Team:Tongji China/Safety P.A.

Safety P.A.

Safety

P. aeruginosa

Pseudomonas aeruginosa is one of ubiquitous gram-negative rods which we can see in many environments. It is also called hospital-acquired infections because it has multidrug-resistance which ensures its survival state. Also, P. aeruginosa is an opportunistic extracellular pathogen to human. Because this bacterium is responsible for some diseases such as acute infections with burn wound and its primary hazard is the infections during surgery.

The way of attenuating

So, won't such a dangerous pathogen do harm to patients via oral approach? Of course not. Description before this paragraph is for the wild-type P. aeruginosa, while the bacterium we use is modified to ensure its safety. This strain is called PAK-JΔ9 and is advanced compared to PAK-JΔ8. PAK-JΔ9 was added with a feature that makes it an auxotrophic bacterium. The μA gene of this strain is mutated so they cannot live without the presence of D-Glutamate. But the human body does not contain D-Glutamate, so this P. aeruginosa strain cannot amplify in the human body or survive for a long time, nor can they survive in the natural environment.

P. aeruginosa Δ8 is a genetically engineered attenuated strain, belonging to the fourth species level with the lowest degree of damage, which is the same level as the vaccine for human body use. The biosafety laboratory level required for the experimental activities of this species is BSL-1/ABSL-1 (laboratory/animal laboratory), which is the lowest biosafety level. According to the classification and packaging requirements of ICAO document Doc9284 "Technical Specifications for the Safe Transport of Dangerous Goods", the species belongs to Class B transport requirements, and the corresponding UN number is UN 3373 for packaging and transportation.

• First, it is rid of exoS, exoT, exoY, and ndk genes, which are exotoxins involved in T3SS. This modification ensures that individuals in this strain lose their partial hazard. [2][3]

• Then, deletion for the xcpQ in this strain blocks T2SS, which is the system for P. aeruginosa to release toxin into the surroundings. And this is an insurance measurement for attenuating. [4]

• Also, PAK-JΔ8's queue sensing is blocked by knocking lasR-Ⅰand rhlR-Ⅰout. The queue sense is equally important to the formal portions. Because it is critical in the formation of biofilm shown in lawns, which is an important structure for these germs to resist the host's immune system or other unfavorable environment and other biosynthesis such as iron-acquiring siderophore. [5]

The toxin genes contains in the original P.A. strain

• Finally, to facilitate the experiment on T3SS, popN is knocked out to enhance the frequency of using T3SS. These modifications block almost all the accesses for P. aeruginosa to the detriment of human beings, and it is proved by many clinical trials. [1]

Other insurances

While determining which kind of bacteria we choose to experiment with, we also analyze the disturbances to the balance of the homeostasis of intestinal environment in oral approach. As we all know, P. aeruginosa is not a kind of inherent germs in our intestine, so it will be eliminated by force from intestinal flora and our own immune system as soon as it is taken in. Also, subsequent study shows that it is easier for P. aeruginosa to attack immune cells in intestinal environment compared to other kind of cells. It is more convenient for us to use this kind of species.

Also, we have designed a complete security system to avoid bio-hazard caused by leaking of this engineered strain. It will be covered in the next portion of our wiki.

References：

[1] Galle, M., Carpentier, I., & Beyaert, R. (2012). Structure and Function of the Type III Secretion System of Pseudomonas aeruginosa. Current Protein & Peptide Science, 13(8), 831–842. http://doi.org/10.2174/138920312804871210
[2] Yang, H., Shan, Z., Kim, J., Wu, W., Lian, W., Zeng, L., … Jin, S. (2007). Regulatory Role of PopN and Its Interacting Partners in Type III Secretion of Pseudomonas aeruginosa . Journal of Bacteriology, 189(7), 2599–2609. http://doi.org/10.1128/JB.01680-06
[3] Neeld, D., Jin, Y., Bichsel, C., Jia, J., Guo, J., Bai, F., … Jin, S. (2014). Pseudomonas aeruginosa injects NDK into host cells through a type III secretion system. Microbiology, 160(Pt 7), 1417–1426. http://doi.org/10.1099/mic.0.078139-0
[4] Bitter, W. , Koster, M. , Latijnhouwers, M. , De Cock, H. and Tommassen, J. (1998), Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Molecular Microbiology, 27: 209-219. doi:10.1046/j.1365-2958.1998.00677.x
[5] Bjarnsholt, T., Tolker-Nielsen, T., Høiby, N., & Givskov, M. (2010). Interference of Pseudomonas aeruginosa signalling and biofilm formation for infection control. Expert Reviews in Molecular Medicine, 12, E11. doi:10.1017/S1462399410001420
[6] Diepold, A., & Armitage, J. P. (2015). Type III secretion systems: the bacterial flagellum and the injectisome. Philosophical Transactions of the Royal Society B: Biological Sciences, 370(1679), 20150020. http://doi.org/10.1098/rstb.2015.0020
[7] Markham, A., Birket, S., Picking, W., Picking, W., & Middaugh, C. (2010). Ph sensitivity of type iii secretion system tip proteins. Proteins Structure Function & Bioinformatics, 71(4), 1830-1842.
[8] Von Klitzing, E., Ekmekciu, I., Bereswill, S., & Heimesaat, M. M. (2017). Intestinal and Systemic Immune Responses upon Multi-drug Resistant Pseudomonas aeruginosa Colonization of Mice Harboring a Human Gut Microbiota. Frontiers in Microbiology, 8, 2590. http://doi.org/10.3389/fmicb.2017.02590