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The Chlamydia pneumoniae Adhesin Pmp21 Forms Oligomers with Adhesive Properties*

Open AccessPublished:August 22, 2016DOI:https://doi.org/10.1074/jbc.M116.728915

      Abstract

      Chlamydiae sp. are obligate intracellular pathogens that cause a variety of diseases in humans. The adhesion of Chlamydiae to the eukaryotic host cell is a pivotal step in pathogenesis. The adhesin family of polymorphic membrane proteins (Pmp) in Chlamydia pneumoniae consists of 21 members. Pmp21 binds to the epidermal growth factor receptor (EGFR). Pmps contain large numbers of FXXN (where X is any amino acid) and GGA(I/L/V) motifs. At least two of these motifs are crucial for adhesion by certain Pmp21 fragments. Here we describe how the two FXXN motifs in Pmp21-D (D-Wt), a domain of Pmp21, influence its self-interaction, folding, and adhesive capacities. Refolded D-Wt molecules form oligomers with high sedimentation values (8–85 S). These oligomers take the form of elongated protofibrils, which exhibit Thioflavin T fluorescence, like the amyloid protein fragment β42. A mutant version of Pmp21-D (D-Mt), with FXXN motifs replaced by SXXV, shows a markedly reduced capacity to form oligomers. Secondary-structure assays revealed that monomers of both variants exist predominantly as random coils, whereas the oligomers form predominantly β-sheets. Adhesion studies revealed that oligomers of D-Wt (D-Wt-O) mediate significantly enhanced binding to human epithelial cells relative to D-Mt-O and monomeric protein species. Moreover, D-Wt-O binds EGFR more efficiently than D-Wt monomers. Importantly, pretreatment of human cells with D-Wt-O reduces infectivity upon subsequent challenge with C. pneumoniae more effectively than all other protein species. Hence, the FXXN motif in D-Wt induces the formation of β-sheet-rich oligomeric protofibrils, which are important for adhesion to, and subsequent infection of human cells.

      Introduction

      Chlamydiae are Gram-negative bacteria with compact genomes, and some species represent significant threats to human health. Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide (
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      The abbreviations used are: EB, elementary body; Pmp, polymorphic membrane protein; EGFR, epidermal growth factor receptor; ThT, Thioflavin T; Aβ42, amyloid β42; SEC, size-exclusion chromatography; MALS, multiangle light scattering; AUC, analytical ultracentrifugation; RFU, relative fluorescent unit; TEM, transmission electron microscopy.
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      ). Several of the C. pneumoniae and all C. trachomatis Pmps have been shown to be located on the chlamydial surface (
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      From the inside out-processing of the chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells.
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      Analysis of pmpD expression and PmpD post-translational processing during the life cycle of Chlamydia trachomatis serovars A, D, and L2.
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      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
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      ).
      All C. trachomatis Pmps as well as Pmp6, Pmp20, and Pmp21 from C. pneumoniae have been found to serve as adhesins, mediating the attachment of chlamydial EBs to human epithelial cells. In addition, blocking experiments using recombinant Pmp proteins have provided direct evidence for the critical role of the Pmp proteins in chlamydial pathogenesis (
      • Mölleken K.
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      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ,
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      All subtypes of the Pmp adhesin family are implicated in chlamydial virulence and show species-specific function.
      ) More recently, the epidermal growth factor receptor (EGFR) was identified as the host receptor for the C. pneumoniae adhesin Pmp21, and binding to EGFR was shown to induce the uptake of the chlamydial EB, thus qualifying Pmp21 as an invasin (
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      ).
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      • Wehrl W.
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      From the inside out-processing of the chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells.
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      The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029.
      ,
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      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
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      Expression of Chlamydia pneumoniae polymorphic membrane protein family genes.
      ). Interestingly, Pmp21 possesses multiple adhesion domains (
      • Vandahl B.B.
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      • Birkelund S.
      The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029.
