YisP is involved in biofilm formation in Bacillus subtilis and has been predicted to produce C30 isoprenoids. We determined the structure of YisP and observed that it adopts the same fold as squalene and dehydrosqualene synthases. However, the first aspartate-rich motif found in essentially all isoprenoid synthases is aspartate poor in YisP and cannot catalyze head-to-head condensation reactions. We find that YisP acts as a phosphatase, catalyzing formation of farnesol from farnesyl diphosphate, and that it is the first phosphatase to adopt the fold seen in the head-to-head prenyl synthases. Farnesol restores biofilm formation in a ?yisp mutant and modifies lipid membrane structure similarly to the virulence factor staphyloxanthin. This work clarifies the role of YisP in biofilm formation and suggests an intriguing possibility that many of the YisP-like homologs found in other bacteria may also have interesting products and functions.
Bacterial adhesion on implants is a first step in the development of chronic foreign body associated infections. Finding strategies to minimize bacterial adhesion may contribute to minimize such infections. It is known that surfaces with oligo-ethylene-glycol (EG3OMe) or poly-ethylene-glycol (PEG2k) terminations decrease unspecific protein adsorption and bacterial adhesion. However, little is known about the influence of serum and its components on bacterial adhesion. We therefore prepared two coatings on gold surface with HS-(CH2)11EG3OMe (EG3OMe) and PEG2k-thiol and studied the role of bovine serum albumin (BSA), ?-globulins, and serum on Staphylococcus aureus adhesion. While BSA and lysozyme showed no adherence even when applied at very high concentrations (100mg/ml), ?-globulins adsorbed already from 10mg/ml on. The adsorption of ?-globulins was, however, significantly decreased when it was mixed with BSA in a ratio of 3:1, as it is in the serum. Pretreatment of EG3OMe and PEG2k coatings with ?-globulins or serum strongly promoted adherence of S. aureus when resuspended in buffer, suggesting that ?-globulins play a pivotal role in promoting S. aureus adhesion by its IgG binding proteins; the finding that a spa-deletion mutant, lacking the IgG binding protein A, showed decreased adherence corroborated this. Similarly, when S. aureus was pretreated with serum or ?-globulins its adherence was also significantly decreased. Our findings show that particularly ?-globulins bind to the coated surfaces thus mediating adherence of S. aureus via its protein A. As pretreatment of S. aureus with serum or ?-globulins significantly decreased adherence, treatment of patients with ?-globulins before implant surgery might lower the risk of implant-associated infections.
The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 ? resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.
Colonization of the human nose by Staphylococcus aureus in one-third of the population represents a major risk factor for invasive infections. The basis for adaptation of S. aureus to this specific habitat and reasons for the human predisposition to become colonized have remained largely unknown. Human nasal secretions were analyzed by metabolomics and found to contain potential nutrients in rather low amounts. No significant differences were found between S. aureus carriers and non-carriers, indicating that carriage is not associated with individual differences in nutrient supply. A synthetic nasal medium (SNM3) was composed based on the metabolomics data that permits consistent growth of S. aureus isolates. Key genes were expressed in SNM3 in a similar way as in the human nose, indicating that SNM3 represents a suitable surrogate environment for in vitro simulation studies. While the majority of S. aureus strains grew well in SNM3, most of the tested coagulase-negative staphylococci (CoNS) had major problems to multiply in SNM3 supporting the notion that CoNS are less well adapted to the nose and colonize preferentially the human skin. Global gene expression analysis revealed that, during growth in SNM3, S. aureus depends heavily on de novo synthesis of methionine. Accordingly, the methionine-biosynthesis enzyme cysteine-?-synthase (MetI) was indispensable for growth in SNM3, and the MetI inhibitor DL-propargylglycine inhibited S. aureus growth in SNM3 but not in the presence of methionine. Of note, metI was strongly up-regulated by S. aureus in human noses, and metI mutants were strongly abrogated in their capacity to colonize the noses of cotton rats. These findings indicate that the methionine biosynthetic pathway may include promising antimicrobial targets that have previously remained unrecognized. Hence, exploring the environmental conditions facultative pathogens are exposed to during colonization can be useful for understanding niche adaptation and identifying targets for new antimicrobial strategies.
The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the Staphylococcus intermedius group of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community.
