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

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1- mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1- strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.

Real-time bioluminescence imaging of mixed mycobacterial infections.

Molecular analysis of infectious processes in bacteria normally involves construction of isogenic mutants that can then be compared to wild type in an animal model. Pathogenesis and antimicrobial studies are complicated by variability between animals and the need to sacrifice individual animals at specific time points. Live animal imaging allows real-time analysis of infections without the need to sacrifice animals, allowing quantitative data to be collected at multiple time points in all organs simultaneously. However, imaging has not previously allowed simultaneous imaging of both mutant and wild type strains of mycobacteria in the same animal. We address this problem by using both firefly (Photinus pyralis) and click beetle (Pyrophorus plagiophthalamus) red luciferases, which emit distinct bioluminescent spectra, allowing simultaneous imaging of two different mycobacterial strains during infection. We also demonstrate that these same bioluminescence reporters can be used to evaluate therapeutic efficacy in real-time, greatly facilitating our ability to screen novel antibiotics as they are developed. Due to the slow growth rate of mycobacteria, novel imaging technologies are a pressing need, since they can they can impact the rate of development of new therapeutics as well as improving our understanding of virulence mechanisms and the evaluation of novel vaccine candidates.