Leukocidins and the Nuclease Nuc Prevent Neutrophil-Mediated Killing of Staphylococcus aureus Biofilms.

Bacterial biofilms are linked with chronic infections and have properties distinct from those of planktonic, single-celled bacteria. The virulence mechanisms associated with Staphylococcus aureus biofilms are becoming better understood. Human neutrophils are critical for the innate immune response to S. aureus infection. Here, we describe two virulence strategies that converge to promote the ability of S. aureus biofilms to evade killing by neutrophils. Specifically, we show that while neutrophils exposed to S. aureus biofilms produce extracellular traps (NETs) and phagocytose bacteria, both mechanisms are inefficient in clearance of the biofilm biomass. This is attributed to the leukocidin LukAB, which promotes S. aureus survival during phagocytosis. We also show that the persistence of biofilm bacteria trapped in NETs is facilitated by S. aureus nuclease (Nuc)-mediated degradation of NET DNA. This study describes key aspects of the interaction between primary human neutrophils and S. aureus biofilms and provides insight into how S. aureus evades the neutrophil response to cause persistent infections.

Mycoplasma hyopneumoniae Mhp597 is a cytotoxicity, inflammation and immunosuppression associated nuclease.

Nucleases are ubiquitously recognized as essential proteins in mycoplasmas because these organisms lack most capacities for de novo synthesis of nucleotides. Some of these proteins were proved to be important pathogenic factors during the infection of mycoplasms. In this study, the protein Mhp597 from M. hyopneumoniae was expressed and purified in Escherichia coli. Analysis of nuclease activity showed that recombinant Mhp597 (rMhp597) was a Ca2+ or Mg2+ dependent thermostable nuclease with very high activity and neutrophil extracellular traps (NETs) induced by M. hyopneumoniae were completely degraded by this nuclease. In addition, when PK15 cells were incubated with different concentrations of rMhp597, their viability was reduced and cell apoptosis was observed in a dose-dependent manner. To further investigate the host immune system response, we report that rMhp597 up-regulated the exression of inflammatory genes showing that TLR4/MyD88/NF-κB signal pathway was involved. On the other hand, rMhp597 down-regulated the expression of type I IFN (IFN-α/ß) and promoted the multiplication of porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant rMhp597δ315-377 lacking C-terminal 63 amino acids exhibited all biological functions mentioned above except for nuclease activity. In summary, Mhp597 is a dynamic secreted nuclease involved in cytotoxicity, inflammation and immunosuppression.

Active immunization with an octa-valent Staphylococcus aureus antigen mixture in models of S. aureus bacteremia and skin infection in mice.

Proteomic studies with different Staphylococcus aureus isolates have shown that the cell surface-exposed and secreted proteins IsaA, LytM, Nuc, the propeptide of Atl (pro-Atl) and four phenol-soluble modulins α (PSMα) are invariantly produced by this pathogen. Therefore the present study was aimed at investigating whether these proteins can be used for active immunization against S. aureus infection in mouse models of bacteremia and skin infection. To this end, recombinant His-tagged fusions of IsaA, LytM, Nuc and pro-Atl were isolated from Lactococcus lactis or Escherichia coli, while the PSMα1-4 peptides were chemically synthesized. Importantly, patients colonized by S. aureus showed significant immunoglobulin G (IgG) responses against all eight antigens. BALB/cBYJ mice were immunized subcutaneously with a mixture of the antigens at day one (5 µg each), and boosted twice (25 µg of each antigen) with 28 days interval. This resulted in high IgG responses against all antigens although the response against pro-Atl was around one log lower compared to the other antigens. Compared to placebo-immunized mice, immunization with the octa-valent antigen mixture did not reduce the S. aureus isolate P load in blood, lungs, spleen, liver, and kidneys in a bacteremia model in which the animals were challenged for 14 days with a primary load of 3 × 10(5) CFU. Discomfort scores and animal survival rates over 14 days did not differ between immunized mice and placebo-immunized mice upon bacteremia with S. aureus USA300 (6 × 10(5) CFU). In addition, this immunization did not reduce the S. aureus isolate P load in mice with skin infection. These results show that the target antigens are immunogenic in both humans and mice, but in the used animal models do not result in protection against S. aureus infection.

