Manual Microbial Toxins, Vol. 4: Bacterial Endotoxins

Free download. Book file PDF easily for everyone and every device. You can download and read online Microbial Toxins, Vol. 4: Bacterial Endotoxins file PDF Book only if you are registered here. And also you can download or read online all Book PDF file that related with Microbial Toxins, Vol. 4: Bacterial Endotoxins book. Happy reading Microbial Toxins, Vol. 4: Bacterial Endotoxins Bookeveryone. Download file Free Book PDF Microbial Toxins, Vol. 4: Bacterial Endotoxins at Complete PDF Library. This Book have some digital formats such us :paperbook, ebook, kindle, epub, fb2 and another formats. Here is The CompletePDF Book Library. It's free to register here to get Book file PDF Microbial Toxins, Vol. 4: Bacterial Endotoxins Pocket Guide.
Navigation menu
  1. Bacterial Endotoxins (Microbial Toxins) (Volume 4)
  2. Shop with confidence
  3. Ebook Microbial Toxins, Vol. 4: Bacterial Endotoxins
  4. Pathogenic bacteria - Wikipedia
  5. Shop by category

Based on this study bivalent sequential binding model is proposed to d- endotoxin binding Jinkins et al. Receptor binding models constructed based on the BBMV binding studies showed that toxins sharing high homology in the loops of domain II recognize same receptor molecules in larval midgut, which results in cross resistance. Thus, selecting toxins with less homology in the domain II will be a better alternative to delay the resistance development against Bt toxins.

Difference in activity of the toxins is due to difference in affinity for a single binding site and also difference in the concentration of binding sites. It has been supported by X-ray crystallographic data that domain II loops showed immunoglobulin like structural folds Li et al. Structural similarity is observed between d- endotoxin folds and other known carbohydrate binding proteins like plant lectin jacalin Machura pomifora , outer layer protein I from hen's egg.

Carbohydrates are used as recognition epitopes by these folds. So far, three kinds of glycoproteins Amino peptidase N, Cadherin-like proteins and anionic glycoconjugates have been identified as receptor molecules for different insect species Agarwal et al. Genetic engineering studies involving exchange of fragments of C-terminal region of toxic fragment between closely related toxins but having different specificity to tested insects showed that the specificity determining regions are essentially located in domain II Widner and Whiteley, ; Caramori et al.

Hydrophobic interactions between d- endotoxin loops and insect midgut receptor molecules were tested by substituting hydrophobic residues with Alanine residue or replacing positively charged residues with negatively charged residues. Mutations were created in the domain II-loop 2 residues of Cry1Ab toxin.

Positively charged residues in the domain II might help in orientation of the toxin to midgut receptor molecules. Hydrophobic aromatic side chain residue at the position is important for the irreversible binding. When phenylalanine at position was replaced by hydrophilic aliphatic and smaller side chain residues such as Cysteine, Valine, and Serine amino acids toxicity was reduced but not by Tyrosine or Trytophan amino acid substitution Rajamohan et al.

The loss of toxicity was correlated with reduced initial binding Rajamohan et al. Mutants generated using site-directed mutagenesis in the loops of Cry3A toxin were tested against Tenebrio molitor. Loop 2 mutants PA and SA did not show any effect on receptor binding and toxicity. Thus, loop 2 is not involved in toxicity determination against Tenebrio molitor. However, loop 3-block mutant Alanine substitution of QGSRG residues showed enhanced toxicity due to increased irreversible binding Wu and Dean, Recently Gomez et al.

From these studies, it is clear that all loops of domain II are not involved in binding of toxins to the receptor molecules of single insect species. Therefore, a toxin, which became ineffective due to loss of receptor recognition need not be ineffective on other susceptible insects. High homology in the domain II of toxins results in cross-resistance due to sharing of the receptor molecules. It is established that loops in the domain II affect irreversible or reversible binding through hydrophobic interactions with receptor molecules.

