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Streptomyces Coelicolor Strains Lacking Polyprenol Phosphate Mannose Synthase and Protein O-Mannosyl Transferase Are Hyper-Susceptible to Multiple Antibiotics

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Streptomyces Coelicolor Strains Lacking Polyprenol Phosphate Mannose Synthase and Protein O-Mannosyl Transferase Are Hyper-Susceptible to Multiple Antibiotics This is a repository copy of Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/128020/ Version: Accepted Version Article: Howlett, Robert, Read, Nicholas, Varghese, Anpu S. et al. (3 more authors) (2018) Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics. Microbiology. pp. 369-382. ISSN 1465-2080 https://doi.org/10.1099/mic.0.000605 Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. 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[email protected] https://eprints.whiterose.ac.uk/ Microb iology Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics --Manuscript Draft-- Manuscript Number: MIC-D-17-00343R1 Full Title: Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics Article Type: Research Article Section/Category: Physiology and metabolism Corresponding Author: Margaret CM Smith, PhD University of Leeds Leeds, West Yorkshire UNITED KINGDOM First Author: Robert Howlett Order of Authors: Robert Howlett Nicholas Read Anpu Varghese Charles Kershaw Yvette Hancock Margaret CM Smith, PhD Abstract: Polyprenol phosphate mannose (PPM) is a lipid linked sugar donor used by extra- cytoplasmic glycosyl tranferases in bacteria. PPM is synthesised by polyprenol phosphate mannose synthase, Ppm1, and in most Actinobacteria is used as the sugar donor for protein O-mannosyl transferase, Pmt, in protein glycosylation. Ppm1 and Pmt have homologues in yeasts and humans, where they are required for protein O- mannosylation. Actinobacteria also use PPM for lipoglycan biosynthesis. Here we show that ppm1 mutants of Streptomyces coelicolor have increased susceptibility to a number of antibiotics that target cell wall biosynthesis. The pmt mutants also have mildly increased antibiotic susceptibilities, in particular to β-lactams and vancomycin. Despite normal induction of the vancomycin gene cluster, vanSRJKHAX, the pmt and ppm1 mutants remained highly vancomycin sensitive indicating that the mechanism of resistance is blocked post-transcriptionally. Differential RNA expression analysis indicated that catabolic pathways were downregulated and anabolic ones upregulated in the ppm1 mutant compared to the parent or complemented strains. Of note was the increase in expression of fatty acid biosynthetic genes in the ppm1- mutant. A change in lipid composition was confirmed using Raman spectroscopy, which showed that the ppm1- mutant had a greater relative proportion of unsaturated fatty acids compared to the parent or the complemented mutant. Taken together these data suggest that an inability to synthesise PPM (ppm1-) and loss of the glycoproteome (pmt- mutant) can detrimentally affect membrane or cell envelope functions leading to loss of intrinsic and, in the case of vancomycin, acquired antibiotic resistance. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document) 1 Streptomyces coelicolor strains lacking polyprenol phosphate mannose 2 synthase and protein O-mannosyl transferase are hyper-susceptible to multiple 3 antibiotics 4 Robert Howletta,¶, Nicholas Reada,¶, Anpu Vargheseb, Charles Kershawc, Y. 5 Hancockc,d and Margaret C. M. Smitha,b, # 6 7 ¶ Joint first authors. 8 9 a Department of Biology, University of York, York, United Kingdom; 10 b Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom; 11 c Department of Physics, University of York, York, United Kingdom; 12 d York Centre for Complex Systems Analysis, University of York, York, United 13 Kingdomd 14 15 Running Title: Antibiotic hypersensitive mutants of S. coelicolor 16 Abbreviations; PPM; polyprenol phosphate mannose, Ppm1; polyprenol phosphate 17 mannose synthase, Pmt; protein mannosyl transferase, TUFA; total unsaturated fatty 18 acids, TFA; total fatty acids. 