Table of Contents

Abstract: A one-step affinity chromatography method was developed to purify Shiga toxin 2 variants (Stx2) Stx2a, Stx2c, Stx2d and Stx2g from bacterial culture supernatants. Analysis of the purified Stx2 variants by denaturing gel electrophoresis revealed 32 kDa and 7 kDa protein bands, corresponding to the Stx2A- and B-subunits, respectively. However, native gel electrophoresis indicated that purified Stx2c and Stx2d were significantly higher in molecular weight than Stx2a and Stx2g. In a cytotoxicity assay with Hela cells, the 50% cytotoxic dose of Stx2a and Stx2g were 100 pg and 10 pg, respectively, but 1 ng each for Stx2c and Stx2d. Interestingly, analysis of the 50% inhibitory dose in a cell-free translational system from rabbit reticulocyte lysates indicated that Stx2g had a lower capacity to inhibit protein synthesis than the other Stx2 variants. The cytotoxicities in Hela cells were neutralized with an anti-Stx2B antibody and were denatured at 80 °C for 1 h. These findings demonstrated that Stx2 variants exhibited different toxicities, holotoxin structure, and stabilities using distinct systems for assessing toxin activities. The development of a simple method for purification of Stx2 variants will enable further studies of Stx2-mediated toxicity in various model systems.

Abstract: Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5′-triphosphate, uridine 5′-triphosphate and inosine 5′-triphosphate, in addition to adenosine 5′-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3′:5′-monophosphate, cyclic uridine 3′:5′-monophosphate and cyclic inosine 3′:5′-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed.

Abstract: The mechanism of phagosome escape by intracellular pathogens is an important step in the infectious cycle. During the establishment of anthrax, Bacillus anthracis undergoes a transient intracellular phase in which spores are engulfed by local phagocytes. Spores germinate inside phagosomes and grow to vegetative bacilli, which emerge from their resident intracellular compartments, replicate and eventually exit from the plasma membrane. During germination, B. anthracis secretes multiple factors that can help its resistance to the phagocytes. Here the possible role of B. anthracis toxins, phospholipases, antioxidant enzymes and capsules in the phagosomal escape and survival, is analyzed and compared with that of factors of other microbial pathogens involved in the same type of process.

Abstract: Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA₂). The PLA₂ exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V_HH) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V_HH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V_HH, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V_HH purified from the E. coli homogenates neutralized PLA₂ enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V_HH covered the areas around the PLA₂ catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V_HH would ameliorate/abrogate the principal toxicity of the venom PLA₂ (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

Abstract: This work proposes to use the marine algal toxin yessotoxin (YTX) to establish reference model experiments to explore medically valuable effects from induction of multiple cell death pathways. YTX is one of few toxins reported to make such induction. It is a small molecule compound which at low concentrations can induce apoptosis in primary cultures, many types of cells and cell lines. It can also induce a non-apoptotic form of programmed cell death in BC3H1 myoblast cell lines. The present contribution reviews arguments that this type of induction may have principal interest outside this particular example. One principal effect of medical interest may be that cancer cells will not so easily adapt to the synergistic effects from induction of more than one death pathway as compared to induction of only apoptosis.