A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 2a: Grouping cobalt compounds based on their capacity to stabilize HIF-1α in human alveolar epithelial cells in vitro.

Excessive inhalation of cobalt (Co) dust can have harmful effects on the respiratory tract, yet all cobalt substances do not have the same potential for inducing toxicity. The prevalent hypothesis is that the potential of Co substances to release Co2+ ions in the organism and in cells drives their toxicity profile. Here, we explored the possibility of grouping Co substances for predicting inhalation toxicity based on in vitro data using the stabilization of hypoxia-inducible factor (HIF)-1α as a read out for intracellular Co ion content. We evaluated the potential of 11 inorganic Co compounds and two Co metal powder samples to stabilize intracellular HIF-1α in alveolar epithelial cells (A549) after 24 h exposure to 250-1000 µM Co equivalents. Cytotoxic activity of the substances was assessed in parallel after 72 h at the same doses. Two groups were identified: (1) substances with high intracellular bioavailability (n=9), causing cytotoxicity and stabilizing HIF-1α and (2) substances with low intracellular bioavailability (n = 4), and not inducing these effects. This study provides a link between screening-level data (solubility in artificial lung fluids, Tier 1) and hypothesized biological key events.

Identification and Functional Characterization of Two Homologous SpoVS Proteins Involved in Sporulation of Bacillus thuringiensis.

Sporulation is an important part of the life cycle of Bacillus thuringiensis and the basis for the production of parasporal crystals. This study identifies and characterizes two homologous spoVS genes (spoVS1 and spoVS2) in B. thuringiensis, both of whose expression is dependent on the σH factor. The disruption of spoVS1 and spoVS2 resulted in defective B. thuringiensis sporulation. Similar to Bacillus subtilis, B. thuringiensis strain HD(ΔspoVS1) mutants showed delayed formation of the polar septa, decreased sporulation efficiency, and blocked spore release. Different from B. subtilis, B. thuringiensis HD(ΔspoVS1) mutants had disporic septa and failed to complete engulfment in some cells. Moreover, HD(ΔspoVS2) mutants had delayed spore release. The effect of spoVS1 deletion on polar septum delay and sporulation efficiency could be compensated by spoVS2. ß-Galactosidase activity analysis showed that the expression of pro-sigE and spoIIE decreased to different degrees in the HD(ΔspoVS1) and HD(ΔspoVS2) mutants. The different effects of the two mutations on the expression of sporulation genes led to decreases in Cry1Ac production of different levels. IMPORTANCE There is only one spoVS gene in B. subtilis, and its effects on sporulation have been reported. In this study, two homologous spoVS genes were found and identified in B. thuringiensis. The different effects on sporulation and parasporal crystal protein production in B. thuringiensis and their relationship were investigated. We found that these two homologous spoVS genes are highly conserved in the Bacillus cereus group, and therefore, the functional characterization of SpoVS is helpful to better understand the sporulation processes of members of the Bacillus cereus group.

Production and characterization of the 13 C/15 N single-labeled insecticidal protein Cry1Ab/Ac using recombinant Escherichia coli.

