Patent Application
20220220489
The present invention provides a circular DNA vector, which may be used to introduce specific a mutation in a target region of a host cell. The present invention further provides methods for using the circular DNA vector for generating engineered host cells. The circular DNA vector and methods are useful for studying gene functions and generating cells producing recombinant gene products.
Lactobacilli has been extensively used as probiotics and have become increasingly studied as delivery vehicles of medically relevant recombinant proteins to mucosal surfaces. In contrast, the genetic tools (especially mutagenesis) to investigate and enhanced their probiotic activity are rather poorly developed. The traditional genome engineering methods are largely dependent on the bacterial transformation efficiency. This defect can be overcome by the conditional replicate plasmid assisted recombineering, such as plasmids containing a thermosensitive replication origin of pWV01, but the latter may have a limited host range. Therefore, more flexible and effective genome editing strategies need to be developed for a better understanding and application of these health-promoting microorganisms.
A first aspect of the present invention provides a circular DNA vector comprising:
The circular DNA vector is useful as targeting vector for targeted integration of a (gene) sequence in the genome host by homologous recombination. The circular DNA vector is particularly useful for gene targeting in host cells, which are generally known to be difficult to transform, such as species of Lactobacilli and Bifidobacteria.
The circular DNA vector is particularly useful for gene targeting in accordance with the methods of the present invention.
Accordingly, in a second aspect of the present invention provides a method for introducing recombination between a circular DNA vector and a target region of the genome of a host cell, said method comprising the steps of:
In a third aspect, the present invention provides a method for generating a host cell having a mutation in a target gene of the genome of a host cell, said method comprising the steps of:
In a fourth aspect, the invention provides a method for generating a host cell having loss of function in a target gene of the genome of a host cell, said method comprising the steps of:
In a fifth aspect, the invention provides a method for generating a host cell having gain of function in a target gene of the genome of a host cell, said method comprising the steps of:
In a sixth aspect, the invention provides a method for preparing a host cell expressing a recombinant polypeptide, said method comprising the steps of:
A further aspect of the present invention provides a recombinant host cell obtained by any of the methods of the present invention.
In yet a further aspect, the present invention provides the use of the circular vector of the present invention for introducing a gene sequence in the genome of a host cell.
In one further aspect, present invention provides the use of the circular vector of the present invention for increasing tissue adhesion of a host cell.
The physical map of IPSD vector pINTZrec. Cmr, chloramphenicol resistance gene; Rec, recombinase gene β.
Growth of the indicated Lactobacillus strains on MRS agar plate supplemented with 10 μg/ml chloramphenicol and in presence or not of 100 ng/ml SpplP.
(a) L. paracasei BL23, (b) L. acidophilus ATCC 4356, (c) L. plantarum NL42.
Deletion of the upp gene in L. gasseri DSM 14869 by IPSD assisted recombineering.
(a) Single-crossover integration of the upp-Cmr fragment into the chromosome of L. gasseri DSM 14869. Colony PCR using primers pIrec-F and pIrec-R was performed to detect the intact plasmid pINTZrec-upp. Ten colonies without the corresponding band were regarded as candidate single-crossover clones, which were labelled “+”.
(b) The single-crossover event in the ten candidate clones was further confirmed by DNA extraction and PCR using primers uppleft-F and pIrecSC-R (the upper gel), or pIrecSC-F and uppright-R (the lower gel). The correct clones were labelled “+” and the uncertain clones were labelled “?”. (c) Colony PCR using primers uppseq-F and uppseq-R was performed to detect upp double-crossover deletion mutants selected on SDM medium supplemented with 5-FU. M, GeneRuler™ 1 kb DNA ladder; P, DNA of L. gasseri DSM 14869 harboring plasmid pINTZrec-upp; WT, DNA of wild type L. gasseri DSM 14869.
The inducible promoters used to drive the expression of recombinase are not effective in some strains. Growth of the indicated L. sakei NC03 strain (a) and L. rhamnosus GG strain (b) on MRS agar plate supplemented with 10 μg/ml chloramphenicol and in the presence or absence of 100 ng/ml SppIP.
Schematic representation of the EPS gene cluster (A) and protein domains of N506_1778 (B) and N506_1709 (C) in L. gasseri DSM 14869. (A) The EPS gene cluster (N506_0396 to N506_0411) which is located nucleotide number 379, 930-394,580 in genome sequence (CP006803) is shown. The enzyme and gene names were annotated by BLASTP analysis using the strains L. gasseri ATCC 33323 and L. gasseri CECT 5714 (Marcotte et al. 2017). (B) The N506_1778 protein includes a YSIRK signal sequence in N-terminus and a Gram-positive LPxTG (LPQTG) motif in C-terminus. The repetitive region consists of two MucBP-like domains. (C) The N506_1709 protein includes a N-terminal YSIRK signal sequence and a C-terminal LPQTG motif. The repeat region harbors three different Rib/alpha-like repeats including one that is only partial. The N-terminus (ca. 1-900 aa) shows no similarities with other proteins in the databank, while the C-terminus (ca. 900-1456 aa) shows low percentage identity (34-48%) with the C-terminus of proteins from L. johnsonii and L. gasseri (accession number WP_095670316, QYS15157, WP_061400034, OYS08635, OYS05727, OUL52955 and AHA97914). Genes and protein structure are not represented at scale.
(A) Transmission electron microscopy pictures of L. gasseri DSM 14869 wild-type (WT), EPS mutant (ΔEPS) and complemented strains. A dense EPS layer was observed in WT, while the EPS production on the surface was significantly reduced for the EPS mutant strain. The EPS complement strain restored the EPS level to WT. Scale bar=200 nm. (B) Comparison of the thickness of EPS layer of WT, ΔEPS and EPS complement strains. The relative thickness is shown as a percentage relative to WT (set at 100%). Thickness was evaluated in 10 cells for each group and the mean±SD of thickness per cell was determined.
(A) Phenotypic analysis of L. gasseri DSM 14869 (WT) and its mutant derivatives. The overnight culture in MRS medium was vortexed and put at room temperature for 1 h. The mutant showed a cell sediment and a very clear upper solution. In contrast, a relatively homogenous suspension can be seen for the WT and the complemented strain. (B) Quantitative analysis of auto-aggregation of WT and its mutant derivatives. The overnight cultures were washed and resuspended in PBS to an OD600 of 1.5 and were left undisturbed for 5 h. The OD of the upper suspension was measured. The auto-aggregation percentage was calculated by the expression as follows: auto-aggregation (%)=(1−(OD600 5 h/OD600 0 h))×100, where OD600 5 h represents the absorbance at the 5 h time point and OD600 0 h represents the absorbance at 0 h. The auto-aggregation capacities are shown as a percentage relative to wild-type strain (set at 100%). Data represent means±SD of three independent experiments, *P<0.05, ***P<0.001. (C) Quantification of biofilm formation of WT and its mutant derivatives. The biofilms formed on polystyrene plates were assessed after 72 h of incubation in MRS medium using crystal violet staining. The absorbance values at OD570 were read and represent the capacity of biofilm formation. Data represent means±SD of three independent experiments, ***P<0.001.
Comparison of the adhesion ability of L. gasseri DSM 14869 wild-type, EPS mutant and complemented strain. The adhesion rates are shown as a percentage relative to wild-type (set at 100%). Each panel shows the adhesion ability towards (a) Caco-2 cells, (b) HeLa cells, and (c) vaginal epithelial cells. Data represent means±SD of three independent experiments, **P<0.01, ***P<0.001.
The mRNA expression of genes N506_1778 in WT, N506_1778 mutant (Δ1778) and N506_1778 complement (1778 complement) strains (A) and N506_1709 in WT, N506_1709 mutant (Δ1709) and N506_1709 complement (1709 complement) strains (B). The mRNA expression of the two genes in the WT served as a control. Data represent means±SD of three independent experiments.
