RESUMEN
Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.
Asunto(s)
Proteínas Bacterianas/inmunología , Metaloendopeptidasas/inmunología , Fosfotransferasas (aceptor de Grupo Carboxilo)/inmunología , Serina Endopeptidasas/inmunología , Úlcera Cutánea/inmunología , Infecciones Cutáneas Estafilocócicas/inmunología , Staphylococcus aureus/patogenicidad , Animales , Carga Bacteriana , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Quimiocina CCL4/genética , Quimiocina CCL4/inmunología , Femenino , Regulación de la Expresión Génica , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-1alfa/genética , Interleucina-1alfa/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Ratones , Fosfotransferasas (aceptor de Grupo Carboxilo)/deficiencia , Fosfotransferasas (aceptor de Grupo Carboxilo)/genética , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Transducción de Señal , Piel/inmunología , Piel/microbiología , Piel/patología , Úlcera Cutánea/genética , Úlcera Cutánea/microbiología , Úlcera Cutánea/patología , Infecciones Cutáneas Estafilocócicas/genética , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/patología , Staphylococcus aureus/enzimología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factores de Virulencia/genética , Factores de Virulencia/inmunologíaRESUMEN
OBJECTIVE: In order to optimize precursor supply for L-arginine biosynthesis, we constructed a Corynebacterium crenatum 8-193 mutant with gamma-glutamyl kinase gene (proB) in-frame deletion. The effects of proB knock-out on physiological characteristics of the mutant were investigated. METHODS: The upstream and downstream fragments of proB were cloned from C. crenatum 8-193 chromosome and ligated to integration vector. The mutant C. crenatum 8-193-deltaproB was obtained by homologous recombination. The mutant phenotype can be reversed by complementation with proB gene from the expression vector. The physiological characteristics of the mutant were investigated by measurement of the activities of phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PYC). RESULTS: The proB gene in-frame deletion was screened and confirmed by PCR, gamma-glutamyl kinase determination and complementation. The mutant lost the ability of growth on minimal medium without proline addition. The proB knock-out mutant resulted a decrease of cell mass by 9.6% and an increase of L-arginine accumulation by 13.6% compared with that of the parent strain. The analysis of by-products of fermentation broth showed that the concentrations of glutamate-related and aspartate-related amino acids increased, and the concentrations of alpha-ketoglutaric acid, PEP and succinic acid decreased. The specific activities of PEPCx and PYC increased in 8-193-deltaproB. CONCLUSION: The proB gene knock-out of the strain 8-193 blocked branch catabolism of L-glutamate and improved efficiency of the glucose utilization and L-arginine accumulation.