Bacillus thuringiensis Spores and Cry3A Toxins Act Synergistically to Expedite Colorado Potato Beetle Mortality.

The insect integument (exoskeleton) is an effective physiochemical barrier that limits disease-causing agents to a few portals of entry, including the gastrointestinal and reproductive tracts. The bacterial biopesticide Bacillus thuringiensis (Bt) enters the insect host via the mouth and must thwart gut-based defences to make its way into the body cavity (haemocoel) and establish infection. We sought to uncover the main antibacterial defences of the midgut and the pathophysiological features of Bt in a notable insect pest, the Colorado potato beetle Leptinotarsa decemlineata (CPB). Exposing the beetles to both Bt spores and their Cry3A toxins (crystalline δ-endotoxins) via oral inoculation led to higher mortality levels when compared to either spores or Cry3A toxins alone. Within 12 h post-exposure, Cry3A toxins caused a 1.5-fold increase in the levels of reactive oxygen species (ROS) and malondialdehyde (lipid peroxidation) within the midgut - key indicators of tissue damage. When Cry3A toxins are combined with spores, gross redox imbalance and 'oxidation stress' is apparent in beetle larvae. The insect detoxification system is activated when Bt spores and Cry3A toxins are administered alone or in combination to mitigate toxicosis, in addition to elevated mRNA levels of candidate defence genes (pattern-recognition receptor, stress-regulation, serine proteases, and prosaposin-like protein). The presence of bacterial spores and/or Cry3A toxins coincides with subtle changes in microbial community composition of the midgut, such as decreased Pseudomonas abundance at 48 h post inoculation. Both Bt spores and Cry3A toxins have negative impacts on larval health, and when combined, likely cause metabolic derangement, due to multiple tissue targets being compromised.

Cellular damage, including wounding, drives C. elegans stress-induced sleep.

Across animal phyla, sleep is associated with increased cellular repair, suggesting that cellular damage may be a core component of sleep pressure. In support of this notion, sleep in the nematode Caenorhabditis elegans can be triggered by damaging conditions, including noxious heat, high salt, and ultraviolet light exposure. It is not clear, however, whether this stress-induced sleep (SIS) is a direct consequence of cellular damage, or of a resulting energy deficit, or whether it is triggered simply by the sensation of noxious conditions. Here, we show that thermosensation is dispensable for heat-induced sleep, that osmosensation is dispensable for salt-induced sleep, and that wounding is also a sleep trigger, together indicating that SIS is not triggered by sensation of noxious environments. We present evidence that genetic variation in cellular repair pathways impacts sleep amount, and that SIS involves systemic monitoring of cellular damage. We show that the low-energy sensor AMP-activated protein kinase (AMPK) is not required for SIS, suggesting that energy deficit is not the primary sleep trigger. Instead, AMPK-deficient animals display enhanced SIS responses, and pharmacological activation of AMPK reduces SIS, suggesting that ATP-dependent repair of cellular damage mitigates sleep pressure.

Etiologic role of root canal infection in apical periodontitis and its relationship with clinical symptomatology.

Evidence shows the polymicrobial etiology of endodontic infections, in which bacteria and their products are the main agents for the development, progression, and dissemination of apical periodontitis. Microbial factors in necrotic root canals (e.g., endotoxin) may spread into apical tissue, evoking and supporting a chronic inflammatory load. Thus, apical periodontitis is the result of the complex interplay between microbial factors and host defense against invasion of periradicular tissues. This review of the literature aims to discuss the complex network between endodontic infectious content and host immune response in apical periodontitis. A better understanding of the relationship of microbial factors with clinical symptomatology is important to establish appropriate therapeutic procedures for a more predictable outcome of endodontic treatment.

Etiologic role of root canal infection in apical periodontitis and its relationship with clinical symptomatology

Abstract: Evidence shows the polymicrobial etiology of endodontic infections, in which bacteria and their products are the main agents for the development, progression, and dissemination of apical periodontitis. Microbial factors in necrotic root canals (e.g., endotoxin) may spread into apical tissue, evoking and supporting a chronic inflammatory load. Thus, apical periodontitis is the result of the complex interplay between microbial factors and host defense against invasion of periradicular tissues. This review of the literature aims to discuss the complex network between endodontic infectious content and host immune response in apical periodontitis. A better understanding of the relationship of microbial factors with clinical symptomatology is important to establish appropriate therapeutic procedures for a more predictable outcome of endodontic treatment.

