Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis,and Cry10Aa-Cyt1Aa Synergism

Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.The subspecies Bacillus thuringiensis subsp. israelensis (serotype H-14) was discovered by Goldberg and Margalit in 1977 (11). To date, its insecticidal potential has not been overcome by any other bacterium (or any biological control agent) as an effective control measure against mosquito and blackfly larvae (8). Recently, its toxicity spectrum has been expanded to a coleopteran pest, the coffee berry borer (Hypothenemus hampei) (23), indicating that this strain may have potential versatility. Also, the so-called pBtoxis megaplasmid harbored in this strain, containing all the endotoxin-encoding genes found in its parasporal crystal, including cry4A, cry4B, cry10A, cry11A, and cyt1A, was recently sequenced (1). Among many other interesting aspects of this serotype, the occurrence of this mosquitocidal arsenal in one strain and their synergistic interaction make this bacterium scientifically and technologically attractive.The parasporal crystal of B. thuringiensis subsp. israelensis contains large amounts of Cry4A, Cry4B, Cry11A, and Cyt1A toxins (14), and consequently, most of the knowledge about the toxicity of this strain has been focused on these proteins, acting either as a complex (31) or tested separately (6). Although the cry10Aa gene was originally cloned in 1986 (known then as cryIVC) (30), to date, little is known about cry10Aa and the protein it encodes, mostly due to its very low levels of expression (10) in B. thuringiensis subsp. israelensis. Interestingly, cry10Aa is an operon as it includes two open reading frames (ORFs), previously reported as pBt047 and pBt048 (hereafter referred to only as ORF1 and ORF2, respectively), separated by a 48-bp untranslated gap (1). ORF1 contains the complete δ-endotoxin sequence (active toxin), with a coding capacity for a 78-kDa protein. Interestingly, ORF2 shows high identity with the coding sequence of the C-terminal half of Cry4-type proteins, with a coding capacity for a 56-kDa protein. Therefore, it is believed that a putative ancestral cry10Aa gene is similar in size to the cry4-type genes (ca. 4 kbp), but either a small sequence had been inserted in the middle of the coding sequence or site mutations produced end codons (two end codons flank the gap) in this region (1).Previous attempts to clone and express the cry10Aa gene included ORF1 and only part of ORF2 (7, 10, 30). This was a reasonable strategy, as most of the so-called “complete” protoxins are partially digested to become active toxins (δ-endotoxins) (28), and ORF1 included the complete sequence to code the Cry10Aa δ-endotoxin. However, in all these cases, the expression levels were very low, and no parasporal body was formed. Similar results were obtained when the promoter was changed and a stabilizing sequence was added to the construction (13). The low expression levels achieved in these cases led to conclusions that assumed low toxic levels of Cry10Aa when tested against mosquito larvae (30). In spite of the low toxicity of Cry10Aa found against mosquito larvae, a synergistic effect was reported between Cry10Aa and Cry4Ba toxins in Culex (7). Obtaining high levels of expression and crystallization of Cry10Aa are required to properly characterize and understand the toxic spectrum of this protein.In this report, we show the formation of parasporal bodies of Cry10Aa, achieved by cloning the whole Cry10Aa operon under the control of the cyt1A promoter and the STAB-SD sequence. We also show that Cry10Aa is as toxic as most of the other B. thuringiensis subsp. israelensis toxins acting separately, and in synergism with the Cyt1A toxin.     

Effect of a nuclepolyhedrovirus of Autographa californica expressing the enhancin gene of Trichoplusia ni granulovirus on T. ni larvae

A recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) strain showing higher virulence against Trichoplusia ni larvae than the wild-type virus was developed. The 'enhancin' (VEF) gene of T. ni granulovirus (TnGV) and the AcMNPV polyhedrin gene were cloned into the baculovirus transfer vector pAcUW31. This plasmid and AcMNPV BacPAK6 DNA were co-transfected into the BTI-Tn5B1-4 cell line. A recombinant AcMNPV strain (BacVEFPol) was purified, amplified, and bioassayed against T. ni first instar larvae. Its estimated LC₅₀ (0.184 OB/mm²) was 2.18 times lower than the LC₅₀ estimated for the wild-type AcMNPV (0.402 OB/mm²). Likewise, an LT₅₀ of 67.7 h was estimated for the recombinant AcMNPV strain while the LT₅₀ of wild-type AcMNPV was estimated at 81.9 h. This indicates a 17.4% reduction of the time required to kill the larvae. The higher virulence of the recombinant strain, evidenced by its LC₅₀ and LT₅₀ values being lower than those of the wild-type strain, indicates that the VEF protein is expressed properly and may be occluded in the OBs.     