Comparison of the adhesion ability of L. gasseri DSM 14869 wild-type, mutant Δ1778 and Δ1709 strains. The adhesion rates are shown as a percentage relative to wild-type (set at 100%). Each panel shows the adhesion ability towards (a) Caco-2 cells, (b) HeLa cells, (c) vaginal epithelial cells. Data represent means±SD of three independent experiments, *P<0.05, **P<0.01, ***P<0.001.
(A) The mRNA expression of N506_1709 in overexpressed strain L. lactis NZ9000/pNZ8048-1709. The L. lactis harboring empty pNZ8048 plasmid (NZ9000/pNZ8048) served as a control. (B) Adhesion ability of overexpressed strain NZ9000/pNZ8048-1709 to vaginal epithelial cells. The adhesion rates are shown as a percentage relative to NZ9000/pNZ8048 (set at 100%). Data represent means±SD of three independent experiments, ***P<0.001.
Growth curve of L. gasseri DSM 14869 wild type, N506_0400 mutant (ΔEPS), N506_1778 mutant (Δ1778) and N506_1709 mutant (Δ1709) strains in MRS medium.
(A) The plasmid profile of pINTZrec. This plasmid includes a multiple clone site which can be used to integrate homologous fragments; two six sites and a β-recombinase (Rec) that specifically catalyzes the recombination between two six sites that flanked the antibiotic resistance gene and DNA to be integrated. The expression of β-recombinase is controlled by the sakacin-inducible promoter. (B) The plasmid profile of pNZ8048. (C) The plasmid profile of pNZe-Rec.
In a first aspect, the present invention provides a circular DNA vector comprising:
The selectable marker may be any marker suitable for identifying and selecting the host cell expressing in the marker. In one embodiment, the selectable marker is an antibiotic resistance gene, such as an antibiotic resistance gene selected from the group consisting of a chloramphenicol resistance gene, spectinomycin resistance gene, tetracycline resistance gene and erythromycin resistance gene.
The site-specific recombinase may be any site-specific recombinase suitable for genetic engineering. In one embodiment, the site-specific recombinase is a site-specific serine recombinases. In another embodiment, the site-specific recombinase is selected from the group consisting of beta-recombinase, Cre-recombinase, FLP-recombinase and PhiC31 integrase.
The target sites for the site-specific recombinase are typically between 30 and 200 nucleotides in length and consist typically of two motifs with a partial inverted-repeat symmetry, to which the recombinase binds, and which flank a central crossover sequence at which the recombination takes place. Examples of target sites includes six (the target site of beta-recombinase), Lox (the target site of the Cre recombinase) and FRT (the target site of the FLP recombinase). The target sites for the site-specific recombinase may be engineered to facilitate the recombination. Sets of single mutant sites may be used, e.g. lox66 and lox71, which produce a double mutant site as a product of the site-specific recombination. The double mutant site is not substrate for the site specific recombinase and thus reversible site-specific recombination events is prevented.
In a preferred embodiment, circular DNA vector comprises two target sites for said site-specific recombinase are orientated such that the product of site-specific recombination between two target sites for said site-specific recombinase is a first circular DNA product comprising (a) and (b) and a second circular DNA product comprising (c) and (d).
The circular DNA vector comprises a replicon, preferably a prokaryotic replicon sequence, which allows the replication of the vector in a cloning host (e.g. E. coli) from which the vector may be harvested. The replicon is useful expansion of the vector and cloning purposes, e.g. cloning a gene targeting sequence. In one embodiment, the replicon sequence is a replicon sequence that allows replication of said vector in E. coli, such as the replication origin of pBR322. The circular DNA vector comprises a replicon that allows replication of the host cell, which is subject to the targeted insertion of the DNA vector (e.g. a Lactobacilli or a Bifidobacteria). In one embodiment, the circular DNA vector comprises a replicon for the replication of the vector in a cloning host and a further replicon for the replication of the vector in the host cell, which is subject to the targeted insertion of the DNA vector in the genome of the host cell. In another embodiment, the replicon sequence is a replicon sequence that allows replication of said vector in E. coli and a further prokaryotic host cell, such as a host cell selected from the group consisting of Lactobacilli and Bifidobacteria. Thus, in this embodiment, the replicon has a dual function, i.e. replication in cloning host cell, for vector expansion and cloning and replication of the vector in the host cell, which is subject to the targeted insertion of the DNA vector in the genome of the host cell.
In one embodiment, the replicon sequence is a replicon sequence that allows replication of said vector in a least one host cell selected from the group consisting of a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869), Lactobacillus rhamnosus (such as Lactobacillus rhamnosus DSM 14870), Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus vaginalis, Lactobacillus iners, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus curvatus, Lactobacillus delbrueckii and Lactobacillus johnsonii.
The replicon sequence typically encodes a origin of replication (ori) and replication initiator protein (Rep protein). In one embodiment, the replicon comprises repA encoding Regulatory protein RepA or repB encoding RepFIB replication protein A or RepC encoding Replication initiation protein.
The selectable marker gene sequence is operably linked to a first promoter sequence. The first promoter directing the expression of the selectable marker is preferably a promoter that is constitutively active and thus expressing the marker in the host cell which is subject to the targeted insertion of the DNA vector. Any suitable promoter and marker may be used.
In order to control the timing of the site-specific recombination, the site-specific recombinase is conditionally expressed, e.g. using any suitable inducible promoter. The choice of promoter for expression of the recombinase (the second promoter) depends on the host cell. In one embodiment, the second promoter sequence is an inducible prokaryotic promoter. In another embodiment, the second promoter sequence is selected from the group consisting of a sakacin-inducible promoter, a tetracycline-inducible promoter, D-xylose-inducible promoter, lactose-inducible promoter, IPTG-inducible promoter, nisin inducible promoter, a bile inducible promoter, a bacteriocin-inducible promoter and a synthetic inducible promoter.
The circular DNA vector comprises a cloning site, preferable a multiple cloning site, which allows the cloning of a gene targeting sequence in the vector. In a preferred embodiment, the vector comprises a gene targeting sequence inserted in the multiple cloning site. The gene targeting sequence comprises sequences with sequence identity sufficiently high to allow the targeted integration of the vector at the corresponding target region in the host cell. The circular DNA vector comprising the gene targeting sequence inserted at the cloning site is suitable for use in the methods of the present invention. The host cell is preferably a bacterial cell, more preferably a host cell selected from the group consisting of Lactobacilli and Bifidobacteria. The vector may be introduced in the host cell using any suitable method, such as by transformation, such as electro-transformation, conjugation or transduction.
The circular DNA vector preferably comprises a replicon that allows replication of the vector in the host cell, wherein the vector is to be inserted in the genome by homologous recombination. In the episomal state in the host cell, the vector may be re-arranged by expression of the site-specific recombinase, which (i) eliminates the vectors ability to replicate in the host cell and (ii) facilitates the selection of homologous recombination event between the vector and chromosomal DNA. The product of the site-specific recombination event is a first circular DNA product and a second DNA product, where the first circular DNA product comprises the selectable marker and the multiple cloning site in which the targeting sequence is inserted. The second circular DNA product comprises the replicon and the sequences encoding the site-specific recombinase.
Single cross-over genomic integration of the circular DNA vector (first homologous recombination event) may be selected using the selectable marker. Cells comprising the episomal circular DNA vector is limited due to the expression of the site-specific recombinase that eliminates the vectors ability to replicate in the host cell. Target specific integration of the DNA vector (no longer circular) may be verified e.g. by PCR. The host may be propagated to allow a further (second) homologous recombination event, the product of which is the excision of the selectable marker. The host having undergone the two homologous recombination events may be identified by counterselection for loss of the selectable marker, e.g. sensitivity to the antibiotic where an antibiotic resistance marker is used. The correct double cross-overs should preferably by identified using methods such as PCR and/or sequencing.
