Proteome analysis of Chlamydia pneumoniae - proteins at the Chlamydia-host cell interface

This PhD dissertation was accepted by the Faculty of Health Sciences, University of Aarhus and defended on June 4, 2004.

Official opponents: Professor Ian Clarke, England, Vice-president Henning Løwenstein DMSc, Professor Peter Oluf Schiøtz.

Correspondence: Brian Stidsen Vandahl, Skovvangsvej 154 st. tv. DK-8200 Aarhus N. E-mail: vandahl@medmicro.au.dk

Dan Med Bull 2004;51:306.

ABSTRACT

Chlamydiae are obligate intracellular gram-negative bacteria characterized by a unique bi-phasic developmental cycle comprising an extracellular, infectious elementary body (EB) and an intracellular dividing reticulate body (RB). Throughout the intracellular development the bacteria stay inside a vacuole called the chlamydial inclusion. Chlamydia pneumoniae causes respiratory diseases and has been associated with atherosclerosis.

A comprehensive proteome map of C. pneumoniae was established in which 263 protein spots from two-dimensional gels were identified by mass spectrometry. This study provided a novel insight into the protein content of EBs, and for the first time four members of a putative type III secretion system were shown to be expressed by Chlamydia .

The protein composition of the Chlamydia outer membrane complex was likewise investigated by proteome analysis. Polymorphic membrane proteins (Pmps) were found to be abundant constituent of the outer membrane complex and several Pmps were found to be exposed at the bacterial surface by immunofluorescence microscopy.

Important potential mediators of interactions between Chlamydia and its host cell are proteins secreted from Chlamydia into the cell during infection. The identification of such proteins is difficult because the host cell cytoplasm of Chlamydia -infected cells cannot be purified. This problem was circumvented by a comparative proteomics approach, which led to the identification of the first known secreted Chlamydia protein, CPAF. Another research group simultaneously found CPAF to be secreted and showed that its function is to down-regulate host major histocompatibility class I and II presentation.

Also the previoulsy uncharacterized protein Cpn0796 was found to be secreted from C. pneumoniae . Cpn0796 is localized in the membrane of Chlamydia in young inclusions whereas an N-terminal part of Cpn0796 is secreted into the host cell cytoplasm at later times. Cpn0796 was proposed to be an autotransporter and it is the first example of secretion into the host cell cytoplasm of a proposed Chlamydia autotransporter. The finding in the host cell cytoplasm of Cpn0796 suggests that an unknown transport mechanism exists for translocation over the inclusion membrane.