19 20 21 22 #Address correspondence to Margaret C.M. Smith, [email protected] 23 RH and NR contributed equally to this work. 24 25 1 26 Abstract 27 Polyprenol phosphate mannose (PPM) is a lipid linked sugar donor used by extra- 28 cytoplasmic glycosyl tranferases in bacteria. PPM is synthesised by polyprenol 29 phosphate mannose synthase, Ppm1, and in most Actinobacteria is used as the 30 sugar donor for protein O-mannosyl transferase, Pmt, in protein glycosylation. Ppm1 31 and Pmt have homologues in yeasts and humans, where they are required for 32 protein O-mannosylation. Actinobacteria also use PPM for lipoglycan biosynthesis. 33 Here we show that ppm1 mutants of Streptomyces coelicolor have increased 34 susceptibility to a number of antibiotics that target cell wall biosynthesis. The pmt 35 mutants also have mildly increased antibiotic susceptibilities, in particular to - 36 lactams and vancomycin. Despite normal induction of the vancomycin gene cluster, 37 vanSRJKHAX, the pmt and ppm1 mutants remained highly vancomycin sensitive 38 indicating that the mechanism of resistance is blocked post-transcriptionally. 39 Differential RNA expression analysis indicated that catabolic pathways were 40 downregulated and anabolic ones upregulated in the ppm1 mutant compared to the 41 parent or complemented strains. Of note was the increase in expression of fatty acid 42 biosynthetic genes in the ppm1- mutant. A change in lipid composition was confirmed 43 using Raman spectroscopy, which showed that the ppm1- mutant had a greater 44 relative proportion of unsaturated fatty acids compared to the parent or the 45 complemented mutant. Taken together these data suggest that an inability to 46 synthesise PPM (ppm1-) and loss of the glycoproteome (pmt- mutant) can 47 detrimentally affect membrane or cell envelope functions leading to loss of intrinsic 48 and, in the case of vancomycin, acquired antibiotic resistance. 49 50 2 51 Introduction 52 53 In the Actinobacteria, mannose is an important component of extracellular 54 glycoconjugates including lipoglycans and glycoproteins [1, 2]. Lipomannan (LM) 55 and lipoarabinomannan (LAM) are essential constituents of the cell envelope in 56 mycobacteria [3] and mutants of corynebacteria that lack these molecules grow 57 poorly [4]. Membrane and secreted proteins are also modified by mannose residues 58 in the Actinobacteria and species of Mycobacterium, Corynebacterium and 59 Streptomyces have been shown to contain glycoproteins [5-9]. Extra-cytoplasmic 60 glycosyl transferases require a lipid linked sugar donor, which for the transfer of 61 mannose is polyprenol phosphate mannose (PPM). The enzyme polyprenol 62 phosphate mannose synthase, Ppm1, synthesises PPM by catalysing the transfer of 63 mannose from GDP-mannose to polyprenol phosphate on the cytoplasmic face of the 64 plasma membrane and PPM is then flipped in the membrane, transporting the 65 mannose moiety to the periplasm [10] (Fig.1). In mycobacteria, Ppm1, is an essential 66 enzyme, as it is required for lipomannan biosynthesis [11] and Corynebacterium 67 ppm1 mutants have a reduced growth rate [12]. 68 69 We are interested in the role of protein O-glycosylation in bacteria and ultimately we 70 aim to understand how the glycans affect protein function. The protein O- 71 glycosylation pathway in the Actinobacteria is highly reminiscent of the O- 72 mannosylation pathway in yeast and humans [2, 9, 13]. In fungi, mutations that 73 inactivate PMTs lead to loss of fitness and/or avirulent phenotypes [14-19]. In 74 humans loss of PoMT leads to a developmental phenotype [20]. Overall the evidence 3 75 suggests that protein O-mannosylation is a fundamentally important protein 76 modification in prokaryotes and eukaryotes. 77 78 S. coelicolor is a convenient model to study the protein O-glycosylation pathway in 79 bacteria as the pmt and ppm1 genes are not essential [21, 22] and Streptomyces 80 spp. are not known to generate the heavily glycosylated lipomannans and 81 lipoarabinomannans that depend on the activity of Ppm1. In previous work we 82 showed that S. coelicolor Ppm1 (SCO1423) catalyses the transfer of mannose from 83 GDP-mannose to polyprenol phosphate to generate PPM and Pmt (SCO3154) was 84 shown to transfer mannose from PPM to target peptides [9] (Fig.1). Ppm1 and Pmt 85 activities are confined to the membrane fractions. The periplasmic phosphate binding 86 protein, PstS, part of a high affinity phosphate uptake system, was shown to be O- 87 glycosylated with a trihexose [9]. The enzymes Pmt and Ppm1 were first identified as 88 being required for phage ϕC31 infection, suggesting the phage receptor
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