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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.

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Related in: MedlinePlus

mT3 Escherichiacoli secretes anddelivers proteins into mammalian cells. Shigella andmT3 E. coli strains were grown underconditions that induce type 3 secretion system expression. Secretionwas induced by exposure to Congo Red dye, and delivery was inducedby bacterial contact with mammalian cells. (a) Western blot analysisof T3SS apparatus proteins in mT3 E. coli. Whole cell lysate and supernatant proteins were separated by SDS-PAGEand immunoblotted with anti-IpaB or anti-IpaD antibodies. DnaK isa cytoplasmic protein unrelated to type 3 secretion and serves asa loading and bacterial cell lysis control. (b) Plasmids expressingFLAG-tagged versions of native Shigella effectorswere introduced into each strain background, and cell lysate (L) andsecreted proteins (S) were probed with anti-FLAG antibodies. The blotsshown are representative of at least three experiments. Each strainwas transformed with a target protein (substrate) plasmid that expressesan IPTG-inducible construct of an OspB–TEM-1 fusion proteinillustrated in (c). (d) Images of HeLa cells loaded with CCF4/AM exposedto wild-type Shigella or mT3 E. coli strains expressing OspB–TEM-1. (e) Translocation was quantifiedby measuring the percentage of cells that fluoresce blue (cleavedCCF4/AM). Data are expressed as the mean of three independent experimentsperformed in triplicate. Error bars represent the standard error ofthe mean (SEM). At least 600 cells were counted for each sample.
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fig3: mT3 Escherichiacoli secretes anddelivers proteins into mammalian cells. Shigella andmT3 E. coli strains were grown underconditions that induce type 3 secretion system expression. Secretionwas induced by exposure to Congo Red dye, and delivery was inducedby bacterial contact with mammalian cells. (a) Western blot analysisof T3SS apparatus proteins in mT3 E. coli. Whole cell lysate and supernatant proteins were separated by SDS-PAGEand immunoblotted with anti-IpaB or anti-IpaD antibodies. DnaK isa cytoplasmic protein unrelated to type 3 secretion and serves asa loading and bacterial cell lysis control. (b) Plasmids expressingFLAG-tagged versions of native Shigella effectorswere introduced into each strain background, and cell lysate (L) andsecreted proteins (S) were probed with anti-FLAG antibodies. The blotsshown are representative of at least three experiments. Each strainwas transformed with a target protein (substrate) plasmid that expressesan IPTG-inducible construct of an OspB–TEM-1 fusion proteinillustrated in (c). (d) Images of HeLa cells loaded with CCF4/AM exposedto wild-type Shigella or mT3 E. coli strains expressing OspB–TEM-1. (e) Translocation was quantifiedby measuring the percentage of cells that fluoresce blue (cleavedCCF4/AM). Data are expressed as the mean of three independent experimentsperformed in triplicate. Error bars represent the standard error ofthe mean (SEM). At least 600 cells were counted for each sample.

Mentions: To evaluatethe potential of mT3 E. coli as a proteindelivery strain, we first investigatedwhether this strain expresses a functional type 3 secretion system.mT3 E. coli was grown under conditionsthat activate Shigella type 3 secretion: growth at37 °C followed by the addition of the dye Congo Red, an in vitro inducer of type 3 secretion.21 Cell lysate and secreted fractions were examinedfor the presence of IpaB and IpaD, two secreted components of the Shigella translocon apparatus and the outermost proteinsof the machine.6,22 However, in contrast to wild-type Shigella, we observed no evidence of the production or secretionof IpaB or IpaD from mT3 E. coli, suggestingthat an essential type 3 secretion regulator was missing from thisstrain (Figure 3a).


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)

mT3 Escherichiacoli secretes anddelivers proteins into mammalian cells. Shigella andmT3 E. coli strains were grown underconditions that induce type 3 secretion system expression. Secretionwas induced by exposure to Congo Red dye, and delivery was inducedby bacterial contact with mammalian cells. (a) Western blot analysisof T3SS apparatus proteins in mT3 E. coli. Whole cell lysate and supernatant proteins were separated by SDS-PAGEand immunoblotted with anti-IpaB or anti-IpaD antibodies. DnaK isa cytoplasmic protein unrelated to type 3 secretion and serves asa loading and bacterial cell lysis control. (b) Plasmids expressingFLAG-tagged versions of native Shigella effectorswere introduced into each strain background, and cell lysate (L) andsecreted proteins (S) were probed with anti-FLAG antibodies. The blotsshown are representative of at least three experiments. Each strainwas transformed with a target protein (substrate) plasmid that expressesan IPTG-inducible construct of an OspB–TEM-1 fusion proteinillustrated in (c). (d) Images of HeLa cells loaded with CCF4/AM exposedto wild-type Shigella or mT3 E. coli strains expressing OspB–TEM-1. (e) Translocation was quantifiedby measuring the percentage of cells that fluoresce blue (cleavedCCF4/AM). Data are expressed as the mean of three independent experimentsperformed in triplicate. Error bars represent the standard error ofthe mean (SEM). At least 600 cells were counted for each sample.
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fig3: mT3 Escherichiacoli secretes anddelivers proteins into mammalian cells. Shigella andmT3 E. coli strains were grown underconditions that induce type 3 secretion system expression. Secretionwas induced by exposure to Congo Red dye, and delivery was inducedby bacterial contact with mammalian cells. (a) Western blot analysisof T3SS apparatus proteins in mT3 E. coli. Whole cell lysate and supernatant proteins were separated by SDS-PAGEand immunoblotted with anti-IpaB or anti-IpaD antibodies. DnaK isa cytoplasmic protein unrelated to type 3 secretion and serves asa loading and bacterial cell lysis control. (b) Plasmids expressingFLAG-tagged versions of native Shigella effectorswere introduced into each strain background, and cell lysate (L) andsecreted proteins (S) were probed with anti-FLAG antibodies. The blotsshown are representative of at least three experiments. Each strainwas transformed with a target protein (substrate) plasmid that expressesan IPTG-inducible construct of an OspB–TEM-1 fusion proteinillustrated in (c). (d) Images of HeLa cells loaded with CCF4/AM exposedto wild-type Shigella or mT3 E. coli strains expressing OspB–TEM-1. (e) Translocation was quantifiedby measuring the percentage of cells that fluoresce blue (cleavedCCF4/AM). Data are expressed as the mean of three independent experimentsperformed in triplicate. Error bars represent the standard error ofthe mean (SEM). At least 600 cells were counted for each sample.
Mentions: To evaluatethe potential of mT3 E. coli as a proteindelivery strain, we first investigatedwhether this strain expresses a functional type 3 secretion system.mT3 E. coli was grown under conditionsthat activate Shigella type 3 secretion: growth at37 °C followed by the addition of the dye Congo Red, an in vitro inducer of type 3 secretion.21 Cell lysate and secreted fractions were examinedfor the presence of IpaB and IpaD, two secreted components of the Shigella translocon apparatus and the outermost proteinsof the machine.6,22 However, in contrast to wild-type Shigella, we observed no evidence of the production or secretionof IpaB or IpaD from mT3 E. coli, suggestingthat an essential type 3 secretion regulator was missing from thisstrain (Figure 3a).

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