Eiji Tamai

The pathogenesis and infectivity of Gram‐positive bacteria are mediated by many surface proteins covalently attached to the bacterial cell wall. Pili are types of surface appendages that play important roles in the initial adhesion of bacterial cells to host tissues and bacterial colonization. The Gram‐positive bacterium Clostridium perfringens ( C. perfringens ), one of the pathogenic clostridial species causing gas gangrene and food poisoning, has sortase‐mediated pili composed of shaft/major pilin A (CppA) and tip/minor pilin B (CppB). The pilus shaft is formed by covalent polymerization of CppA, and CppB is covalently attached to the tip of the shaft involved in adhesion to the host cell. The formation of covalent bonds between CppB and CppA, as well as between CppA and CppA, is catalyzed by class C sortase (CpSrtC), a member of the cysteine transpeptidase family. Since pili consistently have CppB at their tip, CpSrtC is the enzyme that preferentially catalyzes the attachment of CppB (tip) to CppA (shaft) rather than polymerization of CppAs by an unknown mechanism. We determined the structures of complexes formed by covalently linking CppB and CppA by X‐ray crystallography and cryo‐EM analysis. The complexes have an elongated structure in which β‐sandwich folded domains are sequentially arranged, and an amide bond between Thr688 of CppB and Lys174 of CppA was clearly identified. The determined structures allowed us to construct a three‐dimensional structure model with dynamic motion of C. perfringens pili, and proposed new insights into the mechanism by which CppB is preferentially attached to CppA.

Clostridioides difficile is a major pathogen of pseudomembranous colitis, and new antimicrobial agents are needed for its treatment. Autolysins are peptidoglycan-degrading enzymes that generally reorganize the cell wall during cell division but kill bacteria by bacteriolysis when applied from outside the bacterial cell. Therefore, they have potential as novel therapeutic agents for the treatment of infectious diseases. We surveyed the genome of C. difficile strain 630 and identified two virtually identical autolysin genes, cdCwlT33800 and cdCwlT , with a lysozyme-like domain and endopeptidase domain. The entire region and each domain of the two proteins were expressed, purified, and assayed for bacteriolytic activity. Only the individual endopeptidase domain variants exhibited bacteriolytic activity against C. difficile . We also investigated the optimal pH and salt concentration, the effects of metal ions, thermostability, long-term storage, and the species specificity of the CdCwlT33800 endopeptidase domain (CdCwlT33800CD2). The structure of CdCwlT33800CD2 was elucidated by X-ray crystallography at a resolution of 1.45 Å. The overall structure was spherical and consisted of five helices and eight β-strands, with a 28 Å substrate-binding groove, at the center of which the catalytic residues of Cys242 and His296 were located. The structure of the substrate–enzyme complex was proposed through modeling and mutagenic analyses of CdCwlT33800CD2. IMPORTANCE: Clostridioides difficile is a bacterium that causes severe colitis and life-threatening diarrhea, particularly after antibiotic treatment. Since current therapies are not always effective and resistance to drugs continues to increase, there is an urgent need for new treatment strategies. One promising approach is the use of lytic enzymes, which break down the bacterial cell wall and lead to bacterial death. These enzymes include autolysins, which are produced by bacteria themselves and phage-derived endolysins. In the present study, we identified a novel autolysin from C. difficile and analyzed its biochemical characteristics and structure. The present results provide insights into the development of enzyme-based therapies to combat C. difficile infections and may lead to effective alternatives to conventional antibiotics.

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Clostridioides difficile is the major pathogen of pseudomembranous colitis, and novel antimicrobial agents are sought after for its treatment. Phage-derived endolysins with species-specific lytic activity have potential as novel antimicrobial agents. We surveyed the genome of C. difficile strain 630 and identified an endolysin gene, Ecd09610, which has an uncharacterized domain at the N-terminus and two catalytic domains that are homologous to glucosaminidase and endopeptidase at the C-terminus. Genes containing the two catalytic domains, the glucosaminidase domain and the endopeptidase domain, were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. The purified domain variants showed lytic activity almost specifically for C. difficile, which has a unique peptide bridge in its peptidoglycan. This species specificity is thought to depend on substrate cleavage activity rather than binding. The domain variants were thermostable, and, notably, the glucosaminidase domain remained active up to 100 °C. In addition, we determined the optimal pH and salt concentrations of these domain variants. Their properties are suitable for formulating a bacteriolytic enzyme as an antimicrobial agent. This lytic enzyme can serve as a scaffold for the construction of high lytic activity mutants with enhanced properties.

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

Autolysin is a lytic enzyme that hydrolyzes peptidoglycans of the bacterial cell wall, with a catalytic domain and cell wall‐binding (CWB) domains, to be involved in different physiological functions that require bacterial cell wall remodeling. We identified a novel autolysin, Acd24020, from Clostridioides (Clostridium) difficile (C. difficile), with an endopeptidase catalytic domain belonging to the NlpC/P60 family and three bacterial Src‐homology 3 domains as CWB domains. The catalytic domain of Acd24020 (Acd24020‐CD) exhibited C. difficile‐specific lytic activity equivalent to Acd24020, indicating that Acd24020‐CD has full‐function as a lytic enzyme by itself. To elucidate the specific peptidoglycan‐recognition and catalytic reaction mechanisms of Acd24020‐CD, biochemical characterization, X‐ray structure determination, a modeling study of the enzyme/substrate complex, and mutagenesis analysis were performed. Acd24020‐CD has an hourglass‐shaped substrate‐binding groove across the molecule, which is responsible for recognizing the direct 3–4 cross‐linking structure unique to C. difficile peptidoglycan. Based on the X‐ray structure and modeling study, we propose a dynamic Cys/His catalyzing mechanism, in which the catalytic Cys299 and His354 residues dynamically change their conformations to complement each step of the enzyme catalytic reaction.

Bacteria often show resistance against antibiotics due to various mechanisms such as the expression of efflux pumps, biofilm formation, or bacterial quorum sensing (QS) controls. For successful therapy, the discovery of alternative agents is crucial. The objective of this study was to evaluate the efflux pump, anti-biofilm, and QS inhibiting, as well as antibacterial effects of 2-trifluoroacetonylbenzoxazole ligands (1–3) and their metal complexes (4–12) in bacteria. The ligand 2 and its Zn(II) complex 5, and furthermore the Cu(II) complex 7 of ligand 1, exerted remarkable antibacterial activity on the Staphylococcus aureus 272123 (MRSA) strain. In the minimum inhibitory concentration (MIC) reduction assay the ligand 3, the Zn(II) complex 5 of ligand 2, and the Cu(II), Ni(II), Mg(II), Fe(III) complexes (7, 8, 9, 12) of ligand 1 enhanced the antibacterial activity of ciprofloxacin in MRSA. An increased ethidium bromide accumulation was detected for ligand 3 in MRSA while the Fe(III) complex 12 of ligand 1 decreased the biofilm formation of the reference S. aureus ATCC 25923 strain. The Zn(II) and Ag(II) complexes (3 and 4) of ligand 1 and ligand 3 inhibited the QS. Based on our results, the ligands and their metal complexes could be potential alternative drugs in the treatment of infectious diseases.