Project Description

Background

Acne Vulgaris, characterized by pimples, pustules, and lesions induced by the presence of Propionibacterium acne (P. acne), is a prevalent skin condition affecting approximately 9.4% of the global population [1]. This anaerobic, gram-positive bacterium naturally resides in human sebaceous glands, making individuals prone to follicle inflammation, residual scarring and pigmentation. According to the American Academy of Dermatology Association, acne persists chronically and infects up to 85% of people aged 12 to 24. Beyond its physical manifestations, acne inflicts significant psychological and emotional distress, notably impacting self-esteem and overall mental well-being. Individuals with acne are found to have reduced social activities, negative impact on work/study, and interpersonal problems [2]. In response to the challenges posed by the treatment of Acne Vulgaris, we conducted the following experiment for a combination of fast detection of P. acne for targeted audiences and high-specificity prevention of Acne Vulgaris. This research aims to contribute to a better understanding of effective interventions for inhibiting P. acne presence, addressing both its physical and psychological dimensions, with the ultimate goal of improving the well-being of those affected by this common skin condition.

Theory

1. Pathogenesis of acne

The development of acne is intricately linked to the proliferation of Propionibacterium acne (P. acne). As a human skin commensal, P. acne naturally resides in the sebaceous glands, obtains its energy from the fatty acids present in the environment, and undergoes metabolic activity that induces follicular irritation, inflammation, and imbalance of the skin microbiome [3].

2. Conventional Solutions and Detection

The efficacy of current acne treatments on the market is far from uniform. A finding by Z. Draelos [4] discovered that topical treatments for acne, including azelaic acid, salicylic acid, nicotinamide, and alpha-hydroxy acid have shown insignificant improvements in skin conditions. Despite the prevalence of over-the-counter (OTC) acne treatment products, many of which do not even list active ingredients [5]. The potential damage to the skin barrier caused by both acne itself and conventional treatment methods, as outlined in Dermatology Therapy Vol. 3, Issue 6, underscores the need for a more holistic approach to acne treatments. Furthermore, there is an absence of a fast, cost-effective, and efficient method for identifying high-risk individuals by the presence of P. acne on the face, and people often rely on traditional PCR techniques. The PCR techniques mostly detect the specific 16S ribosomal RNA (16S rRNA) of P. acne. The 16S rRNA, ubiquitous in bacteria workable to detect a wide diversity of microbes, can identify, specifically, the P. acne, not other strains in the same genus. However, PCR techniques often lack diagnostic specificity and time efficiency, leaving the room for novel detection methods.

Design

1. BS Kit: L-RCA

Ligation and Rolling Circle Amplification (L-RCA) is a novel detection method to determine the presence and amount of P. acne, which has high specificity, accuracy, and speediness. The primers used in detecting the target gene in L-RCA are padlock probes. The BS United China padlock primers are stranded DNAs which vary in length: 50, 162, or 322 base pairs; however, they contain the same composition: binding region (two ends), amplification region, and random region. In general, L-RCA works in the following description. The two ends of the DNA fragments are complementary to and bind to the 16S rRNA of P. acne due to hydrogen bond interactions. Then, the two ends are held closely to be ligated, and the DNA fragment forms into circular DNA. Next, by amplifying the circular DNA, we can amplify the detection result. Specifically, the process of L-RCA has three steps: phosphorylation, ligation, and rolling circle amplification (RCA). First, the 5’ end should be phosphorylated with a phosphate group to enable the dehydration reaction of forming the phosphodiester bond between two DNA nucleotides. This step is only required for DNA fragments that have only hydroxyl groups attached to the two ends. Second, ligation is finished when the T4 DNA ligase enzyme is used to ligate the two ends of the padlock primer to create a circular DNA. Lastly, from the common region where a primer is attached, RCA amplifies circular DNA molecules using φ29 DNA polymerase. The continuous rolling characteristic of RCA results in the extension of the circular DNA to a length larger than its original length.

In current studies of L-RCA, there are five notable areas for improvement [6]:

1. Absence of controlled experiments, the paper did not discuss or provide information on control experiments.
2. The paper did not give empirical evidence on the usage of electrophoresis to testify L-RCA results.
3. The paper did not give empirical evidence on the use of large-size DNA fragments as padlock probes to detect certain genes.
4. The paper did not testify to the feasibility of a thermostable amplification process of L-RCA
5. The paper did not significantly reduce the time for detection compared with PCR.

