Lif, the lysostaphin immunity factor, complements FemB in staphylococcal peptidoglycan interpeptide bridge formation

The formation of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide chain needs FemA and FemB for the incorporation of glycines Gly2-Gly3, and Gly4-Gly5, respectively. The lysostaphin immunity factor Lif was able to complement FemB, as could be shown by serine incorporation and by an increase in lysostaphin resistance in the wild-type as well as in a femB mutant. However, Lif could not substitute for FemA in femA or in femAB-null mutants. Methicillin resistance, which is dependent on functional FemA and FemB, was not complemented by Lif, suggesting that serine-substituted side chains are a lesser substrate for penicillin-binding protein PBP2′ in methicillin resistanc

Contribution of SecDF to Staphylococcus aureus resistance and expression of virulence factors

Background SecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND) family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined. Results Markerless deletion mutants were constructed to analyze the impact of the so far uncharacterized RND proteins in S. aureus. While the lack of Sa2056 and Sa2339 caused no phenotype regarding growth and resistance, the secDF mutant resulted in a pleiotropic phenotype. The secDF mutant was cold sensitive, but grew normally in rich medium at 37°C. Resistance to beta-lactams, glycopeptides and the RND substrates acriflavine, ethidium bromide and sodium dodecyl sulfate was reduced. The secDF mutant showed an aberrant cell separation and increased spontaneous and Triton X-100 induced autolysis, although the amounts of penicillin-binding proteins in the membrane were unchanged. The impact of secDF deletion on transcription and expression of specific virulence determinants varied: While coagulase transcription and activity were reduced, the opposite was observed for the autolysin Atl. A reduction of the transcription of the cell wall anchored protein A (spa) was also found. The accumulation of SpA in the membrane and lowered amounts in the cell wall pointed to an impaired translocation. Conclusions The combination of different effects of secDF deletion on transcription, regulation and translocation lead to impaired cell division, reduced resistance and altered expression of virulence determinants suggesting SecDF to be of major relevance in S. aureus. Thus SecDF could be a potential target for the control and eradication of S. aureus in the future

Vancomycin-induced deletion of the methicillin resistance gene mecA in Staphylococcus aureus

Objective: To elucidate factors that contribute to the development of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA). Methods: Forty-nine MRSA isolates were subjected to passage selection with vancomycin to isolate mutants with reduced susceptibility to vancomycin. One mutant was chosen for detailed molecular and biochemical characterization. Results: Five vancomycin-resistant mutants (vancomycin MICs, 6-12 mg/L) were obtained in vitro from five MRSA parent isolates. Upon acquisition of vancomycin resistance, all mutants showed a concomitant decrease in oxacillin resistance. In one particular MRSA strain, selection for vancomycin resistance repeatedly produced deletions and rearrangements, including loss of the mecA gene. Pleiotropic phenotypical changes, such as yellow pigment formation, loss of haemolysis, thickened cell wall, increased resistance to lysostaphin and reduced cell wall turnover were observed in this mutant. Conclusion: Acquisition of vancomycin resistance in one MRSA strain triggered mecA deletion suggesting that this deletion, coupled to other rearrangements and/or mutations, may be responsible for the increased vancomycin resistance phenotyp

Variability of SCC mec in the Zurich area

A periodic survey of methicillin-resistant Staphylococcus aureus (MRSA) in Zurich in 2004 and 2006 revealed a consistently low prevalence of MRSA. SCCmec and ccr typing showed fluctuations in the proportions of SCCmec types and in the carriage of mobile virulence determinants. Together with the presence of variant SCCmecs these findings suggest a high clonal diversity and level of SCCmec recombination. The prevalence of a local "drug clone", associated with low-level methicillin resistance and rapid growth, significantly decreased. This clone had spread among intraveneous drug users, steadily increasing from 1994 to 2001 and was dominant in 2001. Apparently, changes in the management of the Zurich drug scene have restricted the spread of this clon

Evaluation of Fourier transform infrared spectroscopy for the rapid identification of glycopeptide-intermediate Staphylococcus aureus

Objectives To evaluate Fourier transform infrared (FTIR) spectroscopy as a rapid method for distinguishing glycopeptide-intermediate Staphylococcus aureus (GISA) from glycopeptide-susceptible methicillin-resistant S. aureus (MRSA) and to compare three data analysis methods. Methods First-derivative normalized spectra of dried films of bacterial growth on Que-Bact® Universal Medium No. 2 were examined by singular value decomposition to identify key spectral regions. Region selection was analysed by principal component analysis (PCA), self-organizing maps (SOMs) and the K-nearest neighbour (KNN) algorithm. The initial data set included 35 GISA (including GISA Mu50 and heterogeneous GISA Mu3) and 25 epidemic MRSA. The regions were then tested using enlarged data sets that included 22 sporadic and 85 additional epidemic MRSA. Results Epidemic MRSA and GISA/hGISA were separated into two distinct clusters on the basis of spectral data from regions 1352-1315 and 1480-1460 cm−1, the former providing 100% correct classification by all three analyses and the latter providing 96.67% correct by PCA, 98.34% by SOM and 100% by KNN. The 1480-1460 cm−1 region was more effective for distinguishing GISA/hGISA from a set combining sporadic and epidemic MRSA, with two GISA/hGISA and four sporadic MRSA misclassified by PCA and SOM (92.69% correct), while the KNN method misclassified three of the four sporadic MRSA (93.90% correct). The addition of 85 other epidemic MRSA this set increased the fraction of correctly classified isolates to 96.41% and 97.01% by PCA, SOM and KNN, respectively. Conclusions As only 6 of 167 isolates were misclassified, FTIR spectroscopy may provide means of rapid and accurate identification of GISA and hGISA among isolates of MRS

MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus

MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureu

LytR-CpsA-Psr proteins in Staphylococcus aureus display partial functional redundancy and the deletion of all three severely impairs septum placement and cell separation

Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced β-lactam resistance; SA0908 protected cells from autolysis; and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell division in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulenc

Variability of SCCmec in the Zurich area

A periodic survey of methicillin-resistant Staphylococcus aureus (MRSA) in Zurich in 2004 and 2006 revealed a consistently low prevalence of MRSA. SCCmec and ccr typing showed fluctuations in the proportions of SCCmec types and in the carriage of mobile virulence determinants. Together with the presence of variant SCCmecs these findings suggest a high clonal diversity and level of SCCmec recombination. The prevalence of a local "drug clone", associated with low-level methicillin resistance and rapid growth, significantly decreased. This clone had spread among intraveneous drug users, steadily increasing from 1994 to 2001 and was dominant in 2001. Apparently, changes in the management of the Zurich drug scene have restricted the spread of this clone

A Peptidoglycan Fragment Triggers β-lactam Resistance in Bacillus licheniformis

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation

Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureus

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling