Chlamydia pneumoniae effector chlamydial outer protein N sequesters fructose bisphosphate aldolase A, providing a benefit to bacterial growth

Abstract

Background: Pathogenic chlamydiae are obligate intracellular pathogens and have adapted successfully to human cells, causing sexually transmitted diseases or pneumonia. Chlamydial outer protein N (CopN) is likely a critical effector protein secreted by the type III secretion system in chlamydiae, which manipulates host cells. However, the mechanisms of its action remain to be clarified. In this work, we aimed to identify previously unidentified CopN effector target in host cells.

Results: We first performed a pull-down assay with recombinant glutathione S-transferase (GST) fusion CopN proteins (GST-CpCopN: Chlamydia pneumoniae TW183, GST-CtCopN: Chlamydia trachomatis D/UW-3/CX) as "bait" and soluble lysates obtained from human immortal epithelial HEp-2 cells as "prey", followed by SDS-PAGE with mass spectroscopy (MS). We found that a host cell protein specifically bound to GST-CpCopN, but not GST-CtCopN. MS revealed the host protein to be fructose bisphosphate aldolase A (aldolase A), which plays a key role in glycolytic metabolism. We also confirmed the role of aldolase A in chlamydia-infected HEp-2 cells by using two distinct experiments for gene knockdown with an siRNA specific to aldolase A transcripts, and for assessment of glycolytic enzyme gene expression levels. As a result, both the numbers of chlamydial inclusion-forming units and RpoD transcripts were increased in the chlamydia-infected aldolase A knockdown cells, as compared with the wild-type HEp-2 cells. Meanwhile, chlamydial infection tended to enhance expression of aldolase A.

Conclusions: We discovered that one of the C. pneumoniae CopN targets is the glycolytic enzyme aldolase A. Sequestering aldolase A may be beneficial to bacterial growth in infected host cells.

Figure 1

Sequence similarity between C. pneumoniae and C. trachomatis CopNs and representative pull-down images. (A) Sequence similarity between C. pneumoniae and C. trachomatis CopNs. Amino acid sequence data were obtained from NCBI genome site (http://www.ncbi.nlm.nih.gov/genome/). C. pneumoniae TW183 CopN, CpB0334 (accession number: NC_005043.1). C. trachomatis UW-3/CX serovar D CopN, CT_089 (accession number: NC_000117.1). Alignment was constructed by web software, UniProt (http://www.uniprot.org/). C. pneumoniae CopN had 48% homology as compared with C. trachomatis CopN. Stars highlighted gray indicate identical amino acids. (B) Protein expression profiles of constructed recombinant GST-fused CopNs. Cpn, C. pneumoniae CopN full length (399 aa) (GST-CpCopN). CtD, C. trachomatis CopN full length (421 aa) (GST-CtCopN). GST, GST alone. (C) Representative pull-down images with GST-fused chlamydial CopNs in HEp-2 cell lysates. Red arrow shows a specific band captured by GST–CpCopN, but not GST–CtCopN, in the HEp-2 cell lysates.

Figure 2

Pull-down assay with MS analysis and Western blotting showing that C. pneumoniae CopN interacts with human aldolase A. (A) MS data with nano-LC-MS/MS. Bold red amino acid sequences, peptide matched sequences. (B) Representative Western blotting pattern with a specific aldolase A antibody for elution obtained from pull-down assay with GST-fused chlamydial CopNs and HEp-2 cell lysates. Input, HEp- 2 cell lysates. GST, GST alone. Cpn, GST-fused C. pneumoniae CopN (GST-CpCopN). CtD, GST-fused C. trachomatis CopN (GST-CtCopN). Red arrow, aldolase A. Asterisk, fragmented proteins.

Figure 3

Changes in chlamydial infectious particle numbers in aldolase A knockdown cells. (A) Representative aldolase A protein expression images in knockdown HEp-2 cells. scRNA, scramble RNA transfected cells. ALDOA, aldolase A targeting siRNA transfected cells. (B) Representative images of inclusions formed in the knockdown HEp-2 cells. Chlamydiae infected knockdown cells at MOI 5, and were incubated for up to 3 days. Morphological changes in the inclusions were assessed under fluorescence microscopy at 3 (for C. pneumoniae) or 2 (for C. trachomatis) days after infection. Green, inclusions. (C, D) Changes in chlamydial infectious particle (IFU) numbers in the knockdown cells. Bacterial IFUs were evaluated by IFU assay. The IFU numbers were also assessed at the same time as above. Data show mean ± SD obtained from at least three independent experiments. *p < 0.05 versus value into scRNA-transfected cells. N.S., not significant. C. pneumoniae (C). C. trachomatis (D).

Figure 4

Representative images showing chlamydial RpoD protein expression and its kinetic change into aldolase A knockdown cells. (A) Representative images showing chlamydial RpoD protein expression in knockdown cells. Aldolase A knockdown cells infected with chlamydiae at MOI 5 were incubated for up to 3 days. RpoD protein expression was evaluated using Western blotting with a specific antibody against chlamydial RpoD. (B) Kinetic change of chlamydial RpoD protein expression in aldolase A knockdown cells at 3 (for C. pneumoniae) or 2 (for C. trachomatis) days after infection. Band-density values were evaluated by NIH Image J software. Data show mean ± SD obtained from at least three independent experiments. N.S., not significant.

Figure 5

Expression of genes encoding glycolytic enzymes including aldolase A, GAPDH and enolase, and the kinetic changes in HEp-2 cells with chlamydial infection. (A) Representative RT-PCR images showing expression of genes encoding aldolase A, chlamydial 16S rRNA and β-actin in infected HEp-2 cells. C. pneumoniae infected cells at MOI 5 were collected at 24, 48 and 60 h. uninf, uninfected HEp-2 cells. (B) Kinetic changes in glycolytic enzyme gene expression in HEp-2 cells with chlamydial infection. The gene expression changes were evaluated by qRT-PCR. The values were normalized to that of β-actin. Data show mean ± SD from at least three independent experiments. N.S., not significant.