Serine protease EpiP from Staphylococcus epidermidis catalyzes the processing of the epidermin precursor peptide.

The function of serine protease EpiP in epidermin biosynthesis was investigated. Epidermin is synthesized as a 52-amino-acid precursor peptide, EpiA, which is posttranslationally modified and processed to the mature 22-amino-acid peptide antibiotic. epiP was expressed in Staphylococcus carnosus with xylose-regulated expression vector pCX15. The cleavage of the unmodified EpiA precursor peptide to leader peptide and proepidermin by EpiP-containing culture filtrates of S. carnosus (pCX15epiP) was followed by reversed-phase chromatography and subsequent electrospray mass spectrometry.     

Staphylococcus aureus and Staphylococcus epidermidis peptide pheromones produced by the accessory gene regulator agr system

The accessory gene regulator (agr) system of staphylococci regulates the expression of virulence factors in response to cell density. The extracellular signaling molecule encoded by this system is a thiolactone-containing pheromone peptide whose primary sequence varies among staphylococcal strains. A post-translational modification of the peptide is believed to be carried out by an enzyme with a novel function, AgrB. Staphylococcal pheromones show cross-inhibiting properties: Pheromones of self and pheromones of non-self induce and suppress the agr response, respectively, and have therefore been proposed as novel anti-staphylococcal drugs. As inhibition of agr leads to diminished expression of toxins, but to increased expression of colonization factors and biofilm formation, their therapeutic potential remains yet to be evaluated in depth.     

Producer self-protection against the lantibiotic epidermin by the ABC transporter EpiFEG of Staphylococcus epidermidis Tü3298

Self-protection of the epidermin-producing strain Staphylococcus epidermidis Tü3298 against the pore-forming lantibiotic epidermin is mediated by an ABC transporter composed of the EpiF, EpiE, and EpiG proteins. We developed a sensitive assay based on HPLC analysis to investigate the capacity of the EpiFEG transporter to release epidermin and analogues from the cell surface to the external fluid. Our results indicate that the EpiFEG transporter works by expelling the lantibiotic from the cytoplasmic membrane into the surrounding medium. Analysis of transporter efficacy using nisin and gallidermin derivatives as substrates revealed a high substrate specificity. Furthermore, we showed that the activity of the gallidermin derivative L6G is enhanced by the presence of EpiE.     

Regulation of antimicrobial peptide production by autoinducer-mediated quorum sensing in lactic acid bacteria

Several lactic acid bacteria produce peptides with antimicrobial activity. During the last few years, cell–cell communication has emerged as the key regulatory mechanism that controls the production of many of these antimicrobial peptides via a regulatory strategy denominated quorum sensing. Quorum sensing allows population-wide synchronised production of antimicrobial peptides as a function of cell density. The cell–cell communication phenomenon required for sensing of the cell density is mediated by secreted signalling molecules. These molecular messengers accumulate in the environment as the cell density increases and activate signal transduction cascades that result in the production of antimicrobial peptides by the stimulated bacterial cell.     

In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis

Abstract

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24?h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24?h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4?h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24?h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.     

Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives.

The agr quorum-sensing system in Staphylococci controls the production of surface proteins and exoproteins. In the pathogenic species Staphylococcus aureus, these proteins include many virulence factors. The extracellular signal of the quorum-sensing system is a thiolactone-containing peptide pheromone, whose sequence varies among the different staphylococcal strains. We demonstrate that a synthetic Staphylococcus epidermidis pheromone is a competent inhibitor of the Staphylococcus aureus agr system. Derivatives of the pheromone, in which the N-terminus or the cyclic bond structure was changed, were synthesized and their biological activity was determined. The presence of a correct N-terminus and a thiolactone were absolute prerequisites for an agr-activating effect in S. epidermidis, whereas inhibition of the S. aureus agr system was less dependent on the original structure. Our results show that effective quorum-sensing blockers that suppress the expression of virulence factors in S. aureus can be designed based on the S. epidermidis pheromone.     

