Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis.

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Bacillus produces many virulence factors that may contribute to retinal damage and robust inflammation. We analyzed Bacillus immune inhibitor A (InhA) metalloproteases in the context of this disease, hypothesizing that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild-type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. The ΔinhA1 and ΔinhA2 mutants entered log-phase growth earlier than WT B. thuringiensis. Proteolysis by the ΔinhA1-3 mutant was decreased, but this strain grew similar to WT in vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of WT B. thuringiensis or InhA mutants. Eyes were analyzed for intraocular Bacillus and myeloperoxidase concentrations, retinal function loss, and gross histological changes. Eyes infected with the ΔinhA1 or ΔinhA2 mutant strains contained greater numbers of bacteria than eyes infected with WT throughout the infection course. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with the WT strain. Eyes infected with the ΔinhA1-3 mutant cleared the infection. Quantitative real-time PCR (qRT-PCR) results suggested that there may be compensatory expression of the other InhAs in the single InhA mutant. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis.

Regulator ThnR and the ThnDE ABC transporter proteins confer autoimmunity to thurincin H in Bacillus thuringiensis.

The structural gene that encodes thurincin H, a bacteriocin produced by Bacillus thuringiensis, is harboured in a genetic cluster (thnP, E, D, R, A1, A2, A3, B, T, I) that controls its synthesis, modification, secretion and autoimmunity. The specific genes in the cassette that confer immunity in B. thuringiensis to thurincin H are unknown. To identify these immunity determinants, we generated constructs that were used to transform a natural thurincin H-sensitive B. thuringiensis strain (i.e. Btk 404), and resistance or susceptibility to the bacteriocin in resultant recombinants was evaluated. When Btk 404/pHT3101-ThnARDEP and Btk 404/pHT3101-ThnABTI were exposed to thurincin H, immunity was demonstrated by the former only, indicating that ThnI does not play a role in resistance to the bacteriocin as previously proposed. Furthermore, we generated different sub-cassettes under the control of divergent promoters pThnR and pThur of the thurincin H locus, and pChi, and using the green fluorescent protein gene as reporter, which demonstrated that all promoters were recognised by ThnR, except pChi. We show for the first time that the small operon composed of thnR, thnD and thnE is required for immunity of B. thuringiensis to thurincin H, and thnI is not necessary for this response.

Cry1A(b)16 toxin from Bacillus thuringiensis: Theoretical refinement of three-dimensional structure and prediction of peptides as molecular markers for detection of genetically modified organisms.

Transgenic maize produced by the insertion of the Cry transgene into its genome became the second most cultivated crop worldwide. Cry gene from Bacillus thuringiensis kurstaki expresses protein derivatives of crystalline endotoxins which confer insect resistance onto the maize crop. Mandatory labeling of processed food containing or made by genetically modified organisms is in force in many countries, so, it is very urgent to develop fast and practical methods for GMO identification, for example, biosensors. In the absence of an available empirical structure of Cry1A(b)16 protein, a theoretical model was effectively generated, in this work, by homology modeling and molecular dynamics simulations based on two available homologous protein structures. Molecular dynamics simulations were carried out to refine the selected model, and an analysis of its global structure was performed. The refined models of Cry1A(b)16 showed a standard fold and structural characteristics similar to those seen in Bacillus thuringiensis Cry1A(a) insecticidal toxin and Bacillus thuringiensis serovar kurstaki Cry1A(c) toxin. After in silico analysis of Cry1A(b)16, two immunoreactive candidate peptides were selected and specific polyclonal antibodies were produced resulting in antibody-peptide interaction. Biosensing devices are expected to be developed for detection of the Cry1A(b) protein as a marker of transgenic maize in food. Proteins 2017; 85:1248-1257. © 2017 Wiley Periodicals, Inc.

Simultaneous production of monoclonal antibodies against Bacillus thuringiensis (Bt) Cry1 toxins using a mixture immunization.