Studies on domain III. Domain III is made of two antiparallel b -sheets into b -sandwich structure. Intermolecular interactions through salt bridges and hydrogen bonding between domains III and I have been identified through X-ray crystallographic studies. Initially it was proposed that maintaining the stability of the protein is the major function of this domain. From the studies on b strand structure of other protein molecules, it could be assumed that domain III b - sandwich of d- endotoxin can take part in other functions such stability as receptor binding, specificity determination and ion channel gating Schnepf et al.

Arginine rich block in the domain III of d- endotoxins is called "arg face", through which domain III makes contacts with domain I and regulates the ion channel conductance. Recent studies involving site-directed mutagenesis of conserved regions of the domain III and domain III exchange between cry genes demonstrated the above-mentioned functions for domain III of d- endotoxins. One mutant was poorly expressed in E.

This may be due to the disturbance of salt bridges between domains III and I as predicted from X-ray crystallographic studies. But these mutants showed reduced conductance in PLB membranes. These mutants displayed one quarter of the maximum conductance recorded for the native Cry1Ac protein. This result is consistent with earlier observation that domain III "arg face" influences the ion channel formation through domain I interactions Chen et al.

Binding studies using reciprocal hybrids made by exchanging a fragment between amino acids of Cry1Aa with that of Cry1Ac on BBMV from Lymantria dispar showed the location of receptor binding in the third domain Lee et al. Therefore, first direct evidence for domain III binding to receptor was established. Interestingly, hybrid with Cry1Aa third domain resulted in binding of kDa receptor molecule, which is not recognized either by Cry1Aa or Cry1Ac, showing that the domain III also influences the receptor binding.

Since loss of receptor binding is attributed as a major reason for the resistance development towards existing toxins, hybrid toxins with differential binding capacity can be used. Chimeric protein constructed by exchanging domain III of Cry1E inactive on Spodoptera exiqua with that of Cry1C most active on Spodoptera exigua showed toxicity level equal to most active toxin, Cry1C.

In heterlogous binding assay, it was demonstrated that hybrid toxin was bound to the receptor that is recognized by Cry1E toxin. Since Cry1E is already capable of binding to S. This shows the involvement of the domain III in receptor binding. Therefore, domain III exchange can be followed for the other weak toxins to make them more active on agronomically important pests.

When they substituted Cry1I domain II in Cry1B background, the toxicity of chimeric protein approached the toxicity level of the most active toxin Cry3A against this insect. This shows optimum combination of domains II and III may have a role in binding the toxin to midgut receptors, there by increasing the toxicity level Samir et al. There is a structural similarity between domain III of Cry3A toxins with that of carbohydrate binding domain of the 1,4- b glucanase from Cellulomonas fimi.

This suggests that domain III of d- endotoxins might have carbohydrate mediated receptor recognition regions Burton et al. The function of this lectin like fold on domain III is not known under in vivo condition. The above-mentioned experimental results on domain III mutants of different toxins provide evidence for the functional role of domain III in channel gating regulation, receptor binding and specificity determination.

Advantages of protein engineering d- endotoxins. Site-directed mutagenesis and domain exchange studies on different d- endotoxins threw light on the function of each domain in binging out toxicity in susceptible insects. Protein engineering not only reveals the mechanism by which d- endotoxins work, but it can generate toxins with enhanced toxicity with or without new BBMV binding properties. Selected list of these new toxins created through protein engineering is given in Table 3. Recent experiments with domain III replacement resulted in improved toxins that recognize different receptors.

De Maagd et al. These toxins could be used in resistant management as alternatives for the toxins already in use to which insects may become resistant by losing receptors Figure 2. Knowledge of d- endotoxins can be utilized to make these inactive toxins active by protein engineering. Most of the mutants created in domain I resulted in low or no toxicity on tested insects. This might be due to domain I being the most conserved among three domains and it is involved in the basic function of the d- endotoxins viz. Variable and hyper variable regions confer differential specificity and differential receptor binding in the target cells.

Many authors reported the failure of expression of mutant proteins in E. Instability of these proteins might be due to exposure of proteolytic cleavage sites because of conformational changes in the mutant toxins. Properties of hybrid toxins cannot be simply predicted form their parental toxins because hybrids toxins showed altered binding property and specificity.

