A Proteomic Analysis In The Intracranial Angiostrongylus Cantonensis Larvae In The Different Hosts

BackgroundsAngiostrongylus cantonensis is a rat lung worm found in 1935 by Chen,which related to causing severe eosinophilic meningitis.Human get infected through the ingestion of raw or undercook snails or vegetables contaminated with infected L3 larvae.Patients can have serious encephalitis and sometimes may become ocular angiostrongyliasis with optic neuritis.Human Angiotrongyliasis have been detected throughout Southeast Asia,Pacific Ocean and some countries in South America and several outbreaks in epidemic areas have been reported in the last decade,especially in mainland China.Rodents are known to serve as its definitive host,whereas its intermediate hosts are mollusc including snails and slugs,and freshwater shrimp and frogs act as reservoir host with the ability of reserve different larval stages of the parasite that the parasite are not appropriate for the parasite to develop further.Humans and other mammalians were reported to be the accidental hosts of the parasite,ingestion of the L3 larvae leading to severe damages to the central nervous system(CNS).Currently,the explosive expansion of the rodents and snails in many places around the world has the potential to increase the transmission ofA.cantonensis.A.cantonensis has been considered a public health problem and cause great concerns.It has been reported that the development of intracranial A.eantonensis larvae in permissive hosts differs from that in non-permissive hosts.In both the permissive and non-permissive hosts,majority of the ingested infective larvae(L3)migrate to the central nervous system(CNS).In the CNS of permissive hosts(e.g,,rats),L3 develops into the fourth-and fifth-stage larvae(L4 and L5)via two molts.Then,they migrate to the pulmonary artery where they develop into adult worms.In contrast,L3 does not complete their life cycle in non-permissive hosts(e.g.,mice and humans)but remain in the CNS until death and thereby cause eosinophilic meningitis.Yet it is not known whether the differentially expressed proteins in the intracranial A.cantonensis larvae in rats(ILR)and the intracranial A.cantonensis larvae in mice(ILM)brains correspond to different developmental time points and pathogenicity in permissive and non-permissive hosts.Therefore,in this study we analyzed and characterized the differentially expressed proteins in the intracranial A.cantonensis larvae in permissive and non-permissive hosts.Differential proteomics is commonly used to select and identify differences and/or changes among proteins in the different parasitic stages to provide new targets and candidates for drug therapy and vaccines against parasite infection.Previously differential proteomics analysis was used to investigate proteins expressed in female and male adult A.cantonensis.Using proteomic analysis,a previous study reported 37 proteins in different A.cantonensiwdevelopmental stages.Furthermore,a comparison of the protein expression profiles in L3 and L5 A.cantonensis larvae revealed that in L3 the cytoskeleton and membrane proteins were up-regulated when compared to L5.Thus far,no study has determined the differential protein expression in the intracranial larvae of A.cantonensis in the permissive and non-permissive hostsby differential proteomics.In a previous study,we found different patterns of protein bands in ILR and ILM via sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE).This study was conducted to analyze and characterize the differentially expressed proteins in the intracranial A.cantonensis larvae in rats(ILR)and mice(ILM)brains by two-dimensional difference gel electrophoresis(2D-DIGE)and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS).In addition,proteomic analyses revealed that act-1 and act-2 were up-regulated in ILM compared to ILR,whereas dim-1 was down-regulated in ILM.These results indicate that the differentially expressed proteins,dim-1 and act-1,could be related to the development and pathogenicity of A.cantonensis in different hosts.The aim of the present study was to identify act-1?dim-1 in ILM and ILR to provide insights into the protein functions during larval development and pathogenicity in permissive and non-permissive hosts.Objectives1.A.cantonesis infection in rats and mice for twenty-first days and the larvae were separated and were to comparatively identify the most abundant and differentially expressed proteins in extracts from in the intracranial A.cantonensis larvae between permissive and non-permissive hosts.2.Analysis the differentially expressed proteins insights to understand the development and pathogenicity of A.cantonensis and the interaction of A.eantonensis with its host.Methods1.The establishment ofA.cantonesis infection animal model in rats and mice.Light microscopy observation of larval structures in rats and mice.1.5g pepsin artificial digestion liquid,37 ?were used to digest the whitejade snail that were infected by the Angiostrongylus,after 2 hours,80 mesh