Introduction

Innate immune cells detect invading bacterial pathogens by the recognition of pathogen-associated molecular patterns (pamps). Toll-like receptors (TLR), but also NACHT-LRRs (NLR) and RNA-helicases are crucial to recognize bacteria. While TLRs induce the production of proinflammatory cytokines and type I interferons, NLRs like NALP, IPAF or NAIP control the release of mature IL-1β via activation of caspase 1. Our group studies the interaction of three different pathogens with innate immune cells in vivo and in vitro: Chlamydophila pneumoniae, Chlamydia trachomatis and uropathogenic Escherichia coli.

Projects

A) Recognition of Chlamydia by Toll-like receptors

Chlamydial inclusion (green)

Chlamydia are obligate intracellular bacteria with an unique replication cycle. Elementary bodies of Chlamydia are metabolically inert, but are able to infect cells. Inside cells elementary bodies develop within a vacuole to reticular bodies, which are metabolically active and multiply. Subsequently, reticular bodies revert back to elementary bodies which leave the host cell to infect neighboring cells. The intracellular bacteria are able to survive within the host cell since they prevent the fusion of lysosomes with the bacteria-containing vacuole. C. trachomatis causes ocular trachoma, inclusion conjunctivitis and urogenital infections. C. pneumoniae is wide-spread within the population since up to 70% of healthy individuals are antibody-positive. Clinically, C. pneumoniae is responsible for a variety of diseases like atypical pneumonia, sinusitis and bronchitis. Furthermore, the organism was implicated in the pathogenesis of artherosclerosis, a conclusion which is heavily debated. Our own studies focus on the host-parasite relationship of this obligate intracellular bacterium with cells of the innate immune

Elementary (EB) and reticulate bodies (RB) of C. pneumoniae

system. We demonstrated previously that bone marrow derived dendritic cells are rapidly activated upon infection with C. pneumoniae. The cells recognize the microorganism via TLR2 and TLR4, up-regulate the expression of MHC class II molecules and co-stimulatory molecules like CD40, CD80, and CD86 and release pro-inflammatory cytokines like TNF and IL-12p40. To define the chlamydial TLR-ligand we were able to show that purified chlamydial endotoxin from C. trachomatis stimulates TNF-secretion in a TLR4-dependent manner. However, the whole microorganism C. trachomatis activated innate immune cells independent from TLR4, therefore we conclude that other chlamydial TLR-ligands are recognized during infection. Another potential pamp of Chlamydia is their heat shock protein 60 (HSP60). Exploration of the stimulatory capacity of purified HSP60 from C. pneumoniae for dendritic cells revealed that the protein stimulates these cells in a TLR2 and TLR4-dependent fashion. In vivo, purified HSP60 was able to induce the secretion of chemokines like KC and to recruit neutrophils upon intra-peritoneal injection. Both host responses were again dependent on TLR2 and TLR4. Unlike LPS, purified HSP60 failed to induce TNF-secretion. Thus, based on the cytokine profile induced, HSP60 stimulates innate immune cells in a special way which differs from LPS. TLR2 and TLR4 as well as the adapter molecule MyD88 were also crucial to detect C. pneumoniae in vivo, since mice double-deficient for TLR2 and TLR4 or deficient for MyD88 were impaired to secrete chemo- and cytokines upon pulmonary infection, failed to recruit polymorphonuclear neutrophils (PMN), were unable to control chlamydial burden during the later stage of the disease and finally succumbed to the infection. Using microarray technology we also reported that 221 genes are not or much weaker induced in infected MyD88-deficient mice compared to wild type mice. We published earlier that PMNs are able to increase chlamydial replication in vivo and in vitro. This surprising result explains why MyD88-deficient mice display a lower chlamydial burden compared to wild type mice at the beginning of the infection, since MyD88-deficient mice are unable to recruit PMNs. Furthermore, very recent data show that MyD88-deficient macrophages are impaired to increase the expression of iNOS and consequently production of nitric oxide (which is known to impede chlamydial replication) is reduced upon infection with C. pneumoniae. Current and future investigations: At present we explore the molecular basis for the PMN-mediated enforcement of chlamydial replication. We established that a soluble factor of PMNs is able to increase chlamydial replication and very recent experiments identified the identity of this factor. We intend to identify the molecular events which are induced in the infected host cell by this factor. To explore this mechanism, a better understanding of basic events concerning chlamydial uptake by host cells and the development of chlamydial inclusions is required. Using different chlamydial proteins which are either secreted or expressed in the cell wall or in the inclusion membrane we develop a potent vaccine which protects against chlamydial infections.

