Selection of anthrax toxin protective antigen variants that discriminate between the cellular receptors TEM8 and CMG2 and achieve targeting of tumor cells

Anthrax toxin, a three-component protein toxin secreted by Bacillus anthracis, assembles into toxic complexes at the surface of receptor-bearing eukaryotic cells. The protective antigen (PA) protein binds to receptors, either tumor endothelial cell marker 8 (TEM8) or CMG2 (capillary morphogenesis protein 2), and orchestrates the delivery of the lethal and edema factors into the cytosol. TEM8 is reported to be overexpressed during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues. To extend prior work on targeting of tumor with modified anthrax toxins, we used phage display to select PA variants that preferentially bind to TEM8 as compared with CMG2. Substitutions were randomly introduced into residues 605-729 of PA, within the C-terminal domain 4 of PA, which is the principal region that contacts receptor. Candidates were characterized in cellular cytotoxicity assays with Chinese hamster ovary (CHO) cells expressing either TEM8 or CMG2. A PA mutant having the substitutions R659S and M662R had enhanced specificity toward TEM8-overexpressing CHO cells. This PA variant also displayed broad and potent tumoricidal activity to various human tumor cells, especially to HeLa and A549/ATCC cells. By contrast, the substitution N657Q significantly reduced toxicity to TEM8 but not CMG2-overexpressing CHO cells. Our results indicate that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.     

Methylation-dependent DNA restriction in Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, is poorly transformed with DNA that is methylated on adenine or cytosine. Here we characterize three genetic loci encoding type IV methylation-dependent restriction enzymes that target DNA containing C5-methylcytosine (m5C). Strains in which these genes were inactivated, either singly or collectively, showed increased transformation by methylated DNA. Additionally, a triple mutant with an ~ 30-kb genomic deletion could be transformed by DNA obtained from Dam⁺Dcm⁺E. coli, although at a low frequency of ~ 10^− 3 transformants/10⁶ cfu. This strain of B. anthracis can potentially serve as a preferred host for shuttle vectors that express recombinant proteins, including proteins to be used in vaccines. The gene(s) responsible for the restriction of m6A-containing DNA in B. anthracis remain unidentified, and we suggest that poor transformation by such DNA could in part be a consequence of the inefficient replication of hemimethylated DNA in B. anthracis.     

Quickening the pace of anthrax research: three advances point towards possible therapies

Anthrax toxin is the dominant virulence factor of Bacillus anthracis and drugs blocking its action could therefore have therapeutic benefit. Three recent papers suggest new ways to inhibit the toxin. Identification of the cell surface toxin receptor could lead to the design of binding competitors and receptor decoys. Determination of the crystal structure of the lethal factor protease will facilitate ongoing efforts to develop protease inhibitors as therapies. Finally, the susceptibility of certain inbred mice to anthrax lethal toxin was associated with mutations in the kinesin-like protein Kif1C, a discovery that could help to explain how anthrax toxin kills animals.     

Heat shock inhibits caspase‐1 activity while also preventing its inflammasome‐mediated activation by anthrax lethal toxin

Anthrax lethal toxin (LT) rapidly kills macrophages from certain mouse strains in a mechanism dependent on the breakdown of unknown protein(s) by the proteasome, formation of the Nalp1b (NLRP1b) inflammasome and subsequent activation of caspase‐1. We report that heat‐shocking LT‐sensitive macrophages rapidly protects them against cytolysis by inhibiting caspase‐1 activation without upstream effects on LT endocytosis or cleavage of the toxin's known cytosolic substrates (mitogen‐activated protein kinases). Heat shock protection against LT occurred through a mechanism independent of de novo protein synthesis, HSP90 activity, p38 activation or proteasome inhibition and was downstream of mitogen‐activated protein kinase cleavage and degradation of an unknown substrate by the proteasome. The heat shock inhibition of LT‐mediated caspase‐1 activation was not specific to the Nalp1b (NLRP1b) inflammasome, as heat shock also inhibited Nalp3 (NLRP3) inflammasome‐mediated caspase‐1 activation in macrophages. We found that heat shock induced pro‐caspase‐1 association with a large cellular complex that could prevent its activation. Additionally, while heat‐shocking recombinant caspase‐1 did not affect its activity in vitro, lysates from heat‐shocked cells completely inhibited recombinant active caspase‐1 activity. Our results suggest that heat shock inhibition of active caspase‐1 can occur independently of an inflammasome platform, through a titratable factor present within intact, functioning heat‐shocked cells.     

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary

Background  

The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR).     

Linear Dispersal of the Filth Fly Parasitoid Spalangia cameroni (Hymenoptera: Pteromalidae) and Parasitism of Hosts at Increasing Distances

Release of parasitic wasps (Hymenoptera: Pteromalidae) as biological control agents for house flies and stable flies in livestock confinements has had variable success. In part, this may reflect a lack of knowledge regarding the optimal distance to be used between parasitoid release stations. In the current study, we assessed the effect of linear distance on host parasitism by the wasp Spalangia cameroni Perkins. In open fields at distances ranging from 1 m to 60 m from a central point, house fly puparia were placed in a mixture of pine shavings soiled with equine manure, urine, and alfalfa hay. Releases of S. cameroni then were made using a 5:1 host: parasitoid ratio. Host pupae were parasitized at all distances, with the highest rate of total parasitism (68.9%) recorded ≤ 5 m from the release site. Analyses of results using non-linear and linear models suggest that S. cameroni should be released in close proximity to host development areas. Additionally, releases may not be suitable in pasture situations where long-distance flight is required for control. However, further testing is needed to examine the effect of density-dependent dispersal and diffusion of S. cameroni.