A second aspect of the present invention provides a method for introducing recombination between a circular DNA vector and a target region of the genome of a host cell, said method comprising the steps of:
The circular DNA vector of the present invention may be used to introduce a specific mutation in a target region of a host cell. The target region may be a coding region, such as a gene encoding a protein. The mutation may be a point mutation that change or disrupt the encoded sequence. The encoded sequence may also be disrupted by mutations that introduces a deletion or insertion in the sequence. Alternatively, the mutation may change (decrease or increase) or restore the activity of the encoded protein.
Accordingly, a third aspect of the present invention provides a method for generating a host cell having a mutation in a target gene of the genome of a host cell, said method comprising the steps of:
The method is useful for e.g. evaluation of the function of specific genes. For example, to evaluate if a specific gene has a function e.g. in bacterial adherence, auto-aggregation and/or biofilm formation.
In a related aspect, the invention provides a method for generating a host cell having loss of function in a target gene of the genome of a host cell, said method comprising the steps of:
This method is useful for e.g. evaluation of the function of specific genes as described above. Functional assay may be used to address the potential impact of the targeted engineering at the targeted region resulting in the loss of gene function on phenotypical characteristics of the host cells (compared to the wild-type host cell). Phenotypical characteristics include, but are not limited to bacterial adherence (e.g. to mammalian tissue), auto-aggregation and/or biofilm formation.
Another aspect of the present invention provides a method for generating a host cell having gain of function in a target gene of the genome of a host cell, said method comprising the steps of:
This method is useful for e.g. evaluation of the reversion of a previous loss of gene function in a host cell restore lost phenotypical characteristics such as bacterial adherence (e.g. to mammalian tissue), auto-aggregation and/or biofilm formation.
The target gene may encode any gene product. The target gene typically encodes a protein. In one embodiment, the target gene encodes a cell surface protein. In one embodiment, the target gene encodes a protein involved in bacterial adherence, auto-aggregation and/or biofilm formation. In another embodiment, the cell surface protein is a sortase dependent protein (SDP) or a S-layer protein or encodes a protein involved in the biosynthesis of a cell surface molecule. In one embodiment, the cell surface molecule is an exopolysaccharide (EPS). In a particular embodiment, the target gene is a gene selected from the group consisting of N506_1709, N506_1778, N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410 and N506_0411.
In one embodiment, the host cell is a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869) and the target gene is selected from the group consisting of N506_1709, N506_1778, N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410 and N506_0411.
The circular DNA vector of the present invention may be used to prepare host cell recombinant expression of recombinant gene products, such as recombinant proteins. In this aspect, the gene targeting sequence will typically comprises sequences having sufficient sequence identity to the targeting region in order to allow target region specific integration; and a sequence encoding the recombinant gene product, e.g. a polypeptide.
Accordingly, in a further aspect, the present invention provides a method for preparing a host cell expressing a recombinant polypeptide, said method comprising the steps of:
In one embodiment, the recombinant polypeptide is selected from the group consisting of antibodies (such as monoclonal antibodies, humanized monoclonal antibodies, chimeric antibodies, single-domain antibodies, camelid antibodies), enzymes, cytokines, hormones and blood-clotting proteins. The host cell obtained by the method may be used as a producer cell for production of the recombinant product.
The gene targeting sequence comprises sequences in the flanking region that has sufficiently high identity to the sequence of the target region in order to allow/facilitate homologous recombination. In one embodiment, the sequences in the flanking region (the flanking sequences) comprises consecutive nucleosides in the range of 200 to 1500, for example at least about 300 consecutive nucleosides, such as at least about 400 consecutive nucleosides, for example at least about 500 consecutive nucleosides, such as at least about 600 consecutive nucleosides, for example at least about 700 consecutive nucleosides, such as at least about 800 consecutive nucleosides, for example at least about 900 consecutive nucleosides, such as at least about 1000 consecutive nucleosides, for example at least about 1100 consecutive nucleosides, such as at least about 1200 consecutive nucleosides, for example at least about 1300 consecutive nucleosides, such as at least about 1400 consecutive nucleosides, for example at least about 1500 consecutive nucleosides.
In another embodiment, the flanking sequences have a sequence identity of at least 85% to the corresponding region of the target gene of the host cell genome, for example at least 95% to the corresponding region of the target gene of the host cell genome, such as at least 97% to the corresponding region of the target gene of the host cell genome, for example at least 98% to the corresponding region of the target gene of the host cell genome, such as at least 99% to the corresponding region of the target gene of the host cell genome, for example 100% to the corresponding region of the target gene of the host cell genome.
In one embodiment, the selection under (iii) uses the selectable marker gene sequence (a) of the circular DNA vector (which selects for host cells having a single cross-over integration of the DNA vector). Host cells comprising a single cross-over integration of the DNA vector may be identified/verified using PCR and/or DNA sequencing. Thus in one embodiment, the selection under (iii) uses PCR and/or DNA sequencing, such as sequencing the sequence junction between the targeting sequence and the target region.
The host may be propagated to allow a further (second) homologous recombination event, the product of which is the excision of the selectable marker. The host having undergone the two homologous recombination events may be identified by counterselection for loss of the selectable marker, e.g. resulting in sensitivity to the antibiotic where an antibiotic resistance marker is used. The correct double cross-overs events should preferably by identified using methods such as PCR and/or DNA sequencing. In a preferred embodiment, the host cells having undergone a first and a second homologous recombination event are selected and/or verified using PCR or DNA sequencing.
The product of the method of the invention (the product of (iv)) is typically a host cell comprising a deletion of the target region, a partial deletion of the target region, a sequence insertion at the target region, a point mutation of the target region, or a sequence replacement of the target region.
The host cell is preferably a prokaryote, preferably a bacteria. In a preferred embodiment, the host cell is a Lactobacilli or Bifidobacteria. In another embodiment, the host cell is selected from the group consisting of a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869), Lactobacillus rhamnosus (such as Lactobacillus rhamnosus DSM 14870), Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus vaginalis, Lactobacillus iners, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus curvatus, Lactobacillus delbrueckii and Lactobacillus johnsonii. In a preferred embodiment, the host cell is a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869).
In a further aspect, the present provides a recombinant host cell obtainable by the method of the present invention. In one embodiment, the host cell is a Lactobacillus or Bifidobacteria. In another embodiment, the host cell is a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869). In another embodiment, the host cell is a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869) engineered to express a gene selected from the group consisting of N506_1709, N506_1778, N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410 and N506_0411. In a preferred embodiment, the Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869) is engineered to express N506_1709 and/or N506_1778,
In one embodiment, the coding region of the gene is operably linked to a recombinant promoter, preferably a recombinant constitutive promoter.
One aspect of the present provides the use of the circular DNA vector of the present invention for introducing a gene sequence in the genome of a host cell. In one embodiment, the use of the circular DNA vector is for increasing tissue adhesion of a host cell, such as for increasing tissue adhesion of a bacterial host cell to vaginal tissue, preferably human vaginal tissue. The host cell is preferably a Lactobacilli or Bifidobacteria. In one embodiment, the host cell selected from the group consisting of a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869), Lactobacillus rhamnosus (such as Lactobacillus rhamnosus DSM 14870), Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus vaginalis, Lactobacillus iners, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus curvatus, Lactobacillus delbrueckii and Lactobacillus johnsonii.
In one embodiment, the use of the circular DNA vector is for introducing a deletion of a target region, partial deletion of a target region, a sequence insertion at a target region, a point mutation of a target region, or a sequence replacement of a target region.
In one embodiment, the use of the circular DNA vector is for targeting a target gene selected from the group consisting of N506_0396, N506_0397, N506_0398, N506_0399, N506_0400, N506_0401, N506_0402, N506_0403, N506_0404, N506_0405, N506_0406, N506_0407, N506_0408, N506_0409, N506_0410 and N506_0411 in a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869) host cell.
