Molecular analysis of the multiple GroEL proteins of Chlamydiae

Abstract

Genome sequencing revealed that all six chlamydiae genomes contain three groEL-like genes (groEL1, groEL2, and groEL3). Phylogenetic analysis of groEL1, groEL2, and groEL3 indicates that these genes are likely to have been present in chlamydiae since the beginning of the lineage. Comparison of deduced amino acid sequences of the three groEL genes with those of other organisms showed high homology only for groEL1, although comparison of critical amino acid residues that are required for polypeptide binding of the Escherichia coli chaperonin GroEL revealed substantial conservation in all three chlamydial GroELs. This was further supported by three-dimensional structural predictions. All three genes are expressed constitutively throughout the developmental cycle of Chlamydia trachomatis, although groEL1 is expressed at much higher levels than are groEL2 and groEL3. Transcription of groEL1, but not groEL2 and groEL3, was elevated when HeLa cells infected with C. trachomatis were subjected to heat shock. Western blot analysis with polyclonal antibodies raised against recombinant GroEL1, GroEL2, and GroEL3 demonstrated the presence of the three proteins in C. trachomatis elementary bodies, with GroEL1 being present in the largest amount. Only C. trachomatis groEL1 and groES together complemented a temperature-sensitive E. coli groEL mutant. Complementation did not occur with groEL2 or groEL3 alone or together with groES. The role for each of the three GroELs in the chlamydial developmental cycle and in disease pathogenesis requires further study.

FIG. 1.

Phylogenetic relationships of chlamydial GroELs. The figure shows an unrooted tree of GroEL homologs in 75 completed genomes (see Materials and Methods for details regarding how the tree was constructed). Branches consisting of GroEL1, GroEL2, and GroEL3 cluster from six chlamydial genomes (C. trachomatis serovar D and biovar MoPn; C. pneumoniae strains AR39, CWL029, and J138; and C. psittaci) are indicated by shaded boxes. Bacterial genera at key branch points are also included.

FIG. 2.

Predicted 3-D structures of C. trachomatis serovar D GroEL1 (A), GroEL2 (B), and GroEL3 (C) (see Materials and Methods for details of how the 3-D prediction was done through homology modeling) and the structural superposition of the three predicted structures for GroEL1, GroEL2, and GroEL3 (D) (represented by green, blue, and yellow, respectively).

FIG. 3.

(A) The top panel shows control PCR amplification of groEL1, groEL2, and groEL3 with serovar D DNA as the template as a measure to compare primer efficiency. The bottom panel shows an RT-PCR analysis of total RNA from HeLa cells infected with C. trachomatis serovar D isolated at 12 h after infection. (B) The top panel shows a Western blot analysis of C. trachomatis serovar D GroEL1, GroEL2, and GroEL3. Boiled samples of purified serovar D C. trachomatis elementary bodies were subjected to SDS-7.5% polyacrylamide gel electrophoresis, and the blotted nitrocellulose membranes were incubated with polyclonal anti-GroEL1 (first lane), anti-GroEL2 (middle lane), or anti-GroEL3 (third lane) antibodies, followed by incubation with alkaline phosphatase-conjugated secondary antibodies and color detection. In the bottom panel is shown a quantification of GroEL1, GroEL2, and GroEL3 by using the procedure described above except that 1-, 10-, and 100-ng portions of recombinant proteins were loaded per lane.

FIG. 4.

Quantification of C. trachomatis serovar D groEL1, groEL2, and groEL3 expression after heat shock by using microarray. (groES and dnaK genes were included for comparison purposes). Heat-shocked and non-heat-shocked RNA samples were reverse transcribed, labeled with Cy3 or Cy5, and hybridized to the microarray. The fluorescence intensity was measured for each spot and then normalized to the average fluorescence intensity for the entire microarray. Data analysis was performed with the GeneSpring software (see Materials and Methods for more details).

FIG. 5.

Complementation of a temperature-sensitive mutant of E. coli groEL by C. trachomatis groEL1 and groES. Two independent transformants of NL441 strains containing the plasmid pairs indicated were streaked onto Luria-Bertani agar plates supplemented with ampicillin (100 μg/ml), chloramphenicol (50 μg/ml), and arabinose (as indicated) and then incubated at the temperatures indicated for 18 h.