Novel role of the antimicrobial peptide LL-37 in the protection of neutrophil extracellular traps against degradation by bacterial nucleases.

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defence mechanism. They consist of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) which are able to engulf and kill pathogens. The AMP LL-37, a member of the cathelicidin family, is highly present in NETs. However, the function of LL-37 within NETs is still unknown because it loses its antimicrobial activity when bound to DNA in the NETs. Using immunofluorescence microscopy, we demonstrate that NETs treated with LL-37 are distinctly more resistant to S. aureus nuclease degradation than nontreated NETs. Biochemical assays utilising a random LL-37-fragment library indicated that the blocking effect of LL-37 on nuclease activity is based on the cationic character of the AMP, which facilitates the binding to neutrophil DNA, thus protecting it from degradation by the nuclease. In good correlation to these data, the cationic AMPs human beta defensin-3 and human neutrophil peptide-1 showed similar protection of neutrophil-derived DNA against nuclease degradation. In conclusion, this study demonstrates a novel role of AMPs in host immune defence: beside its direct antimicrobial activity against various pathogens, cationic AMPs can stabilise neutrophil-derived DNA or NETs against bacterial nuclease degradation.

Structural and thermodynamic insights into the recognition of native proteins by anti-peptide antibodies.

The mechanism by which antibodies elicited against protein-derived peptides achieve cross-reactivity with their cognate proteins remains unknown. To address this question, we have carried out the complete thermodynamic characterization of the association of a monoclonal antibody (260.33.12) raised against a peptide (SNpep) derived from staphylococcal nuclease (SNase) with both eliciting peptide and cognate protein. Although both ligands bind with similar affinity (Kd=0.42 µM and 0.30 µM for protein and peptide, respectively), protein and peptide binding have highly different thermodynamic signatures: peptide binding is characterized by a large enthalpic contribution (ΔH=-7.7 kcal/mol) whereas protein binding is dominated by a large entropic contribution (-TΔS=-7.2 kcal/mol). The structure of the SNpep:Fab complex, determined by X-ray diffraction, reveals that the bound conformation of the peptide differs from the conformation of the corresponding loop region in crystal structures of free SNase. The energy difference, estimated by molecular dynamics simulations between native SNase and a model in which the Ω-loop is built in the conformation of the Fab-bound peptide, shows that the energetic cost of adopting this conformation is compatible with the enthalpic cost of binding the protein vis-à-vis the peptide. These results are compatible with a mechanism by which the anti-peptide antibody recognizes the cognate protein: high affinity is maintained upon binding a non-native conformation by offsetting enthalpic penalties with reduced entropic losses. These findings provide potentially useful guidelines for the identification of linear epitopes within protein sequences that are well suited for the development of synthetic peptide vaccines.

Bispecific antibody-mediated detection of the Staphylococcus aureus thermonuclease.

We report a novel fluorescence-based immunoassay which enables qualitative detection of the Staphylococcus aureus Thermonuclease (TNase) enzyme, thus providing confirmation of the presence of the S. aureus bacterium in vitro. The biomedical problem of chronic wound healing and the continuing emergence of antibiotic-resistant species is addressed in the development of a detection system capable of the rapid, real-time assessment of bacterial load and diversity. The use of bispecific antibodies (BsAb) provides integration of the molecular detection and signal response components of a standard immunoassay due to steric hindrance-mediated release of prebound fluorescent reporter molecules upon specific binding of TNase to adjacent sites. Rhodamine and fluorescein-labeled hemocyanin from Megathura crenulata (KLH) were prepared as effective immunoconjugates containing a sensitive fluorescent reporter moiety. BsAb that both specifically quenched the fluorescence of the reporter conjugate and bound the TNase target antigen were produced using cell fusion techniques. Assays were then performed to analyze the properties attributable to the steric hindrance-mediated release of the fluorescent reporter molecules upon adjacent TNase binding. This was performed by monitoring the intensity of fluorescence emission of the immunogenic reporter conjugate released into an aqueous environment at 578 and 520 nm, respectively.

[Expression and nuclease activity analysis of staphylococcus nuclease in E.coli].

