Biochemical properties and substrate specificity of GOB-38 in Elizabethkingia anophelis

Abstract

The novel pathogen, Elizabethkingia anophelis, has gained attention due to its high mortality rates and drug resistance facilitated by its inherent metallo-β-lactamases (MBLs) genes. This study successfully identified and outlined the functions of the B3-Q MBLs variant, GOB-38, in a clinical sample of E. anophelis. The T7 expression system was employed to stimulate the expression of recombinant protein in Escherichia coli, followed by an analysis of the biochemical properties of purified GOB-38. Our findings indicate that the enzyme GOB-38 displays a wide range of substrates, including broad-spectrum penicillins, 1-4 generation cephalosporins, and carbapenems, potentially contributing to in vitro drug resistance in E. coli through a cloning mechanism. It is important to highlight that GOB-38 exhibits a distinct active site composition compared to GOB-1/18, featuring hydrophilic amino acids Thr51 and Glu141 at both ends of its active center instead of hydrophobic alanine, potentially indicating a preference for imipenem. Furthermore, the co-isolation of Acinetobacter baumannii and E. anophelis, two opportunistic pathogens, from a single lung infection is noteworthy. Our in vitro co-culture experiments suggest that E. anophelis, carrying two MBL genes, may have the ability to transfer carbapenem resistance to other bacterial species through co-infection.

Keywords: Acinetobacter baumannii; Elizabethkingia anophelis; Antibiotic resistance; GOB-38; Metallo-β-lactamases.

Fig. 1

Phylogenetic tree and pangenomes of A. baumannii Ab6-2 and E. anopheles EA-1. Based on the average nucleotide identity (ANI), phylogenetic trees of (A), 60 complete genomes of E.anophelis (including EA-1 and 59 reference strains), and (B), 120 complete genomes of A. baumannii (including Ab6-2 and 119 reference strains) were constructed. (C), The distribution of persistent (softcore), shell (core/shell), and cloud (accessory/dispensable) genes, as well as the pangenome composition of EA-1 and 59 E. anophelis strains, and the pangenomes of Ab6-2 and 119 A. baumannii strains. Pangenome was calculated by PPanGGOLiN.

Fig. 2

MBL phenotype examine and RT-qPCR. (A) MBL phenotype of Ab6-2 and EA-1 pure cultures and cocultures (1:1 ratio). (B–D) RT-qPCR analysis of blaB-29 and blaGOB-38 expression in coculture. Data are expressed as the mean and standard error of three independent biological replicates. (*, p < 0.05; **, p < 0.01 and ****, p < 0.0001; ns, not significant). AMP, Ampicillin; IPM, Imipenem; CAZ, Ceftazidime.

Fig. 3

DNA cloning, the purification of proteins, and the determination of pI. (A) Description of the upstream and downstream sequences of blaGOB-38 and the experimental steps for overexpression of the GOB-38 protein. (B) SDS-PAGE after the different purification steps. M, molecular mass standards; lane 1, crude extract from BL21-DE3; lane 2, Ni-NTA agarose resin eluent; lane 3, STarm Streptactin beads flow through liquid; lane 4, STarm Streptactin beads detergent; lane 5–7, STarm Streptactin beads eluent. (C) GOB-38 pI determination. Red arrow, standard; black arrow, sample.

Fig. 4

The maximum likelihood tree illustrated the relationship between GOB and other functionally characterized B3-MBLs. Bootstrap support for internal nodes was indicated by purple dots, with values ranging from 0.5 to 1. The GOB family was depicted with a pink background, while the B3-Q MBLs, including GOB-1/18/38 and CPS-1, were highlighted in red font. Additionally, the tetramer members L1 and POM-1 were emphasized in purple, while the representative enzyme SPR-1 of B3-RQK was highlighted in green, and the representative enzyme SIE-1 of B3-E was highlighted in yellow.

Fig. 5

The amino acid alignment of GOB-38 (NCBI Reference protein Sequence number: WP_058879139.1), GOB-1 (WP_063860523.1), GOB-18 (WP_063860560.1), FEZ-1 (WP_058468956.1), CPS-1 (WP_063857696.1), LMB-1 (WP_108361548.1), SMB-1 (WP_063864723.1), THIN-B (WP_063842948.1), BJP-1 (WP_011088970.1), AIM-1 (WP_063857820.1), CAU-1 (WP_063859390.1), PJM-1 (WP_213603971.1), PAM-1 (WP_043245728.1), POM-1 (WP_044403015.1), L1 (WP_063842689.1), SIE-1 (WP_007683232.1), SPR-1 (WP_012145975.1) with the secondary structure of GOB-38 was depicted. Conserved residues were indicated by red boxes, while similar residues were indicated by white boxes. The QHH site (Zn1) was highlighted in pink, and the DHH site (Zn2) was highlighted in yellow. The secondary structure elements of GOB-38 were displayed above the alignments. This figure was generated using ESPript.

Fig. 6

Overall and active-site structure of GOB-38. (A) The GOB-38 protein was depicted in a cartoon representation, where the helices were colored in dark green, the strands in pink, and the non-structured loops in dark yellow. Specifically, the two loops, referred to as loop1 and loop2, which covered the active site, were highlighted in dark blue. The presence of zinc atoms was represented by gray spheres. The residues that were the subject of discussion in this paper were visually represented using orange spheres and stick models, with carbon atoms depicted in green, nitrogen atoms in blue, and oxygen atoms in red. (B) The active-site structure of GOB-38 was depicted, with gray representing the zinc atoms, red representing the water molecules (Wat1, Wat2), and dashed lines indicating the coordination bonds. The estimated distance between the two Zn²⁺ ions was 3.5 Å.