Impact of propeptide cleavage on the stability and activity of a streptococcal immunomodulatory C5a peptidase for biopharmaceutical development

Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group AStreptococcus pyogenesC5a peptidase (ScpA) is a multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group AS. pyogenesknown to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed inEscherichia colicells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.</p

Pharmaceutical design of a delivery system for the bacteriocin lacticin 3147

Lacticin 3147 is a dual-acting two-peptide bacteriocin which is generally active against Gram-positive bacteria, including Listeria monocytogenes and antimicrobial-resistant bacteria such as Closteroides diffcile in the colon. L. monocytogenes infections can cause life-long effects in the elderly and vulnerable and can cause severe complications in pregnant women. C. diffcile causes one of the most common healthcare-associated infections and can be fatal in vulnerable groups such as the elderly. Although lacticin 3147 is degraded by intestinal proteases and has poor aqueous solubility, encapsulation of the bacteriocin could enable its use as an antimicrobial for treating these bacterial infections locally in the gastrointestinal tract. Lacticin 3147 displayed activity in aqueous solutions at a range of pH values and in gastric and intestinal fluids. Exposure to trypsin and α-chymotrypsin resulted in complete inactivation, implying that lacticin 3147 should be protected from these enzymes to achieve successful local delivery to the gastrointestinal tract. The amount of lacticin 3147 dissolved, i.e. its solution concentration, in water or buffered solutions at pH 1.6 and 7.4 was low and varied with time but increased and was stabilized in gastrointestinal fluids by the phospholipid and bile salt components present. Thus, the feasibility of a solid lipid nanoparticle (SLN) delivery system for local administration of lacticin 3147 was investigated. Bacteriocin activity was observed after encapsulation and release from a lipid matrix. Moreover, activity was seen after exposure to degrading enzymes. Further optimization of SLN delivery systems could enable the successful pharmaceutical development of active lacticin 3147 as an alternative to traditional antibiotics

Single versus double occupancy solid lipid nanoparticles for delivery of the dual-acting bacteriocin, lacticin 3147

The bacteriocin lacticin 3147 (lacticin) has shown activity against clinically relevant and antimicrobial-resistant  bacteria such as Listeria monocytogenes and Clostridioides difficile. It is composed of two peptides, Ltnα and Ltnβ,  which work together to form pores in the membrane of Gram-positive bacteria. Lacticin possesses poor aqueous  solubility and is degraded by intestinal proteases. In a previous study, peptides encapsulated into solid lipid  nanoparticles (SLNs) displayed activity in aqueous media and were protected from enzyme degradation but  showed a low encapsulation efficiency (EE%) for Ltnα. In this study, however, lacticin was encapsulated into  SLNs both individually (single occupancy, SLNα + SLNβ) and together (double occupancy SLNαβ) via a nano precipitation technique. This achieved SLNs of uniform size with an EE% above 87% for both peptides at loadings  of 9 or 18 mg/g of lipid under single occupancy or double occupancy respectively. SLNαβ dispersions displayed  more potent activity at 3.13 and 1.56 µg/ml lacticin than SLNα + SLNβ dispersions. Thus, the SLNαβ dispersion  was chosen for further analysis. SLNαβ dispersions showed no cytotoxicity to endothelial cells. The SLN release  media (fasted state simulated intestinal fluid; FaSSIF) retained activity at 1 h and 3 h indicating that lacticin may  be sufficiently protected from proteases present in the duodenum. Finally, a reconstituted freeze-dried SLNαβ  dispersion was stable and achieved 99.99% bacterial killing at 3.125 µg/ml lacticin. Thus, an SLN based lacticin  delivery system was developed, potentially enabling oral administration of the bacteriocin to the colon to treat  local infections such as C. difficile.  </p

The development of a solid lipid nanoparticle (SLN)‑based lacticin 3147 hydrogel for the treatment of wound infections

Chronic wounds affect millions of people globally. This number is set to rise with the increasing incidence of antimicrobial-resistant bacterial infections, such as methicillin-resistant Staphylococcus aureus (MRSA), which impair the healing of chronic wounds. Lacticin 3147 is a two-peptide chain bacteriocin produced by Lactococcus lactis that is active against S. aureus including MRSA strains. Previously, poor physicochemical properties of the peptides were overcome by the encapsulation of lacticin 3147 into solid lipid nanoparticles. Here, a lacticin 3147 solid lipid nanoparticle gel is proposed as a topical treatment for S. aureus and MRSA wound infections. Initially, lacticin 3147’s antimicrobial activity against S. aureus was determined before encapsulation into solid lipid nanoparticles. An optimised gel formulation with the desired physicochemical properties for topical application was developed, and the lacticin-loaded solid lipid nanoparticles and free lacticin 3147 aqueous solution were incorporated into separate gels. The release of lacticin 3147 from both the solid lipid nanoparticle and free lacticin gels was measured where the solid lipid nanoparticle gel exhibited increased activity for a longer period (11 days) compared to the free lacticin gel (9 days). Both gels displayed potent activity ex vivo against S. aureus-infected pig skin with significant bacterial eradication (>75%) after 1 h. Thus, a long-acting potent lacticin 3147 solid lipid nanoparticle gel with the required physicochemical properties for topical delivery of lacticin 3147 to the skin for the potential treatment of S. aureus-infected chronic wounds was developed.</p