plug mesh filtration to remove tissue debris,to PBS wash filtrate 3 times,static natural precipitation collecting sediments in larvae,in which50 A.cantonesis infection stage larvae of the successful establishment of A.cantonesis infection of rat and mouse animal model.Rat infection model using SD rats BALB/c mice,model after observation of the two host life condition and infection of symptoms and signs of change;to 21dpi anatomy collection two host brains of larvae,scanning light microscopic observation,two host brain larval body length,width and the analysis of its differences.2.Protein extractionThe A.cantonensis larvae isolated from rats and mice were homogenized in 500 ?l lysis buffer,sonicated on ice-bath and centrifuged at 12,000 rpm for 60 min at 4?.The supernatant was transferred to a clean tube and mixed with four volumes of acetone.The mixture was kept overnight at-20?.The total protein concentration was determined using the Bradford method,and stored at-80 ? until further use.3.Isoelectric focusingBriefly,the extracted protein was dissolved in rehydration buffer and centrifuged at 15,000 rpm and 4? for 60 min to collect the supernatant.Protein concentration was determined by the Bradford method using a 2D Quant kit according to the manufacturer's instructions.Proteins were initially separated using an Ettan IPGphor Isoelectric Focusing system(GE Healthcare)and focused to their isoelectric points on a 24 cm(pH 4-7)Immobiline DryStrip(GE Healthcare).After the isoelectric focusing,the immobilized pH gradient(IPG)strips were equilibrated for 15 min in equilibration buffer containing 2%sodium dodecyl sulphate(SDS),50 mM Tris-Hel(pH 8.8),6 M urea,30%glycerol,and 200 mM DTT(freshly added before use,Amersham),followed by a second wash for 15 min with equilibration buffer containing 250 mM iodoacetamide(freshly added before use,Amersham).These SDS-PAGE-ready IPG strips were stored at-80 ?.4.SDS-PAGEFor the 2D SDS-PAGE,the IPG strips were place on 12.5%polyacrylamide gels and sealed with 1%(w/v)agarose.After electrophoresis,gently pry the two layers of glass,remove the gel and marked with cut away,analytical mass spectrometry compatible silver staining using silver nitrate staining.5.Gel analysisImages of gels were obtained from a gel scanner(Powerlookl100 UMAX)and analyzed using the PD Quest 7.1.0 software.The protein spots were detected by an automatic spot detection program,the spot volumes were calculated relative to the background and normalized,and the proteins in the two gels(permissive host and non-permissive host)were compared.Two protein spots were considered significantly different when the fold difference was more than 2.Therefore,any spot present on one gel but not on the other or any spot that was present in both gels but had a 2-fold or more difference in volumes were then selected and subjected to in-gel tryptic digestion.6.In-gel trypsin digestionThe differentially expressed protein spots were manually excised from the silver stained gels,fixed in NH4HCO3(pH8.0)for 20 min,washed twice with Milli-Q water,dehydrated in acetonitrile twice and vacuum dried in a Speed Vac(Thermo Savant)for 15 min.Next,the samples were incubated in 20 ng/?l trypsin(Sigma)at 37? over night.After enzymolysis,the samples were extracted with a solution containing 5%trifluoroacetic acid(TFA)/50%acetonitrile(ACN).7.MALDI-TOF MS analysisAbout 1 ?l of each trypsin digested protein was loaded onto a target platewith 1 ?l HCCA(Alpha cyano-4-hydroxy cinnamic acid)matrix,dried at room temperature,and analyzed using an ABI voyager-DE STR matrix-assisted laser desorption ionization-time of flight/time of flight(MALDI-TOF/TOF)proteomics analyzer mass spectrometer(Applied Biosystems,USA).8.Quantitative real-time PCRTotal RNA was extracted from intracranial larvae using the Trizol reagent(Invitrogen,USA),according to the manufacturers' instructions.RNA integrity and concentration were assessed by electrophoresis on a 1%(w/v)agarose gel,while RNA purity was determined based on the absorbance ratio at A260/A280 nm using the NanDrop2003(Thermo Scientific,USA).The RNA preparations were then stored at-80? until further use.Total RNA was used to synthesize the first-strand cDNA(Thermo scientific,Lithuania)according to the manufacturer's instructions,and then the cDNA was used as the template in real time PCR performed on an Applied Biosystem 7500 real-time PCR system using SYBR(?)Select Master Mix(Applied Biosystem,USA).Since the A.cantonensis database is not complete at present,we designed primers for the qPCR based on protein sequences of other nematodes.The primer sequences used for real-time PCR and PCR product sizes are listed in Table 1.The first internal transcribed spacers(ITS-1)of 18S rRNA in A.cantonensis(GenBank:GU587760)served as the reference gene.9.Database searchesProteins were identified by comparing the obtained amino acid sequences on the UniprotKB/SwissProt database by peptide mass fingerprint(PMF)in the Mascot database(http://www.matrixscience.com)and against the non-redundant NCBI