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The main virulence determinant of Yersinia entomophaga MH96 is a broad-host-range toxin complex active against
Mark R H Hurst1, Sandra A Jones, Tan Binglin
1BioControl and BioSecurity, AgResearch, Lincoln Research Centre, Private Bag 4749, Christchurch 8140, Canterbury, New Zealand. mark.hurst@agresearch.co.nz
Abstract:
Through transposon mutagenesis and DNA sequence analysis, the main disease determinant of the entomopathogenic bacterium Yersinia entomophaga MH96 was localized to an ~32-kb pathogenicity island (PAI) designated PAI(Ye₉₆). Residing within PAI(Ye₉₆) are seven open reading frames that encode an insecticidal toxin complex (TC), comprising not only the readily recognized toxin complex A (TCA), TCB, and TCC components but also two chitinase proteins that form a composite TC molecule. The central TC gene-associated region (~19 kb) of PAI(Ye₉₆) was deleted from the Y. entomophaga MH96 genome, and a subsequent bioassay of the ΔTC derivative toward Costelytra zealandica larvae showed it to be innocuous. Virulence of the ΔTC mutant strain could be restored by the introduction of a clone containing the entire PAI(Ye₉₆) TC gene region. As much as 0.5 mg of the TC is released per 100 ml of Luria-Bertani broth at 25°C, while at 30 or 37°C, no TC could be detected in the culture supernatant. Filter-sterilized culture supernatants derived from Y. entomophaga MH96, but not from the ΔTC strain grown at temperatures of 25°C or less, were able to cause mortality. The 50% lethal doses (LD₅₀s) of the TC toward diamondback moth Plutella xylostella and C. zealandica larvae were defined as 30 ng and 50 ng, respectively, at 5 days after ingestion. Histological analysis of the effect of the TC toward P. xylostella larva showed that within 48 h after ingestion of the TC, there was a general dissolution of the larval midgut.
Insights
The insecticidal toxin complex (TC) from Yersinia entomophaga MH96, located on a pathogenicity island (PAI), causes larval mortality by dissolving the midgut. This bacterial toxin is temperature-dependent and effective against specific insect pests.
Area of Science:
- Microbiology
- Molecular Biology
- Insect Pathology
Background:
- Yersinia entomophaga MH96 is an entomopathogenic bacterium.
- Pathogenicity islands (PAIs) often harbor virulence factors.
- Insecticidal toxin complexes (TCs) are crucial for bacterial virulence in insects.
Purpose of the Study:
- To identify and characterize the primary disease determinant of Yersinia entomophaga MH96.
- To investigate the role of the insecticidal toxin complex (TC) in bacterial pathogenicity.
- To determine the efficacy and mode of action of the TC against insect larvae.
Main Methods:
- Transposon mutagenesis and DNA sequence analysis to locate the pathogenicity island (PAI).
- Gene deletion (ΔTC mutant) and complementation to assess TC function.
- Bioassays with insect larvae (Costelytra zealandica, Plutella xylostella) to determine virulence and lethal doses (LD₅₀s).
- Temperature-dependent analysis of TC production and activity.
- Histological examination of larval midgut tissues.
Main Results:
- A ~32-kb pathogenicity island (PAI(Ye₉₆)) was identified as the main disease determinant.
- PAI(Ye₉₆) contains seven open reading frames encoding a novel insecticidal toxin complex (TC) with chitinase components.
- Deletion of the TC gene region rendered the bacterium non-pathogenic to Costelytra zealandica larvae.
- TC production is optimal at 25°C and undetectable at 30-37°C.
- The TC caused mortality in Plutella xylostella and C. zealandica larvae with LD₅₀s of 30 ng and 50 ng, respectively.
- Histological analysis revealed rapid dissolution of the larval midgut upon TC ingestion.
Conclusions:
- The insecticidal toxin complex (TC) encoded on PAI(Ye₉₆) is essential for the virulence of Yersinia entomophaga MH96.
- The TC acts by causing rapid midgut dissolution in susceptible insect larvae.
- TC production is regulated by temperature, with optimal activity at lower temperatures (≤25°C).
- This study elucidates a novel insecticidal mechanism with potential applications in pest control.
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