The Chlamydia pneumoniae Adhesin Pmp21 Forms Oligomers with Adhesive Properties*

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.

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 (1). Furthermore, it causes trachoma, a form of ocular conjunctivitis characterized by massive inflammation that leads to scarring of the inner epithelial lining of the eyelid and eventually to blindness (2). Chlamydia pneumoniae is an important respiratory pathogen, causing pneumonia, pharyngitis, sinusitis, and bronchitis. Moreover, it is associated with several chronic diseases including atherosclerosis, central nervous system disorders, and Alzheimers disease (3)(4)(5). Despite the clinical relevance of Chlamydia, no vaccine is available for use in humans (6,7). Chlamydia species have a unique biphasic developmental cycle, alternating between the infectious elementary body (EB) 2 and the intracellular, metabolically active, reticulate body that replicates in eukaryotic cells (8,9).
Polymorphic membrane proteins (Pmps) found in different species of the Chlamydiaceae are adhesion-mediating proteins (10 -13). Bioinformatic analysis has shown that the Pmp protein family is composed of 9 members (PmpA to PmpI) in C. trachomatis and 21 (Pmp1 to Pmp21) in C. pneumoniae (11,14). The pmp gene family has been subdivided, on phylogenetic grounds, into six subtypes (14). The subtypes from both species have retained a significant degree of sequence similarity across species. Thus, the level of identity between the PmpD subtype members PmpD and Pmp21 of C. trachomatis serovar E and C. pneumoniae CWL029, respectively, is 33%, indicating some level of functional similarity across chlamydial species (14).
The various Pmp families show a unique overrepresentation of repeats of the motifs GGA(I/L/V) and FXXN (14,15). For example, the FXXN motif is found on average 11.3 times in the Pmps of C. pneumoniae, whereas its average incidence in the rest of the proteome is 0.84 (14).
Furthermore, Pmp proteins are united by their predicted autotransporter characteristics. Thus all Pmps share an N-terminal Sec-dependent leader sequence followed by a passenger domain and a C-terminal ␤-barrel (14,16). Structure predictions have suggested that a large region of the passenger domain of Pmp6 folds into a parallel ␤-strand in a helical pattern with three faces that form a ␤-helix (17,18). ␤-Helical structures are a characteristic of autotransporters and might be required for their efficient translocation across the outer membrane and for folding (19). Moreover, it has been speculated that Pmp ␤-helices could associate with each other to generate oligomers (20). Like other autotransporter proteins, many Pmps undergo complex proteolytic processing (13,18,(21)(22)(23)(24). Several of the C. pneumoniae and all C. trachomatis Pmps have been shown to be located on the chlamydial surface (10, 13, 18, 21, 22, 24 -26).
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 (22,27) 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 (28).
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 (24).
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 (30,31). First observed in 1989 in fibronectin binding E. coli isolates from bovine fecal samples (32), curli fibers have been shown to mediate interactions between individual bacteria, bacteria and host tissues, and bacteria and inert surfaces like Teflon and stainless steel, which are usually refractory to bacterial colonization (30,(33)(34)(35)(36)(37). Subsequently, curli fibers were shown to be made up of an amyloid-like protein that binds the amyloid-specific dye Thioflavin T (ThT) (38,39). Amyloids are insoluble protein aggregates derived from the conversion of protofibrils into amyloid fibrils, which are formed by proteins characterized by a typical ␤-sheet structure. The commercially available human amyloid ␤ 42 (A␤ 42 ) 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 (22) (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 A␤ 42 . 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.

