Team:Tongji China/P.A.

P.A.

Background

P. aeruginosa

Now, we are concerning about the species and strain of the bacteria involved in our project. We choose Pseudomonas aeruginosa PAK-JΔ9 which is an attenuated strain, for it has many advantages varying from efficiency to safety. To be specific, we will introduce the course of searching for this ideal strain step by step to you.

The Reason that we choose P. aeruginosa

We decided to use Type III secretion system (T3SS) to activate the patient's immune system in the first place, because it seems to be a practical way. So which type of bacteria can we choose? We went through carefully investigation and finally focused our attention on Pseudomonas aeruginosa (P. aeruginosa).

1 We already knew that T3SS is one of the features that belong to Gram-negative bacteria, and there are significant differences between species. As a result, some important species which are widely used in attenuated vaccine such as Listeria monocytogenes are out of the range because of its classification.

2 Referring to a large number of articles, we discovered that P. aeruginosa's T3SS shows little difference associated with the change of pH value, which is a distinct feature of intestinal environment, while others' don't. T3SSs can be divided into two groups by the homologue of its core proteins. IpaD, BipD, and SipD have similar physical properties such as stability and spectroscopic measurements and they fall into one subfamily, while LcrV and PcrV also share these properties together. Proteins in IpaD subfamily manifest three distinct structural forms from pH 3 to 8 which result in enormous decline in ability of T3SS. In contrast, LcrV and PcrV show little differences in structures associated with the change of pH value, as well as its property. Because high pH value is a feature of intestinal environment, we can assume that bacteria whose T3SS is construed with LcrV subfamily as the core can better serve in intestinal environment. And, of course, P. aeruginosa is one of them. [6][7]

3 When it comes to T3SS, there are three bacteria strains that are widely used in research, such as Salmonella spp., Yersinia spp. and also P. aeruginosa. Comparing their pathogenicity, wild type Salmonella and Yersinia are all pathogenic bacteria. Salmonella would cause Gastroenteritis fever and Yersinia would cause Enterocolitis. But the wild type P. aeruginosa is opportunistic pathogenic, which means that it will be safer and less toxic in terms of the human disease treatment.

4 There are already some researches using attenuated P. aeruginosa to deliver some proteins of interest. The common way they use is directly injecting the bacteria into vein. After injection, P. aeruginosa would gather in two organs, liver and spleen, which are the places the immune cells are concentrated. So we indicate that the P. aeruginosa may be more likely to attach the immune cells, which are also our target cells.

5 P. aeruginosa has been proved not belong to the inherent germs in our intestine. So when P. aeruginosa enters the intestine, it would be attacked by the immune system not only in intestinal, but also systemic compartments. Moreover, the inherent germs in our intestine could also be a threat to it. The role of intestinal microbiota in this course is proved by a trial conducted between fecal microbiota transplantation mice and normal mice. The trial shows a significant difference of colonization ratio between these two groups, and we can surmise that it is resulted from the differentiation of intestinal microbiotas. If these microbiotas do make a difference, they are more likely to be regarded of influencing the colonization of P. aeruginosa. So the attenuated P. aeruginosa cannot stay in the intestine for too long and the majority of them will be eliminated in the intestine. [8]

©Dennis Kunkel Microscopy

Electron microscopy picture of Pseudomonas aeruginosa

P. aeruginosa.

Pseudomonas aeruginosa is one of the ubiquitous gram-negative rods which distributes widely in the surroundings. 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 after oral administration? Of course not. Description above is for the wild-type P. aeruginosa, while the bacteria we use is modified to ensure its safety. This strain is called PAK-JΔ9, an advanced type comparing to PAK-JΔ8, whose extra feature makes it auxotrophic. Δ8 P. aeruginosa is a genetically engineered attenuated strain, belonging to the fourth safety level with the lowest degree of damage, which is at the same level as the vaccine for human body use. The strain is attenuated by deleting all its virulence factors by genetic manipulation on the basis of P. aeruginosa. The biosafety laboratory level required for the experimental activities of this species is BSL-1/ABSL-1 (laboratory/animal laboratory), 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. 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 be amplified in the human body and survive for a long time, nor can they survive in the natural environment.

The immunoreaction

By sending the bacterial into the gut, the antigen can then be delivered into several kinds of intestinal mucosa- colonized APCs by T3SS. These cells will cut antigens into proper size and combine with MHC I waiting for the CD8+ T cells’ recognition while traveling to peripheral immune organs. By recognizing specific antigen by TCR, a series complex reactions inside the cell will finally activate both T cells and APCs. After activation, as for CD8+ cell, with the stimulation of different cytokines, will differentiate into several kinds of cells which can enhance the immunological reaction by releasing cytokines and activating other immunological cells who can directly kill the target cell.
Finally, some T cells will become memory T cells with the ability to remember the antigen and accelerate the reaction when meeting the same antigen. This means when attacked by tumor cells, organisms immunized by OCANDY can start the immunological reaction with high efficiency and finally avoid the tumorigenesis on early stage.

To know more about the safety of the P. aeruginosa, you could go to the Safety-P. aeruginosa.

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

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