Showing papers on "Protein A published in 2013"
TL;DR: It is shown that the Fcγ and VH3-type Fab binding activities of staphylococcal protein A (SpA) are essential for S. aureus escape from host immune surveillance in mice and suggest that antibodies and B cells play a key role in the pathogenesis of stAPHylococ CAL infections and provide insights into the development of a vaccine against S.Aureus.
Abstract: Heritable defects in human B cell/antibody development are not associated with increased susceptibility to Staphylococcus aureus infection. Protein A (SpA), a surface molecule of S. aureus, binds the Fcγ domain of immunoglobulin (Ig) and cross-links the Fab domain of V H 3-type B cell receptors (IgM). Here we generated S. aureus spa variants harboring amino acid substitutions at four key residues in each of the five Ig-binding domains of SpA. Wild-type S. aureus required SpA binding to Ig to resist phagocytosis and SpA-mediated B cell receptor cross-linking to block antibody development in mice. The spa KKAA mutant, which cannot bind Ig or IgM, was phagocytosed and elicited B cell responses to key virulence antigens that protected animals against lethal S. aureus challenge. The immune evasive attributes of S. aureus SpA were abolished in µMT mice lacking mature B cells and antibodies. Thus, while wild-type S. aureus escapes host immune surveillance, the spa KKAA variant elicits adaptive responses that protect against recurrent infection. IMPORTANCE Staphylococcus aureus causes recurrent skin and bloodstream infections without eliciting immunity. Heritable defects in neutrophil and T cell function, but not B cell or antibody development, are associated with increased incidence of S. aureus infection, and efforts to develop antibody-based S. aureus vaccines have thus far been unsuccessful. We show here that the Fcγ and V H 3-type Fab binding activities of staphylococcal protein A (SpA) are essential for S. aureus escape from host immune surveillance in mice. The virulence attributes of SpA in mice required mature B cells and immunoglobulin. These results suggest that antibodies and B cells play a key role in the pathogenesis of staphylococcal infections and provide insights into the development of a vaccine against S. aureus.
238 citations
TL;DR: It is demonstrated that higher CP expression renders bacteria more resistant to non-specific opsonophagocytic killing than increased SpA expression, suggesting that the expression of capsular polysaccharides may be the more important immune evasion strategy for S. aureus.
Abstract: Staphylococcus aureus can cause severe life threatening invasive diseases. The principal immune effector mechanism by which humans are protected from Gram positive bacteria such as S. aureus is antigen specific antibody- and complement-dependent opsonophagocytosis. This process can be measured in vitro using the opsonophagocytic antibody assay (OPA), which is a complex assay composed of live S. aureus bacteria, a complement source, phagocytic effector cells such as differentiated HL-60 cells, and test serum. In this report, we investigated the impact on the OPA of S. aureus surface antigens capsular polysaccharides (CP) and protein A (SpA). We demonstrated that higher CP expression renders bacteria more resistant to non-specific opsonophagocytic killing than increased SpA expression, suggesting that the expression of capsular polysaccharides may be the more important immune evasion strategy for S. aureus. Bacteria that were not fully encapsulated were highly susceptible to non-specific killing in the assay in the absence of immune serum. This non-specific killing was prevented by growing the bacteria under conditions that increased capsular polysaccharide levels on the surface of the bacteria. In contrast, the level of SpA expression had no detectable effect on non-specific killing in OPA. Using anti-CP antibodies we demonstrated type-specific killing in OPA of both MRSA and MSSA clinical isolates. SpA expression on the cell surface did not interfere with OPA activity, providing evidence that despite the role of SpA in sequestering antibodies by their Fc region, killing is easily accomplished in the presence of high titered anti-capsular polysaccharide antibodies. This highlights the role of CP as an important immune evasion mechanism and supports the inclusion of capsular polysaccharide antigens in the formulation of multi-component prophylactic vaccines against S. aureus.
104 citations
TL;DR: It is demonstrated that the immobilizing mechanism was effective in enhancing the retention of IgG locally in vivo in the in situ-forming injectable system used to enhance the delivery of immune modulatory antibodies in tumors.