Multiple mechanisms have been correlated with daptomycin-resistance (DAP-R) in Staphylococcus aureus. However, one common phenotype observed in many DAP-R S. Aureus strains is a thickened cell wall (CW). The first evidence for an impact of CW-linked glycopolymers on this phenotype was recently demonstrated in a single, well-characterized DAP-R methicillin-susceptible S. aureus (MSSA) strain. In this isolate the thickened CW phenotype was linked to an increased production and D-alanylation of wall teichoic acids (WTA). In the current report, we extended these observations to methicillin-resistant daptomycin-sensitive/daptomyin-resistant (DAP-S/DAP-R) strain-pairs. These pairs included methicillin-resistant S. aureus (MRSA) isolates with and without single nucleotide polymorphisms (SNPs) in mprF (a genetic locus linked to DAP-R phenotype). We found increased CW dry mass in all DAP-R vs DAP-S isolates. This correlated with an increased expression of the WTA biosynthesis gene tagA, as well as an increased amount of WTA in the DAP-R vs DAP-S isolates. In addition, all DAP-R isolates showed a higher proportion of WTA D-alanylation vs their corresponding DAP-S isolate. We also detected an increased positive surface charge amongst the DAP-R strains (presumably related to the enhanced D-alanylation). In comparing the detailed CW composition of all isolate pairs, substantive differences were only detected in one DAP-S/DAP-R pair. The thickened CW phenotype, together with an increased surface charge most likely contributes to either: i) a charge-dependent repulsion of calcium complexed-DAP; and/or ii) steric-limited access of DAP to the bacterial cell envelope target. Taken together well-defined perturbations of CW structural and functional metrics contribute to the DAP-R phenotype and are common phenotypes in DAP-R S. Aureus isolates, both MSSA and MRSA. Note: Although "daptomycin-nonsusceptibility" is the generally accepted terminology, we have utilized the term "daptomycin resistance" for ease of presentation in this manuscript.
Strains from various staphylococcal species produce bacteriocin peptides, which are thought to play important roles in bacterial competition and offer interesting biotechnological avenues. Many bacteriocins are secreted as inactive prepeptides with subsequent activation by specific proteolytic cleavage. By deletion of the protease gene gdmP in Staphylococcus gallinarum Tü3928, which produces the highly active lanthionine-containing bacteriocin gallidermin (lantibiotic), a strain was created producing inactive pregallidermin. On this basis, a new suicidal mutant selection system in the food-grade bacterium Staphylococcus carnosus was developed. Whereas pregallidermin was inactive against S. carnosus, it exerted potent bactericidal activity toward GdmP-secreting S. carnosus strains. To take advantage of this effect, gdmP was cloned in plasmid vectors used for random transposon mutagenesis or targeted allelic replacement of chromosomal genes. Both mutagenesis strategies rely on rare recombination events, and it has remained difficult and laborious to identify mutants among a vast majority of bacterial clones that still contain the delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2 enabled very fast, easy, and reliable identification of transposon and gene replacement mutants, respectively. Mutant selection in the presence of pregallidermin caused suicidal inactivation of all clones that had retained the plasmids and allowed growth of only plasmid-cured mutants. Efficiency of mutant identification was several magnitudes higher than standard screening for the absence of plasmid-encoded antibiotic resistance markers and reached 100% specificity. Thus, the new pregallidermin-based mutant selection system represents a substantial improvement of staphylococcal mutagenesis methodology.
Many microorganisms excrete typical cytoplasmic proteins into the culture supernatant. As none of the classical secretion systems appears to be involved, this type of secretion was referred to as "nonclassical protein secretion." Here, we demonstrate that in Staphylococcus aureus the major autolysin plays a crucial role in release of cytoplasmic proteins. Comparative secretome analysis revealed that in the wild type S. aureus strain, 22 typical cytoplasmic proteins were excreted into the culture supernatant, although in the atl mutant they were significantly decreased. The presence or absence of prophages had little influence on the secretome pattern. In the atl mutant, secondary peptidoglycan hydrolases were increased in the secretome; the corresponding genes were transcriptionally up-regulated suggesting a compensatory mechanism for the atl mutation. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic indicator enzyme, we showed that all clinical isolates tested excreted this protein. In the wall teichoic acid-deficient tagO mutant with its increased autolysis activity, GAPDH was excreted in even higher amounts than in the WT, confirming the importance of autolysis in excretion of cytoplasmic proteins. To answer the question of how discriminatory the excretion of cytoplasmic proteins is, we performed a two-dimensional PAGE of cytoplasmic proteins isolated from WT. Surprisingly, the most abundant proteins in the cytoplasm were not found in the secretome of the WT, suggesting that there exists a selection mechanism in the excretion of cytoplasmic proteins. As the major autolysin binds at the septum site, we assume that the proteins are preferentially released at and during septum formation.
In mammalian host cells staphylococcal peptidoglycan (PGN) is recognized by Nod2. Whether PGN is also recognized by TLR2 is disputed. Here we carried out PGN co-localization and stimulation studies with TLR2 and Nod2 in wild type and mutant host cells. To exclude contamination with lipoproteins, polymeric staphylococcal PGN (PGN(pol)) was isolated from Staphylococcus aureus ?lgt (lacking lipidated prelipoproteins). PGN(pol) was biotinylated (PGN-Bio) for fluorescence monitoring with specific antibodies. Keratinocytes from murine oral epithelium (MK) readily internalized PGN-Bio in an endocytosis-like process. In wt MK, PGN(pol) induced intracellular accumulation of Nod2 and TLR2 and co-localized with Nod2 and TLR2, but not with TLR4. In TLR2-deficient MK Nod2 and in Nod2-deficient MK TLR2 was induced, indicating that PGN(pol) recognition by Nod2 is independent of TLR2 and vice versa. In both mutants IL-6 and IL-1B release was decreased by approximately 50% compared to wt MK, suggesting that the immune responses induced by Nod2 and TLR2 are comparable and that the two receptors act additively in MK. In TLR2-transfected HEK293 cells PGN(pol) induced NFkB-promoter fused luciferase expression. To support the data, co-localization and signaling studies were carried out with SHL-PGN, a lipase protein covalently tethered to PGN-fragments of varying sizes at its C-terminus. SHL-PGN also co-localized with Nod2 or TLR2 and induced their accumulation, while SHL without PGN did not. The results show that staphylococcal PGN not only co-localizes with Nod2 but also with TLR2. PGN is able to stimulate the immune system via both receptors.