The detections of Cry1 toxins are mainly dependent on immunoassays based on specific monoclonal antibodies (mAb). In the present study, a mixture immunization with seven Cry1 toxins was administered. The results showed that five mAbs with different characteristics, especially one mAb named 5-E8 which could recognize all the seven Cry1 toxins were obtained. Based on the 5-E8 mAb, a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) which can specifically detect the seven Cry1 toxins without cross-reactivity to Cry2A and vip3 was developed with the limit of detection (LOD) and limit of quantification (LOQ) of 6.37-11.35 ng mL-1 and 17.36-24.48 ng mL-1, respectively. The recovery tests showed that the recoveries ranged from 78% to 110% within the quantitation range (LOQ-100 ng mL-1). The established DAS-ELISA can be a useful tool for monitoring the Cry1 toxins in agricultural products. Mixture immunization opens a new path for producing diverse mAbs simultaneously in a single immunization circle.

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10-8 M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL-1, respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL-1), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Intraperitoneal Immunization with Cry1Ac Protoxin from Bacillus thuringiensis Provokes Upregulation of Fc-Gamma-II/and Fc-Gamma-III Receptors Associated with IgG in the Intestinal Epithelium of Mice.

In humans, intestinal epithelial FcRn is expressed throughout life and mediates the bidirectional transport of IgG, but in mice, it is markedly expressed in neonatal intestine. In adults, its expression is only faintly upregulated after intestinal IgG induction such as that elicited by i.p. immunization with Cry1Ac protoxin (pCry1Ac) Bacillus thuringiensis. This led us to suggest that additional Fcγ receptors (Fcγ-R) may be participating in epithelial IgG uptake. So, first we determined whether CD16/32 [an epitope shared by Fcγ-RII (CD32) and Fcγ-RIII (CD16)] was expressed in the intestinal epithelia of mice. Using confocal microscopy and flow cytometry, we detected co-localization of IgG and CD16/32 in epithelial cells, whose frequency was increased by immunization with pCry1Ac. Western blot and cross-immunoprecipitation results with anti-CD16/32 and IgG antibodies in epithelial cell extracts suggested that epithelial cells bear both Fcγ-RII and Fcγ-RIII and contained IgG associated with Fcγ-RII/RIII. Using anti-CD32 and anti-CD16 antibodies, we confirmed by Western blot, confocal microscopy and flow cytometry that both Fcγ-RII and Fcγ-RIII were expressed and suggested that upregulation occurred upon immunization in intestinal epithelia. Finally, we examined the in vitro effect of anti-CD16/32, anti-CD16 and anti-CD32 antibodies on IgG uptake and transport by intestinal epithelial cells and found that it was partially reduced. Although further studies are still required, our results suggest that Fcγ-RII and Fcγ-RIII might participate in the uptake and/or transport of IgG through the intestinal epithelia of adult mice.

Contributions of cellular and humoral immunity of Galleria mellonella larvae in defence against oral infection by Bacillus thuringiensis.

In this study the cellular and humoral immune reactions of the Greater wax moth Galleria mellonella have been investigated during bacterial infection caused by oral administration of Bacillus thuringiensis. Two different dose strengths were investigated to assess the contribution of immune parameters to induced Bt resistance. Low-dose (sublethal LC15) infection resulted in significantly elevated haemolymph phenoloxidase and lysozyme-like activity, enhanced phagocytic activity of haemocytes, and increased encapsulation responses in infected larvae at 48 and 72 h post infection. Higher doses of Bt (half-lethal LC50) also triggered significantly elevated haemolymph phenoloxidase and lysozyme-like activity, but decreased the coagulation index and activity of phenoloxidase in haemocytes of infected larvae. In both types of infection, the pool of circulating haemocytes became depleted. The importance of cellular and humoral immune reactions in induced insect resistance to intestinal bacterial infection Bt is herein discussed.

Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster.

Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.

Granulocyte dynamics: a key player in the immune priming effects of crickets.

