA (propionate and acetate SCFA) induce apoptotic death whereas at pH e =5.5, they induce necrotic death in HT29 human colon cancer cells (Lan et al., Apoptosis, 2007). In vivo, these food-quality (GRAS: generally regarded as safe) bacteria adapt and survive in the digestive tract of animals and humans with an efficiency that, although strain-dependent, exceeds that of other probiotics (Hervé et al., 2007). Moreover, they express in the intestine enzymatic activities characteristic of their fermentative metabolism by producing an increase in SCFA concentrations (Lan et al., Br. J. Nutr., 2007) and induce an increase in apoptosis in the mucosa of the colon of rats treated with 1,2-dimethylhydrazine (Lan et al., 2008). The Inventors have further shown a synergistic action with TRAIL in vitro as illustrated in the examples below.
A particularly preferred recombinant vector is the vector pFB4:TRAIL, wherein the amino acid sequence of interest is the sequence from amino acids 114 to 281 of the TRAIL C-terminal extracellular domain. This vector is hosted by type strain CIP103027 of P. freudenreichii subsp. shermanii , deposited on Jul. 23, 2009 under number 1-4213 with the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France).
Another particularly preferred recombinant vector is the vector pCHH04: TRAIL wherein the peptide of interest is the sequence from amino acids 114 to 281 of the TRAIL C-terminal extracellular domain, said sequence being translationally fused to the signal peptide of the Surface layer protein A of the Propionibacterium freudenreichii CIRM BIA 118, under the control of the promoter of the Surface layer protein A of the Propionibacterium freudenreichii CIRM BIA 118. A P. freudenreichii CIRM-BIA-118 comprising said vector pCHH04: TRAIL has been deposited on Nov. 13, 2012 under number CNCM 1-4692 with the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France).
The present invention further relates to a recombinant propionibacterium comprising at least one recombinant vector according to the invention as previously defined.
The propionibacterium comprising at least one recombinant vector according to the invention can be selected in the group consisting of P. freudenreichii, P. jensenii, P. thoenii, P. acidipropionicii, P. acnes, P. granulosum, P. avidum, P. propionicum and P. microaerophilum.
In particular, the propionibacterium comprising at least one recombinant vector according to the invention can be a P. freudenreichii selected in the group consisting of P. freudenreichii freudenreichii and P. freudenreichii shermanii.
Advantageously, the propionibacterium comprising at least one recombinant vector according to the invention can be a P. freudenreichii , in particular chosen in the group consisting of CIRM BIA 512 strain, CIRM BIA 125 strain, CIRM BIA 118 strain, CIRM BIA 129 strain and CIRM BIA 122 strain, more particularly CIRM BIA 512 strain, CIRM BIA 125 strain, CIRM BIA 118 strain and CIRM BIA 129 strain, even more particularly CIRM BIA 512 strain, CIRM BIA 125 strain, CIRM BIA 118 strain and even more particularly CIRM BIA 512 strain and CIRM BIA 118 strain and even more particularly CIRM BIA 118 strain.
Advantageously, the recombinant vector will be carried by the chromosome of the propionibacterium according to the invention. The vector can be integrated, for example, in the chromosome of the host cell by homologous recombination.
One particularly preferred propionibacterium is type strain CIP103027 of P. freudenreichii subsp. shermanii , deposited with the CNCM on Jul. 23, 2009 under number I-4213.
Another particularly preferred propionibacterium is the propionibacterium deposited on Nov. 13, 2012 under number CNCM I-4692 with the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France).
If the peptide or protein of interest to be secreted lends itself to such a use (an anorexiant peptide, for example), the propionibacterium according to the invention could be used as a probiotic food or dietary supplement for mammals, in particular humans. Advantageously, the propionibacterium will be integrated in the mammal's food in the form of a fermented dairy product (e.g., fermented milk, fermented whey, cheese).
The present invention further relates to a recombinant vector or a recombinant propionibacterium as defined above, for the use of same as a drug.
In other words, the invention also relates to a recombinant vector or a recombinant propionibacterium according to the invention, for its use as a drug, in particular for the prevention and/or treatment of a disease, in particular cancer and more particularly colorectal cancer.
The present invention further relates to a drug (or pharmaceutical composition) comprising an effective quantity (amount) of at least one vector according to the invention and/or at least one propionibacterium according to the invention and at least one pharmaceutically acceptable carrier (or excipient).
Such therapeutically effective amount can be determined by one skilled in the art by routine tests including assessment of the effect of administration of said components (vector according to the invention and/or propionibacterium according to the invention) on the pathologies and/or disorders which are sought to be prevent and/or to be treated by the administration of said drug (or pharmaceutical composition).
For example, such tests can be implemented by analyzing both quantitative and qualitative effect of the administration of different amounts of said aforementioned components (vector according to the invention and/or propionibacterium according to the invention) on a set of markers (biological and/or clinical) characteristics of said pathologies and/or of said disorders, in particular from a biological sample of a subject.
In said drug, the vector and/or the propionibacterium of the invention are advantageously used as therapeutic agents. A drug according to the invention can be manufactured in a conventional way. A drug in accordance with the invention can moreover include one or more pharmaceutically acceptable excipients or additives such as diluents, adjuvants, anti-foaming agents, stabilizers, dispersants, colorants, preservatives, etc. Inert excipients or adjuvants can be used in such a way that, in the drugs according to the present invention, the only therapeutic agents will be the vector and/or the propionibacterium . Nevertheless, the drug according to the present invention can include one or more other therapeutically or prophylactically active agents, in addition to the vector and/or the propionibacterium . Advantageously, the combination of several therapeutic agents, including at least the vector and/or the propionibacterium , will have a better therapeutic or prophylactic action than when the vector and/or the propionibacterium are the only therapeutic agents present in the drug. This better action can be, among others:
a better dose-effect relationship; a therapeutic or prophylactic effect that is more stable or longer lasting over time; a better administration of the drug; a synergy of action between at least two therapeutic agents present in the drug.

