Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects

Dalhammar, Gunnel; Steiner, Håkan

doi:10.1111/j.1432-1033.1984.tb08000.x

Cited by 138 publications

(104 citation statements)

References 26 publications

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“…It was also sensitive to InA, an enzyme isolated from Bacillus thuringiensis, which is known to cleave cecropin A in several places (15), and furthermore it was inactivated by serum. In sharp contrast, all-D-cecropin A was very resistant to degradation by trypsin, InA, and serum.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

All-D amino acid-containing channel-forming antibiotic peptides.

Wade

Boman

Wåhlin

et al. 1990

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

652

492

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The D enantiomers of three naturally occurring antibiotics-cecropin A, magainin 2 amide, and melittin-were synthesized. In addition, the D enantiomers of two synthetic chimeric cecropin-melittin hybrid peptides were prepared. Each D isomer was shown by circular dichroism to be a mirror image of the corresponding L isomer in several solvent mixtures. In 20% hexafluoro-2-propanol the peptides contained 43-75% a-helix. The all-D peptides were resistant to enzymatic degradation. The peptides produced single-channel conductances in planar lipid bilayers, and the D and L enantiomers caused equivalent amounts of electrical conductivity. All of the peptides were potent antibacterial agents against representative Gram-negative and Gram-positive species. The D and L enantiomers of each peptide pair were equally active, within experimental error. Sheep erythrocytes were lysed by both D-and L-melittin but not by either isomer of cecropin A, magainin 2 amide, or the hybrids cecropin A-(1-13)-melittin-(1-13)-NH2 or cecropin A-(1-8)-melittin-(1-18)-NH2. The infectivity of the bloodstream form of the malaria parasite Plasmodium falciparum was also inhibited by the D and L hybrids. It is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.The cecropins (1, 2) and several other antibiotic peptides of the animal kingdom, including defensins (3), magainins (4), and the bee venom toxin melittin (5), are thought to function through the formation of ion channels in lipid membranes. This idea has been based on recent studies of electrical conductivity in artificial lipid bilayers (3,(6)(7)(8), where activity is a function of the structure of the peptide and the composition of the membrane lipids. The bilayer lipids and cell membranes are chiral and contain many asymmetric centers. It has been generally assumed that the chirality of the membrane would require a specific chirality ofthe peptide for it to be active, in much the same way that peptide hormones are required to fit with the conformation of their natural receptors or for a substrate and enzyme to form a tight stereospecific complex. However, we have suggested that these peptide antibiotics can exert their effect without requiring a specific target receptor on the cell membrane (7, 9).The purpose of the present study was to test this assumption by the synthesis of the all-D enantiomers of several natural, all-L peptide antibiotics and some of their active analogs. These D stereoisomers would be expected to assume equivalent, but mirror image, conformations when placed in the same environment as the all-L peptides. If a close molecular contact with the chiral components of the cell membrane is required, the D enantiomers would be expected to be inactive. However, if the interaction of the peptide with the membrane is only between achiral...

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Section: Discussionmentioning

confidence: 99%

“…Stock solutions of enzymes containing trypsin at 5 mg/ml or immune inhibitor A (InA) (15) Electrical Conductivity Measurements. The equipment and procedures were similar to those described previously (7,16 …”

Section: Methodsmentioning

confidence: 99%

All-D amino acid-containing channel-forming antibiotic peptides.

Wade

Boman

Wåhlin

et al. 1990

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

652

492

View full text Add to dashboard Cite

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“…Its amino acid sequence contains the zinc-binding and catalytic active site residues common to these various metalloproteases. In vitro, InhA is secreted and selectively hydrolyses cecropins and attacins, two antibacterial proteins found in the haemolymph of Hyalophora cecropia (Dalhammar & Steiner, 1984). InhA is toxic to adult Drosophila (Siden et al, 1979).…”

Section: Introductionmentioning

confidence: 99%

Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF287346.

2001

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The immune inhibitor A (InhA) metalloprotease from Bacillus thuringiensis specifically cleaves antibacterial proteins produced by the insect host, suggesting that it may contribute to the overall virulence of B. thuringiensis. The transcriptional regulation of the inhA gene in both B. thuringiensis and Bacillus subtilis was investigated. Using a transcriptional inhA'-lacZ fusion, it was shown that inhA expression is activated at the onset of sporulation.However, the transcriptional start site of inhA is similar to σ A -dependent promoters, and deletion of the sporulation-specific sigma factors σ F or σ E had no effect on inhA expression in B. subtilis. The DNA region upstream from inhA contains two genes encoding polypeptides similar to the SinI and SinR regulators of B. subtilis. SinR is a DNA-binding protein regulating gene expression and SinI inhibits SinR activity. Overexpression of the sin genes affects the expression of the inhA'-lacZ transcriptional fusion in B. thuringiensis : early induction of inhA expression was observed when sinI was overexpressed, whereas inhA expression was reduced in a strain overexpressing sinR, suggesting that inhA transcription is repressed, directly or indirectly, by SinR. inhA transcription was greatly reduced in B. thuringiensis and B. subtilis spo0A mutants. Analysis of the inhA'-lacZ expression in abrB and abrB-spo0A mutants of B. subtilis indicates that the Spo0A-dependent regulation of inhA expression depends on AbrB, which is known to regulate expression of transition state and sporulation genes in B. subtilis.

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“…Like mammals, insects possess a complex innate immune system 1 . Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides 2,3 . In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems.…”

mentioning

confidence: 99%

The Insect <em>Galleria mellonella</em> as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Ramarao¹,

Nielsen-LeRoux²,

Lereclus³

2012

JoVE

246

233

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The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system 1 . Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides 2,3 . In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals 1 . Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis [4][5][6][7] . Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model [8][9][10][11] . Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C 12,13 , allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD 50 14 , measurement of bacterial survival 15,16 and examination of the infection process 17 . Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial...

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Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects

Cited by 138 publications

References 26 publications

All-D amino acid-containing channel-forming antibiotic peptides.

All-D amino acid-containing channel-forming antibiotic peptides.

Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF287346.

The Insect <em>Galleria mellonella</em> as a Powerful Infection Model to Investigate Bacterial Pathogenesis

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