Pseudomonas Donghuensis HYS Pantothenic Acid Contributes To Pathogenicity Toward Caenorhabditis Elegans By 7-Hydroxytropolone

Pseudomonas donghuensis HYS is an environmental microorganism isolated from the water of Donghu Lake in Wuhan.Many strains of the same genus have been found all over the world.The HYS strain has the ability to produce siderophores.Under the same culture conditions,it shows stronger toxicity to C.elegans than human opportunistic pathogens Pseudomonas aeruginosa PAO1 and PA14.At present,there are few studies on the toxicity mechanism of HYS.There is evidence in previous studies that the Cbr/Crc pathways play a role in HYS toxity,but it is not the only toxic pathway,implying the existence of other pathogenic mechanisms.Pseudomonas donghuensis HYS produces high level of7-Hydroxytropoone(7-HT).7-HT is able to chelate iron,and has anti-fungal,anti-bacterial activities.previous studies show that addition 7-HT to NGM medium would change the lifespan of C.elegans.However,whether 7-HT can act as a virulence factor remains to be studied.1.determine whether 7-Hydroxytropolone(7-HT)in HYS is a toxic factor for C.elegans.The 7-HT synthesis gene was knocked out by homologous recombination,and a 7-HT synthesis defective strain?orf9 was constructed.This study tested the toxicity of7-HT to nematodes from genetic aspects.On the one hand,the Pseudomonas-worms interaction model was used to test the chronic lethal toxicity of the?orf9.The LT₅₀ of the C.elegans increased from 3.43±0.12 days to 5.03±0.20 days,when treated with HYS or?orf9.Suggesting 7-HT defective strain is less toxic than HYS to C.elegans.The?orf9/orf9 complement strain was constructed and tested,the LT₅₀ of feeding C.elegans was restored to 3.81±0.12 days.On the other hand,the 7-HT extract was obtained by separation and purification,and 50?M 7-HT extract was added to the?orf9 strain,which can restore the C.elegans LT₅₀ to 3.96±0.17 days,and the toxicity is significantly restored.At the same time,ferrous ion can be used as a regulating factor of 7-HT to affect the toxicity to nematodes.After adding 30?M Fe SO4,the LT₅₀ of feeding nematodes increased from 3.55±0.12 days to 4.33±0.15 days.These evidences indicate that 7-HT can be used as a bacterial virulence factor to participate in the chronic lethal effect of P.donghuenesis HYS on C.elegans.2.Testing the relationship between the synthesis of pantothenic acid in HYS and its toxicity to C.elegans.In this study,through the third-generation genome sequencing technology,a fine map of the entire HYS genome was obtained,which supplemented missing regions of some gene sequences in the previous study.In order to obtain more toxicity-related genes,we constructed a random transposon insertion mutation library of HYS.A mutant strain with greatly reduced repellency was isolated through the repellent experiment of nematodes.Through bioinformatic analysis,it is found that the gene is highly homologous to pan B.To study the role of pan B in toxitity to C.elegans.Firstly,the?pan B-deficient strain of HYS was obtained by homologous recombination.The growth inhibition test on the medium was used to detect that the growth of?pan B and found that was inhibited.However the growth rate of?pan B could be restored by complementation.When 10?M or100?M pantothenic acid was added to culture the?pan B mutant,the growth of the?pan B mutant could also be restored to normal level.In this study,the effects of pantothenic acid synthesis on the toxicity of HYS to nematodes were tested through genetical aprroaches.Through bacteria-nematode interaction model testing,it was found that the LT₅₀ of?pan B fed to nematodes could reach 8.96±0.25 days,which was 5.5 days longer than that of wild type.By replenishing the pan B gene,the LT₅₀ of feeding nematodes can be restored to 3.51±0.09 days.By adding 10?M or 100?M pantothenic acid,the nematode LT₅₀ can be restored to about6.9 days and 3.9 days,respectively.In addition,detection of genes in the pathway of pantothenic acid synthesis,such as?pan C and?pyd X,also reduced the toxicity of HYS to nematodes.This result indicates that the pantothenic acid synthesis pathway plays an important role in nematode toxicity of HYS.3.It is determined that the synthesis of pantothenic acid in HYS is related to the synthesis of 7-HT and pyoverdine.Some studies have shown that there is a certain correlation between pantothenic acid and siderophore synthesis in fungi.In order to study the effect of pantothenic acid synthesis system on 7-HT siderophore production in HYS,we used CAS-NGM plate to detect the siderophores produced by?pan B strain.The fluorescent siderophore pyoverdine and the non-fluorescent siderophore 7-HT produced in HYS,combined with the observation under natural light and ultraviolet light,found that the production of both siderophores in the pantothenic acid synthetic mutants was limited.The transcription levels of the key gene orf9 for 7-HT synthesis and the regulatory gene orf1 were all down-regulated in the?pan B strain.The production of 7-HT was verified by separating and purifying on NGM medium,and detecting the characteristic absorption peaks at 330 nm and 392 nm.Both the pan B gene complemented and the addition of exogenous pantothenic acid by genetics can restore the production of 7-HT,indicating that pantothenic acid synthesis mutations in HYS strains will limit the production of 7-HT,which will affect the toxicity of HYS to nematodes.4.Tests whether the conservative virulence factors in other strains affect.Through the toxicity of HYS to C.elegans bioinformatic analysis,there are many conservative virulence genes in Pseudomonas of other species are present in HYS genome.Mutant strains were obtained by knocking out related genes,and the pathogenic phenotypes of protease,hydrocyanic acid detection,T6SS system and motility were detected using nematode pathogenic models.However,no one system has an impact on the nematode toxicity of HYS.In addition,our preliminary data showed detected that the transcriptional expression of scot1 gene was regulated by pantothenic acid through fluorescence quantitative technology,and the toxicity of the scot1 mutant to nematodes was significantly reduced compared of HYS.On the other hand,the accumulation of phenylacetic acid in pantothenic acid synthetic mutants was detected by HPLC,which verified that the accumulation of phenylacetic acid can also reduce the toxicity of HYS to nematodes.These studies indicate that in HYS strains,apart from pantothenic acid which causes nematode toxicity through 7-HT,there are still other toxic factors that have not yet been discovered.In summary,this study tested the toxic effects of 7-HT in HYS on nematodes through genetics and biochemistry.Screening of transposon insertion mutation libraries revealed that the pantothenic acid synthesis system is closely related to nematode toxicity.Verifying that the pantothenic acid synthesis system can participate in the toxic effects on nematodes through the synthesis of siderophores;bioinformatics analysis has expanded the nematode toxicity factors in other HYS.It is preliminarily proved that pantothenic acid can participate in the chronic lethal effect of nematodes through 7-HT,which also provides a direction for the improvement of other toxic factors in HYS.