Recombinant Pmp21-D Can Exist in Monomeric and Diverse
Homo-oligomeric Forms-When we performed an automated structural characterization based on the C. pneumoniae Pmp21 protein sequence, we found that the processing product M-Pmp21 is predicted to form a long, right-handed ␤-helix domain, which could provide a rigid platform on which multiple adhesive sites can be presented and could self-associate to generate oligomers (Fig. 1B). To test this hypothesis experimentally, we expressed Pmp21-D (D-Wt; Fig. 1E), an adhesive C-terminal fragment of M-Pmp21 (13,18), in E. coli. We then purified the recombinant protein under denaturing conditions via its N-terminal His tag and allowed it to refold (at a concentration of 3.1 mg ml Ϫ1 ) before elution from the affinity column (see "Experimental Procedures"). The eluate was first analyzed by SDS-PAGE, which revealed that the major species migrated at ϳ30 kDa as expected for the monomeric D-Wt protein (M, Fig. 2A). However, a small amount of the SDS-resistant dimer was also detected (D, ϳ60 kDa; Fig. 2A). Western blotting using an anti-His antibody confirmed the identity of the ϳ30 kDa and ϳ60 kDa bands as recombinant D-Wt (Fig. 2D).
We then analyzed the homogeneity of the purified and refolded protein by size-exclusion chromatography (SEC). A broad peak emerging near the void volume (46 -70 ml) was observed together with one distinct later peak eluting at around  (13,14,18,22). B, structure of M-Pmp21 as predicted by I-Tasser (C-score: Ϫ1.58) (80 -82). The ␤-sheets are displayed in yellow, and random coils are in gray. C, predominant forms of Pmp21 in vivo, as detected by proteome analysis (13,18,22,29).  We additionally repeated the experiment by eluting denatured D-Wt from the nickel column and refolded it by dialysis for 36 h in PBS before SEC. In this SEC we observed the same elution profile as for the previously described "on column refolding" procedure (data not shown).
To determine the molecular mass of the protein species in the second peak, analytical size-exclusion chromatography coupled to multiangle light scattering detection (SEC-MALS) was performed. The data revealed that this peak (    AUC assay has a running time of several hours, we first determined the stability of D-Wt-O 1 by analytical SEC on the time scale required for AUC sample preparation and measurement. Even after an incubation period for 20 h in PBS at 4°C, D-Wt-O 1 was still stable and had not dissociated into lower molecular weight species (Fig. 4A).
The peak fraction of monomeric D-Wt-M ( Fig. 3) was analyzed by measuring its sedimentation velocity with AUC at 40,000 rpm and 20°C. Subsequent data analysis by SEDFIT, assuming a continuous c(s) distribution model, indicated particles of 1.6S with a small side peak at 2.6S and an overall frictional ratio f/f 0 of 1.9 (Fig. 4, B and C). Similarly, D-Wt-O 1 was analyzed at a sedimentation velocity of 20,000 rpm. The results suggested that D-Wt-O 1 accounted for ϳ87.4% of D-Wt, with a size distribution ranging from 8S to 85S, with a mean size of 23.8S and a frictional ratio of f/f 0 ϭ 2.8 (

Oligomers of Pmp21-D Exhibit Amyloid-specific ThT
Fluorescence-The benzothiazole dye ThT is a sensitive probe for amyloid fibril detection. The ThT test is based on its unique ability to form highly fluorescent complexes with amyloid and amyloid-like proteins (44,45). To ask whether the Pmp protofibrils detected by EM (Fig. 5, A and B) had amyloid-like characteristics, all SEC fractions studied by EM (except D-Wt-O 3 , in which protein concentrations were too low) were tested for ThT fluorescence. BSA, which served as the negative control, the positive control amyloid ␤ 42 (A␤ 42  The FXXN Motif Strongly Promotes the Formation of D-Wt Oligomers-In previous studies we found that mutating the two FXXN motifs present in D-Wt to SXXV (D-Mt) results in complete loss of adhesive capacity (22). Thus we speculated that these motifs might also be relevant for oligomer formation. Therefore, recombinant D-Wt and D-Mt were refolded at four different concentrations (0.7, 1.7, 2.4, and 3.1 mg ml Ϫ1 ), centrifuged, and analyzed by SEC. The area under the oligomer peaks was then expressed as a percentage of the total area under the curve. To get an impression of the ratios easily, the monomer peaks were set to 1 (Fig. 6A). At a concentration of 0.7 mg ml Ϫ1 22% of total D-Wt protein eluted as oligomeric D-Wt-O. The figure for oligomeric mutant D-Mt-O was only 4%. Increasing the total concentration of either monomer in the assay also increased the percentage of oligomers formed (Fig. 6B). However, D-Wt always gave rise to a significantly higher proportion of oligomers than the same concentration of the mutated D-Mt protein. This difference was very pronounced at a concentration of 1.7 mg ml Ϫ1 , at which the mutant form D-Mt produced ϳ10-fold fewer oligomers (5%) compared with D-Wt (53%). Thus, these data demonstrate that both D-Wt and D-Mt can form oligomers and that the capacity to form oligomers is significantly enhanced by the two FXXN motifs.
