Patent Application
20240392305
The present invention relates to genetically modified bacteria capable of inducibly expressing a nucleic acid sequence of interest, genetic constructs, vectors and uses thereof.
Lactic acid bacteria (LAB) are attractive candidates for the production of molecules, in particular for therapeutic purposes. Indeed, they are bacteria with “GRAS” (Generally Recognized As Safe) status and, as such, they benefit from a healthy reputation. They are relatively easy to manipulate genetically and, therefore, potentially capable of expressing heterologous proteins through controlled expression systems. Moreover, they can be administered orally or intranasally and they can target many pathologies, including digestive ones, since they remain metabolically active during their passage through the digestive tract.
Among model LAB, Lactococcus lactis represents the reference bacterium in both academic studies and recombinant protein production platforms. For example, a controlled expression system based on the NisRK regulatory genes is widely used in L. lactis. This system involves a promoter positively controlled by the 2-component system NisRK and it is inducible via the nisin peptide. However, notwithstanding its usefulness, this expression system is not that much controlled, i.e. the promoter remains active at a significant level in the absence of the inducer. In contrast, only a few academic studies exist for S. thermophilus despite that this bacterium shows some advantages over L. lactis, such as its optimum growth temperature of 37° C. or its natural competence for transformation. Indeed, this later property facilitates genetic manipulation and allows S. thermophilus to naturally incorporate exogenous DNA into its chromosome by homologous recombination. However, there is no satisfactory system for the controlled expression of proteins of interest in S. thermophilus.
There thus remains a genuine need for an effective system allowing the controlled expression of proteins of interest in lactic acid bacteria such as S. thermophilus.
The present invention is believed to meet such need by providing a new inducible expression system based on the use of a quorum sensing cellular communication mechanism discovered in streptococci.
In some previous studies, the Inventors discovered a new quorum sensing system present in some Streptococcus thermophilus strains of industrial interest (Fleuchot et al., PLOS ONE 8 (6): e66042, 2013). In the strain LMD-9, they showed that the shp1358 gene encodes an auto-inductive small hydrophobic peptide (SHP) matured and secreted in culture supernatants. They then identified a membrane protease (Eep) as well as a peptide transporter (PptAB) essential for the production of a mature form of SHP in the extracellular environment. They also showed that an Ami oligopeptide transporter allows the import of the mature SHP. Finally, they demonstrated an interaction between the mature SHP peptide and a transcriptional regulator of the Rgg family and showed that the activity of Rgg is positively controlled by its interaction with the mature SHP. The SHP1358/Rgg1358 (STER_RS06695 according to the new NCBI nomenclature) complex controls the expression of two groups of target genes: the shp1358 gene encoding the SHP precursor peptide and the ster_1357-ster_1355 operon (STER_RS10575, STER_RS06690 and STER_RS06685), involved in the production and secretion of a cyclic peptide, named streptide, the function of which remains unknown. A schematic representation of the quorum sensing mechanism involving the transcriptional regulator Rgg1358 and the hydrophobic peptide SHP1358 is shown in
In an original way, the Inventors designed a new inducible expression system based on the elements of this quorum sensing cellular communication mechanism. The expression system of the invention can be implemented in two variations: a complete version that is auto-regulated (self-induction) and an incomplete version that is regulated by the addition of a SHP peptide (external induction).
In the complete version, all the genes of the locus are present and/or have been introduced into the bacterium used to produce the protein. When the bacterium is cultivated in a medium devoid of hydrophobic peptides, the system is self-induced by its own SHP production and allows the strong expression of the protein of interest at the end of the exponential growth phase.
In the incomplete version, the bacterium does not contain the gene encoding the SHP peptide. In this version, the expression of the protein can be triggered during the growth of the bacterium by adding the SHP peptide to the culture medium. This strategy allows to disconnect the bacterial growth and the production of proteins of interest. This is particularly advantageous to avoid yield losses in the production of proteins affecting bacterial growth.