  1. Full Text - Protein engineering of d-endotoxins of Bacillus thuringiensis.
  2. Dona Gracia of the House of Nasi.
  3. The regulation of non-bank financial institutions: the United States, the European Union, and other countries!
  4. Microbial Toxins, Vol. 5: Bacterial Endotoxins - AbeBooks: .
  5. Electronic Counters.
  6. Bacterial Endotoxin Testing | Lonza!
  7. Ebook Microbial Toxins Vol 4 Bacterial Endotoxins!

Mutation which alter the hydrophobicity of domain I results in complete loss of toxicity Wu et al. Neutral or positive amino acid substitution has little effect on Domain I function than negatively charged amino acid, which completely abolishes the toxicity. Domain I inserts itself into the lipid bilayer of the membrane. Therefore, hydrophobicity in this domain is important. For construction of hybrid toxins, parental toxins should be selected such a way that they should differ in toxicity significantly on tested insects. This will be helpful in assessing the hybrid toxins specificity clearly.

Introduction of culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering. Applied and Environmental Microbiology , September , vol. Directed Mutagenesis of selected Regions of Bacillus thuringeinsis entomocidal proteins. Applied and Environmental Microbiology , June , vol. Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene. Journal of Bacteriology, July , vol. Applied and Environmental Microbiology. May , vol. These findings support that CTB causes an increase in membrane permeability upon binding to C.

The G. To test the potential impact of CTB on C. When C. This indicates that CTB binding to C. Error bars represent the standard error within each group. Chickens are a common host for C. At the termination of the experiment, cecal contents from all birds were serially diluted and plated onto Karmali agar to confirm the chickens were not colonized with C. To determine whether chicken intestinal epithelial cells or chicken gut bacteria other than C. CTB bound strongly to the apical surface of the epithelium, to the mucus layer and to mucin-filled goblet cells Fig.

CTB also bound to commensal bacteria present within the cecal lumen Fig. Cholera toxin CT binds to goblet cells, mucins and commensal bacteria in the chicken cecum. Arrows in a , b indicate goblet cells and mucus.

Bacterial Endotoxins (Microbial Toxins) (Volume 4)

Given the evidence that AB 5 toxins bind to a subset of bacteria present in the chicken intestine, we next examined if such toxins impact the composition of the chicken intestinal microbiota. We focused this experiment on LTB since enterotoxin producing E. These findings demonstrate that LTB has a major effect on the chicken gut microbiota. AB 5 toxins cause shifts in the gut microbiome of chickens by affecting diversity and composition. Whiskers in b represent minimum and maximum values. Lines and bars in d represent means and standard error of means. Although statistical significance could not be obtained, we observed similar trends to those obtained with LTB Supplementary Fig.

Overall, our findings indicate that AB 5 toxins affect more than just C. This indicates that only a fraction of the culture expresses the potential GM1 mimics in vivo , reminiscent of potential phase-variation as observed in C. It has long been considered that the primary role of bacterial toxins is to target vertebrate hosts and cause disease Another assumption, that has recently been disputed, is that GM1 gangliosides in the human gut are necessary for CT to induce disease in humans However, since CT is capable of binding to C.

Furthermore, C. In endemic areas, C.

Shop with confidence

AB 5 toxin-producing bacteria have also been found in chickens, a common commensal host for C. As a result, C. In addition, we have shown that when fed to chickens, LTB and CTB alter the microbial composition of the chicken intestinal tract, suggesting that these toxins and others like it may indeed play a role in inter-bacterial warfare.

After confirming that CT binding to C. Interestingly, we found that the B-subunit was sufficient for the binding and growth clearance phenotype. This was unexpected, since the B-subunit of these toxins, while crucial for adhesion and internalization by eukaryotic cells, as well as for immune activation in humans and animals, is not toxic to eukaryotic tissues without the enzymatically active A-subunit In contrast, the antibacterial effect we observed appears to be mediated through binding by the toxin.

