Patent Application: US-59671505-A

Abstract:
the invention relates to a recombinant lactobacillus strain , with limited growth and viability in the environment . more particularly , it relates to a recombinant lactobacillus that can only survive in a medium where thymidine is present . by this strict dependency upon thymidine , thymidineless death is rapidly induced in this recombinant strain . a preferred embodiment is a lactobacillus that may only survive in a host organism where thymidine is present , but cannot survive outside the host organism in absence of this medium compound . moreover , the lactobacillus strain can be transformed with prophylactic and / or therapeutic molecules and can , as such , be used to treat diseases such as , but not limited to , inflammatory bowel diseases .

Description:
unless otherwise stated , lactobacillus strains were cultivated in mrs ( merck ). special media used were : bm9 : 1 liter of 50 mm co 3 − buffer at ph 8 . 5 supplemented with 6 g of na 2 hpo 4 / 3 g of kh 2 po 4 1 g of nh 4 cl / 0 . 5 g of nacl / 1 mmol of mgso 4 / 0 . 1 mmol of cacl 2 / 0 . 5 % of glucose / 0 . 5 % of casitone ( difco ) mrsδt ( mrs devoid of thymidine ): mrs powder ( merck ) is dissolved in an appropriate ( according to the manufacturer ) volume of distilled water . the solution is heated to 100 ° c . for one minute and allowed to cool to room temperature . 1 . 2 units of thymidine phosphorylase ( sigma ) are added per ml . the solution is incubated at 37 ° c . for 20 hours and autoclaved subsequently . lactobacillus salivarius ucc118 ( dunne et al ., 2001 ) was used as recipient strain to construct the thya mutant . the construction of the l . salivarius thya mutant was essentially carried out as described for lactococcus lactis ( steidler et al ., 2003 ), with modifications . the construction is summarized in fig1 . the thya region of l . salivarius subsp . salivarius strain ucc118 was sequenced , including the upstream and downstream sequences of the coding sequence . the knowledge of these sequences is of critical importance for the genetic engineering of any lactobacillus strain in a way as described below , as the strategy will employ double homologous recombination in the areas 1000 bp at the 5 ′ end and 1000 bp at the 3 ′ end of thya , the “ thya target .” in strain ucc118 , the thya gene is replaced by a synthetic gene encoding a protein that has a secretion leader , functional in lactobacillus fused to a protein of identical amino acid sequence than the mature part of hil - 10 in which proline at position 2 had been replaced with alanine , operably linked to the lactococcus lactis thya promoter ( pthya , genbank af462070 ). any combination of a promoter and the hil - 10 gene is called a hil - 10 expression cassette . transformation was by electroporation , at 1 . 5 kv , 25 mf , 400ω , 2 mm gap length . the thya replacement was performed by homologous recombination , essentially as described by biwas et al . ( 1993 ). suitable replacements in a plasmid borne version of the thya target are made , as described below . the strategy involves a helper plasmid ( carrying a chloramphenicol selection marker ), which is brought in the target lactobacillus strain beforehand , and a carrier plasmid ( carrying an erythromycin - resistance marker ), encoding the hil - 10 expression cassette flanked by upstream and downstream sequences of the chromosomal thya gene , as described above . the helper plasmid ptgb019 is a modified version of pve6007 . to construct ptgb019 , a 3221 bp insert was generated by pcr amplification from pkd20 using the oligonucleotides gcgaagcttcaaataggggttccgcgc ( seq id no : 17 ) and gcgactagtgggaaaactgtccataccc ( seq id no : 18 ) and cut with hindiii and spei . this fragment encodes the red γ , β and exo genes under the control of the e . coli arabinose promoter and was ligated in the hindiii - spei opened pve6007 . this expression system , however , showed not to be functional in lb . salivarius . the addition of arabinose to a strain carrying myc tag - labeled versions of the various red recombinase genes did not show any expression when revealed by western blot , neither did a lactobacillus carrying ptgb019 show expression of either one of the red genes as judged by intracellular protein analysis though sds - page and coomassie brilliant blue staining . the insert will rather render the helper plasmid ptgb019 more unstable for replication in lactobacillus when compared to pve6007 . the carrier plasmid was electroporated into the lactobacillus strain that holds ptgb019 . both plasmids do not stably coexist . it is at this time unclear how the mechanism of integration functions . the electroporation mixture is plated on solid agar mrs plates containing erythromycin at 10 μg / ml and thymidine at 200 μm and incubated at 42 ° c . for 24 hours . the carrier plasmid is unable to replicate in lactobacillus . therefore , the only way to transfer the erythromycin resistance to a given strain is when a first homologous recombination , at either the 5 ′ 1000 bp or at the 3 ′ 1000 bp of the thya target is taking place . erythromycin - positive colonies were checked by pcr for the occurrence of such homologous recombination , as indicated in fig1 . a subset of the erythromycin - resistant clones still carries ptgb019 . these clones are utilized to isolate clones that show the second cross over . appropriate dilutions were plated on mrs solid agar plates at 42 ° c . and from these colonies , erythroymycin - and chloramphemicol - sensitive clones were screened for the incapacity to grow in thymidine - free mrs , for the presence of both the upstream and downstream recombination , as well as for the absence of the thya gene . a second homologous recombination at the 3 ′ 1000 bp or at the 