Preferably, a drug in accordance with the present invention is intended to prevent and/or to treat at least one disease selected from allergies, hypertension (e.g., the peptide or protein of interest has a hypotensive activity), obesity (e.g., the peptide or protein of interest has an anorexiant activity), cancers, in particular colorectal cancers (e.g., the peptide or protein of interest has a proapoptotic activity) and inflammatory colon diseases, in particular Crohn's disease (wherein the peptide or protein of interest is advantageously an anti-inflammatory cytokine, for example IL-10), etc. . . . Alternately, a drug of the invention is intended to prevent at least one microbial infection, for example a viral, bacterial, fungal or parasitic infection, etc. In this case, the peptide or protein of interest will be an antigen or an epitope, for example. Thus, said drug advantageously will be a vaccine, in which case the pharmaceutically acceptable carrier could be an immune adjuvant.
The various means of the present invention (vector, propionibacterium , drug) as described above are preferably administered to a mammal for the secretion of the peptide and/or protein of interest in the small intestine and/or the colon, preferably the colon, of said mammal.
The term “mammal” is defined in its usual sense. Examples of mammals include bovines; pigs; goats; sheep; horses; rodents such as mice, rabbits, rats and hamsters; felines and canines, including domestic animals such as cats and dogs. A preferred mammal in the context of the invention is a human.
The means of the invention (vector, propionibacterium , drug) can be administered by any suitable conventional route, in particular selected from the oral, subcutaneous, intramuscular, intravenous, intrarectal, enema and intratracheal routes. Oral administration is preferred, wherein the drug is in the form of tablets, hard gelatin capsules (e.g., gastroprotective gelatin capsules), soft capsules, powders for direct use or for dilution (e.g., lyophilisates), syrups, gels, etc. Said means can be administered in a single or repeated dose one or more times spaced over a certain interval of time. The suitable administration route and dosing schedule can vary according to various parameters, such as the subject to be treated and/or the peptide and/or protein of interest.
The invention further relates to a method of therapeutic or prophylactic treatment, wherein a therapeutically effective quantity of a vector and/or a propionibacterium and/or a drug according to the invention is administered to a subject in need of such a treatment.
In other words, the invention also relates to a method for therapeutic or prophylactic treatment of a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of at least one compound selected from the group consisting of:
a vector according to the invention; a propionibacterium according to the invention; and a drug according to the invention.

The invention also relates to a method for therapeutic or prophylactic treatment of a disease, in particular a cancer, more particularly a colorectal cancer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of at least one compound selected from the group consisting of:
a vector according to the invention; a propionibacterium according to the invention; and a drug according to the invention;

wherein the peptide or protein of interest is the proapoptotic TRAIL protein or the C-terminal extracellular domain of the TRAIL protein (in particular having the sequence from amino acids 114 to 281 of the protein TRAIL sequence set forth in SEQ ID NO: 58).
The present invention further relates to the use of a vector and/or a propionibacterium in accordance with the preceding description to produce and secrete, preferably into the extracellular medium, one or more peptides and/or proteins of interest.
The present invention further relates to a method for producing and secreting into the extracellular medium, by a propionibacterium according to the invention, at least one amino acid sequence of interest (at least one peptide or protein of interest) as defined above, wherein said method comprises at least:
culturing said propionibacterium under suitable conditions; recovering the culture medium containing said amino acid sequence of interest (said peptide or protein of interest) (since it is secreted in the culture medium by the propionibacterium ); and optionally, purifying said amino acid sequence of interest (said peptide or protein of interest).

