Study Of The Toxicity Of Bacillus Cereus On Silkworm(Bombyx Mori) And The Relevant Proteome

Bacillus cereus,a Gram-positive bacterium,is an aerobic or facultative anaerobic type.Given differences in growth environments,the different contents of the plasmid and the toxicity factors.Some B.cereus may be added as probiotics to food and feed in the animal husbandry and food industry,whereas some strains are conditional pathogens,which are potentially hazardous to food health and environment.Twenty-six strains of bacteria were obtained from a local hospital in Zhenjiang City(Jiangsu Province,China)by random sampling previously in our lab.The most pathogenic strain was identified as Bacillus cereus through toxicity assessment of silkworms.The pathological changes caused by the infection were observed by Histopathological examination.The differential protein spots of silkworms in the treatment group and control group were detected by two-dimensional electrophoresis.This study evaluated toxicity of environmental pathogenic microorganisms and provided theoretical basis for the pathogenicity of the microorganisms at the molecular level,not only for silkworms but also for other organism.The main research results are listed as follows:1.When it comed to the individual level,we injected 5?L of Bacillus cereus bacterium suspension into the hemolymph which is located on the conjuctivum of the Bombyx mori.In contrast with those silkworms that were injected with nomal saline,it turned out that all five instar larvae that were injected with the Bacillus cereus bacterium suspension died within 12 h after the injection.After bacterial infection,symptoms observed on silkworms were similar to those occurred on human patients,including vomiting and diarrhea,the majority of the larvae started ceasing taking food,they moved slower and began to swell,part of the larval heads became green and inflated.Through the dissection,we observed that the silkworms mulberry leaves were all distributed on the upper half of the midgut part,while the midgut turned apparently thinner,the unique folding structure disappeared,the ektexine got thinner and partly dissolved.Twelve hours later,all of the silkworm larvae died,the body blackened slowly and became soft while some larval limbs become stiff after the body swelled,the anatomical midgut tissue dissolved completely as well.Twenty-four hours after the injection,the larval body became all black,followed with the swollen heads and the outflow of black liquid.2.When it comed to the tissue level,we utilized the histological section tenique to observe the continuous pathological changes of the midgut tissues which were injected with the Bacillus cereus bacterium suspension,via the microscopic examination,in different periods.We found that the nuclei of the midgut tissue didn't change distinctly,but the pillar cells decreased in amount and size,the obvious folding structure of the midgut disappeared and the ektexine turned thinner,gradually cracked,eventually dissolved.Some evident lesions such as midgut-tissue-dissolving were detected both in histological section and individual level,which illustrated that this bacterium did huge harm to silkworm's tissue and organ,and the severe injury and lesion of the midgut tissue maybe the direct reason that directed the silkworm's death.3.When it comed to the molecular level,we extracted five instar larval total protein at 6h from the treatment group and control group and used two dimensional gel electrophoresis to find out proteins that have wide differences in expression quantity.In the end,we screened 5 different protein spots via the mass spectrometry and the bio-information retrieval analysis.According to the biological function analysis,down-regulated protein ribonuclease is concerned with the metabolic capacity and neurotransmitter system of the parasitifer.The low molecular mass 30 kD from up-regulated protein is related to the relevant immunized response,anti-inflammatory reaction and apoptosis or other reactions in the parasitifer.Proteosome functioned mainly in controlling over embryo life period and degradation of the extracellular pertains.14-3-3zeta is the inhibitor of the apoptosis.In order to verify the consistency between these genes' expression quantity changes and protein level,we extracted silkworm's adipose tissue at 1,2,4,6,8,10 h to conduct the PCR test,and turns out that changes of silkworm's gene expression quantity in transcriptional level is consistent with that in protein level.4.In addition,to study the Bacillus cereus toxity against the midgut tissue,in the molecular level,we extracted colon total protein from the experimental group and the control group at 6h,screened 13 different protein spots via the combination of mass spectrometry and two dimensional gel electrophoresis in the same way.By means of biological analysis we know that the up-regulated proteins are tubulin,acetylhydrolase,glutamine synthetase and 14-3-3zeta,respectively functions in phagocytosis,metabolic detoxification,immune system and nervous system.The down-regulated proteins are blebbing ATP synthases,protein EF-domain and annexin,respectively functions in nerve conduction,biological membrane and stability of the physiological activities.And then in the gene level,we verified that the consequence was basically coincident.According to the research above,we entirely illustrated the pathological changes that Bacillus cereus affected on Bombyx mori in different periods,both in individual and molecular level,which offers theoretical basis to the prevention against this bacterium in hospital.Meanwhile,we screened some proteins that have distinct expression quantity from the silkworm's fat body and midgut tissue.These proteins are relevant to immunity and metabolism and we believe that they can cast the role of warning genes in the antibacterial agents' study,which can lay the foundation for the futher research.