Search

Cyt1 Aa is a [delta]-endotoxin protein that is produced by Bacillus thuringiensis subsp, israelensis. It is a membrane pore-forming toxin that is lethal to insect larvae and is broadly cytolytic to vertebrate as well as invertebrate cells. Cyt1 Aa is produced as a protoxin of 27 kDa. Proteolytic activation results in a reduction of the molecular mass to approximately 23-24 kDa and a threefold increase in activity. In this research, Cyt1 Aa crystals were purified from B. thuringiensis IPS78/11 harbouring the expression vector pHT-cyAp20. The activity of the activated form of Cyt1 Aa (23-24 kDa) was examined on a pathogenic strain of the Gram-negative Escherichia coil and the Gram-positive species Staphylococcus aureus. The Cyt1 Aa minimal inhibitory concentration for E. coil and S. aureus was 1.25 and 5 [micro]g [ml.sup.-1], respectively. Cyt1Aa was found to be bactericidal for E. coli, whereas it was bacteriostatic for S. aureus. Furthermore, Cyt1 Aa increased the lethal effect when acting in combination with antibiotics. The association of Cyt1 Aa with cells of these two bacteria was demonstrated by Western blot analysis using antibodies against the whole [delta]-endotoxin crystal. Scanning electron microscopy displayed damage to Cyt1 Aa-treated cells. Ion imbalance due to damage of the cell walls and membranes was confirmed by X-ray microanalysis. These experiments show that Cyt1 Aa has an antibacterial effect on pathogenic species and demonstrate, apparently for the first time, that exogenous Cyt1 Aa has a bactericidal effect upon Gram-negative bacteria.

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode ~1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28[degrees]C and 37[degrees]C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coil BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coil BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multinucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coil BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 ceils, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

Certain amino acids, and cysteine in particular, promptly blocked toxin expression in Clostridium difficile strain VPI 10463 when added to late-exponential-phase peptone-yeast cultures, i.e. prior to normal induction of toxins A and B. Glucose reduced toxin yields by 80-fold, but only when supplemented at inoculation. Forty upregulated C. difficile proteins were identified during maximum toxin expression, and most of these were enzymes involved in energy exchange, e.g. succinate, CO/folate and butyrate metabolism. Transcription of tcdA (toxin operon) and folD (CO/ folate operon) was induced by 20- and 10-fold, respectively, and with strikingly similar kinetics between OD 0.8 and 1.2. The sigma factors tcdR and sigH were upregulated simultaneously with tcdA and folD (3.5-fold increase of mRNA level), whereas transcription of tcdC, codY, sigB and sigL showed little or no correlation with that of tcdA and folD. The results suggest a connection between toxin expression, alternative energy metabolism and initial sporulation events in C. difficile.

Author(s): Weiguo Hu [1, 2]; Sean P Ferris [2]; Rodney K Tweten [3]; Gongxiong Wu [2]; Svetlana Radaeva [4]; Bin Gao [5]; Roderick T Bronson [6]; Jose A Halperin [1, [...]

The multifrequency EPR work on colicin A mutants gives new insights into the formation of membrane channels by means of bacterial toxins. This leads to further studies for measuring effects of microenvironments of site specific spin labeled proteins at work.

A study was conducted to identify the residues lining the anthrax protective antigen channel. The results reveal alternating reduction and absence of reduction of conductance in consecutive residues over two stretches. The findings provide support for the model of conversion of the prepore to a 14-stranded Beta barrel pore and fortify the foundation for future research on the mechanism of translocation by anthrax toxin.

A research was conducted to study the crystal structure of the Shiga-like toxin I (SLT-I) B-pentamer complexed with a Gb3 analogue. Molecular replacement techniques was utilized to determine the structure of the complex while analyzing the model at different stages using the program O. Results showed that the complex had more exact 5-fold symmetry than the native SLT-I B-pentamer and shared similarities with the B-pentamers in the asymmetric unit.

Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules and activate T cells in a V[Beta]-restricted fashion. We recently identified subsets of HLA-DR1 molecules that show selectivity for SAgs. Here, we extend these observations by showing that different cell lineages demonstrate distinct SAg-binding specificities although they all express HLA-DR1. Indeed, B cells bind staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) with high affinity while staphylococcal enterotoxin B (SEB) binding is barely detectable. In contrast, DR1-transfected HeLa cells show efficient binding of SEB, but not of SEA or TSST-1. We investigated the class II maturation events required for efficient interaction with SAgs and found that the ability of cells to bind and present the toxins can be drastically modulated by coexpression of the class II-associated invariant chain (Ii) and HLA-DM. SEA binding to DR1 molecules required coexpression of Ii, whereas TSST-1 binding was selectively enhanced by DM. Binding of SEB was affected by cell type-specific factors other than Ii or DM. The selectivity of SAgs for different MHC class II populations was minimally affected by HLA-DR intrinsic polymorphism and could not be explained by binding to alternative sites on DR molecules. Our results indicate that SAgs are sensitive to structural heterogeneity in class II molecules, which is consequent to the differential regulation of expression of antigen processing cofactors. Therefore, we speculate that Staphylococcus aureus have retained the ability to express numerous SAgs in adaptation to the micro-heterogeneity displayed by MHC class II molecules and that this may relate to their ability to infect different tissues.

Flow cytometric analysis with biotilynated Clostridium perfringens enterotoxin COOH-terminal fragment peptide probe was used in Escherichia coli DE3 strain to describe the molecular expression cloning. Isolation of clonal L929pyT18 cell lines expressing the Clostridium perfringens enterotoxin receptor (CPE-R) was used to delineate the functional characteristics of the receptor. Results have shown that CPE-R is composed of four putative transmembrane segments and that it binds specifically with CPE in the initial step of CPE-induced reaction.

A hybrid Bordetella pertussis adenylate cyclase toxin (CyaA) with narrower target cell specificity compared to the native toxin was constructed and analyzed to determine the structure-function relationship of CyaA. Analysis of the hybrid CyaA toxin in Escherichia coli host cells indicated the absence of toxic activity due to changes in the CyaA C-terminal region which mediated the delivery of the enzymatic domain across the target cell membrane. Furthermore, amino acids in the C-terminal region and hydrophobic domain of CyaA were important for toxic activity.

The Bacillus thuringiensis-produced Cry1Ab and the Cry1Ac toxins fail to show any differences in their binding to the protein of ca.80-kDa in Plodia interpunctella strains resistant to the toxins. The binding protein is necessary for toxicity and differs in size from the aminopeptidase N antigens responsible for binding in other insects. The toxins bind to the membranes of susceptible larvae but not to those of resistant larvae. This indicates that the resistant P. interpunctella strains have altered accessibility of Cry1A-binding protein rather than altered binding properties.

The anaerobic bacterial strain, Clostridium bifermentans subsp. malaysia CH18, has been widely used in the production of biological insecticides because of its toxicity to mosquito larvae. Cloning and expression of the gene encoding the protein for toxicity identified a 71,128-kilodalton protein termed Cbm71. Protein analysis indicated that the mosquitocidal protein is homologous to the first four blocks conserved in Bacillus thuringiensis delta-endotoxins, but they are not immunologically related. Bioassays of mosquito larvae showed that the toxins are effective against the species Aedes aegypti, Culex pipiens and Anopheles stephensi.

The nucleotide sequence and cloning analysis of 8.7kb fraction from the 60 kb plasmid Lactobacillus sake Lb706 shows that signal sequence-independent transport system of HlyB/HlyD is necessary for the secretion of sakacin A. Deletion analyses and frameshift mutations shows that two different transcribed operons namely sapK and sapR are required for sakacin production and immunity. Northern blot analysis shows that SapK/SapR system activates both the operons.

Mutagenesis, and nucleotide sequence and complementation analyses of the genetic structure of Enterococcus faecalis plasmid pAD1 cytolysin determinant reveal that the structural arrangement of E. faecalis cytolysin is similar to that of lantibiotics. The structural similarity indicates that E. faecalis cytolysin is the first member of a novel family to encode toxin activity. Four open reading frames (orfs) are essential for expressing the cytolysin precursor, with the products of two orfs constituting the cytolysin precursor.

Analysis of Rhizobium isolates from leguminous tree nodules that contain a toxin, mimosine, reveals that the mimosine catabolic pathway enzymes are inducible by mimosine. The enzymes are present on the soluble region of the cell extracts, and the toxin reduces to carbon dioxide and biomass after the induction of mimosine catabolism. Isolates capable of proliferating with mimosine as the nitrogen and carbon source are also capable of using the toxic mimosine degradation intermediate, 3-hydroxy-4-pyridone.