Preferably, the method according to the invention makes it possible to produce and secrete peptides and/or proteins on a large scale, that is, on an industrial scale (in protein production plants).
The “suitable conditions” (in terms of the composition of the culture medium, temperature, time, ventilation, stirring, etc.) for culturing propionibacteria are known to those persons skilled in the art (see in particular documents US 20090312425 in the name of Meiji Dairies Corp. and CN 101045910 in the name of Nanjing University of Technology). Once again, these bacteria are robust, are able to grow on particular substrates such as whey or molasses and are able to adapt to non-standard culture conditions, characteristics which a large number of other bacteria do not share.
Peptide, protein purification calls upon the general knowledge of those persons skilled in the art and can be carried out without any difficulty using classic techniques.
The invention further relates to pharmaceutical products containing at least one propionibacterium , preferably one non-recombinant propionibacterium , and at least one peptide or protein of interest as a combination product for prophylactic or therapeutic use, in mammals, that is simultaneous, separated or sequential over time.
The invention also relates to a combination product, which comprises:
at least one vector according to the invention and/or at least one propionibacterium comprising said vector; and at least another active agent, in particular chosen in the group consisting of an anti-tumoral agent, an anti-inflammatory agent and immunomodulatory agent, more particularly an anti-tumoral agent;

for simultaneous, separate or sequential use a medicament.
Said active agent can be a short-chain fatty acid (SCFA), preferably propionate or acetate.
The pharmaceutical products according to the invention can comprise the peptide or protein itself or a nucleotide sequence encoding said peptide or protein, wherein said nucleotide sequence is optionally carried by a suitable expression vector.
For example, the pharmaceutical products in accordance with the present invention are intended to prevent and/or to treat at least one disease selected from allergies, hypertension (e.g., the peptide or protein of interest has a hypotensive activity), obesity (e.g., the peptide or protein of interest has an anorexiant activity), colorectal cancers (e.g., the peptide or protein of interest has a proapoptotic activity) and inflammatory colon diseases, in particular Crohn's disease (wherein the peptide or protein of interest is advantageously an anti-inflammatory cytokine, for example IL-10), etc. Alternately, said products can be intended to prevent at least one microbial infection, for example a viral, bacterial, fungal or parasitic infection, etc. In this case, the peptide or protein of interest will be an antigen or an epitope, for example.
Advantageously, for an antineoplastic therapy, in particular for the treatment of colorectal cancers, the pharmaceutical products according to the present invention preferably comprise:
at least one non-recombinant propionibacterium such as P. freudenreichii , in particular subsp. shermanii , and at least the TRAIL protein or the peptide of sequence from amino acids 114 to 281 of the TRAIL C-terminal extracellular domain, in particular set forth in SEQ ID NO: 58.

In said pharmaceutical products, the propionibacterium can be administered in the form of a probiotic which will be added to the mammal's food and which will serve, for example, as an adjuvant TRAIL-based chemotherapy. In the latter case, it is recalled that, as being typically administered systemically, conventional chemotherapeutic treatments have a lot of undesirable side-effects, in particular due to a high dosage regime and a lack of specificity. Thus, by orally administering the probiotic propionibacterium as an adjuvant to a TRAIL-based chemotherapy, the bacteria and TRAIL will be able to exhibit an enhanced, advantageously synergistic, anti-tumoral activity in the colon, resulting in noticeably reduced (or even prevented) side-effects by reducing the required dosages and increasing the specificity of the treatment.
Alternatively, still in reference to an antineoplastic therapy, in particular for the treatment of colorectal cancers, the pharmaceutical products according to the present invention comprise:
a culture supernatant of a non-recombinant propionibacterium such as P. freudenreichii , in particular subsp. shermanii , wherein said supernatant has optionally undergone one or more suitable conventional treatments to improve its harmlessness and/or preservation and/or physicochemical properties etc., and at least the TRAIL protein or the peptide of sequence from amino acids 114 to 281 of the TRAIL C-terminal extracellular domain, in particular set forth in SEQ ID NO: 58.

As the examples illustrate below, the Inventors indeed have shown a synergy of proapoptotic action on HT29 colon cancer cells between propionibacterium and/or propionibacterium culture supernatants (containing SCFA, in particular propionate and/or acetate SCFA) and TRAIL.
Alternately again, and still in reference to an antineoplastic therapy, in particular for the treatment of colorectal cancers, the pharmaceutical products according to the present invention comprise:
one or more SCFA, in particular propionate and/or acetate SCFA, advantageously obtained from the culture supernatant of one or more non-recombinant propionibacterium such as P. freudenreichii , in particular subsp. shermanii , and at least the TRAIL protein or the peptide of sequence from amino acids 114 to 281 of the TRAIL C-terminal extracellular domain, in particular set forth in SEQ ID NO: 58.

The invention further concerns a method for treating a cancer in a mammal in need thereof, comprising administering to said mammal:
at least one short-chain fatty acid (SCFA), preferably propionate and/or acetate, advantageously obtained by fermentation of at least one Propionibacterium such as Propionibacterium freudenreichii , and

proapoptotic TNF-related apoptosis inducing ligand (TRAIL/Apo-2 ligand), or a functional fragment thereof.
Said mammal is as defined above.
Said cancer is preferably a colorectal cancer.
Said functional fragment of TRAIL preferably comprises amino acid sequence from position 114 to position 281 of the TRAIL C-terminal extracellular domain, in particular set forth in SEQ ID NO: 58.
Said SCFA and said TRAIL or functional fragment thereof may be administered to said mammal simultaneously, separately or sequentially.



