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Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

Montanaro J, Inic-Kanada A, Ladurner A, Stein E, Belij S, Bintner N, Schlacher S, Schuerer N, Mayr UB, Lubitz W, Leisch N, Barisani-Asenbauer T - Drug Des Devel Ther (2015)

Bottom Line: In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed.Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability.These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

View Article: PubMed Central - PubMed

Affiliation: Laura Bassi Centres of Expertise, OCUVAC - Centre of Ocular Inflammation and Infection, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Vienna, Austria.

ABSTRACT
To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

No MeSH data available.


Related in: MedlinePlus

Cell lysates of EcN (pBGKB-MOMP, pGLysivb and pBGKB-N-PmpC, pGLysivb) were separated on a 4%–12% polyacrylamide gel and c-Myc-tagged MOMP and N-PmpC protein levels were determined by Western blot.Notes: Samples for lanes 1–3 were taken at sequential intervals (−90, −60, and −30 minutes) during exponential growth. Lane M shows molecular weight marker. Upon induction with L-arabinose (time point −10 minutes, lane 4) expression of MOMP and N-PmpC is induced. To induce E-lysis temperature is increased to 42°C (time point 0 minutes, lane 5). The process to complete transformation of live EcN to EcN BGs is consecutively monitored in 30 minutes intervals after induction of E-lysis (time point from 30 minutes to 120 minutes; lanes 6–9). After complete E-lysis, EcN BGs are washed and samples are taken after removal of growth media (W1) and subsequently after PBS wash (W2) (lanes 10 and 11). Lanes 12 and 14 are negative controls; and lane 13 shows positive MOMP and N-PmpC expression after β-Propiolactone treatment to inactivate remnant nucleic acids.Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; N-PmpC, N-terminal part of polymorphic membrane protein C; PBS, phosphate-buffered saline.
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f4-dddt-9-3741: Cell lysates of EcN (pBGKB-MOMP, pGLysivb and pBGKB-N-PmpC, pGLysivb) were separated on a 4%–12% polyacrylamide gel and c-Myc-tagged MOMP and N-PmpC protein levels were determined by Western blot.Notes: Samples for lanes 1–3 were taken at sequential intervals (−90, −60, and −30 minutes) during exponential growth. Lane M shows molecular weight marker. Upon induction with L-arabinose (time point −10 minutes, lane 4) expression of MOMP and N-PmpC is induced. To induce E-lysis temperature is increased to 42°C (time point 0 minutes, lane 5). The process to complete transformation of live EcN to EcN BGs is consecutively monitored in 30 minutes intervals after induction of E-lysis (time point from 30 minutes to 120 minutes; lanes 6–9). After complete E-lysis, EcN BGs are washed and samples are taken after removal of growth media (W1) and subsequently after PBS wash (W2) (lanes 10 and 11). Lanes 12 and 14 are negative controls; and lane 13 shows positive MOMP and N-PmpC expression after β-Propiolactone treatment to inactivate remnant nucleic acids.Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; N-PmpC, N-terminal part of polymorphic membrane protein C; PBS, phosphate-buffered saline.

Mentions: Expression of c-Myc- and poly-histidine-tagged chlamydial antigens after the induction of protein expression with L-arabinose was confirmed via Western blot (Figure 4). The cell lysate pellets prepared for Western blot analysis were taken after removal of growth media (W1) and subsequently after PBS wash (W2), contained less protein than sequential samples taken before washing steps.


Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface.

Montanaro J, Inic-Kanada A, Ladurner A, Stein E, Belij S, Bintner N, Schlacher S, Schuerer N, Mayr UB, Lubitz W, Leisch N, Barisani-Asenbauer T - Drug Des Devel Ther (2015)

