Team:Tongji China/T3SS
Background
Type III secretion system
Type III secretion system (T3SS) is a highly coordinated multi-protein system which consists of structural, regulatory and secreted proteins. The structure of the type III secretion nanomachine is highly conserved among Gram-negative bacteria, which is defined as "injectisome". To make good use of its infecting potential, T3SS can be an amazing tool to deliver proteins. The moment we found this surprising tool in literature, we decided to utilize this system to deliver our item antigens.[1]
Structure - injectisome
In fact, the structure of T3SS looks quite like a needle tubing, and its function is like a needle tubing as well. That is why we called it "injectisome". The T3SS injectisome is composed of a needle complex, an inner membrane export apparatus, and a cytoplasmic platform that energizes the secretion process and selectively sorts substrates for their orderly delivery to the secretion machine.
The needle complex is composed of a multi-ring cylindrical base with ~26 nm in diameter that is anchored on the bacterial envelope and a needle-like structure that projects ~60 nm from the bacterial surface. The entire structure is traversed by a channel ~2 nm in diameter that serves as a conduit for the passage of proteins injected through the type III secretion machinery. An ATPase at the cytoplasmic sorting platform provides energy for the protein export.
Figure1 (left). Injectisome structure
Figure2 (right). Delivery process
Process - delivery
The bacterial T3SS has been exploited to deliver antigenic peptides and proteins into various target cells. Type III effectors were shown efficiently injected into a wide range of host cells, including professional antigen presenting cells (APCs), such as macrophages and dendritic cells (DCs). First T3SS comes into being by attaching with host cells, and the attachment triggers the formation of the translocon. A set of pore-forming proteins are transported through the needle and are inserted into the eukaryotic cell membrane to form the translocon. Following the pore formation, type III secretion regulatory protein (repressor) is secreted, resulting in transcriptional activation of the whole T3SS regulon genes.[2]
Then viral and bacterial epitopes, as well as peptides from human tumors, have been translated into protein and delivered by the bacterial T3SS with the aim to elicit immune response (vaccination) or cancer immunotherapy.
"Polar translocation" vs "Non-polar translocation"
Secretion of the antigens can be activated in two ways. One is to form the host cell contact. As is described above, when a contact signal has been sensed by the bacteria, a rapid production and specific insertion into the translocon follows and the antigens can successfully be injected into the host cell cytosol, without wasting them into the culture supernatant. This way is called "polar translocation". Another way is triggered with low calcium environment, such as in the presence of calcium chelator EGTA. It can trigger the bacteria to release the antigens into the culture medium without the formation of the T3SS translocon. This way works without the presence of host cells and is defined as "non-polar translocation". We use both of these activation methods to test the efficiency of our system.
P. aeruginosa's Type III secretion system
The Type III secretion system (T3SS) of P. aeruginosa is an important virulence determinant. Transcription of the T3SS is highly regulated and coupled to the activity of the type III secretion channel (pore). The pore is generally closed, and transcription of the effector protein is repressed. Inducing signals such as calcium depletion and host cell contact, can open the secretion pore and activate transcription of the T3SS. The coupling of transcription with secretion requires many cytoplasmic regulatory proteins. And some of them play an important role in the whole process. Taking the example of host cell contact induction and the T3SS system we used, let us take a deep view of how this cascade control mechanism works.
The cytoplasmic protein ExsE, ExsC, ExsD and ExsA are four proteins that control the coupling of transcription and secretion. ExsA is a DNA-binding protein required for transcriptional activation of the entire T3SS. The second regulatory protein, ExsD, functions as an anti-activator by directly binding to ExsA. ExsC functions as an anti-anti-activator by directly binding to and inhibiting ExsD.[3] ExsE functions as a direct inhibitor of ExsC and provides an initiating signal for the whole process. Antigens are cloned and expressed on the Escherichia-Pseudomonas shuttle expression plasmid, which encodes the T3S effector ExoS promoter with N-terminal ExoS1–54 signal sequence, followed by a FLAG tag and a multiple cloning site (MCS). We knock out the ExoS toxin gene (a kind of toxins which wild type P. aeruginosa injects into the host cell) in the attenuated P. aeruginosa we use, and use ExoS1-162 to act as a secretion signal.
When there is no inducing signal, the T3SS pore remains closed. ExsE binds to the ExsC and ExsD binds to the ExsA to directly inhibit its activation. It seems that there might be some ExsCs binding to ExsDs, but Exscs prefer binding with ExsEs to bingding with ExsDs.
When a host cell contact has been sensed by the bacteria, a set of pore-forming proteins are transported through the needle and are inserted into the host cell membrane, called translocon. The formation of the translocon will enable the T3SS pore to open and the protein injecting channel comes into being.
The open of the T3SS pore results in the secretion of repressor protein ExsE. The secretion of ExsEs releases ExsCs and they subsequently bind with the ExsDs. ExsAs are set free and derepressed. Free ExsAs bind to the promoter region of our expression plasmid and start the transcription of item antigens.[4]
Free ExsAs bind to the promoter region of our expression plasmid and start the transcription of item antigens.
Item antigens go through the channel and enter the host cell membrane. The whole process of the P. aeruginosa T3SS protein secretion is finished. The antigens will undergo various processes and finally be presented to the CD8+ T cells. Want to know more about the antigen presenting? You can go to visit the Background-P. aeruginosa
References:
[1] Bai F, Li Z, Umezawa A et al. Bacterial type III secretion system as a protein delivery tool for a broad range of biomedical applications. Biotechnol Adv 2018;36:482–93.
[2]Galle M, Carpentier I, Beyaert R. Structure and function of the Type III secretion system of Pseudomonas aeruginosa. Curr Protein Pept Sci. 2012;13:831–42. doi: 10.2174/138920312804871210.
[3]Arne Rietsch, Isabelle Vallet-Gely, Simon L. Dove, and John J. Mekalanos ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa, PNAS, 2005, 102 (22) 8006-8011
[4]Mark L. Urbanowski, Guinevere L. Lykken, and Timothy L. Yahr, A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system, PNAS July 12, 2005, 102, (28) 9930-9935.
Return to the Background Overview[1] Bai F, Li Z, Umezawa A et al. Bacterial type III secretion system as a protein delivery tool for a broad range of biomedical applications. Biotechnol Adv 2018;36:482–93.
[2]Galle M, Carpentier I, Beyaert R. Structure and function of the Type III secretion system of Pseudomonas aeruginosa. Curr Protein Pept Sci. 2012;13:831–42. doi: 10.2174/138920312804871210.
[3]Arne Rietsch, Isabelle Vallet-Gely, Simon L. Dove, and John J. Mekalanos ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa, PNAS, 2005, 102 (22) 8006-8011
[4]Mark L. Urbanowski, Guinevere L. Lykken, and Timothy L. Yahr, A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system, PNAS July 12, 2005, 102, (28) 9930-9935.
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