In another embodiment, the use of the circular DNA vector is for introducing and expression of said gene sequence in said host cell. In yet another embodiment, said gene sequence blocks expression of an endogenous host gene. In a further embodiment, the gene sequence replaces a corresponding endogenous host gene.
In one embodiment, the use of the circular DNA vector is for increasing tissue adhesion of a bacterial host cell, such as increasing tissue adhesion of a bacterial host cell (such as a Lactobacilli or Bifidobacteria) to vaginal tissue, preferably human vaginal tissue. In one embodiment, the host cell is selected from the group consisting of a Lactobacillus gasseri (such as Lactobacillus gasseri DSM 14869), Lactobacillus rhamnosus (such as Lactobacillus rhamnosus DSM 14870), Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus vaginalis, Lactobacillus iners, Lactobacillus reuteri, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus curvatus, Lactobacillus delbrueckii and Lactobacillus johnsonii.
Provided is a novel vector, which may be conditionally destructed facilitating the selection of homologous recombination event between the vector and chromosomal DNA. Essentially, the replicon and the antibiotic resistance gene were separated by two oriented six fragments. Upon addition of the inducer and expression of the site-specific recombinase (β), the vector can recombine and lose function due to the excision of the replicon (
Previous work by the inventors showed that the transformation efficiency of L. gasseri DSM 14869 was very low possibly due to the thickness of EPS covering the cell surface or the presence of two resident plasmids, and therefore, generating mutation in this strain using existing methods was not possible (Marcotte, H. et al. (2017) and unpublished data). In order to demonstrate IPSD assisted recombineering in L. gasseri DSM 14869, we targeted upp, a non-essential gene encoding uracil phosphoribosyltransferase (UPRTases) that is commonly used as a counterselection marker. A recombinant plasmid pINTZrec-Rupp containing homologous regions up- and downstream of upp gene was constructed and introduced into L. gasseri DSM 14869. The transformant was induced by SpplP and the single-crossover integration event was selected using colony PCR (
The major advantage of IPSD assisted bacterial recombineering is that it does not depend on transformation (or conjugation) efficiency. However, it needs two pre-requirements: 1) a functional replicon allowing the plasmid to replicate in the host bacteria; 2) a tightly controlled expression element used to drive recombinase gene expression. The inducible promoters used in this study are not effective in some strains, either due to strong background expression (L. sakei NC03) (
In conclusion, the inventors have shown that the IPSD plasmid can be used for genome engineering in lactobacilli, with the potential to be extend to other bacterial species. The IPSD strategy could be used in a range of applications in both the food and pharmaceutical industry such as identification of probiotic genes, metabolic engineering and delivery of therapeutics, thus opening new avenues for the engineering of biotherapeutic agents with enhanced health-promoting functional features.
Methods Bacterial strains, plasmids and growth conditions Bacterial strains and plasmids used in this study are listed in Table1. Lactobacillus strains were generally cultured at 37° C. in deMan Rogosa Sharpe (MRS) medium (Difco, BD BioSciences). For upp-based double-crossover event selection, the Lactobacillus strain was grown on semi-defined medium (SDM) agar plates (Kimmel et al.). Escherichia coli strains were cultured in Luria-Bertani broth at 37° C. with rotary shaking at 200 rpm, or on LB agar plates. When needed, antibiotics were supplemented at the following concentrations: 10 μg/ml chloramphenicol for Lactobacillus strains and E. coli VE7108 strain.
Plasmid Construction
The primers used in this study are listed in Table 2. For construction of IPSD vector pINTZrec used for lactobacilli recombineering, the two six DNA fragments were amplified from site-specific integration vector pEM76 by using the primers pairs SIX-F1&R1 and SIX-F2&R2, respectively, and inserted into plasmid pNZ8048 on each flank of the Cm′ expression cassette generating pNZ8048-SIX. The orientation of the insertion was confirmed by sequencing. A multiple cloning sites was introduced into pNZ8048-SIX by inserting a linker between PstI and BgIII, resulting in the plasmid pNZmcs-SIX. The β-recombinase gene was amplified from plasmid pEM94 and inserted into plasmid pVPL3017 downstream of the sakacin-inducible promoter PorfX generating pVPL3017-rec. Subsequently, the PorfX-rec expression cassette was digested using SalI and HindIII, and inserted into similarly digested pNZmcs-SIX plasmid to obtain the final plasmid pINTZrec.
For construction of the plasmid used for L. gasseri DSM 14869 upp gene deletion, two 1090 bp DNA fragments, upstream and downstream of upp gene, were amplified from genomic DNA of L. gasseri DSM 14869 using the primer pairs upp-up-F/upp-up-R and upp-down-F/upp-down-R, respectively. The upstream DNA fragment was inserted into pMD19-T Simple vector by TA cloning generating pMD19-upp-up, then the downstream DNA fragment was inserted into pMD19-upp-up between SacI and SphI, generating pMD19-4upp. Δupp was digested with Δpal and SphI and inserted into similarly digested pINTZrec generating pINTZrec-Δupp.
Transformation
The plasmids pINTZrec and pINTZrec-Δupp were electrotransformed into L. gasseri DSM 14869 and other Lactobacillus strains according to De Keersmaecker et al. The plasmid pNZ8048 was used as controls. The transformants were confirmed by colony PCR followed by PCR on DNA extracted from pure cultures.
Recombineering
The single colonies of L. gasseri DSM 14869 harboring the plasmid pINTZrec or its derivatives were grown overnight in MRS broth containing 10 μg/ml chloramphenicol. The cultures were inoculated (1%, v/v) into MRS broth without antibiotics and grown at 37° C. until OD600nm reached ˜0.30, then supplemented with 100 ng/ml sakacin P (SpplP) (Genscript). The cultures were allowed to grow overnight, and serial dilutions were plated on MRS agar supplemented with 10 μg/ml chloramphenicol and 100 ng/ml SpplP. The single-crossover events were detected by colony PCR followed by PCR on DNA extracted from pure cultures. For double-crossover selection, the single-crossover clones were grown overnight in MRS broth in absence of antibiotics, followed by spreading serial dilutions on MRS agar or SDM agar supplement of 100 μg/ml 5-Fluorouracil (5-FU). The colonies from MRS agar were replicated to MRS agar containing 10 μg/ml chloramphenicol, the Cms colonies were selected and detected by PCR on extracted DNA.
All the mutants generated in this study were confirmed by PCR and sequencing.
E.
coli DH5a
E.
coli VE7108
L.
gasseri DSM 14869
L.
paracasei BL23
L.
plantarum NL42
L.
acidophilus ATCC
L.
sakei NC03
L.
gasseri DSM 14869
L.
gasseri DSM 14869 upp
aKmr, kanamycin resistance; Cmr, chloramphenicol resistance; Smr, spectinomycin resistance.
aThe restriction sites are underlined.
Lactobacilli play an important role for the maintenance of a healthy vaginal microbiota, and some select species are widely used as probiotics. Vaginal isolates of Lactobacillus gasseri DSM 14869 and L. rhamnosus DSM 14870 were previously selected to develop the probiotic EcoVag® capsules and were shown to have therapeutic effects in women with bacterial vaginosis (BV). However, the molecular basis and mechanisms involved in their probiotic activity are largely unknown. In this study, we identified three cell surface molecules in L. gasseri DSM 14869 that promote adhesion to vaginal epithelial cells by constructing dedicated knock-out mutants, including exopolysaccharides (EPS), a protein containing MucBP-like domains (N506_1778) and a putative novel adhesin (N506_1709) with Rib/alpha-like domain repeats. EPS knock-out mutants revealed a 20-fold and 14-fold increase in adhesion to Caco-2 and HeLa cells, respectively, compared to wild-type, while the adhesion to vaginal cells was reduced 30% by the mutation, suggesting that EPS might mediate tissue tropism for vaginal cells. A significant decrease in adhesion to Caco-2, HeLa and vaginal cells was observed in the N506_1778 knock-out mutant. The N506_1709 mutant showed no significant difference for the adhesion to Caco-2 and HeLa cells compared to WT; in contrast, the adhesion to vaginal cells revealed a significant decrease (42%), suggesting that N506_1709 might mediate specific binding to stratified squamous epithelial cells and this putative novel adhesin was annotated as Lactobacillus vaginal epithelium adhesin (LVEA). Thus, for the first time, the inventors have discovered an important role for EPS and a novel adhesin LVEA in the adhesive capacity of a vaginal probiotic Lactobacillus strain.