database(http://www.ncbi.nlm.nih.gov/BLAST).The GO analyses of these predicted proteins revealed that some of the proteins potentially had important biological functions.10.Statistical analysisThe length and width of ILR and ILM at 21 dpi were measured through a microscope and analyzed using independent-samples T tests.The differences in act-1 and dim-1 expression between ILM and ILR were also assessed by independent-samples T tests.All data are expressed as the mean ± standard deviation(SD).Statistical analyses were performed using SPSS 13.0 and p<0.05 was considered as statistically significant.RESULTS1.Comparative proteomic analysis using 2-DETo identify differential protein expression in ILM and ILR,total proteins from both samples were independently fractioned on 2-DE and analyzed.About 650 spots were visualized on these gels by silver staining.Spots with more than 2-fold increase or decrease in their relative abundance were considered to represent differentially expressed proteins.Among these,less than 0.05%of the spots(29 spots)showed significant differential expression patterns between the two hosts.Most of these protein spots were located between pI 4-7,and their molecular weights ranged from 10 kDa to 170 kDa.Among the 29 differentially expressed protein spots,17 displayed abundant expression in ILM and 12 among them were more than 10-fold higher in ILM than in ILR.Among the remaining 12,six were up-regulated 10 times higher in ILR than in ILM.Some proteins had the isoelectric points(pI)and molecular weights(MW)predicted by the Mascot and NCBI databases.Figure 2 shows the amplified protein spots differentially expressed in ILM and ILR analyzed on 2-DE.2.Identification and interpretation of differentially expressed proteinsIn an attempt to better characterize the differentially expressed proteins in the intracranial larvae in rat and mouse brains,the differentially expressed proteinspots were excised and analyzed by MALDI-MS.All 29 protein spots that were differentially expressed were then identified through the Mascot Database using the peptide sequences obtained from the MS analysis.Twenty two proteins were successfully identified with high confidence scores(>95%),while 7 spots were unnamed proteins.Then,using the Gene Ontology(GO)database we found that the 22 identified proteins were involved in ATP catabolic process,ATP binding,positive regulation of multicellular organism growth,pharyngeal pumping,and reproduction.The differentially expressed proteins were mainly located in the membrane and cytoplasm.It should be noted that no nuclear genes of A.cantonensis is available in the NCBI nr database.Therefore,the peptide sequences we obtained from the MS analysis were compared with the C.elegans protein database.Among the proteins,three were successfully identified as dim-1,act-1,and act-2,which were associated with the Gene Ontology Biological Process terms:"ATP catabolic process" and "ATP binding".Act-1 and act-2 were up-regulated in ILM,and dim-1 was up-regulated in ILR.3.Quantitative real-time PCR of differentially expressed proteinsThe expression of act-1 and dim-1 were analyzed by qPCR using the first ITS-1 of A.cantonensis 18S rRNA as an internal control.Act-1 was expressed significantly higher in BALB/c mice(p<0.05);whereas,dim-1 displayed significantly higher expression in SD rats(p<0.01).These results were consistent with the respective protein levels observed by 2-DE analysis,indicating that the differentially expressed proteins were regulated at the transcriptional level.4.The length and width of A.cantonensis larvaeThe length and width of A.cantonensis larvae in ILR and ILM 21 dpi were measured through a microscope.Mean lengths of the female larvae in rats and mice were 11.049 ± 0.348 and 8.438 ± 0.371(p<0.001),respectively and meanlengths of the male larvae were 9.333 ± 0.404 and 6.909 ± 0.36(p<0.001),respectively.Mean width of the female larvae in the rats and mice 21 dpi were 0.116± 0.003 and 0.092 ± 0.005(p<0.001),respectively while that of male larvae were 0.104± 0.003 and 0.076 ± 0.003(p<0.001),respectively.Evidently,the larvae recovered from rats were significantly longer and wider than those from mice.Conclusions1.This study revealed differences in the proteomic profiles of ILR and ILM,the possible relationship between the differentially expressed proteins and their likely role in the development ofA.cantonensis larvae.In total,29 proteins spots with pI 4-7 and MW 10-170 kDa were expressed differentially in the intracranial larvae of A.cantonensis.Among the 29 differentially expressed protein spots,17 displayed abundant expression in ILM and 12 among them were more than 10-fold higher in ILM than in ILR.2.qPCR analysis of act-1 and dim-1(spot 29,spot 9)with the first ITS of A.cantonensis 18S rRNA as the reference gene were consistent with the findings of DIGE analysis,indicating that act-1 and dim-1(protein and protein 29 points 9 points)may play a role in the development and pathogenic mechanism of A.cantonensis and its interaction with hosts.