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Overview

In our mission to address S. aureus skin infection, we decided to provide a new treatment for the disease. Inspired by existing treatments using the antimicrobial peptides (AMPs) to target the S. aureus. Moreover, we came up with the antimicrobial peptides complex, abbreviated to AMPC, and designed the specific cleavage sites to change inactive and active states for the AMP.

Fig.1 The AMPC is inactive, but can be activated upon cleavaged by V8 protease.

Antimicrobial Peptides Selection

When choosing AMP, we give priority to two major directions. First, we look for the original AMP of human beings. Next, we considered AMP that is not possessed by humans, but has a good antimicrobial effect on S. aureus. Furthermore, has a synergistic effect to increase the antimicrobial effect.

Information of Antimicrobial Peptides (AMPs)

  1. Cathelicidin LL-37
    AMP of human origin can reduce undesired immune response, and have good activity against S. aureus biofilm.
  2. Human Beta Defensin-3 (hBD3)
    AMP of human origin can reduce undesired immune response, and reasonable effective, well characterized, two Glu residues can reduce the chance of non-specific digestion by V8 protease.
  3. Human neutrophil peptide 1 (HNP1)
    AMP of human origin can reduce undesired immune response, and reasonable effective, well characterized, one Glu residues can reduce the chance of non-specific digestion by V8 protease.
  4. Histatin 5 (Hst 5)
    AMP of human origin can reduce undesired immune response, and show a high bactericidal effect on MRSA .
  5. Lysostaphin
    AMP of non-human origin can be produced by S. aureus simulans. Lysostaphin is a single polypeptide chain (approximately 27 kDa, aa 248-452) , and it shows synergistic effect after combined with some AMP.
  6. Ranalexin
    AMP from the skin of the bullfrog, which shows a high antimicrobial effect on MRSA. Also it shows synergistic anti-staphylococcal activity after combined with Lysostaphin .
  7. LcCCL28
    AMP of non-human origin is antimicrobial peptide from shrimp Litopenaeus vannamei , it can be showed good antimicrobial activity.

Construction of AMPC

To attain the goal of specific detection and sterilization of S. aureus, first, we designed the HAMA coating on microneedles to release the AMP while encountering S. aureus. Consistent with the specificity design of the microneedle, the professor suggested us to change the construction of AMP inside to let it also have specific anti-S. aureus efficacy. Here is where the concept of AMPC comes from. We decided to combine AMP with the linker, which is the cleavage site of a protease secreted by S. aureus. Hence, we expect AMPC achieves the following purpose:

  1. It can be produced by E. coli without causing any detrimental effects. It might be difficult for E. coli to produce simple AMP effectively while AMP might kill E. coli or be eaten as a nutrient..
  2. AMPC will remain inactive if the linker exists. The antimicrobial effect appears only when encountering S. aureus.
  3. The specificity of AMP release can reduce multi-drug resistance and normal microflora disruption.
  4. AMPC has the property of slow drug release, it may prolong the half-life of AMP and improve the convenience for patient usage so that patients don’t need to change their medicament often. Additionally, at the point of safety, AMPC can avoid large amounts of harmful antimicrobial peptide released at the same time if AMP has negative effect on the patient.
  5. The high positive charges of AMPC allow the absorption of toxic microbial components such as lipoteichoic acid to reduce inflammation of the wounds.

We investigated the protease which only secreted by S. aureus, for example, Staphopain A, V8 protease, SplA, SplB, SplD, and aureolysin. Considering the working environment and the protease is common, we finally chose the V8 protease cleavage site as linker of AMPC. V8 protease specifically cleaves peptides at glutamate residues in ammonium bicarbonate buffer, pH 7.8. So we use ammonium bicarbonate buffer to dialysis the purified protein, hoping to increase the success probability of our experiment.

AMPC Gene Construction

In order to express protein better, we chose pET28a as a plasmid vector, which carries T7 promoter, lac operator, and poly-histidine purification tag coding sequence. Separating the design into a few parts, the first is the linker sequence design. As just mentioned, we chose glutamate residues, which is the V8 protease cleavage site, as the linker. Also, we checked that there aren’t any EN, EE, and EP sequence at the cleavage site, which dislikes by V8 protease.

We ordered the sequence from IDT company as the pattern of linker [AMP sequence] linker [AMP sequence] linker [AMP sequence]. However, the redundancy sequence can’t be produced by IDT company. In addition, we found that some AMPs such as LL37 and Ranalexin have synergistic anti-staphylococcal activity. Therefore, we designed AMPC by combining two different AMPs together. Detail sequence is mentioned on the Parts page.

See Parts page for detail information.

The main AMP in AMPC is lysostaphin, and by linking it with other AMP as mentioned previously such as LL37, we made eight combinations of AMPC. Also, we used the same AMP but synonymous codons to avoid repetitive sequences and NdeI and XhoI restriction sites for digestion. Using NdeI and XhoI restriction enzyme, we digested our target gene from the pUCIDT (Amp) Golden Gate vector provided by IDT company. Afterward, we inserted it into the pET28a vector and transformed the plasmid into E. coli TOP10 for storage.

Fig.2 The flowchart of gene cloning.

Protein Production, Purification and Antimicrobial Activity Test

After cloning, we inserted target gene into pET28a vector and transformed the plasmid into E. coli TOP10 for storage. Then we extracted the plasmid and transformed it into pET28a. Induce the bacteria by IPTG overnight for protein expression, we purified the protein by nickel column. Lastly, we test the antimicrobial activity by disc diffusion assay.

Fig.2 The flowchart of gene cloning.

Major Experiments & Results

See Result - AMPC page for detail information.

Reference

  1. Carmona C, Gray GL. Nucleotide sequence of the serine protease gene of Staphylococcus aureus, strain V8. Nucleic Acids Res. 1987 Aug 25;15(16):6757. doi: 10.1093/nar/15.16.6757. PMID: 3306605; PMCID: PMC306157.
  2. Houmard J, Drapeau GR. Staphylococcal protease: a proteolytic enzyme specific for glutamoyl bonds. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3506-9. doi: 10.1073/pnas.69.12.3506. PMID: 4509307; PMCID: PMC389807.
  3. Sharma R, Sharma PR, Choudhary ML, Pande A, Khatri GS. Cytoplasmic expression of mature glycylglycine endopeptidase lysostaphin with an amino terminal hexa-histidine in a soluble and catalytically active form in Escherichia coli. Protein Expr Purif. 2006 Jan;45(1):206-15. doi: 10.1016/j.pep.2005.07.025. Epub 2005 Aug 19. PMID: 16181789.
  4. Sadeghi S, Bakhshandeh H, Ahangari Cohan R, Peirovi A, Ehsani P, Norouzian D. Synergistic Anti-Staphylococcal Activity Of Niosomal Recombinant Lysostaphin-LL-37. Int J Nanomedicine. 2019 Dec 10;14:9777-9792. doi: 10.2147/IJN.S230269. PMID: 31849468; PMCID: PMC6911324.
  5. Chen S, He FT, Dong YL, Li RF, Gao HG, Chen M, Peng JH. [The cloning, high level expression in Escherichia coli of human beta-defensin 3 and its antimicrobial activity analysis]. Sheng Wu Gong Cheng Xue Bao. 2004 Jul;20(4):490-5. Chinese. PMID: 15968976.