Cytocidal activity of Cry41Aa, an anticancer toxin from Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Amongst these parasporins, parasporin-3 most closely resembles the commercially used insecticidal toxins and presents the narrowest activity spectrum, showing moderate cytotoxicity against only two cancer cell lines, HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells). Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards these two cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made at the N- or C-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60 respectively) which we hypothesize occlude a binding motif. Preliminary work carried out on binding showed a lack of correlation between binding and toxicity since toxin binds to both susceptible and non-susceptible cancer cell lines. In an attempt to better understand the mechanism of action of Cry41Aa against these cells, we evolved resistance in HepG2 through repeated exposure to increasing doses of the toxin. Morphological, physiological and genetic characteristics of the resistant cell line were compared with susceptible cells. Toxin was shown to bind to resistant HepG2 similarly to the susceptible line. RNA sequencing identified AQP9 as a potential mediator of resistance but extensive investigations failed to show a direct link. The involvement of certain intracellular signalling pathways were also investigated in order to understand cell responses to the toxin and showed that in response to the toxin p38, but not ERK1/2, is activated and in a dose dependent manner

Synthesis of novel heteroleptic delocalised cationic pyrazole gold complexes as potent HepG2 cytotoxic agents

A new series of cationic gold(I) pyrazole complexes were prepared in excellent yields as their perchlorate salts. Results of cell viability assays show that these novel complexes have good cytotoxic properties against the human HepG2 cancer cell line. These complexes showed promising anti-cancer activities and to our knowledge, pyrazoles have never been tested against this cell line. The regioselectivity of the complexation is also discussed in regards to the substitution pattern of the pyrazoles

Differential proteolytic activation of the Bacillus thuringiensis Cry41Aa parasporin modulates its anticancer effect

Bacillus thuringiensis (Bt) is a gram positive spore forming bacterium which produces intracellular protein crystals toxic to a wide variety of insect larvae and is the most commonly used biological pesticide worldwide. More recently, Bt crystal proteins known as parasporins have been discovered, that have no known insecticidal activity but target some human cancer cells exhibiting strong cytocidal activities with different toxicity spectra and varied activity levels. Parasporin-3, also called Cry41Aa, has only been shown to exhibit cytocidal activity towards HL-60 (Human promyelocytic leukemia cells) and HepG2 (Human liver cancer cells) cell lines after being proteolytically cleaved. In order to understand this activation mechanism various mutations were made in the N-terminal region of the protein and the toxicity against both HepG2 and HL-60 cell lines was evaluated. Our results indicate that only N-terminal cleavage is required for activation and that N-terminally deleted mutants show some toxicity without the need for proteolytic activation. Furthermore we have shown that the level of toxicity towards the two cell lines depends on the protease used to activate the toxin. Proteinase K-activated toxin was significantly more toxic towards HepG2 and HL-60 than trypsin-activated toxin. N-terminal sequencing of activated toxins showed that this difference in toxicity is associated with a difference of just two amino acids (serine and alanine at positions 59 and 60 respectively) which we hypothesize occlude a binding motif

Probing the mechanism of action of Cry41Aa on HepG2 through the establishment of a resistant subline

Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium Bacillus thuringiensis that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin. Concentrations of Cry41Aa that killed over 50% of the parental HepG2 cells had no significant effect on the viability of the resistant cells and did not induce either pore formation or p38 phosphorylation (both characteristic features of pore-forming toxins). Preliminary RNA sequencing data identified AQP9 as a potential mediator of resistance, but extensive investigations failed to show a causal link and did not support an enhanced cell repair process as the resistance mechanism

Assessing the construct validity of a theory of mind battery adapted to Tunisian school-aged children

Background: Theory of mind (ToM) refers to the ability to understand others’ states of mind, desires, emotions, beliefs, and intentions to predict the content of their mental representations. Two major dimensions within ToM have been studied. The first is the type of inferred mental state, which can be cognitive or affective. The second comprises the types of processes involved according to their degree of complexity (first- and second-order false belief and advanced ToM). ToM acquisition is fundamental—a key component in the development of everyday human social interactions. ToM deficits have been reported in various neurodevelopmental disorders through various tools assessing disparate facets of social cognition. Nevertheless, Tunisian practitioners and researchers lack a linguistically and culturally appropriate psychometric tool for ToM assessment among school-aged children. Objective: To assess the construct validity of a translated and adapted French ToM Battery for Arabic-speaking Tunisian school-aged children. Methods: The focal ToM Battery was designed with neuropsychological and neurodevelopmental theory and composed of 10 subtests distributed evenly in three parts: Pre-conceptual, cognitive, and affective ToM. Translated and adapted to the Tunisian sociocultural context, this ToM battery was individually administered to 179 neurotypical Tunisian children (90 girls and 89 boys) aged 7–12 years. Results: After controlling for the age effect, construct validity was empirically confirmed on two dimensions (cognitive and affective) via structural equation modeling (SEM) analysis, demonstrating that this solution has a good fit. The results confirmed that the age affected differentially the performance obtained on ToM tasks based on the two components of the battery. Conclusion: Our findings confirm that the Tunisian version of the ToM Battery has robust construct validity for the assessment of cognitive and affective ToM in Tunisian school-aged children; hence, it could be adopted in clinical and research settings.Open Access funding provided by Qatar National Library

Probing the Mechanism of Action of Cry41Aa on HepG2 through the Establishment of a Resistant Subline

Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium Bacillus thuringiensis that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin. Concentrations of Cry41Aa that killed over 50% of the parental HepG2 cells had no significant effect on the viability of the resistant cells and did not induce either pore formation or p38 phosphorylation (both characteristic features of pore-forming toxins). Preliminary RNA sequencing data identified AQP9 as a potential mediator of resistance, but extensive investigations failed to show a causal link and did not support an enhanced cell repair process as the resistance mechanism

Selecting for infectivity across metapopulations can increase virulence in the social microbe <i>Bacillus thuringiensis</i>

Passage experiments that sequentially infect hosts with parasites have long been used to manipulate virulence. However, for many invertebrate pathogens, passage has been applied naively without a full theoretical understanding of how best to select for increased virulence and this has led to very mixed results. Understanding the evolution of virulence is complex because selection on parasites occurs across multiple spatial scales with potentially different conflicts operating on parasites with different life histories. For example, in social microbes, strong selection on replication rate within hosts can lead to cheating and loss of virulence, because investment in public goods virulence reduces replication rate. In this study, we tested how varying mutation supply and selection for infectivity or pathogen yield (population size in hosts) affected the evolution of virulence against resistant hosts in the specialist insect pathogen Bacillus thuringiensis, aiming to optimize methods for strain improvement against a difficult to kill insect target. We show that selection for infectivity using competition between subpopulations in a metapopulation prevents social cheating, acts to retain key virulence plasmids, and facilitates increased virulence. Increased virulence was associated with reduced efficiency of sporulation, and possible loss of function in putative regulatory genes but not with altered expression of the primary virulence factors. Selection in a metapopulation provides a broadly applicable tool for improving the efficacy of biocontrol agents. Moreover, a structured host population can facilitate artificial selection on infectivity, while selection on life-history traits such as faster replication or larger population sizes can reduce virulence in social microbes