Abstract: The performance of an in situ-forming injectable membrane designed to retain antibody molecules in vivo is described. The system entails an aqueous mixture of peptide amphiphiles (referred to as"EAK16-II" and "EAKH6") and intermediate proteins (anti-H6 antibody and protein A/G) through which therapeutic IgG molecules are colocalized and oriented. Scanning electron micrographs show IgG molecules localized on the EAK16-II/EAKH6 membrane. IgG were captured via specific interactions and remained biologically active in vitro. Upon administration into mice subcutaneously, the amphiphilic peptides coassembled into stable His-tags displaying materials locally. The system was shown to retain in vivo a fluorescent dye-labeled IgG in two epithelial tumor lines. IgG coadministered with the system were found to remain in 4T1 mouse mammary tumors for up to 120 h, while free antibody was cleared within the first 24 h. Decreased clearance was also found in B16 melanoma established in mouse footpads. These studies demonstrated that the immobilizing mechanism was effective in enhancing the retention of IgG locally in vivo. The injectable system may be used to enhance the delivery of immune modulatory antibodies in tumors.
84 citations
TL;DR: These studies are the first to demonstrate the importance of the TNFR-1-SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction.
Abstract: Staphylococcus aureus is the major pathogen among the staphylococci and the most common cause of bone infections. These infections are mainly characterized by bone destruction and inflammation, and are often debilitating and very difficult to treat. Previously we demonstrated that S. aureus protein A (SpA) can bind to osteoblasts, which results in inhibition of osteoblast proliferation and mineralization, apoptosis, and activation of osteoclasts. In this study we used small interfering RNA (siRNA) to demonstrate that osteoblast tumour necrosis factor receptor-1 (TNFR-1) is responsible for the recognition of and binding to SpA. TNFR-1 binding to SpA results in the activation of nuclear factor kappa B (NFκB). In turn, NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin 6 (IL-6). Silencing TNFR-1 in osteoblasts or disruption of the spa gene in S. aureus prevented both NFκB activation and IL-6 release. As well as playing a key role in proinflammatory reactions, IL-6 is also an important osteotropic factor. Release of IL-6 from osteoblasts results in the activation of the bone-resorbing cells, the osteoclasts. Consistent with our results described above, both silencing TNFR-1 in osteoblasts and disruption of spa in S. aureus prevented osteoclast activation. These studies are the first to demonstrate the importance of the TNFR-1–SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction. Anti-inflammatory drug therapy could be used either alone or in conjunction with antibiotics to treat osteomyelitis or for prophylaxis in high-risk patients.
80 citations
TL;DR: Findings provide strong support for the idea that SpA promotes S. aureus immune evasion in vivo and form the foundation for a new approach in the efforts to develop a vaccine that prevents severe S.aureus infections.
Abstract: Staphylococcus aureus is a prominent cause of human infections worldwide and is notorious for its ability to acquire resistance to antibiotics. Methicillin-resistant S. aureus (MRSA), in particular, is endemic in hospitals and is the most frequent cause of community-associated bacterial infections in the United States. Inasmuch as treatment options for severe MRSA infections are limited, there is need for a vaccine that protects against such infections. However, recent efforts to generate a staphylococcal vaccine have met with little success in human clinical trials. These failures are somewhat puzzling, since the vaccine antigens tested promote opsonophagocytosis in vitro and confer protection in animal infection models. One possibility is that the pathogen inhibits (and/or fails to elicit) the development of protective immunity in humans. Indeed, S. aureus produces numerous molecules that can potentially promote immune evasion, including protein A (SpA), an immunoglobulin (Ig)-binding protein present on the bacterial surface and freely secreted into the extracellular environment. SpA binds the Fc region of antibody and the Fab regions of the B-cell receptor, processes that are known to block opsonophagocytosis and cause B-cell death in vitro . In a recent study, Falugi et al. [F. Falugi, H. K. Kim, D. M. Missiakas, and O. Schneewind, mBio 4(5):e00575-13, 2013] showed that vaccination with spa mutant S. aureus strains lacking antibody Fc- and/or Fab-binding capacity protects against subsequent challenge with the USA300 epidemic strain. The findings provide strong support for the idea that SpA promotes S. aureus immune evasion in vivo and form the foundation for a new approach in our efforts to develop a vaccine that prevents severe S. aureus infections.
