Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Manganese-containing superoxide dismutase and its gene from Candida albicans

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.     

Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5''and 3''ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.     

Trials for the co-expression of the merozoite surface protein-1 and circumsporozoite protein genes of Plasmodium vivax

Merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen, and circumsporozoite protein (CSP), a component of sporozoites that includes a Plasmodium vivax B-cell epitope, are strong candidates for use in a malaria vaccine. A chimeric recombinant gene containing portions of both msp-1 and csp from P. vivax separated by Pro-Gly linker motif was generated. The construct gene was named mlc (msp-1, linker, and csp). The MLC chimeric recombinant protein had a molecular weight of approximately 25 kDa when expressed in Escherichia coli, as determined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The purified chimeric protein reacted with the sera of patients infected with P. vivax but not with the sera of uninfected patients according to western blot analysis. The chimeric protein reacted well with sera of malaria patients (109/115, 94.78%) as assessed with enzyme-linked immunosorbent assay (ELISA). BALB/c mice that were orally immunized with the MLC chimeric recombinant protein successfully produced antigen-specific antibodies. Additionally, levels of the Th1-associated cytokines IL-12(p40), TNF-α, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Therefore, the E. coli-expressed MLC chimeric recombinant protein might be used as a valuable vaccine candidate for oral immunization against vivax malaria.     

Enhancement of adipose tissue formation by implantation of adipogenic-differentiated preadipocytes

Engineered adipose tissue could be used for the reconstruction or augmentation of soft tissues lost due to mastectomy or lumpectomy in plastic and reconstructive surgery. Preadipocytes are a feasible cell source for adipose tissue regeneration. However, the enhancement of the in vivo adipogenic conversion of preadipocytes remains a major task. In vitro, the adipogenic differentiation of preadipocytes prior to implantation might enhance the adipose tissue regeneration. In the present study, we investigated whether implantation of adipogenic-differentiated preadipocytes enhances the adipose tissue formation compared with implantation of undifferentiated preadipocytes. We also investigated whether basic fibroblast growth factor (bFGF) further enhances the adipose tissue formation mediated by the implantation of adipogenic-differentiated preadipocytes. A fibrin matrix containing human preadipocytes cultured in adipogenic differentiation-inducing conditions with (group 1) or without (group 2) bFGF was injected into the subcutaneous spaces of athymic mice. Fibrin matrices containing undifferentiated human preadipocytes with (group 3) or without (group 4) bFGF were also implanted. Six weeks after implantation, the implanted cells formed new tissues in all groups. Importantly, the implantation of adipogenic-differentiated preadipocytes resulted in more extensive adipogenesis than the implantation of undifferentiated preadipocytes, as evaluated by adipose tissue area and human adipocyte-specific gene expression in the newly formed tissues. In addition, bFGF enhanced neovascularization in the newly formed tissues and further enhanced the adipogenesis mediated by the adipogenic-differentiated preadipocytes. The present study demonstrates that the implantation of adipogenic-differentiated preadipocytes enhances adipose tissue regeneration, as compared with the implantation of undifferentiated preadipocytes, and that cell transplantation-mediated adipogenesis can be further enhanced by the delivery of bFGF.     

Structural determinants of conformationally selective, prion-binding aptamers

We have recently described the isolation of 2'-fluoropyrimidine-substituted RNA aptamers that bind selectively to disease-associated beta-sheet-rich forms of the prion protein, PrP, from a number of mammalian species. These aptamers inhibit the accumulation of protease-resistant forms of PrP in a prion-seeded, in vitro conversion assay. Here we identify the minimal portions of two of these aptamers that retain binding specificity. We determine their secondary structures by a combination of modeling and solution probing. Finally, we identify an internal site for biotinylation of a minimized, synthetic aptamer and use the resultant reagent in the detection of abnormal forms of PrP in vitro.     

Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion

We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2'-fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrPSc than for recombinant PrPC and inhibited the accumulation of PrPres in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from non-specific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, alpha-helix-rich and beta-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the beta-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the alpha-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the alpha-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is beta-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.     

The binding of the molecular chaperone Hsc70 to the prion protein PrP is modulated by pH and copper

Conformational transitions in the prion protein (PrP) are thought to be central to the pathogenesis of the transmissible spongiform encephalopathies (TSE), such as Creutzfeldt-Jacob disease and bovine spongiform encephalopathy. Studies of prion phenomena in yeast have shown that molecular chaperones play an important role in prion related conformational transitions. Here, we investigated the interaction of the molecular chaperone Hsc70 (HSPA8) with recombinant PrP in vitro using an ELISA based assay. Hsc70 bound to PrP in a saturable manner over a range of temperatures and binding was greatest at low pH. Surprisingly, Hsc70 bound more avidly to native recombinant PrP than to denatured PrP or other potential clients, such as denatured luciferase or rhodanese. Hsc70 binding to native PrP was enhanced by incubation with Cu²⁺ at low pH. The Hsc70 binding sites in PrP were analysed using a synthetic PrP-derived peptide array. The binding of Hsc70 to PrP was reminiscent of the published ovine PrP to bovine PrP binding data and included two potential regions of binding that correspond to the proposed ‘protein X’ binding sites in PrP. Synthetic peptides corresponding to these sites specifically inhibited the Hsc70 interaction with native PrP, further demonstrating that Hsc70 might interact with PrP via this epitope. The data suggest that molecular chaperones could modulate important PrP conformational transitions or protein–protein interactions in TSE pathogenesis.