Neutrophil chemotaxis by pathogen-associated molecular patterns--formylated peptides are crucial but not the sole neutrophil attractants produced by Staphylococcus aureus

The chemotactic migration of phagocytes to sites of infection, guided by gradients of microbial molecules, plays a key role in the first line of host defence. Bacteria are distinguished from eukaryotes by initiation of protein synthesis with formyl methionine. Synthetic formylated peptides (FPs) have been shown to be chemotactic for phagocytes, leading to the concept of FPs as pathogen-associated molecular patterns (PAMPs). However, it remains unclear whether FPs are major chemoattractants released by bacteria and whether further chemoattractants are produced. A Staphylococcus aureus mutant whose formyltransferase gene was inactivated (Deltafmt) produced no FPs and the in vitro and in vivo ability of Deltafmt culture supernatants to recruit neutrophils was considerably reduced compared with those of the parental strain. However, some chemotactic activity was retained, indicating that bacteria produce also unknown, non-FP chemoattractants. The activity of these novel PAMPs was sensitive to pertussis toxin but insensitive to the formyl peptide receptor inhibitor CHIPS. Deltafmt culture supernatants caused reduced calcium ion fluxes and reduced CD11b upregulation in neutrophils compared with wild-type supernatants. These data demonstrate an important role of FPs in innate immunity against bacterial infections and indicate that host chemotaxis receptors recognize a larger set of bacterial molecules than previously thought.     

The role of peptide deformylase in protein biosynthesis: a proteomic study

Recently we investigated the influence of classical and emerging antibiotics on the proteome of Bacillus subtilis including in our studies actinonin, a potent novel inhibitor of peptide deformylase. The protein synthesis pattern under actinonin treatment changed so dramatically that a direct comparison to the control pattern was impossible. Dual channel imaging revealed that actinonin treatment caused the majority of newly synthesised proteins to accumulate in spots different from the ones usually observed, indicating a more acidic isoelectric point. Two strategies were used to investigate the nature of the charge shift. In the first place, protein patterns of a conditional peptide deformylase mutant under nonrepressing and repressing conditions were compared. Secondly, several protein pairs excised from two-dimensional (2-D) gels of the peptide deformylase mutant, exponentially growing untreated wild-type and the actinonin treated wild-type were investigated with matrix-assisted laser desorption/ionization and electrospray ionization (ESI) time of flight mass spectrometry (TOF MS) for the existence of N-terminal formylation. Under nonrepressing conditions the mutant protein pattern resembled that of the wild-type. The loss of peptide deformylase activity under repressing conditions led to the same pI shift observed for actinonin treatment in the wild-type. Quadrupole TOF-MS on 11 protein pairs proved that the remaining N-terminal formyl residue was indeed responsible for the charge shift. Eight of these protein pairs were also present on 2-D gels of exponentially growing B. subtilis, where the more acidic, still formylated protein species represented the smaller parts.     

Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors: A NEW MECHANISM FOR SENSING PATHOGENS*

Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.     

A new human peptide deformylase inhibitable by actinonin

Peptide deformylases (PDFs) have been investigated as potential specific targets for antibiotics, but the possible existence of a functional human PDF (HsPDF) presents a potential hurdle to the design of specific drugs. We have expression cloned a HsPDF that has deformylase activity, although it is a slower and catalytically less active enzyme than bacterial or plant PDFs. A cobalt-substituted form of HsPDF (but not nickel or zinc) is active, and the enzyme appears to be active at a pH between 6.0 and 7.2, a temperature range of 25-50 degrees C, and in a low KCl ionic strength buffer. Actinonin inhibits HsPDF activity with an IC50 of 43 nM and kills Daudi and HL60 human cancer cell lines with an LC50 of 5.3 and 8.8 microM, respectively. The inhibition of HsPDF may provide an explanation for the mechanism by which actinonin is cytotoxic against various human tumor cell lines.     