Synthetic Cry1Ab/Ac proteins expressed by genetically modified (GM) crops have a high potential to control insect pests without utilizing large amounts of chemical insecticides. Before these crops are used in agriculture, the environmental fate and interactions in the soil must be understood. Stable isotope-labeled Cry1Ab/Ac protein is a highly useful tool for collecting such data. We developed a protocol to produce 13 C/15 N single-labeled Cry proteins. The artificially synthesized gene Cry1Ab/Ac of Bt rice Huahui No. 1, which has been certified by the Chinese government to be safe for human consumption, was subcloned into pUC57, and the expression vector pET-28a-CryAb/Ac was constructed and transformed into Escherichia coli BL21 (DE3) competent cells. Next, 0.2 mM isopropyl thiogalactoside (IPTG) was added to these cells and cultured at 37°C for 4 h to induce the synthesis and formation of inclusion bodies in M9 growth media containing either [U-13 C] glucose (5% 13 C-enriched) or [15 N] ammonium chloride (5% 15 N-enriched). Then, Cry inclusion bodies were dissolved in urea and purified by affinity chromatography under denaturing conditions, renatured by dialysis, and further detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The purities of 13 C/15 N-labeled Cry proteins reached 99% with amounts of 12.6 mg/L and 8.8 mg/L, respectively. The δ 13 C and ä 15 N values of 13 C-labeled Cry protein and 15 N-labeled Cry protein were 3,269‰ and 2,854‰, respectively. A bioassay test revealed that the labeled Cry1Ab/Ac proteins had strong insecticidal activity. The stable isotope-labeled insecticidal Cry proteins produced for the first time in this study will provide an experimental basis for future metabolic studies on Cry proteins in soil and the characteristics of nitrogen (N) and carbon (C) transformations. Our findings may also be employed as a reference for elucidating the environmental behavior and ecological effects of BT plants and expressed products.

Bacillus thuringiensis endotoxin production: a systematic review of the past 10 years.

Bacillus thuringiensis (Bt) is one of the most promising biological control agents used commercially. Its products can contribute to reducing ecological and environmental problems associated with the use of chemical pesticides. Among the limiting factors of using Bt as bioinsecticide are the costs and ensuring its biological activity, which may vary according to the strain and culture conditions. This systematic review aimed to collect state-of-the-art information on the production of Bt endotoxins and to score the methodological feasibility of the data obtained, thus highlighting possible incoherencies. In order to consolidate recent findings and guide future studies, a total of 47 original articles from the last 10 years was analysed, with special attention being given to corroborating data, identifying inconsistencies and suggesting future adjustments so as to increase data reliability. With a maximum score of 8 points, three production parameters were classified on the following scale: preferable (score: 2), adequate (score: 1) and inadequate (score: 0), and another two parameter were classified as adequate (score: 1) or inadequate (score: 0). No article scored more than 6 out of the maximum of 8, thus reflecting the need for more detailed studies regarding Bt endotoxin production. The lack of standardization of methods and units of measurement also have made a comparison of results and an overall analysis difficult. Standards are suggested in the present study. The inclusion of bioassays and quantifying toxin via alkaline dilution are strongly recommended for studies of this nature, along with LC50 expressed in mg/L. Sixteen articles (34%) did not use either of these suggested methods, which indicates the need for further supporting studies. These findings reinforce the need for robust studies in this area, which could include the development of more affordable and effective bioinsecticides, thus increasing their competitiveness against insecticides derived from unsustainable sources.

The signal peptide of Cry1Ia can improve the expression of eGFP or mCherry in Escherichia coli and Bacillus thuringiensis and enhance the host's fluorescent intensity.

BACKGROUND: The signal peptides (SPs) of secretory proteins are frequently used or modified to guide recombinant proteins outside the cytoplasm of prokaryotic cells. In the periplasmic space and extracellular environment, recombinant proteins are kept away from the intracellular proteases and often they can fold correctly and efficiently. Consequently, expression levels of the recombinant protein can be enhanced by the presence of a SP. However, little attention has been paid to the use of SPs with low translocation efficiency for recombinant protein production. In this paper, the function of the signal peptide of Bacillus thuringiensis (Bt) Cry1Ia toxin (Iasp), which is speculated to be a weak translocation signal, on regulation of protein expression was investigated using fluorescent proteins as reporters. RESULTS: When fused to the N-terminal of eGFP or mCherry, the Iasp can improve the expression of the fluorescent proteins and as a consequence enhance the fluorescent intensity of both Escherichia coli and Bt host cells. Real-time quantitative PCR analysis revealed the higher transcript levels of Iegfp over those of egfp gene in E. coli TG1 cells. By immunoblot analysis and confocal microscope observation, lower translocation efficiency of IeGFP was demonstrated. The novel fluorescent fusion protein IeGFP was then used to compare the relative strengths of cry1Ia (Pi) and cry1Ac (Pac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of Pi at the same level of sensitivity. The fluorescent signals of E. coli and Bt cells expressing the Iasp fused mCherry (ImCherry) were also enhanced. Importantly, the Iasp can also enhanced the expression of two difficult-to-express proteins, matrix metalloprotease-13 (MMP13) and myostatin (growth differentiating factor-8, GDF8) in E. coli BL21-star (DE3) strain. CONCLUSIONS: We identified the positive effects of a weak signal peptide, Iasp, on the expression of fluorescent proteins and other recombinant proteins in bacteria. The produced IeGFP and ImCherry can be used as novel fluorescent protein variants in prokaryotic cells. The results suggested the potential application of Iasp as a novel fusion tag for improving the recombinant protein expression.