The Inventors validated both variations of the expression system with a marker protein (a luciferase) and a secreted protein of interest (elafin, which inhibits protease activity in patients with diseases inflammatory bowels).
Similar loci comprising a gene encoding a SHP, a gene encoding a regulator transcriptional Rgg and a promoter positively controlled by the complex SHP/Rgg can be found in many streptococci in one or several copies, and can therefore be used according to the present invention.
In an aspect, the present invention thus concerns a genetically modified bacterium capable of inducibly expressing a nucleic acid sequence of interest, wherein:
A “genetically modified bacterium” as used herein, refers to a bacterium in which the genetic material of the bacterium has been altered using genetic engineering techniques. In particular, the term “genetically modified” refers to a bacterium with a modification, such as a gene knockout, or wherein an exogenous gene is introduced for expression of a protein. Those skilled in the art will appreciate that there are multiple ways to produce genetically modified bacteria such as by introducing heterologous nucleic acids and/or by generating mutations. In some embodiments a nucleic acid (e.g. a gene or portion thereof) is introduced into a bacterium using a suitable technique. In some embodiments a bacterium is transformed with a nucleic acid by a suitable technique. Non-limiting examples of suitable techniques for introducing a nucleic acid into a bacterium include electroporation, transduction (e.g., injection of a nucleic acid by a bacteriophage), microinjection, by inducing competence (e.g., by addition of alkali cations, cesium, lithium, polyethylene glycol, competence-inducing peptide or by osmotic shock), the like or combinations thereof. A nucleic acid can be introduced into a bacterium in the form of a linear or circular plasmid, for example. In some embodiments, transformed bacterium are selected for integration of a nucleic acid into the genome of the bacterium by using a suitable selection method (e.g. a selection marker).
As used herein, a “genetic construct” refers to an artificially-designed segment of DNA that is used to introduce genetic material into a target, namely a bacterium.
The “inducible expression” means that the protein can be regulated. By inducibly expressing a nucleic acid sequence of interest is meant herein that expression of a nucleic acid sequence of interest is at least in part influenced by at least one inducer, i.e. a SHP peptide. Hence, by regulating the amount of inducer that is administered to said expression system, one is capable of regulating the amount of expression of said nucleic acid sequence of interest. Preferably, expression of a nucleic acid sequence of interest is dependent on the presence of an inducer. This means that said nucleic acid is expressed in the presence of an inducer, while it is expressed to a significant lesser extent, or not expressed, in the absence of said inducer. In a preferred embodiment, said nucleic acid sequence is essentially not expressed in absence of said inducer.
In principle, any nucleic acid sequence of interest is inducibly expressed by a genetically modified bacterium according to the present invention. In particular, suitable applications for an inducible gene expression system according to the present invention are the production of a protein with therapeutic properties or health benefits or the production of proteins in the fields of white biotechnology and green chemistry. In an embodiment, the invention relates to a genetically modified bacterium as defined above, with the proviso that the nucleic acid sequence of interest does not encode a luciferase.
As used herein, “SHP” refers to a small hydrophobic peptide, i.e. a secreted peptide signaling molecule (or pheromone), which can regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, the SHP binds to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Generally, the sequence of the SHP peptide is from 5 to 12 amino acids in length, has a D or E in position 1, has a G in positions 7, 8 and/or 9, has at least two I and contains at least 40% of amino acids I, L and V.
As used herein, “Rgg” refers to a regulatory protein, i.e. a transcription factor. Cytoplasmic transcription factors known as “Rgg proteins” are peptide pheromone receptors ubiquitous in Firmicutes, in particular in Streptococcus genus.
