Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

Abstract

Inhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors in Staphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as ␣-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated with S. aureus pathogenicity. Consequently, S. aureus FMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-type S. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection. In bacteria, protein synthesis initiates with formyl-methionyltRNAi, and therefore, all newly synthesized polypeptides contain an N-formyl-methionine terminal end that, in most cases, is not retained in mature proteins. As the polypeptides emerge from the ribosome, the N-formyl group is hydrolyzed by peptide deformylase (PDF), and subsequently, methionine aminopeptidase removes the N-terminal methionine. Deformylation plays a crucial role in protein maturation and has been shown to be essential for bacterial growth (1-4), as methionine aminopeptidase cannot hydrolyze N-blocked peptides (5). The ubiquitous nature of PDF, together with the fact that this function is not required for cytoplasmic protein synthesis in eukaryotes, has made this an attractive target for the development of new antibacterial agents. The successful purification and characterization of the native form of Escherichia coli PDF (6), as well as the discovery that the natural product actinonin is an inhibitor of PDF with weak antibacterial activity (7-9), triggered the search for additional PDF inhibitors. Since then, a large number of structurally diverse PDF inhibitors have been identified, including several compounds with demonstrated in vivo efficacy and good safety profiles (10). Three PDF inhibitors have progressed to clinical trials (11, 12), and one of them, GSK1322322, is in phase II clinical development for the treatment of respiratory tract and skin infections.While mutations in FolD and GlyA, two enzymes involved in the synthesis of 10-formyl-tetrahydrofolate, have been described (13, 14), loss-of-function mutations in the gene encoding formylmethionyl transferase (FMT), the enzyme that catalyzes the formylation of the initiator methionyl-tRNA, are the most common cause of resistance to PDF inhibito...

Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor GSK1322322 in Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae

“…Therefore, although resistance to GSK1322322 can occur through mutations in the FMT protein in S. pyogenes and even S. pneumoniae, the mutations are associated with severe in vitro fitness costs, and it can be anticipated that the mutants would not be able to subsist under more challenging in vivo conditions. In addition, although the loss of FMT function in S. aureus does not seem to have a pronounced fitness cost in vitro, recent studies have demonstrated that FMT mutants are nonhemolytic and cannot cause productive infections in animal models (20). Mutations in the PDF protein, in contrast, could be expected to yield fitter stable mutants.…”

“…Plates were incubated at 35°C, and growth and resistance were evaluated at 24 and 48 h. In cases in which a large number of mutants were obtained, only a representative set of colonies was tested. In the cases of S. aureus and S. pyogenes, colonies were also analyzed for production of hemolysin in blood agar plates, as it is known that S. aureus fmt mutants are nonhemolytic (20).…”

“…aureus. Mutations in the S. aureus fmt gene are associated with a number of phenotypic features, including lack of hemolysin production, high-level resistance to PDF inhibitors, a small-colony phenotype, and hypersensitivity to pleuromutilins (20). Therefore, in order to easily identify FMT mutants, FoR studies were performed with four methicillin-resistant hemolytic S. aureus strains.…”

“…Such mutants are highly resistant to PDF inhibitors, but they display compromised in vitro (12,13,(15)(16)(17)(18) and in vivo (12,16,20) growth. In Staphylococcus aureus, FMT mutants also show drastic reductions in the production of virulence factors and restricted ability to produce invasive infections (20).…”

“…Such mutants are highly resistant to PDF inhibitors, but they display compromised in vitro (12,13,(15)(16)(17)(18) and in vivo (12,16,20) growth. In Staphylococcus aureus, FMT mutants also show drastic reductions in the production of virulence factors and restricted ability to produce invasive infections (20). In organisms in which formylation seems to be essential, such as Streptococcus pneumoniae and Haemophilus influenzae, resistance to PDF inhibitors involves mutations in, or increased production of, the target gene (21)(22)(23) or efflux (24).…”