The present invention is illustrated by the following figures:
FIG. 1 : Graphs illustrating the synergy observed in vitro between TRAIL and P. freudenreichii metabolites. HT29 colon cancer cells were treated with sublethal doses of TRAIL (25 ng/ml, 50 ng/ml and 100 ng/ml). Various doses of SCFA (propionate/acetate SCFA, FIG. 1A ) or of P. freudenreichii supernatant ( FIG. 1B ) were used in co-treatment. Viability of the HT29 cells was determined after 24 hours of treatment.
FIG. 2 : Identification of the protein secreted in the majority by P. freudenreichii , PF963. A: growth of two strains of P. freudenreichii , one autolytic (□) and the other nonlytic (◯). B and C: electrophoretic analysis (SDS-PAGE) of proteins secreted by a strain of lytic (B) and nonlytic (C) P. freudenreichii . Protein PF963 was identified by mass spectrometry.
FIG. 3 : Diagram detailing the cloning strategy to obtain the pFB4:TRAIL plasmid (deposited with the CNCM on Jul. 23, 2009 under number I-4213). A: the promoter region and signal peptide of the P. freudenreichii protein PF963 were amplified by PCR with introduction (PCR mutagenesis) of restriction sites Nde1 and HindIII. The active extracellular portion of TRAIL (residues 114 to 281) was amplified by PCR with introduction of restriction sites HindIII and Pst1. B and C: the two PCR products were purified and linked in order to obtain the ligation product SP-TRAIL. D: plasmid pK705 was opened by digestion using two enzymes Nde1 and Pst1. The ligation product SP-TRAIL was introduced into the open plasmid. The new plasmid pFB4 includes a promoter and a signal peptide enabling the secretion, in particular into the extracellular medium, by P. freudenreichii of a heterologous protein. The arrows and the scissors represent PCR primers and restriction sites, respectively.
FIG. 4 : Map of the pFB4:TRAIL plasmid (deposited with the CNCM on Aug. 13, 2009 under number 1-4213).
FIG. 5 : Sequence of the fusion protein coded for by the pFB4:TRAIL plasmid. In this sequence, the underlined region corresponds to the PF963 protein signal sequence and the region in bold corresponds to the TRAIL extracellular domain sequence (residues 114 to 281).
FIG. 6 : Detection by Western blot of the fusion protein coded for by the pFB4:TRAIL plasmid. The samples deposited were culture supernatants of wild P. freudenreichii CIP103027 (1) or of P. freudenreichii CIP103027 carrying the pFB4:TRAIL plasmid (2 and 3). A solution of SuperKillerTRAIL™ (Alexis Biochemicals, Coger, France) was deposited as positive control (4). The Western blot was developed using a commercial “PAb to TRAIL” antibody (Alexis Biochemicals).
FIG. 7 : Map of the pCHH04: TRAIL plasmid (deposited with the CNCM on Nov. 13, 2012 under number CNCM I-4692).
FIG. 8 : Proteomic comparative analysis of Propionibacterium freudenreichii exoproteomes. Strains CIRM BIA 1, 129, 118, 456, 127, 508, 516 and 512 were grown in YEL medium and the culture supernatants analyzed by SDS PAGE followed by Coomassie Blue staining. The reference strain CIRM BIA 1, the first sequenced in 2010, secretes 4 proteins with equivalent efficiency, including PF963. Other strains, sequenced since then (unpublished data), secrete a major extracellular 60 kDa protein abundantly. This was identified in strain 118 as SlpA.
FIG. 9 : Western Blot detection. The plasmids pFB4:TRAIL and pCHH04:TRAIL are compared within the same CIRM BIA 118 strain with respect to TRAIL secretion into the extracellular medium. Secretion of TRAIL into the extracellular medium is then compared in different strains harboring the pCHH04:TRAIL plasmid:The pCHH04:TRAIL plasmid was transformed into different strains of Propionibacterium freudenreichii and the resulting supernatants were analyzed with respect to TRAIL secretion by western blotting.



