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

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.

Romo1-Derived Antimicrobial Peptide Is a New Antimicrobial Agent against Multidrug-Resistant Bacteria in a Murine Model of Sepsis.

To overcome increasing bacterial resistance to conventional antibiotics, many antimicrobial peptides (AMPs) derived from host defense proteins have been developed. However, there are considerable obstacles to their application to systemic infections because of their low bioavailability. In the present study, we developed an AMP derived from Romo1 (AMPR-11) that exhibits a broad spectrum of antimicrobial activity. AMPR-11 showed remarkable efficacy against sepsis-causing bacteria, including multidrug-resistant strains, with low toxicity in a murine model of sepsis after intravenous administration. It seems that AMPR-11 disrupts bacterial membranes by interacting with cardiolipin and lipid A. From the results of this study, we suggest that AMPR-11 is a new class of agent for overcoming low efficacy in the intravenous application of AMPs and is a promising candidate to overcome multidrug resistance.IMPORTANCE Abuse of antibiotics often leads to increase of multidrug-resistant (MDR) bacteria, which threatens the life of human beings. To overcome threat of antibiotic resistance, scientists are developing a novel class of antibiotics, antimicrobial peptides, that can eradicate MDR bacteria. Unfortunately, these antibiotics have mainly been developed to cure bacterial skin infections rather than others, such as life-threatening sepsis. Major pharmaceutical companies have tried to develop antiseptic drugs; however, they have not been successful. Here, we report that AMPR-11, the antimicrobial peptide (AMP) derived from mitochondrial nonselective channel Romo1, has antimicrobial activity against Gram-positive and Gram-negative bacteria comprising many clinically isolated MDR strains. Moreover, AMPR-11 increased the survival rate in a murine model of sepsis caused by MDR bacteria. We propose that AMPR-11 could be a novel antiseptic drug candidate with a broad antimicrobial spectrum to overcome MDR bacterial infection.

Hepatitis C Virus p7 Induces Membrane Permeabilization by Interacting with Phosphatidylserine.

Hepatitis C virus (HCV) p7 is known to be a nonselective cation channel for HCV maturation. Because the interaction of HCV proteins with host lipids in the endoplasmic reticulum membrane is crucial for the budding process, the identification of p7-lipid interactions could be important for understanding the HCV life cycle. Here, we report that p7 interacts with phosphatidylserine (PS) to induce membrane permeabilization. The interaction of p7 with PS was not inhibited by Gd3+ ions, which have been known to interact with negatively charged lipids, but channel activity and p7-induced mitochondrial depolarization were inhibited by Gd3+ ions. From the present results, we suggest that the p7-PS interaction plays an essential role in regulating its ion channel function and could be a potential molecular target for anti-HCV therapy.

Hepatitis B virus X protein induces size-selective membrane permeabilization through interaction with cardiolipin.

Hepatitis B virus X protein (HBx) functions in a variety of cellular events during the HBV life cycle. In a previous study, we reported that the HBx protein is sufficient to induce mitochondrial membrane permeabilization; however, the exact mechanism of HBx-induced mitochondrial membrane permeabilization has been not proposed. In this study, we report that HBx specifically targets cardiolipin (CL) and induces membrane permeabilization depending on CL concentration in mitochondrial outer membrane-mimic artificial liposomes. Interestingly, HBx-induced membrane permeabilization was enhanced by liposomes containing phosphatidylethanolamine, which plays a crucial role in forming a negative curvature on the membrane. We also show that the 68-117 region of HBx, which interacts with mitochondria, is necessary for membrane permeabilization. We examined the size of the pores formed by HBx and found that HBx permeates fluorescent dyes depending on the hydrodynamic diameter with a pore size of approximately 10 nm. The results of this study suggest that CL is necessary for HBx-induced membrane permeabilization and provide important information that suggests a new strategy for anti-HBV therapy.

Romo1 is a mitochondrial nonselective cation channel with viroporin-like characteristics.

Reactive oxygen species (ROS) modulator 1 (Romo1) is a nuclear-encoded mitochondrial inner membrane protein known to regulate mitochondrial ROS production and to act as an essential redox sensor in mitochondrial dynamics. Although its physiological roles have been studied for a decade, the biophysical mechanisms that explain these activities of Romo1 are unclear. In this study, we report that Romo1 is a unique mitochondrial ion channel that differs from currently identified eukaryotic ion channels. Romo1 is a highly conserved protein with structural features of class II viroporins, which are virus-encoded nonselective cation channels. Indeed, Romo1 forms a nonselective cation channel with its amphipathic helical transmembrane domain necessary for pore-forming activity. Notably, channel activity was specifically inhibited by Fe2+ ions, an essential transition metal ion in ROS metabolism. Using structural bioinformatics, we designed an experimental data-guided structural model of Romo1 with a rational hexameric structure. We propose that Romo1 establishes a new category of viroporin-like nonselective cation channel in eukaryotes.

Hepatitis C virus p7 induces mitochondrial depolarization of isolated liver mitochondria.

Hepatitis C virus (HCV)­encoded protein p7 is a viroporin that acts as an ion channel and is indispensable for HCV particle production. Although the main target of HCV p7 is the endoplasmic reticulum, it also targets mitochondria. HCV­infected cells show mitochondrial depolarization and ATP depletion; however, the function of HCV p7 in mitochondria is not fully understood. The present study demonstrated that treatment of isolated mouse liver mitochondria with the synthesized HCV p7 protein induced mitochondrial dysfunction. It also demonstrated that HCV p7 targeted isolated mouse liver mitochondria and induced mitochondrial depolarization. In addition, HCV p7 triggered matrix acidification and, ultimately, a decrease in ATP synthesis in isolated mitochondria. These findings indicate that targeting of mitochondria by HCV p7 in infected cells causes mitochondrial dysfunction to support HCV particle production. The present study provided evidence for the role of HCV p7 in mitochondria, and may lead to the development of novel strategies for HCV therapy.