Chimeric fab fragments of antibodies to recombinant Ebola virus glycoprotein

Previously, we have determined the nucleotide and amino acid sequences of the variable domains of three mouse monoclonal antibodies specific to the individual epitopes of the Ebola virus glycoprotein: GPE118 (IgG), GPE325 (IgM) and GPE534 (IgG) [1]. In the present paper, chimeric Fab fragments of Fab118, Fab325, and Fab534 antibodies were obtained based on the variable domains of murine antibodies by attaching CH1 and CL constant regions of human kappa-IgG1 to them. The recombinant chimeric Fab fragments were synthesized in the heterologous expression system Escherichia coli, isolated and purified using metal chelate affinity chromatography. The immunochemical properties of the obtained Fab fragments were studied by immunoblotting techniques as well as indirect and competitive ELISA using recombinant Ebola virus proteins: EBOV rGPdTM (recombinant glycoprotein of Ebola hemorrhagic fever virus without the transmembrane domain), NP (nucleoprotein) and VP40 (structural protein). The identity of recombinant chimeric Fab fragments, as well as their specificity to the recombinant glycoprotein of Ebola hemorrhagic fever virus (EBOV GP) was proved. The results of indirect ELISA evidence the absence of immunological cross-reactivity to NP and VP40 proteins of Ebola virus. The dissociation constants of the antigen-antibody complex K _d equal to 5.0, 1.0 and 1.0 nM for Fab118, Fab325 and Fab534, respectively, were determined; they indicate high affinity of the obtained experimental samples to EBOV GP. The epitope specificity of Fab fragments was studied using a panel of commercial neutralizing antibodies. It was found that all studied antibodies to EBOV GP are targeted to different epitopes, while the epitopes of the recombinant chimeric Fab fragments and original murine monoclonal antibodies (mAbs) coincide. All the obtained and studied mAbs to EBOV GP are specific to epitopes that coincide or overlap the epitopes of three commercial neutralizing mAbs to Ebola virus: epitopes Fab118 and Fab325 overlap the epitope of the known commercial mAb h13F6; Fab325 epitope also overlaps mAb c6D8 epitope; Fab534 epitope is located near mAb KZ52 conformational epitope, in the formation of which amino acid residues of GP1 and GP2 domains of EBOV GP are involved.     

Flexible docking-based molecular dynamics simulation of natural product compounds and Ebola virus Nucleocapsid (EBOV NP): a computational approach to discover new drug for combating Ebola

Background

Ebola still remains as one of the most problematic infectious diseases in Africa with a high rate of mortality. Although this disease has been known for an almost half-century, there are no vaccines and drugs available in the market to treat Ebola. Zaire ebolavirus (EBOV), a single-stranded RNA virus which belongs to Filoviridae family and Mononegavirales order, is one of the virus causing Ebola. As one of seven proteins that EBOV encodes, Ebola virus nucleoprotein (EBOV NP) plays an imperative role in EBOV proliferation cycle. Therefore, the development of a new Ebola treatment can be targeted towards EBOV NP.

Results

In this work, we screened about 190,084 natural product compounds from ZINC15 database through in silico virtual screening and flexible docking simulation. Furthermore, the bioavailability and toxicity prediction have been conducted as well. Two best ligands according to the simulation and prediction tests were progressed into the molecular dynamics simulation.

Conclusion

In the end, we found that our proposed ligands, namely α-lipomycin (ZINC56874155) and 3-(((S)-1-amino-1,2,3,4-tetrahydroisoquinolin-5-yl)methyl)-5-((5-((5R,7S)-5,7-dihydroxy-3-oxodecyl)-2-hydroxyphenoxy) methyl)pyrrolo[3,4-b]pyrrol-5-ium (ZINC85628951), showed the promising results to be developed as a lead compounds for treating Ebola. Therefore, an experimental study is required to validate their inhibition activities against EBOV NP.

     

A new strategy for full-length Ebola virus glycoprotein expression in E.coli

Ebola virus(EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein(GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses.However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 ℃. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.     