“Identity” with respect to percent amino acid sequence “identity” for peptides and proteins is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the target sequences after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Percent sequence identity is determined by conventional methods. Briefly, two amino acid sequences are aligned to optimize the alignment scores using the ClustalW algorithm (Thompson et al., Nuc. Ac. Res. 22:4673-4680, 1994) and PAM250 weight matrix (Dayhoff et al., “Atlas of Protein Sequence and Structure.” National Biomedical Research Foundation. Washington, DC 5:345-358, 1978) and default parameters as provided by the program MegAlign (DNASTAR, Inc.; Madison, WI). The percent identity is then calculated as: [Total number of identical matches×100] divided by [length of the longer sequence+number of gaps introduced into the longer sequence in order to align the two sequences].
In an embodiment, the Rgg protein has at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
In an embodiment, the promotor regulated by the protein complex SHP/Rgg comprises a DNA-binding site selected from the group consisting of: GCAAATAGGGGAATA (SEQ ID NO: 14), GCAAATAGGGGAATT (SEQ ID NO: 15), GCATATAGGGGAATA (SEQ ID NO: 16), GCATATAGGGGAATT (SEQ ID NO: 17), GCAAATATGGGAATA (SEQ ID NO: 18), GCAAATATGGGAATT (SEQ ID NO: 19), GCATATATGGGAATA (SEQ ID NO: 20), GCATATATGGGAATT (SEQ ID NO: 21), AATTGCGTATAAGGGAAA (SEQ ID NO: 22), AATTGCITATAAGGGAAA (SEQ ID NO: 23) and ATTTCATATCTTCAATTTT (SEQ ID NO: 3).
In an embodiment, the SHP peptide is selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, preferably SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.
S. thermophilus strains (LMD-9, CNRZ1066,
EGIIVIVVG (SEQ ID NO: 24)
EGIIVILVG (SEQ ID NO: 25)
ESIIVIAVG (SEQ ID NO: 27)
DIIIFPPFG (SEQ ID NO: 28)
PFG
DIIIIVGG (SEQ ID NO: 29)
EIIIIIAL (SEQ ID NO: 30)
CIYTIVGGV (SEQ ID NO: 31)
CIYIIVGGV (SEQ ID NO: 32)
As shown by the Inventors, different SHP/Rgg quorum sensing systems can be present in the same bacterium and SHP peptides cross-react with different Rgg proteins, and inversely.
In an embodiment, the invention thus relates to a genetically modified bacterium as defined above, wherein:
In an embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the bacterium expresses a native form of the SHP peptide.
As used herein, the “precursor” or “native form” of the SHP peptide refers to a full-length precursor peptide comprising the sequence of the SHP peptide. The SHP peptide is then produced after a cleavage by a bacterial protease, in particular by a membrane protease. When the genetically modified bacterium expresses the native form of the peptide, the expression is auto inducible.
In an embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the bacterium does not express a native form of the SHP peptide.
When the genetically modified bacterium does not express the native or mature form of the peptide, the expression is externally inducible. In such an embodiment, the SHP peptide must be added to the culture medium of the bacterium to induce the expression of the nucleic acid sequence of interest.
In an embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the genetic construct further comprises a nucleic sequence encoding the Rgg protein.
In an embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the genetic construct further comprises a nucleic sequence encoding the SHP peptide.
In an embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the genetic construct is plasmid-borne or is integrated in the genome of said bacterium.
In an embodiment, the genetically modified bacterium according to the invention is a lactic acid bacterium, preferably from the genus Streptococcus.
In an embodiment, the genetically modified bacterium according to the invention is selected from the group consisting of: S. thermophilus, S. salivarius, S. agalactiae, S. pyogenes, S. pneumoniae, S. suis, S. mutans and S. mitis, preferably S. thermophilus or S. salivarius.
In an embodiment, the genetically modified according to the invention is a non-pathogenic streptococcus, such as S. thermophilus or S. salivarius.
In an embodiment, the genetically modified according to the invention is a pathogenic streptococcus, such as S. agalactiae, S. pyogenes, S. pneumoniae, S. mitis, S. mutans and S. mitis.