The fact that D-Mt has a strongly reduced propensity to form oligomers points to structural differences between the monomers and possibly their respective oligomeric forms too. Therefore, the secondary structure of wild-type and mutant monomer and oligomer forms was determined by circular dichroism (CD) spectroscopy (Fig. 6C). Data analysis using the program CONTINLL revealed that D-Wt-O 1 consists of ϳ11.5% ␤-sheets and ϳ3.4% ␣-helices, whereas D-Wt-M displayed a random-coil structure similar to that of a disordered protein FXXN-induced Oligomerization of Pmp21-D Is Crucial for Its Ability to Adhere to Human Cells-In previous studies, Pmp6, -20, and -21 from C. pneumoniae and all nine Pmps from C. trachomatis serovar E, were characterized as bacterial adhesins that are important for the infection of human epithelial cells (22,27). The motifs GGA(I/L/V) and FXXN are characteristic for Pmps and essential for adhesion (22). Our results so far have demonstrated that these motifs are additionally responsible for protein oligomerization (Fig. 6, A and B). To analyze which of the different protein species represents the adhesion-competent conformation and whether adhesion is mediated by the FXXN motifs alone, we performed bead-based adhesion assays with all D-Wt and D-Mt protein species (Fig. 7A). Proteincoated green fluorescent latex beads were incubated with human epithelial HEp-2 cells. The coating efficiency estimated by immunoblotting revealed only slight differences in the amount of each protein coupled to the beads (data not shown). The negative control, BSA-coated beads, only bound to 15.9 Ϯ 3% HEp-2 cells. Importantly, D-Wt-O 1 showed a significant adhesion capacity with 50 Ϯ 4.3% HEp-2 cells carrying bound beads. In contrast, the monomeric protein species D-Wt-M and D-Mt-M as well as the oligomeric mutant form D-Mt-O 1 mediated comparatively little binding of beads to HEp-2 cells (20.6 Ϯ 3.2%, 20.5 Ϯ 2.1%, and 20.3 Ϯ 5%, respectively). Hence, the bead assay suggests that only the oligomeric wild-type protein species D-Wt-O 1 exhibits strong adhesion capacity. This  Fig. 2B). Samples were applied to carbon-coated copper grids, negatively stained with 1% uranyl acetate, and imaged at 50,000ϫ magnification (D) and 85,000ϫ magnification (E, scale bar ϭ 100 nm). The images shown are representative for the whole grids (independent replicates n ϭ 2).  OCTOBER 21, 2016 • VOLUME 291 • NUMBER 43 implies that both the formation of oligomers as well as presence of FXXN motifs is crucial for binding to HEp-2 cells.

