Study On Mechanism Of Fibrinolytic System Activation And Larva Invasion By Trichinella Spiralis Enolase

Trichinella spiralis is an important foodborne parasitic nematode.Their adult worms parasitized in the small intestinal mucosa of its host.The critical variable in the establishment of T.spiralis infection is whether the larvae invade the small intestinal mucosa to develop further.However,the mechanism of larval invasion is unclear to date.Morphological studies have shown that there is no tooth（spear）like structure in the oral orifice of the invading larvae.Therefore,the invasion of the intestinal mucosa by the larvae is probably mediated by the larval protease rather than a mechanical force.Enolase is not only a key enzyme in glycolysis,but also a multifunctional protein.Enolase plays an important role in the activation of the fibrinolytic system,muscle regeneration,cell stress,cancer cell metastasis and infection.Studies have shown that enolase participates in infection process,and plays a key role during the infectious organisms infecting the host.Our previous LC-MS/MS results of infective larval proteins showed that T.spiralis enolase（Ts ENO）was present at very high levels after co-culture with intestinal epithelial cells（IECs）in vitro.The relative transcription level of the T.spiralis enolase gene（Ts-eno）was upregulated in larvae cultured with IECs.High expression of Ts ENO was also identified in a screen of T.spiralis intestinal infective larvae（IIL）surface proteins.Previous studies indicate that Ts ENO may promote larval penetration of the tissue barrier during invasion,with an unknown moluclar mechanism.Up to now,enolase binded with plasminogen（PLG）has been observed during the infection process of various pathogens（bacteria,protozoa,helminthes and insects）.Enolase promoting the activation of the fibrinolytic system and accelerating the pathogen invasion.The fibrinolytic system not only exists in the blood vessels,but also widely distributed in the tissue fluid.Under physiological conditions,due to mutual restriction between PLG activators（PAs）,PLG activator inhibitor type-1（PAI-1）andα₂-Antiplasmin（α₂-AP）,the fibrinolytic system maintains a dynamic balance between coagulation and fibrinolytic.It is suggested that enolase serves as a PLG receptor in the process of infection,breaking the balance and utilizes fibrinolytic system to degradate the extracellular matrix（ECM）,facilating the larval invasion,but the molecular mechanism is still unclear.Does Ts ENO act as a receptor for host PLG?Can Ts ENO bind and interact with PLG in host tissue fluid?What is the molecular mechanism that Ts ENO breaks the equilibrium state of the fibrinolytic system and promotes its activation?Does Ts ENO facilitate larval invasion of the host?These are the key scientific questions that I want to answered through this study.Bioinformatics technology was applied to analyze the biological characteristics of Ts ENO in this study.The 3D molecular model of Ts ENO was constructed by I-TASSER server,and docked with human PLG（PDB ID:4DUR）by ZDOCK.The molecular mechanism of Ts ENO-PLG interaction was analyzed by using a series of bioinformatics software tools.Recombinant Ts ENO（r Ts ENO）and site-mutated r Ts ENO（M-r Ts ENO）were expressed and purified in vitro based on the systematic bioinformatics studies.Anti-r Ts ENO and anti-M-r Ts ENO serum was prepared respectively.The relative transcription level of Ts-eno and the localization of Ts ENO in T.spiralis different developmental stages were analyzed.The binding ability of r Ts ENO to small intestinal mucosa and PLG was identified,respectively.The enzyme activity of r Ts ENO was analyzed,and the molecular mechanism of the interaction between Ts ENO and PLG was verified by PLG activation experiment and competitive combination experiment.The scientific hypothesis that Ts ENO activating fibrinolytic system and facilitating larval invasion,was verified by larvae-IECs invasion model,RNA interference（RNAi）and animal experiments,respectively.Materials and methods1.Trichinella spiralis,experimental animals,expression plasmids and cell linesT.spiralis（T1）isolated strain was separated from infected pigs in Nanyang city of Henan province,and