Toxin-antitoxin (TA) systems regulate key cellular functions in bacteria. Here, we report a unique structure of the Streptococcus pneumoniae HigBA system and a novel antimicrobial agent that activates HigB toxin, which results in mRNA degradation as an antibacterial strategy. In this study, protein structure-based peptides were designed and successfully penetrated the S. pneumoniae cell membrane and exerted bactericidal activity. This result represents the time during which inhibitors triggered S. pneumoniae cell death via the TA system. This discovery is a remarkable milestone in the treatment of antibiotic-resistant S. pneumoniae, and the mechanism of bactericidal activity is completely different from those of current antibiotics. Furthermore, we found that the HigBA complex shows a crossed-scissor interface with two intermolecular β-sheets at both the N and C termini of the HigA antitoxin. Our biochemical and structural studies provided valuable information regarding the transcriptional regulation mechanisms associated with the structural variability of HigAs. Our in vivo study also revealed the potential catalytic residues of HigB and their functional relationships. An inhibition study with peptides additionally proved that peptide binding may allosterically inhibit HigB activity. Overall, our results provide insights into the molecular basis of HigBA TA systems in S. pneumoniae, which can be applied for the development of new antibacterial strategies.
Gaegurin 4 (GGN4) is a cationic peptide of 37 amino acids (MW 3748) isolated from the skin of Rana rugosa. It has shown a broad spectrum antimicrobial activity in vitro against Gram-negative and -positive bacteria, fungi and protozoa. To understand its mechanism of antimicrobial action, we examined the effect of GGN4 on the membrane conductance and the electrical properties of GGN4-induced pores in planar lipid bilayers under voltage clamp. Natural and synthetic GGN4 (0.01-1 microg/mL) increased the membrane conductance in a concentration-dependent manner, but GGN4 (1-23), an N-terminal fragment of the peptide with little antimicrobial activity, failed to increase the conductance. At symmetrical 100 mM KCI, unitary conductances of about 120 pS were frequently observed. Their current-voltage relations were linear and open state probabilities were close to 1, but longer closing events were seen more frequently at negative voltages. In addition, GGN4-induced pores were selective for cation over anion, the permeability ratio of K+ to Cl- being 6: 1 in neutral and 7: 1 in acidic lipid bilayers. In conclusion, our results indicate that GGN4 forms voltage-dependent and cation-selective pores in planar lipid bilayers. The ionophoric property of GGN4 is likely to contribute to its antimicrobial activity.
The structure-function relationships of toxin-antitoxin (TA) systems from Mycobacterium tuberculosis have prompted the development of novel and effective antimicrobial agents that selectively target this organism. The artificial activation of toxins by peptide inhibitors can lead to the growth arrest and eventual death of bacterial cells. Optimizing candidate peptides by hydrocarbon α-helix stapling based on structural information from the VapBC TA system and in vitro systematic validation led to V26-SP-8, a VapC26 activator of M. tuberculosis. This compound exhibited highly enhanced activity and cell permeability owing to the stabilizing helical propensity of the peptide. These characteristics will increase its efficacy against multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. Similar approaches utilizing structural and biochemical information for new antibiotic targets opens a new era for developing TB therapies.
Toxin–antitoxin (TA) systems have been considered essential factors for bacterial survival. During our drug development program aimed against tuberculosis (TB), we discovered certain peptides that mimic the binding of the VapBC30 complex, leading to the arrest of bacterial cell growth and eventually cell death. Herein, we optimized these candidate peptides based on a hydrocarbon stapling strategy and performed biological in vitro evaluations. The V30-SP-8 peptide successfully penetrated Mycobacterium smegmatis cell membranes and exerted bactericidal activity at a minimum inhibitory concentration that inhibited 50% of the isolates (MIC50) < 6.25 μM. With the aid of structural and biochemical information for the VapBC30 TA system from M. tuberculosis, we suggest potential antimicrobial agents that could provide a platform to establish a novel antibacterial strategy. Reflecting the limited number of therapeutic agents targeting TA systems, we believe that this study not only provides chemical tools for exploring the biological events relevant to TA systems but also opens a new gateway toward TB drug discovery.
The Mw 4.9 Offshore Jeju Island, Korea, earthquake of 14 December 2021 is the most significant event that occurred close to the young intraplate volcano on the continental shelf south of the Korean Peninsula. About 185 small earthquakes occurred during nine days following the mainshock. We accurately located 39 events despite limited station azimuthal coverage. We found that the mainshock ruptured along a ∼1.4 km long fault striking east–west (275°) and triggered two distinct clusters in the north–northeast direction. The mainshock is pure strike-slip faulting on the vertical plane. Its horizontal P axis strikes 50°, which differs substantially from the average trend of ∼70° for earthquakes in the southern Korean Peninsula. However, it is consistent with the P-axes orientation of shallow earthquakes in the offshore western Kyushu and along Okinawa trough. The 2021 Offshore Jeju Island earthquake may be the first significant earthquake in southern Korea–East China Sea continental shelf to date, whose causative fault is well constrained by accurate aftershock locations and detailed space–time analysis of the earthquake sequence. This earthquake may represent intraplate deformation in response to the tectonic evolution of the Philippine Sea plate, which is manifested through volcanic activities.
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