Molecular Microbiology (1991)5(1). 109-115 0950382X9100014H Chlamydia trachomatis contains a protein similar to the Legionella pneumophiia mip gene product A. G. Lundemose,* S. Birkelund, S. J. Fey, P. Mose Larsen and G. Christiansen Institute of Medical Microbiology. Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmarl<. Summary A 27 kDa Chlamydia trachomatis L2 protein was characterized by the use of monoclonal antibodies and by two-dimensional gel electrophoresis. The pro- tein was shown to be located in the membrane of reticulate bodies as well as elementary bodies. Its synthesis could be detected from 10 hours post-infec- tion. Cloning and sequence analysis of the distal part of the gene revealed an open reading frame of 175 amino acids. Comparison of the deduced amino acid sequence with the NBRF data base revealed signifi- cant homology between the 27kOa chlamydial mem- brane protein and the product of the macrophage infectivity potentiator (mip) gene of Legionella pneu- mophiia. Introduction Chlamydia trachomatis is an obligate intraceilular microorganism with a unique biphasic life cycle (Schach- ter, 1988). The elementary bodies (EBs) are infectious but metabolically inactive. Prior to the infection, the EB adsorbs to the susceptible host cell and becomes inter- nalized. In the host cell the chlamydial EB is located within the phagosome. During the first 8 to 10 hours after internalization, transformation to the metabolically active, replicating reticulate bodies (RBs) occurs. Factors neces- sary for establishment of an intraoeilular chlamydial infec- tion are: (i) adhesins, which mediate adsorption of EB to the host cell; (ii) factor(s) that mediates internalization of the EB: (iii) factor(s) that inhibits fusion between phago- some and lysosomes; (iv) factor(s) that inhibits hostile cellular components which try to eliminate the intraceilular infectious particle. After the intracellular infection is estab- lished the RBs replicate during the next 20-30 hours within the cellular inclusion before they reorganize into EBs. Forty-eight hours after the start of infection, EBs burst the host cell {Moulder et al., 1984). Received 20 June. 1990; revised 18 August. 1990. "For correspondence. Tel. 86139711; Fax 86191277. Hackstadt (1986) and Wenman and Meuser (1986) have described two surface-focated proteins of 32 and 18 kilodaltons (kDa) which bind HeLa cell-surface components. These proteins are probably identical to the two lectin-binding proteins described by Swanson and Kuo (1990) and may be involved in the attachment to the host cell. The exact roie that these proteins play in the chiamydial life cycle, however, remains to be determined. Factors that mediate internalization and factors that inhibit fusion between phagosome and lysosomes are still unknown. Recently, heat-shock factors have been described as proteins that may play a protective role during estab- lishment of chlamydial intracellular growth {Birkelund ef ai. 1990; Lundemose etai, 1990; Danilition etai. 1990; Morrison e( a/., 1989). In this paper we characterize a 27 kDa membrane protein and describe cloning and sequencing of part of its gene. Data base homology search revealed strong homo- logy between the deduced amino acid sequence for the 175 C-terminal amino acids of the 27 kDa chlamydial polypeptide and the gene product of the macrophage infectivity potentiator (mip) gene of Legionelia pneumo- phiia (Engleberg etai, 1989). The m/pgene product is, in L pneumophiia, necessary for optimal intracellular infection in macrophages (Cianciotto et ai. 1989). Results Characterization and localization of the 27kDa chiamydial protein Characterization of the 27 kDa chlamydial protein by use of monoclonal antibodies {mAbs) 112.3 and 28.2 is shown in Table 1. Two mAbs from our library of monoclonal antibodies against C. traohomatis L2 reacted with a 27kDa protein determined by sodium dodecyl sulphate/ potyacrylamide gel eiectrophoresis (SDS-PAGE) and immunoblotting. The 27kDa protein was shown to be membrane associated. By Triton X-114 phase partitioning analysis {Birkelund etai, 1989; Bordier, 1981) integral membrane proteins will become incorporated into the Triton X-114 micelles while hydrophilic proteins become dissolved in the aqueous phase. SDS-PAGE-separated silver-stained chlamydial proteins present in each of the two phases are shown in Fig. 1. Monoclonal antibody 28.2 reacted with an antigen only present in the Triton X-114 phase (Fig. 1).