Designing a less immunogenic nattokinase from Bacillus subtilis subsp. natto: a computational mutagenesis.

Nattokinase is an enzyme produced by Bacillus subtilis subsp. natto that contains strong fibrinolytic activity. It has potential to treat cardiovascular diseases. In silico analysis revealed that nattokinase is considered as an antigen, thus hindering its application for injectable therapeutic protein. Various web servers were used to predict B-cell epitopes of nattokinase both continuously and discontinuously to determine which amino acid residues had been responsible for the immunogenicity. With the exclusion of the predicted conserved amino acids, four amino acids such as S18, Q19, T242, and Q245 were allowed for mutation. Substitution mutation was done to lower the immunogenicity of native nattokinase. Through the stability of the mutated protein with the help of Gibbs free energy difference, the proposed mutein was S18D, Q19I, T242Y, and Q245W. The 3D model of the mutated nattokinase was modeled and validated with various tools. Physicochemical properties and stability analysis of the protein indicated that the mutation brought higher stability without causing any changes in the catalytic site of nattokinase. Molecular dynamics simulation implied that the mutation indicated similar stability, conformation, and behavior compared to the native nattokinase. These results are highly likely to contribute to the wet lab experiment to develop safer nattokinase.

Human spermatozoa anti-proprotein convertase subtilisin/kexin type 4 synthesis using New Zealand rabbit for novel immunocontraception in males.

Purpose: Proprotein convertase subtilisin/kexin type 4 (PCSK4, a 54-kDa protease) is expressed in the plasma membrane of the acrosome in human spermatozoa. It plays a critical role in penetrating the zona pellucida. Synthesis of human anti-PCSK4 might be important for novel male immunocontraception. Materials and Methods: We used semen from adult males as the source of antigen (acrosomal PCSK4). Isolation and antigen characterization were done by immunohistochemistry followed by electrophoresis. Purification of PCSK4 was done by the electroelution method, followed by immunization with an animal model (Oryctolagus cuniculus). Antibody was collected, purified, and tested by using Western blot and dot blot. Antibody in vitro potential testing was performed on human spermatozoa by laser scanning microscopy with rhodamine stain under a light microscope and on rat spermatozoa. Results: Human anti-PCSK4 bound with PCSK4 on the head of human spermatozoa and could interfere with rat spermatozoa activity to penetrate the oocyte. Conclusions: The result of this study can be used as a basis to develop new immunocontraceptives targeted towards males. This study shows that antibodies from induction of PCSK4 from spermatozoa may hinder fertilization.

Cryptosporidium parvum Subtilisin-Like Serine Protease (SUB1) Is Crucial for Parasite Egress from Host Cells.

Despite the severity and global burden of Cryptosporidium infection, treatments are less than optimal, and there is no effective vaccine. Egress from host cells is a key process for the completion of the life cycle of apicomplexan parasites. For Plasmodium species, subtilisin-like serine protease (SUB1) is a key mediator of egress. For Toxoplasma species, calcium-dependent protein kinases (CDPKs) are critical. In this study, we characterized Cryptosporidium SUB1 expression and evaluated its effect using an infection model. We found increased expression between 12 and 20 h after in vitro infection, prior to egress. We induced silencing of SUB1 (ΔSUB1) mRNA using SUB1 single-stranded antisense RNA coupled with human Argonaute 2. Silencing of SUB1 mRNA expression did not affect parasite viability, excystation, or invasion of target cells. However, knockdown led to a 95% decrease in the proportion of released merozoites in vitro (P < 0.0001). In contrast, silencing of CDPK5 had no effect on egress. Overall, our results indicate that SUB1 is a key mediator of Cryptosporidium egress and suggest that interruption of the life cycle at this stage may effectively inhibit the propagation of infection.

Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance.

Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.

C-terminal cleavage of human Foxp3 at a proprotein convertase motif abrogates its suppressive function.