      ), and truncation experiments have demonstrated (Fig. 1, D and E) that adhesion critically depends on the presence of the repetitive GGA(I/L/V) and FXXN motifs. Thus, targeted mutagenesis has revealed that at least one GGA(I/L/V) and one FXXN motif (present in Pmp21-A) (Fig. 1E) or two FXXN motifs (present in Pmp21-D) (Fig. 1D) are required and sufficient for significant adhesion to HEp-2 cells (
      • Mölleken K.
      • Schmidt E.
      • Hegemann J.H.
      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ).
      Figure thumbnail gr1
      FIGURE 1.A, schematic representation of Pmp21. The full-length protein is depicted with the N-terminal signal sequence (SS) and the C-terminal β-barrel domain (β-barrel). Each of the tetrapeptide motifs GGA(I/L/V) (in yellow) and FXXN (in red) in the central passenger domain is marked. Two known proteolytic cleavage sites in Pmp21 are indicated by arrows (
      • Wehrl W.
      • Brinkmann V.
      • Jungblut P.R.
      • Meyer T.F.
      • Szczepek A.J.
      From the inside out-processing of the chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells.
      ,
      • Grimwood J.
      • Stephens R.S.
      Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae.
      ,
      • Vandahl B.B.
      • Pedersen A.S.
      • Gevaert K.
      • Holm A.
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      • Christiansen G.
      • Birkelund S.
      The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029.
      ,
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      • Schmidt E.
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      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ). B, structure of M-Pmp21 as predicted by I-Tasser (C-score: −1.58) (
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      ). The β-sheets are displayed in yellow, and random coils are in gray. C, predominant forms of Pmp21 in vivo, as detected by proteome analysis (
      • Wehrl W.
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      • Jungblut P.R.
      • Meyer T.F.
      • Szczepek A.J.
      From the inside out-processing of the chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells.
      ,
      • Vandahl B.B.
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      The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029.
      ,
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      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ,
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      Expression of Chlamydia pneumoniae polymorphic membrane protein family genes.
      ). D and E, the recombinant full-length Pmp21 passenger domain (PD-Pmp21), the processed forms N-Pmp21 and M-Pmp21, and the truncated subdomains Pmp21-A to Pmp21-D (D-Wt) mediate adhesion. A mutant form of Pmp21-D (D-Mt) in which each FXXN motif is replaced by SXXV was found to be incapable of mediating adhesion (
      • Mölleken K.
      • Schmidt E.
      • Hegemann J.H.
      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ).
      Strikingly, immunoaffinity enrichment of PmpD, the C. trachomatis homologue of Pmp21, from infectious EBs resulted in the isolation of high molecular weight structures, which included full-length PmpD and two proteolytically processed forms. The functional significance of these structures remains unknown (
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      Chlamydia trachomatis polymorphic membrane protein D is an oligomeric autotransporter with a higher-order structure.
      ).
      High molecular weight structures with adhesive characteristics have been identified on the surface of a number of pathogenic bacteria. In Enterobacteriaceae, including Escherichia coli, highly aggregative surface fibers called curli have been found (
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      Curli, fibrous surface proteins of Escherichia coli, interact with major histocompatibility complex class I molecules.
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      • Bokranz W.
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      ,
      • Uhlich G.A.
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      • Zogaj X.
      • Bokranz W.
      • Nimtz M.
      • Römling U.
      Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract.
      ). Subsequently, curli fibers were shown to be made up of an amyloid-like protein that binds the amyloid-specific dye Thioflavin T (ThT) (
      • Barnhart M.M.
      • Chapman M.R.
      Curli biogenesis and function.
      ,
      • Wang X.
      • Smith D.R.
      • Jones J.W.
      • Chapman M.R.
      In vitro polymerization of a functional Escherichia coli amyloid protein.