78 citations
TL;DR: A new biological entity is described that is designed to treat the brain of humans with MLD following non‐invasive, intravenous infusion of an IgG‐ASA fusion protein.
Abstract: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder of the brain caused by mutations in the gene encoding the lysosomal sulfatase, arylsulfatase A (ASA). It is not possible to treat the brain in MLD with recombinant ASA, because the enzyme does not cross the blood-brain barrier (BBB). In the present investigation, a BBB-penetrating IgG-ASA fusion protein is engineered and expressed, where the ASA monomer is fused to the carboxyl terminus of each heavy chain of an engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor-mediated transport on the endogenous BBB insulin receptor, and acts as a molecular Trojan horse to ferry the ASA into brain from blood. The HIRMAb-ASA is expressed in stably transfected Chinese hamster ovary cells grown in serum free medium, and purified by protein A affinity chromatography. The fusion protein retains high affinity binding to the HIR, EC50 = 0.34 ± 0.11 nM, and retains high ASA enzyme activity, 20 ± 1 units/mg. The HIRMAb-ASA fusion protein is endocytosed and triaged to the lysosomal compartment in MLD fibroblasts. The fusion protein was radio-labeled with the Bolton-Hunter reagent, and the [(125) I]-HIRMAb-ASA rapidly penetrates the brain in the Rhesus monkey following intravenous administration. Film and emulsion autoradiography of primate brain shows global distribution of the fusion protein throughout the monkey brain. These studies describe a new biological entity that is designed to treat the brain of humans with MLD following non-invasive, intravenous infusion of an IgG-ASA fusion protein.
67 citations
TL;DR: Domains of Vipp1 from Arabidopsis thaliana involved in homo-oligomerization as well as association with chloroplast inner envelope membranes are elucidated and structural differences between VIP1 and PspA are highlighted, which might be important in regard to their different function in thylakoid biogenesis and bacterial stress response.
Abstract: The vesicle-inducing protein in plastids 1 (Vipp1) is an essential component for thylakoid biogenesis in cyanobacteria and chloroplasts. Vipp1 proteins share significant structural similarity with their evolutionary ancestor PspA (bacterial phage shock protein A), namely a predominantly α-helical structure, the formation of oligomeric high molecular weight complexes (HMW-Cs) and a tight association with membranes. Here, we elucidated domains of Vipp1 from Arabidopsis thaliana involved in homo-oligomerization as well as association with chloroplast inner envelope membranes. We could show that the 21 N-terminal amino acids of Vipp1, which form the first α-helix of the protein, are essential for assembly of the 2 MDa HMW-C but are not needed for formation of smaller subcomplexes. Interestingly, removal of this domain also interferes with association of the Vipp1 protein to the inner envelope. Fourier transform infrared spectroscopy of recombinant Vipp1 further indicates that Escherichia coli lipids bind tightly enough that they can be co-purified with the protein. This feature also depends on the presence of the first helix, which strongly supports an interaction of lipids with the Vipp1 HMW-C but not with smaller subcomplexes. Therefore, Vipp1 oligomerization appears to be a prerequisite for its membrane association. Our results further highlight structural differences between Vipp1 and PspA, which might be important in regard to their different function in thylakoid biogenesis and bacterial stress response, respectively.
57 citations
TL;DR: It is shown that staphylococcal protein A induces T cell–independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the VH3+ BCR and that S. aureus exploits pDC and TLR to establish B cell–mediated immune tolerance.
Abstract: Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.
52 citations
TL;DR: The results obtained using a protein A-deficient mutant and tnfr1 −/− mice strongly suggest that the increased levels of soluble TNFR1 present during experimental S. aureus infection may neutralize circulating TNF-α and impair the host inflammatory response.
Abstract: Staphylococcus aureus infections are an important public health concern due to their increasing incidence and high rates of mortality. The success of S. aureus as a pathogen is highly related to its enormous capacity to evade the host immune response. The critical role of tumor necrosis factor alpha (TNF-) in the initial host defense against systemic staphylococcal infection has been demonstrated in experimental models and may partially explain the lack of significant benefits observed in clinical trials attempting to neutralize this cytokine in septic patients. S. aureus protein A plays a key role in regulating inflammation through its ability to bind and signal through the TNF- receptor 1 (TNFR1). In this study, we demonstrate that S. aureus, via protein A-mediated signaling, induces early shedding of TNFR1, which precedes the secretion of TNF- in vitro and in vivo. The results obtained using a protein A-deficient mutant and tnfr1 / mice strongly suggest that the increased levels of soluble TNFR1 present during experimental S. aureus infection may neutralize circulating TNF- and impair the host inflammatory response. Early shedding of TNFR1 induced by protein A may constitute a novel mechanism by which S. aureus subverts the host immune response.