This study investigates immune priming effects associated with granulocytes in crickets through a comprehensive analysis. Kaplan-Meier survival analysis reveals a significant contrast in survival rates, with the heat-killed Bacillus thuringiensis (Bt)-primed group exhibiting an impressive ~80% survival rate compared to the PBS buffer-primed group with only ~10% survival 60 hours post live Bt infection. Hemocyte analysis underscores elevated hemocyte counts, particularly in granulocytes of the killed Bt-primed group, suggesting a correlation between the heat-killed Bt priming and heightened immune activation. Microscopy techniques further explore granulocyte morphology, unveiling distinctive immune responses in the killed Bt-primed group characterized by prolonged immune activation, heightened granulocyte activity, phagocytosis, and extracellular trap formation, contributing to enhanced survival rates. In particular, after 24 hours of injecting live Bt, most granulocytes in the PBS buffer-primed group exhibited extracellular DNA trap cell death (ETosis), while in the killed Bt-primed group, the majority of granulocytes were observed to maintain highly activated extracellular traps, sustaining the immune response. Gene expression analysis supports these findings, revealing differential regulation of immune-related genes such as antibacterial humoral response, detection of bacterial lipopeptides, and cellular response to bacteria lipopeptides. Additionally, the heat-killed Bt-primed group, the heat-killed E. coli-primed group, and the PBS-primed group were re-injected with live Bt 2 and 9 days post priming. Two days later, only the PBS-primed group displayed low survival rates. After injecting live Bt 9 days later, the heat-killed E. coli-primed group surprisingly showed a similarly low survival rate, while the heat-killed Bt-primed group exhibited a high survival rate of ~60% after 60 hours, with actively moving and healthy crickets. In conclusion, this research provides valuable insights into both short-term and long-term immune priming effects in crickets, contributing to our understanding of invertebrate immunity with potential applications in public health.

Tribolium castaneum Apolipophorin-III acts as an immune response protein against Bacillus thuringiensis Cry3Ba toxic activity.

In this study, a 2.1-fold Apolipophorin-III mRNA up-regulation was found in Tribolium castaneum larvae challenged with Bacillus thuringiensis Cry3Ba spore-crystal mixture. Knockdown of Apolipophorin-III by RNAi resulted in increased T. castaneum larvae susceptibility following Cry3Ba spore-crystal treatment, demonstrating Apolipophorin-III involvement in insect defense against B. thuringiensis. We showed that Apolipophorin-III participates in T. castaneum immune response to B. thuringiensis activating the prophenoloxidase cascade since: (i) phenoloxidase activity significantly increased after Cry3Ba spore-crystal treatment compared to untreated or Cry1Ac spore-crystal treated larvae and (ii) phenoloxidase activity in Cry3Ba spore-crystal treated Apolipophorin-III silenced larvae was 71±14% lower than that of non-silenced intoxicated larvae.

Development of a high-resolution melting-based approach for efficient differentiation among Bacillus cereus group isolates.

Strains belonging to Bacillus cereus Group include six different species, among which are Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus cereus sensu stricto, a causative agent of food poisoning. Sequence of the panC-housekeeping gene is used for B. cereus Group affiliation to seven major phylogenetic groups (I-VII) with different ecological niches and variations in thermal growth range and spore heat resistance of B. cereus Group microorganisms varies among phylogenetic groups. We assigned a selection of B. cereus sensu stricto strains related to food poisoning from the Spanish cultivar Collection (Valencia) to Group IV strains based on panC gene sequence. Thermal inactivation assays revealed variability of spore heat resistance within these Group IV strains. Adequate food sanitizing treatments therefore require fast and reliable identification of particular strains. In the present study, feasibility of genotyping via high-resolution melting (HRM) analysis was examined. HRM analysis of amplified polymorphic 16S-23 intergenic spacer region (ISR) region proved to be discriminatory for B. cereus sensu stricto strain typing, while two other polymorphic regions within the bacterial rRNA operon allowed differentiation between Bacillus species, demonstrating its applicability for discrimination on the species and strain level within B. cereus Group.

Dietary administration of Bacillus thuringiensis on the cellular innate immune response of African catfish (Clarias gariepinus) against Aeromonas hydrophila.