The following non-limiting examples, which refer to the figures above, illustrate the embodiments and advantages of the present invention.
EXAMPLES
I-Induction of the Intrinsic Mitochondrial Pathway of Apoptosis by Propionibacterium freudenreichii
During preliminary studies, the Inventors showed that certain selected strains of P. freudenreichii surviving the stresses undergone during intestinal transit in humans (Hervé et al., 2007), as well as in the rat (Lan et al., 2007), express the genes coding for fermentative metabolism enzymes and produce propionate and acetate short-chain fatty acids (SCFA) in situ in the colon (Lan et al., 2007).
Furthermore, this bacterium induced the apoptosis of human colon adenocarcinoma cells in vitro via these SCFA which act on cancer cell mitochondria (Jan et al., 2002). The mitochondrial pathway of apoptosis induction has been clearly identified in the triggering of programmed cell death of HT29 cells by dairy propionibacteria (Jan et al., 2002; Lan et al., 2007). Said SCFA cause the opening of mitochondrial permeability transition pores (PTP), the depolarization of mitochondria, the leaking of proapoptotic mitochondrial proteins and the activation of effector caspases.
Such an induction of apoptosis was then researched in vivo in a rat model of human digestive flora. Rats were treated or not treated by the carcinogen dimethylhydrazine (DMH) for the purpose of causing the appearance of damaged colonic epithelial cells likely to develop into colon cancer. These rats received by gavage, or did not receive, the P. freudenreichii bacterium. Colonic epithelial cell apoptosis and proliferation were quantified by anatomopathological analysis of histological sections of colon. The administration in healthy rats of P. freudenreichii had no effect on these parameters. On the other hand, a significant increase in apoptosis was observed in the rats treated with DMH (Lan et al., 2008). It thus appears that a specific apoptosis of cancer cells can be induced by dairy propionibacteria.
II-Induction of the Extrinsic Pathway of Apoptosis by TRAIL Via Death Receptors, Synergy with Propionibacterium freudenreichii
TRAIL is a cytokine capable of inducing the apoptosis of human colon cancer cells by binding to death receptors. TRAIL thus induces a different apoptotic pathway on the cellular and molecular levels and potentiates the action of other proapoptotic molecules used in cancer chemotherapy (Lacour et al., 2001; Lacour et al., 2003; Meurette et al., 2005; Meurette et al., 2006). Cell death induced by TRAIL or by SCFA is promoted by an acidic environment (Meurette et al., 2007; Lan et al., 2007).
By viability tests ( FIG. 1 ), and by in vitro apoptosis quantification methods (Hoechst staining and caspases activity, data not shown), the Inventors showed a synergistic proapoptotic effect of the cytokine TRAIL in combination with the propionate/acetate mixture or the propionibacteria culture supernatant in HT29 human colon cancer cells ( FIG. 1 ). More precisely, the viability illustrated in FIG. 1 was determined using the following cytotoxicity test. HT29 human colon cancer cells (ATCC, Biovalley) were cultured in 96-well plates (30,000 cells/well) for 24 hours. They were then treated with TRAIL (0 ng/ml, 25 ng/ml, 50 ng/ml and 100 ng/ml) (SuperKillerTRAIL™, Alexis Biochemicals, Coger, France) in the presence of increasing concentrations of propionate/acetate (7.5 mmol/3.5 mmol; 15 mmol/7.5 mmol; 30 mmol/15 mmol; 60 mmol/30 mmol) or of bacterial supernatant ( P. freudenreichii bacteria) (⅙, ¼, ½, pure). At the end of treatment (24 hours), the medium was discarded and the adherent cells were washed three times with 1× PBS (100 μl/well) and fixed in 99% ethanol (100 μl/well) for 30 minutes. After discarding the ethanol, the fixed cells were air dried and then stained for 30 minutes with methylene blue (diluted in l× borate buffer). After three washings in water and drying (roughly 30 minutes), 100 μl of hydrochloric acid (0.1 N) was added to the wells. The plates were then analyzed by spectrophotometer at a wavelength of 620 nm (iEMS Reader MF; Lab-systems, Helsinki, Finland).
FIG. 1 shows that sublethal doses of TRAIL (25 ng/ml, 50 ng/ml and 100 ng/ml) do not significantly induce cell death during the treatment period. Moreover, the smallest doses of SCFA alone induce little or no cell death, but induce massive death in the presence of TRAIL ( FIGS. 1A and 1B ). These results show a synergy of proapoptotic action on human colon cancer cells between SCFA metabolites produced by PB and TRAIL.
III-Development of a First Recombinant Propionibacterium with the Goal of Inducing Both the Intrinsic and Extrinsic Apoptotic Pathways
III.1 Summary
The Inventors sought to make a bacterium, harmless to healthy cells, produce inducers of the two apoptotic pathways. These inducers are the SCFA produced by P. freudenreichii for the intrinsic pathway and TRAIL for the extrinsic pathway. Since propionibacteria have a positive tropism for the mucosa of the colon, said recombinant bacterium will not only be likely to produce TRAIL in situ in the colon, but also to carry SCFA and TRAIL toward colon epithelial cells.
