| Research BackgroundPathogen adhesion to host tissue components is a critical initial step of the process that leads to the establishment of infection.For this purpose,bacteria have evolved surface-exposed proteins that specifically interact with the host ECM fibrous proteins(such as laminin,FN,collagen,and elastin)that together with proteoglycans constitute the main ECM components.Human FN is among the most common targets for bacterial adhesins.Fibronectin is however a glycoprotein of the ECM with two forms:plasma and cellular FN.The latter plays a major role in cell adhesion,growth,migration,and differentiatio.Although similar in structure,chemical composition,and biological activities,plasma and cellular FN sequences differ in three regions,one located near the gelatin-binding site,while the others are near the heparin-binding sites of the molecule.For the syphilis agent,the ability to bind FN was reported over three decades ago by Peterson et al.In their early work,Fitzgerald et al.reported that T.pallidum adherence to coverslips coated with FN,laminin,collagen ? and ?,and that treponemal attachment to such components could be inhibited by prior pathogen exposure to IRS.Thomas et al.subsequently explored the specificity of this interaction.In those studies,it was shown that 1)attachment of T.pallidum to FN-coated slides was inhibited by antibodies directed against the cell-binding(also known as integrin-binding)domain of FN and,to a lesser extent,by antibodies targeting the heparin-binding domain,that 2)the tetrapeptide sequence RGDS(arg-gly-asp-ser)of the cell-binding domain was critical for T.pallidum binding to FN.After these initial reports the interest on T.pallidum adhesion to host ECM abated,until it was re-ignited by the sequencing of the T.pallidum Nichols strain genome,which allowed the use of in silico tools to identify T.pallidum putative surface-exposed proteins and,among them,the FN receptors of the syphilis spirochete.Work by Cameron et al.led to the identification of TP0155 and TP0483 proteins as FN receptors.Based on preliminary unpublished data by Patterson et al.,a similar role was reported for TP0136 by Brinkman et al.TP0136,initially annotated as a hypothetical protein of unknown function and subsequently shown by Brinkman et al.to bind human plasma FN,as well as its derivative super-FN(a combination of human plasma FN and recombinant human FN fragment ?-C mixed together to facilitate cross-linking of FN molecules into multimers resembling matrix fibrils)and,in minor extent,also laminin.Whether TP0136 would recognize also cellular FN in addition to the plasma isoform and which region recognize FN best was not investigated?Also suggestive of an important role of TP0136 in the pathogenesis of syphilis was the analysis of T.pallidum transcriptome performed by Smajs et al.on treponemes harvested at peak orchitis from the rabbit host.Results showed that during experimental infection TP0136 is highly expressed compared to most T.pallidum genes.However,whether TP0136 message level would differ in treponemes inoculated intradermally(ID)compared to treponemes grown intratesticularly(IT)and whether TP0136 transcription would vary over time in primary dermal lesions was not explored,TP0136 heterogeneity among two syphilis strains(Nichols and SS14)and among these syphilis isolates and two T.pallidum subsp.pertenue isolates(SamoaD and F),which cause the treponemal disease known as yaws,and T.paraluiscuniculi Cuniculi A strain,was also reported by Brinkman.This prompted us to further investigate the presence of antigenic variants of TP0136 in seven additional historical syphilis strains as well as 17 clinical strains of very recent isolation.Research purposesAdherence-mediated colonization plays an important role in pathogenesis of microbial infections,in particular those caused by extracellular pathogens responsible for multisystemic diseases,such as Treponema pallidum subsp.pallidum(T.pallidum),the agent of syphilis.Among T.pallidum putative outer membrane proteins(OMPs)currently identified as adhesins,TP0136 is known to bind human plasma fibronectin(FN)and its derivative super-FN.In a time where syphilis incidence is raising globally,our data on TP0136 activity and heterogeneity will help in the quest to identify suitable targets for development of a much needed vaccine against this important disease.Materials and Methods1.Skin lesion biopsies for quantification of TP0136 message were obtained from three rabbits experimentally infected with the Nichols