After the amplification of DNA, we suggest two main methods of detection of amplified DNA. Firstly, DNA electrophoresis can simply be applied using an electrophoresis machine to obtain images indicating the abundance of DNA at specific base pairs. Through L-RCA, the amplified DNA is larger than just a single padlock primer base pair length due to the property of the rolling circle amplification (RCA). Moreover, if P. acne’s abundance is higher, indicating that the patient’s risk of acne vulgaris is higher, the rolling circle amplification would be more frequent and active, acquiring higher base pair lengths. On the other end with a lower abundance of P. acne, little to no padlock primers will even be guided and ligated, and RCA activity will decrease, causing base pair lengths that are lower or merely the base pair length of a single padlock primer. Secondly, the method that we suggest is SYBR-based real-time quantitative Ligation Rolling Circle Amplification (qL-RCA). Its name derives from SYBR-based real-time qPCR, involving the use of double-stranded DNA-binding fluorescence for detection. With more amplified DNA and higher base pair length through rolling circle amplification, there will be more double-stranded DNA that SYBR can bind to. The opposite is true in that less amplified double-stranded DNA means less SYBR activity [7]. Subsequently, due to the role of P. acne abundance in yielding more amplified DNA, the detection of fluorescence emitted by SYBR can provide insight into P. acne abundance. Whether the fluorescence level surpassed a pre-determined cycle threshold (CT) value or not predicts whether the patient is at risk of acne vulgaris, while the speed in which it overcomes the CT value quantitatively assess P. acne abundance, as more P. acne leads to more amplified double-stranded DNA, allowing for faster SYBR Green binding. Additionally, the characteristic of the SYBR binding and fluorescence detection while phi29 DNA polymerase amplifies the padlock primer significantly decreases the time of detection. The time required is around 10 minutes for qL-RCA.

2. Hypothetical Procedure of L-RCA

3. Basic Cleanser: Use of Caf1/AMP Protein

To first capture and hold the P. acne in one place, we inserted Capsular antigen fragment 1 (Caf1) that is combined with aloe vera gel, which serves as a transporter of Caf1. Caf1 is a small subunit of 15 kDa that derives from Yersinia pestis bacteria, which can form stronger non-covalent interactions. Through cooling, Caf1 monomers polymerize into a complex and tight shape. When heated, the polymer will subsequently unfold [8]. The Caf1 subunits can also bind to AMP and histidine tags, which have important characteristics that can be utilized. Firstly, the histidine tags bound to the Caf1 proteins can assist the polymers, which are formed by Caf1 proteins in a cool environment, in forming a net shape. This property is due to the fact that his-tag antibodies are able to bind to the individual his-tags, linking the polymers together. This net shape can effectively capture the P. acne for later inhibition.
Subsequently, we can use the AMP to inhibit P. acne. The use of antimicrobial peptides (AMPs) is a general method of inhibiting bacteria that involves either disruption to cell membrane or intramolecular components.
Later, the properties of Caf1 assist in the clean removal of our proteins and bacterial fragments from the pores. The antibodies connected to the his-tags bind to protein A/G, which are subsequently bound to protein A/G magnetic beads. With this, we can utilize magnets to effectively remove our Caf1 protein combined with a variety of parts aiming to inhibit P. acne. This procedure is important to ensure that our pores are clean of any remains of proteins and P. acne bacteria from the cleansing process.

4. Enhanced Cleanser: Use of TurboID-FGB and GPX7

Apart from our established method of P. acne inhibition, we also developed an enhancer protein with additional parts: TurboID-FGB and GPX7.
TurboID-Fibrinogen (TurboID-FGB) is utilized to specifically target P. acne as we received feedback that AMP is a general bacterial inhibition technique. The TurboID-FGB method is modified from the TurboID method of BS_United_China 2022 [9]. TurboID binds to the signaling peptides of a protein related to the bacteria P. acne. TurboID biotinylates the protein and the bacteria through ligation using biotin catalyzed with ATP [10]. The beta units of human fibrinogen strongly bind to P. acne's surface protein. In this way, the fibrinogen can lead TurboID to be near to P. acne and biotinylate it. P. acne. We then utilize streptavidin phycoerythrin which has high affinity with biotin to further encapsulate the protein. These two blocking mechanisms prevent P. acne from its normal functions in quorum sensing, which is essential for a bacteria to communicate, increase virulence, and undergo various pathogenic behaviours. Therefore, the use of TurboID-FGB successfully inhibits P. acne.
Additionally, we employ the glutathione peroxidase 7 (GPX7) part to nurture facial pores after inhibition of P. acne. Upon killing the bacteria, it releases oxidative stress in the form of hydrogen peroxide (H2O2), which can be bad for the pore in increasing the aging of cells, damaging cell membranes, disrupting cell DNA, and even causing cell death through apoptosis [11]. GPX7 can be utilized to reduce hydrogen peroxide to water, preventing secondary damage to the cells.

