Background Information
Staphylococcus aureus (S. aureus) is a highly prevalent and pathogenic bacterium that poses a significant public health concern. It is responsible for a wide spectrum of infections, affecting various body systems such as internal organs, endovascular sites, and the skin and soft tissues. Surveys have revealed that a substantial portion of the population carries S. aureus asymptomatically, with approximately 30% of adults harboring the bacterium in their nasal passages and 20% on their skin, even in the absence of apparent illness. (van Hal et al., 2012)
Figure 1. The statistics data of S. aureus's affections
Furthermore, S. aureus infections can have severe consequences, extending beyond superficial skin infections. They can lead to life-threatening conditions such as infective endocarditis (heart valve infection), pneumonia, and osteomyelitis (bone infection). The emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains has added to the gravity of the situation. According to statistics from the World Health Organization (WHO), MRSA accounts for 35.86% of bloodstream infections among patients seeking medical care. This highlights the urgency in addressing the impact of S. aureus infections on global health. It is estimated that there are approximately 140 million individuals, including 100 million children, affected by S. aureus infections worldwide. (Stoltenburg et al., 2016)
Problem faced
S. aureus originates primarily in the human intestinal lumen. Its presence in this location can lead to severe skin infections. Currently, serologic tests based on antigen-antibody interactions are widely used for S. aureus detection. However, these tests have limitations in terms of being semi-quantitative and lacking specificity and sensitivity. Molecular biological methods, such as PCR, can also be employed for S. aureus detection. (Wu et al., 2016)
Nevertheless, routine microbial screenings, including S. aureus, often involve expensive and time-consuming procedures, making them less accessible to the general population. Furthermore, S. aureus has emerged as one of the earliest bacterial species to develop resistance against antibiotics. Over the years, it has acquired resistance to multiple antibiotics, such as Methicillin, leading to the emergence of methicillin-resistant Staphylococcus aureus (MRSA). (Lee et al., 2018) This ability of S. aureus to evolve and acquire resistance poses a significant challenge in the treatment of infections caused by this bacterium. As MRSA infections can be prevalent in hospitals and the community to cause from minor skin infection to life-threatening disease like pneumonia, it is important to find a convenient and efficient way to eradicate the bacteria in human body. It is suggested that the dominating habitat of S.aureus may be the intestine rather than the nose in humans. Frequent anorectal-nasopharyngeal transmission results in nasal colonization through re-inoculation from a permanently S. aureus-colonized intestine. Several evidences supported that human intestinal colonization, the much more extended reservoir than nasal colonization, plays an important role in leading to infections. For example, in children infected with MRSA strains, staphylococcal skin and soft tissue infections are linked to rectal colonization instead of nasal colonization. Additionally, the failure of nasal decolonization measures could be explained by intestinal S. aureus colonization. (Piewngam & Otto, 2019)
These findings emphasize the urgent need for effective prevention, diagnosis, and treatment strategies to combat S. aureus infections. Further research and innovative approaches are necessary to address the challenges posed by this pathogen and mitigate its impact on public health.
Our solutions
Our project is focused on the development of an accurate, cost-effective, and non-resistable detection and elimination kit for S. aureus in the human lumen. The kit comprises three essential components: in vivo detection of S. aureus, in vivo elimination of the bacteria, and in vitro confirmation of elimination.
1. S. aureus in vivo detection
The Quorum sensing (QS) pathway in S. aureus plays a crucial role in the detection and response to extracellular communication molecules -- autoinducer peptides (AIPs). Through the Agr locus and the P2 promoter, the sensor kinases in S. aureus detect AIPs and trigger a series of events. AIPs bind to AgrC, the histidine kinase, leading to the activation of AgrA, the response regulator. Phosphorylated AgrA then binds to the P2 promoter, resulting in the auto-induction of the Agr operon. (Tan et al., 2018) By utilizing the properties of AgrC, AgrA, and the p2 promoter, we can establish a bacterial detection system using the QS mechanism. (Wang et al., 2014) When AIPs are present, the P2 promoter initiates the conditionally expressed endolysin SPN1S_Lys RZ (spanin), leading to the lysis of the genetically modified E. coli. This lysis event allows for the release of S.aureus-specific endolysins and antimicrobial peptide (AMP) into the extracellular environment.
Figure 2. Quorum sensing pathway of S.aureus
While there may be differences in membrane structure between gram-negative and gram-positive bacteria, previous studies have shown the establishment of interspecies signaling pathways between E. coli. and other gram-positive species such as Bacillus megaterium. These studies have demonstrated that AIPs can cross the membrane of E. coli. with the assistance of AgrD, which is part of the Agr gene cluster responsible for AIP generation in gram-positive bacteria. This provides initial confirmation that Agr can function in the membrane of E. coli. (Marchand & Collins, 2013) Additionally, we have also utilized Bacillus Subtilis as an engineered bacteria to implement the Agr circuits in our project.