To this end, a recombinant propionibacterium expressing TRAIL fused with a secretion signal peptide was developed for in situ production in experimental models of cancer colon.
Briefly, the major protein secreted by P. freudenreichii during its growth and in the absence of lysis, named PF963, was identified. The experimental procedure (e.g., electrophoresis, trypsinolysis, nano-LC and MS/MS) which led to the identification of PF963 is similar to that which had previously enabled the Inventors to identify GAPDH (Tarze et al., 2007). Very briefly, the supernatant of the nonlytic strain of P. freudenreichii was analyzed by electrophoresis. The gel fragment containing the major protein secreted was removed, rinsed and then subjected to “in gel” trypsin proteolysis. The resulting peptides were separated by nano-LC and then analyzed with tandem mass spectrometry (MS/MS).
PF963 is an enzyme secreted via the machinery of the “Sec” pathway which recognizes and cleaves a signal peptide (SP). By genetic engineering, said SP was fused with the active C-terminal extracellular domain of TRAIL. This construction was carried out in E. coli on a cloning plasmid. The fusion thus obtained was introduced into an expression vector (pK705) previously developed for the cloning and expression of propionibacterial genes in dairy propionibacteria and efficient in P. freudenreichii (Kiatpapan et al., 2000) in order to express the fusion protein. The expression and the extracellular addressing of the fusion protein were then analyzed by Western blot.
According to FIG. 2A , the growth of P. freudenreichii shows that certain strains are lysed (□) and others not (◯). In the latter case, protein PF963 is secreted in the medium ( FIG. 2C ) without leakage of cytoplasmic proteins as in the case of spontaneous bacterial lysis ( FIG. 2B ). The upstream portion of the PF963 gene, comprising the promoter and the signal peptide, was amplified by PCR and fused with the C-terminal portion of TRAIL ( FIGS. 3A to 3C ). The following step consisted of its introduction in a P. freudenreichii expression plasmid ( FIG. 3D ).
III.2 Obtaining the Strain Propionibacterium freudenreichii Subsp. Shermanii CIP103027 (TL34) Carrying the pFB4:TRAIL Plasmid.
III.2.1 Identification of the Protein PF963 Secreted by Propionibacterium freudenreichii Subsp. Shermanii.
In order to identify a secreted protein, strains were screened on the basis of aptitude for autolysis. Indeed, it is known that certain strains of said bacterium make use of a programmed cell suicide, autolysis. In this case, cytoplasmic proteins are released in the surrounding medium. On the other hand, other strains, including strain CIP103027, do not undergo autolysis and on the contrary make use of a tolerance reaction with respect to various stresses, called the starvation-induced multi-tolerance response. In principle, these nonlytic strains thus only release actively secreted proteins and do not release proteins by accident. FIG. 2A shows the evolution of the bacterial population for an autolytic strain (□) and for a nonlytic strain (◯) of Propionibacterium freudenreichii subsp. shermanii.
FIG. 2C shows the electrophoretic analysis (SDS-PAGE) of proteins secreted by a nonlytic strain, CIP103027. This analysis reveals several secreted proteins, including protein PF963, identified in the culture supernatant of all the nonlytic strains tested. This protein was cut out of a preparative SDS-PAGE gel and subjected to digestion by trypsin. The resulting peptides were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) on a hybrid triple quadrupole time-of-flight apparatus (QSTAR®XL, Applied Biosystems) according to a standard laboratory procedure (Science and Technology of Milk and Eggs) described in Tarze et al. (2007).
By this analysis, the Inventors identified protein PF963, a secreted bacterial wall peptidase belonging to the NlpC/P60 family. The complete sequence of protein PF963 (SEQ ID NO 36; table I) can be deduced after determination of the complete sequence of the genome of strain CIP103027 by the Inventors.
III.2.2 Fusion of the N-Terminal Portion of Protein PF963 with the C-Terminal Portion of TRAIL
The presence of a signal peptide at the N-terminal end of PF963 indicates that this enzyme is secreted via the Sec secretion pathway. The sequence of said signal peptide is SEQ ID NO 36. PCR primers were designed to amplify the DNA sequence corresponding to the promoter and to the signal peptide of protein PF963 ( FIG. 3 ). Another pair of primers was designed to amplify the sequence of the human cytokine TRAIL. Only the active extracellular sequence Val 114 -Gly 281 was amplified. The primer sequences are indicated in the following Table IV.