Pmp21 Forms Functional Oligomeric Structures
The Oligomeric Form of Pmp21-D Is Important for the Binding to the EGFR-In previous studies the EGF receptor was identified as the receptor for the M-Pmp21 protein domain, which occurs naturally (28). To analyze whether the monomeric and oligomeric D-Wt species have identical or different affinities for EGFR, we performed pulldown assays with D-Wt-O 1 and D-Wt-M. As positive control we used recombinant M-Pmp21 (28), whereas other controls were recombinant GST, the C. pneumoniae adhesin OmcB-BD (46), and the C. trachomatis adhesin and invasin Ctad1 (47) (Fig. 8A). After biotinylation the recombinant proteins were incubated with human epithelial HEp-2 cells, cross-linked, and affinity-purified using a streptavidin resin. Interaction partners were eluted after cross-link removal. Western blots of whole cell lysates cross-linked to the recombinant proteins revealed the presence of identical amounts of EGFR (Fig. 8B). After pulldown the EGFR signals were found to be of similar strength for the positive control M-Pmp21 and the oligomeric D-Wt-O 1 (1.03 and 1.0, respectively) (Fig. 8C). In contrast, the pulldown using the monomeric D-Wt-M brought down only ϳ50% of the amount brought down by the oligomeric form (0.5 relative intensity). Recombinant GST as well as OmcB-BD failed to bring down detectable amounts of EGFR. A very weak signal was observed for Ctad1. These results provide evidence that both D-Wt-M and D-Wt-O 1 can interact with EGFR; however, the efficiency of the oligomeric species is twice as high as that of monomeric species. This again strongly suggests the relevance of oligomer formation for the infection process.
The Oligomeric Form of Pmp21-D Is Important for Infection-Next we asked whether the adhesion-competent oligomeric D-Wt-O protein species is also relevant for a chlamydial infection. To this end, soluble recombinant monomeric or oligomeric D-Wt and D-Mt protein species were preincubated with HEp-2 cells before infection. ϳ48 h post infection the efficiency of infection was measured by counting the numbers of inclusions formed (Fig. 9, A and B). Pretreatment with the negative control BSA did not significantly reduce the infection upon subsequent exposure to C. pneumoniae EBs, whereas the positive control heparin reduced infectivity by 99%. Interestingly, D-Wt-O 1 had the strongest effect, blocking the subsequent infection by almost 50% compared with the PBS control (53.5 Ϯ 2.3% inclusions). D-Wt-M (82.9 Ϯ 5.7% inclusions) and D-Mt-O 1 (83 Ϯ 1.7% inclusions) had a much weaker effect, reducing infectivity by only ϳ17%, which is not significantly different from the value for the PBS control. The least effective of the protein species tested was D-Mt-M (92.7 Ϯ 2.9%), which inhibited infection to about the same extent as the negative control BSA (96.7 Ϯ 8.8%) (Fig. 9, A and B). These data also show that the wild-type and mutant oligomeric forms of Pmp21-D inhibit infection more effectively than the corresponding monomeric forms.

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 (48). Chla-mydiae enter cells via multiple routes, using mechanisms that are poorly understood (49). 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 (22,27). Pmps are known to share characteristic features with Type V autotransporters, including proteolytic processing (13,14,16,18,22). 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 (28). Interestingly, the characteristic FXXN and GGA(I/L/V) motifs, which are known to occur in multiple copies exclusively in chlamydial Pmp proteins (14), have been shown to be crucial for Pmp21mediated adhesion (22). Structure prediction programs have indicated that the passenger domains of Pmps are dominated by parallel ␤-strands disposed in a helical pattern with three faces that form a ␤-helix (17,18). It has, therefore, been speculated that these ␤-helices could associate with each other to generate Pmp oligomers (20).
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 (24). Moreover, it was reported that Pmps of Chlamydia psittaci also occur in supramolecular complexes (50).
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/f 0 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 (M r 330) with an S value of 7.6S and an f/f 0 ϭ 2.3 and myosin (M r 570) with an S value of 6.4S and an f/f 0 ϭ 3.6 (51). 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 (ϳ10 nm) but different lengths. One might speculate that the largest oligomer W-Wt-O 1 (ϳ60 nm) could be the most mature form produced in vitro, whereas the others could represent intermediates. Interestingly, the D-Wt protofibrils exhibited amyloidlike characteristics, as they bind the dye thioflavin T and strongly enhance its fluorescence, as does the prototypical ␤-sheet-rich A␤ 42 (Fig. 5E). Interestingly and in agreement with the theory, the longest Pmp oligomers (D-Wt-O 1 ) yielded a significantly higher ThT fluorescence emission than the shorter D-Wt-O 2 . In contrast monomeric D-Wt at time point 0 only showed background ThT fluorescence, indicating the absence of ␤-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 (31,(52)(53)(54). However, dimer formation has also been observed for amyloid-like proteins upon sample preparation for SDS-PAGE (55). Moreover, in vitro ThT-induced amyloid aggregation has been described, and this might also contribute to the formation of oligomeric D-Wt (56).
