PROJECT DESCRIPTION
Background
Staphylococcus aureus (S. aureus) is a common pathogen that the enterotoxins released by it is a sever pathogenic factor of food poisoning cases. S. aureus doesn’t need restricted conditions to grow; It has great resistance to arid, heated and saliferous conditions, therefore spreading and surviving widely in the environment around people. S. aureus is ubiquitous in nature which can be found in air, water, dust and human and animal’s waste. As a result, People can easily be exposed to S. aureus and get infected. Nowadays, S. aureus has become a worldly health problem. Reported by the U.S. Centers of Disease Control, about 25% food poisoning cases are caused by S. aureus infection in China, about 33% in the U.S., and even about 45% in Canada. As people infected by S. aureus, it will cause detrimental diseases as pneumonia, pericarditis, sepsis and death. In the hospital or communities, people catch S. aureus through contact infection and the morbidity rate in a developed country is between 100,000 to 300,000 people each year. According to data from the Emerging Infections program (EIP) and Cerner Electronic Health Record databases, 19,832 associated deaths occurred in an estimated 119,247 S. aureus bloodstream infections in 2017. To understand the spreading range of S. aureus, we construct the following experiments.
Theory
Why S. aureus can cause disease and the mechanism?
S. aureus, one of the most widespread pathogens, causes mainly pneumonia and other respiratory infections. The cases of patients getting infected by S. aureus can reach to a unbelievable number in America, which represents a huge public health burden. Since S. aureus isolates appear to be occasionally antibiotic resistant, the problems of infecting S. aureus prove to be especially important, among which the methicillin-resistant S. aureus (MRSA) receives more attention clinically. Compared with methicillin-sensitive S. aureus (MSSA), infections by MRSA are accompanied by increases in death rate, morbidity, and hospital stay.
The virulence factors of S. aureus often encode on the accessory genome of the pathogens, which differs from the code genome that encodes “housekeeping” functions. The accessory genome involves mobile genetic elements (MGE), such as plasmids, transposons, sequences and pathogenicity islands. Apart from virulence factors, it also contains antibiotic resistant determinants. The different MGE take the responsibility for coding different poisons, including TSST-1 or the food poisoning toxins.
Quorum sensing system in S. aureus
The regulation of virulence in S. aureus is highly complicated, consisting of large amounts of regulatory systems and parts, so that because of the size of the article, we only depict the most important ones. S. aureus virulence determinants is regulated by a large range of influences, including regulations by local-specific regulations, such as the icaR gene that is connected to ica operator, is subject to many impacts. Here, we just choose some specific condition. Apart from Agr, the exact model of the systems is hard to predict till now. Agr, or accessory gene regulator, is a quorum-sensing system that levels up the contents of many poisons and virulence determinants when the density of cells raise to a certain amount. Within a phagosome, factors controlled by Agr are expressed possibly by “diffusion sensing” to activates the quorum-sensing system. As predicted, inflammation caused by S. aureus are led by Agr mutants in many animal infection models. Because of the enlarging of the biofilm formed by Agr mutants, the cells turn to have a stronger resistance towards neutrophil attacks, leading to biofilm-related infections.
Design
Why we choose TurboID to block QS in S. aureus?
Before talking about TurboID itself, we are necessary to know that biotin has already been verified to be useful in the process of blocking surface receptors. Here is a concrete example, cited the information from Macromolecular Research, Vol. 15, No. 7, pp 646-655 (2007), the biotin-conjugated PEG/PCL block copolymer itself evidenced no significant adverse effects on human cells regardless of the cell types. Apparently, as TurboID is also a biotin, we can briefly conclude that TurboID is useful no matter what.
Then, we should now focus on TurboID itself. What is TurboID? TurboID a new pair of biotin ligases, was designed to overcome the problem of slow existing biotin ligases using yeast display directed evolution techniques, the labeling time will be shorted to less than 10 minutes. This enables probing of dynamic biological processes with higher temporal resolution. In addition to the shorter time of the labeling time, TurboID can also retain catalytic activity at low temperatures. In this case, we can clearly know that TurboID really is the one we should choose to use in our project as it is a biotin with very good performance.