      ). Amyloids are insoluble protein aggregates derived from the conversion of protofibrils into amyloid fibrils, which are formed by proteins characterized by a typical β-sheet structure. The commercially available human amyloid β42 (Aβ42) is an amyloid-like peptide that can also form amyloid fibrils in vitro.
      In this study we focus on Pmp21-D (D-Wt), a C-terminal fragment derived from the naturally occurring M-Pmp21. It is the smallest Pmp21 fragment identified thus far that exhibits adhesion and infection blocking capacity (
      • Mölleken K.
      • Schmidt E.
      • Hegemann J.H.
      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ) (Fig. 1E). Our data demonstrate that the monomer (D-Wt-M) forms oligomers that adopt an amyloid-like structure. The Pmp21-D oligomers (collectively referred to as D-Wt-O) are comparable in size and shape to protofibrils of Aβ42. Comparison of the oligomerization capacity of D-Wt with that of a previously analyzed mutant form (D-Mt) with poor adhesion properties revealed that the FXXN motif in D-Wt contributes significantly to the formation of oligomers. Interestingly, both the adhesion to its host cell receptor EGFR and the neutralization capacity of D-Wt require its oligomerization.

      Discussion

      Successful infection of host cells by C. pneumoniae depends on a variety of virulence factors. These include specialized surface structures, which mediate uptake by host cells (
      • Kline K.A.
      • Fälker S.
      • Dahlberg S.
      • Normark S.
      • Henriques-Normark B.
      Bacterial adhesins in host-microbe interactions.
      ). Chlamydiae enter cells via multiple routes, using mechanisms that are poorly understood (
      • Campbell L.A.
      • Kuo C.-C.
      Interactions of Chlamydia with the host cells that mediate attachment and uptake.
      ). In previous studies we identified the Pmp proteins of C. pneumoniae and C. trachomatis as adhesins that are essential for the successful infection of human cells (
      • Mölleken K.
      • Schmidt E.
      • Hegemann J.H.
      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ,
      • Becker E.
      • Hegemann J.H.
      All subtypes of the Pmp adhesin family are implicated in chlamydial virulence and show species-specific function.
      ). Pmps are known to share characteristic features with Type V autotransporters, including proteolytic processing (
      • Wehrl W.
      • Brinkmann V.
      • Jungblut P.R.
      • Meyer T.F.
      • Szczepek A.J.
      From the inside out-processing of the chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells.
      ,
      • Grimwood J.
      • Stephens R.S.
      Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae.
      ,
      • Henderson I.R.
      • Lam A.C.
      Polymorphic proteins of Chlamydia spp.: autotransporters beyond the proteobacteria.
      ,
      • Vandahl B.B.
      • Pedersen A.S.
      • Gevaert K.
      • Holm A.
      • Vandekerckhove J.
      • Christiansen G.
      • Birkelund S.
      The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029.
      ,
      • Mölleken K.
      • Schmidt E.
      • Hegemann J.H.
      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ). Recently, Pmp21 was shown to bind to the host's EGF receptor and to induce its own uptake; hence, Pmp21 acts both as an adhesin and as an invasin (
      • Mölleken K.
      • Becker E.
      • Hegemann J.H.
      The Chlamydia pneumoniae invasin protein Pmp21 recruits the EGF receptor for host cell entry.
      ). Interestingly, the characteristic FXXN and GGA(I/L/V) motifs, which are known to occur in multiple copies exclusively in chlamydial Pmp proteins (
      • Grimwood J.
      • Stephens R.S.
      Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae.
      ), have been shown to be crucial for Pmp21-mediated adhesion (
      • Mölleken K.
      • Schmidt E.
      • Hegemann J.H.
      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ). Structure prediction programs have indicated that the passenger domains of Pmps are dominated by parallel β-strands disposed in a helical pattern with three faces that form a β-helix (
      • Bradley P.
      • Cowen L.
      • Menke M.
      • King J.
      • Berger B.
      BETAWRAP: successful prediction of parallel β-helices from primary sequence reveals an association with many microbial pathogens.