In an embodiment, the genetically modified bacterium according to the invention is a probiotic strain.
In an embodiment, the invention relates to a genetically modified bacterium as defined above, with the proviso that the genetically modified bacterium is not, or is not derived from, S. thermophilus LMD-9 or S. thermophilus LMG18311/ATCC BAA-250.
In an embodiment, the genetically modified bacterium according to the invention is from the genus
In an embodiment, the genetically modified bacterium according to the invention is selected from the group consisting of E. faecalis and E. faecium, preferably E. faecalis.
In some cases, the production of proteins of interest by bacteria can be rendered difficult due to the surface proteolysis, especially in Gram positive bacteria. Indeed, surface proteolysis leads to a degradation of these proteins during and/or after their export, leading to a drop in yield and/or a deterioration of the structure and activity of the protein of interest.
In an embodiment, the invention thus relates to a genetically modified bacterium as defined above, wherein the bacterium has no or low surface proteolytic activity.
Preferably, a bacterium having no or low surface proteolytic activity is a bacterium of the species Streptococcus thermophilus, wherein an endogenous surface protease homologous to the protein designated STR_RS07745 in Streptococcus thermophilus CNRZ1066 has a reduced or abolished expression and/or activity. Bacteria having no or low surface proteolytic activity to be used in the present invention and method to produce such bacteria are described in details in PCT/EP2021/055561.
Unexpectedly, the Inventors also observed that, in some cases, a residual expression of the protein of interest can be detected even in the absence of the peptide SHP. This leak of the controlled system can be explained by the fact that other loci encoding similar quorum sensing components, including similar SHP peptides, are naturally present in many streptococci.
In order to overcome this issue, the Inventors have further developed an improved version of the expression system that avoids any expression of the sequence of interest unless the peptide SHP is added to the culture medium.
In such embodiments, the ABC-type transporter that enables export of the SHP peptide to the extracellular medium and/or the membrane protease that enables cleavage of the native form of the SHP peptide, are not functional or inactivated. In these embodiments, the similar SHP peptides that can be naturally produced by the bacterium are not matured and thus, cannot affect the expression system of the present invention.
In an embodiment, the invention relates to a genetically modified bacterium as defined above, wherein an ABC-type transporter called “PptAB”, which enables export of the SHP peptide to the extracellular medium, is not functional or is inactivated.
In an embodiment, said PptAB comprises or consists in the amino acid sequence SEQ ID NO: 42 and SEQ ID NO: 43 or any variants thereof.
S. thermophilus strain CNRZ1066.
In an embodiment, the invention relates to a genetically modified bacterium as defined above, wherein a transmembrane protease called “Eep”, which enables cleavage of the native form of the SHP peptide, is not functional or is inactivated.
In an embodiment, said Eep comprises or consists in the amino acid sequence SEQ ID NO: 44 or any variants thereof.
In another aspect, the invention relates to a genetic construct comprising:
In an embodiment, the invention relates to a genetic construct as defined above, wherein said genetic construct further comprises genetic elements allowing the integration of the nucleic acid sequence of interest into the genome of a bacterium.
In an embodiment, the invention relates to a genetic construct as defined above, with the proviso that the nucleic acid sequence of interest does not encode a luciferase
In another aspect, the invention relates to a genetic vector comprising a genetic construct as defined above.
In an embodiment, the invention relates to a genetic vector as defined above, said vector being a plasmid, in particular a replicative plasmid or an integrative plasmid.
In another aspect, the invention relates to a the use of a genetically modified bacterium as defined above, a genetic construct as defined above or a genetic vector as defined above, for producing a protein of interest, said protein of interest being encoded by the nucleic acid sequence of interest.
In another aspect, the invention relates to a method for producing a protein of interest comprising a step of culturing a genetically modified bacterium as defined above.