How do we apply the TurboID to our project?
In our project, we use protein fusion technology to achieve blocking. However, we first need to know that we are capable to use this technology. One of the essential prerequisites of using this technology is that we're able to unraveling the mechanisms by which proteins fold into their correct three dimensional. We noticed that periplasmic cavity can ensure this prerequisite to happen. It is worth mentioning that a relatively well understood quality control machinery is presented in the cytoplasm. Apparently, we can know that we can successfully and correctly fold the protein under this kind of control. But the truth will be lots of the proteins are destined to encode in the cell envelope, and this is not what we expected to happen. According to scientific research, we can gain the knowledge that periplasmic cavity, which is a viscous and oxidizing compartment that contains a thin layer of peptidoglycan and represents 10 to 20% of the total cell volume, can prohibit the target protein to leak out of the cell envelope so that proteins can be successfully unraveled and folded. In this case, we are able to use this technology. In the last section, we need to focus on the technology of fusing proteins itself. The clear process of the fusion is shown on the graph beneath.
Fig1. Schematic model of Part1 system
If there is no IPTG TurboID will not be activated. If we add IPTG, it will activate TurboID and eventually complete the hole process.
As for our basic materials, biotin and ATP, we thought we could use engineered Escherichia coli (E.coli) to produce what we needed. Since there is already more than enough ATP in an organism, we only need to increase the production of biotin. We have two ways to achieve this. One is by increasing the production of biotin in one specific metabolic pathway, and the other is that we can block the other channel. However, blocking other channels will lead to a decrease in the growth rate and productivity of the bacteria, so we chose to increase the amount of the catalyst, which in turn produces more biotin. We will be increasing the catalyst of these two catalysts as shown. In this stage, we will put the artificially modified plasmid back into E.coli to express the target product so as to increase the biotin synthase and increase the biotin production. After that, we will use D-caspr 9 technique to block the pathway after the biotin has formed to prevent it from continuing disintegration.
Hypothesis for Inhibition of S. aureus by TurboID-AIP Specific Channel Protein Biotinylation
Here, we invent a new method to block the quorum sensing system in S. aureus. At level 0 which is the rest state, autoinducer protein (AIP) can interact with AgrC, followed by downstream activation of AgrA. AgrA is a quorum-sensing system that levels up the contents of many poisons and virulence determinants when the density of cells raise to a certain amount. AgrA can also amplify the quorum sensing signaling by initiating the expression of downstream gene cluster named agrBCDA. The AgrD protein can interacted with AgrB, then AgrD will be spliced into three parts and AIP can release into the environment to enhance quorum sensing signaling. At level 1, we purified the protein named TurboID-N-AgrD-AIP and these protein can have high affinity on interacting with AgrA through AIP domain and also have high affinity on interacting with AgrD through N-AgrD domain. At level 2, the chemical reaction (other named Protein proximity labeling) happens when we apply the system with biotin and ATP. TurboID will catalyze the biotin and ATP with the lysine residue on the surface of AgrA and AgrB. Those channel proteins have a lot of lysine exposed and will be prevented by interaction with AIP. As such, we can use this specific way to block the the quorum sensing system in S. aureus.
At level 3, we also hypothesis that if we can link the drug with the streptavidin. Compare with the traditional drug functioning in the body, the interaction between streptavidin with biotinylated AgrA and AgrB. We help the drug target the S. aureus and kill the bacteria at the same time.
Other application has been performed when we collaborate with Jilin_China team (Level 4). The invent a protein which can eliminate the Pb2+ in the environment. However, they can not qualify how many bacteria put into the environment can be enough to clean out Pb2+. How to qualify the interaction of their LPP-OmpA-pbrR bacteria with Pb2+. During the discussion with them, they are quite interesting with our design, so we invent a TurboID-pbrR which is a tool to evaluate how many empty LPP-OmpA-pbrR bacteria. The TurboID-pbrR will help to label the bacteria which have not been absorb with Pb2+. In this way, we help them qualify their engineering success at the molecular level.