A Novel Peptide Derived from the Transmembrane Domain of Romo1 Is a Promising Candidate for Sepsis Treatment and Multidrug-Resistant Bacteria

Abstract

The emergence of multidrug-resistant (MDR) bacteria through the abuse and long-term use of antibiotics is a serious health problem worldwide. Therefore, novel antimicrobial agents that can cure an infection from MDR bacteria, especially gram-negative bacteria, are urgently needed. Antimicrobial peptides, part of the innate immunity system, have been studied to find bactericidal agents potent against MDR bacteria. However, they have many problems, such as restrained systemic activity and cytotoxicity. In a previous study, we suggested that the K58–R78 domain of Romo1, a mitochondrial protein encoded by the nucleus, was a promising treatment candidate for sepsis caused by MDR bacteria. Here, we performed sequence optimization to enhance the antimicrobial activity of this peptide and named it as AMPR-22 (antimicrobial peptide derived from Romo1). It showed broad-spectrum antimicrobial activity against 17 sepsis-causing bacteria, including MDR strains, by inducing membrane permeabilization. Moreover, treatment with AMPR-22 enabled a remarkable survival rate in mice injected with MDR bacteria in a murine model of sepsis. Based on these results, we suggest that AMPR-22 could be prescribed as a first-line therapy (prior to bacterial identification) for patients diagnosed with sepsis.

Identification of Bacterial Membrane Selectivity of Romo1-Derived Antimicrobial Peptide AMPR-22 via Molecular Dynamics

“…Construction of the Representative AMPR-11 or AMPR-22 Structure via Molecular Dynamics AMPR-11 is composed of 21 amino acids (K58-R78 region of Romo1) and AMPR-22 was constructed by substituting four polar amino acids (2THR, 5GLN, 6SER, and 9THR) with lysine and deleting 18MET to increase both amphipathicity and net positive charge (Figure 1A). Both AMPR-11 and AMPR-22 predominantly formed alpha-helical structures in circular dichroism (CD) (60.8% for AMPR-11 and 71.2% for AMPR-22) [18,20] when they were both dissolved in 50% hexafluoro-2-propanol (HFIP), which has been used for a membrane mimetic environment [27]. Here, we used MD simulations to explore how AMPR-22 acts on membranes with low hemolytic toxicity.…”

“…AMPR-11 is composed of 21 amino acids (K58-R78 region of Romo1) and AMPR-22 was constructed by substituting four polar amino acids (2THR, 5GLN, 6SER, and 9THR) with lysine and deleting 18MET to increase both amphipathicity and net positive charge (Figure 1A). Both AMPR-11 and AMPR-22 predominantly formed alpha-helical structures in circular dichroism (CD) (60.8% for AMPR-11 and 71.2% for AMPR-22) [18,20] when they were both dissolved in 50% hexafluoro-2-propanol (HFIP), which has been used for a membrane mimetic environment [27]. As AMPR-11 and AMPR-22 are intermediate AMPs that need to be optimized further, we performed a cost-effective MD simulation to predict their representative structures instead of using nuclear magnetic resonance (NMR) spectroscopy.…”

“…As AMPR-11 and AMPR-22 are intermediate AMPs that need to be optimized further, we performed a cost-effective MD simulation to predict their representative structures instead of using nuclear magnetic resonance (NMR) spectroscopy. Based on the alpha-helical proportion determined by the CD [18,20], the initial structures of AMPR-11 and AMPR-22 were modeled using Modeller. Each structure was simulated for 2 µs under a CHARMM36m force field with 60 molecules of dodecylphosphocholine (DPC) micelles, As AMPR-11 and AMPR-22 are intermediate AMPs that need to be optimized further, we performed a cost-effective MD simulation to predict their representative structures instead of using nuclear magnetic resonance (NMR) spectroscopy.…”

“…Each structure was simulated for 2 µs under a CHARMM36m force field with 60 molecules of dodecylphosphocholine (DPC) micelles, As AMPR-11 and AMPR-22 are intermediate AMPs that need to be optimized further, we performed a cost-effective MD simulation to predict their representative structures instead of using nuclear magnetic resonance (NMR) spectroscopy. Based on the alphahelical proportion determined by the CD [18,20], the initial structures of AMPR-11 and AMPR-22 were modeled using Modeller. Each structure was simulated for 2 µs under a CHARMM36m force field with 60 molecules of dodecylphosphocholine (DPC) micelles, which have been widely used to solubilize amphipathic peptides or hydrophobic proteins for NMR (Figure 1B).…”

“…To improve the efficacy of AMPR-11, AMPR-22 was constructed by replacing the polar residues (2THR, 5GLN, 6SER, and 9THR) in an amphipathic helix with lysine [19] and deleting 18MET. Compared to AMPR-11, AMPR-22 showed an enhanced antimicrobial effect in a murine model of sepsis induced by MDR bacteria with decreased hemolysis [20], which is a wellknown limitation of intravenously administered AMP for therapeutics. How AMPR-22 shows higher efficacy and lower hemolytic toxicity over AMPR-11 has not been established, and this limits the optimization strategy.…”