5 ′ 1000 bp of the thya target yielded the desired strain . selection for the second recombination was carried out by repeated growth in absence of erythromycin and in presence of 50 μg / ml thymidine . colonies were tested by pcr as indicated on fig1 . the resulting strains were called tgb078 ( human il - 10 ) and tgb092 ( human el - 10 operably linked to the thya promoter ). the primary confirmation of the lactobacillus colonies carrying a hil - 10 insert was done by pcr testing , as presented in fig2 . several sets of primers were used for the detection of thya ( fig2 , pcr1 ) of il - 10 ( fig2 , pcr2 ) of the flanking sequences of il - 10 ( fig2 , pcr3 through pcr6 ) and of the flanking sequences of thya ( fig2 , pcr7 and pcr8 ). the results show clearly that in the mutant strains tgb072 and tgb092 , the coding sequence of thya has been replaced by the human il - 10 sequence . to ensure that there are no thya or il - 10 copies present elsewhere in the genome , the integration was tested by southern blot . from the different lactobacillus strains , a genomic dna preparation was made . the genomic lactobacillus dna was digested by ecori and southern blotted . the blot was revealed with digoxygenin - labeled probes for identifying thya ( thya probe , obtained with pcr primer pair 1 ) or hil - 10 ( hil - 10 probe , obtained with pcr primer pair 2 ). as expected on the basis of the pcr results , the thya probe signal is negative and the hil - 10 probe signal on the blot is positive for the mutants , whereas the thya probe signal is positive and the hil - 10 signal is negative for the parental strain . the results are summarized in table 2 . to evaluate the hil - 10 secretion , single colonies of each strain were grown in mrs supplemented with 50 μg / ml thymidine . after 40 hours of growth at 37 ° c ., the bacteria were harvested by centrifugation and resuspended in buffered m9 ( bm9 ) supplemented with 50 μg / ml thymidine . the suspension was incubated for five hours at 37 ° c ., and then the prevalence of human il - 10 was determined by elisa ( becton dickinson ). the results are summarized in fig4 . both strains comprising the human il - 10 coding sequence do produce il - 10 , but the production is far higher when the human il - 10 coding sequence is operably linked to the lactococcus lactis thya promoter . although the production of hil - 10 is lower than what is described for lactococcus lactis ( steidler et al ., 2003 ), the amount is sufficiently high to be effective in vivo for the treatment of chronic intestinal inflammation . survival in thymidine - free medium was tested for the two mutant strains and the parental strain . survival was measured as colony forming units ( cfu ) per ml of culture , in function of the time . the results are presented in fig5 and 6 . single colonies of all strains were inoculated in mrsδt supplemented with 25 μg / ml of thymidine and incubated for 20 hours at 37 ° c . bacteria were harvested by centrifugation , washed twice with 1v mrsδt , resuspended in 1v of mrsδt , diluted 1 : 20 in mrsδt and incubated at 37 ° c . at relevant time points , cfu per ml were determined by plating on mrs solid agar plates supplemented with 50 μg / ml of thymidine . as can be seen , the cfu is reduced by more than 2 log units after 500 minutes . a reduction of 3 log units is obtained after less than 1000 minutes . these results are far better than those obtained by steidler et al . ( 2003 ) for lactococcus lactis , where about twice the time is needed to obtain a reduction with 2 log units and 50 hours is needed to obtain a reduction with 3 log units . it is important to note that these results are obtained in presence of thymine . indeed , the thymidine is removed from the medium by enzymatic treatment , converting the thymidine in thymine . notwithstanding the remaining concentration of thymine , the death induced by thymidine starvation is extremely fast , indicating that the strain cannot be rescued by the presence of thymine . lactobacillus salivarius ucc118 ( thya wild - type ), tgb078 and tgb092 ( both thya deficient ) were grown in mrs , mrs with 200 μm thymidine ( mrstd ) or mrs with 800 μm thymine ( mrstm ). the optical density at 600 nm was measured after 29 hours of growth at 37 ° c . the data obtained ( fig7 ) show that ucc118 reaches a comparable optical density irrespective of the growth medium . the concentration of thymidine in mrs is limiting the growth of tgb078 and tgb092 . when 200 μm thymidine is added to mrs , tgb078 and tgb092 reach the same optical density as ucc118 . the addition of 800 μm thymine to mrs is unable to support the growth of tgb078 and tgb092 to higher optical densities . as can be appreciated from fig7 , mrs contains a substantial amount of thymidine . thymidine can be converted to thymine with thymidine phosphorylase . mrs digested with thymidine phosphorylase thus gives mrsδt . lactobacillus salivarius ucc118 ( thya wild - type ), tgb078 and tgb092 ( both thya deficient ) were grown in mrsδt with a range of thymidine or thymine concentrations added . after 24 hours of growth at 37 ° c ., the cultures reach saturation . the od 600 at 24 hours was plotted against thymidine or thymine concentration ( fig8 and 9 ). these results show that both thya - deficient strains can use exogenous thymidine but not thymine for growth , whereas wild - type growth is not influenced by addition of either thymidine or thymine ( fig1 ), proving that the lack of growth is not due to thymine toxicity . ahmad s . i ., s . h . kirk and a . eisenstark ( 1998 ). thymine metabolism and thymineless death in prokaryotes and eukaryotes . annu . rev . microbiol . 52 : 591 - 625 . biswas i ., a . gruss , s . d . ehrlich , et al . 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