RESUMO
The prevalence of multidrug-resistant highly virulent Klebsiella pneumoniae (MDR-hvKP) requires the development of new therapeutic agents. Herein, a novel lytic phage vB_KpnS_ZX4 against MDR-hvKP was discovered in hospital sewage. Phage vB_KpnS_ZX4 had a short latent period (5 min) and a large burst size (230 PFU/cell). It can rapidly reduce the number of bacteria in vitro and improve survival rates of bacteremic mice in vivo from 0 to 80 % with a single injection of 108 PFU. LysZX4, an endolysin derived from vB_KpnS_ZX4, exhibits potent antimicrobial activity in vitro in combination with ethylenediaminetetraacetic acid (EDTA). The antimicrobial activity of LysZX4 was further enhanced by the fusion of KWKLFKI residues from cecropin A (LysZX4-NCA). In vitro antibacterial experiments showed that LysZX4-NCA exerts broad-spectrum antibacterial activity against clinical Gram-negative bacteria, including MDR-hvKP. Moreover, in the mouse model of MDR-hvKP skin infection, treatment with LysZX4-NCA resulted in a three-log reduction in bacterial burden on the skin compared to the control group. Therefore, the novel phages vB_KpnS_ZX4 and LysZX4-NCA are effective reagents for the treatment of systemic and local MDR-hvKP infections.
Assuntos
Antibacterianos , Bacteriófagos , Camundongos , Animais , Antibacterianos/farmacologia , Endopeptidases/farmacologia , Klebsiella pneumoniaeRESUMO
The bacterial expression and purification of human glutathione S-transferase P1-1(hGST P1-1), as a hexahistidine-tagged polypeptide was performed. Site-directed mutagenesis was used to construct mutants in which alanine replaced two (C47A/C101A), three (C14A/C47A/C101A) or all four (C14A/C47A/ C101A/C169A) cysteine residues using the plasmid for the wild type enzyme. Analysis of their catalytic activities and kinetic parameters suggested that cysteins are not essential for the catalytic activity but may contribute to some extent to the catalytic efficiency. Moreover, on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, hexahistidine-tagged hGST P1-1 (His(6)-hGST P1-1) treated with 1 mM H(2)O(2) showed at least three extra bands, in addition to the native His(6)-hGST P1-1 subunit band. These extra bands were not detected in the cysteinyl mutants. Thus, it indicated that disulfide bonds were formed mainly within subunits between cysteine residues, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed.