Peptides

Introduction

Antimicrobial peptides have an action against different types of bacteria: but what about the vaginal cells and the vaginal flora ? It’s has been shown that most of antimicrobials peptides (AMPs) are hydrophobics and cationics peptides, they bind to the anionic phospholipids on the outer surface of bacterial cell membrane [1]. The affinity for the mammalian cells is on the contrary really low because the outer surface is composed of zwitterionic phospholipids. As a consequence, AMPs will not hurt the vaginal epithelial cells. Moreover, AMPs have an higher affinity for spermatozoa membranes which are composed of anionic phospholipids such as phosphatidylglycerol and phosphatidylserine in the plasma membrane, and caused the lost of motility of spermatozoa and prevent the fecondation. Lastly, all the AMPs studied are not harmful for the lactobacilli in the vaginal microbiota [2]. All of this reasons make some AMPs promising targets for being used for contraception.

The different AMPs

Subtilosin A

Subtilosin A (SboA) is an antimicrobial peptide of 32 amino acids naturally produced by Bacillus subtilis. This AMP is a very good candidate since it also is involved in various antimicrobial activity against pathogenic organisms such as Gardnerella vaginalis [3] involved in Bacterial vaginosis.

SboA is produced and then secreted by B. subtitles thanks to a very complex operon made of 8 genes called the Alb operon. (figure 1)

Figure 1 : The alb operon

Production of Subtilosin A

In Bacillus subtilis, the subtilosin A is produced by the expression of an operon of 8 genes (figure 1). However, It is possible to get a correctly folded - and therefore active - protein with only SboA and AlbA genes [4]. Indeed, SboA is the Subtilosin A precursor of 32 amino acids, and Alba is part of radical SAM proteins superfamily proteins that use [4Fe-4S]+ cluster to reductively cleave S-adenosyl-L-methionine (SAM) to generate a radical and catalyze the formation of 3 thioether bonds in SboA making it mature.

Iron Sulfur Cluster Complementation

It’s known that the production of mature SboA is more efficient when the SUF genes (Iron Sulfur Cluster) are added to the host organism. It is due to the [4Fe-4S]+ cluster, a cofactor of AlbA. [4] These SUF genes are expressed by numerous prokaryotic cells under stress conditions. And this is no exception of B. subtilis. Also, to make sure that our peptide is well produced and in a sufficient amount, we added the suf genes of B. subtilis to our construction, so that the cluster is produced even in non stressing conditions.

Lacticin 3147

This Class I bacteriocin is composed by two peptides lantibiotic: Ltn⍺ and Ltnbeta. (Figure 2). It’s produced by Lactoccocus lactis subsp. lactis [5]. This bacterium contains a 60,232 bp conjugative plasmide, pMRC01 which presents the 6 gene cluster (ltnA1, A2, M1, T and M2) to synthesize lacticin 3147 [6].

Figure 2 : Lacticin 3147 structure. LtnA1 and LtnA2 peptides in their mature active form. “Carine Dortu & Philippe Thonart : Bacteriocins from lactic acid bacteria: interest for food products biopreservation.”

ltnA1 and ltnA2 encode LtnA1 and LtnA2 who in their mature active form are termed Ltn⍺ and Ltnꞵ. It was study that ltnM1 and ltnM2 show homology to genes involved in the production and export of lantibiotics. That’s why we used ltnA1, ltnA2, ltnM1 and ltnM2 in our circuit [7]. Some experiments have proved lacticin exhibit spermicidal activity against horse/pony, bovine, boar and rat sperm immobilizing them within 30 s by using peptides LtnA1 and Ltn2 together [3].

LL37

LL-37 is an antimicrobial peptide from human macrophages and leukocytes. It is the only member from the human cathelicidin family. This peptide appears to have very important spermicidal effects. “Human cathelicidin LL-37 is the most promising spermicidal AMP to be further developed for vaginal use” [2] LL-37 has shown effect in vivo in mice. Females naturally cycling to estrous phase failed to become pregnant when in injected with LL-37 and sperm. LL-37 also showed in vitro effect on human sperm which makes it a very good potential peptide for contraception. The fact that this peptide is naturally produced by human and moreover in the vagina after intercourse adds to the peptide the proof of harmlessness for the vaginal flora. Lastly this peptide is only 37 amino acids long so it is easy to produce and doesn’t need post translational modification.

Design

General design

Each of our constructions contained rpsU promoter [8] which is a Lactobacillus jensenii strong promoter. This RpsU sequence also contains the putative sequence for the RBS We added spacers to all of our constructions to unable easier use of the sequence and separation of the different genes of the sequences. We used two Terminators to our sequences :BBa_B0014 & BBa_B0015 to ensure the stopping of the transcription. Our constructions were assembled in the Plem415 vector by Gibson Assembly method. Plem 415 is a plasmid that works in Lactobacilli species but it’s not specific to L. jensenii [9]

Figure 3 : legend of the designs

SubtilosinA

Figure 4 : Design of the sequence coding the Subtilosin protein with the RpsU promoter.

Figure 5 : Design of the sequence coding the Iron Sulfur Cluster with the RpsU promoter.

Lacticin 3147

This circuit was made from 2 native genes of Lactococcus Lactis ltA1 and ltnA that express Lacticin peptide. Also, the design contains Lacticin-post-transcriptional regulator ltM1 and M2. A promoter orthogonal was used : ptsH and differents spacer taken from igem_parts.

Figure 6 : Design of the sequence coding the Lacticin 3147

LL-37

The design of LL-37 is simpler than the one of the other peptides. Indeed, the protein is coded only with the well-named gene LL-37.

Figure 7 : Design of the sequence coding the LL-37 protein with the RpsU promoter.

Figure 8 : Design of the sequence coding the LL-37 protein with the pHyperSpank promoter.