Application of monoclonal antibodies generated against Panton-Valentine Leukocidin (PVL-S) toxin for specific identification of community acquired methicillin resistance Staphylococcus aureus

Panton-Valentine Leukocidin (PVL) produced by community acquired methicillin Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing pneumonia comprised of two fractions, namely PVL S and PVL F. In the present study, three monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against recombinant PVL-S (35 kDa) protein of S. aureus. All the three MAbs specifically reacted to confirm PVL-S positive strains of S. aureus recovered from clinical samples in Western blot analysis. Similarly all the three MAbs did not show any binding to other tested 14 different pathogenic bacteria belonging to other genera and species in Western blot analysis. Furthermore, a simple dot-ELISA method was standardized for the identification of PVL-S toxin containing S. aureus strains. Initially in dot-ELISA, Protein A (Spa) of S. aureus posed background noise problems due to the non-specific binding of antibodies resulting in false positive reactions. With the addition of 10 mM diethylpyrocarbonate (DEPC) along with 5% milk in PBS in the blocking step prevented this non-specific binding of Spa to mouse anti-PVL monoclonal antibodies in dot-ELISA. Once standardized, this simple dot-ELISA was evaluated with nine PVL positive strains recovered from food, environmental and clinical samples and the results were compared with PCR assay for the presence of PVL toxin genes and also with Western blot analysis. A 100% correlation was found between dot-ELISA, PCR assay and Western blot analysis. Collectively our results suggest the newly developed simple dot-ELISA system can be of immense help in providing, rapid detection of the PVL toxin containing S. aureus strains at a relatively low cost and will be a valuable tool for the reliable identification of CA-MRSA.     

Intramammary immunization with live-attenuated Staphylococcus aureus protects mice from experimental mastitis

Female mice were immunized by the intramammary route with live-attenuated Staphylococcus aureus according to different schedules and challenged with virulent S. aureus. Immunization in late pregnancy or early lactation induced a significant decrease (P<0.05) in the number of S. aureus CFU recovered from glands after the challenge and a significant increase (P<0.05) in the levels of milk and serum specific IgG and IgA antibodies. Mice immunized before pregnancy were not protected from S. aureus challenge. Immunization did not increase the number of somatic cells in milk when compared with control mice. Protection from S. aureus intramammary infection may be achieved if mice are locally immunized during late pregnancy or early lactation.     

Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

Background

Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.

Methods/Principal Findings

We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures.

Conclusion/Significance

Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.     

Electrical protein array chips for the detection of staphylococcal virulence factors

A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.     

Isolation of Highly Active Monoclonal Antibodies against Multiresistant Gram-Positive Bacteria

Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL) of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA). At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium), a mouse peritonitis model (using S. aureus Newman and LAC) and a rat endocarditis model (using E. faecalis 12030) and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.     

PIA and rSesC Mixture Arisen Antibodies Could Inhibit the Biofilm-Formation in Staphylococcus aureus

Background:Staphylococcus aureus as a causative agent of hospital-acquired infections has been considered as the primary concern in biomaterial-related infections (BAIs).Methods:Following the purification of polysaccharide intercellular adhesion (PIA) as an efficient macromolecule in biofilm formation in the native condition, recombinant S. epidermidis surface-exposed rSesC protein, with the most homology to clumping factor A (ClfA) in S. aureus was cloned and expressed in a prokaryotic host as well. Fourier transform infrared spectrometry (FTIR) and Western blotting procedure analyzed purified PIA and protein, respectively. Then, the immune response was evaluated by measuring total IgG titers. Moreover, the capacity of Anti-biofilm forming activity of arisen antibodies to a biofilm-forming S. aureus strains was assessed by the semi-quantitative micro-plate procedure.Results:Data showed that the total IgGs were boosted in mice immunized sera. By performing an inhibition assay, the biofilm inhibitory effect of secreted antibodies to test strain was observed. Arisen antibodies against the mixture significantly were more potent than PIA and rSesC, when comparing individual antigens in a biofilm inhibition assay.Conclusion:immunization of mice with mentioned antigens especially a mixture of them, could eliminate the biofilm formation process in S. aureus. Hopefully, this study corresponds to the suggestion that the immunization of mice with PIA and rSesC candidate vaccines could protect against S. aureus infection.Key Words: PIA, Purification, Staphylococcus aureus, rSesC, Vaccine candidates     

Glycoepitopes of Staphylococcal Wall Teichoic Acid Govern Complement-mediated Opsonophagocytosis via Human Serum Antibody and Mannose-binding Lectin

Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or β-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, β-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that β-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact β-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-β-GlcNAc-specific WTA IgGs, anti-β-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.     

