RESUMEN
Staphylococcus aureus can cause both acute and recurrent persistent infections such as peritonitis, endocarditis, abscesses, osteomyelitis, and chronic wound infections. Effective therapies to treat persistent disease are paramount. However, the mechanisms of S. aureus persistence are poorly understood. In this study, we performed a comprehensive and unbiased high-throughput mutant screen against a transposon-insertion mutant library of S. aureus USA300 and focused on the role of argJ encoding an acetyltransferase in the arginine biosynthesis pathway, whose transposon insertion caused a significant defect in persister formation using multiple drugs and stresses. Genetic complementation and arginine supplementation restored persistence in the argJ transposon insertion mutant while generation of mutations on the active site of the ArgJ protein caused a defect in persistence. Quantitative RT-PCR analysis showed that the genes encoded in the arg operon were over-expressed under drug stressed conditions and in stationary phase cultures. In addition, the argJ mutant had attenuated virulence in both mouse and C. elegans. Our studies identify a new mechanism of persistence mediated by arginine metabolism in S. aureus. These findings provide not only novel insights about the mechanisms of S. aureus persistence but also offer novel therapeutic targets that may help to develop more effective treatment of persistent S. aureus infections.
Asunto(s)
Acetiltransferasas/genética , Proteínas Bacterianas/genética , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Infecciones Estafilocócicas/microbiología , Factores de Virulencia/genética , Acetiltransferasas/metabolismo , Animales , Antibacterianos/farmacología , Arginina/biosíntesis , Proteínas Bacterianas/metabolismo , Vías Biosintéticas/genética , Caenorhabditis elegans , Elementos Transponibles de ADN/genética , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Femenino , Biblioteca de Genes , Genes Bacterianos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Staphylococcus aureus Resistente a Meticilina/enzimología , Staphylococcus aureus Resistente a Meticilina/genética , Ratones , Mutación/efectos de los fármacos , Virulencia/efectos de los fármacos , Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Familial amyotrophic lateral sclerosis (FALS) has been modeled in transgenic mice by introducing mutated versions of human genomic DNA encompassing the entire gene for Cu,Zn superoxide dismutase (SOD1). In this setting, the transgene is expressed throughout the body and results in mice that faithfully recapitulate many pathological and behavioral aspects of FALS. By contrast, transgenic mice made by introducing recombinant vectors, encoding cDNA genes, that target mutant SOD1 expression to motor neurons, only, or astrocytes, only, do not develop disease. Here, we report that mice transgenic for human SOD1 cDNA with the G37R mutation, driven by the mouse prion promoter, develop motor neuron disease. In this model, expression of the transgene is highest in CNS (both neurons and astrocytes) and muscle. The gene was not expressed in cells of the macrophage lineage. Although the highest expressing hemizygous transgenic mice fail to develop disease by 20 months of age, mice homozygous for the transgene show typical ALS-like phenotypes as early as 7 months of age. Spinal cords and brain stems from homozygous animals with motor neuron disease were found to contain aggregated species of mutant SOD1. The establishment of this SOD1-G37R cDNA transgenic model indicates that expression of mutant SOD1 proteins in the neuromuscular unit is sufficient to cause motor neuron disease. The expression levels required to induce disease coincide with the levels required to induce the formation of SOD1 aggregates.