Antimicrobial activity of an abiotic host defense peptide mimic

Bacterial drug resistance is emerging as one of the most significant challenges to human health. Antimicrobial peptides (AMPs), which are produced by many tissues and cell types of invertebrates, insects, and humans, as part of their innate immune system, have attracted considerable interest as alternative antibiotics. Interest in novel mimics of AMPs has increased greatly over the last few years. This report details a new AMP mimic, based on phenylene ethynylene, with improved antimicrobial activity and selectivity. Screening against a large set of bacterial and other organisms demonstrates broad spectrum antimicrobial activity including activity against antibiotic resistant bacterial like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) as well as activity against yeast (Candida albicans) and fungus (Stachybotrys chartarum). Bacterial resistance development studies using Staphylococcus aureus show a rapid increase in MIC for conventional antibiotics, ciprofloxacin and norfloxacin. In sharp contrast, no change in MIC was observed for the AMP mimic. Cytotoxicity experiments show that the AMP mimic acts preferentially on microbes as opposed to mammalian red blood cells, 3T3 fibroblasts, and HEPG2 cells. In vivo experiments determined the maximum tolerated dose (MTD) to be 10 mg/kg suggesting a therapeutic window is available. These studies indicate that nonpeptidic amphiphilic AMP mimics could be developed as potential new treatments for antibiotic-resistant bacterial infections.     

Multicolor Whole-Cell Bacterial Sensing Using a Synchronous Fluorescence Spectroscopy-Based Approach

The wide collection of currently available fluorescent proteins (FPs) offers new possibilities for multicolor reporter gene-based studies of bacterial functions. However, the simultaneous use of multiple FPs is often limited by the bleed-through of their emission spectra. Here we introduce an original approach for detection and separation of multiple overlapping fluorescent signals from mixtures of bioreporters strains. The proposed method relies on the coupling of synchronous fluorescent spectroscopy (SFS) with blind spectral decomposition achieved by the Canonical Polyadic (CP) decomposition (also known as Candecomp/Parafac) of three-dimensional data arrays. Due to the substantial narrowing of FP emission spectra and sensitive detection of multiple FPs in a one-step scan, SFS reduced spectral overlap and improved the selectivity of the CP unmixing procedure. When tested on mixtures of labeled E. coli strains, the SFS/CP approach could easily extract the contribution of at least four overlapping FPs. Furthermore, it allowed to simultaneously monitor the expression of three iron responsive genes and pyoverdine production in P. aeruginosa. Implemented in a convenient microplate format, this multiplex fluorescent reporter method provides a useful tool to study complex processes with different variables in bacterial systems.     

Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts

Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 PDF specific activity to values similar to those of bacterial PDFs. We next show that Vp16 PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.     

Variable responses of formyl peptide receptor haplotypes toward bacterial peptides

The chemoattractant neutrophil formyl peptide receptor (FPR) binds bacterial and mitochondrial N-formylated peptides, which allows the neutrophils to find the bacterial source and/or site of tissue damage. Certain inflammatory disorders may be due in part to an impaired innate immune system that does not respond to acute bacterial damage in a timely fashion. Because the human FPR is encoded by a large number of different haplotypes arising from ten single-nucleotide polymorphisms, we examined the possibility that some of these haplotypes are functionally distinct. We analyzed the response of three common FPR haplotypes to peptides from Escherichia coli, Mycobacterium avium ssp. paratuberculosis, and human mitochondria. All three haplotypes responded similarly to the E. coli and mitochondrial peptides, whereas one required a higher concentration of the M. avium peptide fMFEDAVAWF for receptor downregulation, receptor signaling, and chemotaxis. This raises the possibility of additional bacterial species differences in functional responses among FPR variants and establishes a precedent with potentially important implications for our innate immune response against bacterial infections. We also investigated whether certain FPR haplotypes are associated with rheumatoid arthritis (RA) by sequencing FPR1 from 148 Caucasian individuals. The results suggested that FPR haplotypes do not significantly contribute toward RA. George J. Saari, Deceased.     

