Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release.

For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, Bacillus thuringiensis, a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of B. thuringiensis with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two loxP sites were inserted, one on each side of the spo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted spo0A during sporulation, producing spores that lacked the spo0A gene. In addition, two major α/ß-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced in vivo safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment.IMPORTANCEB. thuringiensis has recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features of B. thuringiensis as a simulant in environmental studies. Since spores can persist in the environment for years after release, environmental contamination is a big problem, especially when genetically engineered strains are used. To solve these problems, we report here the development of B. thuringiensis simulant strains that are capable of forming yellow colonies for easy detection, incapable of forming spores more than once due to a genetic circuit, and lacking in two major SASP genes. The genetic circuit to produce a spore without sporulation capability, together with the deletion of SASP genes, ensures the environmental and human safety of the simulant strains developed in this study. All of these features will allow wider use of B. thuringiensis as a simulant for Bacillus anthracis in environmental release studies.

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.

Complete Genome Sequence of Bacillus subtilis Strain WB800N, an Extracellular Protease-Deficient Derivative of Strain 168.

Bacillus subtilis WB800N is a genetically engineered variant of B. subtilis 168, such that all extracellular proteases are disrupted, which enables WB800N to be widely used for the expression of secretory proteins. Here, we report the 4.2-Mb complete genome sequence of WB800N and present all of the disrupted gene structure.

Generation of a specific marker to discriminate Gacillus anthracis from other bacteria of the Bacillus cereus group.

Bacillus anthracis is a soil pathogen capable of causing anthrax that is closely related to several environmental species, including B. cereus, B. mycoides, and B. thuringiensis. DNA homology studies showed that B. anthracis, B. cereus, B. mycoides, and B. thuringiensis are closely related, with a high sequence homology. To establish a method to specifically detect B. anthracis in situations such as environmental contamination, we initially performed RAPD-PCR with a 10-mer random primer and confirmed the presence of specific PCR bands only in B. anthracis species. One region specific for B. anthracis was cloned and sequenced, and an internal primer set was designed to amplify a 241-bp DNA fragment within the sequenced region. The PCR system involving these specific primer sets has practical applications. Using lyses methods to prepare the samples for PCR, it was possible to quickly amplify the 241-bp DNA segment from samples containing only a few bacteria. Thus, the PCR detection method developed in this study is expected to facilitate the monitoring of environmental B. anthracis contamination.

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.

Significance of CD133 as a cancer stem cell markers focusing on the tumorigenicity of pancreatic cancer cell lines.

PURPOSE: The cancer stem cell hypothesis states that the capacity of a cancer to grow and propagate is dependent on a small subset of cells. To determine the significances of the cancer stem cell markers CD133, CD44, and CD24 using a comparative analysis with a focus on tumorigenicity. METHODS: Four pancreatic cancer cell lines, Capan-1, Mia-PACA-2, Panc-1, and SNU-410 were analyzed for the expressions of CD133, CD44, and CD24 by flow cytometry. The tumorigenicity was compared using tumor volumes and numbers of tumors formed/numbers of injection in nonobese diabetic severe combined deficiency mice. Fluorescence-activated cell sorting (FACS) analysis was used to confirm that xenograft explants originated from human pancreatic cancer cells. RESULTS: CD133 was positive in only Capan-1, CD44 positive in all, CD24 partially positive in Panc-1. After injecting 2 × 10(6) cells, all mice administered Capan-1 or Mia-Paca-2 developed tumors, 3 of 5 administered Panc-1 developed tumors, but no mouse administered SNU-410 developed any tumors. The volumes of Capan-1 tumors were seven times larger than those of Mia-Paca-2 tumors. When 2 × 10(5) or 2 × 10(4) of Capan-1 or Mia-Paca-2 was injected, tumors developed in all Capan-1 treated mice, but not in Mia-Paca-2 treated mice. Furthermore, xenograft explants of Capan-1 expressed CD133+CD44+ and Capan-1 injected mice developed lung metastasis. FACS analysis showed that xenograft explants originated from human pancreatic cancer cell lines. CONCLUSION: CD133 positive cells have higher tumorigenic and metastatic potential than CD44 and CD24 positive cells, which suggests that CD133 might be a meaningful cell surface marker of pancreatic cancer stem cells.

