Streptomyces coelicolor strains lacking polyprenol phosphate mannose synthase and protein O-mannosyl transferase are hyper-susceptible to multiple antibiotics

Abstract

Polyprenol phosphate mannose (PPM) is a lipid-linked sugar donor used by extra-cytoplasmic glycosyl tranferases in bacteria. PPM is synthesiszed 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 synthesize 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.

Genome-Wide Mutagenesis Links Multiple Metabolic Pathways with Actinorhodin Production in Streptomyces coelicolor

“…Mannosylation of the high-affinity phosphate-binding protein PstS by Pmt has been reported in Streptomyces (54), and insertions in SCO4142 ( pstS ) increased ACT production, suggesting that the decreased ACT production seen in the mannosylation mutants may be, at least in part, due to altered regulation of phosphate uptake. In addition, as a pmt mutant lost intrinsic functions of the cell envelope (55), the decreased ACT production of our pmt mutant may be caused by cell envelope damage.…”

Disruption of the GDP-mannose synthesis pathway in Streptomyces coelicolor results in antibiotic hyper-susceptible phenotypes

“…The synthesis of polyprenol phosphate mannose by Ppm1 is therefore an important activity and ppm1 mutants are considerably less fit than the parent strains [ 11, 12 ]. In the case of S. coelicolor, ppm1 - mutants have a small colony growth phenotype and are hyper-susceptible to multiple antibiotics, most of which inhibit cell wall biogenesis suggesting that these mutants are pleiotropically deficient in membrane and/or periplasmic function (Howlett et al , [ 4 ]). Mutants lacking Ppm1 or Pmt are also resistant to phage infection and we have proposed that φC31 uses a glycoprotein(s) as its receptor [ 6, 7 ].…”

“…We recently showed that strains of S. coelicolor lacking the ability to synthesise polyprenol phosphate mannose due to mutations in polyprenol phosphate mannose synthase (Ppm1) were hyper-sensitive to multiple antibiotics (Howlett et al ., [ 4 ]). We used RNA-seq and Raman spectroscopy to demonstrate that the strains had undergone changes to the membrane phospholipids, with possible subsequent changes to membrane functions.…”

“…One of these is a protein mannosyl transferase (Pmt), which glycosylates periplasmic and membrane proteins in Streptomyces [ 5, 6 ]. Pmt defective strains also show increased antibiotic susceptibility compared to the parent strain, but to fewer antibiotics and to a lower level than the ppm1 - mutants (Howlett et al [ 4 ]). Loss of protein glycosylation is therefore likely to contribute in part to the antibiotic hyper-susceptible phenotype of the ppm1 - mutants.…”

“…The protein O-glycosylation pathway is present in most Actinobacteria and Pmt in Mtb has been shown to be important for virulence [ 13, 14 ]. In Streptomyces coelicolor other putative glycosyl transferases are also likely to use polyprenol phosphate mannose as a sugar donor and some of these are described in Howlett et al [ 4 ].…”