Foxp3 plays a critical role in the development and function of regulatory T cells (Tregs). Differences in translational and post-translational processing of murine and human Foxp3 have been recently reported. Human Foxp3 exists as four isoforms generated by alternative splicing. Mouse Foxp3 only exists as one isoform, but can be proteolytically cleaved by N-terminal and/or C-terminal proprotein convertase subtilisin/kexins (PCSKs). Here, we show by transcriptome analysis that the proprotein convertases PCSK7, PCSK5 and Furin are present in human CD4(+) T cells with different expression patterns. Notably, after in vitro activation, only PCSK7 and Furin are expressed in Tregs and T effector cells (Teffs), with overexpression of PCSK7 in Tregs compared to Teffs. Human Foxp3 protein displays specific motifs that can be potentially cleaved by convertases. Consequently, we transduced human CD4(+) cells with Foxp3-expressing lentiviral vectors and assessed the generation of proteolytically cleaved Foxp3 forms by Western blot. Three different Foxp3 forms were detected, indicating that human Foxp3 can also be subjected to proteolytic cleavage at the N-terminal and C-terminal ends. These results prompted us to assess the suppressive activity associated with each forms. We observed that full length and N-cleaved Foxp3-transduced CD4(+) T cells similarly suppressed the in vitro proliferation of Teffs. However, the C-cleaved or N&C-cleaved Foxp3 forms afforded almost no suppressive function, indicating a crucial role of the human Foxp3 C-terminal region in Tregs suppressive activity, in marked contrast with the report of a superior suppressive activity for the C-cleaved murine Foxp3 compared to the full length.

Differential effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on chemokine and proinflammatory cytokine expression in human macrophage, colonic epithelial, and brain microvascular endothelial cell lines.

Subtilase cytotoxin (SubAB) is the prototype of a recently emerged family of AB5 cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). Its mechanism of action involves highly specific A-subunit-mediated proteolytic cleavage of the essential endoplasmic reticulum (ER) chaperone BiP. Our previous in vivo studies showed that intraperitoneal injection of purified SubAB causes a major redistribution of leukocytes and elevated leukocyte apoptosis in mice, as well as profound splenic atrophy. In the current study, we investigated selected chemokine and proinflammatory cytokine responses to treatment with SubAB, a nontoxic derivative (SubAA272B), or Shiga toxin 2 (Stx2) in human macrophage (U937), brain microvascular endothelial (HBMEC), and colonic epithelial (HCT-8) cell lines, at the levels of secreted protein, cell-associated protein, and gene expression. Stx2 treatment upregulated expression of chemokines and cytokines at both the protein and mRNA levels. In contrast, SubAB induced significant decreases in secreted interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in all three tested cell lines and a significant decrease in secreted IL-6 in HBMECs. The downregulation of secreted chemokines or cytokines was not observed in SubAA272B-treated cells, indicating a requirement for BiP cleavage. The downregulation of secreted chemokines and cytokines by SubAB was not reflected at the mRNA and cell-associated protein levels, suggesting a SubAB-induced export defect.

Antibody and T cell responses in reciprocal prime-boost studies with full-length and truncated merozoite surface protein 1-42 vaccines.

The P. falciparum Merozoite Surface Protein 1-42 (MSP1-42) is one of the most studied malaria subunit vaccine candidates. The N-terminal fragment of MSP1-42, MSP1-33, is primarily composed of allelic sequences, and has been shown to possess T helper epitopes that influence protective antibody responses toward the C-terminal region, MSP1-19. A truncated MSP1-42 vaccine, Construct 33-I, consisting of exclusively conserved T epitope regions of MSP1-33 expressed in tandem with MSP1-19, was previously shown to be a more effective immunogen than the full-length MSP1-42 vaccine. Here, by way of reciprocal priming/boosting immunization regimens, we studied the immunogenicity of Construct 33-I in the context of recognition by immune responses induced by the full-length native MSP1-42 protein, in order to gauge the effects of pre- and post-exposures to MSP1-42 on vaccine induced responses. Judging by immune responsiveness, antibody and T cell responses, Construct 33-I was effective as the priming antigen followed by full-length MSP1-42 boosting, as well as the boosting antigen following full-length MSP1-42 priming. In particular, Construct 33-I priming elicited the broadest responsiveness in immunized animals subsequently exposed to MSP1-42. Moreover, Construct 33-I, with its conserved MSP1-33 specific T cell epitopes, was equally well recognized by homologous and heterologous allelic forms of MSP1-42. Serum antibodies raised against Construct 33-I efficiently inhibited the growth of parasites carrying the heterologous MSP1-42 allele. These results suggest that Construct 33-I maintains and/or enhances its immunogenicity in an allelic or strain transcending fashion when deployed in populations having prior or subsequent exposures to native MSP1-42s.

