Protein expression pattern of murine macrophages treated with anthrax lethal toxin.

Anthrax lethal toxin (LeTx; a combination of protective antigen and lethal factor) secreted by the vegetative cells of Bacillus anthracis is cytotoxic for certain macrophage cell lines. The role of LeTx in mediating these effects is complicated largely due to the difficulty in identifying and assigning functions to the affected proteins. To analyze the protein profile of murine macrophages treated with LeTx, we employed two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF MS, and interpreted the peptide mass fingerprint data relying on the ProFound database. Among the differentially expressed spots, cleaved mitogen-activated protein kinase kinase 1 acting as a negative element in the signal transduction pathway, and glucose-6-phosphate dehydrogenase playing a role in the protection of cells from hyperproduction of active oxygen were up-regulated in the LeTx-treated macrophages.

Bacillus anthracis spores influence ATP synthase activity in murine macrophages.

Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be downregulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.

Isolation and characterization of a neutralizing antibody specific to internalization domain of Clostridium botulinum neurotoxin type B.

Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. In this study, a neutralizing mouse monoclonal antibody against botulinum neurotoxin serotype B (BoNT/B), named BTBH-N1, was developed from mice immunized with BoNT/B toxoid without non-toxic components, which are generally associated with the toxin. Western blot analysis, using recombinant toxin fragments containing light (L), N-terminal half of heavy (HN) and C-terminal half of heavy chains, indicated that BTBH-N1 recognizes linear epitopes located on the HN domain. An in vivo neutralization assay with mice, was conducted to characterize the neutralization capacity of the BTBH-N1. Only 10 microg of BTBH-N1 completely neutralized 20 units (1 unit = one 50% lethal dose) of BoNT/B. Even though the Mab (up to 100 microg) failed to protect mice challenged with 100 units, it significantly prolonged the time to death in a dose dependent manner. BTBH-N1, the first neutralizing antibody against BoNT/B, could be further developed as effective biological therapeutics for preventing and treating botulism, as well as other diseases caused by BoNT/B.

Genetic diversity of Korean Bacillus anthracis isolates from soil evaluated with a single nucleotide repeat analysis.

Bacillus (B.) anthracis, the etiological agent of anthrax, is one of the most genetically monomorphic bacteria species in the world. Due to the very limited genetic diversity of this species, classification of isolates of this bacterium requires methods with high discriminatory power. Single nucleotide repeat (SNR) analysis is a type of variable-number tandem repeat assay that evaluates regions with very high mutation rates. To subtype a collection of 21 isolates that were obtained during a B. anthracis outbreak in Korea, we analyzed four SNR marker loci using nucleotide sequencing analysis. These isolates were obtained from soil samples and the Korean Center for Disease Control and Prevention. The SNR analysis was able to detect 13 subgenotypes, which allowed a detailed evaluation of the Korean isolates. Our study demonstrated that the SNR analysis was able to discriminate between strains with the same multiple-locus variable-number tandem repeat analysis genotypes. In summary, we obtained SNR results for four SNR marker loci of newly acquired strains from Korea. Our findings will be helpful for creating marker systems and help identify markers that could be used for future forensic studies.

Genetic populations of Bacillus anthracis isolates from Korea.

Bacillus (B.) anthracis is the pathogen that causes fatal anthrax. Strain-specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial population genetics. In the present study, we employed molecular approaches including multiple-locus variable-number tandem repeat analysis (MLVA) and canonical single-nucleotide polymorphism (canSNP) analysis to perform molecular typing of B. anthracis strains isolated in Korea. According to the MLVA, 17 B. anthracis isolates were classified into A3a, A3b, and B1 clusters. The canSNP analyses subdivided the B. anthracis isolates into two of the three previously recognized major lineages (A and B). B. anthracis isolates from Korea were found to belong to four canSNP sub-groups (B.Br.001/2, A.Br.005/006, A.Br.001/002, and A.Br.Ames). The A.Br.001/002 and A.Br.Ames sub-lineages are closely related genotypes frequently found in central Asia and most isolates were. On the other hand, B. anthracis CH isolates were analyzed that belonged to the B.Br.001/002 sub-group which found in southern Africa, Europe and California (USA). B.Br.001/002 genotype is new lineage of B. anthracis in Korea that was not found before. This discovery will be helpful for the creation of marker systems and might be the result of human activity through the development of agriculture and increased international trade in Korea.

Comparative proteome analysis of Bacillus anthracis with pXO1 plasmid content.

Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4-7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.