No More Tears: Mining Sequencing Data for Novel Bt Cry Toxins with CryProcessor.

Bacillus thuringiensis (Bt) is a natural pathogen of insects and some other groups of invertebrates that produces three-domain Cry (3d-Cry) toxins, which are highly host-specific pesticidal proteins. These proteins represent the most commonly used bioinsecticides in the world and are used for commercial purposes on the market of insecticides, being convergent with the paradigm of sustainable growth and ecological development. Emerging resistance to known toxins in pests stresses the need to expand the list of known toxins to broaden the horizons of insecticidal approaches. For this purpose, we have elaborated a fast and user-friendly tool called CryProcessor, which allows productive and precise mining of 3d-Cry toxins. The only existing tool for mining Cry toxins, called a BtToxin_scanner, has significant limitations such as limited query size, lack of accuracy and an outdated database. In order to find a proper solution to these problems, we have developed a robust pipeline, capable of precise 3d-Cry toxin mining. The unique feature of the pipeline is the ability to search for Cry toxins sequences directly on assembly graphs, providing an opportunity to analyze raw sequencing data and overcoming the problem of fragmented assemblies. Moreover, CryProcessor is able to predict precisely the domain layout in arbitrary sequences, allowing the retrieval of sequences of definite domains beyond the bounds of a limited number of toxins presented in CryGetter. Our algorithm has shown efficiency in all its work modes and outperformed its analogues on large amounts of data. Here, we describe its main features and provide information on its benchmarking against existing analogues. CryProcessor is a novel, fast, convenient, open source (https://github.com/lab7arriam/cry_processor), platform-independent, and precise instrument with a console version and elaborated web interface (https://lab7.arriam.ru/tools/cry_processor). Its major merits could make it possible to carry out massive screening for novel 3d-Cry toxins and obtain sequences of specific domains for further comprehensive in silico experiments in constructing artificial toxins.

Effect of Prebiotic Complex on Gut Microbiota and Endotoxemia in Female Rats with Modeled Heart Failure.

We studied the levels endotoxin and microbial markers in the blood of female rats with experimental heart failure and the effects of preliminary treatment with a prebiotic complex based on fermented wheat bran and inactivated Saccharomyces cerevisiae culture on these parameters. The concentrations of endotoxin, markers of lactobacilli, and opportunistic microorganisms were found to increase in rats with experimental heart failure and significantly decreased against the background of treatment with prebiotic complex. The dynamics of markers of bifidobacteria, eubacteria, and propionibacteria were reciprocal. The observed effect of the prebiotic complex effect on gut microbiota in rats with experimental heart failure suggests that this complex can be used for the correction of intestinal dysbiosis and endotoxemia in this clinical condition.

Selection of the Bacillus thuringiensis Berliner strain to produce a parasporin with cytotoxic activity against MCF-7 breast cancer cells.

