Structure of the Mosquitocidal Toxin from Bacillus sphaericus

Reinert, D.J.; Carpusca, Irina; Aktories, Klaus; Schulz, Georg E.

doi:10.1016/j.jmb.2006.01.025

Cited by 26 publications

(37 citation statements)

References 47 publications

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“…3,4 MTX is produced as a single polypeptide chain of 870 residues consisting of a putative signal sequence (residues 1-29), a catalytic domain (residues 30-264), a linker (265-295) and a further 575 residues. [3][4][5][6][7][8] The catalytic domain is homologous to those of other toxins such as cholera toxin, pertussis toxin, Escherichia coli heat-labile enterotoxin, Pseudomonas exotoxin A and diphtheria toxin [9][10][11][12][13][14][15] as well as to ADPribosylating enzymes such as poly(ADP-ribosyl) polymerase (PARP) and ART2.2. 16,17 The catalytic domain of MTX ADP-ribosylates arginines of various proteins.…”

Section: Introductionmentioning

confidence: 99%

Structure and Mode of Action of a Mosquitocidal Holotoxin

Treiber

Reinert

Carpusca

et al. 2008

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 99%

Structure and Mode of Action of a Mosquitocidal Holotoxin

Treiber

Reinert

Carpusca

et al. 2008

Journal of Molecular Biology

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“…The 3D structures of several protein ADP-ribosyltransferases have been reported including chicken poly(ADP-ribose) polymerase (PARP)-1 (14), rat ART2.2 (15), mosquitocidal toxin from Bacillus sphaericus (16), diphtheria toxin (17), cholera toxin (18), domain III of Pseudomonas aeruginosa exotoxin A (19), iota-toxin from Clostridium perfringens (20), pertussis toxin (21), C2-toxin (22), and C3-toxin (23) from Clostridium botulinum.…”

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confidence: 99%

Rifamycin antibiotic resistance by ADP-ribosylation: Structure and diversity of Arr

Baysarowich

Koteva

Hughes

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

164

196

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The rifamycin antibiotic rifampin is important for the treatment of tuberculosis and infections caused by multidrug-resistant Staphylococcus aureus. Recent iterations of the rifampin core structure have resulted in new drugs and drug candidates for the treatment of a much broader range of infectious diseases. This expanded use of rifamycin antibiotics has the potential to select for increased resistance. One poorly characterized mechanism of resistance is through Arr enzymes that catalyze ADP-ribosylation of rifamycins. We find that genes encoding predicted Arr enzymes are widely distributed in the genomes of pathogenic and nonpathogenic bacteria. Biochemical analysis of three representative Arr enzymes from environmental and pathogenic bacterial sources shows that these have equally efficient drug resistance capacity in vitro and in vivo. The 3D structure of one of these orthologues from Mycobacterium smegmatis was determined and reveals structural homology with ADP-ribosyltransferases important in eukaryotic biology, including poly(ADP-ribose) polymerases (PARPs) and bacterial toxins, despite no significant amino acid sequence homology with these proteins. This work highlights the extent of the rifamycin resistome in microbial genera with the potential to negatively impact the expanded use of this class of antibiotic.crystallography ͉ resistome ͉ rifampin ͉ ribosyltransferase

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“…N-terminal sequencing of bands from the 1-h incubation yielded the residues G-S-M-A-Sϳ (for the smaller peptide, corresponding to the N terminus of the thrombin-cleaved Mtx1 product (27), and I-L-D-L-Dϳ, indicating cleavage after residue Phe264 in the region known to be involved in the processing of Mtx1 into its two functional toxin derivatives (27). The recently solved crystal structure of a catalytically inactive and truncated form of Mtx1 clearly shows that this part of Mtx1 forms a surfaceexposed region that is an obvious target for proteolytic activity (19). The degradation by the above-described extracts was completely inhibited by preincubation of the proteinase source with phenylmethylsulfonyl fluoride (PMSF) (Fig.…”

Section: Resultsmentioning

confidence: 98%

Proteolytic Stability of Insecticidal Toxins Expressed in Recombinant Bacilli

Yang

Wang

Gaviria

et al. 2007

Appl Environ Microbiol

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The production of the vegetative mosquitocidal toxin Mtx1 from Bacillus sphaericus was redirected to the sporulation phase by replacement of its weak, native promoter with the strong sporulation promoter of the bin genes. Recombinant bacilli developed toxicity during early sporulation, but this declined rapidly in later stages, indicating the proteolytic instability of the toxin. Inhibition studies indicated the action of a serine proteinase, and similar degradation was also seen with the purified B. sphaericus enzyme sphericase. Following the identification of the initial cleavage site involved in this degradation, mutant Mtx1 proteins were expressed in an attempt to overcome destructive cleavage while remaining capable of proteolytic activation. However, the apparently broad specificity of sphericase seems to make this impossible. The stability of a further vegetative toxin, Mtx2, was also found to be low when it was exposed to sphericase or conditioned medium. Random mutation of the receptor binding loops of the Bacillus thuringiensis Cry1Aa toxin did, in contrast, allow production of significant levels of spore-associated protein in the form of parasporal crystals. The exploitation of vegetative toxins may, therefore, be greatly limited by their susceptibility to proteinases produced by the host bacteria, whereas the sequestration of sporulation-associated toxins into crystals may make them more amenable to use in strain improvement. B. sphaericus produces binary toxin (Bin) during its sporulation phase and Mtx toxins (Mtx1, Mtx2, and Mtx3) (14, 28, 29) during vegetative growth. Due to their low level of expression and their susceptibility to a subtilisin-like serine proteinase (32) also known as sphericase (2), Mtx1 and Mtx2 are not able to contribute to the toxicity of the insecticidal spore formulations used in mosquito control programs. However, these toxins have mechanisms of action distinct from that of the Bin toxin and have significant activity against Aedes aegypti larvae (27) that show low sensitivity to Bin toxins (7). Recent studies have shown that a vegetative insecticidal protein from B. thuringiensis can be redirected in its time of production (6), and a similar retargeting of Mtx1 might enhance B. sphaericus strain activity. In this study, we have examined the possibility of supplementing the binary toxins of B. sphaericus with Mtx toxins from this species and attempted to direct the production of Mtx1 during sporulation and to produce mutants that are proteolytically stable. In addition, the stability of Mtx2 to proteolytic degradation was also examined. Strains of Bacillus sphaericus andWith the importance of proteolytic stability in toxin production and increasing attempts to modify other insecticidal proteins for the selection of novel activities, for instance by phage display (12,16,17,31), we also considered it to be of interest to analyze mutants of a B. thuringiensis Cry toxin to determine whether specific mutation of receptor binding regions might also introduce proteolytic sensitivi...

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Structure of the Mosquitocidal Toxin from Bacillus sphaericus

Cited by 26 publications

References 47 publications

Structure and Mode of Action of a Mosquitocidal Holotoxin

Structure and Mode of Action of a Mosquitocidal Holotoxin

Rifamycin antibiotic resistance by ADP-ribosylation: Structure and diversity of Arr

Proteolytic Stability of Insecticidal Toxins Expressed in Recombinant Bacilli

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