48 citations
TL;DR: Biolog phenotype microarrays have been used to show that a ΔcstA mutant has a reduced ability to utilize a number of di- and tri-peptides as nitrogen sources, suggesting that C. jejuni CstA is important not only for peptide utilization, but also it may influence host-pathogen interactions.
Abstract: Campylobacter jejuni is the most frequent cause of severe gastroenteritis in the developed world. The major symptom of campylobacteriosis is inflammatory diarrhoea. The molecular mechanisms of this infection are poorly understood compared to those of less frequent disease-causing pathogens. In a previous study, we identified C. jejuni proteins that antibodies in human campylobacteriosis patients reacted with. One of the immunogenic proteins identified (Cj0917) displays homology to carbon starvation protein A (CstA) from Escherichia coli, where this protein is involved in the starvation response and peptide uptake. In contrast to many bacteria, C. jejuni relies on amino acids and organic acids for energy, but in vivo it is highly likely that peptides are also utilized, although their mechanisms of uptake are unknown. In this study, Biolog phenotype microarrays have been used to show that a ΔcstA mutant has a reduced ability to utilize a number of di- and tri-peptides as nitrogen sources. This phenotype was restored through genetic complementation, suggesting CstA is a peptide uptake system in C. jejuni. Furthermore, the ΔcstA mutant also displayed reduced motility and reduced agglutination compared to WT bacteria; these phenotypes were also restored through complementation. Murine dendritic cells exposed to UV-killed bacteria showed a reduced IL-12 production, but the same IL-10 response when encountering C. jejuni ΔcstA compared to the WT strain. The greater Th1 stimulation elicited by the WT as compared to ΔcstA mutant cells indicates an altered antigenic presentation on the surface, and thus an altered recognition of the mutant. Thus, we conclude that C. jejuni CstA is important not only for peptide utilization, but also it may influence host–pathogen interactions.
47 citations
TL;DR: Tetrameric gp350(1-470) is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.
TL;DR: Chicken egg yolk IgY over mammalian IgG as capture antibody prevents both soluble and surface bound protein A from causing false positives quantified by chicken anti-protein A antibodies, demonstrated by development of sandwich ELISA for detection of alpha hemolysin toxin from culture supernatants of S. aureus.
TL;DR: The E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressive AMCase and can be used to elucidate detailed biomedical functions of the mouse AMCase.
Abstract: Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.
Patent•
31 Jul 2013
TL;DR: In this paper, the authors proposed constant regions incorporating a cysteine mutation and linked to a µ tailpiece and antibodies or fusion proteins incorporating the same, which can be used to form multivalent complexes, e.g., pentameric or hexameric structures.
Abstract: The invention provides constant regions incorporating a cysteine mutation and linked to a µ tailpiece and antibodies or fusion proteins incorporating the same. The constant regions include at least CH2 and CH3 regions of an IgG heavy chain constant region including a cysteine mutation and µ tailpiece. Antibodies or fusion proteins incorporating the constant regions gains the ability to form multivalent complexes, e.g., pentameric or hexameric structures. Antibodies or fusion proteins incorporating the constant regions also retain IgG properties including specific binding to protein G, which facilitates purification and may exhibit pH-dependent FcRn binding, which is associated with a relatively long in vivo half-life. Depending on the isotype and subtype, the nature of the antigen and presence of an additional IgG hinge domain, such antibodies or fusion proteins may also have properties of specific binding to protein A, and effector functions such as ADCC, CDC and opsonization.
TL;DR: The notion that even a single mutation on the originally alkali‐stable C domain would improve its alkaline stability supports the notion that an engineered protein A based on this C domain is expected to show remarkable performance as an affinity ligand for immunoglobulin.
