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* This work was supported in part by a Ph.D. fellowship from the NRW Graduate School BioStruct-Biological Structures in Molecular Medicine and Biotechnology (to S. E. T. L.). The authors declare that they have no conflicts of interest with the contents of this article.
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 (
). 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 (
). 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 (
). 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 (
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 β-barrel (
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 (
) 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 (
Pmp21 occurs on the surface of infectious EBs in various processed forms, referred to as N-M-C-Pmp21, N-M-Pmp21, N-Pmp21 (31–670 amino acids (aa)), M-Pmp21 (671–1145 aa), and C-Pmp21 (1146–1609 aa) (Fig. 1C) (
), 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 (
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 (
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 (
), 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 (
). 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 (
) (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.
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 (
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 (
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 (
). 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 (
). 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 (
). 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 (
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 (
), 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 (
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 (
). 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 (
). 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 (
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.
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.