Plasmalogen synthesis is regulated via alkyl-dihydroxyacetonephosphate-synthase by amyloid precursor protein processing and is affected in Alzheimer's disease

Lipids play an important role as risk or protective factors in Alzheimer's disease, which is characterized by amyloid plaques composed of aggregated amyloid-beta. Plasmalogens are major brain lipids and controversially discussed to be altered in Alzheimer's disease (AD) and whether changes in plasmalogens are cause or consequence of AD pathology. Here, we reveal a new physiological function of the amyloid precursor protein (APP) in plasmalogen metabolism. The APP intracellular domain was found in vivo and in vitro to increase the expression of the alkyl-dihydroxyacetonephosphate-synthase (AGPS), a rate limiting enzyme in plasmalogen synthesis. Alterations in APP dependent changes of AGPS expression result in reduced protein and plasmalogen levels. Under the pathological situation of AD, increased amyloid-beta level lead to increased reactive oxidative species production, reduced AGPS protein and plasmalogen level. Accordingly, phosphatidylethanol plasmalogen was decreased in the frontal cortex of AD compared to age matched controls. Our findings elucidate that plasmalogens are decreased as a consequence of AD and regulated by APP processing under physiological conditions.     

Sensitivity of the quorum sensing system is achieved by low pass filtering

Autoinducer sensing, also known as quorum sensing, is the communication of bacteria by autoinducer (small signaling molecules). Cells respond on extremely low concentrations of autoinducer: only one or two molecules per cell are sufficient. At this signal level a high degree of noise is inherent. We ask for the mechanism that is able to overcome the stochasticity of the signal. By means of a model and parameter fitting we show that the sensing module acts as a low pass filter, representing the biochemical equivalent of a moving average. It is shown that the system works most sensitive in the range of 0-50 nM autoinducer. Moreover, the time scale of the reaction depends on the signal strength in a crucial manner. Nonlinear feedback is able to further enhance the sensitivity. The biological implications of the low pass filter property are discussed.     

Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants.

The plasmid-encoded epidermin biosynthetic gene epiC of Staphylococcus epidermidis Tü3298 was expressed in Escherichia coli by using the T7 RNA polymerase-promoter system, and the gene product EpiC was identified by Western blotting (immunoblotting) with an anti-EpiC-peptide antiserum. EpiC was a hydrophobic but soluble protein. EpiC was purified by hydrophobic-interaction chromatography. The determined amino-terminal amino acid sequence was M I N I N N I .... The electrophoretic migration behavior of EpiC depended on the oxidation state of the enzyme, indicating the formation of an intramolecular disulfide bridge between C-274 and C-321. The cysteine residues in the motifs WC-274YG and C-321HG of EpiC are conserved in all lantibiotic enzymes of the C type (so-called LanC proteins) and in the CylM protein. Mutated epiC genes from S. epidermidis epiC mutants were cloned and expressed in E. coli. Sequence analysis revealed that the mutations occurred in the two motifs -S-X-X-X-G-X-X-G- and -N-X-G-X-A-H-G-X-X-G-, which are conserved in all LanC proteins. For the investigation of EpiC-EpiA interactions, precursor peptide EpiA was coupled to N-hydroxysuccinimide-activated Sepharose High Performance Material (HiTrap). Under reducing conditions, EpiC was retarded on the EpiA-HiTrap column. In the incubation experiments, EpiC did not react with EpiA, with proepidermin, or with oxidative decarboxylated peptides.     

Ribosomal-protein synthesis is not autogenously regulated at the translational level in Xenopus laevis

Whether ribosomal-protein synthesis in Xenopus laevis is autogenously controlled at the translational level as is known to occur in prokaryotes has been studied. For this purpose ribosomal (r) proteins were prepared from X. laevis ribosomal subunits and group fractionated by ion-exchange chromatography. They were then added to an in vitro translation system directed by an oocyte mRNA fraction which contains template activity for r proteins. The synthesized radioactive products were analyzed by 2D gel electrophoresis and compared with controls. Similarly in vivo experiments were performed by microinjection of the fractionated proteins into the cytoplasm of Xenopus oocytes followed by incubation with [35S]methionine for different times. In all the experiments no evident effect of r proteins on the translation of their own mRNA was observed.     

Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system.