Abstract: Protein A affinity chromatography is the standard purification process for the capture of therapeutic antibodies. The individual IgG-binding domains of protein A (E, D, A, B, C) have highly homologous amino acid sequences. From a previous report, it has been assumed that the C domain has superior resistance to alkaline conditions compared to the other domains. We investigated several properties of the C domain as an IgG-Fc capture ligand. Based on cleavage site analysis of a recombinant protein A using a protein sequencer, the C domain was found to be the only domain to have neither of the potential alkaline cleavage sites. Circular dichroism (CD) analysis also indicated that the C domain has good physicochemical stability. Additionally, we evaluated the amino acid substitutions at the Gly-29 position of the C domain, as the Z domain (an artificial B domain) acquired alkaline resistance through a G29A mutation. The G29A mutation proved to increase the alkaline resistance of the C domain, based on BIACORE analysis, although the improvement was significantly smaller than that observed for the B domain. Interestingly, a number of other amino acid mutations at the same position increased alkaline resistance more than did the G29A mutation. This result supports the notion that even a single mutation on the originally alkali-stable C domain would improve its alkaline stability. An engineered protein A based on this C domain is expected to show remarkable performance as an affinity ligand for immunoglobulin.
TL;DR: Two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively are studied, illustrating how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.
Abstract: Affibody molecules are engineered binding proteins, in which the three-helix bundle motif of the Z domain derived from protein A is used as a scaffold for sequence variation. We used phage display to select Affibody binders to staphylococcal protein A itself. The best binder, called ZpA963, binds with similar affinity and kinetics to the five homologous E, D, A, B and C domains of protein A, and to a five-domain protein A construct with an average dissociation constant, K(D), of ~20 nM. The structure of ZpA963 in complex with the Z domain shows that it interacts with a surface on Z that is identical in the five protein A domains, which explains the multi-domain affinity. This property allows for high-affinity binding by dimeric Affibody molecules that simultaneously engage two protein A domains in a complex. We studied two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively. The dimers both bind protein A with high affinity, very slow off-rates and with saturation-dependent kinetics that can be understood in terms of dimer binding to multiple sites. The head-to-tail (ZpA963)2htt dimer binds with an off-rate of k(off) ≤ 5 × 10(-6) s(-1) and an estimated K(D) ≤ 16 pM. The results illustrate how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.
TL;DR: IgG is sensitive to acid treatment; however, it can be highly purified with retention of high affinity by using a TRPA column, which can be used on an industrial scale for the purification of antibody drugs.
Patent•
25 Sep 2013
TL;DR: In this paper, the authors describe novel hetero-dimeric immunoglobulinvariants or fragments thereof, which have reduced or eliminated binding to Protein A, Protein G or both Protein A and Protein G.
Abstract: The present invention describes novel hetero-dimeric immunoglobulinvariants or fragments thereof, which have reduced or eliminated binding to Protein A, Protein G or both Protein A and Protein G. Also encompassed in the present invention are methods for the selective purification of hetero-dimeric immunoglobulins or fragments thereof using Protein A and Protein G.
TL;DR: A bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation is presented.
Abstract: We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine-HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.
TL;DR: The findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocyte proliferation in normal and pathological human skin.
TL;DR: It is demonstrated that S. aureus isolates with truncated SpA, not anchored to the cell wall, can still be found in bacteraemia, infection and among carriers.
Abstract: Staphylococcus aureus is a major human pathogen and a multitude of virulence factors enables it to cause infections, from superficial lesions to life-threatening systemic conditions. Staphylococcal protein A (SpA) is a surface protein contributing to S. aureus pathogenesis by interfering with immune responses and activating inflammation. Seven isolates with frameshift mutations in the spa repeat region were investigated to determine whether these mutations lead to truncation and secretion of SpA into the extracellular environment. Five isolates originated from blood cultures, one from an MRSA infection and one from a persistent nasal carrier. Full-length spa genes from the seven isolates were sequenced, and Western blot experiments were performed to localize SpA. Three isolates had identical deviating 25-bp spa repeats, but all isolates displayed different repeat successions. The DNA sequence revealed that the frameshift mutations created premature stop codons in all seven isolates, resulting in truncated SpA of different lengths, however, all lacking the XC region with the C-terminal sorting signal. SpA was detected by Western blot in six of the seven isolates, mainly extracellularly. Our findings demonstrate that S. aureus isolates with truncated SpA, not anchored to the cell wall, can still be found in bacteraemia, infection and among carriers.