Molecular determinants for subcellular trafficking of the malarial sheddase PfSUB2.

The malaria merozoite invades erythrocytes in the vertebrate host. Iterative rounds of asexual intraerythrocytic replication result in disease. Proteases play pivotal roles in erythrocyte invasion, but little is understood about their mode of action. The Plasmodium falciparum malaria merozoite surface sheddase, PfSUB2, is one such poorly characterized example. We have examined the molecular determinants that underlie the mechanisms by which PfSUB2 is trafficked initially to invasion-associated apical organelles (micronemes) and then across the surface of the free merozoite. We show that authentic promoter activity is important for correct localization of PfSUB2, likely requiring canonical features within the intergenic region 5' of the pfsub2 locus. We further demonstrate that trafficking of PfSUB2 beyond an early compartment in the secretory pathway requires autocatalytic protease activity. Finally, we show that the PfSUB2 transmembrane domain is required for microneme targeting, while the cytoplasmic domain is essential for surface translocation of the protease to the parasite posterior following discharge from micronemes. The interplay of pre- and post-translational regulatory elements that coordinate subcellular trafficking of PfSUB2 provides the parasite with exquisite control over enzyme-substrate interactions.

An extracellular subtilase switch for immune priming in Arabidopsis.

In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity.

Protein expressions and their immunogenicity from Riemerella anatipestifer cultured in iron restriction medium.

Riemerella anatipestifer was cultured in both iron restriction media and normal media. Two-dimensional gel electrophoresis identified 23 proteins that significantly increased in the iron restriction media. Of them 12 proteins were analyzed with mass spectrography. Nine of 12 proteins belong to 6 different protein families: fibronectin type iii domain protein, secreted subtilase family protein, phosphoglycerate kinase, translation elongation factor, leucine-rich repeat-containing protein, and Galactose-binding domain-like protein. Other 3 proteins were novel with unknown function. Two novel proteins (Riean_1750 and Riean_1752) were expressed in prokaryotic expression systems. The specificities of these 2 novel proteins to R. anatipestifer were confirmed by western-blotting analysis. The ducks immunized with either protein had low mortality challenged by R. anatipestifer, 33.3% and 16.7%, respectively. The ducks developed 100% immunity when immunized with combined Riean_1750 and Riean_1752 proteins. The data suggested 2 novel proteins play important roles in the bacterial survival in the iron restricted environment. They could be used as subunit vaccines of R. anatipestifer.

Immunohistochemical analysis of the proteolytic enzyme subtilase in the digestive organs of the sea urchin Strongylocentrotus intermedius.

Subtilase, a major protease in the short-spined sea urchin (Strongylocentrotus intermedius), was isolated and used as antigen for the subsequent production of a specific polyclonal antibody. Immunoreactive cells were observed by immunohistochemical analysis in granules in the anterior and posterior stomach and the anterior intestine. These granules, which were most numerous in the anterior stomach, also stained intensely with methylene blue-Azure II. However, granules in cells of the esophagus, posterior intestine, and rectum were not stained by this antibody. We conclude that subtilase mainly localizes in the stomach and anterior intestine of the sea urchin.

Identification of Toxoplasma gondii SUB1 antigen as a marker for acute infection by use of an innovative evaluation method.

By the separation of Toxoplasma lysate using two-dimensional gel electrophoresis and its analysis with human serum samples and mass spectrometry, the subtilisin-like protein (SUB1) was identified to be a potential marker for acute toxoplasmosis. Following expression of the C-terminal domain of SUB1 in Escherichia coli, it was tested in a line blot assay using a total of 80 human serum samples. Two computer programs based on different evaluation strategies were used for judgment of the line blot results: (i) a time-dependent method with a predefined cutoff value and (ii) a fixed-time-point method with a calculated cutoff. Thereby, SUB1 was proven to be rather reactive with specific immunoglobulin A (IgA), IgM, and IgG of patients with an acute infection. This finding makes this antigen an attractive candidate for improving diagnosis of toxoplasmosis and demonstrates that not only the selection of respective antigens but also the evaluation method chosen are important for the evaluation of new diagnostic markers.