Lactobacilli are known to contribute to the maintenance of a healthy vaginal microbiota and some are selected as probiotics for prevention or treatment of urogenital diseases such as bacterial vaginosis. However, the molecular mechanisms for these health-promoting effects are poorly understood. Here, we functionally identified three cell surface factors of a Lactobacillus gasseri strain potentially involved in its adhesion to vaginal epithelial cells, including exopolysaccharides (EPS) and two sortase-dependent proteins (N506_1778 and N506_1709). The inventors have demonstrated for the first time the tissue specific adhesion of EPS to vaginal cells and that N506_1709 might be a novel adhesin specifically mediating bacterial binding to stratified squamous epithelial cells. The results provide important new information on the molecular mechanisms of vaginal Lactobacillus adhesion.
The vaginal microbiota of a healthy woman is usually dominated by lactobacilli and the most frequently occurring species are Lactobacillus crispatus, L. gasseri, L. jensenii, L. vaginalis, and L. iners (Pendharkar et al. 2013; Ravel et al. 2011; Vasquez et al. 2002). These bacteria maintain the normal vaginal microbiota by adhering to vaginal epithelial cells (VEC) and prevent the growth of pathogenic organisms (Ronnqvist et al. 2006). Once the balance of the local microbiota is broken, it can predispose women to urogenital infections, such as bacterial vaginosis (BV) (Danielsson et al. 2011). Supplying selected lactobacilli might be a rational therapeutic strategy in restoring a healthy microbiota and preventing infections (Bolton et al. 2008; Reid et al. 2009). To meet this challenge, commercial EcoVag® vaginal capsules which consist of two strains (L. gasseri DSM 14869 and L. rhamnosus DSM 14870, 108 CFU for each strain) isolated from healthy women vaginal epithelial cells have previously been developed and vaginal administration of EcoVag® capsules can eliminate the symptoms of BV in 90% of the patients (Stray-Pedersen et al. 2003, unpublished data). Supplement treatment with vaginal EcoVag® capsules can also effectively improve the therapeutic effect of antibiotic treatment of BV (Larsson et al. 2008; 2011; Pendharkar et al. 2015). In spite of the wealth of clinical data showing the health benefits of EcoVag® strains in humans, there is still a lack of understanding of the cell surface factors or molecular mechanisms underlying their probiotic activities.
Studies of gastrointestinal lactobacilli suggest that health-promoting effects of probiotics could be related to their capacity to adhere to intestinal epithelial cells and/or mucus, as it can promote colonization, pathogen exclusion and interactions with host (Lebeer et al. 2008; 2010). Thus a high adhesive ability to intestinal surface is considered to be a major distinguishing feature for the selection of probiotic strains. Bacterial adherence to host epithelial surface is often mediated by the cell surface components, including sortase-dependent proteins (SDPs) and other cell surface molecules, such as exopolysaccharides (EPS), lipoteichoic acid and S-layer proteins (Lebeer et al. 2008) and extracellular appendages, such as pili, fimbriae and flagella (Juge, 2012).
EPS contribute significantly to lactobacilli-host interactions, especially with intestinal mucosa and epithelial cells, thus contributing to the strain-specific probiotic characteristics (Lebeer et al., 2008). Bacterial polysaccharides vary in sugar composition, position of branches and modifications, contributing to the wide diversity of surface structures (Ruas-Madiedo et al. 2002). EPS have been reported to be involved in aggregation, biofilm formation, adhesive properties and immunomodulation by probiotic strains (Lebeer et al. 2009; Dertli et al. 2015; Lee et al. 2016; Živkoviĉ et al. 2016).
SDPs are an important group of cell surface proteins in Gram positive bacteria, which are best characterized in lactobacilli and suggested to play a key role in bacterial adhesion (Boekhorst et al. 2005). These SDPs share a common structure including a YSIRK signal peptide that promotes secretion (Bae & Schneewind, 2003), a C-terminal LPxTG anchoring motif, followed by a transmembrane helix and a positively charged tail (Lebeer et al. 2008; Jensen et al. 2014). After the surface protein precursor is transferred to the plasma membrane, it will be cleaved and covalently anchored to the cell wall by sortase A (Marraffini et al. 2006). Different SDPs have been identified in lactobacilli, including the mucus-binding pilin SpaC in L. rhamnosus GG (Kankainen et al. 2009), Lactobacillus epithelium adhesion (LEA) in L. crispatus ST1 (Edelman et al. 2012), mucus binding protein A (CmbA) in L. reuteri ATCC PTA6475 (Jensen et al. 2014) and mannose-specific adhesin Msl in L. plantarum CMPG5300 (Malik et al. 2016). However, until now, very few SDPs have been identified in human vaginal lactobacilli.
In previous studies, the inventors have found that the EcoVag® strains shown a highly efficient adhesion ability to vaginal epithelial cells. We have also performed complete genome sequencing and characterization of the two strains and found several genes potentially involved in bacterial probiotic activity (Marcotte et al. 2017). In particular, production of a thick (40 nm) EPS layer and a putative new adhesin containing three rib/alpha-like repeats was found in L. gasseri DSM 14869 (Marcotte et al. 2017). In this study, the inventors aimed to characterize the L. gasseri DSM 14869 surface molecules that mediate adhesion to the human vaginal mucosa, including the EPS, a protein (N506_1778) with mucus-binding like domain, and a putative novel protein (N506_1709) with rib/alpha-like repeat domains. The results suggest that the genes encoding EPS, N506_1778 and N506_1709 contribute to the ability of L. gasseri DSM 14869 to adhere to vaginal epithelial cells in vitro.
Identification of a putative EPS gene cluster in L. gasseri DSM 14869. The genome of L. gasseri DSM 14869 (Marcotte et al. 2017) harbors a putative EPS cluster composed of 16 genes (N506_0396 to N506_0411) (
Deletion of the N506_0400 gene influences the total level of EPS. TEM pictures clearly showed that the N506_0400 gene deletion mutant strain (ΔEPS) produce significantly (p<0.001) less EPS layer around the cell surface compared to the wild type (WT) strain (
Deletion of the N506_0400 gene results in increased auto-aggregation and biofilm formation. When the N506_0400 mutant strain L. gasseri DSM 14869-ΔN506_0400 (ΔEPS) was grown in liquid medium, no significant difference in the growth rate was observed as compared to WT L. gasseri DSM 14869 (
However, the mutant showed a cell sediment and a very clear upper solution while the WT strain showed a relatively homogenous suspension (
Impact of EPS on adhesion of L. gasseri DSM 14869 to different epithelial cells. Previous studies have shown that EPS from intestinal lactobacilli is involved in adhesion to intestinal epithelial cells (Lebeer et al. 2009; Lee et al. 2016; Živković et al. 2016). In this study, we investigated the role of EPS in the adhesive capacity of the vaginal strain L. gasseri DSM 14869. As observed in
Sequence analysis of putative adhesion proteins in L. gasseri DSM 14869. Surface proteins are very important molecules involved in bacterial adhesion to mucus and epithelial cells. In the genome of L. gasseri DSM 14869, 22 predicated ORFs with putative adhesion-related domains have previously been identified (Marcotte et al. 2017). However, only two ORFs (N506_1778 and N506_1709) encode both a YSIRK-type signal peptide (PF04650) and a LPXTG anchor motif (PF00746), which would result in a protein secreted and covalently anchor to the cell wall (
The N506_1778 gene in L. gasseri DSM 14869 is 5.055 kb long and encodes a protein of 1684 amino acid residues with a predicted molecular weight of 186.7 kD. The N-terminus is a YSIRK-type signal peptide and the C-terminus contains a LPQTG (LPXTG-like cell wall anchor) motif which belongs to the gram-positive LPXTG anchoring superfamily. The N506_1778 protein also contains 2 non-identical repeats (amino acid 986-1092 and 1384-1490), showing 66% amino acid identity (
L.