Seattle strain.Rabbits were inoculated ID on ten sites on their clipped backs with 106 cells/site.2.TP0136 message quantification was performed according to real-time qPCR protocols that use a relative quantification approach with external standards and normalize the mRNA level of the treponemal target gene to the mRNA level of TP0574 gene(encoding the 47 kDa lipoprotein)in the same sample.3.Eight T.pallidum subsp.pallidum strains(Nichols Houston,Nichols Seattle,Nichols Dallas,Dal-1,MexicoA,Bal73-1,Seattle81-4,and SS14)were propagated in New Zealand White rabbits and treponemal DNA from 17 clinical strains collected from blood or cerebrospinal fluid(CSF)of 16 syphilis patients.Partial TP0136 ORF from the 8 T.pallidum subsp.pallidum strains for cloning into the pEXP-5-CT expression vector,and prior to sequencing,amplicons were cleaned form residual primers and dNTPs by using the Exo-SAP-IT reagent.Results were analyzed with BioEdit4.The recombinant proteins of the full length TP0136 from Nichols Houston(TP0136flH)and from Nichols Seattle(TP0136flS)were expressed for our studies.Their concentration evaluated using a bicinchoninic acid(BCA)assay kit.5.The recombinant proteins of TP0136 Fragment 1 and 2(TP0136F1/F2,identical in both the Houston and Seattle variants,and corresponding to the protein NH2-terminal and central regions,respectively),TP0136 Fragment 3 from Nichols Houston(TP0136F3H),and Nichols Seattle(TP0136F3S)were expressed for our studies.Their concentration evaluated using a BCA assay kit.6.All TP0136 recombinant proteins(two full length variants and four fragments,ranging from 100 pmol to 1,000 pmol)were tested in FN binding assays.7.The capacity of recombinant TP0136 proteins with either 2000 or 8000 nmol to inhibit T.pallidum adhesion to cellular and plasma FN was tested by Lab-Tek ? chamber.8.Anti-recombinant TP0136flH and TP0136flS antibodies were obtained by immunizing six adult NZW rabbits(three per antigen)with a total of 300 ?g of purified recombinant proteins per rabbit resuspended in TiterMax Gold Adjuvant.Reactivity of immune sera against all immunogens was tested by ELISA.9.Antibody-mediated inhibition of treponemal adherence to human plasma and cellular FN was tested by Lab-Tek ? chamber.Results1.On day 6,rabbits showed a diameter of 0.5 cm,clear boundary,hard red pimples.As time increases,papules gradually increased above the skin,and the color deepened.On day 19,the rash was increased to about 2.1 cm,the ulcer appeared about 0.2 cm.Lasts about 4-5 days,the rash becomes smaller,the color fades,and disappeared.2.Quantitative analysis of TP0136 mRNA expression in primary dermal lesions of rabbits infected with Nichols Seattle revealed that TP0136 transcription is elevated but steady during early lesion development(day 6-18 post-infection),to subsequently increase(day 21-30 post-infection).TP0136 message levels calculated at day 27 and 30 were statistically significant with respect to earlier time points,but not with respect to each other(F=5.164,P<0.001).TP0574 absolute message level,although not normalized,reflects the known evidence that total treponemal burden in experimental lesions steeply declines following day 15 post-infection,due to the host's adaptive immune response against T.pallidum.3.Predicted amino acid sequences of TP0136(without signal peptide)from eight historical T.pallidum strains(Nichols Houston,Nichols Seattle,Nichols Dallas,Dal-1,MexicoA,Bal73-1,Seattle81-4,and SS14)and 17 clinical isolates.Dal-1 sequence is identical to Nichols Seattle,while Bal73-1 and UW126C sequences are identical to Nichols Houston.Nichols Dallas is identical to Nichols Seattle with the exception of a 32 amino acid deletion.Twelve(out of 17)sequences from clinical isolates are identical to the SS14 sequence.UW125B sequence is identical to MexicoA.With the exception of 1 amino acid change,UW189B,UW259B,and UW279B sequences are all identical to Nichols Houston.Seattle81-4 sequence is unique to this strain.The Nichols Seattle TP0136 gene that causes a shift in the reading frame of the gene and early termination.The nucleotide sequence of the insertion was previously shown to contain the donor sites for the variable gene tprK.4.After dialysis,the purification Nichols Houston and Nichols Seattle TP0136 ORF protein(referred to TP0136flH and TP0136flS)were tested by SDS-PAGE and Western Blot.The molecular weight of TP0136flH and TP0136flS were 51.7KDa and 46.1KDa.After BCA assay,purified protein concentration of TP0136flH protein was 1312.4?g/ml,TP0136flS protein