The SspA subtilisin-like protease of Streptococcus suis triggers a pro-inflammatory response in macrophages through a non-proteolytic mechanism.

BACKGROUND: Streptococcus suis is a major swine pathogen worldwide that causes meningitis, septicemia, arthritis, and endocarditis. Using animal models, a surface-associated subtilisin-like protease (SspA) has recently been shown to be an important virulence factor for S. suis. In this study, we hypothesized that the S. suis SspA subtilisin-like protease may modulate cytokine secretion by macrophages thus contributing to the pathogenic process of meningitis. RESULTS: Phorbol 12-myristate 13-acetate-differentiated U937 macrophages were stimulated with recombinant SspA prior to monitor cytokine secretion by ELISA. Our results indicated that the recombinant SspA was able to dose-dependently induce IL-1ß, IL-6, TNF-α, CXCL8 and CCL5 secretion in macrophages. The heat-inactivated protease was still able to induce cytokine secretion suggesting a non-proteolytic mechanism of macrophage activation. Using specific kinase inhibitors, evidence were bought that cytokine secretion by macrophages stimulated with the recombinant SspA involves the mitogen-activated protein kinase signal transduction pathway. While stimulation of macrophages with low concentrations of recombinant SspA was associated to secretion of high amounts of CCL5, the use of recombinant SspA at a high concentration resulted in low amounts of CCL5 detected in the conditioned medium. This was found to be associated with a proteolytic degradation of CCL5 by SspA. The ability of SspA to induce cytokine secretion in macrophages was confirmed using a mutant of S. suis deficient in SspA expression. CONCLUSION: In conclusion, this study identified a new mechanism by which the S. suis SspA may promote central nervous system inflammation associated with meningitis.

Use of murine models to detect the allergenicity of genetically modified Lactococcus lactis NZ9000/pNZPNK.

By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of pepsin digestion stability and murine model serologic identity. Subjecting to the GM strain and host to pepsin digestion, the soluble fractions and cell debris were fast degraded completely. Feeding with ovalbumin resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, the serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than that of the OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induce allergenicity in BALB/c mice. On the basis of the results, the new GM lactic acid bacterium is regarded as safe to use.

Fatal hemorrhage induced by subtilase cytotoxin from Shiga-toxigenic Escherichia coli.

Subtilase cytotoxin (SubAB) is an AB(5) type toxin produced by a subset of Shiga-toxigenic Escherichia coli. The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone BiP. The B subunit binds to a receptor on the cell surface. Although SubAB is lethal for mice, the cause of death is not clear. In this study, we demonstrate in mice that SubAB induced small bowel hemorrhage and a coagulopathy characterized by thrombocytopenia, prolonged prothrombin time and activated partial thromboplastin time. SubAB also induced inflammatory changes in the small intestine as detected by ¹8F-fluoro-2-deoxy-d-glucose positron emission tomography imaging and histochemical analysis. Using RT-PCR and ELISA, SubAB was shown to increase interleukin-6 in a time-dependent manner. Thus, our results indicate that death in SubAB-treated mice may be associated with severe inflammatory response and hemorrhage of the small intestine, accompanied by coagulopathy and IL6 production.

MSP-1p42-specific antibodies affect growth and development of intra-erythrocytic parasites of Plasmodium falciparum.

BACKGROUND: Antibodies are the main effector molecules in the defense against blood stages of the malaria parasite Plasmodium falciparum. Understanding the mechanisms by which vaccine-induced anti-blood stage antibodies work in protecting against malaria is essential for vaccine design and testing. METHODS: The effects of MSP-1p42-specific antibodies on the development of blood stage parasites were studied using microscopy, flow cytometry and the pLDH assay. To determine allele-specific effects, if present, allele-specific antibodies and the various parasite clones representative of these alleles of MSP-1 were employed. RESULTS: The mode of action of anti-MSP-1p42 antibodies differs among the parasite clones tested: anti-MSP-1p42 sera act mainly through invasion-inhibitory mechanisms against FVO parasites, by either preventing schizonts from rupturing or agglutinating merozoites upon their release. The same antibodies do not prevent the rupture of 3D7 schizonts; instead they agglutinate merozoites and arrest the development of young parasites at the early trophozoite stage, thus acting through both invasion- and growth inhibitory mechanisms. The second key finding is that antibodies have access to the intra-erythrocytic parasite, as evidenced by the labeling of developing merozoites with fluorochrome-conjugated anti-MSP-1p42 antibodies. Access to the parasite through this route likely allows antibodies to exert their inhibitory activities on the maturing schizonts leading to their inability to rupture and be released as infectious merozoites. CONCLUSION: The identification of various modes of action by which anti-MSP-1 antibodies function against the parasite during erythrocytic development emphasizes the importance of functional assays for evaluating malaria vaccines and may also open new avenues for immunotherapy and vaccine development.

