Gene Regulation Pathways Affect Toxin Gene Expression, Sporulation and Pigment Generation in Bacillus Anthracis and Bacillus Cereus

B. anthracis alters its virulence gene expression profile in response to a number of environmental signals, including levels of bicarbonate and CO2. Virulence plasmid pXO1 is important to Bacillus anthracis pathogenicity as it carries the genes encoding the anthrax toxin and virulence regulatory factors. Induction of toxin and other virulence genes requires the pXO1-encoded AtxA regulatory protein. The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. pXO1 also carries a gene encoding an Hfq-like protein, pXO1-137. Loss of pXO1-137 results in significant growth defects and reductions in toxin gene expression only when grown under toxin inducing conditions. Similarly, loss of a small RNA on pXO1, sRNA-1, results in similar growth defects and reductions in toxin gene production. Both increased and decreased expression of pXO1-137 and sRNA-1 result in growth defects suggesting narrow functional set points for Hfq and sRNA levels

GENE REGULATION PATHWAYS AFFECT TOXIN GENE EXPRESSION, SPORULATION AND PIGMENT GENERATION IN BACILLUS ANTHRACIS AND

B. anthracis alters its virulence gene expression profile in response to a number of environmental signals, including levels of bicarbonate and CO2. Virulence plasmid pXO1 is important to Bacillus anthracis pathogenicity as it carries the genes encoding the anthrax toxin and virulence regulatory factors. Induction of toxin and other virulence genes requires the pXO1-encoded AtxA regulatory protein. The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. pXO1 also carries a gene encoding an Hfq-like protein, pXO1-137. Loss of pXO1-137 results in significant growth defects and reductions in toxin gene expression only when grown under toxin inducing conditions. Similarly, loss of a small RNA on pXO1, sRNA-1, results in similar growth defects and reductions in toxin gene production. Both increased and decreased expression of pXO1-137 and sRNA-1 result in growth defects suggesting narrow functional set points for Hfq and sRNA levels

Loss of Homogentisate 1,2-Dioxygenase Activity in Bacillus anthracis Results in Accumulation of Protective Pigment.

Melanin production is important to the pathogenicity and survival of some bacterial pathogens. In Bacillus anthracis, loss of hmgA, encoding homogentisate 1,2-dioxygenase, results in accumulation of a melanin-like pigment called pyomelanin. Pyomelanin is produced in the mutant as a byproduct of disrupted catabolism of L-tyrosine and L-phenylalanine. Accumulation of pyomelanin protects B. anthracis cells from UV damage but not from oxidative damage. Neither loss of hmgA nor accumulation of pyomelanin alter virulence gene expression, sporulation or germination. This is the first investigation of homogentisate 1,2-dioxygenase activity in the Gram-positive bacteria, and these results provide insight into a conserved aspect of bacterial physiology

Complementation analysis of Δ<i>hmgA</i> mutant strain.

<p><b>A.</b> Pigment production in strains grown on LB-agar for 48 hours at 37°C. <b>B.</b> Pigment production in supernatants of strains grown in LB for 72 hours at 37°C. For both: 1. 34F2 + pAW285 (empty vector); 2. 34F2 + pAW444 (<i>hmgA</i>); 3. AW-A127 (Δ<i>hmgA</i>) + pAW285 (empty vector); 4. AW-A127 (Δ<i>hmgA</i>) + pAW444 (<i>hmgA</i>).</p

Growth and pigment production in defined medium.

<p><b>A.</b> Cell growth of parental and mutant strains grown in R-Bic medium at 37°C. -■- 34F2 in R-bic; -◆- AW-A127 (Δ<i>hmgA</i>) in R-bic; -▲- 34F2 in R-bic without L-tryptophan, L-phenylalanine, or L-tyrosine; -●- AW-A127 in R-bic without L-tryptophan, L-phenylalanine, or L-tyrosine. <b>B.</b> Pigment production in 34F2 supernatants. <b>C.</b> Pigment production in AW-A127 supernatants. 1, R-bic without L-tryptophan, L-phenylalanine, or L-tyrosine; 2, R-bic without L-tryptophan or L-phenylalanine; 3, R-bic without L-tryptophan or L-tyrosine; 4, R-bic without L-phenylalanine or L-tyrosine.</p

Genetic organization of BAS0228 (<i>hmgA</i>) region.

<p>Numbers above specify size of intergenic regions. Predicted transcriptional terminator indicated downstream of <i>hmgA</i>.</p

Pigment production in cell-free extracts.

<p>Cell-free extracts from cells grown in LB at 37°C for 6 hours. Amino acids then added to cell-free extracts, as indicated, and incubated for an additional 24 hours at 37°C. 1, 34F2, no added amino acids; 2, 34F2 with L-tyrosine; 3, 34F2 with L-phenylalanine; 4, 34F2 with L-tryptophan; 5, AW-A127 (Δ<i>hmgA</i>), no added amino acids; 6, AW-A127 with L-tyrosine; 7, AW-A127 with L-phenylalanine; 8, AW-A127 with L-tryptophan.</p

Epitope Mapping of Metuximab on CD147 Using Phage Display and Molecular Docking

Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23–30), I37, D45, E84, V88, EPMGTANIQLH (92–102), VPP (131–133), Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer

Simplified Seismic Loss Estimation of RC Frame using Component-performance-based Methodology

A framework of simplified probabilistic seismic loss estimation of RC frame using component- performance -based methodology was presented in this paper. Firstly, component- performance -based methodology was introduced, and component performance level was measured using elasto-plastic rotation angle of structural component. Then structural performance level was determined by the statistics of damaged components distribution. Additionally, the relationship between the structural performance level and loss ratio of RC frame was established. Last, expected seismic loss ratio of 3-storey RC frame was studied in detail. This study reveals that the simplified probabilistic framework proposed herein could be used in RC frame