Nutritional limitation of iron and methionine. Physiological consequences and new evasion strategies in staphylococci

Gram-positive cocci are a major cause of healthcare associated infections. Staphylococcus aureus and Stapylococcus lugdunensis represent prominent species. Additionally, Gram-negative bacteria as Pseudomonas aeruginosa and Escherichia coli frequently cause infections. With the emerge of multi drug resistant strains, the development of new antibacterial agents against Gram-positive and Gram-negative pathogens becomes increasingly urgent. To maintain proliferation, bacteria need to adapt to different parameters as e.g. varying nutrient availability. During invasive disease, the access to trace nutrients such as iron (Fe) is actively limited by the host. This strategy is referred to as “nutritional immunity”. Besides trace metals like Fe, several metabolites as e.g. the amino acid methionine, are essential for bacterial growth and their availability in the human host is scarce. During infection bacteria either need efficient acquisition systems or must rely on the de novo synthesis of such nutrients. Here we provide insight into the unique role of an iron regulated ABC transporter from the energy-coupling factor type (ECF) in S. lugdunensis which is involved in heme acquisition. We showed that the Lha transporter is specific for heme and recombinant substrate-specific protein LhaS accepts heme from various hemoproteins. By creating isogenic mutants and recombinant expression of Lha we showed that its function is independent of the well-studied heme acquisition system Isd and allows usage of human cells as a source of iron. Our investigations revealed a new strategy of nutritional heme acquisition to overcome host-induced iron limitation. Additionally we investigated the importance of the methionine biosynthesis pathway for bacterial growth and biofilm formation. We found methionine auxotrophic mutants of S. aureus, E. coli and P. aeruginosa to depend on exogenous concentrations of methionine exceeding those reported for human serum. Growth characteristics and biofilm formation was impaired in auxotrophic strains of S. aureus and P. aeruginosa. Our studies suggest primary metabolic pathways as interesting targets for antimicrobial therapy

Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

The emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products is Amycolatopsis. However, Amycolatopsis japonicum does not produce an antibiotic under standard laboratory conditions. In contrast to most Amycolatopsis strains, A. japonicum is genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, the bbr gene from Amycolatopsis balhimycina (bbr(Aba)), into A. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing of A. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed the in silico prediction that the recombinant A. japonicum/pRM4-bbr(Aba) synthesizes ristomycin A