AIM: To express staphylococcus nuclease (SN) in E.coli and prepare rabbit antisera against SN. METHODS: The SN gene was amplified by high-fidelity PCR from plasmid pPLC-SN and then subcloned into expression vector pLEX to obtain the recombinant plasmid pLEX-SN. The expression of recombinant protein was induced by tryptophan. The expressed SN was used to immunize a rabbit to prepare specific antibody. RESULTS: SDS-PAGE analysis showed that the relative molecular mass (M(r)) of the expressed SN was about 17,000 and the expressed SN accounted for about 37% of total bacterial proteins. The prepared antisera were specific to react with recombinant SN. CONCLUSION: Expression vector of SN has been successfully constructed and rabbit antibody against SN was prepared. These results lay the foundation for developing SN as antiviral protein.

Improvement of bovine beta-lactoglobulin production and secretion by Lactococcus lactis.

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Improvement of bovine ß-lactoglobulin production and secretion by Lactococcus lactis

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 æg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Characterization of a Lactococcus lactis strain that secretes a major epitope of bovine beta-lactoglobulin and evaluation of its immunogenicity in mice.

Bovine beta-lactoglobulin (Blg) is one of the major cow's milk allergens. Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope. The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacterium Lactococcus lactis and the characterization of its immunogenicity in mice. We constructed a recombinant strain of L. lactis capable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc). The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction. At this time, up to 75% of Blg41-60::Nuc was secreted. When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities. Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinant L. lactis resulted in the induction of specific anti-Blg41-60 IgG2a and IgG1. The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response. Similar administrations of the killed Blg41-60::Nuc-producing L. lactis strain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producing L. lactis strain.

An automated method for the detection of staphylococcal heat stable deoxyribonuclease in dairy products.

An automated sandwich immunoassay with specific polyclonal antibodies for the detection of Staphylococcus aureus thermostable nuclease (DNase) is described. To evaluate this assay, different quantities of purified S. aureus nuclease were added to dairy products. Additionally, staphylococcal counts and nuclease activity of milk samples inoculated with S. aureus were determined. Different extraction procedures were performed and compared. The results indicated that the automated test was a reliable method for detecting DNase activity in milk products. The procedure was completed in 2 h and detected 1 ng of DNase ml-1. Detection of the DNase was especially useful in cheeses and could be used to confirm positive enterotoxin results.

A monoclonal antibody selected for probing the folding of staphylococcal nuclease and its N-terminal fragments.

Monoclonal antibody McAb2C9 against Staphylococcal nuclease (SNase R) and its N-terminal fragments was produced and characterized. It was observed that the intact enzyme SNase R and its seven fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) differed in their interactions with McAb2C9. However, the fragments with weak immunoreactivity, such as SNR141 and SNR110, increased ability reacting with McAb2C9 in their partially unfolded state. It suggests that the differences of immunoreactivity among the fragments are due to diverse extent of the exposure of the specific epitope and the conformation of the peptide fragment. The monoclonal antibody McAb2C9 could be a useful probe to investigate the mechanism of folding of SNase R and its N-terminal fragments.

The immunodominant region of Staphylococcal nuclease is represented by multiple peptide sequences.

Several published reports have lead to the characterization of naturally processed peptides that are presented in association with either class I or class II MHC molecules. Most peptides isolated from class II molecules are heterogeneous in length and exhibit ragged amino and carboxy termini. An intriguing finding was that one region of a molecule was often represented by many distinct peptides, rather than by a single dominant peptide species. Each of the peptides representing this dominant region exhibited a common core of amino acids, suggesting that this core may play a significant role in the binding of the peptide to class II and the recognition by peptide-specific T cells. Work from our laboratory has focused on the mechanisms involved in the immunodominance of antigenic determinants using the bacterial antigen Staphylococcal nuclease (Nase) as a model. Using truncated synthetic peptides, we have identified the immunodominant determinant of Nase to be located within the region 81-100 with a minimal antigenic core of 91-100 as determined. Addition of five residues to the carboxy terminus of this peptide had a negative effect on T cell recognition of this region. The present studies were undertaken in an effort to determine the sequence of the naturally processed immunodominant Nase determinant(s) presented in association with I-Ek class II. Our results indicate that the dominant region of the Nase molecule is represented by at least four distinct peptide species that are predicted to lie between residues 86 and 106 with a common core sequence of 91-96. These results indicate that the negative effects of flanking regions are dependent upon length and amino acid composition, and thus the use of truncated peptides to study minimal antigenic determinants may be misleading.

