Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Kita-ku, Okayama 700-8530, Japan.
Department of Molecular Biology, Daiichi University of Pharmacy, Tamagawa-machi, Minami-ku, Fukuoka 815-8511, Japan.
Gene. 2023 Aug 20;878:147543. doi: 10.1016/j.gene.2023.147543. Epub 2023 Jun 16.
RND-type multidrug efflux systems in Gram-negative bacteria protect them against antimicrobial agents. Gram-negative bacteria generally possess several genes which encode such efflux pumps, but these pumps sometimes fail to show expression. Generally, some multidrug efflux pumps are silent or expressed only at low levels. However, genome mutations often increase the expression of such genes, conferring the bacteria with multidrug-resistant phenotypes. We previously reported mutants with increased expression of the multidrug efflux pump KexD. We aimed to identify the cause of KexD overexpression in our isolates. Furthermore, we also examined the colistin resistant levels in our mutants.
A transposon (Tn) was inserted into the genome of Klebsiella pneumoniae Em16-1, a KexD-overexpressing mutant, to identify the gene(s) responsible for KexD overexpression.
Thirty-two strains with decreased kexD expression after Tn insertion were isolated. In 12 of these 32 strains, Tn was identified in crrB, which encodes a sensor kinase of a two-component regulatory system. DNA sequencing of crrB in Em16-1 showed that the 452nd cytosine on crrB was replaced by thymine, and this mutation changed the 151st proline into leucine. The same mutation was found in all other KexD-overexpressing mutants. The expression of crrA increased in the mutant overexpressing kexD, and the strains in which crrA was complemented by a plasmid showed elevated expression of kexD and crrB from the genome. The complementation of the mutant-type crrB also increased the expression of kexD and crrA from the genome, but the complementation of the wild-type crrB did not. Deletion of crrB decreased antibiotic resistance levels and KexD expression. CrrB was reported as a factor of colistin resistance, and the colistin resistance of our strains was tested. However, our mutants and strains carrying kexD on a plasmid did not show increased colistin resistance.
Mutation in crrB is important for KexD overexpression. Increased CrrA may also be associated with KexD overexpression.
革兰氏阴性菌中的 RND 型多药外排系统可保护其免受抗菌药物的侵害。革兰氏阴性菌通常具有多个编码此类外排泵的基因，但这些泵有时未能表达。通常，一些多药外排泵处于沉默或仅低水平表达状态。然而，基因组突变经常会增加这些基因的表达，使细菌具有多药耐药表型。我们之前报道了多药外排泵 KexD 表达增加的突变体。我们旨在确定我们分离株中 KexD 过表达的原因。此外，我们还检查了我们突变体的多粘菌素耐药水平。
将转座子（Tn）插入产酸克雷伯菌 Em16-1 的基因组中，该突变体为 KexD 过表达突变体，以鉴定导致 KexD 过表达的基因。
插入 Tn 后，有 32 株菌的 kexD 表达降低。在这 32 株菌中，有 12 株菌的 Tn 插入到 crrB 中，crrB 编码一个双组分调控系统的感应激酶。Em16-1 中 crrB 的 DNA 测序显示，crrB 的第 452 位胞嘧啶被胸腺嘧啶取代，该突变将第 151 位脯氨酸突变为亮氨酸。在所有其他 KexD 过表达的突变体中均发现了相同的突变。过表达 kexD 的突变体中 crrA 的表达增加，并且通过质粒互补 crrA 的菌株显示基因组中 kexD 和 crrB 的表达升高。突变型 crrB 的互补也增加了基因组中 kexD 和 crrA 的表达，但野生型 crrB 的互补则没有。crrB 的缺失降低了抗生素耐药水平和 KexD 的表达。CrrB 被报道为多粘菌素耐药的一个因素，我们测试了我们菌株的多粘菌素耐药性。然而，我们的突变体和携带质粒上 kexD 的菌株并没有表现出增加的多粘菌素耐药性。
crrB 中的突变对于 KexD 的过表达很重要。CrrA 的增加也可能与 KexD 的过表达有关。
