Problem
Staphylococcus aureus is a type of bacteria commonly found in hospitals, sports facilities, and even the bodies of healthy individuals (1). As many as one third of the population carries S. aureus in their nose, and about 20% of people carry S. aureus on the skin (1). While S. aureus does not normally cause severe problems, it can cause infections of the skin, blood, and soft tissues, with approximately 20,000 deaths reported in the United States in 2017 as a result of S. aureus infection (2). Methicillin-resistant Staphylococcus aureus, or MRSA, is a potent strain of S. aureus that is resistant to common antibiotic treatments (1). This resistance to conventional treatment makes MRSA infections difficult to combat, turning S. aureus into a much more deadly pathogen. To overcome antibiotic resistance, the medical community must find new ways to combat bacterial infection.
Inspiration
Our team was inspired by a 2013 paper by Hwang et al. titled “Reprogramming Microbes to be Pathogen-Seeking Killers” (3). Hwang et al. engineered E. coli to detect and fight Pseudomonas aeruginosa infections using a seek-and-kill technique. Their design is modular, which makes it possible to use their approach to target different bacteria. We found that the rise of antibiotic resistant bacteria makes it increasingly vital to find new treatments, which inspired us to adapt the system devised by Hwang et al. to target methicillin-resistant Staphylococcus aureus, or MRSA.
Our modified version of the system, designed to target and treat Staphylococcus aureus.
Sensing
Quorum sensing is a method of cell communication that uses small molecules to regulate gene expression (4). Gram-positive and Gram-negative use different small molecules to accomplish quorum sensing; Gram-positive species use autoinducing peptides (AIP) while Gram-negative species use acyl homoserine lactones (AHL) (4). Our target species, Staphylococcus aureus, uses four forms of AIP in the accessory gene regulator (agr) quorum sensing system. In the agr system, the genes agrA and agrC code for proteins that detect AIP produced by neighboring S. aureus and activate the P2 promoter in response (5).
Our project builds on BBa_K1022100, a BioBrick that combines AgrA and AgrC from the agr sensing system with green fluorescent protein under a pBAD promoter. We found that the wild-type AgrA and AgrC proteins contain amino acids not present in BBa_K1022100, and we aim to modify and potentially improve the existing part by introducing these missing amino acids. The P2 promoter used in BBa_K1022100 is also missing base pairs found in the binding site region of the wild-type P2 sequence. We plan to reintroduce these binding sites by using a more complete P2 promoter sequence to improve GFP production in the original BioBrick. Lastly, SarA is a transcriptional activator found in S. aureus that is believed to activate expression of genes in the agr system (6). We will introduce the SarA gene to further improve the effectiveness of the agr system.
Motility
Chemotaxis is the movement of a cell towards or away from a chemical stimulus based on concentration, either from high to low or from low to high (7). In our system, AIP will provide the chemical gradient necessary to move E. coli in the direction of S. aureus. Chemotaxis is influenced by the post-translation modulation of the CheY and CheZ proteins; however, there is an issue of potential overexpression that can lead to the inhibition of chemotaxis (3). In order to combat this issue, we will be implementing a degron, a sequence of amino acids responsible for protein degradation, to control CheZ expression (3). We will attach the YbaQ degron to CheZ’s C-terminus, much like the application used by the Hwang et al. study in 2013 (3). To demonstrate the functionality of chemotaxis in our system, we will first use a pBAD promoter to trigger chemotaxis. If successful, we will replace the pBAD promoter with a P2 promoter which is activated by the agr system. This will ignite chemotaxis in the presence of AIP, moving the E. coli towards S. aureus.