The crystal structure of the antibody N10-staphylococcal nuclease complex at 2.9 A resolution.

The three-dimensional structure of the antibody N10 Fab fragment complexed with staphylococcal nuclease (SNase) has been determined to 2.9 A resolution. Eighteen residues from six complementarity-determining regions (CDR) recognize an epitope of five distinct SNase segments with a total of 17 residues. The overall shape of the antibody-antigen interface is U-shaped rather than the more or less rectangular interface seen in other antibody-protein antigen interfaces. Despite the U-shaped interface, the amount of surface buried in the complex, 828 A2 for SNase and 793 A2 for N10, is typical of antibody-protein antigen complexes. Contributing to the shape of the interface is the shortest antibody heavy chain-CDR3 loop reported to date, which probably allows access of bulk solvent in the center of the "U" interface. Another unusual feature of the N10 antibody is the 15 residue antibody light chain-CDR1, a length seen in only three other reported antibodies. Antibody light chain-CDR1 displays a previously unobserved conformation in its distal portion. Finally, although some of the movement observed in the antibody-bound SNase may be due to crystal contacts, it is clear that some side-chain rearrangements are the result of antigen-antibody interaction.

Comparison of tests designed to identify Staphylococcus aureus thermostable nuclease.

The thermonuclease test has been employed for same-day identification of Staphylococcus aureus in blood cultures and a seroinhibition test to confirm S. aureus thermonuclease (TNase) by using polyclonal anti-S. aureus TNase antiserum. However, strains of non-S. aureus staphylococci may produce TNases which are neutralized by the antiserum. This study evaluated alternative reagents and confirmatory tests for the S. aureus TNase. The tests included seroinhibition by a monoclonal antibody (MAb) against S. aureus TNase, MAb-based detection of the TNase in a sandwich ELISA, and a polymerase chain reaction for amplification of the nuc gene encoding the S. aureus TNase. All these tests discriminated between TNase produced by S. aureus and TNase produced by strains of the species S. caprae, S. carnosus, S. simulans, S. capitis, and S. intermedius. Specificity for the S. aureus TNase was confirmed by an inhibition ELISA for two out of three MAbs tested. Thus, the MAb- and nuc-based tests will be useful to discriminate between TNases from S. aureus and non-S. aureus staphylococci.

Interference with the binding of a naturally processed peptide to class II alters the immunodominance of T cell epitopes in vivo.

T lymphocytes elicited in response to an immunizing Ag usually recognize only one or a few immunodominant peptides. The mechanisms governing this process are poorly understood. This study examines the consequences of peptide competition on immunodominance. Immunization of B10.A mice with the native Staphylococcus aureus nuclease protein primes T cells to the dominant 86-100 peptide presented in association with I-Ek class II molecules. To render the 86-100 peptide incapable of binding to the class II molecule, single amino acid substitutions were introduced in the native Staphylococcus aureus nuclease protein within a putative I-Ek class II binding motif. Introduction of residue changes at positions 89 and 91 in the protein prevents 86-100-specific T cell clone recognition of the protein in vitro. Competition studies demonstrate that substitutions at residues 89 or 91 decreased the I-Ek binding affinity of the 86-100 peptide. Immunization of B10.A mice with the L89F or Y91S mutant proteins does not prime T cells to the dominant 86-100 peptide; T cells are primed instead to I-Ek-restricted subdominant peptide(s) encompassed by the residues 111-135. In vitro binding studies demonstrate that both the 111-130 and 116-135 synthetic peptides compete with a labeled I-Ek-binding peptide 20-fold less efficiently than the dominant 86-100 peptide, suggesting that these subdominant peptides may be of lower binding affinity than the dominant 86-100 peptide. These results support the hypothesis that dominance is dependent on peptide binding affinity for the appropriate class II molecule and the ability to compete with other peptides, derived from the same Ag, for class II binding.