      ,
      • Vandahl B.B.
      • Pedersen A.S.
      • Gevaert K.
      • Holm A.
      • Vandekerckhove J.
      • Christiansen G.
      • Birkelund S.
      The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029.
      ). It has, therefore, been speculated that these β-helices could associate with each other to generate Pmp oligomers (
      • Hegemann J.H.
      • Moelleken K.
      Chlamydial adhesin and adhesins.
      ).
      Our initial characterization of refolded recombinant Pmp21-D, an adhesion-competent subdomain of Pmp21, by SEC revealed that homo-oligomeric forms (D-Wt-O) were dominant, whereas the monomer (D-Wt-M) made up only a relatively small fraction of the whole (Fig. 2, B and C). Interestingly, we found D-Wt-O to be very stable, as no disaggregation was observed after prolonged incubation in PBS buffer (Fig. 4A). In contrast, with time, the monomeric D-Wt-M (1.6S) gave rise to a new stable species at 2.6S, probably a dimer, which may nucleate the formation of higher order oligomers (Fig. 4C). The in vitro formation of Pmp21-D oligomers is in agreement with earlier findings which indicated that the C. trachomatis homologue of Pmp21, PmpD, is part of a protein complex on the EB cell surface (
      • Swanson K.A.
      • Taylor L.D.
      • Frank S.D.
      • Sturdevant G.L.
      • Fischer E.R.
      • Carlson J.H.
      • Whitmire W.M.
      • Caldwell H.D.
      Chlamydia trachomatis polymorphic membrane protein D is an oligomeric autotransporter with a higher-order structure.
      ). Moreover, it was reported that Pmps of Chlamydia psittaci also occur in supramolecular complexes (
      • Tanzer R.J.
      • Longbottom D.
      • Hatch T.P.
      Identification of polymorphic outer membrane proteins of Chlamydia psittaci 6BC.
      ).
      Analysis of the sizes of the oligomeric D-Wt-O yielded remarkable large S values of up to 85S with an average of 23.8S, which may reflect the formation of amorphous aggregates by refolding intermediates. However, determination of the frictional ratio f/f0 of 2.75 for the D-Wt sample argues that large elongated structures are formed (Fig. 4E). Typical examples for highly elongated proteins are human fibrinogen (Mr 330) with an S value of 7.6S and an f/f0 = 2.3 and myosin (Mr 570) with an S value of 6.4S and an f/f0 = 3.6 (
      • Tanford C.
      ). However, in contrast to those proteins, Pmp21-D is very small (23 kDa), supporting the idea that it might form long homo-oligomeric protein species.
      Indeed, EM analysis revealed that the D-Wt-O oligomers form protofibril-like structures (Fig. 5, A and B). Interestingly, the three differently sized D-Wt-O species isolated by SEC correspond to protofibrils with almost identical widths (∼10 nm) but different lengths. One might speculate that the largest oligomer W-Wt-O1 (∼60 nm) could be the most mature form produced in vitro, whereas the others could represent intermediates. Interestingly, the D-Wt protofibrils exhibited amyloid-like characteristics, as they bind the dye thioflavin T and strongly enhance its fluorescence, as does the prototypical β-sheet-rich Aβ42 (Fig. 5E). Interestingly and in agreement with the theory, the longest Pmp oligomers (D-Wt-O1) yielded a significantly higher ThT fluorescence emission than the shorter D-Wt-O2. In contrast monomeric D-Wt at time point 0 only showed background ThT fluorescence, indicating the absence of β-sheet-rich oligomer structures. However, over time D-Wt-M also yielded significant ThT fluorescence, likely due to spontaneous protein oligomerization (as observed also in Fig. 4C). Indeed, the folding of Pmp21-D is remarkably stable, as dimers can be detected by SDS-PAGE (Fig. 2A). Such high stability is characteristic for a number of amyloid-like proteins (
      • Chapman M.R.
      • Robinson L.S.
      • Pinkner J.S.