TABLE IV







Nucleotides





hybridizing

PCR

Tm
Total
with the

primer
Sequence
(° C.)
nucleotides
matrix



P963Fw
ATACATATGCCACCGTGAG
70
27
18


CTGCACCT





(SEQ ID NO 38)






P963Rv
GCAAGCTTTCGGCCTGTGC
71
27
19


AAGTGGTG





(SEQ ID NO 39)






TRAILFw
GCAAGCTTAGTGAGAGAAA
70
37
28


GAGGTCCTCAGAGAGTAG





(SEQ ID NO 40)






TRAILRev
ACTGCAGTTAGCCAACTAA
70
39
32


AAAGGCCCCGAAAAAACTG





G





(SEQ ID NO 41)



































The construction resulting from the fusion between 1) the promoter and the signal peptide of PF963 and 2) the Val 114 -Gly 281 sequence of TRAIL was introduced in cloning vector pPK705 (Kiatpapan et al., 2000). The new pFB4:TRAIL expression plasmid is presented in FIG. 4 .
III.2.3 Verification of the Genetic Construction
The sequence of the pFB4:TRAIL plasmid was verified (SEQ ID NO 42). The portion corresponding to the fusion protein ranges from nucleotides 8451 to 9070. This portion is translated in FIG. 5 (SEQ ID NO 43): the sequence corresponding to the PF963 peptide signal protein is underlined and the Val 114 -Gly 281 sequence of TRAIL appears in bold.
The fusion protein has a sequence of 205 amino acid residues corresponding to a mass of 23,190 Da and an isoelectric point of 9.08. The elimination of the signal peptide leads to a sequence of 171 amino acid residues corresponding to a mass of 19,822 Da and an isoelectric point of 8.60.
Expression and secretion of the fusion protein were verified by Western blot using a commercial anti-TRAIL polyclonal antibody (Pab to TRAIL, Alexis Biochemicals). This antibody recognizes the monomeric form (31 kDa) as well as the dimeric form (63 kDa) of TRAIL in the SuperKillerTRAIL™ preparation ( FIG. 6 ; lane 4). In the supernatant of the two clones of the transformed P. freudenreichii strain carrying the plasmid, a protein of 22 kDa, corresponding to the expected size, was detected by this antibody. This protein was absent in the supernatant of the wild strain.
IV-Development of a Second Recombinant Propionibacterium with the Goal of Inducing Both the Intrinsic and Extrinsic Apoptotic Pathways
The inventors have optimized the plasmid pFB4: TRAIL by changing both the promoter region and the signal peptide to obtain the pCHH04: TRAIL plasmid, allowing secretion into the extracellular medium, of a higher amount of TRAIL into the supernatant.
IV.1 Materials and Methods
The construction of pCHH04:TRAIL is described in FIG. 7 . The pFB4:TRAIL plasmid, which is a shuttle plasmid, was transformed into Escherichia coli . The propionibacterial insert containing the promoter PF963 and the signal peptide PS963 were excised using the appropriate restriction endonucleases. A new propionibacterial DNA fragment (set forth in SEQ ID NO: 69), containing the promoter region and the signal peptide of protein slpA of the Propionibacterium freudenreichii CIRM BIA 118 strain, was introduced in order to put the signal peptide in frame with the TRAIL coding sequence. The corresponding sequence was amplified by PCR (PSlpA_fwd_BamH1, which has the sequence set forth in SEQ ID NO: 70 and PS_SlpA-EcoRV-Rev_bis, which has the sequence set forth in SEQ ID NO: 71) from Propionibacterium freudenreichii genomic sequence, which was previously sequenced and annotated (Falentin et al., 2010a). This new plasmid, pCHH04:TRAIL, was then transformed into different strains of Propionibacterium freudenreichii . The corresponding supernatants were then analyzed by western blot with respect to TRAIL secretion as above described. Proteomic experiments, including gel electrophoresis and mass spectrometry, was conducted as previously described (Leverrier et al., 2004). TRAIL concentration was further quantified by Enzyme-Linked ImmunoSorbent Assay (ELISA) in supernatant of Propionibacterium freudenreichii transformed with pFB4:TRAIL or pCHH04:TRAIL plasmid according to the manufacturer's instructions (R&D System Europe, Lille, France). Human TRAIL standard concentration-response curves were used to quantify TRAIL levels in supernatants. TRAIL concentrations were determined using an automatic plate reader associated with genesis software (LabSystems Spectrophotometer, Cambridge, UK) and data were expressed in ng/ml.
IV.2 Results
In order to investigate secretion abilities in Propionibacterium freudenreichii , supernatants of several strains were analyzed by SDS-PAGE electrophoresis. FIG. 8 shows great variability, between strains, in terms of both number and amount of secreted proteins. In particular, Propionibacterium freudenreichii CIRM BIA 118 secreted elevated amounts of a 60 kDa protein identified by mass spectrometry as slpA. A translational fusion including slpA signal peptide and TRAIL C-terminal active extracellular part was then obtained ( FIG. 7 , pCHH04:TRAIL). This new construct was compared to pBF4:TRAIL in terms of TRAIL secretion in the CIRM BIA 118 strain. Western blot analysis of the corresponding culture supernatants revealed the presence of a 21 kDa polypeptide identified as TRAIL by immunoblotting. As shown in FIG. 9 (left panel), secretion into the extracellular medium of TRAIL was higher using the pCHH04:TRAIL construct, compared to pFB4:TRAIL. Furthermore, different strains of Propionibacterium freudenreichii were transformed using pCHH04:TRAIL and secreted TRAIL ( FIG. 9 , right panel). The amount of TRAIL secreted by Propionibacterium freudenreichii CIRM BIA 118 (pBF4:TRAIL) was 0.178 ng/ml while it was 22 ng/ml in Propionibacterium freudenreichii CIRM BIA 118 (pCHH04:TRAIL) (Table V). This evidences a 100-fold increase in secretion efficiency. Moreover, similar secretion efficiency was demonstrated in different strains of Propionibacterium freudenreichii (including CIRM BIA 512, 125, 118, 129 and 122 strains, FIG. 9 right panel and Table V).





