Dataset Information

Structural and functional characterization of fosfomycin resistance conferred by FosB from Enterococcus faecium.

ABSTRACT: The Gram-positive pathogen Enterococcus faecium is one of the leading causes of hospital-acquired vancomycin resistant enterococci (VRE) infections. E. faecium has extensive multidrug resistance and accounts for more than two million infections in the United States each year. FosB is a fosfomycin resistance enzyme found in Gram-positive pathogens like E. faecium. Typically, the FosB enzymes are Mn²⁺ -dependent bacillithiol (BSH) transferases that inactivate fosfomycin through nucleophilic addition of the thiol to the antibiotic. However, our kinetic analysis of FosB^Ef shows that the enzyme does not utilize BSH as a thiol substrate, unlike the other well characterized FosB enzymes. Here we report that FosB^Ef is a Mn²⁺ -dependent L-cys transferase. In addition, we have determined the three-dimensional X-ray crystal structure of FosB^Ef in complex with fosfomycin at a resolution of 2.0 Å. A sequence similarity network (SSN) was generated for the FosB family to investigate the unexpected substrate selectivity. Three non-conserved residues were identified in the SSN that may contribute to the substrate selectivity differences in the family of enzymes. Our structural and functional characterization of FosB^Ef establishes the enzyme as a potential target and may prove useful for future structure-based development of FosB inhibitors to increase the efficacy of fosfomycin.

SUBMITTER: Wiltsie V

PROVIDER: S-EPMC8862413 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Structural and functional characterization of fosfomycin resistance conferred by FosB from Enterococcus faecium.

Wiltsie Vanessa V Travis Skye S Shay Madeline R MR Simmons Zachary Z Frantom Patrick P Thompson Matthew K MK

Protein science : a publication of the Protein Society 20211222 3

The Gram-positive pathogen Enterococcus faecium is one of the leading causes of hospital-acquired vancomycin resistant enterococci (VRE) infections. E. faecium has extensive multidrug resistance and accounts for more than two million infections in the United States each year. FosB is a fosfomycin resistance enzyme found in Gram-positive pathogens like E. faecium. Typically, the FosB enzymes are Mn<sup>2+</sup> -dependent bacillithiol (BSH) transferases that inactivate fosfomycin through nucleoph ...[more]

PMID: 34882867

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Project description:The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M(2+)-dependent thiol tranferases that catalyze nucleophilic addition of either L-cysteine (L-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosB(Bc)). The overall structure of FosB(Bc), at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosB(Bc) cocrystallized with fosfomycin and a variety of divalent metals, including Ni(2+), Mn(2+), Co(2+), and Zn(2+), indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosB(Bc) structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate L-Cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel, positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosB(Bc) in complex with the L-Cys-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn(2+) metal in the active site have been determined. The L-Cys moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosB(Bc) indicated that the enzyme has a preference for BSH over L-Cys when activated by Mn(2+) and is inhibited by Zn(2+). The fact that Zn(2+) is an inhibitor of FosB(Bc) was used to obtain a ternary complex structure of the enzyme with both fosfomycin and L-Cys bound.

Project description:Vancomycin-resistant E. faecium (VREfm) is a significant public health concern because of limited treatment options. Genomic surveillance can be used to monitor VREfm transmission and evolution. Genomic analysis of VREfm has not been reported for the Dallas/Fort Worth/Arlington, TX, area, which is currently the 4th largest metropolitan area in the United States. Our study aimed to address this gap in knowledge by analyzing the genomes of 46 VREfm strains and 1 vancomycin-sensitive comparator collected during routine fecal surveillance of high-risk patients upon admission to a Dallas, TX, hospital system (August to October 2015). Thirty-one complete and 16 draft genome sequences were generated. The closed VREfm genomes possessed up to 12 extrachromosomal elements each. Overall, 251 closed putative plasmid sequences assigned to previously described and newly defined rep family types were obtained. Phylogenetic analysis identified 10 different sequence types (STs) among the isolates, with the most prevalent being ST17 and ST18. Strikingly, all but three of the VREfm isolates encoded vanA-type vancomycin resistance within Tn1546-like elements on a pRUM-like (rep17) plasmid backbone. Relative to a previously reported typing scheme for the vanA-carrying Tn1546, new variants of the Tn1546 were identified that harbored a combination of 7 insertion sequences (IS), including 3 novel IS elements reported here (ISEfa16, ISEfa17, and ISEfa18). We conclude that pRUM-like plasmids are important vectors for vancomycin resistance in the Dallas, TX, area and should be a focus of plasmid surveillance efforts. IMPORTANCE Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium. Among E. faecium strains, vancomycin resistance genes can be disseminated by plasmids with different host ranges and transfer efficiencies. Surveillance of resistance plasmids is critical to understanding antibiotic resistance transmission. This study analyzed the genome sequences of VREfm isolates collected from the Dallas, TX, area, with particular focus on the mobile elements associated with vancomycin resistance genes. We found that a single plasmid family, the pRUM-like family, was associated with vancomycin resistance in the majority of isolates sampled. Our work suggests that the pRUM-like plasmids should continue to be studied to understand their mechanisms of maintenance, transmission, and evolution in VREfm.

Dataset Information

Structural and functional characterization of fosfomycin resistance conferred by FosB from Enterococcus faecium.

Publications

Structural and functional characterization of fosfomycin resistance conferred by FosB from Enterococcus faecium.

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