First vertebrate BRICHOS antimicrobial peptides: ß-hairpin host defense peptides in limbless amphibia lung resemble those of marine worms.

Innate immunity of invertebrates offers potent antimicrobial peptides (AMPs) against drug-resistant infections. To identify new worm ß-hairpin AMPs, we explored the sequence diversity of proteins with a BRICHOS domain, which comprises worm AMP precursors. Strikingly, we discovered new BRICHOS AMPs not in worms, but in caecilians, the least studied clade of vertebrates. Two precursor proteins from Microcaecilia unicolor and Rhinatrema bivittatum resemble SP-C lung surfactants and bear worm AMP-like peptides at C-termini. The analysis of M. unicolor tissue transcriptomes shows that the AMP precursor is highly expressed in the lung along with regular SP-C, suggesting a different, protective function. The peptides form right-twisted ß-hairpins, change conformation upon lipid binding, and rapidly disrupt bacterial membranes. Both peptides exhibit broad-spectrum activity against multidrug-resistant ESKAPE pathogens with 1-4 µM MICs and remarkably low toxicity, giving 40-70-fold selectivity towards bacteria. These BRICHOS AMPs, previously unseen in vertebrates, reveal a novel lung innate immunity mechanism and offer a promising antibiotics template.

Bioengineering the Antimicrobial Activity of Yeast by Recombinant Thanatin Production.

The global spread of antibiotic resistance marks the end of the era of conventional antibiotics. Mankind desires new molecular tools to fight pathogenic bacteria. In this regard, the development of new antimicrobials based on antimicrobial peptides (AMPs) is again of particular interest. AMPs have various mechanisms of action on bacterial cells. Moreover, AMPs have been reported to be efficient in preclinical studies, demonstrating a low level of resistance formation. Thanatin is a small, beta-hairpin antimicrobial peptide with a bacterial-specific mode of action, predetermining its low cytotoxicity toward eukaryotic cells. This makes thanatin an exceptional candidate for new antibiotic development. Here, a microorganism was bioengineered to produce an antimicrobial agent, providing novel opportunities in antibiotic research through the directed creation of biocontrol agents. The constitutive heterologous production of recombinant thanatin (rThan) in the yeast Pichia pastoris endows the latter with antibacterial properties. Optimized expression and purification conditions enable a high production level, yielding up to 20 mg/L of rThan from the culture medium. rThan shows a wide spectrum of activity against pathogenic bacteria, similarly to its chemically synthesized analogue. The designed approach provides new avenues for AMP engineering and creating live biocontrol agents to fight antibiotic resistance.

Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers.

Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.

Selective Eradication of Staphylococcus aureus by the Designer Genetically Programmed Yeast Biocontrol Agent.

Staphylococcus aureus is a common human pathogen that is particularly often associated with antibiotic resistance. The eradication of this ubiquitous infectious agent from its ecological niches and contaminated surfaces is especially complicated by excessive biofilm formation and persisting cells, which evade the antibacterial activity of conventional antibiotics. Here, we present an alternative view of the problem of specific S. aureus eradication. The constitutive heterologous production of highly specific bacteriolytic protease lysostaphin in yeast Pichia pastoris provides an efficient biocontrol agent, specifically killing S. aureus in coculture. A yeast-based anti-S. aureus probiotic was efficient in a high range of temperatures and target-to-effector ratios, indicating its robustness and versatility in eliminating S. aureus cells. The efficient eradication of S. aureus by live lysostaphin-producing P. pastoris was achieved at high scales, providing a simple, biocompatible and cost-effective strategy for S. aureus lysis in bioproduction and surface decontamination. Future biomedical applications based on designer yeast biocontrol agents require evaluation in in vivo models. However, we believe that this strategy is very promising since it provides highly safe, efficient and selective genetically programmed probiotics and targeted biocontrol agents.