RESUMEN
Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to ß-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/química , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/metabolismo , Dominios Proteicos/fisiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/genética , Cromatografía Líquida de Alta Presión , Mutagénesis Sitio-Dirigida , Peptidoglicano/metabolismo , Dominios Proteicos/genética , Estabilidad Proteica , Staphylococcus aureus/genéticaRESUMEN
Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.
Asunto(s)
Antranilato Sintasa/metabolismo , Antranilato Sintasa/ultraestructura , Transferasas de Grupos Nitrogenados/metabolismo , Transferasas de Grupos Nitrogenados/ultraestructura , Staphylococcus aureus/enzimología , Antibacterianos/análisis , Proteínas Bacterianas/análisis , Ligasas de Carbono-Nitrógeno , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/metabolismo , Dominio Catalítico , Pared Celular/química , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Bacterias Grampositivas , Complejos Multienzimáticos , Peptidoglicano/química , Infecciones Estafilocócicas , Staphylococcus aureus/metabolismoRESUMEN
The enzymes responsible for peptidoglycan amidation in Staphylococcus aureus, MurT and GatD, were recently identified and shown to be required for optimal expression of resistance to beta-lactams, bacterial growth, and resistance to lysozyme. In this study, we analyzed the impact of peptidoglycan amidation in representative strains of the most widespread clones of methicillin resistant S. aureus (MRSA). The inhibition of the expression of murT-gatD operon resulted in different phenotypes of resistance to beta-lactams and lysozyme according to the different genetic backgrounds. Further, clonal lineages CC1 and CC398 (community-acquired MRSA [CA-MRSA]) showed a stronger dependency on MurT-GatD for resistance to beta-lactams, when compared to the impact of the impairment of the cell wall step catalyzed by MurF. In the remaining backgrounds similar phenotypes of beta-lactam resistance were observed upon the impairment of both cell-wall-related genes. Therefore, for CA-related backgrounds, the predominant beta-lactam resistance mechanism seems to involve genes associated with secondary modifications of peptidoglycan. On the other hand, the lack of glutamic acid amidation had a more substantial impact on lysozyme resistance for cells of CA-MRSA backgrounds, than for hospital-acquired MRSA (HA-MRSA). However, no significant differences were found in the resistance level of the respective peptidoglycan structure, suggesting that the lysozyme resistance mechanism involves other factors. Taken together, these results suggested that the different genetic lineages of MRSA were able to develop different molecular strategies to overcome the selective pressures experienced during evolution.
Asunto(s)
Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Muramidasa/farmacología , Péptido Sintasas/metabolismo , Peptidoglicano/metabolismo , beta-Lactamas/farmacología , Adaptación Fisiológica/genética , Amidas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Transporte Biológico , Pared Celular/química , Pared Celular/efectos de los fármacos , Pared Celular/genética , Pared Celular/metabolismo , Expresión Génica , Redes y Vías Metabólicas , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Pruebas de Sensibilidad Microbiana , Operón , Péptido Sintasas/genéticaRESUMEN
In this communication, we describe evidence demonstrating the capacity of Atl, the major Staphylococcus aureus autolytic enzyme to bind DNA. Electrophoretic mobility shift assays (EMSA) show that both the Atl protein and the endo-ß-N-acetylglucosaminidase (GL) domain were able to bind DNA of nonspecific sequence. The implications of this unexpected observation for the physiology of S. aureus remain to be explored.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Hexosaminidasas/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Staphylococcus aureus/enzimología , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Unión ProteicaRESUMEN
The glutamic acid residues of the peptidoglycan of Staphylococcus aureus and many other bacteria become amidated by an as yet unknown mechanism. In this communication we describe the identification, in the genome of S. aureus strain COL, of two co-transcribed genes, murT and gatD, which are responsible for peptidoglycan amidation. MurT and GatD have sequence similarity to substrate-binding domains in Mur ligases (MurT) and to the catalytic domain in CobB/CobQ-like glutamine amidotransferases (GatD). The amidation of glutamate residues in the stem peptide of S. aureus peptidoglycan takes place in a later step than the cytoplasmic phase--presumably the lipid phase--of the biosynthesis of the S. aureus cell wall precursor. Inhibition of amidation caused reduced growth rate, reduced resistance to beta-lactam antibiotics and increased sensitivity to lysozyme which inhibited culture growth and caused degradation of the peptidoglycan.
Asunto(s)
Genes Bacterianos/genética , Ácido Glutámico/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Northern Blotting , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A murF conditional mutant was used to evaluate the effect of suboptimal transcription of this gene on the transcriptome of the methicillin-resistant Staphylococcus aureus strain COL. The mutant was grown in the presence of optimal and suboptimal concentrations of the inducer, and the relative levels of transcription of genes were evaluated genome wide with an Affymetrix DNA microarray that included all open reading frames (ORFs) as well as intergenic sequences derived from four sequenced S. aureus strains. Using a sensitivity threshold value of 1.5, suboptimal expression of murF altered the transcription of a surprisingly large number of genes, i.e., 668 out of the 2,740 ORFs (close to one-fourth of all ORFs), of the genome of S. aureus strain COL. The genes with altered transcription were distributed evenly around the S. aureus chromosome, and groups of genes involved with distinct metabolic functions responded in unique and operon-specific manners to modulation in murF transcription. For instance, all genes belonging to the isd operon and all but 2 of the 35 genes of prophage L54a were down-regulated, whereas all but one of the 21 members of the vraSR regulon and most of the 79 virulence-related genes (those for fibronectin binding proteins A and B, clumping factor A, gamma hemolysin, enterotoxin B, etc.) were up-regulated in cells with suboptimal expression of murF. Most importantly, the majority of these altered gene expression profiles were reversible by resupplying the optimal concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) to the culture. The observations suggest the coordinate regulation of a large sector of the S. aureus transcriptome in response to a disturbance in cell wall synthesis.