Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis

Abstract

Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins). Incs represent 7-10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH) method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF, and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia.

Keywords: BACTH; Chlamydia; Inc protein; bacterial two-hybrid system; inclusion membrane proteins; protein-protein interactions.

Figure 1

BACTH analysis of C. trachomatis IncA and its sub-domains interactions. (A) Schematic representation of the different domains of IncA (Ct119), with numbers indicating the amino acid residues. TM designates the transmembrane domain and SL1 and SL2 the two SNARE-like motifs. (B) The β-galactosidase activity of DHT1 co-expressing the indicated fusion proteins was measured in liquid cultures as described in “Materials and Methods.” The reported values, expressed in relative units (RU), correspond to the average obtained from eight clones tested for each interaction with the standard deviation given in parentheses. The “-” corresponds to the empty vectors. (C) Western blot analysis of the subcellular localization of the T18-IncA protein in E. coli DHT1. Exponentially growing DHT1 cells, co-expressing the T25-IncA and T18-IncA fusion proteins, were collected, lysed by sonication, and fractionated by ultracentrifugation (see Material and Methods). Proteins from the soluble (2), membrane (3) or insoluble (4) fractions were separated by gel electrophoresis on a 12% SDS-gel, transferred onto a PVDF membrane, and revealed with an anti-T18 monoclonal antibody (3D1). Positions of molecular weight markers (in kDa) are indicated on the left of the Figure while the expected position of T18-IncA is indicated by a blue arrow. In lane 1, a polypeptide corresponding to a 65 kDa fragment of CyaA adenylate cyclase (AC65) was run as a positive control for the anti-T18 antibody detection.

Figure 2

BACTH analysis of interactions between Ct222 sub-domains. (A) Schematic representation of the different domains of Ct222 with numbers indicating the amino acid residues. FL means full-length proteins. (B) BACTH interaction assays between the different domains of Ct222 and Ct850 are listed in this table and expressed in relative units (RU). All Inc proteins were fused to the C-terminus of T25 or T18. NT: not tested. ND: not detected as the corresponding transformants formed only white colonies on indicator plates and therefore should display only background levels of β-galactosidase activity (<10 RU).

Figure 3

BACTH analysis of the homotypic interactions of C. trachomatis Incs. DHT1 bacteria were co-transformed with plasmids expressing the T25 and T18 fusions to the indicated inc proteins. The β-galactosidase activity values correspond to the average obtained from eight independent colonies. The positive control C+ corresponds to the interaction between the T25-FtsW and T18-FtsI proteins of E. coli (Karimova et al., 1998), while the negative control C- to cells harboring the empty vectors pST25 and pUT18C.

Figure 4

Quantitative transcriptional analysis of inc genes. The developmental transcription profile of various inc genes was assessed. Total RNA was isolated over a time course of infection with C. trachomatis L2, and transcription of individual inc genes was quantified by RT-qPCR normalized to genomic DNA (ng cDNA/DNA). (A) indicates early genes with euo as a marker. (B) indicates mid-cycle genes with ftsK as a marker. (C) indicates late genes with omcB as a marker. No inc genes were transcribed late. (D) Summary of the transcriptional profiles of inc genes (see also Figure 5).

Figure 5

Interaction network among Inc proteins. Schematic overview of the interactions between the Inc proteins that are colored according to their developmental expression pattern: orange for early genes and blue for mid genes. The interactions identified in this work are indicated with black arrows (the arrow pointing toward the T18-fusion indicates an interaction with a corresponding T25-fusion), and the homo-oligomerizations are shown by green arrows. Proteins reported to co-localize in Mital et al.'s experiment are surrounded with a red line.