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 (22). 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 (57). 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-O 1 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-O 1 harbors some ␤-sheet structure, whereas D-Mt-O 1 shows a certain shift toward random coils, suggesting that the FXXN motifs play a role in protein folding. The ␤-sheets may instead adopt ␤-helical structures. Amyloids and ␤-helices in general are suggested to share similar motifs (58). Indeed, ␤-helical amyloids are already known for other organisms in various contexts (59 -62).
The results presented here document a functional role for Pmp21-D oligomers during the C. pneumoniae infection. The oligomer Pmp21-D (D-Wt-O 1 ) shows significant adhesion to human epithelial cells, in contrast to monomer and mutant species (Fig. 7). Moreover, D-Wt-O 1 interacts significantly more strongly with the Pmp21 receptor EGFR than the monomeric D-Wt (Fig. 8). Finally, recombinant D-Wt-O 1 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 ␤1 (47). As it is known that the integrin and EGFR receptors exhibit intensive cross-talk (63), 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 (38). Curli shares all of the biophysical properties of amyloids, including the propensity to form ordered ␤-sheet-rich fibers with a capacity to bind the dye ThT (31), and produces extracellular proteinaceous fibers that contribute to biofilm formation, host colonization, immune activation, and cell invasion (64 -66).
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 ␤-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 (67,68). 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 (24). 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 (69 -71). 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. 11 and 14) 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 (14,20).
DNA Manipulations and Plasmid Construction-Plasmids were generated in Saccharomyces cerevisiae as described in Mölleken et al. (22), and their structures were verified by sequence analysis (GATC).
Protein Expression and Affinity Purification of His 6 -tagged Proteins-Growth for protein expression in BL21 (DE3) was performed in LB media ϩ 0.8% glucose at 37°C. Cells were induced with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) at an A 600 of 0.6 -1 and grown for a further 4 h. The cells were then harvested by centrifugation in a JLA10.500 rotor (Beckman) at 5000 rpm for 10 min at 4°C.
The cell pellets were lysed with 10 ml of lysis buffer (20 mM Tris-HCl, pH 8, 6 M guanidine HCl, 0.5 M NaCl, 1 mM ␤-mercaptoethanol, 5 mM imidazole) per gram of cell pellet overnight. Cell debris was removed by centrifugation in a Type 45Ti rotor (Beckmann) at 42,000 rpm for 1 h at 4°C.
Purification under denaturing conditions and refolding of the His 6 -tagged D-Wt and D-Mt was performed with HiTrap Chelating Nickel-IDA columns (5 ml column volume) at 4°C using an ÄKTA Prime plus (GE Healthcare). The system was equilibrated with lysis buffer. Subsequently the cleared whole cell lysate was applied to the column. After washing with 50 ml of lysis buffer, the column was washed with 10 ml of washing buffer (20 mM Tris-HCl, pH 8, 8 M urea, 0.5 M NaCl, 1 mM ␤-mercaptoethanol, 5 mM imidazole). Thereafter washing buffer was gradually replaced with 30 ml with refolding buffer (20 mM Tris-HCl, pH 8, 0.5 M NaCl, 1 mM ␤-mercaptoethanol, 5 mM imidazole). Proteins were eluted at 4°C by applying a 40-ml imidazole gradient (0.005-0.5 M in the same buffer), and the eluate was collected in 1-ml fractions. The whole process was monitored by absorbance at 280 nm. All subsequent steps were also performed at 4°C. The protein-containing fractions were centrifuged at 10,000 ϫ g for 10 min. The two peak fractions were pooled, and protein concentrations were determined with the Bradford assay. Within 2 h after elution from the HiTrap column, the samples were analyzed by preparative SEC. Different concentrations of refolded protein were generated by starting with different amounts of whole cell lysate.