In Silico Derived Small Molecules Bind the Filovirus VP35 Protein and Inhibit Its Polymerase Cofactor Activity

The Ebola virus (EBOV) genome only encodes a single viral polypeptide with enzymatic activity, the viral large (L) RNA-dependent RNA polymerase protein. However, currently, there is limited information about the L protein, which has hampered the development of antivirals. Therefore, antifiloviral therapeutic efforts must include additional targets such as protein–protein interfaces. Viral protein 35 (VP35) is multifunctional and plays important roles in viral pathogenesis, including viral mRNA synthesis and replication of the negative-sense RNA viral genome. Previous studies revealed that mutation of key basic residues within the VP35 interferon inhibitory domain (IID) results in significant EBOV attenuation, both in vitro and in vivo. In the current study, we use an experimental pipeline that includes structure-based in silico screening and biochemical and structural characterization, along with medicinal chemistry, to identify and characterize small molecules that target a binding pocket within VP35. NMR mapping experiments and high-resolution x-ray crystal structures show that select small molecules bind to a region of VP35 IID that is important for replication complex formation through interactions with the viral nucleoprotein (NP). We also tested select compounds for their ability to inhibit VP35 IID–NP interactions in vitro as well as VP35 function in a minigenome assay and EBOV replication. These results confirm the ability of compounds identified in this study to inhibit VP35–NP interactions in vitro and to impair viral replication in cell-based assays. These studies provide an initial framework to guide development of antifiloviral compounds against filoviral VP35 proteins.     

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin‐derived protein

New anti‐infective approaches are much needed to control multi‐drug‐resistant (MDR) pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S. aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S. aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S. aureus were reduced 2.4‐ to 23.4‐fold as response to V12CBD. More significantly, V12CBD could activate macrophages through NF‐κB pathway and enhance phagocytosis against S. aureus. As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS. aureus infections.     

Risk Factors Associated with Ebola and Marburg Viruses Seroprevalence in Blood Donors in the Republic of Congo

BackgroundEbola and Marburg viruses (family Filoviridae, genera Ebolavirus and Marburgvirus) cause haemorrhagic fevers in humans, often associated with high mortality rates. The presence of antibodies to Ebola virus (EBOV) and Marburg virus (MARV) has been reported in some African countries in individuals without a history of haemorrhagic fever. In this study, we present a MARV and EBOV seroprevalence study conducted amongst blood donors in the Republic of Congo and the analysis of risk factors for contact with EBOV.Conclusions/SignificanceThis MARV and EBOV serological survey performed in the Republic of Congo identifies a probable role for environmental determinants of exposure to EBOV. It highlights the requirement for extending our understanding of the ecological and epidemiological risk of bats (previously identified as a potential ecological reservoir) and birds as vectors of EBOV to humans, and characterising the protection potentially afforded by EBOV-specific antibodies as detected in blood donors.     

PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans

Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in colonization and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was constructed in a pneumococcal TIGR4 background. When the mutant and wild type strains were compared for their adherence to D562 and A549 cell lines, a reduction in adherence by the mutant was observed (p = 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host Escherichia coli resulting in augmented adherence to D562 (p = 0.002) and A549 (p = 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p = 0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p = 0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.     

Preparing a highly specific inert immunomolecular-magnetic beads for rapid detection and separation of <Emphasis Type="Italic">S. aureus</Emphasis> and group G <Emphasis Type="Italic">Streptococcus</Emphasis>

The rapid detection and separation of Staphylococcus aureus and group G Streptococcus was based on the affinity chromatography interactions between Fc fragment of human IgG and protein A/G (located on the cell wall of S. aureus and group G Streptococcus). In this case, immobilization of antibodies had to take place in a different and complementary way than in the case of conventional immunosensors. In this study, three different kinds of immunomolecular-magnetic beads (IMB) were prepared for rapid detection and separation of S. aureus and group G Streptococcus (GGS). The Fc regions of the immobilized antibodies were fully accessible to adsorb protein A or protein G. On the contrary, conventional immunosensors had to have fully accessible Fab regions to facilitate the antigen–antibody recognition. It was suggested that the worse method of immobilization of the antibodies for conventional use would yield the better results for this specific use. In this study, we also perfectly solved the nonspecific adsorptions and interaction problems, which were the most serious critical problems for all kinds of sensors. It was achieved by blocking the excess surface groups of aldehyde IMB and the Fab region of the immobilized antibodies with aldehyde-dextran.     

A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus

Background

Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the ‘bush-meat’ trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.

Methodology/Principal Findings

We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a ‘proof-of-concept’ for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8⁺ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP_CTL). MCMV/ZEBOV-NP_CTL induced high levels of long-lasting (>8 months) CD8⁺ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.