The Role of Typhoid Toxin in Salmonella Typhi Virulence
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Unlike many of the nontyphoidal Salmonella serovars such as S. Typhimurium that cause restricted gastroenteritis, Salmonella Typhi is unique in that it causes life-threatening typhoid fever in humans. Despite the vast difference in disease outcomes that S. Typhi and S. Typhimurium cause in humans, there are few genomic regions that are unique to S. Typhi. Of these regions, the most notable is the small locus encoding typhoid toxin, an AB toxin that has several distinct characteristics that contribute to S. Typhi's pathogenicity. As a result, typhoid toxin and its role in S. Typhi virulence have been studied in an effort to gain insight into potential treatment and prevention strategies. Given the rise of multidrug-resistant strains, research in this area has become increasingly important. This article discusses the current understanding of typhoid toxin and potential directions for future research endeavors in order to better understand the contribution of typhoid toxin to S. Typhi virulence.

Implications of microbiota and bile acid in liver injury and regeneration.

Studies examining the mechanisms by which the liver incurs injury and then regenerates usually focus on factors and pathways directly within the liver, neglecting the signaling derived from the gut-liver axis. The intestinal content is rich in microorganisms as well as metabolites generated from both the host and colonizing bacteria. Through the gut-liver axis, this complex "soup" exerts an immense impact on liver integrity and function. This review article summarizes data published in the past 30 years demonstrating the signaling derived from the gut-liver axis in relation to liver injury and regeneration. Due to the intricate networks of implicated pathways as well as scarcity of available mechanistic data, it seems that nutrigenomic, metabolomics, and microbiota profiling approaches are warranted to provide a better understanding regarding the interplay and impact between nutrition, bacteria, and host response in influencing liver function and healing. Therefore elucidating the possible molecular mechanisms that link microbiota alteration to host physiological response and vice versa.

Structural basis for inhibition of TLR2 by staphylococcal superantigen-like protein 3 (SSL3).

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.

Nitric oxide participates in the toxicity of Bacillus thuringiensis Cry1Ab toxin to kill Manduca sexta larvae.

Nitric oxide (NO) produced by the nitric oxide synthase (NOS) enzyme is a reactive oxygen molecule widely considered as important participant in the immune system of different organisms to confront microbial infections. In insects the NO molecule has also been implicated in immune response against microbial pathogens. Bacillus thuringiensis (Bt) is an insect-pathogenic bacterium that produces insecticidal proteins such as Cry toxins. These proteins kill insects because they form pores in the larval-midgut cells. Here we show that intoxication of Manduca sexta larvae with Cry1Ab activates expression of NOS with a corresponding increase in NO. This effect is not observed with a non-toxic mutant toxin Cry1Ab-E129K that is affected in pore formation. The increased production of NO triggered by intoxication with LC50 dose of Cry1Ab toxin is not associated with higher expression of antimicrobial peptides. NO participates in Cry1Ab toxicity since inhibition of NOS by selective l-NAME inhibitor prevented NO production and resulted in reduced mortality of the larvae. The fact that mortality was not completely abolished by L-NAME indicates that other processes participate in toxin action and induction of NO production upon Cry1Ab toxin administration accounts only for a part of the toxicity of this protein to M. sexta larvae.

Mesenteric lymph reperfusion after superior mesenteric artery occlusion shock exacerbates endotoxin translocation in brain.

PURPOSE: To determine the role of mesenteric lymph reperfusion (MLR) on endotoxin translocation in brain to discuss the mechanism of brain injury subjected to superior mesenteric artery occlusion (SMAO) shock. METHODS: Twenty-four rats were randomly assigned to MLR, SMAO, MLR+SMAO and sham groups. MLR was performed by clamping the mesenteric lymph duct (MLD) for 1 h and then allowing reperfusion for 2 h in the MLR group; SMAO involved clamping the superior mesenteric artery (SMA) for 1 h, followed by reperfusion for 2 h in the SMAO group; occlusion of both the SMA and MLD for 1 h was followed by reperfusion for 2 h in the MLR+SMAO group rats. RESULTS: SMAO shock induced severe increased levels of the endotoxin, lipopolysaccharide receptor, lipopolysaccharide-binding protein, intercellular adhesion molecule-1 and tumor necrosis factor-α. Concurrently, MLR after SMAO shock further aggravates these deleterious effects. CONCLUSION: Mesenteric lymph reperfusion exacerbated the endotoxin translocation in brain; thereby increased inflammatory response occurred, suggesting that the intestinal lymph pathway plays an important role in the brain injury after superior mesenteric artery occlusion shock.

