Host cell Golgi anti-apoptotic protein (GAAP) and growth of Chlamydia pneumoniae.

Chlamydia pneumoniae protein CPn0809 is a type three secretion system substrate, the exact function of which in infection pathogenesis has remained unknown. In this study, we identified by yeast two-hybrid screening a potential host cell interaction partner of CPn0809, Golgi anti-apoptotic protein (GAAP), a conserved protein found in eukaryotic cells. GAAP gene is expressed at relatively constant levels and its expression remained stable also after C. pneumoniae infection. The interaction between GAAP and C. pneumoniae was suggested by transfection studies. GAAP knock-down by siRNA in infected A549 cells resulted in an increased number of C. pneumoniae genomes and growth of the bacteria as judged by quantitative PCR and inclusion counts, respectively. Silencing of GAAP did not make the A549 cells more susceptible to apoptosis per se, and infection with C. pneumoniae prevented staurosporin-induced apoptosis also in transfected cultures. Taken together, the proposed interaction between C. pneumoniae and GAAP modulates bacterial growth in A549 cells.

Flotillin-1 (Reggie-2) contributes to Chlamydia pneumoniae growth and is associated with bacterial inclusion.

Chlamydiae are obligate intracellular pathogens replicating only inside the eukaryotic host. Here, we studied the effect of human flotillin-1 protein on Chlamydia pneumoniae growth in human line (HL) and A549 epithelial cell lines. RNA interference was applied to disrupt flotillin-1-mediated endocytosis. Host-associated bacteria were detected by quantitative PCR, and C. pneumoniae growth was evaluated by inclusion counts. C. pneumoniae attachment to host cells was unaffected, but bacterial intracellular growth was attenuated in the flotillin-1-silenced cells. By using confocal microscopy, we detected flotillin-1 colocalized with the inclusion membrane protein A (IncA) in the C. pneumoniae inclusion membranes. In addition, flotillin-1 was associated with IncA in detergent-resistant membrane microdomains (DRMs) in biochemical fractioning. These results suggest that flotillin-1 localizes to the C. pneumoniae inclusion membrane and plays an important role for intracellular growth of C. pneumoniae.

IFN-gamma induced persistent Chlamydia pneumoniae infection in HL and Mono Mac 6 cells: characterization by real-time quantitative PCR and culture.

Growth of Chlamydia pneumoniae during gamma interferon (IFN-gamma) induced persistent infection in epithelial (HL) and monocyte-macrophage (Mono Mac 6) cell lines was studied by a quantitative real-time PCR and passage. When HL cultures were treated with IFN-gamma (25 U/ml), the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited, and in contrast to the untreated cultures, no second cycle of infection was observed. The estimated doubling time of C. pneumoniae genomes was 6-7 h in both IFN-gamma treated and untreated HL cultures. At 72 h post inoculation, most infectious EBs were released from untreated cultures, whereas in IFN-gamma treated HL cells >90% of C. pneumoniae genomes were in non-infectious form. A higher dose (1000 U/ml) of IFN-gamma was needed to restrict growth of C. pneumoniae in Mono Mac 6 cells. In untreated Mono Mac 6 cultures, the growth curve of C. pneumoniae resembled that observed in HL cells, except that no second cycle of infection could be detected. In IFN-gamma treated Mono Mac 6 cultures, the number of infectious C. pneumoniae EBs recovered decreased gradually after 3 days post inoculation, while C. pneumoniae genome load remained unaltered suggesting persistence of C. pneumoniae also in these cells.

Production of Chlamydia pneumoniae proteins in Bacillus subtilis and their use in characterizing immune responses in the experimental infection model.

Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the alpha-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the alpha-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.

No evidence of acute Chlamydia pneumoniae infection in patients with Bell's palsy.

The cause of Bell's palsy remains unknown even though available evidence suggests that infection could be a factor. In recent studies, Chlamydia pneumoniae has been associated with neurologic diseases such as multiple sclerosis. In the present study, the association of C pneumoniae with Bell's palsy was studied with the use of serology and polymerase chain reaction to test tear fluid and peripheral blood mononuclear cells from 21 patients with Bell's palsy and 21 control subjects. C pneumoniae DNA was detected from tear fluid samples in 1 patient with Bell's palsy and in 2 healthy control subjects. Whether this indicates earlier disease or subclinical infection remains to be studied. However, an association between Bell's palsy and acute C pneumoniae infection could not be shown.