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Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium.

ABSTRACT: Multidrug-resistant (MDR) bacteria have become a growing threat to public health. The gram-positive Enterococcus faecium is classified by WHO as a high-priority pathogen among the global priority list of antibiotic-resistant bacteria. Peptidoglycan-degrading enzymes (PDEs), also known as enzybiotics, are useful bactericidal agents in the fight against resistant bacteria. In this work, a genome-based screening approach of the genome of E. faecium allowed the identification of a putative PDE gene with predictive amidase activity (EfAmi1; EC 3.5.1.28) in a prophage-integrated sequence. EfAmi1 is composed by two domains: a N-terminal Zn²⁺-dependent N-acetylmuramoyl-L-alanine amidase-2 (NALAA-2) domain and a C-terminal domain with unknown structure and function. The full-length gene of EfAmi1 was cloned and expressed as a 6xHis-tagged protein in E. coli. EfAmi1 was produced as a soluble protein, purified, and its lytic and antimicrobial activities were investigated using turbidity reduction and Kirby-Bauer disk-diffusion assays against clinically isolated bacterial pathogens. The crystal structure of the N-terminal amidase-2 domain was determined using X-ray crystallography at 1.97 Å resolution. It adopts a globular fold with several α-helices surrounding a central five-stranded β-sheet. Sequence comparison revealed a cluster of conserved amino acids that defines a putative binding site for a buried zinc ion. The results of the present study suggest that EfAmi1 displays high lytic and antimicrobial activity and may represent a promising new antimicrobial in the post-antibiotic era.

SUBMITTER: Premetis GE

PROVIDER: S-EPMC10167349 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium.

Premetis Georgios E GE Stathi Angeliki A Papageorgiou Anastassios C AC Labrou Nikolaos E NE

Scientific reports 20230508 1

Multidrug-resistant (MDR) bacteria have become a growing threat to public health. The gram-positive Enterococcus faecium is classified by WHO as a high-priority pathogen among the global priority list of antibiotic-resistant bacteria. Peptidoglycan-degrading enzymes (PDEs), also known as enzybiotics, are useful bactericidal agents in the fight against resistant bacteria. In this work, a genome-based screening approach of the genome of E. faecium allowed the identification of a putative PDE gene ...[more]

PMID: 37156923

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Project description:A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faecium clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faecium and Escherichia coli, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutans SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes (41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagA gene, like the S. mutans homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sagA gene is essential for E. faecium growth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.

Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Dataset Information

Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium.

Publications

Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium.

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