Mesenteric lymph reperfusion after superior mesenteric artery occlusion shock exacerbates endotoxin translocation in brain

PURPOSE: To determine the role of mesenteric lymph reperfusion (MLR) on endotoxin translocation in brain to discuss the mechanism of brain injury subjected to superior mesenteric artery occlusion (SMAO) shock. METHODS: Twenty-four rats were randomly assigned to MLR, SMAO, MLR+SMAO and sham groups. MLR was performed by clamping the mesenteric lymph duct (MLD) for 1 h and then allowing reperfusion for 2 h in the MLR group; SMAO involved clamping the superior mesenteric artery (SMA) for 1 h, followed by reperfusion for 2 h in the SMAO group; occlusion of both the SMA and MLD for 1 h was followed by reperfusion for 2 h in the MLR+SMAO group rats. RESULTS: SMAO shock induced severe increased levels of the endotoxin, lipopolysaccharide receptor, lipopolysaccharide-binding protein, intercellular adhesion molecule-1 and tumor necrosis factor-α. Concurrently, MLR after SMAO shock further aggravates these deleterious effects. CONCLUSION: Mesenteric lymph reperfusion exacerbated the endotoxin translocation in brain; thereby increased inflammatory response occurred, suggesting that the intestinal lymph pathway plays an important role in the brain injury after superior mesenteric artery occlusion shock. .

Septic acute kidney injury: molecular mechanisms and the importance of stratification and targeting therapy.

The most common cause of acute kidney injury (AKI) in hospitalized patients is sepsis. However, the molecular pathways and mechanisms that mediate septic AKI are not well defined. Experiments performed over the past 20 years suggest that there are profound differences in the pathogenesis between septic and ischemic AKI. Septic AKI often occurs independently of hypoperfusion, and is mediated by a concomitant pro- and anti-inflammatory state that is activated in response to various pathogen-associated molecular patterns, such as endotoxin, as well as damage-associated molecular patterns. These molecular patterns are recognized by Toll-like receptors (TLRs) found in the kidney, and effectuate downstream inflammatory pathways. Additionally, apoptosis has been proposed to play a role in the pathogenesis of septic AKI. However, targeted therapies designed to mitigate the above aspects of the inflammatory state, TLR-related pathways, and apoptosis have failed to show significant clinical benefit. This failure is likely due to the protean nature of septic AKI, whereby different patients present at different points along the immunologic spectrum. While one patient may benefit from targeted therapy at one end of the spectrum, another patient at the other end may be harmed by the same therapy. We propose that a next important step in septic AKI research will be to identify where patients lie on the immunologic spectrum in order to appropriately target therapies at the inflammatory cascade, TLRs, and possibly apoptosis.

Periodontitis contributes to initiation, progress and aggravation of septic shock; a feasible hypothesis.

This hypothesis states that active aggressive periodontitis is an important source of putative endotoxin contributing to the onset of septic shock syndrome [SSS]. Consequently patients with periodontitis may be more prone to developing SSS in predisposing conditions.

Correlation between clinical/radiographic features and inflammatory cytokine networks produced by macrophages stimulated with endodontic content.

INTRODUCTION: Macrophages are highly activated by endodontic contents. This study investigated the correlation between different clinical signs/symptoms and radiographic features according to the levels of interleukin (IL)-1ß, tumor necrosis factor α (TNF-α), IL-6, IL-10, prostaglandin E(2) (PGE(2)), and their networks produced by endodontic content-stimulated macrophages collected from primary endodontic infection with apical periodontitis (PEIAP). METHODS: Samples were taken from 21 root canals with PEIAP by using paper points. The presence of exudate (EX), pain on palpation (POP), tenderness to percussion (TTP), and the size of the radiographic lesion (SRL) were recorded. Polymerase chain reaction (16S rDNA) was used for bacterial detection and limulus amebocyte lysate (LAL) assay for endotoxin measurement. Raw 264.7 macrophages were stimulated with bacterial contents during 24 hs. The amounts of IL-1ß, TNF-α, IL-6, IL-10 and PGE(2) were measured by enzyme-linked immunosorbent assay. Log-based data were correlated by multiple logistic regression (P < .05). RESULTS: Bacteria and endotoxin were detected in 100% of the samples. IL-6 and TNF-α were positively correlated with SRL and EX, respectively (P < .05). Clinical signs/symptoms and radiographic findings were set as dependent variables for EX-positive correlations between PGE(2), IL-1ß, and TNF-α (P < .05), whereas IL-6 and PGE(2) were positively correlated to each other in POP but negatively correlated in SRL (P < .05). When POP and TTP-POP were set as dependent variables, different cytokine networks were found. CONCLUSIONS: Our findings suggest different roles for each cytokine in the development of apical periodontitis, whose effects of overlapping networks depend on the signs/symptoms and radiographic features found in endodontic infection.