BACKGROUND: Bacillus thuringiensis (Bt) is a Gram-positive bacterium that is known worldwide for its entomopathogenic properties. Recent studies indicate that bacteria produces protein inclusions called parasporins (PSs) that have anti-cancer activity against several types of tumor cells. OBJECTIVE: The present work aimed to select a Bt strain that produces an active PS against MCF-7 breast cancer cells, and to provide an initial quantification of its toxicity and protein concentration. METHODS: Two batches of Bt strains were fermented, and the parasporins were produced and isolated. In vitro tests were performed in 96-well plates and analyzed by a spectrophotometer. RESULTS: Most peptides did not have any cytopathic effect, but the A14d2 strain produces a PS with high toxicity to cancer cells. In the MTT test, the A14d2 strain PS was efficient with an LD50 of 14.83 µg/mL and a protein concentration of 520 µg/mL. At the end of the experiments, this PS was added to bacterial cells that produce other biologically active bacterial toxins against MCF-7 cells, which allowed it to be produced by a safe and inert microorganism to humans. CONCLUSION: PSs represent a potential tool to treat this form of breast cancer by providing peptides that may be useful in therapy.

Endotoxin Producers Overgrowing in Human Gut Microbiota as the Causative Agents for Nonalcoholic Fatty Liver Disease.

Gut microbiota-derived endotoxin has been linked to human nonalcoholic fatty liver disease (NAFLD), but the specific causative agents and their molecular mechanisms remain elusive. In this study, we investigated whether bacterial strains of endotoxin-producing pathogenic species overgrowing in obese human gut can work as causative agents for NAFLD. We further assessed the role of lipopolysaccharide (LPS)-Toll-like receptor 4 (TLR4) cross talk in this pathogenicity. Nonvirulent strains of Gram-negative pathobionts were isolated from obese human gut and monoassociated with C57BL/6J germfree (GF) mice fed a high-fat diet (HFD). Deletion of waaG in the bacterial endotoxin synthetic pathway and knockout of TLR4 in GF mice were used to further study the underlying mechanism for a causal relationship between these strains and the development of NAFLD. Three endotoxin-producing strains, Enterobacter cloacae B29, Escherichia coli PY102, and Klebsiella pneumoniae A7, overgrowing in the gut of morbidly obese volunteers with severe fatty liver, induced NAFLD when monoassociated with GF mice on HFD, while HFD alone did not induce the disease in GF mice. The commensal Bacteroides thetaiotaomicron (ATCC 29148), whose endotoxin activity was markedly lower than that of Enterobacteriaceae strains, did not induce NAFLD in GF mice. B29 lost its proinflammatory properties and NAFLD-inducing capacity upon deletion of the waaG gene. Moreover, E. cloacae B29 did not induce NAFLD in TLR4-deficient GF mice. These nonvirulent endotoxin-producing strains in pathobiont species overgrowing in human gut may work as causative agents, with LPS-TLR4 cross talk as the most upstream and essential molecular event for NAFLD.IMPORTANCE Recent studies have reported a link between gut microbiota and nonalcoholic fatty liver disease (NAFLD), showing that germfree (GF) mice do not develop metabolic syndromes, including NAFLD. However, the specific bacterial species causing NAFLD, as well as their molecular cross talk with the host for driving liver disease, remain elusive. Here, we found that nonvirulent endotoxin-producing strains of pathogenic species overgrowing in obese human gut can act as causative agents for induction of NAFLD and related metabolic disorders. The cross talk between endotoxin from these specific producers and the host's TLR4 receptor is the most upstream and essential molecular event for inducing all phenotypes in NAFLD and related metabolic disorders. These nonvirulent endotoxin-producing strains of gut pathogenic species overgrowing in human gut may collectively become a predictive biomarker or serve as a novel therapeutic target for NAFLD and related metabolic disorders.

Differences between two populations of bollworm, Helicoverpa zea (Lepidoptera: Noctuidae), with variable measurements of laboratory susceptibilities to Bt toxins exposed to non-Bt and Bt cottons in large field cages.