gasseri
L.
gasseri
L
gasseri K7
L.
gasseri
L.
gasseri
L.
gasseri
L.
gasseri
L.
gasseri
L.
gasseri
L.
gasseri
The N506_1709 gene in L. gasseri DSM 14869 consists of a 4.371 kb sequence encoding a large surface protein of 1456 amino acids with a predicted molecular weight of 158.9 kD. This protein includes a YSIRK signal peptide, a N-terminal region (amino acids 42 to 1233), an internal repeat region (amino acids 892 to 1372) harboring three repeats (the first one is partial) which shows similarity to rib/alpha-like repeats domain (PF08428) in Pfam analysis, and a LPQTG anchoring motif in C-terminus (
Construction of N506_1778 and N506_1709 knockout mutants. As shown in
N506_1778 Mediated Binding of L. gasseri DSM 14869 to Caco-2, HeLa and Human Vaginal Cells.
The growth rate of the N506_1778 mutant strain was not altered under the growth conditions used in the study (
N506_1709 mediated binding of L. gasseri DSM 14869 to human vaginal cells but not to Caco-2 and HeLa cells. In order to determine the contribution of N506_1709 to bacterial adhesion, a N506_1709 knockout mutant was constructed. The mutant strain showed the same growth rate with wild-type (
Overexpression of N506_1709 in Lactococcus lactis NZ9000 increases adhesion to vaginal epithelial cells. To confirm tissue specific adhesion by N506_1709, the protein N506_1709 was overexpressed in L. lactis NZ9000 using the pNZ8048 vector and its nisin-inducible expression system. L. lactis NZ9000 transformed with the empty plasmid pNZ8048 was used as a control. After nisin induction of N506_1709 expression in L. lactis NZ9000, the overexpressed strain showed a 5000-fold increase of the mRNA transcription compared to control (
L. gasseri is one of the major species isolated in the vaginal microbiota (Pendharkar et al. 2013; Ravel et al. 2011) and several health benefits have been reported in L. gasseri strains (Marcotte et al. 2017; Parolin et al. 2015). However, the molecular mechanisms underlying the health-promoting effects, such as the adhesion factors that allow optimal adhesion of lactobacilli to this niche, are in general not yet well understood. In this study, we genetically identified and functionally analyzed three genes that may be involved in adhesion of the probiotic strain L. gasseri DSM 14869 to vaginal epithelial cells.
First, the function of the identified EPS gene cluster in L. gasseri DSM 14869 was evaluated by mutation of the N506_0400 gene, which encodes the putative priming glycosyltransferase. According to the literature, the gene encoding priming glycosyltransferase is highly conserved (Jolly and Stingele, 2001) and priming glycosyltransferase plays an essential role in the first step of EPS biosynthesis by transferring the first sugar to the UndP-lipid carrier (Lebeer et al. 2009). Mutation of the genes encoding priming glycosyltransferase of L. johnsonii, L. rhamnosus or L. paracasei abolished or reduced heteropolysaccharide production (Lebeer et al. 2009; Horn et al. 2012; Živković et al. 2016). In this study, the increased auto-aggregation ability and reduced expression of surface-associated polysaccharide of the N506_0400 mutant strain versus the wild-type also indicate a crucial role for the priming galactosyltransferase N506_0400 in the biosynthesis of EPS of L. gasseri DSM 14869. At the same time, the EPS mutant of L. gasseri DSM 14869 showed a significant increase of biofilm formation when grown in MRS medium, while the complemented strain restored part of the biofilm formation capacity to wild-type (
Furthermore, the role of EPS on adhesion was studied using Caco-2, HeLa and vaginal epithelial cells. The EPS mutant showed a significant increase adhesive capacity to colon carcinoma cells Caco-2 and HeLa cervical carcinoma cells but a slightly reduced adhesion to vaginal epithelial cells. The increased adhesion to Caco-2 and HeLa could potentially be attributed to a better exposure of adhesins because of the absence of EPS. These data are consistent with studies of L. rhamnosus GG showing that deprivation of galactose-rich EPS increased adhesion to Caco-2 cells possibly because of an enhanced exposure of adhesins (Lebeer et al. 2009) and additional studies about characterization of the specific adhesins of L. rhamnosus GG such as pili substantiate this “shielding hypothesis” (Lebeer et al. 2012). Similar results were also reported in the L. johnsonii NCC533 (Denou et al. 2008), L. rhamnosus E/N (Polak-Berecka et al. 2014) and L. plantarum Lp90 strains (Lee et al. 2016). However, the adhesive capacity of the EPS mutant to vaginal epithelial cells was slightly decreased (30%), suggesting that EPS may possibly promote adhesion of L. gasseri DSM 14869 to vaginal epithelial cells. The role of EPS on adhesion is strain-specific and could probably be related to differences in structural characteristics of the EPS, as well as to the cell surface characteristics of the strain. For example, the mutation of cps clusters in the L. plantarum strains WCFS1 and SF2A35B has no significant influence on adhesion to Caco-2 cells (Lee et al. 2016). Živković et al. (2016) reported that the presence of the EPS-SJ P2 increases adhesion to Caco-2 cells which may be attributed to the molecular structure of EPS-SJ P2 matrix. Hence, specific increase in L. gasseri DSM 14869 adhesion to vaginal cells by EPS may be due to a different molecular structure of EPS or different receptors in vaginal cells compared to Caco-2 and HeLa cells. How the EPS promotes bacterial adhesion to vaginal cells need to be further studied. To the best of our knowledge, this is the first report demonstrating that the EPS of vaginal lactobacilli might provide tissue tropism adhesion. This will help to better understand its specific contribution in probiotics-host interactions and its role in adaptation to this vaginal niche.
Increased adhesion to the intestinal mucosal layer by some lactobacilli has been suggested to be mediated by cell surface proteins with a mucus-binding capacity (Kankainen et al. 2009; Rojas et al. 2002; Ossowski et al. 2011; Jensen et al. 2014). The N506_1778 protein, harboring 2 repeats with homology to MucBP domain, was the only predicated cell wall anchored protein that includes MucBP-like domain in L. gasseri DSM 14869. Our results showed that the N506_1778 protein could promote adherence of L. gasseri DSM 14869 to Caco-2, HeLa and human vaginal epithelial cells. N506_1778 homologues were found in all the known L. gasseri strains isolated from different niches (Table 3), suggesting that N506_1778 is an important cell surface protein for L. gasseri species to adapt to different host niches. This is the first time that a protein with MucBP-like domain has been reported to also be involved in adhesion to vaginal epithelial cells.