AIM: An experiment was conducted to evaluate the use of Bacillus thuringiensis (Bt) as a probiotic to enhance the cellular innate immune response of the African catfish (Clarias gariepinus) challenged with a bacterial fish pathogen, Aeromonas hydrophila. MATERIALS AND METHODS: The bacterium (Bt) was administered orally at three different dietary doses of 0.5 x 10(7) (T2), 1.0 x 10(7) (T3) and 1.5 x 107 (T4) cfu/g feed to the fingerlings of African catfish twice daily for 15 days. The positive control group (T1) and negative control group (T5) was fed without Bt for the same period. On the 16th day, blood and serum samples were withdrawn to determine the differential leukocyte count (DLC) and serum bactericidal activity. After 15 days feeding, the treatment groups (T2, T3 and T4) and positive control (T1) were challenged intraperitoneally with the bacterial suspension of 0.2 ml (1 x 10(8) cfu/ml). The negative control group (T5) was injected intraperitoneally with phosphate buffered saline (PBS). On the 3rd day of post challenge, the DLC was assessed in all the treatment groups. RESULTS: The fish fed with Bt incorporated diet showed significantly higher (P < 0.05) bactericidal activity during the pre-challenge compared with the control groups. The highest serum bactericidal activity was observed in T4, the group fed with diet containing highest concentration of Bt. Highest survival rates (90%) were recorded in fish fed with Bt supplemented group (T3 and T4). Granulocyte counts on the other hand were higher in treatment groups compared to the control in both pre and post challenge. CONCLUSIONS: The result demonstrates that Bt can enhance certain innate immune responses in African catfish.

Molecular characterization and expression patterns of heat shock proteins in Spodoptera littoralis, heat shock or immune response?

The Egyptian cotton leaf worm, Spodoptera littoralis (Boisd.), is a major agricultural lepidopterous pest causing extensive damage in a variety of crops including vegetable, cotton, fodder, and fiber crops. Heat shock protein (HSP) family members play important roles in protecting insects against environmental stressors. In this study, we characterized three putative heat shock proteins (SpliHsp70, SpliHsp90, and SpliHSF) from S. littoralis and analyzed their expression levels in response to heat, cold, ultraviolet irradiation, Bacillus thuringiensis, and Spodoptera littoralis nucleopolyhedrovirus treatments. Significant upregulation of SpliHsp70 was observed in female pupae, while the highest expression levels of SpliHsp90 and SpliHSF were found in female adults. Heat shock triggered increases in SpliHsp levels compared to cold treatment. SpliHsp90 exhibited the highest expression levels during the first 30 min of UV treatment. Both bacterial and viral pathogenic agents effected the regulation of Hsps in S. littoralis. These findings suggest that SpliHsp genes might play significant roles in the response to biotic and abiotic stress, as well as in the regulation of developmental stages.

Differential capability of Bacillus thuringiensis Cry1Ac protoxin and toxin to induce in vivo activation of dendritic cells and B lymphocytes.

The insecticidal Bacillus thuringiensis protein Cry1Ac is produced as a protoxin and becomes activated to a toxin when ingested by larvae. Both proteins are immunogenic and able to activate macrophages. The proposed mechanism of immunostimulation by Cry1Ac protoxin has been related to its capacity to activate antigen-presenting cells (APC), but its ability to activate dendritic cells (DC) has not been explored. Here we evaluated, in the popliteal lymph nodes (PLN), spleen and peritoneum, the activation of DC CD11c+ MHC-II+ following injection with single doses (50 µg) of Cry1Ac toxin or protoxin via the intradermal (i.d.) and intraperitoneal (i.p.) routes in C57BL/6 mice. In vivo stimulation with both Cry1Ac proteins induced activation of DC via upregulation of CD86, primarily in PLN 24 h after i. d. injection. Moreover, this activation was detected in DC, displaying CD103+, a typical marker of migratory DC, while upregulation of CD80 was uniquely induced by toxin. Tracking experiments showed that Cy5-labeled Cry1Ac proteins could rapidly reach the PLN and localize near DC, but some label remained in the footpad. When the capacity of Cry1Ac-activated DC to induce antigen presentation was examined, significant proliferation of naïve T lymphocytes was induced exclusively by the protoxin. The protoxin elicited a Th17-biased cytokine profile. Moreover, only the Cry1Ac toxin induced a pronounced proliferation of B cells from both untreated and Cry1Ac-injected mice, suggesting that it acts as a polyclonal activator. In conclusion, Cry1Ac protoxin and toxin show a distinctive capacity to activate APCs.