It is important to note however, that we have not excluded the possibility that the A and B subunits could have a synergistic effect to cause increased clearance when the two are together in the holotoxin configuration. The fact that the binding subunits of CTB and LTB alone are sufficient for clearance shows that the ADP-ribosylating action of the toxins is not necessary for the activity.

The observations that this exposure does not alter individual cell morphology Supplementary Fig. This is in contrast to lysis buffer, which causes visible clearing within minutes of spotting onto C. In Cryptococcus neoformans , growth inhibition has been observed as a result of antibodies directed against its capsular polysaccharide; the antibodies completely surround the cells and prevent it from dividing by encapsulating the organism Since the outer leaflet of the outer membrane acts as a permeability barrier, helping bacteria to regulate flow of harmful compounds, as well as beneficial nutrients 47 , we postulated that disruption of this barrier may cause the bacteriostatic effect that we observe.

Indeed, the results of our EtBr accumulation assay suggest that the mechanism is related to an increase in membrane permeability upon toxin binding. To determine if increased EtBr accumulation was an effect of the toxin blocking active efflux of EtBr, a mutant in the periplasmic component of the Campylobacter multidrug efflux pump, cmeA was inactivated.

This mutant still showed an increase in EtBr permeability even in the absence of an active efflux pump, suggesting that CTB might act directly on the membrane. Interestingly, although CTB bound extensively to E. This could be due to inherent differences between these bacteria in their membrane architecture. Alterations in these structures has been shown to reduce the susceptibility of C. As well, even small changes in LOS have been shown to have a profound effect on C.

Endotoxin - lipopolysaccharide or LPS

By encoding a polyguanosine tract in the cgtB gene, strain C. Interestingly, we also found that pre-incubation with CTB for less than two cell divisions was sufficient to decrease the virulence of C. This not only suggests that expression of the GM1 ganglioside in wax moth larvae is important for virulence, but also shows that CTB binding to C. Since our studies demonstrated that there is an abundance of ganglioside structures in the chicken intestine, it was unexpected, but reasonable to find many ganglioside-mimicking bacteria existing in this environment.

In support of this hypothesis, fucose is known to be abundant in mucins and in goblet cells producing mucin Ganglioside mimicry is a phenomenon thought to be rare in bacteria, and as described earlier, can lead to autoimmune disease in humans. Therefore, the observation that other ganglioside-mimicking bacteria are prevalent in food animals warrants further investigation into their identity and into the nature of the glycoconjugates they display.

Given the broad distribution of GM1 ganglioside-mimicking bacteria among the gut microbiota of chickens, we hypothesized that the administration of AB 5 toxins would alter the microbial composition. The most striking impact we observed was the decrease in microbes in the Firmicutes phylum, which represents a major component of the chicken gut microbiome. We were also able to isolate a member of the Firmicutes, E. Interestingly, E.

Other researchers have also observed gut microbiome changes following V. Hsiao et al.

In the published studies, it is not possible to distinguish between direct toxin effects on the gut microbiota vs consequences of the pathological process; however, the sole administration of the toxin B-subunits used in our studies does not induce pathology 45 , suggesting that these AB 5 toxins could directly provide a competitive advantage for those that produce them through growth inhibition or simply reduced colonization. It may be particularly important for AB 5 toxin-producers to target ganglioside-mimicking bacteria due to the fitness that mimicry imparts in dealing with host defenses 37 , This would make AB 5 toxins particularly useful tools in the microbiome and supports the notion that these toxins did not only or primarily evolve to target the host, but instead increase competitiveness of the producers as colonizers of the gastrointestinal tract.

The evidence that other gut bacteria have the capacity to mimic GM1 gangliosides and that AB 5 toxins may influence these species has other implications for human health. As mentioned above, ganglioside mimicry is closely linked to the development of GBS, but not all persons infected with GM1-mimicking C. It is possible that other ganglioside-mimicking bacteria could be present in our gut or appear transiently through ingestion of poultry products and could impact individual disease susceptibility by either immune tolerizing or training. Further studies into the extent to which other bacteria presenting these antigens contribute to the manifestation of GBS are warranted.