“One feels anger to know there is no one to help us!”. Perceptions of mothers of children with Zika virus-associated microcephaly in Caribbean Colombia: A qualitative study

BackgroundThe epidemic of Zika virus (ZIKV) was associated with a sudden and unprecedented increase in infants born with microcephaly. Colombia was the second most affected country by the epidemic in the Americas. Primary caregivers of children with ZIKV-associated microcephaly, their mothers mainly, were at higher risk of suffering anxiety and depression. Often, these women were stigmatized and abandoned by their partners, relatives, and communities.Methodology/Principal findingsThis study aimed to understand the perceptions about ZIKV infection among mothers of children born with microcephaly during the ZIKV epidemic in Caribbean Colombia, and the barriers and facilitators affecting child health follow-up. An exploratory qualitative study, based on Phenomenology and Grounded Theory, was conducted in Caribbean Colombia. Data were collected through In-Depth Interviews (IDI) from women who delivered a baby with microcephaly during the ZIKV epidemic at Clínica Salud Social, Sincelejo, Sucre District (N = 11). The themes that emerged during the interviews included experiences from their lives before pregnancy; knowledge about ZIKV; experiences and perceptions when diagnosed; considering a possible termination of pregnancy, and children’s clinical follow-up. In some cases, women reported having been told they were having a baby with microcephaly but decided not to terminate the pregnancy; while in other cases, women found out about their newborn’s microcephaly condition only at birth. The main barriers encountered by participants during children’s follow-up included the lack of psychosocial and economic support, the stigmatization and abandonment by some partners and relatives, and the frustration of seeing the impaired development of their children.ConclusionsThis study contributed to identifying the social, medical, psychological, and economic needs of families with children affected by the ZIKV epidemic. Commitment and action by local and national governments, and international bodies, is required to ensure sustained and quality health services by affected children and their families.     

Sequencing and Characterization of Plasmid pUIBI-1 from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Serovar <Emphasis Type="Italic">entomocidus</Emphasis> LBIT-113

Plasmid pUIBI-1 from Bacillus thuringiensis svr. entomocidus was sequenced and its replication mechanism analyzed. Sequence analysis revealed that pUIBI-1 contains 4671 bp and a 32% GC content. Plasmid pUIBI-1 also includes at least seven putative open reading frames (ORFs) encoding for proteins ranging from 5 to 50 kDa. ORF-1 encodes for a putative 16-kDa Rep protein, which lacks homology with proteins of similar function. ORF2 encodes for a protein of 50 kDa and shows homology with Mob proteins of plasmids pLUB1000 from Lactobacillus hilgardii (32.2%) and pGI2 from B. thuringiensis (33.7%). Detection of single-stranded DNA (ssDNA) intermediates indicated that pUIBI-1 replicates by the rolling-circle replication mechanism, as demonstrated by S1 treatment and Southern hybridization under non-denaturing conditions.     

Synthesis and X-ray crystal structure analysis of 1:1 and 1:2 complexes of cisplatin with the model nucleobase 9-methyladenine in its protonated form and a unique HNO3 adduct of cis-[(NH3)2Pt(9-MeAH-N7)2]

The 1:1 and 1:2 complexes of cis-(NH₃)₂Pt^II with 9-methyladeninium cations, 9-MeAH⁺, have been prepared and characterized by X-ray crystallography: cis-[(NH₃)₂Pt(9-MeAH-N7)Cl](NO₃)₂ (1) and cis-[(NH₃)₂Pt(9-MeAH-N7)₂](NO₃)₄ · 2HNO₃ · 2H₂O (2). The pK_a values for 9-MeAH⁺ in H₂O are 1.7 in 1 as well as 0.4 (pK_a1) and 1.3 (pK_a2) for 2, as determined by pD dependent ¹H NMR spectroscopy. Compound 2 is special in that it crystallizes with two equivalents of HNO₃ per Pt entity. The HNO₃ molecules are stacked in rectangular channels provided by cis-(NH₃)₂Pt^II units, 9-methyladeninium ligands and nitrate anions, which form a porous network of hydrogen bonds.