A study of the pathogenicity of environmental Escherichia coli isolates by triplex PCR amplification of shiga-like toxin sequences (SLT) I and II, and heat-labile toxin (LT) sequences reveals that toxigenic E. coli strains are rare in coastal waters. However, detection of SLT II toxigenic sequences in near shore areas indicates the presence of enterohemorrhagic E. coli strains. Studies with mouse Y-1 adrenal cells and human vero cell line confirm the production of toxins in positive E. coli isolates in environmental waters.

Identical classifications of RTX-toxins of Actinobacillus pleuropneumoniae and their genes were proposed after studying three diverse pore-forming RTX-toxins of this organism. The RTX-toxin derived from the reference strains for serotypes 1, 5a, 5b, 9, 10 and 11 were termed Apxl. This protein was found to be highly haemolytic and reveals increased cytotoxic activity toward pig alveolar macrophages and neutrophils, and has a molecular mass between 105 and 110kDa.

The drought of 1988 at Homer Lake, Illinois yielded a harvest of 12 hepatotoxins from threeMicrocystis blue-green algae, M. aeruginosa, M. viridis and M. wesenbergii. Of the 12, microcystins-RR, -YR and -LR (a very potent toxin) are previously known The nine others are newly isolated, of which the seven have been assigned as (9-O-demethyl-Adda)microcystin-LR, (dehydroalanine)microcystin-LR, microcystin-FR, microcystin-AR, microcystin-M(O)R, (N-methylserine)microcystin-LR and microcystin-WR (the 12th in the series). The 10th microcystin has so far been identified only through its four amino acids. The 11th so far has shown no toxicity in bioassays.

Two genes were isolated from Bacillus thuringiensis subsp. thompsoni which coded for two novel crystal proteins. Gel electrophoresis showed that the two proteins had molecular masses of 40 and 34 kDa. Sequence analysis showed that the two proteins were not similar to each other or to any other crystal protein. Toxicity assays on a few lepidopteran species revealed that the 34 kDa protein is insecticidal, making it the smallest mature toxin reported to date. The 40 kDa did not display any insecticidal activity, but may be involved in scaffolding of the toxic cuboidal cyrstals.

Analysis of selection with Bacillus thuringiensis subsp. kurstaki, consisting of CryII and CryIA toxins, reveals that this induces a 200-fold cross-resistance to the CryIF toxin in the diamondback moth. CryIB is toxic while CryIE is nontoxic to the unselected and selected moth larvae. The reduction in the susceptibility of bacterial strains to the toxins indicates the extremely high levels of cross-resistance in B. thuringiensis CryI toxins.

Disruption of the cryIVD gene in the 72 MDa resident plasmid was conducted by in vivo recombination. Characterization of the larvicidal activity of the cryIVD protein-free inclusions against Aedes aegypti, Culex pipiens and Anopheles stephensi larvae was also conducted. Toxicity of cryIVD protein free inclusions resembled those of wild-type crystals of Anopheles stephensi larvae, but was half of that of the wild-type toxicity of Culex pipens and Aedes aegypti larvae.

A study is conducted to determine the potential of chromatophore cells as a biodetectors for function-based detection of chemically and biologically toxic substances. Oncorhynchus tshawytscha (chinook salmon) melanophores, a chromatophore cell type containing brown pigment exhibit ability to rapidly detect the salmonid pathogens Aeromonas salmonicida, Yersinia ruckeri, and Flavobacterium psychrophilum and the human pathogen Bacillus cereus.

Several bacterial protein toxins act by forming oligomers on the cell surface. A novel approach to the prevention of the damage caused by oligomeric pore-forming toxins has recently been discovered using one such protein: the protective antigen (PA) of Bacillus anthracis. Some PA mutants act as dominant-negative effectors of the function performed by the oligomer. In fact, the PA mutant co-oligomerizes with native PA, but the mixed oligomer is nonfunctional. This finding might turn out to be of general validity.

The dairy industry is helping to ensure safety of refrigerated foods in the US by developing a new database to help identify and characterize various forms of microbial isolates. Information on 1000+ strains of bacteria has been developed. Dairy Management Inc. will manage it. Researchers can access the database to obtain characteristics of a bacterial strains to determine the best method of processing to prevent growth of achieve elimination. An example would be Listeria strains. Harmful strains of L. monocytogenes can cause serious outbreaks of food-related illness. Rapid detection of hazardous foodborne microbes helps to pinpoint a contamination source or to refute that dangerous food came from a given plant.