Construction and characterization of<italic> lpxC </italic>deletion strain based on CRISPR/Cas9 in <italic>Acinetobacter baumannii</italic>

Zong-ti Sun; You-wen Zhang; Hai-bin LI; Xiu-kun Wang; Jie YU; Jin-ru Xie; Peng-bo Pang; Xin-xin HU; Tong-ying Nie; Xi LU; Jing Pang; Lei Hou; Xin-yi Yang; Cong-ran LI; Lang Sun; Xue-fu You

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Construction and characterization of lpxC deletion strain based on CRISPR/Cas9 in Acinetobacter baumannii

VernacularTitle:基于CRISPR/Cas9技术构建鲍曼不动杆菌lpxC缺失株及其表型研究
Author: Zong-ti SUN ¹ ; You-wen ZHANG ^{1
,

2} ; Hai-bin LI ¹ ; Xiu-kun WANG ^{1
,

2} ; Jie YU ¹ ; Jin-ru XIE ¹ ; Peng-bo PANG ¹ ; Xin-xin HU ^{1
,

2} ; Tong-ying NIE ^{1
,

2} ; Xi LU ^{1
,

2} ; Jing PANG ^{1
,

2} ; Lei HOU ³ ; Xin-yi YANG ^{1
,

2} ; Cong-ran LI ^{1
,

2} ; Lang SUN ^{1
,

2} ; Xue-fu YOU ^{1
,

2}
Author Information

1. Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
2. Division for Medicinal Microorganisms Related Strains, CAMS Collection Center of Pathogenic Microorganisms, Beijing 100050, China
3. Experimental Animal Center, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Publication Type:Research Article
Keywords: italic>Acinetobacter baumannii; gene editing; CRISPR/Cas9 system; italic>lpxC gene; polymyxin; resistance; lipopolysaccharide
From: Acta Pharmaceutica Sinica 2024;59(5):1286-1294
CountryChina
Language:Chinese
Abstract: Lipopolysaccharides (LPS) are major outer membrane components of Gram-negative bacteria. Unlike most Gram-negative bacteria, Acinetobacter baumannii can still survive and acquire polymyxin resistance after complete loss of LPS. Previous studies of LPS-deficient Acinetobacter baumannii mostly focused on LPS-deficient strains induced by polymyxin, whose background was complex and unstable. To investigate LPS loss-mediated polymyxin resistant-Acinetobacter baumannii, this study constructed a stable and clear background LPS-deficient strain by knocking out lpxC gene in Acinetobacter baumannii ATCC 19606 with CIRSPR/Cas9, and then studied the phenotypic changes of the lpxC-deficient strain including morphology, growth rate, antibiotic susceptibility, virulence, membrane permeability, and membrane potential. Animal experiments were approved by the Animal Care and Welfare Committee Institute of Medicinal Biotechnology, CAMS and PUMC (approval number: IMB-20240119D₉). The results indicated that the lpxC gene of Acinetobacter baumannii ATCC 19606 was successfully knocked out. After losing the lpxC, the strain underwent the morphological change from rod-shaped to spherical. Furthermore, it leads to reduced growth rate, enhanced membrane permeability, decreased membrane potential, lower virulence, and increased antibiotic susceptibility to β-lactams, quinolones, aminoglycosides, macrolides, glycopeptides. The lpxC deletion results in significant changes in membrane homeostasis and adaptability of Acinetobacter baumannii. Understanding the phenotypic changes of colistin-resistant Acinetobacter baumannii mediated by LPS loss is useful for exploring the resistance mechanism of Acinetobacter baumannii and developing new therapeutic strategies.