Publications:

1. Prebeck, S., C. Kirschning, S. Dürr, C. da Costa, B. Donath, K. Brand, V. Redecke, H. Wagner and T. Miethke. Predominant role of toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells. 2001. J. Immunol. 167: 3316-23

2. Prazeres da Costa, C., C.J. Kirschning, D. Busch, S. Dürr, L. Jennen, U. Heinzmann, S. Prebeck, H. Wagner and T. Miethke. Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae. 2002. Eur. J. Immunol. 32: 2460-70

3. Prazeres da Costa, C., F.-J. Neumann, A. Kastrati. I. Stallforth, M. Schmid, N. Jogetai, S. Prebeck, H. Wagner and T. Miethke. Role of IgG-seropositivity in early thrombotic events after coronary stent placement. 2003. Atherosclerosis. 166: 171-6

4. Prebeck, S., Brade, H., Kirschning, C.J., Prazeres da Costa, C., Dürr, S., Wagner, H. and T. Miethke. The gram-negative bacterium Chlamydia trachomatis L2 stimulates TNF-secretion independently of its endotoxin. 2003. Microbes Inf. 5: 463-70.

5. Da Costa CU, N. Wantia, C.J. Kirschning, D.H. Busch, N. Rodriguez, H. Wagner and T. Miethke. Heat shock protein 60 from Chlamydia pneumoniae elicits an unusual set of inflammatory responses via Toll-like receptor 2 and 4 in vivo. 2004. Eur. J. Immunol. 34:2874-84.

6. Rodriguez N., F. Fend, L. Jennen, M. Schiemann, N. Wantia, C. U. Prazeres da Costa, S. Dürr, U. Heinzmann, H. Wagner and T. Miethke. Polymorphonuclear neutrophils improve replication of Chlamydia pneumoniae in vivo upon MyD88-dependent attraction. 2005. J. Immunol. 174:4836-4844.

7. Rodriguez N., N. Wantia, F. Fend, S. Durr, H. Wagner and T. Miethke. Differential involvement of TLR2 and TLR4 in host survival during pulmonary infection with Chlamydia pneumoniae. 2006. Eur. J. Immunol. 36:1145-1155.

8. Rodriguez N., J. Mages, H. Dietrich, N. Wantia, H. Wagner, R. Lang and T. Miethke. MyD88-dependent changes in the pulmonary transcriptome after infection with Chlamydia pneumoniae. 2007. Physiol Genomics. 30:134-45.

B) Bacterial Toll/Interleukin-1 resistance proteins: analysis of their function

FlAsH-tagged TcpC (green) in the cytosol of macrophages

A number of human bacterial pathogens like uropathogenic E. coli strains, Brucella spp. and the S. aureus strainMSSA476 possess genes which encode for proteins containing a Toll/Interleukin 1 receptor (TIR) domain. Based on their aminoacid sequence and their predicted tertiary structure we hypothesized that these proteins might interfere with TLR-signaling. Our published data show, that the TIR-containing protein from the E. coli strain CFT073 (TcpC) and from B. melitensis (TcpB) lower the secretion of pro-inflammatory cytokines like TNF or IL-6. Both proteins bind to the adapter molecule MyD88 and impair signaling of TLRs like TLR2, TLR4, TLR5, TLR9, but not TLR3. CFT073 benefits from TcpC in that the bacteria accumulate intracellularly to higher numbers than a tcpC-deficient strain. TcpC is secreted by CFT073 and the protein is subsequently taken up by host cells into the cytosol. Due to these properties it is sufficient to add TcpC to the culture supernatant of innate immune cells in order to switch off TLR-signaling. The potency of this new virulence factor was explored in vivo. Compared to the tcpC-deficient strain we observed that the bacterial burden of CFT073 in the urine and kidneys is 100-1000 fold higher. Furthermore, CFT073 causes kidney abscesses while this complication was not detected with the tcpC-deficient strain. The secretion of TcpC by CFT073 can be prevented by the efflux pump inhibitor PAβN. This result provides the basis of a new treatment strategy which inactivates this potent new virulence factor and could be applied in addition to antibiotics. Current and future investigations: Presently we explore the mechanism how TLR-signaling is switched off upon binding of Tcps to MyD88. We also investigate, whether other recognition systems of innate immune cells are influenced by Tcps. Furthermore, we intend to define the bacterial secretion and eukaryotic uptake mechanisms which allow TcpC to move from the bacterium to the cytosol of host cells.

Publications:

1. Cirl C., A. Wieser, M. Yadav, S. Duerr, S. Schubert, H. Fischer, D. Stappert, N. Wantia, N. Rodriguez, H. Wagner, C. Svanborg and T. Miethke. Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins. 2008. Nat Med. 14:399-406.