| The development of simple animal models is very important for the study of host-pathogen interactions.Caenorhabditis elegans and Drosophila melanogaster possess many attractive features,such as short life span,easy cultivation and maintenance,and genetic tractability,making them the appropriate systems for investigating interactions between host and animal pathogens.Currently,they are utilized to identify the virulence factors,pathogenetic mechanisms,host-pathogen interactions,biofilm formation and drug resistance of bacteria such as Salmonella typhimurium,Pseudomonas aeruginosa,Staphylococcus aureus,and so on.Listeria monocytogenes is non-spore-forming,gram-positive pathogen that is ubiquitous in nature.It can cause listeriosis if people and animals ingest the food that contaminated L.monocytogenes,giving rise to serious invasive infections including meningitis,osteomyelitis,myocarditis,miscarriage and puerperal infection.With a mean mortality rate in humans of 20 to 30%or higher despite early antibiotic treatment,the World Health Organization(WHO)make it as one of the important foodborne pathogens.Several research laboratories have generated some contradictory results on Listeria’s ability to kill C.elegans,making additional interaction studies more attractive.PrfA(Positive regulatory factor A)can regulate transcription of the majority of virulence gene and play an important role in the infection of the host.Deletion of the prfA gene will significantly attenuate the virulence of the strain.In order to examine the pathogenicity of L.monocytogenes for C.elegans and Drosophila,we carried out a series of killing assays in a systematic manner using different Listeria strains(the wild-type EGDe and its three prfA mutation).Bacillus cereus is Gram-positive bacteria,aerobic but can grow well under anaerobic conditions,widely distributed in soil,air,dust,sewage,animal intestines and various cooked foods.According to China’s food contamination and foodborne disease surveillance network statistics,food poisoning caused by B.cereus accounted for 4.0%of all incidents of bacterial food poisoning.In this study,a B.cereus that isolated from laboratory was identified by morphological and biochemical methods.Its toxicity was studied by C.elegans and Drosophila melanogaster.1.Listeria monocytogenes is not pathogenic to Caenorhabditis elegansIn order to examine the pathogenicity of L.monocytogenes in soil nematode C.elegans,the development period,life pan and brood size of C.elegans were tested when fed on a L.monocytogenes wild type strain EGDe,an attenuated strain △prfA and a highly virulent strain +prfA*.Two nonpathogenic bacteria,Escherichia coli OP50 and Listeria innocua,were chosen as the control.Our results show that compared to OP50,the development period and life span of C.elegans were significantly extended(P≤0.05)when fed on L.innocua,L.monocytogenes wild-type EGDe and its derived PrfA mutation strains,although the brood size was slightly decreased.Furthermore,a large number of live bacteria cells were recovered from the worms’ surface and digestive tract,but only few bacteria were detected in the worms’ excrements.In summary,L.monocytogenes is not pathogenic to C.elegans.Thus,C.elegans is not a good model to study Listeria pathogenesis.C.elegans can feed on L.monocytogenes cells,which would facilitate L.monocytogenes spreading in the soil environment.Meanwhile,in order to study whether the environment of the C.elegans impact the survival of L.monocytogenes,we fed the C.elegans on wild-type EGDe and △prfA,isolated bacteria from worms’ excrements and fed the C.elegans again.Such operation was repeated 10 generations.At last the bacteria were pick up and the gene of sigB was sequenced.SigB is a major stress response factor in many gram-positive,food-borne pathogens,which can be activated by many stress conditions such as high temperature,low temperature,low pH,oxidizing environment,high osmolarity,bile acid salts and other environmental variation.Our results showed there were three clones with two mutations in the SigB of the wild-type EGDe:one is phenylalanine replaced by leucine,and another is histidine replaced by tyrosine.However,the prediction tertiary structure of these mutant SigB protein was not changed.ForAprfA,five clones with arginine replaced by lysine were found in the amino acid sequence of SigB.Tertiary structure prediction showed that the mutation caused a great change in protein structure of SigB,which might change the protein function.The mutation in amino acid sequence of SigB may be the result of bacterial adjustments to adapt the pressure in the intestine of C.elegans.The possible mechanism needs further study.2.Listeria monocytogenes is pathogenic to Drosophila melanogasterIn order to comprehensively understand the interaction of L.monocytogenes and Drosophila melanogaster,two ways(feeding and microinjection)were employed to infect Drosophila melanogaster by L.monocytogenes.Feeding method is simple and economical,but only few bacteria can infect Drosophila melanogaster by this way.Microinjection method requires special equipment and certain skills,but can get a high infection rate.Therefore,most pathogens infect Drosophila through microinjection method.In this study,we compared the pathogenicity of L.monocytogenes to Drosophila melanogaster using feeding and microinjection method.In the feeding experiments,L.monocytogenes wild type strain EGDe,an attenuated strain △prfA and a highly virulent strain +prfA*were cultured on the modified medium to feed Drosophila melanogaster W1118.The results show that compared with the Drosophila melanogaster which fed on the normal medium,the development period of the Drosophila which fed on the modified medium with L.monocytogenes was significantly longer and the brood size was significantly reduced(P<0.01),but the fresh weight of fly and pupae as well as the hatching rate of the pupae had not any change(P>0.05),while the infection rate of L.monocytogenes has no difference among different virulent strains,indicating the efficiency of L.monocytogenes to infect the Drosophila by feeding method is not obvious.However,when Drosophila was microinjected with the wild type strain EGDe,△prfA,+prfA*,△prfA can not infect the Drosophila efficiently,while the wild-type EGDe and +prfA*can infect the Drosophila efficiently and kill the Drosophila melanogaster.The survival time of Drosophila melanogaster microinjected by EGDe was(8.5±0.9)days,and the bacteria can reach to(1 ±0.2)X 1010 CFU after 72 hours,When the Drosophila melanogaste was microinjected by +prfA*,the survival time of Drosophila was(6.9±0.6)days,and the bacteria reach to(4.3±0.5)X 1010 CFU after 72 hours,These showed that L.monocytogenes can infect the Drosophila efficiently by microinjection method and the infection efficiency increased with enhanced virulence factors.The study results demonstrated that Drosophila can serve as host model to study the pathogenesis of L.monocytogenes by microinjection method but not by the feeding method.3.Isolation and identification of a pathogenic Bacillus cereusBacillus cereus is most commonly foodborne pathogenic microorganisms which detected in food.They bring about a serious imperil on food safety in China.In this study,we isolated and identified a B.cereus strain in the laboratory based on their morphological,physiological,biochemical and molecular biological characteristics.Colonies formed by this bacterium in LB were irregular and flat with an undulate margin and presented frosted glass shaped or waxy.They can form spores,but not spore crystal,are able to ferment glucose,but not mannitol,reduce nitrogen,and synthesize catalase enzyme,yet are unable to split indole from the amino acid tryptophan.Furthermore,the similarity of 16S rDNA was above 99%when compared with that of B.cereus ZM01,AY01 and L90 published in NCBI.The specific virulent genes of B.cereus including enterotoxin FM gene(entFM),non hemolytic enterotoxin Nhe gene(nheA,nheB and nheC),the rotation of the highly specific enzyme B subunit gene(gyrB),and cytotoxic K(cytK)gene were found in this isolated strain by PCR.Meanwhile,the pathogenicity of this isolated strain was tested using C.elegans and Drosophila as the infection model.The results showed that Drosophila died within 5-7 days after microinjected by the isolated strain and C.elegans N2 died within 50 hours after feeding this strain.These results show that this strain not only have highly toxicity,but also indicate that C.elegans and Drosophila can serve as a model to study the pathogenic mechanism of the bacteria. |
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