Construction of a bivalent vaccine against anthrax and smallpox using the attenuated vaccinia virus KVAC103.

BACKGROUND: Anthrax and smallpox are high-risk infectious diseases, and considered as potential agents for bioterrorism. To develop an effective countermeasure for these diseases, we constructed a bivalent vaccine against both anthrax and smallpox by integrating a gene encoding protective antigen (PA) of Bacillus anthracis to the genome of the attenuated vaccinia virus strain, KVAC103. RESULTS: Immunization with this bivalent vaccine induced antibodies against both PA and vaccinia virus in a mouse model. We also observed that the efficacy of this vaccine can be enhanced by combined immunization with immunoadjuvant-expressing KVAC103. Mouse groups co-immunized with PA-expressing KVAC103 and either interleukin-15 (IL-15) or cholera toxin subunit A (CTA1)-expressing KVAC103 showed increased anti-PA IgG titer and survival rate against B. anthracis spore challenge compared to the group immunized with PA-expressing KVAC103 alone. CONCLUSIONS: We demonstrated that the attenuated smallpox vaccine KVAC103 is an available platform for a multivalent vaccine and co-immunization of immunoadjuvants can improve vaccine performance.

A therapeutic human antibody against the domain 4 of the Bacillus anthracis protective antigen shows protective efficacy in a mouse model.

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR.

Peroxide operon regulator (PerR) is a broadly conserved hydrogen peroxide sensor in bacteria, and oxidation of PerR at its regulatory metal-binding site is considered irreversible. Here, we tested whether this oxidation specifically targets PerR for proteolysis. We find that oxidizing conditions stimulate PerR degradation in vivo, and LonA is the principal AAA+ (ATPases associated with diverse cellular activities) protease that degrades PerR. Degradation of PerR by LonA is recapitulated in vitro, and biochemical dissection of this degradation reveals that the presence of regulatory metal and PerR-binding DNA dramatically extends the half-life of the protein. We identified a LonA-recognition site critical for oxidation-controlled PerR turnover. Key residues for LonA-interaction are exposed to solvent in PerR lacking metal, but are buried in the metal-bound form. Furthermore, one residue critical for Lon recognition is also essential for specific DNA-binding by PerR, thus explaining how both the metal and DNA ligands prevent PerR degradation. This ligand-controlled allosteric mechanism for protease recognition provides a compelling explanation for how the oxidation-induced conformational change in PerR triggers degradation. Interestingly, the critical residues recognized by LonA and exposed by oxidation do not function as a degron, because they are not sufficient to convert a nonsubstrate protein into a LonA substrate. Rather, these residues are a conformation-discriminator sequence, which must work together with other residues in PerR to evoke efficient degradation. This mechanism provides a useful example of how other proteins with only mild or localized oxidative damage can be targeted for degradation without the need for extensive oxidation-dependent protein denaturation.

Structural basis for the specialization of Nur, a nickel-specific Fur homolog, in metal sensing and DNA recognition.

Nur, a member of the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and anti-oxidative response in Streptomyces coelicolor. Here we report the 2.4-A resolution crystal structure of Nur. It contains a unique nickel-specific metal site in addition to a nonspecific common metal site. The identification of the 6-5-6 motif of the Nur recognition box and a Nur/DNA complex model reveals that Nur mainly interacts with terminal bases of the palindrome on complex formation. This contrasts with more distributed contacts between Fur and the n-1-n type of the Fur-binding motif. The disparity between Nur and Fur in the conformation of the S1-S2 sheet in the DNA-binding domain can explain their different DNA-recognition patterns. Furthermore, the fact that the specificity of Nur in metal sensing and DNA recognition is conferred by the specific metal site suggests that its introduction drives the evolution of Nur orthologs in the Fur family.

Evaluation of the protective efficacy of recombinant protective antigen vaccine (GC1109)-immunized human sera using passive immunization in a mouse model.

The protective efficacy of human sera from vaccinated individuals with a new recombinant protective antigen anthrax vaccine (GC1109) against lethal spore challenge was evaluated in a mouse model. Eighteen human sera were selected from the vaccinated individuals based on their toxin neutralizing assay (TNA) titer (ED50 of 55 to 668). The selected sera were diluted and passively transferred to A/J mice and the mice were subsequently challenged with 100 × LD50 of Bacillus anthracis Sterne spores. The correlation between the survival rate of passively immunized mice and the TNA ED50 of transferred sera was presented (r = 0.873, P-value < 0.001). The estimated TNA titer for 50% survival rate against lethal challenge was 197 (95% confidence interval of 149 and 260). The result suggest that GC1109 is protective against exposure to B. anthracis and the TNA titer of vaccinated serum can be an indicator for protective efficacy.

Crystallization and preliminary X-ray crystallographic analyses of Nur, a nickel-responsive transcription regulator from Streptomyces coelicolor.

Nickel ions serve in the correct folding and function of microbial enzymes implicated in metabolic processes. Although nickel ions are indispensable for the survival of cells, the intracellular level of nickel ions needs to be properly maintained as excessive levels of nickel ions are toxic. Nur, a nickel-uptake regulator belonging to the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and antioxidative response in Streptomyces coelicolor. Nur was purified and crystallized at 295 K. A 2.4 A native data set and a 3.0 A Ni-MAD data set were collected using synchrotron radiation. The Nur crystals belong to space group P3(1), with unit-cell parameters a = b = 78.17, c = 50.39 A. Assuming the presence of two molecules in the asymmetric unit, the solvent content is estimated to be about 54.7%.

Regulation of a nickel-cobalt efflux system and nickel homeostasis in a soil actinobacterium Streptomyces coelicolor.

