Mutation in crrB encoding a sensor kinase increases expression of the RND-type multidrug efflux pump KexD in Klebsiella pneumoniae

Hayata Dodan; Makoto Hiromura; Rui Ting Ni; Futoshi Matsubara; Teruo Kuroda; Wakano Ogawa

doi:10.1016/j.gene.2023.147543

Mutation in crrB encoding a sensor kinase increases expression of the RND-type multidrug efflux pump KexD in Klebsiella pneumoniae

Gene. 2023 Aug 20:878:147543. doi: 10.1016/j.gene.2023.147543. Epub 2023 Jun 16.

Authors

Hayata Dodan¹, Makoto Hiromura², Rui Ting Ni³, Futoshi Matsubara⁴, Teruo Kuroda⁵, Wakano Ogawa⁶

Affiliations

¹ Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Kita-ku, Okayama 700-8530, Japan.
² Department of Molecular Biology, Daiichi University of Pharmacy, Tamagawa-machi, Minami-ku, Fukuoka 815-8511, Japan.
³ Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Kita-ku, Okayama 700-8530, Japan.
⁴ Department of Microbiology and Biochemistry, Daiichi University of Pharmacy, Tamagawa-machi, Minami-ku, Fukuoka 815-8511, Japan.
⁵ Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Kita-ku, Okayama 700-8530, Japan; Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Kita-ku, Okayama 700-8530, Japan; Department of Microbiology, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
⁶ Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Kita-ku, Okayama 700-8530, Japan; Department of Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Kita-ku, Okayama 700-8530, Japan; Department of Microbiology and Biochemistry, Daiichi University of Pharmacy, Tamagawa-machi, Minami-ku, Fukuoka 815-8511, Japan. Electronic address: wogawa@daiichi-cps.ac.jp.

PMID: 37331490
DOI: 10.1016/j.gene.2023.147543

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: Mutation in crrB is important for KexD overexpression. Increased CrrA may also be associated with KexD overexpression.

Keywords: Colistin; CrrAB; Multidrug resistance; RND-type multidrug efflux pump KexD.

MeSH terms

Anti-Bacterial Agents* / metabolism
Anti-Bacterial Agents* / pharmacology
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Colistin* / metabolism
Drug Resistance, Multiple, Bacterial / genetics
Klebsiella pneumoniae / genetics
Microbial Sensitivity Tests
Mutation

Substances

Colistin
Anti-Bacterial Agents
Bacterial Proteins
EM 16