2. S. aureus in vivo elimination
In our project, we employ three proteins, namely Endolysin ClyC, LysDZ25, and antimicrobial peptide (AMP) LL-37, to specifically target and eliminate S. aureus without promoting resistance. (Moon et al., 2006) The design of the in-vivo detection followed by the in-vivo elimination can protect endolysins and AMPs from intestinal environment and release them in the early stage of colonization formation, allowing our product to prevent infections by being taken beforehand.These proteins are continuously produced within the cytoplasm of the recovered freeze-dried E.coli. As we mentioned before, once AIPs are detected, the E.coli. undergoes self-lysis, releasing the endolysins and AMP to effectively eliminate S. aureus.
To ensure efficacy in the intestinal lumen environment, which can be sensitive, we have chosen different endolysins with specific functionalities. Endolysins are chosen to fit the intestinal environment where pH ranges from 4 to 8 and salinity level varies due to food ingestion. LysDZ25 demonstrates effectiveness in alkaline environments and even under high salinity conditions. (Chang et al., 2023) On the other hand, ClyC shows efficacy in low salinity environments. (Lee et al., 2021) By combining a protective capsule and controlled release mechanisms, we aim to prevent any potential harmful effects on the intestine. Furthermore, we have incorporated a strict regulation system using the RHa promoter to minimize any possible harm to the intestine after the capsule is ingested.
3. S. aureus in vitro detection
In our project, we aimed to find a specific and high-affinity single-strand DNA aptamer targeting the S. aureus specific transport protein Protein A. According to previous study, we found PA#2/8 has the specificity to Protein A.
Figure 3. 3-D model of Protein a
To validate the binding interaction between Protein A and PA#2/8, we performed an electrophoretic mobility shift assay (EMSA). The results of the EMSA confirmed the formation of a specific complex between the aptamer and the target protein, indicating proper binding. Additionally, we sought to optimize the aptamer design by generating truncated variants of PA#2/8 and evaluating their affinity towards Protein A. This optimization process was carried out using an Enzyme-Linked Oligonucleotide Assay (ELONA), which allowed us to quantitatively measure the binding affinity of the truncated aptamers to Protein A. We carefully considered both the affinity and specific characteristics of the truncated aptamers to identify the most optimal candidate with strong binding affinity and remarkable specificity for Protein A.
Figure 4. The secondary structure of PA#2/8
Other concerns
To optimize our therapeutic bacteria for medical use, we encapsulate them in a protective enteric capsule. When these bacteria detect S. aureus, they release antibacterial substances to inhibit its growth. The release is tightly controlled to ensure precise action. Additionally, we have incorporated a cold-inducible system that triggers self-destruction of the bacteria when excreted and exposed to lower temperatures. This strategy enhances the effectiveness and safety of the treatment.
References
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Stoltenburg, R., Krafčiková, P., Víglaský, V., & Strehlitz, B. (2016). G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA. Scientific Reports, 6 (1). https://doi.org/10.1038/srep33812
Wu, S., Duan, N., Gu, H., Hao, L., Ye, H., Gong, W., & Wang, Z. (2016). A Review of the Methods for Detection of Staphylococcus aureus Enterotoxins. Toxins, 8(7), 176. https://doi.org/10.3390/toxins8070176
Lee, A. S., de Lencastre, H., Garau, J., Kluytmans, J., Malhotra-Kumar, S., Peschel, A., & Harbarth, S. (2018). Methicillin-resistant Staphylococcus aureus. Nature Reviews Disease Primers, 4(1). https://doi.org/10.1038/nrdp.2018.33
Tan, L., Li, S. R., Jiang, B., Hu, X. M., & Li, S. (2018). Therapeutic Targeting of the Staphylococcus aureus Accessory Gene Regulator (agr) System. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.00055
Wang, B., Zhao, A., Novick, R., & Muir, T. (2014). Activation and Inhibition of the Receptor Histidine Kinase AgrC Occurs through Opposite Helical Transduction Motions. Molecular Cell, 53(6), 929–940. https://doi.org/10.1016/j.molcel.2014.02.029
Marchand, N., & Collins, C. H. (2013). Peptide-based communication system enables Escherichia colito Bacillus megaterium interspecies signaling. Biotechnology and Bioengineering, 110(11), 3003–3012. https://doi.org/10.1002/bit.24975
Lee, C., Kim, J., Son, B., & Ryu, S. (2021). Development of Advanced Chimeric Endolysin to Control Multidrug-Resistant Staphylococcus aureus through Domain Shuffling. ACS Infectious Diseases, 7(8), 2081–2092. https://doi.org/10.1021/acsinfecdis.0c00812
Chang, Y., Li, Q., Zhang, S., Zhang, Q., Liu, Y., Qi, Q., & Lu, X. (2023). Identification and Molecular Modification of Staphylococcus aureus Bacteriophage Lysin LysDZ25. ACS Infectious Diseases, 9(3), 497–506. https://doi.org/10.1021/acsinfecdis.2c00493
Moon, J. Y., Henzler-Wildman, K. A., & Ramamoorthy, A. (2006). Expression and purification of a recombinant LL-37 from Escherichia coli. Biochimica Et Biophysica Acta (BBA) - Biomembranes, 1758(9), 1351–1358. https://doi.org/10.1016/j.bbamem.2006.02.003
Piewngam, P., & Otto, M. (2019, March 26). Probiotics to prevent Staphylococcus aureus disease? PubMed Central (PMC). https://doi.org/10.1080/19490976.2019.1591137