Innate immune sensing of Staphylococcus aureus unravels basic mechanisms leading to either effective antibacterial immune responses or harmful inflammation. The nature and properties of S. aureus-derived pathogen-associated molecular pattern (PAMPs) are still not completely understood. We investigated the innate immune sensing of peptidoglycan (PGN) structures and subsequent immune consequences. Macromolecular PGN (PGN(polymer)) preparations activated NF-?B through human Toll-like receptors 2 (TLR2), as shown by luciferase reporter assays, and induced murine dendritic cell (DC) maturation and cytokine production. In contrast, PGN(polymer) from lgt-mutant S. aureus failed to stimulate human TLR2, demonstrating that lipoproteins within the macromolecular structures of PGN(polymer), but not PGN itself, activate TLR2. Thus, HPLC-purified monomeric PGN (PGN(monomer)) structures were investigated. Strikingly, PGN(monomer) completely lacked NF-?B activation, lacked TLR2 activity, and failed to functionally activate murine DCs. However, PGN(monomer) in concert with various TLR ligands most effectively stimulated DCs to up-regulate IL-12p70 and IL-23 by ?3- to 5-fold. Consequently, DCs coactivated by PGN(monomer) markedly up-regulated Th1 and Th17 while suppressing Th2 cell priming. Notably, PGN(monomer) failed to coactivate NOD2(-/-) DCs. This demonstrates that PGN(monomer) is a natural ligand of NOD2, which was previously only demonstrated for synthetic compounds like muramyl dipeptide. Interestingly, murine DCs lacking TLR2 remained mute in response to the combinative immune sensing of S. aureus-derived PAMPs, including PGN(monomer), providing for the first time an explanation of why S. aureus can colonize the nasal mucosa in the absence of inflammation. This is very likely based on the lack of TLR2 expression in mucosal epithelial cells under normal conditions, which determines the unresponsiveness to S. aureus PAMPs.
Due to their abilities to form strong biofilms, Staphylococcus aureus and Staphylococcus epidermidis are the most frequently isolated pathogens in persistent and chronic implant-associated infections. As biofilm-embedded bacteria are more resistant to antibiotics and the immune system, they are extremely difficult to treat. Therefore, biofilm-active antibiotics are a major challenge. Here we investigated the effect of the lantibiotic gallidermin on two representative biofilm-forming staphylococcal species. Gallidermin inhibits not only the growth of staphylococci in a dose-dependent manner but also efficiently prevents biofilm formation by both species. The effect on biofilm might be due to repression of biofilm-related targets, such as ica (intercellular adhesin) and atl (major autolysin). However, gallidermins killing activity on 24-h and 5-day-old biofilms was significantly decreased. A subpopulation of 0.1 to 1.0% of cells survived, comprising "persister" cells of an unknown genetic and physiological state. Like many other antibiotics, gallidermin showed only limited activity on cells within mature biofilms.
Peptidoglycan (PGN), a component of bacterial cell wall and belonging to "Microbe-Associated Molecular Patterns" (MAMP) triggers host reactions contributing to the pathophysiology of infectious disease. Host cell responses to PGN exposure include apoptosis. Bacterial infections may result in activation of blood platelets and thrombocytopenia. The present study explored, whether HPLC-purified fractions of PGNs from Staphylococcus aureus 113 triggers apoptosis of platelets. To this end platelets were exposed to PGN fractions and annexin-V binding determined to depict cell membrane scrambling, DiOC6 fluorescence to estimate depolarization of mitochondrial potential, Fluo-3AM staining for intracellular Ca(2+) activity ([Ca(2+)](i)) and immunofluorescence to quantify protein abundance of active caspase-3. As a result, a 30 min exposure to monomeric fraction (mPGN) (?50 ng/ml) was followed by annexin-V binding, paralleled by increase of [Ca(2+)](i), mitochondrial depolarization, caspase-3 activation and integrin ?(IIb)?(3) upregulation. The annexin-V binding was significantly blunted by anti-TLR-2 antibodies, in absence of extracellular Ca(2+), and by pancaspase inhibitor zVAD-FMK (1 ?M). In conclusion, PGN triggers apoptosis of platelets in activation-dependent manner, characterized by mitochondrial depolarization, caspase-3 activation and cell membrane scrambling.
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