      • Roth R.
      • Heuser J.
      • Hammar M.
      • Normark S.
      • Hultgren S.J.
      Role of Escherichia coli curli operons in directing amyloid fiber formation.
      ,
      • Epstein E.A.
      • Reizian M.A.
      • Chapman M.R.
      Spatial clustering of the curlin secretion lipoprotein requires curli fiber assembly.
      ,
      • Podlisny M.B.
      • Ostaszewski B.L.
      • Squazzo S.L.
      • Koo E.H.
      • Rydell R.E.
      • Teplow D.B.
      • Selkoe D.J.
      Aggregation of secreted amyloid β-protein into sodium dodecyl sulfate-stable oligomers in cell-culture.
      • Walsh D.M.
      • Klyubin I.
      • Fadeeva J.V.
      • Rowan M.J.
      • Selkoe D.J.
      Amyloid-β oligomers: their production, toxicity and therapeutic inhibition.
      ). However, dimer formation has also been observed for amyloid-like proteins upon sample preparation for SDS-PAGE (
      • Pujol-Pina R.
      • Vilaprinyo-Pascual S.
      • Mazzucato R.
      • Arcella A.
      • Vilaseca M.
      • Orozco M.
      • Carulla N.
      SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer's disease: appealing for ESI-IM-MS.
      ). Moreover, in vitro ThT-induced amyloid aggregation has been described, and this might also contribute to the formation of oligomeric D-Wt (
      • D'Amico M.
      • Di Carlo M.G.
      • Groenning M.
      • Militello V.
      • Vetri V.
      • Leone M.
      Thioflavin T promotes Aβ(1–40) amyloid fibrils formation.
      ).
      Previous work has demonstrated that the FXXN motifs in D-Wt (Pmp21-D) are essential for adhesion and for the ability of soluble Pmp21 fragments to block infection (
      • Mölleken K.
      • Schmidt E.
      • Hegemann J.H.
      Members of the Pmp protein family of Chlamydia pneumoniae mediate adhesion to human cells via short repetitive peptide motifs.
      ). In the present study we have now shown that the two FXXN motifs in D-Wt are also crucial for its ability to form protofibrils, as the capacity for oligomerization is strongly reduced when these motifs are mutated (D-Mt) (Fig. 6B). It is well known that specific protein sequences are involved in the induction of amyloid-like structures (
      • Maurer-Stroh S.
      • Debulpaep M.
      • Kuemmerer N.
      • Lopez de la Paz M.
      • Martins I.C.
      • Reumers J.
      • Morris K.L.
      • Copland A.
      • Serpell L.
      • Serrano L.
      • Schymkowitz J.W.
      • Rousseau F.
      Exploring the sequence determinants of amyloid structure using position-specific scoring matrices.
      ). Thus the FXXN motif is very probably an essential part of the amyloid-promoting sequence within Pmp21-D. However, it is worth mentioning that the D-Mt-O1 oligomers formed again exhibited a rod-shaped structure (Fig. 6C), similar to those seen for the corresponding Wt oligomers. Thus the structures of Wt and Mt oligomers are identical, yet the tendency to be formed is highly increased for D-Wt.
      Interestingly, our CD analysis showed that D-Wt-O1 harbors some β-sheet structure, whereas D-Mt-O1 shows a certain shift toward random coils, suggesting that the FXXN motifs play a role in protein folding. The β-sheets may instead adopt β-helical structures. Amyloids and β-helices in general are suggested to share similar motifs (
      • Tsai H.H.
      • Gunasekaran K.
      • Nussinov R.
      Sequence and structure analysis of parallel β helices: implication for constructing amyloid structural models.
      ). Indeed, β-helical amyloids are already known for other organisms in various contexts (
      • Govaerts C.
      • Wille H.
      • Prusiner S.B.
      • Cohen F.E.
      Evidence for assembly of prions with left-handed β 3-helices into trimers.
      • Smirnovas V.