Resistance of Trichoplusia ni to Bacillus thuringiensis toxin Cry1Ac is independent of alteration of the cadherin-like receptor for Cry toxins.

Alteration of binding sites for Bacillus thuringiensis (Bt) toxins in insect midgut is the major mechanism of high-level resistance to Bt toxins in insects. The midgut cadherin is known to be a major binding protein for Bt Cry1A toxins and linkage of Bt-resistance to cadherin gene mutations has been identified in lepidopterans. The resistance to Bt toxin Cry1Ac evolved in greenhouse populations of Trichoplusia ni has been identified to be associated with the down-regulation of an aminopeptidase N (APN1) gene by a trans-regulatory mechanism and the resistance gene has been mapped to the locus of an ABC transporter (ABCC2) gene. However, whether cadherin is also involved with Cry1Ac-resistance in T. ni requires to be understood. Here we report that the Cry1Ac-resistance in T. ni is independent of alteration of the cadherin. The T. ni cadherin cDNA was cloned and the cadherin sequence showed characteristic features known to cadherins from Lepidoptera. Various T. ni cadherin gene alleles were identified and genetic linkage analysis of the cadherin alleles with Cry1Ac-resistance showed no association of the cadherin gene with the Cry1Ac-resistance in T. ni. Analysis of cadherin transcripts showed no quantitative difference between the susceptible and Cry1Ac-resistant T. ni larvae. Quantitative proteomic analysis of midgut BBMV proteins by iTRAQ-2D-LC-MS/MS determined that there was no quantitative difference in cadherin content between the susceptible and the resistant larvae and the cadherin only accounted for 0.0014% (mol%) of the midgut BBMV proteins, which is 1/300 of APN1 in molar ratio. The cadherin from both the susceptible and resistant larvae showed as a 200-kDa Cry1Ac-binding protein by toxin overlay binding analysis, and nano-LC-MS/MS analysis of the 200-kDa cadherin determined that there is no quantitative difference between the susceptible and resistant larvae. Results from this study indicate that the Cry1Ac-resistance in T. ni is independent of cadherin alteration.

[Effects of transgenic Bt rice on soil dissolved organic carbon and nitrogen contents and microbiological properties].

A two-year field experiment (2009 and 2010) was conducted to evaluate the effects of three transgenic Bt rice lines (KMD, HH1, and BtSY63) and their non-Bt lines (XSD, MH63, and SY63) on soil dissolved organic carbon (DOC) and nitrogen (DON) and microbiological properties. All the measured indices changed significantly with sampling time. Comparing with their corresponding non-Bt lines, the test transgenic Bt lines had little effects on the soil DOC, DON, and microbial biomass nitrogen (MBN). The transgenic Bt lines had significant effects on the soil microbial biomass carbon (MBC), basal respiration (BR), and microbial metabolic quotient (qCO2) in certain periods of time in the first year, but no effects in the second year. Among the soils planted with the three non-Bt rice lines, no difference was observed in the DOC, DON, and microbiological properties, whereas in the soil planted with BtSY63, the MBC and BR were significantly higher, but the qCO2 was significantly lower, as compared with those in the soils planted with KMD and HH1. In sum, two years' planting transgenic Bt rice had little effects on the soil DOC, DON, and microbiological properties, but the differences of soil microbiological properties induced by the planting of different transgenic Bt rice lines were larger than those induced by the planting of different non-Bt lines, implying that long term monitoring would help to reveal the effects of transgenic Bt rice on the structure and function of soil ecosystem.

Identification of a novel aminopeptidase P-like gene (OnAPP) possibly involved in Bt toxicity and resistance in a major corn pest (Ostrinia nubilalis).

Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study, we identified 10 aminopeptidase-like genes, which have previously been identified as putative Bt toxin receptors in other insects and examined their expression in relation to Cry1Ab toxicity and resistance. Expression analysis for the 10 aminopeptidase-like genes revealed that most of these genes were expressed predominantly in the larval midgut, but there was no difference in the expression of these genes in Cry1Ab resistant and susceptible strains. This suggested that altered expression of these genes was unlikely to be responsible for resistance in these ECB strains. However, we found that there were changes in two amino acid residues of the aminopeptidase-P like gene (OnAPP) involving Glu(305) to Lys(305) and Arg(307) to Leu(307) in the two Cry1Ab-resistant strains as compared with three Cry1Ab-susceptible strains. The mature OnAPP contains 682 amino acid residues and has a putative signal peptide at the N-terminus, a predicted glycosylphosphatidyl-inositol (GPI)-anchor signal at the C-terminal, three predicted N-glycosylation sites at residues N178, N278 and N417, and an O-glycosylation site at residue T653. We used a feeding based-RNA interference assay to examine the role of the OnAPP gene in Cry1Ab toxicity and resistance. Bioassays of Cry1Ab in larvae fed diet containing OnAPP dsRNA resulted in a 38% reduction in the transcript level of OnAPP and a 25% reduction in the susceptibility to Cry1Ab as compared with larvae fed GFP dsRNA or water. These results strongly suggest that the OnAPP gene could be involved in binding the Cry1Ab toxin in the ECB larval midgut and that mutations in this gene may be associated with Bt resistance in these two ECB strains.

Overexpression of pulmonary extracellular superoxide dismutase attenuates endotoxin-induced acute lung injury.

PURPOSE: Superoxide is produced by activated neutrophils during the inflammatory response to stimuli such as endotoxin, can directly or indirectly injure host cells, and has been implicated in the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We wished to determine the potential for pulmonary overexpression of the extracellular isoform of superoxide dismutase (EC-SOD) to reduce the severity of endotoxin-induced lung injury. METHODS: Animals were randomly allocated to undergo intratracheal instillation of (1) surfactant alone (vehicle); (2) adeno-associated virus (AAV) vectors containing a null transgene (AAV-null); and (3) adeno-associated virus vectors containing the EC-SOD transgene (AAV-EC-SOD) and endotoxin was subsequently administered intratracheally. Two additional groups were randomized to receive (1) vehicle or (2) AAV-EC-SOD, and to undergo sham (vehicle) injury. The severity of the lung injury was assessed in all animals 24 h later. RESULTS: Endotoxin produced a severe lung injury compared to sham injury. The AAV vector encoding EC-SOD increased lung EC-SOD concentrations, and enhanced the antioxidant capacity of the lung. EC-SOD overexpression decreased the severity of endotoxin-induced ALI, reducing the decrement in systemic oxygenation and lung compliance, decreasing lung permeability and decreasing histologic injury. EC-SOD attenuated pulmonary inflammation, decreased bronchoalveolar lavage neutrophil counts, and reduced interleukin-6 and CINC-1 concentrations. The AAV vector itself did not contribute to inflammation or to lung injury. CONCLUSIONS: Pulmonary overexpression of EC-SOD protects the lung against endotoxin-induced ALI.

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Cry15Aa.

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542, in a crystal together with a 40 kDa accompanying protein, is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this study we examined the role of the C-terminal part of Cry15Aa and of the 40 kDa protein in crystal formation in recombinant B. thuringiensis. The contribution of the 40 kDa protein and of the Cry15Aa carboxy-terminal sequence for crystal formation, crystal solubilization, and insecticidal properties was assessed. No significant differences in toxicity against Cydia pomonella, before or after in vitro solubilization of crystal-spore preparations, were found. Although the 40 kDa protein significantly contributes to in vitro solubility and in vivo crystal formation of Cry15Aa, no direct evidence for involvement of the 40 kDa protein in toxicity of Cry15Aa was found.

Endotoxin in endodontic infections: a review.

Gram-negative bacteria play an essential role in primary endodontic infections. They have several virulence factors such as endotoxin, a large molecule that plays a role in the initiation and perpetuation of apical periodontitis. This paper reviews the role of gram-negative bacteria in endodontic infections, structure and mechanisms of action of endotoxin, endotoxin in infected root canals, effects of calcium hydroxide and polymixin B on endotoxin, and applications of endotoxin to measure leakage.