Ramoplanin is a lipoglycodepsipeptide antimicrobial active against clinically important Gram-positive bacteria including methicillin resistant Staphylococcus aureus. To proactively examine ramoplanin resistance, we subjected S. aureus NCTC 8325-4 to serial passage in the presence of increasing concentrations of ramoplanin, generating the markedly resistant strain RRSA16. Susceptibility testing of RRSA16 revealed the unanticipated acquisition of cross-resistance to vancomycin and nisin. RRSA16 displayed phenotypes, including a thickened cell wall and reduced susceptibility to Triton X-100 induced autolysis, which are associated with vancomycin intermediate resistant S. aureus strains. Passage of RRSA16 for 18 days in drug-free medium yielded strain R16-18d with restored antibiotic susceptibility. The RRSA16 isolate may be used to identify the genetic and biochemical basis for ramoplanin-resistance and further our understanding of the evolution of antibiotic cross-resistance mechanisms in S. aureus.

Gram-positive pathogens possess external pili or fimbrae with which they adhere to host cells during the infection process. Unusual dual intramolecular isopeptide bonds between Asn and Lys side chains within the N-terminal and C-terminal domains of the pilus subunits have been observed initially in the Streptococcus pyogenes pilin subunit Spy0128 and subsequently in GBS52 from Streptococcus agalactiae, in the BcpA major pilin of Bacillus cereus and in the RrgB pilin of Streptococcus pneumoniae among others. Within each pilin subunit, intramolecular isopeptide bonds serve to stabilize the protein. These bonds provide a means to withstand large external mechanical forces, as well as possibly assisting in supporting a conformation favored for pilin subunit polymerization via sortase transpeptidases. Genome-wide analyses of pili-containing Gram-positive bacteria are known or suspected to contain isopeptide bonds in pilin subunits. For the autocatalytic formation of isopeptide crosslinks, a conservation of three amino acids including Asn, Lys, and a catalytically important acidic Glu (or Asp) residue are responsible. However, the chemical mechanism of how isopeptide bonds form within pilin remains poorly understood. Although it is possible that several mechanistic paths could lead to isopeptide bond formation in pili, the requirement of a conserved glutamate and highly organized positioning of residues within the hydrophobic environment of the active site were found in numerous pilin crystal structures such as Spy0128 and RrgB. This suggests a mechanism involving direct coupling of lysine side chain amine to the asparagine carboxamide mediated by critical acid/base or hydrogen bonding interactions with the catalytic glutamate residue. From this mechanistic perspective, we used the QM/MM minimum free-energy path method to examine the reaction details of forming the isopeptide bonds with the Spy0128 as a model pilin, specifically focusing on the role of the glutamate in catalysis. It was determined that the reaction mechanism likely consists of two major steps: the nucleophilic attack on Cγ by nitrogen in the unprotonated Lys ε-amino group and then two concerted proton transfers occur during the formation of the intramolecular isopeptide bond to subsequently release ammonia. More importantly, within the dual active sites of Spy0128, Glu117 and Glu258 residues function as crucial catalysts for each isopeptide bond formation, respectively, by relaying two proton transfers. This study also suggests that domain-domain interactions within Spy0128 may modulate the reactivity of residues within each active site. Our results may hopefully shed light on the molecular mechanisms of pilin biogenesis in Gram-positive bacteria.

The mechanism of oxidation of a peptide substrate by the flavoprotein lysine-specific demethylase (LSD1) has been examined using the effects of pH and isotopic substitution on steady-state and rapid-reaction kinetic parameters. The substrate contained the N-terminal 21 residues of histone H3, with a dimethylated lysyl residue at position 4. At pH 7.5, the rate constant for flavin reduction, kred, equals kcat, establishing the reductive half reaction as rate-limiting at physiological pH. Deuteration of the lysyl methyls results in identical kinetic isotope effects of 3.1 ± 0.2 on the kred, kcat and kcat/Km values for the peptide, establishing CH bond cleavage as rate-limiting with this substrate. No intermediates between oxidized and reduced flavin are detectable by stopped-flow spectroscopy, consistent with the expectation for a direct hydride transfer mechanism. The kcat/Km value for the peptide is bell-shaped, consistent with a requirement that the nitrogen at the site of oxidation be uncharged and that at least one of the other lysyl residues be charged for catalysis. The D(kcat/Km) value for the peptide is pH-independent, suggesting that the observed value is the intrinsic deuterium kinetic isotope effect for oxidation of this substrate.

cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile. 4-Vinylsulfonyl 5-phenyl prolinates inhibit S. aureus sortase SrtA irreversibly by modification of the enzyme Cys184 and could be used as hits for the development of antibacterials and antivirulence agents.

NOD2 (the nucleotide-binding oligomerization domain containing protein 2) is known to be involved in host recognition of bacteria, although its role in the host response to Staphylococcus aureus infection is unknown. NOD2-deficient (Nod2−/−) mice and wild-type (WT) littermate controls were injected intraperitoneally with S. aureus suspension (107 bacteria/g of body weight), and their survival was monitored. Cultured bone marrow-derived neutrophils were harvested from Nod2−/− and WT mice and tested for cytokine production and phagocytosis. Compared to WT mice, Nod2−/− mice were significantly more susceptible to S. aureus infection (median survival of 1.5 days versus >5 days; P = 0.003) and had a significantly higher bacterial tissue burden. Cultured bone marrow-derived neutrophils from Nod2−/− and WT mice had similar levels of peritoneal neutrophil recruitment and intracellular killing, but bone marrow-derived neutrophils from Nod2−/− mice had significantly reduced ability to internalize fluorescein-labeled S. aureus. Nod2−/− mice had significantly higher levels of Th1-derived cytokines in serum (tumor necrosis factor alpha, gamma interferon, and interleukin-2 [IL-2]) compared to WT mice, whereas the levels of Th2-derived cytokines (IL-1β, IL-4, IL-6, and IL-10) were similar in Nod2−/− and WT mice. Thus, mice deficient in NOD2 are more susceptible to S. aureus. Increased susceptibility is due in part to defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden.

A facile synthetic route to substituted trans-2-arylcyclopropylamines was developed to provide access to mechanism-based inhibitors of the human flavoenzyme oxidase lysine-specific histone demethylase LSD1 and related enzyme family members such as monoamine oxidases A and B.