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.
Shaw
Allan C
AC
Department of Medical Microbiology and Immunology, University of Aarhus, The Bartholin Building, DK-8000 Aarhus C, Denmark.
Vandahl
Brian B
BB
Larsen
Martin R
MR
Roepstorff
Peter
P
Gevaert
Kris
K
Vandekerckhove
Joël
J
Christiansen
Gunna
G
Birkelund
Svend
S
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D001483:Base Sequence; D002460:Cell Line; D002692:Chlamydia trachomatis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D016993:Chlamydophila pneumoniae / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D010450:Endopeptidases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000494:pharmacology; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000907:Antibodies, Bacterial; D004269:DNA, Bacterial; D010446:Peptide Fragments; D011480:Protease Inhibitors; D010450:Endopeptidases