5. Hypothetical Procedure of BS Cleanser (Basic + Enhanced Version)

6. Extension of L-RCA: Worm L-RCA

Upon our collaboration with Professor Haitao Zhou at Luoyang Central Hospital, a particular type of disease was brought to our attention, in which the disease DNA consists of numerous triplet repeats. A disease in this category for example includes myotonic dystrophy type 1, a prevalent muscular dystrophy affecting muscles and many organs in the body. Currently, Triplet Repeat Primed PCR (TP-PCR) with flanking primers and fluorescence to signal detection is used to detect these types of diseases, but this method is flawed in precision, time taken, and feasibility with longer triplet repeat length [12]. Moreover, L-RCA is found to not be sufficient for detecting the DNA of this disease, as the repeated triplets are variable and often very long, causing difficulties in forming a definite circular DNA with a constant padlock primer. Worm L-RCA is then proposed in that we modify the usual method of L-RCA to bind to the template DNA. The two sides of the padlock primer that bind to the template DNA, though, bind to the left and right of the triplet repeats, essentially flanking them. Then, 12 base pair (bp) primers consisting of 4 complementary triplet repeats are added to the reaction mixture and bound to the triplets. T4 DNA ligase ligates all of the primers, including padlock and the 12 bp primers together. The circular DNA can then be amplified by phi29 DNA polymerase per L-RCA for precise detection.

7. Hypothetical Procedure of Worm L-RCA

Bibliography

[1] Tan, J. K. L., and K. Bhate. “A Global Perspective on the Epidemiology of Acne.” The British Journal of Dermatology, vol. 172 Suppl 1, 2015, pp. 3–12, https://doi.org/10.1111/bjd.13462.
[2] “Skin Conditions by the Numbers.” American Academy of Dermatology, www.aad.org/media/stats-numbers. Accessed 4 Oct. 2023.
[3] KIRSCHBAUM, J. O. “The pathogenic role of Corynebacterium acnes in acne vulgaris.” Archives of Dermatology, vol. 88, no. 6, 1 Dec. 1963, p. 832, https://doi.org/10.1001/archderm.1963.01590240156026.
[4] Draelos, Zoe Diana. “Examining Over-the-Counter Acne Treatments.” (2010).
[5] Liu, Haibo, et al. “Evidence‐based topical treatments (azelaic acid, salicylic acid, nicotinamide, sulfur, zinc, and fruit acid) for acne: An abridged version of a Cochrane Systematic Review.” Journal of Evidence-Based Medicine, vol. 13, no. 4, 9 Oct. 2020, pp. 275–283, https://doi.org/10.1111/jebm.12411.
[6] Qi, X. (2001). L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs). Nucleic Acids Research, 29(22), 116e116. https://doi.org/10.1093/nar/29.22.e116.
[7] “Universal SYBR Green qPCR Protocol.” Universal SYBR GREEN QPCR Protocol, www.sigmaaldrich.com/JP/ja/technical-documents/protocol/genomics/qpcr/sybr-green-qpcr. Accessed 3 Oct. 2023.
[8] Dura, Gema, et al. “A thermally reformable protein polymer.” Chem, vol. 6, no. 11, 5 Nov. 2020, pp. 3132–3151, https://doi.org/10.1016/j.chempr.2020.09.020.
[9] “Project Description.” | BS_United_China - iGEM 2022, 2022.igem.wiki/bs-united-china/description. Accessed 3 Oct. 2023.
[10] Cho, Kelvin F., et al. “Proximity labeling in mammalian cells with turboid and split-turboid.” Nature Protocols, vol. 15, no. 12, 2 Nov. 2020, pp. 3971–3999, https://doi.org/10.1038/s41596-020-0399-0.
[11] Pizzino, Gabriele et al. “Oxidative Stress: Harms and Benefits for Human Health.” Oxidative medicine and cellular longevity vol. 2017 (2017): 8416763. doi:10.1155/2017/8416763
[12] Warner, J P, et al. “A general method for the detection of large CAG repeat expansions by fluorescent PCR.” Journal of Medical Genetics, vol. 33, no. 12, 1 Dec. 1996, pp. 1022–1026, https://doi.org/10.1136/jmg.33.12.1022.