NF-kappaB and inhibitor of apoptosis proteins are required for apoptosis resistance of epithelial cells persistently infected with Chlamydophila pneumoniae.

Infection with Chlamydophila pneumoniae (Cpn) renders host cells resistant to apoptosis induced by a variety of stimuli. While modulation of apoptosis has been extensively studied in cells acutely infected with Cpn, very little is known on how persistent chlamydial infection influences host cell survival. Here we show that epithelial cells persistently infected with Cpn resist apoptosis induced with TNFalpha or staurosporine. Cpn induced the activation of nuclear factor kappa B (NF-kappaB) and inhibition of NF-kappaB with a chemical inhibitor or by silencing expression of the p65 subunit sensitized infected cells for apoptosis induction by staurosporine or TNFalpha. Persistent infection resulted in the upregulation of the NF-kappaB regulated inhibitor of apoptosis protein 2 (cIAP-2) but not inhibitor of apoptosis protein 1 (cIAP-1). Interestingly, silencing of either cIAP-1 or cIAP-2 sensitized infected cells, suggesting that IAPs play an important role in the apoptosis resistance of persistently infected cells.

Presentation of differentially regulated proteins within a web-accessible proteome database system of microorganisms.

Web-accessible proteome databases represent indispensable tools for quantitative and comparative proteomics research. The majority of two-dimensional gel electrophoresis (2-DE) databases contains clickable 2-DE gel images and descriptive textual information such as protein name, Mr/pI values, methods of identification, cellular localization and other information on proteins. Although a great part of the work in comparative proteomics consists of the analysis of 2-DE gels using image analysis approaches, most proteome databases lack the ability to present protein abundance data and their alterations within experiments via the web. Now, differentially regulated proteins detected in microbial experiments by quantitative gel image analysis are presented in a web-accessible relational database DIFF (Differentially Regulated Proteins). The DIFF database is a part of the proteome database system for microbial research available at http://www.mpiib-berlin.mpg.de/2D-PAGE.

Action and reaction: Chlamydophila pneumoniae proteome alteration in a persistent infection induced by iron deficiency.

Chlamydophila pneumoniae is an obligate intracellular pathogen implicated in a variety of acute and chronic diseases. Long-term infections are associated with a persistent life stage, in which bacteria can stay for years. They are less accessible to antibiotic treatment but still prone to sustain an inflammatory response. Different in vitro models have been established to mimic and characterize chlamydial persistency. For C. pneumoniae and Chlamydia trachomatis, altered metabolic activities and changed antigenic profiles compared to acute infections have been reported. Most studies including transcriptome and proteome analyses describe persistency induced by IFNgamma treatment. Here, we use iron depletion of the infected cell culture that also leads into persistent infection. We describe differently regulated proteins found by subtractive proteome analysis comparing two early stages of infection with and without addition of the iron chelator deferoxamine-mesylate. While only one bacterial protein was up-regulated during iron deficiency up to 24 h post infection (p.i.), 11 were found to be up-regulated and eight to be down-regulated from 24-48 h p.i. Two down-regulated proteins could be identified by peptide mass fingerprinting as thioredoxin reductase and chromosome partitioning protein (ParB). The latter is involved in chromosome segregation. Thus, using a comparative approach we identified on a proteome level down-regulation of ParB in persistent chlamydial forms, which is in agreement with previous results describing changes in cell division and atypical altered morphology of persistent Chlamydiae.

From the inside out--processing of the Chlamydial autotransporter PmpD and its role in bacterial adhesion and activation of human host cells.

Polymorphic membrane protein (Pmp)21 otherwise known as PmpD is the longest of 21 Pmps expressed by Chlamydophila pneumoniae. Recent bioinformatical analyses annotated PmpD as belonging to a family of exported Gram-negative bacterial proteins designated autotransporters. This prediction, however, was never experimentally supported, nor was the function of PmpD known. Here, using 1D and 2D PAGE we demonstrate that PmpD is processed into two parts, N-terminal (N-pmpD), middle (M-pmpD) and presumably third, C-terminal part (C-pmpD). Based on localization of the external part on the outer membrane as shown by immunofluorescence, immuno-electron microscopy and immunoblotting combined with trypsinization, we demonstrate that N-pmpD translocates to the surface of bacteria where it non-covalently binds other components of the outer membrane. We propose that N-pmpD functions as an adhesin, as antibodies raised against N-pmpD blocked chlamydial infectivity in the epithelial cells. In addition, recombinant N-pmpD activated human monocytes in vitro by upregulating their metabolic activity and by stimulating IL-8 release in a dose-dependent manner. These results demonstrate that N-PmpD is an autotransporter component of chlamydial outer membrane, important for bacterial invasion and host inflammation.