In Streptomyces coelicolor, a soil actinobacterium capable of morphological differentiation and complex secondary metabolism, nickel deficiency is sensed by Nur, a Ni-specific Fur family regulator that controls nickel uptake systems (NikABCDE and NikMNOQ) and both Fe-containing and Ni-containing superoxide dismutases (SodF and SodN). On the other hand, the nickel efflux system and its regulator have not been elucidated. In this study, we demonstrate that an ArsR/SmtB family metalloregulator NmtR, a close homologue of NmtR from Mycobacterium tuberculosis, controls a putative efflux pump of P1-type ATPase (NmtA) in S. coelicolor. NmtR binds to the nmtA promoter region to repress its transcription, and is dissociated in the presence of Ni(ii) and Co(ii). Disruption of the nmtA gene makes cells more sensitive to nickel and cobalt, consistent with its predicted role in encoding a Ni-Co-efflux pump. Growth of S. coelicolor in complex YEME medium is only marginally inhibited by up to 0.5 mM Ni(ii), with significant growth retardation at 1 mM. Nur-regulated sodF and nikA genes are repressed at less than 0.1 µM added NiSO4 whereas NmtR-regulated nmtA transcription is induced at 0.5 mM or more Ni(ii). This reveals the extreme sensitivity of S. coelicolor to nickel deficiency as well as tolerance to surplus nickel. How this organism and possibly other actinomycetes have evolved to develop such a highly Ni-tolerant physiology and how the highly sensitive regulator Nur and the obtuse regulator NmtR achieve their characteristic Ni-sensitivity are interesting questions to solve in the future.

Nur, a nickel-responsive regulator of the Fur family, regulates superoxide dismutases and nickel transport in Streptomyces coelicolor.

Nickel serves as a cofactor for various microbial enzymes including superoxide dismutase (SOD) found in Streptomyces spp. In Streptomyces coelicolor, nickel represses and induces production of Fe-containing and Ni-containing SODs, respectively, primarily at the transcriptional level. We identified the nickel-responsive regulator (Nur), a Fur (ferric-uptake regulator) homologue, which binds to the promoter region of the sodF gene encoding FeSOD in the presence of nickel. Disruption of the nur gene caused constitutive expression of FeSOD and no induction of NiSOD in the presence of nickel. The intracellular level of nickel was higher in a Deltanur mutant than in the wild type, suggesting that Nur also regulates nickel uptake in S. coelicolor. A putative nickel-transporter gene cluster (nikABCDE) was identified in the genome database. Its transcription was negatively regulated by Nur in the presence of nickel. Purified Nur protein bound to the nikA promoter region in a nickel-dependent way. These results support the action of Nur as a regulator of nickel homeostasis and antioxidative response in S. coelicolor, and add a novel nickel-responsive member to the list of versatile metal-specific regulators of the Fur family.

Regulation of sigmaB by an anti- and an anti-anti-sigma factor in Streptomyces coelicolor in response to osmotic stress.

sigmaB, a homolog of stress-responsive sigmaB of Bacillus subtilis, controls both osmoprotection and differentiation in Streptomyces coelicolor A3 (2). Its gene is preceded by rsbA and rsbB genes encoding homologs of an anti-sigma factor, RsbW, and its antagonist, RsbV, of B. subtilis, respectively. Purified RsbA bound to sigmaB and prevented sigmaB-directed transcription from the sigBp1 promoter in vitro. An rsbA-null mutant exhibited contrasting behavior to the sigB mutant, with elevated sigBp1 transcription, no actinorhodin production, and precocious aerial mycelial formation, reflecting enhanced activity of sigmaB in vivo. Despite sequence similarity to RsbV, RsbB lacks the conserved phosphorylatable serine residue and its gene disruption produced no distinct phenotype. RsbV (SCO7325) from a putative six-gene operon (rsbV-rsbR-rsbS-rsbT-rsbU1-rsbU) was strongly induced by osmotic stress in a sigmaB-dependent manner. It antagonized the inhibitory action of RsbA on sigmaB-directed transcription and was phosphorylated by RsbA in vitro. These results support the hypothesis that the rapid induction of sigmaB target genes by osmotic stress results from modulation of sigmaB activity by the kinase-anti-sigma factor RsbA and its phosphorylatable antagonist RsbV, which function by a partner-switching mechanism. Amplified induction could result from a rapid increase in the synthesis of both sigmaB and its inhibitor antagonist.

The pqrAB operon is responsible for paraquat resistance in Streptomyces coelicolor.

Paraquat (methyl viologen)-resistant mutants of Streptomyces coelicolor A3(2) that grew and sporulated normally in the presence of paraquat were isolated. Based on the positions of the mutant loci in the genetic map, we isolated the pqr (paraquat resistance) gene whose mutation (pqr501) caused a dominant paraquat-resistant phenotype. The pqr locus consists of two genes (pqrA and pqrB) that form a transcription unit. The pqrA gene encodes a protein with a TetR-like DNA-binding motif, and the pqrB gene encodes a putative efflux pump of the major facilitator superfamily. The pqr501 mutation was a base substitution changing arginine-18 to glutamine (R18Q) near the helix-turn-helix motif in PqrA. A pqrA null mutant exhibited similar paraquat resistance, and an increase in the amount of pqrA promoter-driven transcripts of about eightfold was observed for the pqrA501 mutant. These results suggest that PqrA is a negative regulator of its own operon. Deletion of the pqrAB operon caused cells to be very sensitive to paraquat, consistent with the prediction that PqrB may function as a paraquat-efflux pump. Purified PqrA protein specifically bound to the pqrA promoter region, whereas mutant R18Q protein did not, indicating that PqrA is a direct autoregulator of its own operon.