Interpreting variable laboratory measurements of Helicoverpa zea Boddie susceptibility to toxins from Bacillus thuringiensis Berliner (Bt) has been challenging due to a lack of clear evidence to document declining field control. Research that links laboratory measurements of susceptibility to survival on Bt crops is vital for accurate characterization and any subsequent response to the occurrence of an implied H. zea resistance event. In this study, H. zea survival and the resultant damage to plant fruiting structures of non-Bt, Bollgard II, and Bollgard III cottons from two insect colonies with differing levels of laboratory susceptibility to Bt toxins were evaluated in large field cages. Laboratory bioassays revealed resistance ratios of 2.04 and 622.14 between the two H. zea colonies for Dipel DF and Cry1Ac, respectively. Differences between the two H. zea colonies measured via bioassays with Bollgard II and Bollgard III cotton leaf tissue in the laboratory were not statistically discernable. However, there was 17.6% and 5.3% lower larval mortality in Bollgard II and Bollgard III for the feral relative to the laboratory colony of H. zea, respectively. Although H. zea larval numbers in cages infested with the laboratory susceptible colony did not differ between the two Bt cottons, there were fewer larvae per 25 plants in Bollgard III than in Bollgard II cotton in cages containing tolerant insects. Cages infested with tolerant H. zea moths had higher numbers of total larvae than those containing the laboratory susceptible colony in both Bollgard II and Bollgard III cottons. Bollgard II and Bollgard III cottons received 77.4% and 82.7% more total damage to total plant fruiting structures in cages infested with tolerant insects relative to those containing the laboratory susceptible colony. The damage inflicted to fruiting structures on Bollgard III cotton by a feral H. zea colony with decreased measurements of laboratory susceptibility to Dipel DF and Cry1Ac indicate that the addition of Vip3A to third generation Bt cottons may not provide sufficient control in situations where infestations levels are high.

Expression of cry genes in Bacillus thuringiensis biotechnology.

Bacillus thuringiensis is a gram-positive, spore-forming bacterium that produces insecticidal crystal proteins during sporulation. The production of these crystals results primarily from the expression of cry genes. In this review, we focus on the expression and application of cry genes directed by both cry gene promoters and non-cry gene promoters in different hosts. However, not all cry genes and niches are compatible with B. thuringiensis. New delivery systems offsetting the current limitations in B. thuringiensis application are needed to improve Cry production, niche fitness, and persistence. This review examines currently available research and highlights areas in need of further research and development for more effective production and utilization of Cry insecticidal proteins.

Expression of ChiA74∆sp and its truncated versions in Bacillus thuringiensis HD1 using a vegetative promoter maintains the integrity and toxicity of native Cry1A toxins.

Our objective was to determine whether a recombinant chitinase ChiA74∆sp of Bacillus thuringiensis and its truncated versions (ChiA74∆sp-60, ChiA74∆sp-50) could be produced in B. thuringiensis HD1 with no detrimental effect on the size and insecticidal activity of the native bipyramidal Cry crystal. chiA-p, the promoter used to drive chitinase gene expression, was active during vegetative growth of Cry-B. HD1 recombinants showed increases from ~7- to 12-fold in chitinase activity when compared with parental HD1 and negligible or no effect on the volume of bipyramidal crystals was observed. HD1/ChiA74∆sp-60 showed increases from 20% to 40% in the yield of Cry1A per unit of culture medium when compared with parental HD1 and HD1/ChiA74∆sp-50, HD1/ChiA74∆sp. Inclusion bodies presumably composed of the enzyme attached to native Cry1A crystals of recombinant strains were observed; these inclusions were likely responsible for the enhancements in chitinase activity. Western blot analysis using polyclonal anti-ChiA74∆sp showed a weak signal with proteins of ~50 kDa in sporulated and lysed cells of recombinant strains. Bioassays against Spodoptera frugiperda using sporulated/lysed samples of the recombinant strains did not show statistically significant differences in LC50s when compared with HD1.

Phage on Tap: A Quick and Efficient Protocol for the Preparation of Bacteriophage Laboratory Stocks.