TL;DR: The strong fluorescent label of these bioparticles in combination with covalent display of protein functions provides a molecular toolbox for the design of self-assembled microparticles suitable for antibody-capture or ligand binding based diagnostic assays as well as the high affinity purification of target compounds such as antibodies.
TL;DR: Results suggest that surface display of functional βG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.
Abstract: Extracellular activation of hydrophilic glucuronide prodrugs by β-glucuronidase (βG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). βG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane βG (mβG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mβG/lpp). Both mβG/AIDA and mβG/lpp were expressed on the bacterial surface, but only mβG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mβG/AIDA-BL21cells was 2.6-fold greater than by pβG-BL21 cells, which express periplasmic βG. Human colon cancer HCT116 cells that were incubated with mβG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard β-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 μM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 μM), indicating that mβG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional βG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.
TL;DR: A modified peptide reagent useful for the rapid, native elution of protein complexes containing a Protein-A-tagged component and ideal for eluting protein complexes after Protein A / IgG affinity isolation when protease cleavage is not possible or not desirable and facile depletion of the elution reagent is needed.
Abstract: Here we report a modified peptide reagent useful for the rapid, native elution of protein complexes containing a Protein-A-tagged component. We tested this reagent for the elution of tagged endogenous protein complexes from yeast (Nup53p/Nup170p dimer; Nup1p/Kap95p/Kap60p trimer; pentameric GINS complex) and bacteria (RNAP holoenzyme). The majority of the affinity-isolated material is released within 15 minutes under mild conditions, and the elution reagent itself is readily depleted from the elution mixture by simple spin column gel filtration. This reagent is ideal for eluting protein complexes after Protein A / IgG affinity isolation when protease cleavage is not possible or not desirable and facile depletion of the elution reagent is needed.
TL;DR: For the first time, this study shows that fusion with maltose-binding protein (MBP), N-utilization substance protein A, and protein disulfide isomerase enabled the soluble overexpression of EC-SOD in the cytoplasm of E. coli.
Abstract: Extracellular superoxide dismutase (EC-SOD) is the only enzyme that removes superoxide radical in the extracellular space. The reduction of EC-SOD is linked to many diseases, suggesting that the protein may have therapeutic value. EC-SOD is reported to be insoluble and to make inclusion bodies when overexpressed in the cytoplasm of Escherichia coli. The refolding process has the advantage of high yield, but has the disadvantage of frequent aggregation or misfolding during purification. For the first time, this study shows that fusion with maltose-binding protein (MBP), N-utilization substance protein A, and protein disulfide isomerase enabled the soluble overexpression of EC-SOD in the cytoplasm of E. coli. MBP-tagged human EC-SOD (hEC-SOD) was purified by MBP affinity and anion exchange chromatography, and its identity was confirmed by MALDI-TOF MS analysis. The purified protein showed good enzyme activity in vitro; however, there was a difference in metal binding. When copper and zinc were incorporated into hEC-SOD before MBP tag cleavage, the enzymatic activity was higher than when the metal ions were bound to the purified protein after MBP tag cleavage. Therefore, the enzymatic activity of hEC-SOD is associated with metal incorporation and protein folding via disulfide bond.
TL;DR: Methods for extracellular protein microarray production, screening, and analysis are described for enhanced detection of low‐affinity interactions by generating multivalent complexes using Fc‐fusion bait proteins and protein A microbeads, along with a statistical method for hit scoring and identification of nonspecific interactions.
Abstract: Functional protein microarrays offer the capability for high-throughput protein interaction analysis and have long promised to be a powerful tool for understanding protein interactions at the proteome scale. Although popular techniques for protein-protein interaction mapping like yeast-two-hybrid and affinity-purification mass spectrometry have performed well for identifying intracellular protein-protein interactions, the study of interactions between extracellular proteins has remained challenging for these methods. Instead, the use of protein microarrays appears to be a robust and efficient method for the identification of interactions among the members of this class of protein. This unit describes methods for extracellular protein microarray production, screening, and analysis. A protocol is described for enhanced detection of low-affinity interactions by generating multivalent complexes using Fc-fusion bait proteins and protein A microbeads, along with a statistical method for hit scoring and identification of nonspecific interactions.
