Chlamydia trachomatis is a human pathogen causing sexual transmitted diseases and trachoma. C. trachomatis serovars L1-L3 cause lymphogranuloma venereum, an invasive form that results in lymphadenitis of regional lymph nodes. C. trachomatis serovars A-K only grows within epithelial cells, serovars A-C cause trachoma and serovars D-K are sexual transmitted infections. C. trachomatis is an obligate intracellular bacterium with a unique biphasic developmental cycle, in which the elementary body (EB) (300 nm) induces its own uptake by the host cell into a phagosome. Chlamydia prevent the fusion of the phagosome with lysosomes . EB transform to reticulate bodies (RB) (1000 nm) and an inclusion is formed inside the host cell cytoplasm. RB divides and after 24-48 hours transform to EB, the cell bursts and the EB can infect new cells.

 Both EB and RB secrete effector proteins across the inclusion membrane by the type III secretion system (T3S) whereby proteins can be translocated from the chlamydial cytoplasm to the host cell cytoplasm. The chlamydial inclusion membrane is modified by chlamydial inclusion membrane proteins (Inc), which are secreted via T3S, and they subsequently interact with the inclusion membrane from the cytoplasmic side. By searching the C. trachomatis L2 genome for Inc proteins having a characteristic hydrophobic bilobic domain of at least 50 amino acids with a strong hydrophobicity index, Bannantine et al. identified 46 candidate Inc proteins. When predicted proteins in C. trachomatis serovar D genome were inspected for presence of the bilobic hydrophobic signature 59 proteins were found. And all Inc genes are transcribed. The predicted Inc proteins can be divided into 3 types; type I where the hydrophobic stretch is in the N-terminal end; type II with the hydrophobic stretch is in the C-terminal part; and type III where two or more hydrophobic stretches are present. Type I is the most common type. Proteins with this motif are rarely found outside the order Chlamydiales . and Inc proteins encoded from a specific genome do not share primary protein sequence similarities.

The high number of Inc proteins of which at least 21 have been shown to be present in the inclusionla membrane suggests that they play a central role in the unique biology of the chlamydial inclusion formation. However, identification of expressed Inc proteins is difficult due to their type III secretion into the inclusion membrane. By combining ultrahigh pressure liquid chromatography (UHPLC) and a mass spectrometer with a dynamic range of 105 and sub-ppm mass-accuracy, Wiśniewski et al., identified 7,093 HeLa cell proteins using LC coupled with tandem mass spectrometry (MS/MS). On the background of this number of HeLa cell proteins that were speculated whether they would be able to identify additional Inc proteins by analyzing the entire protein content of infected host cells by LC-MS/MS. It was therefore characterized expression of Inc proteins in C. trachomatis L2 infected HeLa cells 24 and 43 hours post infection (hpi).

 Using this strategy we identified expression but not localization of 19 Inc proteins of which six previously were described as hypothetical.

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