Chapter 1
Improving E. coli Bactofection by Expression
of Bacteriophage ΦX174 Gene E
Dongwon Park, Andrew Hill, and Blaine A. Pfeifer
Abstract
Bactofection, a bacterial-mediated form of genetic transfer, is highlighted as an alternative mechanism for
gene therapy. A key advantage of this system for immune-reactivity purposes stems from the nature of the
bacterial host capable of initiating an immune response by attracting recognition and cellular uptake by
antigen-presenting cells (APCs). The approach is also a suitable technique to deliver larger genetic
constructs more efficiently as it can transfer plasmids of varying sizes into target mammalian cells. Given
these advantages, bacterial vectors are being studied as potential carriers for the delivery of plasmid DNA
into target cells to enable expression of heterologous proteins. The bacteria used for bactofection are
generally nonpathogenic; however, concerns arise due to the use of a biological agent. To overcome such
concerns, enhanced bacterial degradation has been engineered as an attenuation and safety feature for
bactofection vectors. In particular, the ΦX174 lysis E (LyE) gene can be repurposed to both minimize
bacterial survival within mammalian hosts while also improving overall gene delivery. More specifically, an
engineered bacterial vector carrying the LyE gene showed improved gene delivery and safety profiles when
tested with murine RAW264.7 macrophage APCs. This chapter outlines steps taken to engineer E. coli for
LyE expression as a safer and more effective genetic antigen delivery bactofection vehicle in the context of
vaccine utility.
Key words E. coli, Bactofection, ΦX174 Gene E, Lysis E (LyE), Gene delivery
1 Introduction
The efficiency of gene therapy relies on the ability to deliver genetic
material in both a safe and efficient manner. Following the initial
successes in transferring bacterial genes into mammalian cells, bactofection emerged as an alternative method of engineered delivery
of DNA into human cells [1]. An underlying advantage of bactofection is the potential to leverage natural invasion and survival
strategies developed by microbial entities in the native transfer of
DNA and RNA.
An example is the cytosolic localization capabilities of Listeria
monocytogenes through use of an endosome-perforating listeriolysin
O (LLO) protein, which activates upon the development of the
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3
Improving E. coli Bactofection by Expression
of Bacteriophage ΦX174 Gene E
Dongwon Park, Andrew Hill, and Blaine A. Pfeifer
Abstract
Bactofection, a bacterial-mediated form of genetic transfer, is highlighted as an alternative mechanism for
gene therapy. A key advantage of this system for immune-reactivity purposes stems from the nature of the
bacterial host capable of initiating an immune response by attracting recognition and cellular uptake by
antigen-presenting cells (APCs). The approach is also a suitable technique to deliver larger genetic
constructs more efficiently as it can transfer plasmids of varying sizes into target mammalian cells. Given
these advantages, bacterial vectors are being studied as potential carriers for the delivery of plasmid DNA
into target cells to enable expression of heterologous proteins. The bacteria used for bactofection are
generally nonpathogenic; however, concerns arise due to the use of a biological agent. To overcome such
concerns, enhanced bacterial degradation has been engineered as an attenuation and safety feature for
bactofection vectors. In particular, the ΦX174 lysis E (LyE) gene can be repurposed to both minimize
bacterial survival within mammalian hosts while also improving overall gene delivery. More specifically, an
engineered bacterial vector carrying the LyE gene showed improved gene delivery and safety profiles when
tested with murine RAW264.7 macrophage APCs. This chapter outlines steps taken to engineer E. coli for
LyE expression as a safer and more effective genetic antigen delivery bactofection vehicle in the context of
vaccine utility.
Key words E. coli, Bactofection, ΦX174 Gene E, Lysis E (LyE), Gene delivery
1 Introduction
The efficiency of gene therapy relies on the ability to deliver genetic
material in both a safe and efficient manner. Following the initial
successes in transferring bacterial genes into mammalian cells, bactofection emerged as an alternative method of engineered delivery
of DNA into human cells [1]. An underlying advantage of bactofection is the potential to leverage natural invasion and survival
strategies developed by microbial entities in the native transfer of
DNA and RNA.
An example is the cytosolic localization capabilities of Listeria
monocytogenes through use of an endosome-perforating listeriolysin
O (LLO) protein, which activates upon the development of the
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021
3