AasP autotransporter protein of Actinobacillus pleuropneumoniae does not protect pigs against homologous challenge.

Actinobacillus pleuropneumoniae is a major respiratory pathogen of pigs; current vaccines provide only limited protection. AasP, a subtilisin-like serine protease, is a conserved outer membrane-localised autotransporter protein. We hypothesized that AasP would induce protective immunity and may thus constitute a useful component of a vaccine against A. pleuropneumoniae infection. Here we confirm experimentally that AasP is an antigenic in vivo-expressed protein. In pig protection studies, a detectable specific antibody response was induced in response to recombinant AasP. However, the vaccine was not capable of protecting pigs from colonization, infection or severe clinical disease resulting from challenge with the homologous A. pleuropneumoniae strain.

Applicability of multigene family-specific antibodies toward studies of the subtilases in Arabidopsis thaliana.

Polyclonal antibodies were made to two synthetic peptides with sequences patterned after conserved regions in a multigene family of 56 subtilisin-related proteolytic enzymes in Arabidopsis thaliana. GST-fusion proteins encompassing full-length or partial cDNAs bearing putative epitope regions were cloned and expressed in Escherichia coli. Immunoblots showed that the antibodies bound 12 of 13 fusion proteins tested. About 27 more subtilase genes code for regions with sequences very similar to the epitope regions of the subtilases that were visualized on the immunoblots. Subtilases in rosette and cauline leaves, stems, flowers, and siliques could be distinguished by the antibodies; some binding the two antibodies to similar extents, while others bind preferentially or almost exclusively to one or the other antibody. When antibodies were used to monitor ion-exchange fractionation of seedling extracts, one specific subtilase was identified by LC-MS-MS. The specificity of the antibodies extended to subtilases in soybean. These studies show that multigene family-specific antibodies can be applied to the study of gene families, where sequence similarities make it difficult to produce antibodies specific for each individual protein in the group.

Anti-idiotypic antibody mimics proteolytic function of parent antigen.

Functional imaging of subtilisin Carlsberg active center by the idiotypic network yielded a catalytic anti-idiotypic antibody with endopeptidase, amidase, and esterase activities. A monoclonal antibody inhibitory to subtilisin (Ab1 5-H4) was employed as the template for guiding the idiotypic network to produce the catalytic anti-idiotypic Ab2 6B8-E12. Proteolytic activity of 6B8-E12 was demonstrated by zymography using self-quenched fluorescein-BSA conjugate and in a coupled assay detecting Ab2-dependent RNase A inactivation. Cleavage of peptide substrates by 6B8-E12 revealed distinct patterns of hydrolysis with high preference for aromatic residues before or after the scissile bond. Catalytic activity of Ab2 was inhibited by phenylmethylsulfonyl fluoride, a mechanism-based inhibitor of serine hydrolases. 5-H4 and 6B8-E12 were cloned, produced in Escherichia coli as single-chain variable fragments (scFvs), and purified. Kinetic parameters for amidolytic and esterolytic activities were similar in Ab2 and its scFv derivative. Although the antigen-specific portion of 6B8-E12 possesses no primary structure similarity to subtilisin, it mimics proteolytic and amidolytic functions of the parental antigen, albeit with 4 orders of magnitude slower acceleration rates. The lack of detectable endopeptidase activity of 6B8-E12 scFv raises interesting issues concerning general evolution of catalytic activity. The in silico 3D models of Ab1 and Ab2 revealed strong structural similarity to known anti-protease antibodies and to abzymes, respectively. These results indicate that the idiotypic network is capable, to a significant extent, of reproducing catalytic apparatus of serine proteases and further validate the use of imaging of enzyme active centers by the immune system for induction of abzymes accelerating energy-demanding amide bond hydrolysis.