Characterization of Fifty Putative Inclusion Membrane Proteins Encoded in theChlamydia trachomatisGenome

Abstract

Although the Chlamydia trachomatis genome is predicted to encode 50 inclusion membrane proteins, only 18 have been experimentally localized in the inclusion membrane of C. trachomatis-infected cells. Using fusion proteins and anti-fusion protein antibodies, we have systematically evaluated all 50 putative inclusion membrane proteins for their localization in the infected cells, distribution patterns, and effects on subsequent chlamydial infection when expressed ectopically, as well as their immunogenicity during chlamydial infection in humans. Twenty-two of the 50 proteins were localized in the inclusion membrane, and 7 were detected inside the inclusions, while the location of the remaining 21 was not defined. Four (CT225, CT228, CT358, and CT440) of the 22 inclusion membrane-localized proteins were visualized in the inclusion membrane of Chlamydiainfected cells for the first time in the current study. The seven intra-inclusion-localized proteins were confirmed to be chlamydial organism proteins in a Western blot assay. Further characterization of the 50 proteins revealed that neither colocalization with host cell endoplasmic reticulum nor inhibition of subsequent chlamydial infection by ectopically expressed proteins correlated with the inclusion membrane localization. Interestingly, antibodies from women with C. trachomatis urogenital infection preferentially recognized proteins localized in the inclusion membrane, and the immunodominant regions were further mapped to the region predicted to be on the cytoplasmic side of the inclusion membrane. These observations suggest that most of the inclusion membrane-localized proteins are both expressed and immunogenic during C. trachomatis infection in humans and that the cytoplasmic exposure may enhance the immunogenicity.

CPAF selectively degrades chlamydial T cell antigens for inhibiting antigen presentation

“…GST-fusion recombinant proteins of D111, D628, D622, D702, D509, and D694 were expressed in a pGEX expression system (GST was fused to the N terminus of the chlamydial proteins) and purified by glutathione-conjugated agarose beads (Amersham Biosciences, Pittsburgh, USA) as described previously [41][42][43]. To monitor the processing of chlamydia T cell antigens by CPAF, a cell-free pre-incubation assay was used as described elsewhere [36].…”

Cloning, Expression, and Biophysical Investigations of Truncated Inclusion Protein A (IncA) from Chlamydia trachomatis

“…28 The common feature distinguishing inclusion proteins, however, is their 50-to 80-amino-acid, bi-lobed, hydrophobic domain that is localized to the inclusion body membrane. 19,23,24,27,29 Currently, at least 9 proteins are identified with this feature, and each inclusion protein has a unique function in the development process of the pathogen. 16,28 Several inclusion proteins have been investigated in terms of function and localization.…”

“…24 Second, bioinformatics and modeling suggest that the polar residue located in the center of the motif may organize the coiled coils in the hydrophilic domain into a single amphipathic structure. 27,37 SNAREs have a helix formed with either a glutamine or arginine residue located in the center.…”

“…2, 4, 5, 7, 9-14, 16, 19-21, 23 The inclusion body has two functions: (1) to protect Chlamydiae during replication and (2) to serve as a gate while the pathogen interacts with its host. 16,24 Using the inclusion, the pathogen successfully eludes endosomes that target foreign matter for degradation by separating from the endocytic pathway early in its development. Chlamydial species are true obligate pathogens because they are unable to synthesize adenosine triphosphate (ATP).…”

“…3,23,27 Despite 36 to 59 putative inclusion proteins identified in the C. trachomatis genome, only 23 have been identified between five species (muridarum, cavie, felis, pneumonia, and trachomatis). 23,24 Effector proteins that modify the inclusion body, and are secreted by T3SS, include IncA proteins. IncA from C. trachomatis is the focus of this investigation and described later.…”