In another aspect, the invention relates to a method for producing a protein of interest comprising a step of genetically modifying a bacterium with a genetic construct or a genetic vector as defined above.
In an embodiment, the invention relates to a method as defined above, wherein said genetically modified bacterium is cultivated in a culture medium supplemented with the SHP peptide, preferably during the exponential phase of growth.
In an embodiment, the invention relates to a method as defined above, wherein said genetically modified bacterium is cultivated in a culture medium in the absence of SHP.
In an embodiment, the invention relates to a method as defined above, with the proviso that the genetically modified bacterium is not, or is not derived from, S. thermophilus LMD-9 or S. thermophilus LMG18311/ATCC BAA-250.
In another aspect, the invention relates to a genetic vector for inducibly expressing a nucleic acid sequence of interest comprising:
In another aspect, the invention relates to a kit for inducibly expressing a nucleic acid sequence of interest comprising:
The following Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the Inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
S. thermophilus wild-type strain LMD-9 (Makarova et al., Proc. Natl. Acad. Sci. USA 103 (42): 15611-6, 2006) was used as a chromosomal DNA donor to retrieve the shp/rgg1358 system by PCR (
Streptococcus thermophilus strains used in this study.
aKm, Spec, Cm and Erm = resistance to kanamycin, spectinomycin, chloramphenicol and erythromycin, respectively.
bThe arrows indicate construction via transformation with chromosomal DNA or a plasmid.
Restriction enzymes, T4 DNA ligase (New England Biolabs) and Phusion DNA polymerase (Finnzymes) were used in accordance with the manufacturers' instructions. Standard methods were used to carry out DNA purification, restriction digestion, PCR, ligation, and sequencing. S. thermophilus strain CNRZ1066 or its derivatives were transformed using natural competent cells (Gardan et al., J. Bacteriol. 191:4647-4655, 2009) prepared with the addition of synthetic competence peptide (ComS, LPYFAGCL (SEQ ID NO: 45) at a concentration of 1 μM. L. lactis electrocompetent cells were prepared and transformed as described previously (Holo H & Nes I F, Appl. Environ. Microbiol. 55:3119-3123, 1989). The plasmids used are listed in Table 6.
luminescens and an kanamycin resistance cassette surrounded by two
aErm, Km and Cm = resistance to erythromycin, kanamycin and chloramphenicol, respectively.
SHP1358 (EGIIVIVVG, SEQ ID NO: 24), EGIIVILVG (SEQ ID NO: 25), EGIIVIGVG (SEQ ID NO: 26), ESIIVIAVG (SEQ ID NO: 27) and ComS have been synthesized. Purity were greater than 95%. Peptides were reconstituted as 1 mM stock in dimethyl sulfoxide for SHP1358 and water for ComS. Subsequent dilutions (100 μM) were made in water for both peptides before their final dilution in CDM at 1 or 0.5 μM. All stock solutions were stored à−20° C.
The overlapping PCR method was used to delete the sepM (STR_RS07745) and htrA (STR_RS09505) genes in strain CNRZ1066 and replace them with a spectinomycin (spec) and a kanamycin cassette (aphA3), respectively as previously described (Gardan et al., 2009). Briefly, the spec and the aphA3 cassettes were amplified via PCR using pAT28 and pKa plasmids respectively, as DNA template. Upstream and downstream fragments of the sepM and htrA genes were amplified using chromosomal DNA from strain CNRZ1066 as a template. The upstream fragments, the cassettes and the downstream fragments were fused by overlapping PCR. The resulting PCR fragments were used to transform strain CNRZ1066 leading to the construction of strain TIL1523 (sepM::spec) and strain TIL1535 (htrA::aphA3). The TIL1536 (sepM::spec htrA::aphA3) was constructed by transforming strain TIL1523 with chromosomal DNA from strain TIL1535. The same method was used to delete the pptAB genes (STR_RS07560-STR_RS07565) and replace them with an erythromycin cassette. The erm cassette was amplified via PCR using pG+host9 plasmid as a template. The protocol was the same except that chromosomal DNA of strain CNRZ1066 was used for the amplification of the upstream and downstream fragments. The resulting product of the overlapping PCR was used to transform strain CNRZ1066 and strain TIL1525 leading to the construction of strain TIL1568 (ΔpptAB::erm) and strain TIL1566 (blp::rgg1358-Pster1357-luxAB ΔpptAB::erm), respectively.