Size-exclusion Chromatography-SEC was performed on an ÄKTA Prime plus (GE Healthcare) equipped with a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) at 4°C. SEC was performed at a flow rate of 0.5 ml/min in phosphate-buffered saline (PBS) (10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4), and 1.2-ml fractions were collected. The void volume of the column was determined by using blue dextran (BD Biosciences), and the separation range of the column was verified by standard proteins for gel filtration (Sigma). Within 6 h after elution from the SEC column, the sample were used for further analysis. In the meantime the samples were kept on 4°C.
Analytical size-exclusion chromatography was performed on an ÄKTA Prime plus (GE Healthcare) equipped with a Superdex 200 HR 10/30 column (GE Healthcare) at 4°C. Analytical SEC was performed at a flow rate of 0.3 ml/min in PBS, and 0.3-ml fractions were collected. In both cases the elution profiles were analyzed with the PrimeView software package (GE Healthcare).
Combined Analytical SEC and MALS-Analytical SEC-MALS was performed on a HPLC system from Agilent Technologies using a Superdex 200 10/300 GL column (GE Healthcare), equilibrated, and run at a flow rate of 0.3 ml/min in PBS at 4°C. For MALS analysis, the eluate was monitored with a miniDAWN TREOS triple-angle light-scattering detector in combination with an OPTILab T-rEX differential refractive index detector (both from Wyatt Technology). Typically, 100 l of purified D-Wt-M (0.5 mg/ml) was loaded onto the Superdex 200 10/300 GL column, and the elution data were analyzed with the ASTRA software package (Wyatt Technology).
AUC-AUC was performed in an Optima XL-A analytical ultracentrifuge (Beckman Coulter) with absorbance optics using an An-50 Ti for D-Wt and an An-60 Ti rotor for D-Wt-M. Sedimentation velocity centrifugation was done at 20,000 rpm and 20°C for D-Wt and at 40,000 rpm and 20°C for D-Wt-M. The signal intensity for D-Wt was recorded at 280 nm and for D-Wt-M at 230 nm over the 7-h duration of the analysis. Data were fitted to the continuous distribution (c(s)) Lamm equation model with a partial specific volume of 0.7286 cm 3 /g (based on the D-Wt sequence) using the software package Sedfit. The density and viscosity of the buffer were 1.0053 and 0.01019, respectively, according to SEDNTERP (Sedimentation Interpretation Program, Version 20120828, University of New Hampshire) (73). A resolution of 200 was chosen for the S value. The relative amounts of the different species in the D-Wt-M sample were derived from the c(s) distribution exclusive of the area below 0.6 S, which contains a baseline deconvolution artifact. The S values determined were corrected for PBS at 20°C.
Transmission Electron Microscopy (TEM)-D-Wt samples were diluted to 1 M, and 10-l aliquots were incubated for 5 min at room temperature on freshly glow-discharged S162 Formvar/carbon-coated copper grids (Plano). The grids were then washed 3 times with 10 l of H 2 O and subsequently incubated for 1 min with 10 l of 1% aqueous uranyl acetate for negative staining, then air-dried for 5 min at room temperature.
The samples were examined with an E902 electron microscope (Zeiss) operating at 80 kV.
ThT Fluorescence Assay-For fluorescence measurements, ThT (Sigma) was added at a final concentration of 10 M to protein samples containing 10 M D-Wt or BSA in PBS or 48 h of preincubated A␤ 42 (Bachem) in a final volume of 100 l of 10 mM sodium phosphate, 50 mM NaCl, pH 7.4. ThT was allowed to bind at 37°C in a (stationary) round-bottomed 96-well black plate (Nunc) for 48 h. Fluorescence was excited at 442 and measured at an emission wavelength of 484 nm in an Infinite 200pro plate reader (Tecan). The slit width was 10 nm.
