A new generation of recombinant polypeptides combines multiple protein domains for effective antimicrobial activity

Abstract

Background: Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity.

Results: A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA₂), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM.

Conclusions: Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.

Keywords: Antimicrobial peptides; Antimicrobial resistance; Inclusion bodies; Multidomain protein; Protein nanoclusters; Recombinant production; Solubilization.

Fig. 1

Construct design. Schematic representation of JAMF1 protein construct and protein production format. Inset image: FESEM micrography of purified JAMF1 nanoparticles

Fig. 2

Antibacterial activity of JAMF1 nanoclusters. a Graphic representation of the BacTiter-Glo™ Microbial Cell viability assay. b Bacterial survival (%) of E. coli DH5α in the presence of JAMF1 IBs at a range of 0-10 µM. Different letters describe significant differences (p ≤ 0.01). c Bacterial survival of KPC, qnrA, CMY2, SHV-12, ECF and CTX-M-14 bacterial strains in the presence of 10 µM of JAMF1 IBs. Survival of JAMF1 treated bacterial cells (black bars) is significantly different from the negative control (grey bars) (p ≤ 0.001)

Fig. 3

Anti-biofilm activity of JAMF1 nanoclusters. a Biofilm inhibition assay. Plate wells were incubated for 2 h with JAMF1 IBs and then a diluted (1:200) KPC cell culture with 0.2% glucose was added and incubated for 24 h to allow biofilm formation. b Biofilm formation ability (%) of KPC after treating plastic wells with JAMF1 IBs (black bar) vs non-treated wells (grey bar). ****Indicates significant differences (p ≤ 0.0001)

Fig. 4

Solubilized JAMF1 antibacterial activities. a Schematic representation of JAMF1 IB solubilization at RT. b Bacterial survival (%) of E. coli DH5α and KPC at different concentrations (0, 0.5, 1, 2 and 3 µM) of solubilized JAMF1. Capital letters depict significant differences for E. coli DH5α (p ≤ 0.0001) and lower case for KPC (p ≤ 0.001). Filled circles correspond to E. coli DH5α and empty circles correspond to KPC