Svend Birkelund

Interleukin‐1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection

Chlamydia trachomatis infection is associated with severe Fallopian tube tissue damage leading to tubal infertility and ectopic pregnancy. To explore the molecular mechanisms behind infection an ex vivo model was established from human Fallopian tubes and examined by scanning electron microscopy and immunohistochemistry. Extensive tissue destruction affecting especially ciliated cells was observed in C. trachomatis infected human Fallopian tube organ culture. Interleukin‐1 (IL‐1) produced by epithelial cells was detected after infection. Addition of IL‐1 receptor antagonist (IL‐1RA) completely eliminated tissue destruction induced by C. trachomatis. The anti‐inflammatory cytokine IL‐10 reduced the damaging effect of C. trachomatis infection, however, to a lesser extent than IL‐1RA. Furthermore, IL‐1 was found to induce IL‐8, a neutrophil attractant, using a signal transduction pathway involving p38 MAP kinase. Consequently, IL‐1 has the potential to generate a cellular infiltrate at the site of infection in vivo. Blocking the IL‐1 receptors by IL‐1RA eliminated tissue destruction and cytokine production. Hence, these studies show the importance of IL‐1 in initiating the tissue destruction observed in the Fallopian tube following C. trachomatis infection. Because leukocytes are absent in the ex vivo model, this study strongly indicates that IL‐1 is the initial proinflammatory cytokine activated by C. trachomatis infection.

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection.

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia‐containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule‐associated Chlamydia vesicles also collocated with tyrosine‐phosphorylated McCoy cell proteins. After migration, the Chlamydia‐containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule‐dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule‐dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR‐1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR‐1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.

Proteome analysis of the Chlamydia pneumoniae elementary body.

Chlamydia pneumoniaeis an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniaecultivated in Hep‐2 cells were 35S‐labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two‐dimensional gel electrophoresis. Excised protein spots were in‐gel digested with trypsin and peptides were concentrated on reverse‐phase chromatographic beads for identification analysis by matrix‐assisted laser desorption/ionization‐mass spectrometry. In the pH range from 3–11, 263 C. pneumoniaeprotein spots encoded from 167 genes were identified. These genes constitute 15 % of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.

Characterization of a secreted Chlamydia protease

Chlamydiae are obligate intracellular bacteria that are important human pathogens. The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified. Most likely, effector proteins are secreted in order to promote infection. Effector proteins cannot be identified by motif or similarity searches. As a new strategy for identification of secreted proteins we have compared 2D‐PAGE profiles of [35S]‐labelled Chlamydia proteins from whole lysates of infected cells to 2D‐PAGE profiles of proteins from purified Chlamydia. Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method. Two protein spots were identified among the candidates. These represent fragments of the ‘chlamydial protease‐ or proteasome‐like activity factor’ (CPAF) and were clearly present in 2D‐PAGE profiles of whole lysates of infected cells but absent from purified Chlamydia. CPAF was recently identified by Zhong and colleagues as a secreted protease which cleaves host cell transcription factors essential for MHC class I and II antigen presentation. The identification of CPAF in this paper verifies the applicability of the described method for the identification of secreted proteins. We extend the findings by Zhong et al. by proteome studies of expression and turnover of C. trachomatis CPAF showing that the degradation of C. trachomatis D CPAF in the host cell is very limited. Furthermore, we show that two fragments of CPAF exist in C. pneumoniae as well as in C. trachomatis.

Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram‐negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), gels with purified elementary bodies (EB) and auto‐radiography of gels with 35S‐labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N‐terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2‐D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.

Determination of PCR efficiency in chelex-100 purified clinical samples and comparison of real-time quantitative PCR and conventional PCR for detection of Chlamydia pneumoniae

