A novel major facilitator superfamily-type tripartite efflux system CprABC mediates resistance to polymyxins in Chryseobacterium sp. PL22-22A

Abstract

Background: Polymyxin B (PMB) and polymyxin E (colistin, CST) are polymyxin antibiotics, which are considered last-line therapeutic options against multidrug-resistant Gram-negative bacteria in serious infections. However, there is increasing risk of resistance to antimicrobial drugs. Effective efflux pump inhibitors (EPIs) should be developed to help combat efflux pump-mediated antibiotic resistance.

Methods: Chryseobacterium sp. PL22-22A was isolated from aquaculture sewage under selection with 8 mg/L PMB, and then its genome was sequenced using Oxford Nanopore and BGISEQ-500 platforms. Cpr (Chryseobacterium Polymyxins Resistance) genes encoding a major facilitator superfamily-type tripartite efflux system, were found in the genome. These genes, and the gene encoding a truncation mutant of CprB from which sequence called CprBc was deleted, were amplified and expressed/co-expressed in Escherichia coli DH5α. Minimum inhibitory concentrations (MICs) of polymyxins toward the various E. coli heterologous expression strains were tested in the presence of 2-128 mg/L PMB or CST. The pumping activity of CprABC was assessed via structural modeling using Discovery Studio 2.0 software. Moreover, the influence on MICs of baicalin, a novel MFS EPI, was determined, and the effect was analyzed based on homology modeling.

Results: Multidrug-resistant bacterial strain Chryseobacterium sp. PL22-22A was isolated in this work; it has notable resistance to polymyxin, with MICs for PMB and CST of 96 and 128 mg/L, respectively. A novel MFS-type tripartite efflux system, named CprABC, was identified in the genome of Chryseobacterium sp. PL22-22A. Heterologous expression and EPI assays indicated that the CprABC system is responsible for the polymyxin resistance of Chryseobacterium sp. PL22-22A. Structural modeling suggested that this efflux system provides a continuous conduit that runs from the CprB funnel through the CprC porin domain to pump polymyxins out of the cell. A specific C-terminal α-helix, CprBc, has an activation function on polymyxin excretion by CprB. The flavonoid compound baicalin was found to affect the allostery of CprB and/or obstruct the substrate conduit, and thus to inhibit extracellular polymyxin transport by CprABC.

Conclusion: Novel MFS-type tripartite efflux system CprABC in Chryseobacterium sp. PL22-22A mediates resistance to polymyxins, and baicalin is a promising EPI.

Keywords: Chryseobacterium sp.; MFS transporter; baicalin; efflux pump inhibitor; polymyxins; resistance.

Figure 1

Identification and genomic characteristics of strain PL22-22A. (A) Neighbor-joining phylogenetic tree generated on the basis of 16S rDNA gene sequences. (B) PATRIC annotation of the genome of Chryseobacterium sp. PL22-22A. (C) Subsystem analysis of the genome of Chryseobacterium sp. PL22-22A.

Figure 2

Minimum inhibitory concentrations (MICs) and EC₅₀ of polymyxins toward different strains. (A) MICs of polymyxins toward different strains; (B) EC₅₀ of polymyxins toward different strains. DH5α, Escherichia coli DH5α (control); DT, E. coli DH5α with pMD-18 T (control); EcAB, E. coli DH5α expressing cprA-cprB; EcABC3, E. coli DH5α expressing cprABC; PMB, polymyxin B; CST, colistin. *MIC<2 mg/L.

Figure 3

Structure of each monomer in the tripartite transporter complex CprABC from Chryseobacterium sp. PL22-22A. (A) Structure of CprA monomer. (B) Structure of CprC monomer. (C) Structure of CprB monomer, left: structure viewed in the plane of the membrane; right: topology diagram. IM, inner membrane; OM, outer membrane. AcrAB-TolC (PDB: 2VDD) was used as homology model.

Figure 4

Putative assembly of the CprABC pump. (A) Overall structure of the CprABC pump seen from the periplasmic side. (B) Cross-section through CprABC. (C–E) Gates T, F and G seen from the OM side. IM, inner membrane; OM, outer membrane.

Figure 5

The C-terminal fragment CprBc of CprB contributes to activation of the CprABC complex. (A) Predicted PMB- and CST-binding sites and binding amino acids in CprB. PMB and CST are shown as red and green sticks, respectively; PMB- and CST-binding sites are shown by red and green surfaces; PMB- and CST-binding amino acids are shown by lines. (B) Alignment of amino acid sequences of CprBc and ArcZ. Red arrows indicate conserved glycine sites. (C) Structural comparison of CprBc and ArcZ (from E. coli, PDB: 5nc5). (D) Influence of CprBc deletion on CprABC pumping of polymyxins. DH, E. coli DH5α (control strain); DT, E. coli DH5α with pMD-18 T (control strain); EcABC3, E. coli DH5α expressing Chryseobacterium sp. PL22-22A cprABC; EcABdC3, E. coli DH5α expressing Chryseobacterium sp. PL22-22A cprA, cprC, and cprBd (truncated CprB with CprBc deleted); EcB1, E. coli DH5α expressing Chryseobacterium sp. PL22-22A cprB; EcBd1, E. coli DH5α expressing Chryseobacterium sp. PL22-22A cprBd (truncated CprB with CprBc deleted). *MIC<2 mg/L.

Figure 6

Baicalin decreasing resistance to polymyxins. (A) Effect of efflux pump inhibitors (EPIs) on the MIC of strain EcB1. (B) Effect of efflux pump inhibitors (EPIs) on the MBC of strain EcB1; (C) Growth curves of EcB1 in PMB containing medium in the presence of EPIs; (D) Growth curves of EcB1 in CST containing medium in the presence of EPIs. No EPI, strain EcB1 without EPIs; +, with different EPIs, i.e., CCCP, carbonyl cyanide (m-chlorophenyl)hydrazone; PaβN, phenylalanine-arginine β-naphthylamide dihydrochloride; RES, reserpine; VER, verapamil; BAC, baicalin. *MIC<2 mg/L.

Figure 7

BAC obstruct the passage of polymyxins through the MFS transporters pump. (A) Predicted allosteric sites (AS) of CprB. AS1–AS4 are represented in blue, orange, green, and red, respectively; N, N-terminus; C, C-terminus. (B) Predicted BAC-binding sites (BBS) and binding amino acids in Chryseobacterium sp. PL22-22A CprB. AS1–AS4 are shown by blue, orange, green, and red lines, respectively; BAC in BBS 1–3 is shown by red, orange, and violet sticks, respectively.