Pleiotropic roles of formyl peptide receptors

FPR and FPRL1 belong to the seven-transmembrane, G protein-coupled chemoattractant receptor superfamily. Because of their capacity to interact with bacterial chemotactic formylated peptides, these receptors are thought to play a role in host defense against microbial infection. Recently, a variety of novel agonists have been identified for these receptors, including several host-derived endogenous molecules that are involved in proinflammatory responses. Most notably is the use of FPRL1 by at least three amyloidogenic protein and peptide ligands, the serum amyloid A (SAA), the 42 amino acid form of beta amyloid (Abeta(42)), and the prion peptide PrP106-126, to chemoattract and activate human phagocytic leukocytes. These new findings have greatly expanded the functional scope of the formyl peptide receptors and call for more in-depth investigation of the role of these receptors in pathophysiological conditions.     

The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the structure-based design of antibacterial agents

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.     

Distinctive features of the two classes of eukaryotic peptide deformylases.

Peptide deformylases (PDFs) are essential enzymes of the N-terminal protein processing pathway of eubacteria. The recent discovery of two types of PDFs in higher plants, PDF1A and PDF1B, and the detection of PDF1A in humans, have raised questions concerning the importance of deformylation in eukaryotes. Here, we have characterized fully in vitro and compared the properties of the two classes of eukaryotic PDFs, PDF1A and PDF1B, using the PDFs from Arabidopsis thaliana and Lycopersicon esculentum. We have shown that the PDFs of a given class (1A or 1B) all display similar features, independently of their origin. We also observed similar specificity of all plant PDFs for natural substrate peptides, but identified a number of biochemical differences between the two classes (1A or 1B). The main difference lies at the level of the bound cofactor, iron for PDF1B-like bacterial PDFs, and zinc for PDF1A. The nature of the metal cation has important consequences concerning the relative sensitivity to oxygen of the two plant PDFs. Investigation of the specificity of these enzymes with unusual substrates revealed additional differences between the two types of PDFs, enabling us to identify specific inhibitors with a lower affinity against PDF1As. However, the two plant PDFs were inhibited equally strongly in vitro by actinonin, an antibiotic that specifically acts on bacterial PDFs. Uptake of actinonin by A. thaliana seedlings was used to investigate the function of PDFs in the plant. Because it induces an albino phenotype, we conclude that deformylation is likely to play an essential role in the chloroplast.     

Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor

Clostridium difficile may induce antibiotic‐associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB‐induced rise in intracellular‐free Ca²⁺ and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR‐1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR‐1. Domain analyses revealed that the N‐terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro‐inflammatory ligand effect can be triggered even by cleaved and, thus, non‐cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved ‘non‐cytotoxic’ fragments and (iii) an interaction of the N‐terminal glucosyltransferase domain instead of the C‐terminal receptor binding domain of TcdB with target cells.     

Characterization of peptide deformylase2 from B. cereus

Peptide deformylase (PDF) is a metalloenzyme that removes the N-terminal formyl groups from newly synthesized proteins. It is essential for bacterial survival, and is therefore-considered as a potential target for antimicrobial chemotherapy. However, some bacteria including medically relevant pathogens possess two or more def-like genes. Here we have examined two PDFs from Bacillus cereus. The two share only 32% sequence identity and the crystal structures show overall similarity with PDF2 having a longer C-terminus. However, there are differences at the two active sites, and these differences appear to contribute to the activity difference seen between the two. BcPDF2 is found as a dimer in the crystal form with two additional actinonin bound at that interface.     

Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis elegans Model

Background

Cigarette smoking is the major cause of chronic obstructive pulmonary disease (COPD) and lung cancer. Respiratory bacterial infections have been shown to be involved in the development of COPD along with impaired airway innate immunity.