Isolation of a membrane-associated cathepsin D-like enzyme from the model antigen presenting cell, A20, and its ability to generate antigenic fragments from a protein antigen in a cell-free system.

The biochemical mechanisms and enzymes involved in the processing of protein antigens for presentation by major histocompatibility complex class II molecules are poorly understood. This work describes the purification of a cathepsin D-like enzyme isolated from the murine B lymphoma cell line A20, a model antigen presenting cell. Two forms of cathepsin D-like enzyme were detected. One is soluble and located in the lysosome-enriched subcellular fraction. The other is membrane-associated and located in the endosome-enriched fraction. The membrane-associated form was purified to apparent homogeneity by affinity chromatography on pepstatin A-Sepharose. Its apparent molecular weight is 48,000, and its pH optimum is pH 4.0. Endosomal cathepsins are known to be involved in antigen processing in vivo, and the purified membrane-associated cathepsin D-like enzyme from A20 cells was used to study antigen processing in vitro. The enzyme cleaved a model protein antigen, Staphylococcus aureus nuclease (Nase) and thereby generated antigenic fragments recognized by a Nase-specific T cell hybridoma. Such studies have allowed us to begin to understand the role of protease specificity and T cell determinant selection.

Immunodominance: a single amino acid substitution within an antigenic site alters intramolecular selection of T cell determinants.

The mechanism of immunodominance was studied by mutating a single amino acid residue within an immunodominant determinant of Staphylococcus aureus nuclease (Nase). Residues 81 to 100, which can be further reduced to 86 to 100, were determined to be the immunodominant determinant of Nase in H-2k mice. By introducing selected single amino acid substitutions into the peptide encompassing residues 86 to 100 (p86-100), residue 90 was shown to be one of the critical amino acids for T cell recognition, inasmuch as most of the T cells recognizing p86-100 do not respond to a p86-100 analog with a substitution of leucine for alanine at the residue 90. A mutant of Nase with a replacement of alanine by leucine at residue 90 (A90L) was constructed, and for A90L region 112 to 130, which is a subdominant determinant in Nase, becomes immunodominant. Although unable to respond to Nase, T cells primed in vivo with the peptides covering various cryptic determinants proliferate when challenged with A90L in vitro. Our results suggest that at the protein level there is competition among potential T cell determinants of protein Ag for binding to MHC molecules, and that this competition plays a role in determining which determinant may become immunodominant.

Detection of Staphylococcus aureus with biotinylated monoclonal antibodies directed against staphylococcal TNase complexed to avidin-peroxidase in a rapid sandwich enzyme-linked immunofiltration assay (sELIFA).

For rapid identification of Staphylococcus aureus, a monoclonal antibody (MAb)-biotin-avidin-peroxidase complex, directed against the S. aureus thermostable nuclease (TNase), was formed and used in a rapid three-step sandwich enzyme-linked immunofiltration assay (sELIFA) and a three-step sandwich enzyme-linked immunosorbent assay (sELISA). The MAb-peroxidase complex was formed by incubating the biotinylated MAbs with a streptavidin-peroxidase conjugate and the complex was purified by gel permeation chromatography. When compared with a four-step MAb-based sELISA described previously, this complex permitted one reagent step to be omitted in a three-step sELISA, and the test time was significantly reduced. The test sensitivity was slightly reduced in the three-step ELISA (detection limit 1.0-2.0 ng of TNase/ml) when compared to the four-step sELISA (detection limit 0.5-1.0 ng of TNase/ml). The sELIFA method was based on the filtration of bacterial culture supernates through nitrocellulose membrane disks pre-spotted with a MAb directed against the S. aureus TNase, followed by detection with the MAb-peroxidase complex (three-step sELIFA). A detection limit of 0.5-2.0 ng of TNase/ml was achieved with the three-step sELIFA, depending on the filtrate volume of culture supernates. The total test time was 10-15 min when pre-spotted and blocked membranes were used. A total of 85 bacterial strains was tested in the sELIFA. All the 28 S. aureus strains showed positive results, but none of the 57 non-S. aureus strains did so, although some of these produced thermostable nuclease activity.(ABSTRACT TRUNCATED AT 250 WORDS)