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Glycopeptide sulfation evades resistance.

ABSTRACT: The incidence of antibiotic resistance among pathogenic microorganisms is increasing at an alarming rate. Resistance against front-line therapeutics such as the glycopeptide antibiotic vancomycin has emerged and has spread to highly virulent pathogens, including Staphylococcus aureus. Glycopeptide antibiotics are natural products from the Actinomycetes that have a characteristic heptapeptide core. The chemical diversity of the class is achieved through glycosylation, halogenation, methylation, and acylation of the core, modifications that are implicated in improved solubility, stability, or activity of the molecule. Sulfation is yet another modification observed infrequently in glycopeptides, but its role is not known. Although glycopeptide sulfotransferases are found in the environmental metagenome and must therefore serve an evolutionary purpose, all previous studies have reported decreased antibiotic activity with sulfation. We report that sulfation of glycopeptides has little effect on the compound's ability to bind its target, the d-Ala-d-Ala peptidoglycan precursors of the bacterial cell wall. However, sulfation does impact glycopeptide dimerization, and importantly, sulfated glycopeptides are significantly less potent inducers of the resistance gene cluster vanHAX in actinomycetes. Our results begin to unravel the mystery of the biological role of glycopeptide sulfation and offer a potential new strategy for the development of new antibiotics that avoid resistance.

SUBMITTER: Kalan L

PROVIDER: S-EPMC3536170 | biostudies-other | 2013 Jan

REPOSITORIES: biostudies-other

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Glycopeptide sulfation evades resistance.

Kalan Lindsay L Perry Julie J Koteva Kalinka K Thaker Maulik M Wright Gerard G

Journal of bacteriology 20121026 1

The incidence of antibiotic resistance among pathogenic microorganisms is increasing at an alarming rate. Resistance against front-line therapeutics such as the glycopeptide antibiotic vancomycin has emerged and has spread to highly virulent pathogens, including Staphylococcus aureus. Glycopeptide antibiotics are natural products from the Actinomycetes that have a characteristic heptapeptide core. The chemical diversity of the class is achieved through glycosylation, halogenation, methylation, a ...[more]

PMID: 23104813

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Project description:To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over‑expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper‑mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. Our proteome and transcriptome analyses have been performed during stationary‑phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets. Keywords: Molecular markers, antibiotic resistance, glycopeptides, growth-phase

Project description:Glycopeptides are considered antibiotics of last resort for the treatment of life-threatening infections caused by relevant Gram-positive human pathogens, such as Staphylococcus aureus, Enterococcus spp. and Clostridium difficile. The emergence of glycopeptide-resistant clinical isolates, first among enterococci and then in staphylococci, has prompted research for second generation glycopeptides and a flurry of activity aimed at understanding resistance mechanisms and their evolution. Glycopeptides are glycosylated non-ribosomal peptides produced by a diverse group of soil actinomycetes. They target Gram-positive bacteria by binding to the acyl-D-alanyl-D-alanine (D-Ala-D-Ala) terminus of the growing peptidoglycan on the outer surface of the cytoplasmatic membrane. Glycopeptide-resistant organisms avoid such a fate by replacing the D-Ala-D-Ala terminus with D-alanyl-D-lactate (D-Ala-D-Lac) or D-alanyl-D-serine (D-Ala-D-Ser), thus markedly reducing antibiotic affinity for the cellular target. Resistance has manifested itself in enterococci and staphylococci largely through the expression of genes (named van) encoding proteins that reprogram cell wall biosynthesis and, thus, evade the action of the antibiotic. These resistance mechanisms were most likely co-opted from the glycopeptide producing actinomycetes, which use them to avoid suicide during antibiotic production, rather than being orchestrated by pathogen bacteria upon continued treatment. van-like gene clusters, similar to those described in enterococci, were in fact identified in many glycopeptide-producing actinomycetes, such as Actinoplanes teichomyceticus, which produces teicoplanin, and Streptomyces toyocaensis, which produces the A47934 glycopeptide. In this paper, we describe the natural and semi-synthetic glycopeptide antibiotics currently used as last resort drugs for Gram-positive infections and compare the van gene-based strategies of glycopeptide resistance among the pathogens and the producing actinomycetes. Particular attention is given to the strategy of immunity recently described in Nonomuraea sp. ATCC 39727. Nonomuraea sp. ATCC 39727 is the producer of A40926, which is the natural precursor of the second generation semi-synthetic glycopeptide dalbavancin, very recently approved for acute bacterial skin and skin structure infections. A thorough understanding of glycopeptide immunity in this producing microorganism may be particularly relevant to predict and eventually control the evolution of resistance that might arise following introduction of dalbavancin and other second generation glycopeptides into clinics.

Project description:Background: Vancomycin-intermediate Staphylococcus aureus (VISA) emerges typically in the healthcare-associated methicillin-resistant S. aureus and more rarely in community-acquired S. aureus (CA-MRSA). VISA is a serious concern for public health due to its association with persistent infections, the failure of vancomycin treatment, and poor clinical outcomes. Currently, the burden of VISA is somewhat high, even though vancomycin is the mainstay treatment for severe MRSA infections. The molecular mechanisms of reduced glycopeptide susceptibility in S. aureus are constantly under investigation but have still not yet been fully characterized. Methods: Our goal was to investigate the reduced glycopeptide susceptibility mechanisms emerging in a VISA CA-MRSA versus its vancomycin-susceptible (VSSA) CA-MRSA parents in a hospitalized patient undergoing glycopeptide treatment. Comparative integrated omics, Illumina MiSeq whole-genome sequencing (WGS), RNA-Seq, and bioinformatics were performed. Results: Through a comparison of VISA CA-MRSA vs. its VSSA CA-MRSA parent, mutational and transcriptomic adaptations were found in a pool of genes involved, directly or indirectly, in the biosynthesis of the glycopeptide target conferring or supporting the VISA phenotype, and its cross-resistance with daptomycin. This pool included key genes responsible for the biosynthesis of the peptidoglycan precursors, i.e., D-Ala, the D-Ala-D-Ala dipeptide termini of the pentapeptide, and its incorporation in the nascent pentapeptide, as key targets of the glycopeptide resistance. Furthermore, accessory glycopeptide-target genes involved in the pathways corroborated the key adaptations, and thus, supported the acquisition of the VISA phenotype i.e., transporters, nucleotide metabolism genes, and transcriptional regulators. Finally, transcriptional changes were also found in computationally predicted cis-acting small antisense RNA triggering genes related both to the key or accessory adaptive pathways. Conclusion: Our investigation describes an adaptive resistance pathway acquired under antimicrobial therapy conferring reduced glycopeptide susceptibility in a VISA CA-MRSA due to a comprehensive network of mutational and transcriptional adaptations in genes involved in pathways responsible for the biosynthesis of glycopeptide's target or supporters of the key resistance path.

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Glycopeptide sulfation evades resistance.

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Glycopeptide sulfation evades resistance.

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