was 1074.2?g/ml.5.The molecular weight of TP0136F1 was 19.9 KDa,TP0136F2 was 15.8 KDa,TP0136F3H was 12.5KDa,and TP0136F3S was 8.3KDa.After BCA assay,purified protein concentration of TP0136F1 protein was 251.7?g/mL,TP0136F2 was 707.9 ?g/ML,TP0136F3H protein was1076.7 ?g/ml,TP0136F3S protein was 377.4 ?g/mL.6.When recombinant full-length TP0136 variants from the Houston and Seattle Nichols strains,as well as protein fragments derived from each variant were tested for their ability to bind FN.After factorial design analysis of variance,the main effect of factors,concentration factors of main effects and interactions of both are siganificantly binding to plasma FN(F=346.461,P<0.000;F=392.640,P<0.001;F=43.873,P<0.001)and cellular FN(F=552.241,P<0.000;F=773.098,P<0.001;F=54.849,P<0.001).Six recombinant proteins were found to significantly adhere to both FN compared to the negative control protein a70(P<0.05),and to bind to increasing amounts of plasma and cellular FN in a dose-dependent manner except TP0136F3H.Among the individual fragments tested,however,Fragment 1(shared by both TP0136 variants analyzed here)showed the highest affinity for both plasma and cellular FN.Interestingly,Fragment 3 derived from Nichols Houston only showed significant binding to plasma FN when used at the highest concentration.Significant binding to plasma FN was on the contrary detected when Fragment 3 derived from Nichols Seattle,shown to contain TprK-like sequences,was employed.Binding of recombinant TP0136flS,TP0136F2,TP0136F3H,and TP0136F3S proteins to cellular FN was significantly higher than to plasma FN.7.The assay performed to measure the ability of recombinant TP0136 proteins to inhibit T.pallidum attachment to FN.After factorial design analysis of variance,the main effect of factors,concentration factors of main effects and interactions of both siganificantly inhibit T.pallidum attachment to plasma FN(F=573.058,P<0.001;F=174.410,P<0.001;F=5.248,P<0.001)and cellular FN(F=95.685,P<0.000;F=205.275,P<0.001;F=17.132,P<0.000).The results showed that pre-incubation of FN with both full length recombinant proteins from Nichols Houston and Nichols Seattle,as well as with Fragments 1-3,significantly decreased the number of adherent treponemes to FN-coated slides(P<0.05).As expected,pre-incubation of FN with the negative control recombinant ?70 did not significantly decrease treponemal attachment compared to PBS.Among the individual fragments tested,Fragment 1 limited treponemal adhesion most significantly than the other fragments(P<0.05).Dose-dependent inhibition of binding was seen for all recombinant proteins but Fragment 3 from Nichols Houston when tested with both cellular and plasma FN.Dose-dependence was also not seen with Fragment 3 from Nichols Seattle when tested with cellular FN.8.Following immunization with TP0136flH and TP0136flS,analysis of reactivity of rabbit sera against recombinant full-length proteins and fragments.After factorial design analysis of variance,the main effect of factors,concentration factors of main effects and interactions of both siganificantly against recombinant full-length proteins and fragments(F=1555.268,P<0.000;F=19.144,P<0.001;F=641.447,P<0.000).Results showed that the serum raised against TP0136flH recognized all recombinant TP0136 proteins except TP0136F3S(P<0.05).Vice versa,anti-TP0136flS had no reactivity against TP0136F3H(P<0.05).9.ANOVA results showed the serum raised against TP0136flH and TP0136flS siganificantly inhibit T.pallidum attachment to plasma FN(F=20.170,P<0.001)and cellular FN(F=22.216,P<0.001).Compared to NRS,both anti-TP0136flH and TP0136flS immune sera showed a significant ability to inhibit treponemal attachment to plasma FN(P<0.05)and cellular FN(P<0.05)-coated surfaces.ConclusionOur data revealed the existence of at least seven TP0136 antigenic variants among the examined historical strains and clinical isolates,showed that TP0136 binds more efficiently cellular FN than plasma FN,and that TP0136 conserved NH2-terminus is primarily responsible for FN binding,even though binding activity could be detected for all examined fragments.Furthermore,message quantification experiments supported that TP0136 transcription increases over time in experimental primary lesions in parallel to the host immune pressure on the pathogen.Altogether these data expand our previous knowledge on T.pallidum TP0136 function and suggest an important role for this protein in the pathogenesis of syphilis. |
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