Dokument: Role of adhesin proteins in Chlamydia infection
| Titel: | Role of adhesin proteins in Chlamydia infection | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=42486 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20170530-101434-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Favaroni, Alison [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Hegemann, Johannes H. [Gutachter] Prof. Dr. Pfeffer, Klaus [Gutachter] | |||||||
| Stichwörter: | Chlamydia, Pmps, Yaa3 | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Members of the genus Chlamydia are Gram negative bacteria, which can cause infections in several organisms. C. trachomatis and C. pneumoniae are responsible for widespread infections in humans, causing urogenital and respiratory tract infections, respectively. Chlamydiae exist in two morphological forms, the infectious elementary body (EB) and the metabolically active reticulate body (RB) and are obligate intracellular pathogens; therefore the adhesion is the first and essential step for the establishment of the infection; however the molecular details are ill-defined. In an attempt to identify new adhesin proteins for C. pneumoniae, a yeast display screening was performed and three hypothetical proteins were identified as adhesin candidates (Yaa1, Yaa2 and Yaa3). Yaa1 has been recently characterized as an adhesin and invasin and re-named LIPP, while Yaa2 and Yaa3 are still uncharacterized proteins. Polymorphic membrane proteins (Pmps) form a family of Chlamydia adhesins, with 9 members in C. trachomatis and 21 in C. pneumoniae and are relevant candidates for the development of a vaccine. Pmps are characterized by multiple GGA(I, L, V) and FxxN motifs along the functional passenger domain (PD). The PDs of many Pmps are processed during the infection. PD fragments of all 9 C. trachomatis and of representative C. pneumoniae Pmps harboring a high density of motifs (motif-rich) are characterized as adhesins and are relevant for the infection. Furthermore, a recombinant fragment of C. pneumoniae Pmp21, harboring only two motifs (motif-poor) is able to oligomerize, forming protofibrillar structures.
In the first part of this work, motif-rich and motif-poor protein fragments of the 9 C. trachomatis Pmps were produced and shown to interact with other Pmp proteins, independently of the density of motifs. Motif-poor (Ac, Fc and Gc) and motif-rich (D, H and I) Pmp proteins interacted strongly with each other, forming different species of homomeric and heteromeric high molecular weight (hMW) complexes with different characteristics when analyzed by Blue Native-PAGE, followed by second dimension SDS-PAGE and Size Exclusion Chromatography. Electron microscopy revealed that homomeric complexes formed small protofibrils, while heteromeric protofibrils were significantly longer. Homomeric and heteromeric Pmp oligomers had different adhesion strength to human cells. Interestingly, the adhesion-incompetent Ac Pmp was present in the adhesive fraction when in a heteromeric complex with adhesion-competent Pmps. Moreover, adhesion-competent but not adhesion-incompetent oligomers, pre-incubated with human cells, could block a subsequent C. trachomatis infection, showing their relevance for the infection. Conversely, adhesion-incompetent oligomers loaded onto the EB surface inhibited the infection, possibly by masking naturally exposed functional Pmp structures on the Chlamydia cell surface; while adhesion-competent oligomers loaded onto the EB surface did not impair the infectivity, possibly by binding and substituting the function of naturally exposed Pmp structures on the Chlamydia cell surface. Taken together, these data show the ability of C. trachomatis Pmps to generate homomeric and heteromeric complexes relevant for the infection. C. trachomatis may use Pmp complexes in vivo as adhesin structures, in order to reach the human receptor and, at the same time, using the numerous possible combinations of Pmp heteromeric complexes as a decoy mechanism, in order to escape the immune response. In the second part of this work the hypothetical C. pneumoniae protein Yaa3, identified in the yeast display screening, was analyzed. Yaa3 harbors a DUF720 domain, found only in other two proteins in C. pneumoniae and in the three homologous proteins in other Chlamydia species. The Yaa3 homologue in C. trachomatis harbors a type 3 secretion signal, suggesting that Yaa3 may be type 3 secreted as well. A protocol for heterologous expression, purification and refolding of Yaa3 was established. Recombinant Yaa3 could bind human cells and pre-incubation of human cells with rYaa3 inhibited a C. pneumoniae infection. A polyclonal anti-Yaa3 antibody detected expression of Yaa3 associated with the bacteria at 24 h post infection. These data indicate that Yaa3 might be type 3 secreted within the inclusion and loaded onto the EB cell surface, thus could be used in the next round of infection as an adhesin, relevant for the infection. Analyzing C. trachomatis Pmp oligomers and characterizing a novel C. pneumoniae adhesin candidate, further steps were taken in understanding the complex mechanisms, which Chlamydia adopts in order to establish the infection. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 30.05.2017 | |||||||
| Dateien geändert am: | 30.05.2017 | |||||||
| Promotionsantrag am: | 11.01.2017 | |||||||
| Datum der Promotion: | 23.03.2017 |