Conclusions/Significance

This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for ‘disseminating’ CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.     

Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase

Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability.     

Staphylococcus aureus and Pseudomonas aeruginosa Express and Secrete Human Surfactant Proteins

Surfactant proteins (SP), originally known from human lung surfactant, are essential to proper respiratory function in that they lower the surface tension of the alveoli. They are also important components of the innate immune system. The functional significance of these proteins is currently reflected by a very large and growing number of publications. The objective goal of this study was to elucidate whether Staphylococcus aureus and Pseudomonas aeruginosa is able to express surfactant proteins. 10 different strains of S. aureus and P. aeruginosa were analyzed by means of RT-PCR, Western blot analysis, ELISA, immunofluorescence microscopy and immunoelectron microscopy. The unexpected and surprising finding revealed in this study is that different strains of S. aureus and P. aeruginosa express and secrete proteins that react with currently commercially available antibodies to known human surfactant proteins. Our results strongly suggest that the bacteria are either able to express ‘human-like’ surfactant proteins on their own or that commercially available primers and antibodies to human surfactant proteins detect identical bacterial proteins and genes. The results may reflect the existence of a new group of bacterial surfactant proteins and DNA currently lacking in the relevant sequence and structure databases. At any rate, our knowledge of human surfactant proteins obtained from immunological and molecular biological studies may have been falsified by the presence of bacterial proteins and DNA and therefore requires critical reassessment.     

Characteristics of new Staphylococcus aureus-RBC adhesion mechanism independent of fibrinogen and IgG under hydrodynamic shear conditions

Staphylococcus aureus infection begins when bacterial cells circulating in blood adhere to components of the extracellular matrix or endothelial cells of the host and initiate colonization. S. aureus is known to exhibit extensive interactions with platelets. S. aureus is also known to bind to red blood cells (RBCs) in the presence of plasma proteins, such as fibrinogen and IgG. Herein we report a new binding mechanism of S. aureus to RBC independent of those plasma proteins. To characterize the new adhesion mechanism, we experimentally examine the binding kinetics and molecular constituents mediating the new adhesive interactions between S. aureus and RBCs under defined shear conditions. The results demonstrate that the receptors for fibrinogen (clumping factor A) and IgG (protein A) of S. aureus are not involved in the adhesion. S. aureus binds to RBCs with maximal adhesion at the shear rate 100 s^–1 and decreasing adhesion with increasing shear. The heteroaggregates formed after shear are stable when subjected to the shear rate 2,000 s^–1, indicating that intercellular contact time rather than shear forces controls the adhesion at high shear. S. aureus binding to RBC requires plasma, and 10% plasma is sufficient for maximal adhesion. Plasma proteins involved in the cell-cell adhesion, such as fibrinogen, fibronectin, von Willebrand factor, IgG, thrombospondin, laminin, and vitronectin are not involved in the observed adhesion. The extent of heteroaggregation is dramatically reduced on RBC treatment with trypsin, chymotrypsin, or neuraminidase, suggesting that the receptor(s) mediating the heteroaggregation process is a sialylated glycoprotein on RBC surface. Adhesion is divalent cation dependent and also blocked by heparin. This work demonstrates a new mechanism of S. aureus-RBC binding under hydrodynamic shear conditions via unknown RBC sialoglycoprotein(s). The binding requires plasma protein(s) other than fibrinogen or IgG and does not involve the S. aureus adhesins clumping factor A or protein A. adhesion; red blood cell     

Efficient expression of a Trypanosoma cruzi antigen in Escherichia coli and Staphylococcus aureus and its rapid purification

A Trypanosoma cruzi antigen gene was closed into a fusion vector based on the IgG binding domain of Staphylococcus aureus protein A. This vector transformed into Escherichia coli or Staphylococcus aureus and produced about 12 mg fusion protein/l culture. In E. coli, the product remained intracellular while in S. aureus it was excreted into the growth medium. The hybrid protein was purified by IgG Sepharose affinity chromatography. The presence of a cleavage site for enterokinase between protein A and the T. cruzi antigen in the fusion protein allowed the efficient release of the unfused antigen by enzymatic treatment. Further affinity chromatography through IgG Sepharose resulted in the production of the T. cruzi antigen free of protein A.The authors are with the Department of Molecular Genetics, BioSidus S.A., Constitución 4234, 1254, Buenos Aires, Argentina.