Killing
Bacteriocins are small antimicrobial peptides (AMPs) produced by bacteria to kill or inhibit other bacteria. Many AMPs are currently under study as potential alternatives to antibiotic treatment due to the rise of antibiotic resistance. One such AMP is garvicin KS, a bacteriocin produced by Lactococcus garvieae, a bacterial species found in raw milk. Garvicin KS is effective against S. aureus and other Gram-positive bacteria, and is more potent than many other bacteriocins (8). Mature garvicin KS is composed of three polypeptides encoded by three genes: GakA, GakB, and GakC (9). By placing these genes into an E. coli chassis, we plan to use E. coli to produce garvicin KS. As E. coli is a Gram-negative bacterium, additional steps must be taken to ensure that the garvicin KS peptides can be secreted from the cell. We have chosen a novel signal peptide first constructed in a 2017 paper by Han et al. to assist with secretion of the garvicin KS peptides (10). This peptide tags proteins for secretion by the Sec pathway, a pathway used by E. coli to secrete proteins into the periplasm and outer membrane (10).
Impact
Our system has the potential to be used both in vivo and in vitro to combat MRSA planktonic cells and biofilms. Many bacteria produce biofilms, which are collections of cells attached to a surface and the extracellular matrix that encases these cells (11). Biofilms provide protection to the bacteria within and are often resistant to antibiotic treatment, which further compounds the issue of antibiotic resistance (12). One possible solution is the use of antimicrobial peptides such as garvicin KS, which have been shown to be more effective towards biofilms than traditional antibiotics (13). Our three module system first uses quorum sensing to detect AIP produced by MRSA biofilms and planktonic cells. Detection of AIP simultaneously triggers chemotaxis and the production of garvicin KS. The use of chemotaxis allows our E. coli to seek out and move towards biofilms and planktonic cells, releasing garvicin KS near the source and maximizing effectiveness. Potential applications of the system include detecting and killing MRSA infections in the body as well as disinfecting medical or sports equipment.
References
- Staphylococcal infections [Internet]. Merck Manuals; [updated 2017 Sept; cited 2019 Jun 28]. Available from: https://www.merckmanuals.com/professional/infectious-diseases/gram-positive-cocci/staphylococcal-infections
- Staph infections can kill [Internet]. Centers for Disease Control and Prevention (US); [updated 2019 Mar 22; cited 2019 Jun 28]. Available from: https://www.cdc.gov/vitalsigns/staph/index.html">https://www.cdc.gov/vitalsigns/staph/index.html
- Hwang IY, Tan MH, Koh E, Ho CL, Poh CL, Chang MW. Reprogramming microbes to be pathogen-seeking killers. ACS Synth Biol. 2013;3(4):228-237.
- Rutherford ST, Bassler, B. L. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med. 2012;2(11).
- Tan L, Li SR, Jiang B, Hu, XM, Li S. Therapeutic targeting of the Staphylococcus aureus accessory gene regulator (agr) system. Front Microbiol. 2018;9(55).
- Cheung AL, Zhang G. Global regulation of virulence determinants in Staphylococcus aureus by the SarA protein family. Front Biosci. 2002;7:1825-1842.
- Wang Y, Chen CL, Iijima M. Signaling mechanisms for chemotaxis. Dev Growth Differ. 2011;53(4):495-502.
- Chi H, Holo H. Synergistic antimicrobial activity between the broad spectrum bacteriocin garvicin KS and nisin, farnesol, and polymyxin B against Gram-positive and Gram-negative bacteria. Curr Microbiol. 2018;75(3):272-277.
- Ovchinnikov KV, Chi H, Mehmeti I, Holo H, Nes IF, Diep, DB. Novel group of leaderless multipeptide bacteriocins from Gram-positive bacteria. Appl Environ Microbiol. 2016;82(17):5216-5224.
- Han S, Machhi S, Berge M, Xi G, Linke T, Schoner R. Novel signal peptides improve the secretion of recombinant Staphylococcus aureus alpha toxin H35L in Escherichia coli. AMB Expr. 2017;7(93).
- Lopez D, Vlamakis H, Kolter R. (2010). Biofilms. Cold Spring Harb Perspect Biol. 2010;2(7).
- Ciofu O, Rojo-Molinero E, Macia MD, Oliver A. (2017). Antibiotic treatment of biofilm infections. APMIS. 2017;125(4).
- Mathur H, Field D, Rea MC, Cotter PD, Hill C, Ross RP. (2018). Fighting biofilms with lantibiotics and other groups of bacteriocins. NPJ Biofilms Microbiomes. 2018;4.