Sub-chronic lung inflammation after airway exposures to Bacillus thuringiensis biopesticides in mice.

BACKGROUND: The aim of the present study was to assess possible health effects of airway exposures to Bacillus thuringiensis (Bt) based biopesticides in mice. Endpoints were lung inflammation evaluated by presence of inflammatory cells in bronchoalveolar lavage fluid (BALF), clearance of bacteria from the lung lumen and histological alterations of the lungs. Hazard identifications of the biopesticides were carried out using intratracheal (i.t.) instillation, followed by an inhalation study. The two commercial biopesticides used were based on the Bt. subspecies kurstaki and israelensis, respectively. Groups of BALB/c mice were i.t instilled with one bolus (3.5 × 105 or 3.4 × 106 colony forming units (CFU) per mouse) of either biopesticide. Control mice were instilled with sterile water. BALFs were collected and the inflammatory cells were counted and differentiated. The BALFs were also subjected to CFU counts. RESULTS: BALF cytology showed an acute inflammatory response dominated by neutrophils 24 hours after instillation of biopesticide. Four days after instillation, the neutrophil number was normalised and inflammation was dominated by lymphocytes and eosinophils, whereas 70 days after instillation, the inflammation was interstitially located with few inflammatory cells present in the lung lumen.Half of the instilled mice had remaining CFU recovered from BALF 70 days after exposure. To gain further knowledge with relevance for risk assessment, mice were exposed to aerosols of biopesticide one hour per day for 2 × 5 days. Each mouse received 1.9 × 104 CFU Bt israelensis or 2.3 × 103 CFU Bt kurstaki per exposure. Seventy days after end of the aerosol exposures, 3 out of 17 mice had interstitial lung inflammation. CFU could be recovered from 1 out of 10 mice 70 days after exposure to aerosolised Bt kurstaki. Plethysmography showed that inhalation of Bt aerosol did not induce airway irritation. CONCLUSIONS: Repeated low dose aerosol exposures to commercial Bt based biopesticides can induce sub-chronic lung inflammation in mice, which may be the first step in the development of chronic lung diseases. Inhalation of Bt aerosols does not induce airway irritation, which could explain why workers may be less inclined to use a filter mask during the application process, and are thereby less protected from exposure to Bt spores.

Chemical modulators of the innate immune response alter gypsy moth larval susceptibility to Bacillus thuringiensis.

BACKGROUND: The gut comprises an essential barrier that protects both invertebrate and vertebrate animals from invasion by microorganisms. Disruption of the balanced relationship between indigenous gut microbiota and their host can result in gut bacteria eliciting host responses similar to those caused by invasive pathogens. For example, ingestion of Bacillus thuringiensis by larvae of some species of susceptible Lepidoptera can result in normally benign enteric bacteria exerting pathogenic effects. RESULTS: We explored the potential role of the insect immune response in mortality caused by B. thuringiensis in conjunction with gut bacteria. Two lines of evidence support such a role. First, ingestion of B. thuringiensis by gypsy moth larvae led to the depletion of their hemocytes. Second, pharmacological agents that are known to modulate innate immune responses of invertebrates and vertebrates altered larval mortality induced by B. thuringiensis. Specifically, Gram-negative peptidoglycan pre-treated with lysozyme accelerated B. thuringiensis-induced killing of larvae previously made less susceptible due to treatment with antibiotics. Conversely, several inhibitors of the innate immune response (eicosanoid inhibitors and antioxidants) increased the host's survival time following ingestion of B. thuringiensis. CONCLUSIONS: This study demonstrates that B. thuringiensis infection provokes changes in the cellular immune response of gypsy moth larvae. The effects of chemicals known to modulate the innate immune response of many invertebrates and vertebrates, including Lepidoptera, also indicate a role of this response in B. thuringiensis killing. Interactions among B. thuringiensis toxin, enteric bacteria, and aspects of the gypsy moth immune response may provide a novel model to decipher mechanisms of sepsis associated with bacteria of gut origin.

