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The interaction of Chlamydia pneumoniae with its host cell: adhesion, internalization and intracellular differentiation

Johannes H. Hegemann - BMFZ-Bericht 2007 - 2008


Biological and medical background

Chlamydiaceae are obligate intracellular, Gram-negative bacterial pathogens which cause a variety of important human diseases in animals and humans. Chlamydia trachomatis is responsible for a spectrum of acute ocular and genital tract infections and for chronic diseases that lead to preventable blindness and tubal-factor infertility, such as trachoma and salpingitis, respectively (with more than 90 million new cases each year). Chlamydia pneumoniae is an important respiratory pathogen with an epidemiological prevalence of more than 50 % worldwide and associated with 5 to 10 % of community-acquired cases of pneumonia worldwide. It has also been implicated with the development of atherosclerosis and artery disease and several other chronic diseases. Chlamydiae possess a unique biphasic developmental cycle which is characterized by two extremely different bacterial cell forms: the extracellular infectious Elementary Body (EB) and the intracellular non-infectious metabolically active Reticulate Body (RB). Thus chlamydiae exhibit a twofold differentiation process. Most of the molecular mechanisms involved in these processes are currently unknown. Thus our project aims at the understanding of 2 different aspects of the chlamydial developmental cycle: (i) the adhesion and internalization of the bacteria; (ii) the two different forms of differentiation processes: differentiation of the bacteria and the pathogen-provoked differentiation processes occurring within the infected host cell.



We have characterised the 3 C. pneumoniae cell surface proteins OmcB, Pmp21 and GroEL1. Presence of recombinant protein or of antibodies directed against these proteins results in a reduced infection indicating the relevance of these proteins for the chlamydial infection. The eukaryotic receptor for OmcB adhesion are heparan sulfate-like glycosaminoglycan structures on the human cell surface. Interestingly in C. trachomatis we observed serovar-specific heparin-dependencies: adhesion of the LGV serovar OmcB is heparin-sensitive, while E serovar OmcB adhesion to HEp-2 cells is completely heparin-resistant possibly reflecting changes in cell tropism and disease pattern. The recent identification of the two new proteins Cap1 and Cap2 as potential adhesins suggests a complex repertoire of pathogen - host cell interaction factors.

Our transcriptome analysis of the C. pneumoniae developmental cycle resulted in the identification of a number of operons transcribed late in the infection cycle. We concentrated on the functional analysis of these proteins, as they are possibly involved in the assembly of the new EBs (and the EB cell surface), where they may act as adhesins, or being loaded onto the type three secretion machinery. Thus these late expressed proteins might in fact be necessary early in the next round of infection to establish adhesion and subsequent uptake. Our adhesion studies reveal that some gene products are indeed possibly involved in the adhesion process (e.g. Cap1 and Cap2). In order to identify potential effector proteins we expressed these proteins in the eukaryotic model cell Saccharomyces cerevisiae and looked for growth effects. A series of strong phenotypes observed for several of these proteins suggests a role as effector proteins. The further characterisation of these adhesion and effector proteins will be the main goal during the forthcoming period. We have already begun to study the effector protein Cpn0572 in yeast as a eukaryotic model system. Expression of Cpn0572 in yeast results in a severe growth arrest, which was accompanied by complete loss of actin structures and formation of a big actin clump which completely co-localizes with Cpn0572. Time course experiments suggest that Cpn0572 aggregation has to occur prior to actin accumulation. An in vivo domain analysis identified 3 regions of the protein involved in protein-protein interaction, actin recruitment and bundling. Finally biochemical assays using pyrene-labelled actin reveal that recombinant Cpn0572 can initiate actin polymerization in vitro.

Goals for 2009/2010

In the coming 2 years we would like to focus on the following topics:

We plan to achieve a detailed molecular understanding of the adhesion process of chlamydiae to human cells. Binding and subsequent uptake of infectious EBs by the eukaryotic cell is initiated by adhesin - receptor interactions. We plan to solve the three-dimensional structure of the OmcB adhesins from C. pneumoniae and the two serovars C. trachomatis L1 and E together with a detailed mutational analysis of the GAG binding site. Serovar E OmcB binding to epithelial and endothelial cells is not heparin sensitive, while the other 2 OmcB proteins adhere to heparin-like host cell GAGs. The 3D structure of OmcB will help us to determine the molecular basis for the observed heparin sensitivity/resistance phenotype, which most likely has important consequences for cell tropism and disease pattern. We want to characterise which heparin-like GAG is recognized by OmcB. It is feasible to assume that there might be molecular differences between the GAG structure recognised by OmcB from C. pneumoniae (respiratory pathogen) and C. trachomatis L1 (systemic pathogen) and E (local urogenital tract pathogen).

We plan the characterisation of the newly identified adhesion factors Cap1 and Cap2 and identification of their eukaryotic receptors using the yeast display technology. Moreover we want to characterise the possible combined action of the identified adhesins in different cell types.

Finally we plan to analyse chlamydial effector proteins using the model eukaryote Saccharomyces cerevisiae. In particular we plan a deep analysis of the actin modulating protein Cpn0572 and its interaction partners in yeast and human cells.

Publikations belonging to the topics

Moelleken K, Hegemann JH: The Chlamydia outer membrane protein OmcB is required for adhesion and exhibits biovar-specific differences in glycosaminoglycan binding. Mol Microbiol. 2008; 67:403-19.

Wuppermann FN, Mölleken K, Julien M, Jantos CA, Hegemann JH: Chlamydia pneumoniae GroEL1 protein is cell surface-associated and required for infection of HEp-2 cells. J Bacteriol. 2008; 190:3757-67.



(A) In a close cooperation with Prof. Dr. Köhrer, head of the molecular biology laboratory, we analyzed the data from the transcriptome analysis of the chlamydial infection cycle which we together had previously performed using a PCR based, home-made DNA microarray.

(B) Dr. Metzger, head of the proteome laboratory, identified chlamydial and yeast proteins obtained as interaction partners of chlamydial proteins by mass spectrometry. Furthermore we obtained synthetic peptides from this lab for functional studies of Pmp21.

(C) We have an on-going collaboration with Prof. Dr. B. Royer-Pokora, Institute of Human Genetics. We perform functional studies of hMLH1- und hMSH2- missense variants and develop novel assays (funding by the Deutsche Krebshilfe until 2011).

(D) We collaborated with Prof. Hengge, Skin Clinic, on the molecular characterization of the black skin melanoma. This work has been published:

Nambiar S, Mirmohammadsadegh A, Hassan M, Mota R, Marini A, Alaoui A, Tannapfel A, Hegemann JH, Hengge UR: Identification and functional characterization of ASK/Dbf4, a novel cell survival gene in cutaneous melanoma with prognostic relevance. Carcinogenesis 2007; 28:2501-10.

Nambiar S, Mirmohammadsadegh A, Hassan M, Hegemann JH, Hengge UR.: Transcriptional regulation of ASK/Dbf4 in cutaneous melanoma is dependent on E2F1. Exp Dermatol. 2008; 17:986-91.