Sequence edition of single domains modulates the final immune and antimicrobial potential of a new generation of multidomain recombinant proteins

Abstract

Combining several innate immune peptides into a single recombinant antimicrobial and immunomodulatory polypeptide has been recently demonstrated. However, the versatility of the multidomain design, the role that each domain plays and how the sequence edition of the different domains affects their final protein activity is unknown. Parental multidomain antimicrobial and immunomodulatory protein JAMF1 and several protein variants (JAMF1.2, JAMF2 and AM2) have been designed and recombinantly produced to explore how the tuning of domain sequences affects their immunomodulatory potential in epithelial cells and their antimicrobial capacity against Gram-positive and Gram-negative bacteria. The replacement of the sequence of defensin HD5 and phospholipase sPLA2 by shorter active fragments of both peptides improves the final immunomodulatory (IL-8 secretion) and antimicrobial function of the multidomain protein against antimicrobial-resistant Klebsiella pneumoniae and Enterococcus spp. Further, the presence of Jun and Fos leucine zippers in multidomain proteins is crucial in preventing toxic effects on producer cells. The generation of antimicrobial proteins based on multidomain polypeptides allows specific immunomodulatory and antimicrobial functions, which can be easily edited by modifying of each domain sequence.

Lilium Gray Mold Suppression Conferred by the Host Antimicrobial Protein LsGRP1 Involves Main Pathogen-Targeted Manipulation of the Nonantimicrobial Region LsGRP1N

“…The idea of combining antimicrobials, targeted dormant cellwakening compounds, and virulence inhibitors can be put into practical application, possibly in the form of recombinant macromolecules, which are emerging for antimicrobial compound production. 37,38 For example, a synthetic hybrid peptide with dual functions of antibacterial and plant defense elicitation has been demonstrated to control both bacterial and fungal diseases of tomato. 39 Conidial spore phenotypic heterogeneity in stress resistance, nutrient uptake, secondary metabolite production, and virulence could result from transcriptional gene modulation in response to environmental stimulation occurring before dormancy as well as during germination.…”

“…In addition, the influences of LsGRPP1 N on infection-related gene expression and host-defense-eliciting activity of the target fungus recommend an integration of virulence inhibitors into antimicrobial agents. The idea of combining antimicrobials, targeted dormant cell-wakening compounds, and virulence inhibitors can be put into practical application, possibly in the form of recombinant macromolecules, which are emerging for antimicrobial compound production. , For example, a synthetic hybrid peptide with dual functions of antibacterial and plant defense elicitation has been demonstrated to control both bacterial and fungal diseases of tomato …”

The future of recombinant host defense peptides

“…Unlike denaturing protocols, this last strategy allows to obtain soluble protein from IBs without the need to use unfolding and refolding processes. Among these articles, some of them have already successfully tried this approach using IBs formed by antimicrobial proteins [ 20 , 21 , 37 ]. Indeed, solubilized IB proteins show antimicrobial activity against E. coli in a dose-dependent fashion against carbapenem-resistant K. pneumoniae embedded in biofilms [ 20 ].…”

“…BMAP28, TAP and indolicidin) [ 104 ]. Interestingly, when HDPs are linked to enzymes, such as in the construct JAMF1 or GAPDH-HDP chimeras, both the enzyme and the HDP seem to keep the activities of the individual components, namely catalytic, antimicrobial, and immune-modulatory activities [ 21 , 104 ].

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“…Their well-known characteristics make them amenable to engineering [19][20][21] (Fig. 1A, B), peptide repurposing, such as engineered venoms that can be modified to become non-toxic HDPs [22] (Fig.…”