Pseudomonas aeruginosa as Vaccine-vectors
The potential use of attenuated bacteria as engineered vectors for vaccine development offers several advantages, including the stimulation of innate immunity. During the last few years, the use of type III secretion system-based bacterial vectors for immunotherapy purposes has been assessed in various applications. The genetically modified and attenuated Pseudomonas aeruginosa has been showed to deliver protein antigens to antigen presenting cells via a bacterial type III secretion system.
Introduction of Pseudomonas aeruginosa
Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous Gram-negative bacterium found in soil, water, and many other environmental settings. The P. aeruginosa genome is one of the largest bacterial genomes at approximately 6 million base pairs, encoding 5,500 open reading frames with a significant proportion of the genome dedicated to regulatory genes and genes associated with the breakdown, transport and efflux of organic compounds. Due to its diverse lifestyle and its genetic and metabolic capabilities, P. aeruginosa has been used as a host for many areas of biotechnology, including bioremediation and industrial production of proteins. Clinically, P. aeruginosa is an important human pathogen causing many nosocomial infections, therefore, a genetic study of P. aeruginosa is an important research field of medical microbiology.
Type III Secretion System
In recent years, type III secretion systems (TTSS) from Gram-negative bacteria have been used for antigen delivery in various applications. TTSS, also known as needle complex, is a protein secretion device composed of more than 20 kinds of proteins, forming a basal body anchored in the bacterial membranes and an injection needle through which effectors travel to reach target cell cytoplasm. Typically, a set of three proteins, two hydrophobins and one hydrophilic protein are required to allow the protein to translocate from the bacteria to the cytoplasm of the host cell. These highly sophisticated natural injection devices allow bacteria to deliver effector proteins to the cytoplasm of eukaryotic cells to modulate host cell function.
Our Design for P. aeruginosa Type III Secretion System
P. aeruginosa contains a set of TTSS systems consisting of 36 genes that together provide elaboration and regulation of the supramolecular "needle" structure. These supramolecular "needle" structures are capable of spanning the inner and outer membranes of the cell as well as the peptidoglycan layer and provide a conduit through which the effector protein can be secreted directly from the bacterial cytosol into the host cell. To date, four effector proteins (ExoS, ExoT, ExoU, and ExoY) have been identified in P. aeruginosa whose products are actively transferred into the host cells. Among them, ExoS is a bifunctional toxin encoded by the exoS gene, which is found in about 70-80% of clinical isolates of P. aeruginosa.
The P. aeruginosa TTSS is regulated by exsA, a member of the AraC/XylS family of transcriptional regulators that drive expression of the operon encoding the secretion machinery, chaperones, and effectors. To control fusion protein expression in P. aeruginosa, Creative Biolabs has developed a number of live attenuated bacterial vectors based on P. aeruginosa TTSS through genetic engineering techniques. The N-terminal 54 (N54) amino acids of P. aeruginosa TTSS toxin ExoS are required for efficient secretion and translocation of proteins. Therefore, we cloned the exsA gene encoding the ExoS transcriptional activator into a vector under the control of an IPTG-inducible promoter. The addition of IPTG to the culture resulted in an increase of secreted ExoS N54-fusion proteins. The cytotoxicity of the strain can be reduced by deleting the sequences encoding the two TTSS toxin ExoS and ExoT. Following subcutaneous injection of this vector, the fusion proteins are translocated into different cell types including APCs.
Figure 2. Schematic representation of the TTSS. (Derouazi M, 2010)
Advantages of Pseudomonas aeruginosa as Delivery Vector
- The ability to stimulate the innate immune system
- Simple route of administration
- Properly secrete and transport large proteins
- Can induce an antigen-specific CD8+ T-cell response
With years of experience and advanced vaccine technology platform, Creative Biolabs has developed engineered and attenuated P. aeruginosa vectors based on the type III secretion system that deliver proteins in vitro and in vivo into the cytosol of eukaryotic cells. If you have any need for P. aeruginosa as a delivery vehicle, we are your best choice.
Reference
- Derouazi M., et al. (2010). Optimal epitope composition after antigen screening using a live bacterial delivery vector. Bioengineered Bugs. 1(1), 10.
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