      • Baron G.S.
      • Offerdahl D.K.
      • Raymond G.J.
      • Caughey B.
      • Surewicz W.K.
      Structural organization of brain-derived mammalian prions examined by hydrogen-deuterium exchange.
      ,
      • Tycko R.
      • Wickner R.B.
      Molecular structures of amyloid and prion fibrils: consensus versus controversy.
      • Van Melckebeke H.
      • Wasmer C.
      • Lange A.
      • Ab E.
      • Loquet A.
      • Böckmann A.
      • Meier B.H.
      Atomic-Resolution Three-dimensional structure of HET-s(218–289) amyloid fibrils by solid-state NMR spectroscopy.
      ).
      The results presented here document a functional role for Pmp21-D oligomers during the C. pneumoniae infection. The oligomer Pmp21-D (D-Wt-O1) shows significant adhesion to human epithelial cells, in contrast to monomer and mutant species (Fig. 7). Moreover, D-Wt-O1 interacts significantly more strongly with the Pmp21 receptor EGFR than the monomeric D-Wt (Fig. 8). Finally, recombinant D-Wt-O1 blocks chlamydial infection more efficiently than any other species tested (Fig. 9). These data strongly suggest a functional role of oligomeric Pmp species in the chlamydial infection. Unexpectedly, the pulldown with Ctad1 brought down small amounts of EGFR. We recently reported that Ctad1 binds to integrin β1 (
      • Stallmann S.
      • Hegemann J.H.
      The Chlamydia trachomatis Ctad1 invasin exploits the human integrin β 1 receptor for host cell entry.
      ). As it is known that the integrin and EGFR receptors exhibit intensive cross-talk (
      • Huveneers S.
      • Danen E.H.
      Adhesion signaling: crosstalk between integrins, Src and Rho.
      ), it may be speculated that Ctad1-mediated integrin activation results in the association of both receptors in downstream signaling complexes.
      In conclusion, Pmp21 may belong to the group of functional, oligomeric structures found on microbial cell surfaces, the prototypes of which are CsgA and CsgB, which are secreted by their own apparatus to form the filamentous cell surface structures called curli produced by many Enterobacteriaceae (
      • Barnhart M.M.
      • Chapman M.R.
      Curli biogenesis and function.
      ). Curli shares all of the biophysical properties of amyloids, including the propensity to form ordered β-sheet-rich fibers with a capacity to bind the dye ThT (
      • Chapman M.R.
      • Robinson L.S.
      • Pinkner J.S.
      • Roth R.
      • Heuser J.
      • Hammar M.
      • Normark S.
      • Hultgren S.J.
      Role of Escherichia coli curli operons in directing amyloid fiber formation.
      ), and produces extracellular proteinaceous fibers that contribute to biofilm formation, host colonization, immune activation, and cell invasion (
      • Fernández L.A.
      • Berenguer J.
      Secretion and assembly of regular surface structures in Gram-negative bacteria.
      ,
      • Jonson A.B.
      • Normark S.
      • Rhen M.
      Fimbriae, pili, flagella, and bacterial virulence.
      • Epstein E.A.
      • Chapman M.R.
      Polymerizing the fibre between bacteria and host cells: the biogenesis of functional amyloid fibres.
      ).
      So far, Pmp21 oligomers have not been described for extracellular infectious EBs or dividing reticulate bodies within the inclusion. Either these structures do not survive the harsh fixation protocols used for sample preparation for immunofluorescence and electron microscopy, or the size of the oligomeric structures is well controlled by the Chlamydia, and thus their small in vivo size does not enable their detection. Alternatively, Pmp protofibrils might not form in vivo, because the proteins are physically constrained by their anchorage through their β-barrel in the outer membrane, as has been shown for the Candida albicans adhesins, which are also anchored in the cell wall. Nevertheless, these fungal adhesins form cell surface amyloid patches of arrayed adhesin molecules (“adhesin nanodomains”) 100-1000 nm in size, thus binding ligands with high avidity (
      • Lipke P.N.