A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here, we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010-12 PFU/mL), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations, improve the molecular characterizations of phage, and may be applicable for therapeutic applications. The method consists of five major parts, including phage propagation, phage cleanup by 0.22 µm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of >100 mL, the PoT protocol generated a cleaned, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just 2 days. In contrast, the traditional method took upward of 5 days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays.

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera.

Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80-100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25-80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Cry1Ac production is costly for native plants attacked by non-Cry1Ac-targeted herbivores in the field.

Plants are the primary producers in most terrestrial ecosystems and have complex defense systems to protect their produce. Defense-deficient, high-yielding agricultural monocultures attract abundant nonhuman consumers, but are alternatively defended through pesticide application and genetic engineering to produce insecticidal proteins such as Cry1Ac (Bacillus thuringiensis). These approaches alter the balance between yield protection and maximization but have been poorly contextualized to known yield-defense trade-offs in wild plants. The native plant Nicotiana attenuata was used to compare yield benefits of plants transformed to be defenseless to those with a full suite of naturally evolved defenses, or additionally transformed to ectopically produce Cry1Ac. An insecticide treatment allowed us to examine yield under different herbivore loads in N. attenuata's native habitat. Cry1Ac, herbivore damage, and growth parameters were monitored throughout the season. Biomass and reproductive correlates were measured at season end. Non-Cry1Ac-targeted herbivores dominated on noninsecticide-treated plants, and increased the yield drag of Cry1Ac-producing plants in comparison with endogenously defended or undefended plants. Insecticide-sprayed Cry1Ac-producing plants lagged less in stalk height, shoot biomass, and flower production. In direct comparison with the endogenous defenses of a native plant, Cry1Ac production did not provide yield benefits for plants under observed herbivore loads in a field study.

Anti-endotoxin mechanism of the KW4 peptide in inflammation in RAW 264.7 cells induced by LTA and drug-resistant Staphylococcus aureus 1630.

Drug-resistant microorganism infections cause serious disease and can lead to mortality and morbidity. In particular, Staphylococcus aureus induces pyrogenic and toxigenic infections, and drug-resistance occurs rapidly. Multidrug-resistant S. aureus, such as methicillin-resistant S. aureus and methicillin-sensitive S. aureus, can also cause immunodeficiency and immune deficiency syndrome from lipoteichoic acid. However, antimicrobial peptides, such as KW4, have strong antimicrobial activity, low cytotoxicity, and high neutralization activity against endotoxin substances from Gram-negative bacteria. The objective of this study was to use a synthetic KW4 antimicrobial peptide to evaluate the inhibition of drug-resistance development, antimicrobial activity, and neutralizing activity in S. aureus Gram-positive bacteria. The KW4 peptide showed strong antimicrobial activity against drug-resistant S. aureus strains and significantly increased the anti-neutralizing activity of lipoteichoic acid in S. aureus 1630 drug-resistant bacteria. In addition, S. aureus ATCC 29213 did not develop resistance to KW4 as with other antibiotic drugs. These results suggest that the KW4 peptide is an effective antibiotic and anti-neutralizing agent against multidrug-resistant S. aureus strains.

Host-based lipid inflammation drives pathogenesis in Francisella infection.

Mass spectrometry imaging (MSI) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host-pathogen interface during a septic bacterial infection. Using Francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. Here, we demonstrate detection and distribution of endotoxin in a lethal murine F. novicida infection model, in addition to determining the temporally and spatially resolved innate lipid inflammatory response in both 2D and 3D renderings using MSI. Further, we show that the cyclooxygenase-2-dependent lipid inflammatory pathway is responsible for lethality in F. novicida infection due to overproduction of proinflammatory effectors including prostaglandin E2. The results of this study emphasize that spatial determination of the host lipid components of the immune response is crucial to identifying novel strategies to effectively address highly pathogenic and lethal infections stemming from bacterial, fungal, and viral origins.

Highly potent antimicrobial modified peptides derived from the Acinetobacter baumannii phage endolysin LysAB2.