preserved by Kunming mice in our laboratory.The experimental animals（BALB/c mice,SPF grade,6 weeks old）were purchased from experimental animal center of Henan province.The expression vectors p QE-80L and E.coli BL21 were cryopreserved at-80℃in our laboratory.The intestinal epithelial cells（IECs）were separated from fetal mouse small intestine and primary cultured in our laboratory.2.Molecular characteristics of Ts ENO,the analyzation and verification of its PLG binding abilityThe molecular biological characteristics of Ts ENO（XP＿003371233）were systemically analyzed by bioinformatic tools.Signal peptide was predicted and analyzed by Signal P,and the3D molecular model of Ts ENO was constructed by I-TASSER.The quality of the 3D molecular model of Ts ENO was evaluated and verified by SAVES and Super Pose.Molecular docking between Ts ENO and PLG（PDB ID:4DUR）was performed by ZDOCK,and the results of molecular docking were analyzed by VMD,Lig Plot+（DIMPLOT）and PDBe PISA.After the key residues were identified,I-TASSER was used to construct the M-Ts ENO molecular model of site-directed mutation,and the effects of key residues mutations were compared and analyzed.The recombinant p QE-80L/Ts ENO and p QE-80L/M-Ts ENO were transformed into competent E.coli BL21（DE3）for prokaryotic expression,respectively.Far-western blot and ELISA were used to detect the binding ability between r Ts ENO（or M-r Ts ENO）and PLG.The?-ACA competitive combination test was utilized to validate the effect of key residues mutations on binding.3.The molecular mechanism of fibrinolytic system activation by Ts ENO and its experimental verificationThe interaction between Ts ENO（or M-Ts ENO）and the key tryptophan residue 761（Trp761）,which determine the activity of PLG,was analyzed by VMD,Lig Plot+（DIMPLOT）and PDBe PISA.The changes of interaction area,accessible surface area(A_ASA),solvation energy（ΔⁱG）,hydrogen bond,disulfide bond and hydrophobic interactions were calculated and analysed during the interaction between Trp761 and Ts ENO（or M-r Ts ENO）.The enzyme activity of r Ts ENO and M-r Ts ENO after purification was determined.The PLG activation assay was used to study the combination and activation of PLG by r Ts ENO（or M-r Ts ENO）,and to confirm the influences of PAs on PLG activation and PLM production.The molecular mechanism of the interaction between Ts ENO and the key tryptophan residues（Trp761）of PLG which promotes and activates fibrinolytic system was verified.4.The function of Ts ENO in the process of T.spiralis larval invasionReal-time PCR was used to analyze the transcriptional level of Ts-eno at different stages of T.spiralis,and IFA was used to detect the localization of Ts ENO in T.spiralis.The combination of ML ES antigen and PLG was detected by Far-Western blot.The specific binding between r Ts ENO and intestinal mucosal tissue sections of mice was detected by using IFA.The promoting and inhibiting effects on larval invasion were determined with T.spiralis larvae-IECs invasion model in vitro by using r Ts ENO and anti-r Ts ENO serum,respecitively.Specific small interfering RNA（si RNA）that specifically targeted at Ts-eno was designed and synthesized.The si RNA was introduced into the T.spiralis larvae of by electroporation.Western blot was used to detect the interference of si RNA on the expression level of Ts ENO.In order to verify the function of Ts ENO in larval invasion,sixty BABL/c mice were challenged by T.spiralis larvae（300 larvae per mouse）after immunized with r Ts ENO.The 5 d intestinal adult worms and 42 d muscle larvae were collected,and the recovery number,worm reduction rate,larvae per gram and the reproductive capacity index were statistically analyzed.5.Statistical analysisIBM Statistical Product and Service Solutions 21.0（IBM SPSS 21.0）was used for statistical description,hypothesis test and chart drawing of the research data,and the significant level was set asα=0.05.Results1.Molecular characteristics of Ts ENO and the key residues for PLG bindingThe length of Ts ENO without signal peptide was 473 aa,and its molecular weight was51.95 k Da.The sequence consistency between Ts ENO and common parasites enolase ranged from 62.09%to 98.91%.Ts ENO possessed