To study the relevance of two variants of the inducible expression system with the luciferase encoding genes, two derivatives of pGICB004a plasmid (Table 6), pGICB004a::shp-rgg1358-Pster1357 (complete variant) and pGICB004a::rgg1358-Pster1357 (incomplete variant), were constructed as follows. The shp-rgg1358-Pster1357 fragment was amplified by PCR and chromosomal DNA from strain LMD-9 was used as a template; double digested with the restriction enzymes SpeI and EcoRI; and finally ligated into pGICB004a between the related restriction sites leading to the construction of pGICB004a::shp-rgg1358-Pster1357 plasmid. A similar approach was used for the construction of pGICB004a::rgg1358-Pster1357. Both plasmids were linearized by ScaI. Linearized plasmid pGICB004a::shp-rgg1358-Pster1357 and pGICB004a::rgg1358-Pster1357 were used to transform strain CNRZ1066 leading to strains TIL1524 (blp::shp-rgg1358-Pster1357-luxAB) and strain TIL1525 (blp::rgg1358-Pster1357-luxAB), respectively.
To study the functionality of the two variants of the inducible expression system with the production of elafin, two plasmids pEIa::shp-rgg1358-Pster1357 and pEIa::rgg1358-Pster1357 were constructed by replacing the luxAB genes and the aphA3 cassette of plasmid pGICB004a::shp-rgg1358-Pster1357 and pGICB004a::rgg1358-Pster1357 with the elafin gene and a chloramphenicol cassette (P32Cm), as follows. The elafin gene fused to a signal peptide was constructed and amplified by PCR. The chloramphenicol cassette was amplified by PCR from plasmid pNZ5319. The two fragments were joined by overlapping PCR, double digested with the restriction enzymes SalI and EcoRI; and finally ligated into pGICB004a::shp-rgg1358-Pster1357 and pGICB004a::rgg1358-Pster1357 between the related restriction sites leading to the construction of pEIa::shp-rgg1358-Pster1357 and pEIa::rgg1358-Pster1357. Both plasmids were linearized by ScaI. Linearized plasmid pEIa::shp-rgg1358-Pster1357 and plasmid pGICB004a::rgg1358-Pster1357 were used to transform strain TIL1536 (ΔhtrA::aphA3 ΔsepM::spec) leading to strain TIL1551 (blp::shp-rgg1358-Pster1357-elafin-P32 cm ΔhtrA::aphA3 ΔywdF::spec) and TIL1552 (blp::rgg1358-Pster1357-luxAB ΔhtrA:aphA3 ΔywdF::spec), respectively. Chromosomal DNA of strain TIL1568 (ΔpptAB::erm) was used to transform strain TIL1552 leading to strain TIL1567 strain (blp::rgg1358-Pster1357-elafin-P32 cm ΔhtrA::aphA3 ΔywdF::spec ΔpptAB:erm). All constructions were verified by PCR and sequenced when necessary.