The increase in the prevalence of multidrug-resistant Acinetobacter baumannii (MDRAB) strains is a serious public health concern. Antimicrobial peptides (AMPs) are a possible solution to this problem. In this study, we examined whether AMPs could be derived from phage endolysins. We synthesized four AMPs based on an amphipathic helical region in the C-terminus of endolysin LysAB2 encoded by the A. baumannii phage ΦAB2. These peptides showed potent antibacterial activity against A. baumannii (minimum inhibitory concentration, 4-64 µM), including some MDR and colistin-resistant A. baumannii. Of the four peptides, LysAB2 P3, with modifications that increased its net positive charge and decreased its hydrophobicity, showed high antibacterial activity against A. baumannii but little haemolytic and no cytotoxic activity against normal eukaryotic cells. The results of electron microscopy experiments and a fluorescein isothiocyanate staining assay indicated that this peptide killed A. baumannii through membrane permeabilization. Moreover, in a mouse intraperitoneal infection model, at 4 h after the bacterial injection, LysAB2 P3 decreased the bacterial load by 13-fold in ascites and 27-fold in blood. Additionally, LysAB2 P3 rescued sixty percent of mice heavily infected with A. baumannii from lethal bacteremia. Our results confirmed that bacteriophage endolysins are a promising resource for developing effective AMPs.

[Larvicidal activity of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or B.t.i toxin Cyt2Ba against mosquito larvae and formulations for enhancing the effects].

OBJECTIVE: To assess the larvicidal effects of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or Bacillus thuringiensis subsp israelensis (B.t.i) toxin Cyt2Ba against the second instar larvae of Culex pipiensquinquefasciatus and Aedes albopictus and compare different formulations for their larvicidal effects. METHODS: The AaIT- or Cyt2Ba-coding sequences were cloned into pET28a(+) and the recombinant plasmids were transformed into E. coli BL21(DE3). After induction with IPTG, the recombinant proteins expressed by the recombinant E. coli were detected and identified by SDS-PAGE and Western blotting, respectively. The larvicidal activity of the bacterial suspension was tested at different concentrations against mosquitoes. The effective engineered bacteria were prepared into dry powder with different formulations, and their larvicidal activity was tested. RESULTS: AaIT and Cyt2Ba proteins were successfully expressed in E. coli. The recombinant AaIT protein showed no virulence to the mosquito larvae. The suspension of the recombinant E. coli expressing Cyt2Ba protein exhibited a stronger killing effect on Aedes albopictus larvae than on Culex pipiens quinquefasciatus larvae at 48 h (P<0.001) with LC50 of 3.00×106 cells/mL and 1.25×107 cells/mL, respectively. The dry powder of the engineered bacteria formulated with yeast extract, wheat flour or white pepper powder at the mass ratio of 1:1 showed the strongest killing effect on mosquito larvae (P=0.044), and the formulation with white pepper powder produced a stronger killing effect than formulations with yeast extract or wheat flour (P=0.002). CONCLUSION: The B.t.i Cyt2Ba protein expressed in E. coli BL21(DE3) shows a good larvicidal activity against mosquitoes, and appropriate formulations of the engineered bacteria can enhance its efficiency in mosquito control.

Inhibition of Cronobacter sakazakii Virulence Factors by Citral.

Cronobacter sakazakii is a foodborne pathogen associated with fatal forms of necrotizing enterocolitis, meningitis and sepsis in neonates and infants. The aim of this study was to determine whether citral, a major component of lemongrass oil, could suppress putative virulence factors of C. sakazakii that contribute to infection. Sub-inhibitory concentrations of citral significantly decreased motility, quorum sensing, biofilm formation and endotoxin production. Citral substantially reduced the adhesion and invasion of C. sakazakii to Caco-2 cells and decreased bacterial survival and replication within the RAW 264.7 macrophage cells. Citral also repressed the expression of eighteen genes involved in the virulence. These findings suggest that citral has potential to be developed as an alternative or supplemental agent to mitigate the infections caused by C. sakazakii.