characteristic enolas family MOTIF,and highly conserved structural domains,metal-binding sites,activation sites,and substrates binding pockets.According to the Verify 3D results,82.24%of the residues had a score of≥0.2 in the 3D-1D profile.The overall quality factor was 90.753 by the ERRAT results.The Ramachandran plot revealed that 98.8%of the Ts ENO residues were in allowed regions.The multiple three-dimensional structures alignment by Super Pose showed that the structure of Ts ENO was highly consistent with that of the five resolved crystal parasite enolase structures（3QTP,1OEP,4G7F,3OTR and 5WRO）.The largest root mean square deviation（RMSD）among backbones and all atoms were only 2.03 and 2.18,respectively.The ZDOCK protein-protein docking results indicated that Ts ENO and PLG exhibited surface complementarity in the interface area.These four lysine residues（90,289,291 and 300）of Ts ENO were identified as active residues for PLG binding.Notably,one of the key lysine residues（90）binds to Ser383（located in KR4 of PLG）by forming hydrogen bonds.Ts ENO and M-Ts ENO exhibited similar 3D structures,and the local RMSD between the backbones and the global RMSD were 2.63 and 3.30,respectively.However,when M-Ts ENO was docked with PLG,only one lysine residue（Lys116）of M-Ts ENO appeared on the interaction surface.As confirmed by Far-Western blot analysis,purified r Ts ENO and M-r Ts ENO were specifically recognized by the anti-PLG antibodies.The recognition bands of M-r Ts ENO were significantly weaker than those of r Ts ENO under the same conditions.Compared with r Ts ENO,M-r Ts ENO showed a marked loss of PLG binding ability,with a decrease ranging from 20.04%to 45.37%.Theε-ACA at different concentrations could competitive with r Ts ENO（or M-r Ts ENO）when binding PLG,and the competitive inhibition ofε-ACA showed an ascending linear trend along with the concentration increasing(F_{r Ts ENO}=3532.392,F_{M-r Ts ENO}=3499.730,P<0.01).The competitive inhibitory effect ofε-ACA on r Ts ENO was significantly higher than that of M-r Ts ENO at different concentrations（t=3.411,P<0.05）.When the concentration was25 mmol/L,the competitive inhibition rates ofε-ACA on r Ts ENO and M-r Ts ENO were 64.25%and 33%,respectively.2.Mechanism analysis and verification of fibrinolytic system promoting and activating by Ts ENOThe key residue Trp761（PLG）could bind with Glu303（Ts ENO）by hydrophobic interaction,and the distance between them was 7.28 1.60 in Ts ENO-PLG complex.The A_BSA andΔⁱG of Trp761 were 29.55 ² and 0.47 kcal/mol,and the A_BSA andΔⁱG of Glu303were 33.97 ² and-0.24 kcal/mol.The interaction analysis of M-Ts ENO-PLG complex revealed that Trp761 had hydrophobic interactions with Lys90 and Ser91 of M-Ts ENO（Glu303Ala）,and hydrophobic interactions with Phe48 and Tyr93 of M-Ts ENO（Lys90Ala,Lys289Ala,Lys291Ala and Lys300Ala）,respectively.The distance between Trp761 and Lys90,Trp761 and Ser91 were 5.35 0.43 and 7.27 1.41,respectively.The distance between Trp761 and Phe48,Trp761 and Tyr93 were 5.57 0.51 and 6.76 0.84,respectively.The one-way ANOVA results showed that the difference of the distances among Trp761 and its interacting residues was statistically significant（F=10.546,P<0.01）.According to the analysis of the spatial geometry among the interacting residues,Trp761 has the largest spatial displacement when PLG combined and interacted with Ts ENO（7.28）.The movement of Trp761 results in PLG substrate binding pockets（composed of catalytic residues His603,Asp646 and Ser741）being exposed,which is conducive to the activation of PLG.The glycolytic active sites of Ts ENO（Glu246,Lys382）did not appear on the interaction surface either the complex of Ts ENO-PLG or M-Ts ENO-PLG.The specific enzyme activity of r Ts ENO and M-r Ts ENO were 36.77 20.22 U/mg and34.63 16.86 U/mg when catalyzed forward reaction（2-PGA?PEP）,respectively.When the backward reaction was catalyzed,the enzyme activity of r Ts ENO and M-r Ts ENO were16.73 10.70 U/mg and 17.20 13.82 U/mg.When r Ts ENO and M-r Ts ENO catalyzed the forward reaction（2-PGA?PEP）,their Michaelis constant（Km）value were 0.78 mmol/L and0.80 mmol/L,and their maximum velocity（Vmax）were 0.42μmol/min/mg and 0.40μmol/min/mg,respectively.The Mg²⁺buffer