It is also relevant that CTB is currently administered as part of an increasing number of health-promoting strategies including use in the oral cholera vaccine, as a toxoid adjuvant with unexpected cross-reactivity to the C. Taken together, our studies identify a new role for AB 5 toxins and may explain why some organisms have developed mechanisms to vary their ganglioside mimics. We demonstrate that the chicken gut is rich in ganglioside structures derived from the host and the resident microbiota may impact both bacterial competition and human health. The C. The E.

Ebook Microbial Toxins, Vol. 4: Bacterial Endotoxins

To create the C. Next, for both mutants, C. The resulting colonies were evaluated for insertion of the kanamycin cassette by PCR and sequencing. The cat cassette was obtained from plasmid pRY after SmaI digestion. These primers were then used to amplify cmeA::cat by PCR. The purified PCR product was used to transform strain by natural transformation. Absence of CmeA in the mutant was also confirmed by western blotting with CmeA-specific antibodies. Following growth of C.

Immunogold labeling and transmission electron microscopy were carried out as described in previous reports with minor modifications 19 , The grids were then examined by transmission electron microscopy Philips Morgagni ; FEI Company and images were taken using a charge-coupled camera and controller Gatan and processed using DigitalMicrograph Gatan. The effect of the holotoxins and toxin subunits on bacterial growth was examined by spotting CT, CTA, CTB or LTB onto cell-containing double layer agar plates prepared using the standard overlay agar method commonly used in bacteriophage plaque assays Following a clearance assay with CTB on C.

Excised squares were then washed in 0. The gel was then silver-stained according to the method described by Tsai and Frasch with a few modifications Cells were isolated from inside and outside the clearance zones of agar using the same method as was described for the isolation of LOS. For DNA isolation, once the isolated C. The pellet was resuspended in 0. The ethidium bromide EtBr accumulation assay was performed using the following established protocol Each biological replicate included 3 technical replicates.

the next chapter in endotoxin testing. . .

The Galleria mellonella wax moth model was used as a model to assess C. Commercial broiler chickens Ross , Avigen were obtained on the day of hatch from Lilydale, Edmonton. Upon arrival, birds were divided into groups with up to 9 chickens each. The birds were then euthanized on day 31 and both ceca were removed and processed immediately. All animal studies were carried out in accordance with the protocol approved by the Animal Care and Use Committee at the University of Alberta, protocol number AUP Immunofluorescent staining of the paraffin-embedded sections was conducted using the following established protocols Sequences between and nucleotides long were selected for analysis.

To standardize the sequencing depth across samples, sequences were subsampled to 45, reads using Mothur v.

Pathogenic bacteria - Wikipedia

The number of reads in each taxonomic bin was normalized to the proportion of the total number of reads per each sample for statistical analyses. To achieve normality for the microbiome data that were not normally distributed, values were subjected to log 10 transformations. These spot assays were performed in the same way as the others with minor changes. For the EtBr accumulation and Galleria mellonella data, two-tailed, unpaired t -tests were performed to compare the relative fluorescence upon addition of EtBr, and C.

For microbiome analyses, two-tailed Mann Whitney tests were performed for the CTB dataset comparisons, while two-tailed unpaired t -tests with Welch's correction or multiple t -tests were performed for the LTB comparisons using Graph Pad Prism version 7. All relevant data are available from the corresponding author.

Krinos, C. Extensive surface diversity of a commensal microorganism by multiple DNA inversions. Nature , โ€” Moran, A. Endotoxin Res. Yuki, N. Comstock, L. Bacterial glycans: Key mediators of diverse host immune responses. Cell , โ€” Yi, W.

Shop by category

Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide. Chang, Y. The interplay between Siglecs and sialylated pathogens. Glycobiology 24 , โ€” Stowell, S. Innate immune lectins kill bacteria expressing blood group antigen. Wesener, D. Recognition of microbial glycans by human intelectin Materials provided by Harvard T. Note: Content may be edited for style and length. Science News. Story Source: Materials provided by Harvard T. Allen, David C.