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.

Antibiotic resistance is a global public health concern. The choline-based ionic liquids (ILs) have raised particular attention in the design of "greener" ILs and can exert a broad-spectrum of antimicrobial activity. To improve antimicrobial chemotherapy, we herein tested the antimicrobial activity and toxicity of a wide range of choline-based ILs. Two series of compounds were synthesized -dimethylethanolamine monoquaternary ammonium salts (Series A) and -methyl diethanolamine, diethanolamine and triethanolamine monoquaternary ammonium salts (Series B). The antimicrobial screening re-vealed that compounds N-(2-hydroxyethyl)-N,N-dimethyl-1-tetradecanaminium bromide ([N 1,1,14,2(OH) ]Br), N-(2-hydroxyethyl)-N,N-dimethyl-1-hexadecanaminium bromide ([N 1,1,16,2(OH) ]Br) and N-(2-hydroxyethyl)-N,N-dimethyl-1-octadecanaminium bromide ([N 1,1,18,2(OH) ]Br) are potent antimicrobial agents. The presence of a hydroxyethyl group and as mention previously in the literature, the C 14 to C 16 linker in a choline compound improves the antimicrobial activity and lowers the cytotoxic properties of this class of compounds.

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.

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.

Recently, two related chimeric genetic elements (Tn1207.3 and ⌽10394.4) were shown to carry the macrolide efflux gene mef in Streptococcus pyogenes (group A streptococci [GAS]). The dissemination of elements belonging to the Tn1207.3/⌽10394.4 family in recent isolates of GAS, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus pneumoniae, and Streptococcus agalactiae recovered in Portugal was surveyed. In total, 149 GAS, 18 S. pneumoniae, 4 S. dysgalactiae subsp. equisimilis, and 5 S. agalactiae isolates from infections, presenting the M phenotype of macrolide resistance and containing the mef gene, were screened for the presence of Tn1207.3/ ⌽10394.4 by PCR targeting open reading frames (ORFs) specific for these related elements. All the GAS isolates tested and one of the S. dysgalactiae subsp. equisimilis isolates carried Tn1207.3. However, neither of these elements was found in the isolates of the other streptococcal species. It was also noted that the DNAs of the isolates carrying Tn1207.3 were resistant to cleavage by the endonuclease SmaI. Cloning and expression of ORF12 of Tn1207.3 in Escherichia coli showed that it encoded a methyltransferase that rendered DNA refractory to cleavage by SmaI (M.Spy10394I). Using this characteristic as a marker for the presence of the Tn1207.3/⌽10394.4 family, we reviewed the literature and concluded that these genetic elements are widely distributed among tetracyclinesusceptible GAS isolates presenting the M phenotype from diverse geographic origins and may have played an important role in the dissemination of macrolide resistance in this species.

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 β-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.

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.

The number of cases of failure in the treatment of infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, 7α-Acetoxy-6β-hydroxyroyleanone (AHR), a diterpene isolated from different Plectranthus species, showed antibacterial activity, namely against Methicillin-resistant Staphylococcus aureus (MRSA) strains. The high antibacterial activity and low cytotoxicity render this natural compound an interesting alternative against resistant bacteria. The aim of this study is to understand the mechanism of action of AHR on MRSA, using the MRSA/Vancomycin-intermediate S. aureus (VISA) strain CIP 106760, and to study the AHR effect on lipid bilayers and on the cell wall. Although AHR interacted with lipid bilayers, it did not have a significant effect on membrane passive permeability. Alternatively, bacteria treated with this royleanone displayed cell wall disruption, without revealing cell lysis. In conclusion, the results gathered so far point to a yet undescribed mode of action that needs further investigation.