TABLE V



ELISA quantification of human TRAIL in Propionibacterium

freudenreichii supernatants (corresponding to supernatants

analysed by western blot and shown in FIG. 9, left panel)






ELISA
Human soluble TRAIL (ng/ml)









Cirm 118 WT
0


Cirm 118 pFB4:TRAIL
0.178


Cirm 118 pFB4
0


Cirm 118 pCHH04:TRAIL
22


Cirm 122 pCHH04:TRAIL
Non Determined


Cirm 125 pCHH04:TRAIL
24


Cirm 129 pCHH04:TRAIL
22


Cirm 512 pCHH04:TRAIL
Non Determined


Cirm 118 pCHH04:TRAIL
23



































IV.3 Discussion
The expression and secretion construction pCHH04: TRAIL allows higher amounts of TRAIL secretion into the extracellular medium than the pFB4: TRAIL construction. The slpA promoter, isolated from strain CIRM BIA 118, is most probably stronger than PF963 promoter. The slpA signal peptide allows efficient secretion of the TRAIL human cytokine. Other slp proteins were then identified from the genomic sequence of several Propionibacterium freudenreichii strains. The corresponding signal peptides are presented in Table III and may also be used to allow heterologous protein expression in Propionibacterium freudenreichii.
V-Other Embodiments of the Invention
In the examples above, the Inventors made use of a P. freudenreichii signal peptide.
Advantageously, the present invention is implemented using one or more of the P. freudenreichii signal peptides listed in tables I to III above.
Of course, the present invention can be generalized to the use of any propionibacteria signal peptide. The means and methods described in detail above to obtain a recombinant vector enabling the expression and secretion into the extracellular medium of one or more amino acid sequences of interest (the Val 114 -Gly 281 sequence of TRAIL in particular) are indeed suitable to the use of any propionibacteria signal peptide.
As an example, a recombinant vector in accordance with the present invention can be constructed using a signal peptide selected from the signal peptides of Propionibacterium acnes , whose genome is available in databases. Table II above gives examples of putative P. acnes signal peptide sequences.
One approach to identifying other signal peptides applicable to the present invention in particular involves aligning sequences in search of Propionibacterium sp. sequences homologous (for example, with roughly 80% homology, preferably at least 85%, 90%, 95% or roughly 98% homology) to signal peptides sequences of a propionibacterium used as a reference (such as P. freudenreichii or P. acnes ), or searching for proteins secreted by a given propionibacterium and identifying possible corresponding signal peptides. Current computer software tools make it possible to easily identify putative signal peptide sequences within protein or genomic sequences (for example, the SignalP 3.0 software; Center for Biological Sequence Analysis, CBS; http://www.cbs.dtu.dk/services/SiqnalP/; Emanuelsson et al., 2007).
REFERENCES


Jan et al. 2002. Cell Death Differ. 9:179-188.
Falentin et al. 2010a. Plos One 5:e11748.
Falentin et al. 2010b. Int. J. Food Microbiol. 144:10-19.
Hervé et al. 2007. Int J Food Microbiol. 113:303-314.
Lan et al. 2007. Apoptosis 12:573-591.
Lan et al. 2007. Br J. Nutr. 97:714-724.
Lan et al. 2008. Br J. Nutr. 100:1251-1259.
Lacour et al. 2001. Cancer Res. 61:1645-1651.
Lacour et al. 2003. Oncogene 22:1807-1816.
Leverrier et al. 2004. Arch. Microbiol. 181:215-230.
Meurette et al. 2005. Clin. Cancer Res. 11:3075-3083.
Meurette et al. 2006. Ann N Y Acad. Sci. 1090:209-216.
Meurette et al. 2007. Cancer Res. 67:218-226.
Kiatpapan et al. 2000. Appl. Environ. Microbiol. 66:4688-4695.
Tarze et al. 2007. Oncogene 26:2606-2620.
Ganai et al. 2009. British Journal of Cancer. 101:1683-1691.
Hu et al. 2009. Cancer Gene Therapy. 16:655-663.
Patch J. A. and Barron A. E., 2002. Curr. Opin. Chem. Biol., 6(6):872-877. Review.
Ashkenazi A et al. 1999. J. Clin. Invest. 104:155-162.
Sträter J. et al. 2002. Gastroenterology 122:659-666.
Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3 rd Ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Emanuelsson et al. 2007. Nature Protocols 2:953-971.
Bougle et al. 1999. Scand. J. Gastroenterol. 34:144-148.
Zarate et al. 2000. J. Food Prot. 63:1214-1221.
Zhang et al. 2010. Cancer Gene Therapy 17:334-343
Lortal et al. 1993. Applied and Environmental Microbiology 59(8): 2369.