Cells were grown overnight at 42° C. in CDM. These cultures were then diluted in 50 ml of CDM to a final OD600 of 0.05 and incubated at 42° C. Aliquots of 1 ml of the culture were sampled at regular intervals until the culture reached stationary phase and analyzed as follows: OD600 was measured with 1 ml of culture, then 10 μl of a 0.1% nonyl-aldehyde solution was added and the luminescence was immediately measured with a Junior LB9509 (Berthold technologies). Induction of the expression of the luciferase encoding genes in strain TIL1525 was performed with the addition of SHP peptide (EGIIVIVVG, SEQ ID NO: 24) in the growth medium at a final concentration of 1 μM two hours after the beginning of the culture. Results were reported in Relative Luminescent Units divided by the OD600 (RLU/OD600). Figures shown (
For the assessment of the maximum luciferase activity reached with the addition of different SHPs in the culture media of strain TIL1566 (blp::rgg1358-Pster1357-luxAB-aphA3 ΔpptAB::erm), cells were grown overday at 37° C. in CDM. These cultures were diluted to a final OD600 of 0.05 in 1 ml of CDM. Synthetic SHP peptides were added in the culture medium at a final concentration of 1 μM. 250 μl of these diluted cultures were transferred to the wells of a covered sterile white microplate with a transparent bottom. The cultures' OD600 and luminescence values were monitored at 37° C. every 10 minutes in an Infinite M200 spectroluminometer, as previously described (Fontaine et al., J. Bacteriol. 192:1444-1454, 2010). The maximum of luminescence was recovered from each kinetic. The maximum activity was reported in Relative Luminescent Units divided by the OD600 (RLU/OD600) and measured as the mean of three independent experiments.
For the preparation of the samples, derivatives of strain CNRZ1066 were grown in CDM at 42° C. When necessary, induction was performed with the addition of SHP peptide (EGIIVIVVG, SEQ ID NO: 24) at a final concentration of 1 μM for strain TIL1552 and 0.5 UM for strain TIL1567 in the growth medium at OD600 of 0.2. When the cultures reached the desired OD600, 10 ml were centrifuged at 3200 g for 10 min at room temperature. Filtered supernatants (0.22 μM, Millex GV PVDF, Millipore) were further ultrafiltered through Amicon devices (Ultra-15 [3-kDA cutoff]; Millipore) for 1 hour at 25° C. at 3200 g. Five hundred μl containing the secreted elafin were recovered in the upper compartment of the Amicon device.
For the Western blot experiments, 10 μl of the samples were diluted in Laemmli Buffer (4×) and heated to 95° C. for 5 minutes. Samples were run on Precast Bis-Tris Gel 4-12% (NuPAGE 1.5 mm×10; Invitrogen) and transferred to nitrocellulose membranes (Trans-Blot turbo 0.2 μm PVDF; Biorad). The membrane were blocked two hours at room temperature in a TBST solution (Tris 0.1M pH 7.5, NaCl 1.5 M, Tween 20 1%) containing 5% skimmed milk and then hybridized for 2 hours at room temperature in TBST solution with anti-elafin antibody (Santa Cruz sc-398075, 1/1000 dilution). Four washing steps were performed in TBST solution, one for 15 min and three for 5 min and detection was achieved using secondary antibody coupled to peroxidase (Abliance BI2413C, 1/1000 dilution) during 1 hour. Four washing steps were again performed as previously described before the addition of a solution containing luminol (ECL prime, GE healthcare) for 5 min. Five μl of elafin (RD system, 1/100 dilution) were used as a control. Detection was finally recorded using a Chemidoc (Biorad).
