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Engineering Escherichia coli into a protein delivery system for mammalian cells.

Reeves AZ, Spears WE, Du J, Tan KY, Wagers AJ, Lesser CF - ACS Synth Biol (2015)

Bottom Line: We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells.Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells.This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: †Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts 02139, United States.

ABSTRACT
Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

No MeSH data available.


Related in: MedlinePlus

Generation of mT3 Escherichia coli, the protein delivery strain. Akanamycin-resistance cassette (striped box) was inserted into a nonessential region of the Shigella flexneri virulence plasmid to assist inselection of proper recombination events with the capture vector.A capture vector was constructed that contains regions of homologyto the regions of the Shigella virulence plasmidflanking the type 3 secretion genes, which are represented as grayboxes. Landing pad (LP) sequence, denoted as a green box, flanks thepieces of T3SS gene homology to facilitate downstream integrationinto the E. coli chromosome. An originof transfer (oriT), which can mobilize the plasmidbetween bacterial host strains by conjugation, is represented by ablack oval. λ-Red recombination was then used to introduce theregion of the Shigella virulence plasmid that containsthe T3SS genes onto the capture vector. The resulting 44 kb plasmid(pmT3SS) contains the entire T3SS. When pmT3SS is introduced intoa strain of E. coli harboring an engineeredlanding pad sequence, recombination leads to integration of the interveningsequence, in this case the T3SS operons, into the chromosome, generatingthe strain mT3 E. coli.
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fig2: Generation of mT3 Escherichia coli, the protein delivery strain. Akanamycin-resistance cassette (striped box) was inserted into a nonessential region of the Shigella flexneri virulence plasmid to assist inselection of proper recombination events with the capture vector.A capture vector was constructed that contains regions of homologyto the regions of the Shigella virulence plasmidflanking the type 3 secretion genes, which are represented as grayboxes. Landing pad (LP) sequence, denoted as a green box, flanks thepieces of T3SS gene homology to facilitate downstream integrationinto the E. coli chromosome. An originof transfer (oriT), which can mobilize the plasmidbetween bacterial host strains by conjugation, is represented by ablack oval. λ-Red recombination was then used to introduce theregion of the Shigella virulence plasmid that containsthe T3SS genes onto the capture vector. The resulting 44 kb plasmid(pmT3SS) contains the entire T3SS. When pmT3SS is introduced intoa strain of E. coli harboring an engineeredlanding pad sequence, recombination leads to integration of the interveningsequence, in this case the T3SS operons, into the chromosome, generatingthe strain mT3 E. coli.

Mentions: To capture thisregion of the Shigella virulence plasmid onto a smallerautonomously replicatingplasmid, we utilized a combination of yeast and bacterial homologousrecombination-based approaches to generate pmT3SS (see Figure 2 and Methods for details).Several features of the vector backbone of pmT3SS enable the transferof this large 44 kb plasmid between bacteria as well as the stableintegration of the Shigella operons that it carries onto the E. coli chromosome. First, the backbone of pmT3SSincludes an origin of transfer region (oriT) to facilitatethe transfer of this plasmid from one strain background to anothervia conjugation. Second, the region of the Shigella operons present on pmT3SS is flanked on each end by a defined “landingpad” sequence such that this region of DNA can be integratedonto the chromosome of E. coli engineeredto have the corresponding “landing pad” sequence.20 In this manner, the methodology developed byKuhlman and Cox was adapted to add large captured regions of DNA atspecific chromosomal loci, an approach that can be easily adaptedto capture other large pieces of DNA.20 The introduction of mT3SS into the E. coli chromosome alleviates the need for antibiotic selection, thus resultingin a strain, mT3 E. coli, that shouldbe particularly well-suited for use as an in vivo therapeutic protein delivery system.


Engineering Escherichia coli into a protein delivery system for mammalian cells.

Reeves AZ, Spears WE, Du J, Tan KY, Wagers AJ, Lesser CF - ACS Synth Biol (2015)

Generation of mT3 Escherichia coli, the protein delivery strain. Akanamycin-resistance cassette (striped box) was inserted into a nonessential region of the Shigella flexneri virulence plasmid to assist inselection of proper recombination events with the capture vector.A capture vector was constructed that contains regions of homologyto the regions of the Shigella virulence plasmidflanking the type 3 secretion genes, which are represented as grayboxes. Landing pad (LP) sequence, denoted as a green box, flanks thepieces of T3SS gene homology to facilitate downstream integrationinto the E. coli chromosome. An originof transfer (oriT), which can mobilize the plasmidbetween bacterial host strains by conjugation, is represented by ablack oval. λ-Red recombination was then used to introduce theregion of the Shigella virulence plasmid that containsthe T3SS genes onto the capture vector. The resulting 44 kb plasmid(pmT3SS) contains the entire T3SS. When pmT3SS is introduced intoa strain of E. coli harboring an engineeredlanding pad sequence, recombination leads to integration of the interveningsequence, in this case the T3SS operons, into the chromosome, generatingthe strain mT3 E. coli.
© Copyright Policy - editor-choice
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4487226&req=5

fig2: Generation of mT3 Escherichia coli, the protein delivery strain. Akanamycin-resistance cassette (striped box) was inserted into a nonessential region of the Shigella flexneri virulence plasmid to assist inselection of proper recombination events with the capture vector.A capture vector was constructed that contains regions of homologyto the regions of the Shigella virulence plasmidflanking the type 3 secretion genes, which are represented as grayboxes. Landing pad (LP) sequence, denoted as a green box, flanks thepieces of T3SS gene homology to facilitate downstream integrationinto the E. coli chromosome. An originof transfer (oriT), which can mobilize the plasmidbetween bacterial host strains by conjugation, is represented by ablack oval. λ-Red recombination was then used to introduce theregion of the Shigella virulence plasmid that containsthe T3SS genes onto the capture vector. The resulting 44 kb plasmid(pmT3SS) contains the entire T3SS. When pmT3SS is introduced intoa strain of E. coli harboring an engineeredlanding pad sequence, recombination leads to integration of the interveningsequence, in this case the T3SS operons, into the chromosome, generatingthe strain mT3 E. coli.
Mentions: To capture thisregion of the Shigella virulence plasmid onto a smallerautonomously replicatingplasmid, we utilized a combination of yeast and bacterial homologousrecombination-based approaches to generate pmT3SS (see Figure 2 and Methods for details).Several features of the vector backbone of pmT3SS enable the transferof this large 44 kb plasmid between bacteria as well as the stableintegration of the Shigella operons that it carries onto the E. coli chromosome. First, the backbone of pmT3SSincludes an origin of transfer region (oriT) to facilitatethe transfer of this plasmid from one strain background to anothervia conjugation. Second, the region of the Shigella operons present on pmT3SS is flanked on each end by a defined “landingpad” sequence such that this region of DNA can be integratedonto the chromosome of E. coli engineeredto have the corresponding “landing pad” sequence.20 In this manner, the methodology developed byKuhlman and Cox was adapted to add large captured regions of DNA atspecific chromosomal loci, an approach that can be easily adaptedto capture other large pieces of DNA.20 The introduction of mT3SS into the E. coli chromosome alleviates the need for antibiotic selection, thus resultingin a strain, mT3 E. coli, that shouldbe particularly well-suited for use as an in vivo therapeutic protein delivery system.

Bottom Line: We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells.Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells.This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: †Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts 02139, United States.

ABSTRACT
Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

No MeSH data available.


Related in: MedlinePlus