TL;DR: This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates.
Abstract: We developed a fluorescent assay to conveniently determine the kinetics of protein sulfation, which is essential for understanding interface between protein sulfation and protein–protein interactions. Tyrosylprotein sulfotransferase (TPST) catalyzes protein sulfation using 3′-phosphate 5′-phosphosulfate (PAPS) as sulfuryl group donor. In this report, PAPS was regenerated following sulfuryl group transfer between adenosine 3′,5′-diphosphate and 4-methylumbelliferyl sulfate catalyzed by phenol sulfotransferase (PST). The TPST and PST coupled enzyme platform continuously generated fluorescent 4-methylumbelliferone (MU) that was used to real-time monitor protein sulfation. Using a recombinant N utilization substance protein A fused Drosophila melanogaster tyrosylprotein sulfotransferase, we demonstrated that the activity of TPST determined through MU fluorescence directly correlated with protein sulfation. Kinetic constants obtained with small P-selectin glycoprotein ligand-1 peptide (PSGL-1 peptide, MW 1541) or its large glutathione S-transferase fusion protein (GST-PSGL-1, MW 27833) exhibited significant variation. This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates.
TL;DR: It is suggested that efficient disulfide bond formation using the P. pastoris expression system improves the covalent dimer anti-TNF-VHH-Fc neutralizing activity.
Abstract: Antagonists of tumor necrosis factor alpha (TNFa) have revolutionized the treatment of selected inflammatory diseases. Recombination Camelidae variable heavy-chain domain-only TNFa antibodies (anti-TNF-VHH) have been developed to antagonize the action of human and murine TNFa. Here, we describe a strategy to obtain functional covalent dimer anti-TNF-VHH molecules with the C-terminal fusion of human IgG1 Fc domain named anti-TNF-VHH-Fc. The resulting fusion proteins were separately expressed by use of the pET28a vector in Escherichia coli(Ec) strain BL21 and the pPICZaA vector in Pichia pastoris(Pp) strain GS115, then purified by protein A affinity resin. Fc-engineered anti-EcTNF-VHH-Fc was about 40 kDa and anti-PpTNF-VHH-Fc was about 43 kDa. Monomeric VHH was also cloned and expressed in E. coli strain BL21, with the molecular weight of about 18 kDa. Enzyme-linked immunosorbent assay and L929 cell cytotoxicity assay demonstrated that the fusion protein anti-PpTNF-VHH-Fc blocked TNFa activity more effectively than either anti-EcTNF-VHH-Fc or monomeric anti-EcTNF-VHH protein. We suggest that efficient disulfide bond formation using the P. pastoris expression system improves the covalent dimer anti-TNF-VHH-Fc neutralizing activity.
TL;DR: The results suggest that GPS2 acts as a bridge between NS5A and VAP-A and is required for efficient HCV replication.
Abstract: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of the replication complex associated with various cellular proteins. It has been reported that G protein pathway suppressor 2 (GPS2) is a potential NS5A-binding factor, as identified in a yeast two-hybrid screens of human cDNA library using viral proteins as baits [1]. In this study, we demonstrated the interaction between GPS2 and NS5A in mammalian cells by coimmunoprecipitation analysis and found that both exogenously and endogenously expressed GPS2 interacted with NS5A of genotype 1b and 2a. Mutagenesis study demonstrated that Domain I of NS5A and coiled-coil domain of GPS2 are responsible for the interaction. Knockdown of GPS2 in hepatoma cell lines suppressed the replication of HCV RNA, which can be rescued by the expression of an RNAi-resistant GPS2. Furthermore, overexpression of GPS2 enhanced the association of NS5A with a proviral cellular factor, human vesicle-associated membrane protein-associated protein A (VAP-A), while knockdown of GPS2 disrupted interaction between VAP-A and NS5A. Taken together, our results suggest that GPS2 acts as a bridge between NS5A and VAP-A and is required for efficient HCV replication.
TL;DR: An assay in which ligand binding is detected using a microarray approach with immobilized antibodies on a porous aluminum oxide matrix is developed, indicating the potential of the method for a clinical setting.