N506_1709 is another important cell surface protein mediating adhesion of L. gasseri DSM 14869 to vaginal mucosal cells. It is a newly described sortase-dependent adhesin that shows specific binding to stratified squamous epithelial cells. Interestingly, N506_1709 differs from the previously characterized Lactobacillus adhesins, such as Lsp, Mub and mucus-binding factor (MBF) (Walter et al. 2005; Buck et al. 2005; von Ossowski et al. 2011), as it contains no MuBP domains but instead harbors three repeated regions with homology to Rib/a-like repeats. Rib and alpha proteins were first identified in Streptococcus and were suggested to be involved in pathogen adhesion and biofilm formation (Michel et al. 1992; Wästfelt et al. 1996; Stâlhammar-Carlemalm et al. 1999). Later, proteins showing homology with Rib/a-like repeats were also reported in vaginal lactobacilli, such as protein Rip in L. fermentum and LEA in L. crispatus (Turner et al. 2003; Edelman et al. 2012). These proteins with Rib/a-like repeats domain in lactobacilli were suggested to mediate binding to the stratified squamous epithelial lining of the host (Edelman et al. 2012; Turner et al. 2003). In this study, the N506_1709 mutant showed a significantly reduced adhesive capacity to vaginal epithelial cells (stratified squamous epithelial cells), but not to colon carcinoma cells and cervical carcinoma cells (columnar epithelial cells). This suggests that the N506_1709 protein provides tissue tropism to L. gasseri DSM 14869, likely determined by the presence of different receptors on the cell membrane of vaginal epithelial cells. The over-expression of N506_1709 in L. lactis significantly improved L. lactis adhesion to vaginal epithelial cells, further confirming the adhesive capacity of N506_1709 to vaginal epithelial cells. Taken together, N506_1709 is an important surface protein mediating the adherence of L. gasseri to human vaginal epithelium, which could promote the bacterial colonization in the host and may be of ecological importance.
In conclusion, the current report identifies and functionally analyzes three cell surface molecules including EPS, N506_1778 and N506_1709 as important adhesion factors of L. gasseri DSM 14869 involved in vaginal adhesion. To our knowledge, this is the first report that demonstrates the role of EPS in adherence of a vaginal Lactobacillus strain and that a protein with MucBP-like domains could also be involved in vaginal epithelium adhesion. In addition, N506_1709 might be a novel adhesin specifically mediating bacterial binding to stratified squamous epithelial cells and was annotated as Lactobacillus vaginal epithelium adhesin (LVEA). The results provide important new information on the molecular mechanisms of Lactobacillus adhesion and tissue tropism to mucosal surfaces of various hosts, and could help us to screen for better probiotic candidates in the future. Further studies are still needed to address the specific receptors for EPS and LVEA on vaginal epithelial cells as well as the functional domains of LVEA.
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 4.
E.coli VE7108
L.
gasseri DSM 14869
Lactococcus
lactis NZ9000
L.
lactis NZ9000/pNZ8048
L.
lactis NZ9000 harboring
L.
lactis NZ9000/pNZ8048-
L.
lactis NZ9000 harboring
E.
coli-lactic acid bacteria
aKmr, kanamycin resistant; Cmr, chloramphenicol resistant; Emr, erythromycin resistant.
Escherichia coli VE7108 (Mora et al. 2004) strain was incubated in Luria broth supplemented with 25 μg ml−1 of kanamycin at 37° C. with shaking. Lactobacillus strains were statically grown in MRS broth at 37° C. under anaerobic conditions. When required, the following antibiotics were added: 10 μg ml−1 chloramphenicol (Cm) and 300 μg ml−1 erythromycin (Em) for E. coli; 10 μg ml−1 Cm and 5 μg ml−1 Em for Lactobacillus transformants.
DNA manipulations. Standard DNA protocols were used for DNA manipulations in E. coli (Sambrook et al. 1989). Plasmid DNA of E. coli was extracted using QIAGEN Miniprep Spin Kit (Qiagen, Hilden, Germany). L. gasseri DNA was isolated using QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany). Primers (Table 5) were synthesized by Eurofins Genomics (Ebersberg, Germany). Phusion High-Fidelity DNA polymerase (Finnzymes/Thermo Fisher Scientific, Espoo, Finland) was used for amplification of L. gasseri genomic DNA and GoTaq DNA polymerase (Promega, Fitchburg, Wis., USA) was used for colony PCR. The PCR products were purified by QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The restriction endonucleases were supplied by Thermo Scientific and T4 DNA ligase was from Invitrogen (Carlsbad, Calif.). All of the procedures were conducted according to the manufacturer's instructions. Plasmids were transformed into L. gasseri DSM 14869 by electroporation as described previously (De Keersmaecker et al. 2006).
L. gasseri 16S-F
L. gasseri 16S-R
L. lactis 16S-F
L. lactis 165-R
aThe restriction site is underlined in primer sequence.
Construction of EPS (N506_0400), N506_1778 and N506_1709 knock-out mutants by double homologous recombination. In this study, the replicable plasmid pINTZrec (unpublished data) (
Briefly, the transformed L. gasseri strain was inoculated into MRS medium and when growth reached OD600-0.5, 100 ng ml−1 sakacin was added for overnight induction. Serial dilutions of induced culture were plated on MRS agar plates (with 10 μg ml−1 Cm and 100 ng ml−1 sakacin) and anaerobically grown for 48 h. To obtain the replicable plasmid already excised and single crossover integration, individual colonies with Cm gene and without repA gene were selected and further confirmed by PCR using seq1-F/INTZ-R and INTZ-F/seq1-R primers (table 5). The single crossover strain was grown in 3 ml MRS medium without antibiotics, and two subcultures per day were grown during 2 days. Subsequently, bacterial cultures were diluted and plated into MRS plates without antibiotics for 48 h to obtain single-colony isolates and then, these colonies were replica plated on MRS plates with 10 μg ml−1 Cm. The non-antibiotic-resistant colonies were checked by specific primers seq1-F/seq1-R to obtain N506_0400 gene deletion strain, L. gasseri DSM 14869-ΔN506_0400.
For the deletion of N506_1778 and N506_1709 gene from the genome of L. gasseri DSM 14869, the same method for EPS knockout was used. Briefly, about 1.0 kb up- and downstream fragments flanking the 5′ and 3′ ends of the N506_1778 gene and N506_1709 gene were amplified by PCR using the primers 1778upstream-F/R and 1778downstream-F/R, and 1709upstream-F/R and 1709downstream-F/R, respectively. The generated amplicons were joined by overlap extension strategy using the primer pair 1778upstream-F/downstream-R and 1709 upstream-F/downstream-R (Table 5). The resulting PCR products were digested with Sac I and Bam HI and ligated into similarly digested pINTZrec plasmid and transformed into E. coli VE7108 to obtain the plasmid constructions pINTZrec-N506_1778 and pINTZrec-N506_1709. Plasmids pINTZrec-N506_1778 and pINTZrec-N506_1709 were electrotransformed into DSM 14869, respectively, and the transformed strains were induced by sakacin. Single crossover integrated colonies were screened by PCR using seq2-F/INTZ-R and INTZ-F/seq2-R primers for N506_1778 and seq3-F/INTZ-R and INTZ-F/seq3-R primers for N506_1709. Finally, double crossover integrated strains were checked by specific primers seq2-F/seq2-R to obtain N506_1778 gene deletion strain named L. gasseri DSM 14869-ΔN506_1778 and seq3-F/seq3-R to obtain N506_1709 gene deletion strain named L. gasseri DSM 14869-ΔN506_1709.
Complementation: plasmid construction and transformation. The plasmid pNZe-Rec was used as the starting material to achieve the plasmid required for the complementation of the gene N506_0400 in strain L. gasseri DSM 14869-ΔN506_0400. The gene N506_0400 does not have its own promoter; it shares the promoter of the EPS operon. Thus the promoter of EPS gene cluster was amplified using primers EPS-promoter-F/R (table 5) and N506_0400 gene was amplified with primers N506_0400-F/R. The promoter and N506_0400 gene were joined by overlap extension using the primer pair EPS-promoter-F/N506_0400-R and then digested with Kpn I and Hin dIII and ligated into similarly digested pNZe-Rec, resulting in pNZe-N506_0400. Plasmid pNZe-N506_0400 was electroporated into L. gasseri DSM 14869-ΔN506_0400 to yield an Em-sensitive strain, L. gasseri DSM 14869-ΔN506_0400/pNZe-N506_0400.
