The InhA Metalloproteases of<i>Bacillus cereus</i>Contribute Concomitantly to Virulence

Guillemet, Elisabeth; Cadot, Céline; Tran, Seav-Ly; Guinebretière, Marie-Hélène; Lereclus, Didier; Ramarao, Nalini

doi:10.1128/jb.00264-09

Cited by 105 publications

(100 citation statements)

References 60 publications

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“…In addition, there are six predicted InhA-like metalloproteinases in this strain. Based on the premises that InhA metalloproteinase has toxicity to Lepidoptera (9) and that strain YBT-1518 has high toxicity to nematodes, it would be interesting to test whether they have nematicidal activities and explore their mechanisms of action.…”

Section: Discussionmentioning

confidence: 99%

“…Second, the virulence factor can protect B. thuringiensis from the innate immune system through cleavage of antimicrobial peptides, whereby the insecticidal activity of the Cry protein is enhanced (7). It is lethal to inject immune inhibitor A (InhA) into the Lepidopteran larvae hemocoel (9). Thus, virulence factors are used by B. thuringiensis to target insects.…”

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confidence: 99%

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Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

Luo

Chen

Huang

et al. 2013

Appl Environ Microbiol

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Some Bacillus thuringiensis strains have high toxicity to nematodes. Nematicidal activity has been found in several families of crystal proteins, such as Cry5, Cry6, and Cry55. The B. thuringiensis strain YBT-1518 has three cry genes that have high nematicidal activity. The whole genome sequence of this strain contains multiple potential virulence factors. To evaluate the pathogenic potential of virulence factors, we focused on a metalloproteinase called Bmp1. It encompasses a consecutive N-terminal signal peptide, an FTP superfamily domain, an M4 neutral protease GluZincin superfamily, two Big-3 superfamily motifs, and a Grampositive anchor superfamily motif as a C-terminal domain. Here, we showed that purified Bmp1 protein showed metalloproteinase activity and toxicity against Caenorhabditis elegans (the 50% lethal concentration is 610 ؎ 9.37 g/ml). In addition, mixing Cry5Ba with Bmp1 protein enhanced the toxicity 7.9-fold (the expected toxicity of the two proteins calculated from their separate toxicities) against C. elegans. Confocal microscopic observation revealed that Bmp1 protein was detected from around the mouth and esophagus to the intestine. Striking microscopic images revealed that Bmp1 degrades intestine tissues, and the Cry5Ba causes intestinal shrinkage from the body wall. Thus, the B. thuringiensis Bmp1 metalloproteinase is a nematicidal virulence factor. These findings give a new insight into the relationship between B. thuringiensis and its host nematodes. Bacillus thuringiensis is a rod-shaped, Gram-positive, sporeforming bacterium. It is the most successful insect pathogen used for insect control (1). The action to the insect pest relies on insecticidal toxin and array of virulence factors (2). Upon sporulation, it produces insecticidal crystal inclusion that is formed by a variety of insecticidal proteins called Cry or Cyt proteins. These insecticidal crystal proteins are toxic to insects in the orders Lepidoptera, Dipteran, Coleoptera, Hymenoptera, Homoptera, Orthoptera, and Mallophage (2), and they are also toxic to nematodes. Until now, there are several families of Cry proteins (Cry5, Cry6, Cry12, Cry13, Cry14, Cry21, and Cry55) known to be toxic to the larvae of a number of free-living or parasitic nematodes (3). Besides having crystal proteins that are toxic to insects, B. thuringiensis has many virulence factors that contribute to its pathogenic effects. These virulence factors contain exotoxins and extracellular proteases (2). During the stationary growth phase, some B. thuringiensis strains secrete exotoxins, which are heat-stable, watersoluble, and low-molecular-mass compounds (701 Da). These compounds are highly toxic to a wide range of insect species by the oral route (4, 5). In addition, extracellular proteases, e.g., serine protease, chitinase, and collagenase (6), have been reported to be insect pests virulence factors (2).In the past few years, some insecticidal virulence factors have been isolated from B. thuringiensis. The action mode of virulence factors against insect c...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

Luo

Chen

Huang

et al. 2013

Appl Environ Microbiol

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…, 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.…”

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

Ramarao¹,

Nielsen-LeRoux²,

Lereclus³

2012

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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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“…Cytotoxicity of culture supernatants was evaluated by the trypan blue dye method (17,18). The pathogenic strains (FP and C) were highly toxic to human cells (more than 80% of the strains), whereas cytotoxicity was detected for only 12% of the NP strains (P Ͻ 0.001; Fig.…”

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Pathogenic Potential of Bacillus cereus Strains as Revealed by Phenotypic Analysis

et al. 2013

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The InhA Metalloproteases ofBacillus cereusContribute Concomitantly to Virulence

Cited by 105 publications

References 60 publications

Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

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

Pathogenic Potential of Bacillus cereus Strains as Revealed by Phenotypic Analysis

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