      • Garcia M.C.
      • Alsteens D.
      • Ramsook C.B.
      • Klotz S.A.
      • Dufrêne Y.F.
      Strengthening relationships: amyloids create adhesion nanodomains in yeasts.
      ,
      • Otoo H.N.
      • Lee K.G.
      • Qiu W.
      • Lipke P.N.
      Candida albicans Als adhesins have conserved amyloid-forming sequences.
      ). It is tempting to speculate that the flower-like structures observed by EM in affinity-enriched preparations of endogenous PmpD-containing protein complexes might possibly represent the in vivo version of the Pmp21 protofibrils detected in vitro in this study (
      • Swanson K.A.
      • Taylor L.D.
      • Frank S.D.
      • Sturdevant G.L.
      • Fischer E.R.
      • Carlson J.H.
      • Whitmire W.M.
      • Caldwell H.D.
      Chlamydia trachomatis polymorphic membrane protein D is an oligomeric autotransporter with a higher-order structure.
      ). The data presented here are compatible with the idea that oligomeric Pmp complexes might enhance the chlamydial cell's capacity for adhesion to human epithelial cells and be important for the initial step in infection.
      Finally, there is a striking correlation between chlamydial infection and amyloid formation in the brains of mice (
      • Boelen E.
      • Stassen F.R.
      • van der Ven A.J.
      • Lemmens M.A.
      • Steinbusch H.P.
      • Bruggeman C.A.
      • Schmitz C.
      • Steinbusch H.W.
      Detection of amyloid beta aggregates in the brain of BALB/c mice after Chlamydia pneumoniae infection.
      ,
      • Little C.S.
      • Joyce T.A.
      • Hammond C.J.
      • Matta H.
      • Cahn D.
      • Appelt D.M.
      • Balin B.J.
      Detection of bacterial antigens and Alzheimer's disease-like pathology in the central nervous system of BALB/c mice following intranasal infection with a laboratory isolate of Chlamydia pneumoniae.
      • Little C.S.
      • Hammond C.J.
      • MacIntyre A.
      • Balin B.J.
      • Appelt D.M.
      Chlamydia pneumoniae induces Alzheimer-like amyloid plaques in brains of BALB/c mice.
      ). However, there is currently no evidence for a direct or indirect connection between amyloid formation in the mouse brain and Pmp proteins.
      Future work needs to focus on whether or not the different Pmp family members (21 in C. pneumoniae and 9 in C. trachomatis; Refs.
      • Kalman S.
      • Mitchell W.
      • Marathe R.
      • Lammel C.
      • Fan J.
      • Hyman R.W.
      • Olinger L.
      • Grimwood J.
      • Davis R.W.
      • Stephens R.S.
      Comparative genomes of Chlamydia pneumoniae and C-trachomatis.
      and
      • Grimwood J.
      • Stephens R.S.
      Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae.
      ) can interact with each other to form heteromeric protofibrils. If so, this would provide for greater antigenic complexity and enable Chlamydiae to adapt to a larger range of cellular niches (
      • Grimwood J.
      • Stephens R.S.
      Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae.
      ,
      • Hegemann J.H.
      • Moelleken K.
      Chlamydial adhesin and adhesins.
      ).

      Author Contributions

      J. H. H. conceived and coordinated the study. J. H. H. and S. E. T. L. designed, performed, and analyzed the experiments. S. H. J. S. and L. S. gave general biochemical advice and support. C. D. and L. N.-S. gave advice and support concerning AUC and ThT assay. J. H. H. and S. E. T. L. wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Katja Moelleken, Jan Galle, and Sebastian Haensch for help with human cell culture and chlamydial infection assays. We also thank Klaus Meyer for help with FACS and Marion Nissen for help with the EM. We thank Elisabeth Becker for the initial structural prediction analyses, which suggested the possibility of Pmp oligomerization.

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