Plasmid pNZe-N506_0400 was used to complement the gene N506_1778 in strain L. gasseri DSM 14869-ΔN506_1778. The gene N506_1778 and its promoter were amplified by PCRs using the specific primers N506_1778-F/R. The PCR product was digested with Kpn I and Bam HI and ligated into pNZe-N506_0400 which was digested with Kpn I and Bgl II (Bam HI and Bgl II are isocaudarners), resulting in pNZe-N506_1778. Plasmid pNZe-N506_1778 was electroporated in L. gasseri DSM 14869-ΔN506_1778 to yield an Em-sensitive strain, L. gasseri DSM 14869-ΔN506_1778/pNZe-N506_1778.
Plasmid pNZ8048 was used to complement the gene N506_1709 in strain L. gasseri DSM 14869-ΔN506_1709. N506_1709 gene was ligated into pNZ8048 in two steps. First, the first part of gene N506_1709 (˜2 kb) and its promoter was amplified using primers N506_1709-F1/R1. The PCR product was digested with Bgl II and Kpn I and ligated into similarly digested pNZ8048, generating plasmid pNZ8048-N506_1709-1. Then the second part of gene N506_1709 (˜2.3 kb) was amplified using primers N506_1709-F2/R2 and digested with Kpn I and Sac I and ligated into similarly digested pNZ8048-N506_1709-1, resulting in pNZ8048-N506_1709. Plasmid pNZ8048-N506_1709 was electroporated into L. gasseri DSM 14869-ΔN506_1709 to yield a Cm-sensitive strain, L. gasseri DSM 14869-ΔN506_1709/pNZ8048-N506_1709.
Construction of overexpression constructs of N506_1709 in Lactococcus lactis NZ9000. For heterologous expression of N506_1709 in L. lactis, a nisin-inducible vector pNZ8048 was used. The N506_1709 gene from L. gasseri DSM14869 was amplified using primers N506_1709 Re-F/R (Table 5) and subsequently cloned into the pNZ8048 vector resulting in plasmids pNZ8048-N506_1709 Re. Competent L. lactis NZ9000 cells were transformed with plasmid pNZ8048-N506_1709 Re, resulting into strain L. lactis NZ9000/pNZ8048-N506_1709Re.
RNA extraction and Quantitative Real-Time PCR (qRT-PCR). Total RNA was extracted from 109 bacteria grown in the logarithmic phase using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed using QuantiTect Reverse Transcription Kit (Qiagen), containing 1 μg of total RNA as the template. qRT-PCR was carried out by using a SYBR Green assay kit (Qiagen). Specific primers (Table 5) were designed by Primer Premier 5 software and the internal gene 16S rRNA was used as reference. The relative gene expression was calculated by using the ΔΔCT method (Schmittgen & Livak, 2008).
Analysis of auto-aggregation. Auto-aggregation analysis was performed as previously reported with some modifications (Leccese Terraf1 et al. 2014). Briefly, bacteria were grown overnight (˜16 h) in MRS. The cultures were centrifuged and washed twice with phosphate-buffered saline (PBS) (pH 7.2) and suspended in PBS to an OD600 of 1.5. After incubation for 5 h at room temperature, the OD600 of the upper suspension was measured. The auto-aggregation percentage was calculated by the expression as follows: auto-aggregation (%)=(1−(OD600 5 h/OD600 0 h))×100, where OD600 5 h represents the absorbance at the 5 h time point and OD600 0 h represents the absorbance at 0 h.
Biofilm formation assay. Biofilm formation was performed as described previously (Lebeer et al. 2007) with some modifications. Briefly, the biofilms were grown in MRS medium in 96-well polystyrene microplates at 37° C. for 72 h. Then the wells were washed three times with PBS and stained for 30 min with 0.1% crystal violet. Excess stain was rinsed with water and wells were air dried (1 h). The dye bound to the adherent cells was extracted with 200 μl 30% glacial acetic acid. The OD570 of 135 μl of each well was measured. The experiments were repeated three times, each with eight replicates. Additionally, a sterile MRS medium was used as negative control.
Excess stain was rinsed with 200 II distilled water per well.
Transmission electron microscopy (TEM). Cells of L. gasseri DSM 14869, EPS mutant and complemented strains were grown overnight in MRS and the presence of EPS layer on the surface of L. gasseri strains was analysed by TEM as previously described (Alvarez et al. 2015). The relative thickness was expressed as a percentage relative to WT (set at 100%). Thickness was evaluated in 10 cells for each group and the mean±SD of thickness per cell was determined.
Assay of adhesion to Caco-2 and HeLa cells. Caco-2 and HeLa cells were routinely grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 IU ml−1 penicillin G and 100 μg ml−1 streptomycin. Adhesion assays were performed as previously described with some modifications (Lee et al. 2016). Briefly, cells were seeded in 24-well plates at a concentration of 105 cells per well and cultured for 72 h until confluence. L. gasseri DSM 14869 was grown for 18 h and washed two times with PBS, and resuspended in antibiotic-free Dulbecco's modified Eagle's medium (DMEM) with a concentration of 107 CFU ml−1. 0.8 ml bacterial culture was added to the tissue culture wells and incubated for 2 h. The wells were washed 3 times with PBS to remove unadhered bacteria. Following washing, 0.2 ml trypsin-EDTA (Invitrogen) was added to the wells to detach the cells and then 0.6 ml antibiotic-free DMEM medium was added to stop the digestion of trypsin. Serial dilutions were prepared and plated onto MRS agar plates to count the number of adhered bacteria. The adhesion ratio was calculated as percent of bacteria adhering to Caco-2 or HeLa cells in relation to the total number of bacteria added in the wells. All adhesion experiments were performed in triplicate and repeated three times.
Assay of adhesion to vaginal epithelial cells (VEC). The protocol was approved by the Stockholm ethics committee (Regionala etikprövningsnämnden i Stockholm) (Permit Number: 2018/1090/31). Informed consent was obtained from participants before the start of the study. VEC cells were collected from 4 healthy volunteer donors by gently scraping the vaginal mucosal surface with a sterile cotton swab and suspended in 10 ml DMEM medium. The cells were washed three times with 10 ml DMEM and centrifuged at 800×g for 5 min to remove indigenous bacteria. The cells were adjusted to 105 cells ml−1 in DMEM by using a hemocytometer. L. gasseri DSM 14869 harvested from an 18 h culture, were washed twice with PBS (pH 7.4) and re-suspended in DMEM medium to get a final concentration of 5×107CFU ml−1. In the overexpression adhesion assays, the L. lactis strains were inoculated to OD600˜0.5 and induced with 10 ng mL−1 nisin (50 IU mL−1, Sigma) for 1.5 h. Then the induced cultures were harvested as described above.
Equal volumes (400 μl) of vaginal epithelial cells and Lactobacillus or L. lactis were mixed and incubated at 37° C. for 2 h. After incubation, the cells were then washed five times with PBS to remove non-adherent bacteria. Following the last centrifugation, the cell pellet was transferred to microscope slides, dried, fixed with methanol and stained with 0.1% crystal violet. Three replicates were made for each sample and 100 randomly chosen cells from each replicate were examined using a light microscope under oil immersion and the results expressed as number of bacteria per cell. VEC not incubated with lactobacilli or L. lactis were included as negative control.
Statistical analyses. Data are presented as mean±SD. Significant differences (P<0.05) between means were identified by one-way Analysis of Variance (ANOVA) followed by Duncan's test procedures using SPSS 20. All experiments were performed in triplicate and repeated three times.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19174715.3 | May 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/063668 | 5/15/2020 | WO | 00 |
