The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects

Abstract

Bacillus thuringiensis has been widely used for 40 years as a safe biopesticide for controlling agricultural pests and mosquitoes because it produces insecticidal crystal proteins. However, spores have also been shown to contribute to overall entomopathogenicity. Here, the opportunistic properties of acrystalliferous B. thuringiensis Cry N and Bacillus cereus strains were investigated in an insect species, Galleria mellonella, and in a mammal, BALB/c mice. In both animal models, the pathogenicity of the two bacterial species was similar. Mutant strains were constructed in which the plcR gene, encoding a pleiotropic regulator of extracellular factors, was disrupted. In larvae, coingestion of 10 6 spores of the parental strain with a sublethal concentration of Cry1C toxin caused 70 % mortality whereas only 7 % mortality was recorded if spores of the ∆plcR mutant strain were used. In mice, nasal instillation of 10 8 spores of the parental strain caused 100 % mortality whereas instillation with the same number of ∆plcR strain spores caused much lower or no mortality. Similar effects were obtained if vegetative cells were used instead of spores.The cause of death is unknown and is unlikely to be due to actual growth of the bacteria in mice. The lesions caused by B. thuringiensis supernatant in infected mice suggested that haemolytic toxins were involved. The cytolytic properties of strains of B. thuringiensis and B. cereus, using sheep, horse and human erythrocytes and G. mellonella haemocytes, were therefore investigated. The level of cytolytic activity is highly reduced in ∆plcR strains.Together, the results indicate that the pathogenicity of B. thuringiensis strain 407 and B. cereus strain ATCC 14579 is controlled by PlcR.

Fluctuations in haemocyte density and microbial load may be used as indicators of fungal pathogenicity in larvae of Galleria mellonella

“…The use of insects in place of mammals for routine testing of microbial pathogens (or their mutants) would reduce cost, labour and the need to use the same number of mammals as used at present. It has been demonstrated previously that larval death can be employed as an end point for this type of assay [1][2][3]10,11,13]. The work presented here demonstrates two further end points that may extend the applicability of the G. mellonella model system.…”

“…The ability of G. mellonella larvae to detect differences in the pathogenicity of lipopolysaccharide-deficient mutants of Pseudomonas aeruginosa has also been demonstrated [27], and a strong correlation between the virulence of P. aeruginosa in Galleria larvae and in mice has been demonstrated [2]. Larvae of G. mellonella have been used to assess the virulence of Bacillus thuringiensis and Bacillus cereus, and a good agreement between the results obtained in insects and mice has been established [3]. The survival of larvae following inoculation with microbes has been used as the sole end point for in vivo pathogenicity assays.…”

“…Recent work has demonstrated that a strong correlation exists between the virulence of pathogens when determined in insects and mice [1][2][3]. The immune response of insects to pathogens shares a high degree of homology with the innate immune response of mammals [4][5][6][7], which plays a critical role in protecting the body from infection [8,9].…”

“…At present, larval death is the end point chosen for use in in vivo pathogenicity assays [1][2][3]10]. Some microbes of low pathogen-icity fail to kill larvae [1,10] but may still evoke a low-level immune response, which may provide information on the mechanisms employed by the insect to eradicate infection and also allow a discrimination between pathogens of low pathogenicity.…”

Bacterial strategies to overcome insect defences

“…Global regulators play an important role in virulence towards insects: the GacS/GacA two-component system controls the P. entomophila infectious process 50 , PhoP/PhoQ is the master regulator of virulence in P. luminescens 72 , Lrp regulates both mutualism and pathogenicity in Xenorhabus 73 and PlcR activates the transcription of genes required for virulence in B. thuringiensis 74,75 . Another striking example is found in E. c. carotovora 15, where the evf gene is under the control of Hor, a regulator of genes that are involved in plant pathogenesis 36 .…”