Cell lysates of EcN (pBGKB-MOMP, pGLysivb and pBGKB-N-PmpC, pGLysivb) were separated on a 4%–12% polyacrylamide gel and c-Myc-tagged MOMP and N-PmpC protein levels were determined by Western blot.Notes: Samples for lanes 1–3 were taken at sequential intervals (−90, −60, and −30 minutes) during exponential growth. Lane M shows molecular weight marker. Upon induction with L-arabinose (time point −10 minutes, lane 4) expression of MOMP and N-PmpC is induced. To induce E-lysis temperature is increased to 42°C (time point 0 minutes, lane 5). The process to complete transformation of live EcN to EcN BGs is consecutively monitored in 30 minutes intervals after induction of E-lysis (time point from 30 minutes to 120 minutes; lanes 6–9). After complete E-lysis, EcN BGs are washed and samples are taken after removal of growth media (W1) and subsequently after PBS wash (W2) (lanes 10 and 11). Lanes 12 and 14 are negative controls; and lane 13 shows positive MOMP and N-PmpC expression after β-Propiolactone treatment to inactivate remnant nucleic acids.Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; N-PmpC, N-terminal part of polymorphic membrane protein C; PBS, phosphate-buffered saline.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4516183&req=5

f4-dddt-9-3741: Cell lysates of EcN (pBGKB-MOMP, pGLysivb and pBGKB-N-PmpC, pGLysivb) were separated on a 4%–12% polyacrylamide gel and c-Myc-tagged MOMP and N-PmpC protein levels were determined by Western blot.Notes: Samples for lanes 1–3 were taken at sequential intervals (−90, −60, and −30 minutes) during exponential growth. Lane M shows molecular weight marker. Upon induction with L-arabinose (time point −10 minutes, lane 4) expression of MOMP and N-PmpC is induced. To induce E-lysis temperature is increased to 42°C (time point 0 minutes, lane 5). The process to complete transformation of live EcN to EcN BGs is consecutively monitored in 30 minutes intervals after induction of E-lysis (time point from 30 minutes to 120 minutes; lanes 6–9). After complete E-lysis, EcN BGs are washed and samples are taken after removal of growth media (W1) and subsequently after PBS wash (W2) (lanes 10 and 11). Lanes 12 and 14 are negative controls; and lane 13 shows positive MOMP and N-PmpC expression after β-Propiolactone treatment to inactivate remnant nucleic acids.Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; N-PmpC, N-terminal part of polymorphic membrane protein C; PBS, phosphate-buffered saline.
Mentions: Expression of c-Myc- and poly-histidine-tagged chlamydial antigens after the induction of protein expression with L-arabinose was confirmed via Western blot (Figure 4). The cell lysate pellets prepared for Western blot analysis were taken after removal of growth media (W1) and subsequently after PBS wash (W2), contained less protein than sequential samples taken before washing steps.

Bottom Line: In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed.Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability.These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

View Article: PubMed Central - PubMed

Affiliation: Laura Bassi Centres of Expertise, OCUVAC - Centre of Ocular Inflammation and Infection, Centre for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Vienna, Austria.

ABSTRACT
To target chronic inflammatory ocular surface diseases, a drug delivery platform is needed that is safe, possesses immunomodulatory properties, and can be used either for drug delivery, or as a foreign antigen carrier. A new therapeutic approach that we have previously proposed uses nonliving bacterial ghosts (BGs) as a carrier-delivery system which can be engineered to carry foreign antigens and/or be loaded with therapeutic drugs. The parent strain chosen for development of our BG delivery system is the probiotic Escherichia coli strain Nissle 1917 (EcN), whose intrinsic properties trigger the innate immune system with the flagella and fimbriae used to attach and stimulate epithelial cells. In previous studies, we have shown that EcN BGs are safe for the ocular surface route, but evidence that EcN BGs retain flagella and fimbriae after transformation, has never been visually confirmed. In this study, we used different visualization techniques to determine whether flagella and fimbriae are retained on EcN BGs engineered either for drug delivery or as a foreign antigen carrier. We have also shown by immunoelectron microscopy that EcN retains two foreign antigens after processing to become EcN BGs. Furthermore, we demonstrated that BGs derived from EcN and expressing a foreign antigen attachment to conjunctival epithelial cells in vitro without causing reduced cell viability. These results are an important step in constructing a delivery system based on a nonliving probiotic that is suitable for use in ocular surface diseases pairing immunomodulation and targeted delivery.

No MeSH data available.


Related in: MedlinePlus