system promotes r Ts ENO and M-r Ts ENO to achieve their optimal enzymatic catalytic activity,while Zn²⁺,Mn²⁺,Fe²⁺and Cu²⁺could not promote the enzymatic reaction as much as Mg²⁺.The K⁺,Ni²⁺,Al³⁺,Ca²⁺and Li⁺have very weak initiation effect on the enzymatic reaction,and the Cr³⁺could completely inhibit the enzyme activities of r Ts ENO and M-r Ts ENO.Neither PLG nor t-PA can produce abundant PLM by interacting with r Ts ENO,M-r Ts ENO,soluable antigen or ES.There were no statistic differences of OD₄₀₅ value among different groups(F_PLG=1.355,F_t-PA=1.013,P>0.05).When PLG and t-PA co-exist in the same reaction system,t-PA can activate PLG and produce PLM.The OD₄₀₅ values were significantly difference after different proteins were added into the reaction system(F_PLG+t-PA=344.875,P<0.05).Both of the r Ts ENO,M-Ts ENO,soluble antigen and ES can promote the activation of PLG and increase the production of PLM,and the soluble antigen possessed the highest promotion power,then following with r Ts ENO,M-Ts ENO and ES,orderly.3.The role of Ts ENO in the process of larvae invade into IECs monolayer and intestinal mucosaThe q PCR results revealed that Ts-eno transcription was observed at all stages of T.spiralis development.The Ts-eno relative transcriptional level in ML was significantly higher than that in other developmental stages（IIL,3 d AW,6 d AW and NBL）（F=7.878,P<0.05）.IFA of intact T.spiralis worms and tissue sections detected by anti-r Ts ENO serum showed that bright green immunofluorescence staining was observed spread over the T.spiralis body in all life cycle stages.The Ts ENO was more concentrated in stichosome,epicuticle and embryos than the other parts of the worm.Far-Western blot confirmed that the ES antigen included a 52 k Da protein specifically bound to PLG.The IFA results showed that r Ts ENO could specifically bind to the intestinal mucosal tissue of mice.Larvae-IECs invasion assay showed that the number of invasive larvae in r Ts ENO group were significantly higher than that in PBS group at different concentrations(t₂=4.564,t₄=7.920,t₆=24.588,t₈=19.100,t₁₀=30.237,P<0.05).The invasion rate of larvae increased with the increase of r Ts ENO concentration（F=410.744,P<0.01）.The inhibition rate of anti-r Ts ENO serum on larval invasion was as high as 51.26%,and possessed a decending linear trend along with the serum dilution ratio increasing（F=557.494,P<0.05）.The expression of Ts ENO was inhibited up to 53.56%by si RNA-97,result in an inhibition rate of larvae invasion as 49.82%.After immunized with r Ts ENO,the worm reduction rates of 5 d adult worms and 42 d muscle larvae were 31.79%and 47.15%in BABL/c mice,respectively.The LPG and RCI of r Ts ENO group were lower than that of adjuvant group and PBS control group(F_LPG=39.375,F_RCI=46.533,P<0.01).Conclusions1.Four lysine residues（K90,K289,K291,and K300）of Ts ENO were identified to play a key role in the binding of Ts ENO-PLG.2.After binding with PLG by key lysine residues,Ts ENO interacts with tryptophan 761（Trp761）of PLG by its glutamic acid 303（Glu303）.The physical force generated by the interaction can push Trp761 away from its original position,which result in PLG substrate binding pockets（composed of catalytic residues His603,Asp646 and Ser741）being exposed,and facilating PLG activation.The complex of Ts ENO-PLG can promote the transformation of PLG into PLM.Once PLM appears,it can further accelerate the activation of PLG by its positive feedback regulation,then the dynamic equilibrium of host’s fibrinolytic system will be broken.The Ts ENO active sites（Glu246,Lys382）that catalyze 2-PGA PEP reaction have nothing to do with PLG activation.3.Recombinant Ts ENO can promote larvae invade into the IECs monolayer in vitro,specifically bind to the PLG in small intestinal mucosa,and induce specific immune protection in immunized BALB/c mice.These findings confirm that Ts ENO plays an important role in the process of larval invasion.The host’s fibrinolytic system activation by Ts ENO promotes PLM formation and ECM degradation,which result in larvae invasion facilitating.Ts ENO is a key protein for invasion and can be used as a candidate target for the novel anti-Trichinella vaccine/drug development.