CD Spectroscopy-Far-UV CD spectra were measured on a JASCO J-815 spectropolarimeter equipped with a 1-mm Suprasil quartz cuvette (Hellma) at 20°C. Protein species (D-Wt-O 1 , D-Wt-M, D-Mt-M) were analyzed at a concentration of 10 M in 20 mM NaP i , 75 mM NaF, pH 7.4, or D-Mt-O 1 in PBS. The D-Wt-O 1 spectrum was analyzed on the Dichroweb server using the program CONTINLL (74 -78).
Adhesion Assays with Protein-coated Latex Beads-Adhesion assays with protein-coated latex beads were performed as described (27). Coating efficiency with 100 g/ml His 6 -tagged proteins was estimated by immunoblotting before use. Confluent monolayers of HEp-2, HeLa, or HUVE cells were grown in 24-well plates and incubated with a 10-fold excess of proteincoated latex beads (Polyscience) (diameter 1 m, green fluorescent) for 1 h at 37°C. Cells were washed twice with PBS, detached with cell dissociation solution, fixed with 3% formaldehyde for 20 min at room temperature, and analyzed by flow cytometry using a FACSAria instrument (BD Biosciences).
EGFR Pulldown Assay-The EGFR pulldown assay was performed with 200 g/ml D-Wt, M-Pmp21, GST, Ctad1, or the binding domain of OmcB (OmcB-BD) in a volume of 2 ml. All proteins were biotinylated according to the manufacturer's instructions (Thermo Fisher Scientific). Afterward the biotinylated proteins were incubated with HEp2 cells to allow adhesion. After 1 h of incubation at 37°C the unbound protein was washed away, and the bound proteins were cross-linked with DTSSP (Thermo Fisher Scientific). The HEp2 cells were lysed, and the soluble lysate was incubated for 12 h at 4°C with streptavidin resin to allow binding of biotinylated proteins. The streptavidin resin was washed with PBS (3 ϫ 0.5 ml), and afterward the recombinant proteins and their interaction partners were eluted with 100 mM DTT. The elution fractions were analyzed by Western blot with anti-EGFR antibodies (Thermo Fisher Scientific) and quantified by ImageJ.
Infection Inhibition Assay-These assays were performed as previously described (46). Briefly, HEp-2 cells were grown on glass coverslips (12-mm diameter) for 48 h and incubated (as confluent monolayers) with 250 l of medium containing recombinant protein (100 g/ml in PBS) for 2 h at 37°C. Purified chlamydial EBs (multiplicity of infection 20) were added to the protein suspension and incubated for 2 h at 37°C without centrifugation to avoid any influence of the centrifugation procedure on the adhesion/infection process (46). The cells were subsequently washed 3 times, covered with chlamydial growth medium, and incubated for 48 h at 37°C for C. pneumoniae infection. Subsequently the cells were fixed with 96% methanol for 2 min. Each monolayer was then washed 3 times with PBS. OCTOBER 21, 2016 • VOLUME 291 • NUMBER 43

Pmp21 Forms Functional Oligomeric Structures
For detection of chlamydial inclusions, a monoclonal fluorescein isothiocyanate (FITC)-conjugated antibody directed against chlamydial LPS (Bio-Rad) was used. Cells were viewed using a C2 confocal microscope (Nikon). Inclusions were counted, and the results were expressed as percentages of the number found in PBS-treated control samples.
Immunoblot Analysis-SDS-PAGE and immunoblot analysis were performed as described (79). The PageRuler TM (Thermo Fisher Scientific) set of prestained markers was employed as a molecular weight standard. The His 6 -tagged recombinant proteins purified from E. coli were detected with a anti-His 6 antibody (Qiagen) and visualized with alkaline phosphatase (AP)conjugated anti-mouse antibody (Promega).