BackgroundChlamydia pneumoniae infection has been detected by serological methods, but PCR is gaining more interest. A number of different PCR assays have been developed and some are used in combination with serology for diagnosis. Real-time PCR could be an attractive new PCR method; therefore it must be evaluated and compared to conventional PCR methods.ResultsWe compared the performance of a newly developed real-time PCR with a conventional PCR method for detection of C. pneumoniae. The PCR methods were tested on reference samples containing C. pneumoniae DNA and on 136 nasopharyngeal samples from patients with a chronic cough. We found the same detection limit for the two methods and that clinical performance was equal for the real-time PCR and for the conventional PCR method, although only three samples tested positive. To investigate whether the low prevalence of C. pneumoniae among patients with a chronic cough was caused by suboptimal PCR efficiency in the samples, PCR efficiency was determined based on the real-time PCR. Seventeen of twenty randomly selected clinical samples had a similar PCR efficiency to samples containing pure genomic C. pneumoniae DNA.ConclusionsThese results indicate that the performance of real-time PCR is comparable to that of conventional PCR, but that needs to be confirmed further. Real-time PCR can be used to investigate the PCR efficiency which gives a rough estimate of how well the real-time PCR assay works in a specific sample type. Suboptimal PCR efficiency of PCR is not a likely explanation for the low positivity rate of C. pneumoniae in patients with a chronic cough.

Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry

Chlamydia pneumoniae is a common cause of community-acquired pneumonia and it has been associated with atherosclerosis. C. pneumoniae has usually been diagnosed by serology using a microimmunofluorescence test, but more recently polymerase chain reaction (PCR) has been viewed as an advantageous alternative. We developed a quantitative real-time PCR for detection of C. pneumoniae. Primers were targeted for the pmp4 gene, and the PCR fragment was detected real-time with a fluorescence resonance energy transfer probe set using a LightCycler instrument. The PCR was used on DNA released from 50 microm sections of paraffin-embedded formalin-fixed lung tissue from experimentally infected mice. Thereby, the number of C. pneumoniae genomes was determined. To our knowledge this is the first time quantification of C. pneumoniae DNA has been attempted on paraffin-embedded formalin-fixed tissue. C. pneumoniae-specific immunohistochemistry (IHC) was done on 5 microm sections adjacent to the sections used in PCR, and the number of inclusions were counted in each section. Good correlation was found when comparing results from PCR and IHC, which is in contrast to many previous studies.

The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029

BackgroundChlamydiae are obligate intracellular bacteria, which are important human pathogens. Genome sequences of C. trachomatis and C. pneumoniae have revealed the presence of a Chlamydia specific gene family encoding polymorphic outer membrane proteins, Pmps. In C. pneumoniae the family comprises twenty-one members, which are all transcribed. In the present study, the expression, processing and localisation of the sixteen full-length Pmps in C. pneumoniae strain CWL029 have been further investigated by two-dimensional gel electrophoresis and immunofluorescence microscopy.ResultsTen Pmps were identified in elementary bodies (EBs). Eight of these were investigated with respect to time dependent expression and all were found to be up-regulated between 36 and 48 hours post infection. Antibodies against Pmp6, 8, 10, 11 and 21 reacted with chlamydiae when infected cells were formalin fixed. Pmp6, Pmp20 and Pmp21 were found in cleaved forms, and the cleavage sites of Pmp6 and Pmp21 were identified.ConclusionsThe Pmps are heavily up-regulated at the time of conversion of RB to EB, and at least ten Pmps are present in EBs. Due to their reaction in formalin fixation it is likely that Pmp6, 8, 10, 11 and 21 are surface exposed. The identified cleavage sites of Pmp6 and Pmp21 are in agreement with the theory that the Pmps are autotransporters.

Chlamydia trachomatis Mip-like protein

A 27 kDa Chlamydia trachomatis Mip‐like protein with homology of a 175‐amino‐acid C‐terminal fragment to the surface‐exposed Legionella pneumophila mip‐ gene product has previously been described. In this paper the entire chlamydia Mip‐like sequence of C. trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C‐terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506‐binding proteins. The chlamydial mip‐like gene was detected by polymerase chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip‐like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip‐like protein failed to demonstrate surface‐exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno‐gold electron microscopy. However, a complement‐dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N‐terminal fragment of the Mip‐like protein suggesting that antibody‐accessible epitopes are present on infectious EBs.

Chlamydia trachomatis contains a protein similar to the Legionella pneumophila mip gene product

A 27kDa Chlamydia trachomatis L2 protein was characterized by the use of monoclonal antibodies and by two‐dimensional gel electrophoresis. The protein was shown to be located in the membrane of reticulate bodies as well as elementary bodies. Its synthesis could be detected from 10 hours post‐infection. Cloning and sequence analysis of the distal part of the gene revealed an open reading frame of 175 amino acids. Comparison of the deduced amino acid sequence with the NBRF data base revealed significant homology between the 27kDa chlamydial membrane protein and the product of the macrophage infectivity potentiator (mip) gene of Legionella pneumophila.