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Discovering interrelated natural mutations of efflux pump KmrA from Klebsiella pneumoniae that confer increased multidrug resistance.

ABSTRACT: Klebsiella pneumoniae is a notorious pathogen that can cause multiorgan infections, which is difficult to treat mainly due to the widely distributed efflux pumps. Our previous research discovered the upregulation of efflux pump KmrA conferred enhanced antibiotic resistance, while the export mechanism and its natural mutations across K. pneumoniae isolates remain unclear. Herein, we analyzed the natural mutations of KmrA across 830 K. pneumoniae genomes to discover interrelated amino-acid substitutions (simultaneously occurred substitutions) that increase drug export. We identified two variants that contain triple amino-acid substitutions near the periplasmic side and then confirmed their roles in enhancing multidrug resistance of recombinant K. pneumoniae strains. Molecular dynamics simulations were conducted to illustrate the reason for their promoted export efficiencies. Our data indicated the triple substitutions resulted in KmrA's both stronger hydrophilic interaction with water and hydrophobic interaction with membrane. Moreover, these substitutions promoted the flexibilities of KmrA that could facilitate the conformational switch. In parallel, stronger ionic interactions (salt bridges) at cytoplasmic side also suggested the higher possibilities for the reciprocal movements. Collectively we demonstrated the potential risk of the interrelated natural mutations in efflux pump to antibiotic resistance of K. pneumoniae and provided insights into the mechanism of the enhanced drug export.

SUBMITTER: Li Y

PROVIDER: S-EPMC9083138 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Discovering interrelated natural mutations of efflux pump KmrA from Klebsiella pneumoniae that confer increased multidrug resistance.

Li Ying Y Ge Xizhen X

Protein science : a publication of the Protein Society 20220601 6

Klebsiella pneumoniae is a notorious pathogen that can cause multiorgan infections, which is difficult to treat mainly due to the widely distributed efflux pumps. Our previous research discovered the upregulation of efflux pump KmrA conferred enhanced antibiotic resistance, while the export mechanism and its natural mutations across K. pneumoniae isolates remain unclear. Herein, we analyzed the natural mutations of KmrA across 830 K. pneumoniae genomes to discover interrelated amino-acid substit ...[more]

PMID: 35634772

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Project description:The continued challenges of the COVID-19 pandemic combined with the growing problem of antimicrobial-resistant bacterial infections has severely impacted global health. Specifically, the Gram-negative pathogen Klebsiella pneumoniae is one of the most prevalent causes of secondary bacterial infection in COVID-19 patients, with approximately an 83% mortality rate observed among COVID-19 patients with these bacterial coinfections. K. pneumoniae belongs to the ESKAPE group of pathogens, a group that commonly gives rise to severe infections that are often life-threatening. Recently, K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae has drawn wide public attention, as the mortality rate for this infection can be as high as 71%. The most predominant and clinically important multidrug efflux system in K. pneumoniae is the acriflavine resistance B (AcrB) multidrug efflux pump. This pump mediates resistance to different classes of structurally diverse antimicrobial agents, including quinolones, β-lactams, tetracyclines, macrolides, aminoglycosides, and chloramphenicol. We here report single-particle cryo-electron microscopy (cryo-EM) structures of K. pneumoniae AcrB, in both the absence and the presence of the antibiotic erythromycin. These structures allow us to elucidate specific pump-drug interactions and pinpoint exactly how this pump recognizes antibiotics. IMPORTANCE Klebsiella pneumoniae has emerged as one of the most problematic and highly antibiotic-resistant pathogens worldwide. It is the second most common causative agent involved in secondary bacterial infection in COVID-19 patients. K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae is a major concern in global public health because of the high mortality rate of this infection. Its drug resistance is due, in a significant part, to active efflux of these bactericides, a major mechanism that K. pneumoniae uses to resist to the action of multiple classes of antibiotics. Here, we report cryo-electron microscopy (cryo-EM) structures of the prevalent and clinically important K. pneumoniae AcrB multidrug efflux pump, in both the absence and the presence of the erythromycin antibiotic. These structures allow us to understand the action mechanism for drug recognition in this pump. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

Project description:The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10-7-10-9. Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites.

Dataset Information

Discovering interrelated natural mutations of efflux pump KmrA from Klebsiella pneumoniae that confer increased multidrug resistance.

Publications

Discovering interrelated natural mutations of efflux pump KmrA from Klebsiella pneumoniae that confer increased multidrug resistance.

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