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.

Gastric neuroendocrine carcinoma: clinicopathologic review and immunohistochemical study of E-cadherin and Ki-67 as prognostic markers.

BACKGROUND AND OBJECTIVES: Gastric neuroendocrine carcinoma (NEC) is an uncommon cancer of the stomach. The classification of NEC and its clinical behavior remains controversial, and prognostic markers and their therapeutic guidelines have not been clearly defined. The aim of this study was to evaluate the clinicopathologic characteristics of these tumors and analyze the expression of E-cadherin and Ki-67 as prognostic markers in gastric NECs. METHODS: We retrospectively reviewed 17 cases of gastric NECs. Tumor pathology was reviewed and the tumors were categorized as well-differentiated NEC (n = 5) and poorly differentiated NEC (n = 12) according to the WHO classification. With the aim of evaluating the expression of E-cadherin and Ki-67 and their prognostic role in gastric NEC, immunohistochemical analysis of the tumors was performed. RESULTS: The median survival of patients was 20.0 months (95% confidence interval (CI), 13.2-28.8). There was a statistically significant difference in overall survival between well and poorly differentiated NECs (P = 0.021). However, there was no significant difference in overall survival between patients with poorly differentiated small cell and large cell NEC (P = 0.796). Loss of E-cadherin was correlated with lymph node metastasis (P= 0.044). A high Ki-67 proliferation index (PI) (>60%) was correlated with tumor recurrence (P = 0.013) and histologic differentiation (P= 0.028). CONCLUSIONS: Loss of E-cadherin may predict lymph node metastasis in gastric NECs. A high Ki-67 PI (>60%) could be used as a prognostic marker to predict aggressive gastric NECs in addition to standard pathologic classification.

Rapid profiling of the infection of Bacillus anthracis on human macrophages using SELDI-TOF mass spectroscopy.

Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis, which is mainly present in the environment in the form of highly resistant spores. In order to elucidate a surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy analysis to profile different expressed proteins when B. anthracis spores are infected in human macrophages, we analyzed human macrophage cytosolic fractions for the infection of B. anthracis spores. Eleven different protein peaks were obtained. The 8217.8 kDa was increased specifically in inactivated-Sterne spores at 90 min. At 120 min, the peak of 8552.1 kDa in the inactivate-Sterne spores increased more than fourfold compared to live-Sterne spores. The protein peak at 8552.1kDa suggests that inactivated-Sterne spores could cause the phagolysosome formation of macrophages. And the protein peaks that increased in live-Sterne spores suggest that it could escape from the phagolysosome of the macrophage. These SELDI-TOF profiles assume an important role in human macrophage for the survival and escape of the infected B. anthracis spores.

Targeting of Bacillus anthracis interaction factors for human macrophages using two-dimensional gel electrophoresis.

Bacillus anthracis, a gram-positive, endospore-forming, aerobic rod-shaped bacterium, interacts with macrophages at various stages of the disease. Spore germination and the outgrowth of vegetative bacilli are crucial steps enabling the bacteria to proliferate actively and to synthesize the virulence factors leading to a massive septicemia. In this study, we performed a proteomic analysis and MALDI-TOF/MS were carried out to identify proteins using human macrophages infected with the spores of B. anthracis live-Sterne or inactivated-Sterne. We identified 21 proteins which are related to the infection of B. anthracis spores on human macrophages at the early stage events. These proteins function in processes such as cytoskeleton regulation, apoptosis, cell division, and protein degradation. Proteins such as PAK 2 revealed a relationship to apoptosis in human macrophages. These proteins play an important role in the macrophage survival and death on human macrophages with infected B. anthracis spores.