The Tripartite Interaction of Host Immunity-Bacillus thuringiensis Infection-Gut Microbiota.

Bacillus thuringiensis (Bt) is an important cosmopolitan bacterial entomopathogen, which produces various protein toxins that have been expressed in transgenic crops. The evolved molecular interaction between the insect immune system and gut microbiota is changed during the Bt infection process. The host immune response, such as the expression of induced antimicrobial peptides (AMPs), the melanization response, and the production of reactive oxygen species (ROS), varies with different doses of Bt infection. Moreover, B. thuringiensis infection changes the abundance and structural composition of the intestinal bacteria community. The activated immune response, together with dysbiosis of the gut microbiota, also has an important effect on Bt pathogenicity and insect resistance to Bt. In this review, we attempt to clarify this tripartite interaction of host immunity, Bt infection, and gut microbiota, especially the important role of key immune regulators and symbiotic bacteria in the Bt killing activity. Increasing the effectiveness of biocontrol agents by interfering with insect resistance and controlling symbiotic bacteria can be important steps for the successful application of microbial biopesticides.

Comparative genomic analysis and mosquito larvicidal activity of four Bacillus thuringiensis serovar israelensis strains.

Bacillus thuringiensis serovar israelensis (Bti) is used to control insect vectors of human and animal diseases. In the present study, the toxicity of four strains of Bti, named T0124, T0131, T0137, and T0139, toward Aedes aegypti and Culex quinquefasciatus larvae was analyzed. The T0131 strain showed the highest larvicidal activity against A. aegypti (LC50 = 0.015 µg/ml) and C. quinquefasciatus larvae (LC50 = 0.035 µg/ml) when compared to the other strains. Furthermore, the genomic sequences of the four strains were obtained and compared. These Bti strains had chromosomes sizes of approximately 5.4 Mb with GC contents of ~35% and 5472-5477 putative coding regions. Three small plasmids (5.4, 6.8, and 7.6 kb) and three large plasmids (127, 235, and 359 kb) were found in the extrachromosomal content of all four strains. The SNP-based phylogeny revealed close relationship among isolates from this study and other Bti isolates, and SNPs analysis of the plasmids 127 kb did not reveal any mutations in δ-endotoxins genes. This newly acquired sequence data for these Bti strains may be useful in the search for novel insecticidal toxins to improve existing ones or develop new strategies for the biological control of important insect vectors of human and animal diseases.

Immunization with Cry1Ac from Bacillus thuringiensis increases intestinal IgG response and induces the expression of FcRn in the intestinal epithelium of adult mice.

We have shown that Cry1Ac protoxin from Bacillus thuringiensis is a potent mucosal and systemic immunogen with adjuvant properties. Interestingly, we have observed that Cry1Ac preferentially induces high specific IgG responses in intestinal fluid when it is intraperitoneally administered to mice; therefore, in the present study, we used this protocol, as a model to address the influence of systemic immunization on the induction of the intestinal IgG response. The data shown indicate that upon intraperitoneal immunization with Cry1Ac, significant intestinal specific IgG cell responses were produced in the lamina propria, accompanied by an increased frequency of intestinal IgG+ lymphocytes and epithelial cells containing IgG. Considering that FcRn is the receptor responsible for the transport of IgG in neonatal intestinal epithelia, but it is developmentally downregulated in the rodent intestine, we analysed whether upon intestinal IgG induction, FcRn mRNA expression was induced in intestinal epithelial cells, of adult mice. Whereas in intestinal epithelia of unimmunized adult mice FcRn mRNA was not detected, in Cry1Ac immunized mice it was expressed, although the level was lower in comparison with that found in neonatal epithelia. Then using flow cytometry and immunofluorescence we confirmed that the expression of the protein FcRn was induced in the intestines of adult immunized mice especially in the large intestine. Finally, we found that Cry1Ac also increased FcRn expression in isolated intestinal epithelial cells stimulated in vitro. The outcomes suggest that the expression of FcRn in intestinal epithelium might be reactivated upon immunization, and possibly facilitate IgG transport.

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis.

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.