Elsevier

Microbial Pathogenesis

Volume 46, Issue 2, February 2009, Pages 59-62
Microbial Pathogenesis

Purification of a soil bacteria exotoxin using silkworm toxicity to measure specific activity

https://doi.org/10.1016/j.micpath.2008.10.010Get rights and content

Abstract

Overnight cultures of 16 of 25 species of soil bacterium injected into silkworm hemolymph killed the silkworms. Injection of culture supernatants of five of these 16 bacterial species into the hemolymph also killed silkworms. Analysis of 16S rRNA indicated that the toxic species were Bacillus species. We purified an exotoxin produced by one of the Bacillus species by measuring the specific activity based on silkworm toxicity. The final step of purification revealed that the silkworm-killing activity was due to a 34-kDa protein. LC/MS/MS analysis revealed that the 34-kDa protein is sphingomyelinase C of Bacillus cereus. We propose that the silkworm infection model is useful for identifying pathogenic bacteria from soil and purifying bacterial toxins.

Introduction

Many bacterial species are present in soil, but most of these species have not yet been identified [1]. Many pathogenic bacteria are present in soil, including Clostridium tetanii causing tetanus, Clostridium botulinum and Clostridium perfringens causing food poisoning, Clostridium difficile causing colitis, and Bacillus anthracis causing anthrax. Opportunistic pathogens, such as Pseudomonas aeruginosa and Stenotrophomonas maltophilia are also present in the soil. Bacteria living in the soil could be the source of emerging infectious diseases that infect humans. Therefore, it is important to develop methods to effectively identify pathogenic bacteria in the soil. Animal models are invaluable for evaluating bacterial pathogenicity. The use of mammals, however, is costly, both financially and ethically. We previously reported that silkworms are effective for identifying the virulence genes of Staphylococcus aureus, a human pathogen [2], [3], [4], [5], [6], [7], [8]. Silkworms are easily handled because of their slow movement and are large enough for quantitative evaluation of samples by injecting a precise sample volume of samples into the hemolymph [9], [10], [11]. Thus, compared to other invertebrate animals, such as Caenorhabditis elegans and Drosophila melanogaster, silkworms are advantageous as experimental animals. In the present study, we isolated bacterial strains from soil and examined their pathogenicities and toxin production. Silkworm-killing activity of one of the culture supernatants was measured to purify the active compound, which was identified as sphingomyelinase C of Bacillus cereus.

Section snippets

Pathogenicity of soil bacterium to silkworms

We isolated 25 distinct colonies by spreading soil samples onto brain heart infusion agar plates. Full bacterial growths were injected into silkworm hemolymph, and their pathogenicities were examined. Of the 25 bacterial species, 16 killed silkworms (Table 1). The finding suggests that many soil bacteria have virulence against silkworms. Next, we determined whether the killing activity was caused by exotoxins produced by those bacteria. Five out of 16 culture supernatants had a killing activity

Evaluation of pathogenicity of soil bacterium by using silkworms

More than half of the bacteria randomly isolated from soil killed silkworms. Previously we reported that bacteria that are pathogenic in humans, such as S. aureus and P. aeruginosa, also killed silkworms, whereas laboratory strains of Escherichia coli, which are non-pathogenic to humans, do not [2]. These results suggest that pathogenic bacteria have common virulence mechanisms against invertebrates and humans. We speculate that the soil bacteria determined to be virulent against silkworms

Isolation of soil bacteria and determination of species

Soil samples from the campus of the University of Tokyo were collected, suspended in sterilized water, and the supernatants were spread onto brain–heart infusion agar plates. Colonies were isolated after overnight incubation. The 16S rRNA region was amplified by Colony PCR (40 cycles; 96 °C 30 s, 50 °C 15 s, 60 °C 4 min) using primer pairs f-16S (5′-GAGTTTGATCCTGGCTCAG-3′) and r-16S (5′-AAGGAGGTGATCCAGCC-3′) and was sequenced. A BLAST search against the sequenced 700 bp of 16S rRNA was performed to

Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research (19041022, 20790057, 20390021). This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) and Genome Pharmaceuticals Institute.

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