Methodology/Principal Findings

To address the in vivo impact of cigarette smoke (CS) exclusively on host innate defense mechanisms, we took advantage of Caenorhabditis elegans (C. elegans), which has an innate immune system but lacks adaptive immune function. Pseudomonas aeruginosa (PA) clearance from intestines of C. elegans was dampened by CS. Microarray analysis identified 6 candidate genes with a 2-fold or greater reduction after CS exposure, that have a human orthologue, and that may participate in innate immunity. To confirm a role of CS-down-regulated genes in the innate immune response to PA, RNA interference (RNAi) by feeding was carried out in C. elegans to inhibit the gene of interest, followed by PA infection to determine if the gene affected innate immunity. Inhibition of lbp-7, which encodes a lipid binding protein, resulted in increased levels of intestinal PA. Primary human bronchial epithelial cells were shown to express mRNA of human Fatty Acid Binding Protein 5 (FABP-5), the human orthologue of lpb-7. Interestingly, FABP-5 mRNA levels from human smokers with COPD were significantly lower (p = 0.036) than those from smokers without COPD. Furthermore, FABP-5 mRNA levels were up-regulated (7-fold) after bacterial (i.e., Mycoplasma pneumoniae) infection in primary human bronchial epithelial cell culture (air-liquid interface culture).

Conclusions

Our results suggest that the C. elegans model offers a novel in vivo approach to specifically study innate immune deficiencies resulting from exposure to cigarette smoke, and that results from the nematode may provide insight into human airway epithelial cell biology and cigarette smoke exposure.     

Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N

Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.     

The minimal fusion peptide of simian immunodeficiency virus corresponds to the 11 first residues of gp32.

We had previously predicted successfully the minimal fusion peptides (FPs) of the human immunodeficiency virus 1 (HIV-1) gp41 and the bovine leukemia virus (BLV) gp30 using an original approach based on the obliquity/fusogenicity relationship of tilted peptides. In this paper, we have used the same method to predict the shortest FP capable of inducing optimal fusion in vitro of the simian immunodeficiency virus (SIV) mac isolate and of other SIVs and human immunodeficiency virus (HIV-2) isolates. In each case, the 11-residue-long peptide was predicted as the minimal FP. For the SIV mac isolate, liposome lipid-mixing and leakage assays confirmed that this peptide is the shortest peptide inducing optimal fusion in vitro, being therefore the minimal FP. These results are another piece of evidence that the tilted properties of FPs are important for the fusion process and that our method can be used to predict the minimal FPs of other viruses.     

Mechanism of time-dependent inhibition of polypeptide deformylase by actinonin

Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for the removal of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition of bacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterized using steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observed under steady-state conditions. Progress curve analysis is consistent with a two-step binding mechanism, in which tightening of the initial encounter complex (EI) results in a final complex (EI*) with an extremely slow, but observable, off-rate (t(1/2) for inhibitor dissociation >or=0.77 days). Stopped-flow measurement of PDF fluorescence confirms formation of EI and provides a direct measurement of the association rate. Rapid dilution studies establish that the potency of actinonin is enhanced by more than 2000-fold upon tightening of EI to form EI*, from K(i) = 530 nM (EI) to Ki*相似文献   

Actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor

Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making it an attractive target for the discovery of novel antibiotics. We have identified actinonin, a naturally occurring antibacterial agent, as a potent PDF inhibitor. The dissociation constant for this compound was 0.3 x 10(-)(9) M against Ni-PDF from Escherichia coli; the PDF from Staphylococcus aureus gave a similar value. Microbiological evaluation revealed that actinonin is a bacteriostatic agent with activity against Gram-positive and fastidious Gram-negative microorganisms. The PDF gene, def, was placed under control of P(BAD) in E. coli tolC, permitting regulation of PDF expression levels in the cell by varying the external arabinose concentration. The susceptibility of this strain to actinonin increases with decreased levels of PDF expression, indicating that actinonin inhibits bacterial growth by targeting this enzyme. Actinonin provides an excellent starting point from which to derive a more potent PDF inhibitor that has a broader spectrum of antibacterial activity.     

Structure and Activity of Human Mitochondrial Peptide Deformylase, a Novel Cancer Target

Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 Å), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 Å) identified the substrate-binding site. A defined S1′ pocket, but no S2′ or S3′ substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2′ and S3′ binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2′and P3′ positions of a formylated peptide substrate to turnover.