Subtilin is a lanthionine-containing peptide antibiotic (lantibiotic) which is produced by Bacillus subtilis ATCC 6633. Upstream from the structural gene of subtilin, spaS, three genes (spaB, spaT, and spaC) which are involved in the biosynthesis of subtilin have been identified (C. Klein, C. Kaletta, N. Schnell, and K.-D. Entian, Appl. Environ. Microbiol. 58:132-142, 1992). By using a hybridization probe specific for these genes, the DNA region downstream from spaS was isolated. Further subcloning revealed a 5.2-kb KpnI-HindIII fragment on which two open reading frames, spaR and spaK, were identified approximately 3 kb downstream from spaS. The spaR gene encodes an open reading frame of 220 amino acids with a predicted molecular mass of 25.6 kDa. SpaR shows 35% similarity to positive regulatory factors OmpR and PhoB. The spaK gene encodes an open reading frame of 387 amino acids with a predicted molecular mass of 44.6 kDa and was highly similar to histidine kinases previously described (PhoM, PhoR, and NtrB). Hydrophobicity blots suggested two membrane-spanning regions. Thus, spaR and spaK belong to a recently identified family of environmentally responsive regulators. These results indicated a regulatory function of spaR and spaK in subtilin biosynthesis. Indeed, batch culture experiments confirmed the regulation of subtilin biosynthesis starting in the mid-logarithmic growth phase and reaching its maximum in the early stationary growth phase. Gene deletions within spaR and spaK yielded subtilin-negative mutants, which confirms that subtilin biosynthesis is under the control of a two-component regulatory system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor

Toxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing—dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor (EDF), a linear pentapeptide with the sequence NNWNN. In spite of the extensive research on the mazEF system and the involvement of the quorum sensing factor EDF, the effect of EDF itself on bacteria has not yet been studied. In this research, we determined the effect of EDF and variants on cell growth in the Gram-negative bacterium E. coli and the Gram-positive Bacillus globigii. By aligning the zwf gene (from where EDF originates) of different bacterial species, we found 27 new theoretical variants of the peptide. By evaluating growth curves and light microscopy we found that three EDF variants reduced bacterial cell size in B. globigii, but not in E. coli. The D-peptides did not affect cell size, indicating that the effect is stereospecific. Peptides wherein tryptophan was substituted by alanine also did not affect cell size, which indicates that the effect seen is mediated by an intracellular target.     

The Cek1 MAPK is a short-lived protein regulated by quorum sensing in the fungal pathogen Candida albicans

Mitogen activated protein kinase (MAPK) cascades are signal transduction mechanisms present in eukaryotic cells that allow adaptation to environmental changes. MAPK activity is mainly regulated by dual phosphorylation in a TXY motif present in the kinase subdomain VIII as well as dephosphorylation by specific phosphatases. The Cek1 MAPK is involved in filamentous growth in Candida albicans and is an important determinant of virulence in this microorganism; its activation is controlled by the Sho1 adaptor protein. Here we show that Cek1 phosphorylation is regulated by quorum sensing (QS). Cek1 phosphorylation is prevented by farnesol, a compound that also regulates the dimorphic transition in this fungus. Farnesol also induced the activation of Mkc1, the MAPK of the cell integrity pathway. The role of farnesol in Cek1 phosphorylation is independent of the Chk1 histidine kinase, a putative QS sensor, as revealed by genetic analysis. In addition, Cek1, not Hog1, is degraded by proteasome, as revealed by the use of a conditional lethal protein degradation mutant. Our data therefore describe two different mechanisms (QS and protein degradation) that control a MAPK pathway that regulates virulence in a fungal pathogen.     

Cutting edge: mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide

Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils and some epithelia and can act as natural antibiotics by directly killing a wide range of microorganisms. We hypothesized that caths are expressed in mast cells (MCs), because these cells have been previously associated with inherent antimicrobial activity. Cultured murine MCs contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cath, and this expression was inducible by LPS or lipoteichoic acid. Human skin MCs also expressed cath as detected by immunohistochemical analysis for the human cath LL-37. The functional significance of this expression was shown by comparing MCs cultured from normal mice to MCs from littermates deficient in the cathelin-related AMP gene (Cnlp(-)). MCs derived from Cnlp(-/-) animals had a 50% reduction in their ability to kill group A STREPTOCOCCUS: These MCs expressed equivalent amounts of mRNA for murine beta-defensin-4, a beta-defensin AMP. Thus, different antimicrobials can be identified in MCs, and the presence of cath is necessary for efficient bacterial killing. These observations suggest that the presence of cath is vital to the ability of mammalian MCs to participate in antimicrobial defense.