The S. thermophilus pangenome contains several SHP/Rgg systems. Some strains have accumulated up to 6 systems, like strain LMD-9. Among these different SHP/Rgg systems, SHP/Rgg1358 (comprising the promoter sequences SEQ ID NO: 18 and SEQ ID NO: 20 and the sequences encoding the Rgg protein of SEQ ID NO: 6 and the mature SHP of SEQ ID NO: 24) is the most well-known (Fleuchot et al., 2011). The rgg gene is annotated STER_RS06695 in NCBI but was designed as rgg1358 in previous publications based on a primary annotation of strain LMD-9 (Makarova et al., 2006). The shp gene is not annotated but is located in front of the rgg1358 gene in a divergent orientation. This gene encodes a 24 amino acid peptide (MKKQILLTLLLVVFEGIIVIVVG, SEQ ID NO: 33) which is matured by cleavage between the phenylalanine and the glutamic acid residues by the membrane protease Eep and secreted in the extracellular medium by the transporter PptAB before being reimported by the Ami oligopeptide permease. Once inside the cellular compartment, the mature SHP interacts with Rgg1358 to positively control the expression of the shp gene itself and of a polycistronic operon whose the three first genes are involved in the production and secretion of a cyclic peptide called streptide (
Two variants were assessed (
It is highly probable that in strain TIL1525, one (or more) of these SHPs interacts with the Rgg1358 Of strain LMD-9 to control its activity. To confirm this hypothesis, we introduced the ΔpptAB::erm in strain TIL1525 (blp::rgg1358-Pster1357-luxAB) creating strain TIL1566. In a ΔpptAB::erm mutant, none of the SHPs encoded by CNRZ1066 genome can be exported. The positive feedback loops at the origin of the production of the SHPs is blocked and consequently, the SHPs are not synthesized. Indeed, in strain TIL1566, no luminescence could be measured (
The elafin protein is a protease inhibitor of human origin found in the gut and known to have a protective effect against inflammatory bowel disease. The gene encoding this protein was chosen to test the relevance of the inducible system for the production of a secreted protein with therapeutic properties. For that purpose, the luxAB genes and the aphA3 kanamycin cassette of the two plasmids pGICB004a::shp-rgg1358-Pster1357 and pGICB004a::rgg1358-Pster1357 were replaced with the elafin gene fused downstream of a DNA fragment encoding the secretion signal peptide of USP45, the main secreted protein of L. lactis and a P32Cm cassette conferring resistance to chloramphenicol (
These results were confirmed with assays of porcine pancreatic elastase. Elafin is an inhibitor of the elastase. As expected, when supernatant of strain 1551 (blp::shp-rgg1358-Pster1357-elafin-P32 cm ΔhtrA::aphA3 ΔywdF::spec) was recovered at OD600 1, concentrated and used in this assay to compare its activity to the one of supernatant of strain 1536 (ΔhtrA::aphA3 ΔywdF::spec), a 21% reduction of the activity of the elastase was observed. Similarly, the activity of the supernatant of strain 1552 (blp::rgg1358-Pster1357-elafin-P32 cm ΔhtrA::aphA3 ΔywdF::spec) sampled at OD600 1 in a medium containing SHP1358 was 32% reduced compared to the one of strain 1536 (
The fact that endogenous SHPs of strain CNRZ1066 can activate the SHP/Rgg1358 system in strain TIL1525 (blp::rgg1358-Pster1357-luxAB) indicates that other SHPs than SHP1358 can be potentially useful to control the activity of Rgg1358. In order to assess their efficiency, we used their synthetic mature form to record the maximum luciferase activity of strain TIL1566 (blp::rgg1358-Pster1357-luxAB ΔpptAB::erm) in CDM and compared it to the one obtained with SHP1358. We chose the mature SHPs of strain CNRZ1066 similar to the mature SHP1358 (EGIIVIVVG, SEQ ID NO: 24): the SHP associated to Rgg STR_RS07375 (EGIIVILVG, SEQ ID NO: 25), the SHP associated to Rgg STR_RS09145 (EGIIVIGVG, SEQ ID NO: 26) and the SHP associated to Rgg STR_RS04530 (ESIIVIAVG, SEQ ID NO: 27). The most efficient peptide was ESIIVIAVG (SEQ ID NO: 27) with a maximum relative luciferase activity that reached 95% of the one obtained with SHP1358. The two other peptides were less efficient with percentage of 86% with EGIIVILVG (SEQ ID NO: 25) and only 27% with EGIIVIGVG (SEQ ID NO: 26).
| Number | Date | Country | Kind |
|---|---|---|---|
| 21305953.8 | Jul 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/067224 | 6/23/2022 | WO |
