Functional Interaction between Type III-Secreted Protein IncA of Chlamydophila psittaci and Human G3BP1

Chlamydophila (Cp.) psittaci, the causative agent of psittacosis in birds and humans, is the most important zoonotic pathogen of the family Chlamydiaceae. These obligate intracellular bacteria are distinguished by a unique biphasic developmental cycle, which includes proliferation in a membrane-bound compartment termed inclusion. All Chlamydiaceae spp. possess a coding capacity for core components of a Type III secretion apparatus, which mediates specific delivery of anti-host effector proteins either into the chlamydial inclusion membrane or into the cytoplasm of target eukaryotic cells. Here we describe the interaction between Type III-secreted protein IncA of Cp. psittaci and host protein G3BP1 in a yeast two-hybrid system. In GST-pull down and co-immunoprecipitation experiments both in vitro and in vivo interaction between full-length IncA and G3BP1 were shown. Using fluorescence microscopy, the localization of G3BP1 near the inclusion membrane of Cp. psittaci-infected Hep-2 cells was demonstrated. Notably, infection of Hep-2 cells with Cp. psittaci and overexpression of IncA in HEK293 cells led to a decrease in c-Myc protein concentration. This effect could be ascribed to the interaction between IncA and G3BP1 since overexpression of an IncA mutant construct disabled to interact with G3BP1 failed to reduce c-Myc concentration. We hypothesize that lowering the host cell c-Myc protein concentration may be part of a strategy employed by Cp. psittaci to avoid apoptosis and scale down host cell proliferation

The decrease in c-Myc protein concentration can be ascribed to the interaction between IncA and G3BP1.

<p>(A) Schematic structure of the <i>Cp. psittaci</i> IncA (aa 121-383) double mutant (N159S; S237G) which is no longer able to interact with G3BP1 in the yeast two-hybrid system. Mutagenesis was performed in <i>S. cerevisiae</i> using error-prone PCR. (B) Yeast two-hybrid interaction assay of mutated IncA with G3BP1. Interactions were quantitatively assessed by β-galactosidase liquid culture activity assays. β-galactosidase values represent the mean numbers and standard deviations of three independent experiments. (1) GAL4 BD-IncA (aa 121-383)-EGFP + pGADT7 (negative control), (2) GAL4 BD-IncA (aa 121-383)-EGFP + GAL4 AD-G3BP1 (aa 221-451) (positive control), (3) GAL4 BD-IncA (aa 121-383, N159S; S237G)-EGFP + GAL4 AD-G3BP1 (aa 221-451). (C) Immunoblots of whole HEK293 cell lysates transfected with pcDNA3 (1), pcDNA3-HA-IncA (aa 121-383) (2) or pcDNA3-HA-IncA (aa 121-383, N159S; S237G) (3) and probed with anti-HA, anti-c-Myc and anti-β-actin antibodies. Bar diagram below the immunoblots represent a quantification of c-Myc protein concentration related to β-actin. Experiments were performed in triplicate. (D) Immunoblots of whole Hep-2 cells untreated (1), transfected with non-targeting siRNA (2) or transfected with ON-TARGET G3BP1 siRNA pool and probed with anti-G3BP1, anti-c-Myc and anti-β-actin antibodies. Bar diagrams show knock-down rate of G3BP1 and c-Myc standardized to β-actin and the <i>c-myc</i> mRNA concentrations estimated by qRT-PCR. Experiments were performed in triplicate.</p

Infection of Hep-2 cells with <i>Cp. psittaci</i> leads to a decrease in c-Myc protein concentration.

<p>Hep-2 cells were infected with <i>Cp. psittaci</i> at a MOI of 3, with heat-inactivated <i>Cp. psittaci</i> or chloramphenicol-treated after infection as controls, and at different hours postinfection (hpi) cells were lysed for RNA or protein analysis. (A, above) Semi-quantitative (sq) PCR shows the unchanged expression of <i>c-myc</i> mRNA in infected (middle column), infected and chloramphenicol-treated (right) and control (left) Hep-2 cells. <i>β-actin</i> served as an internal control gene. (A, below) Quantitative Real-Time (qRT) PCR. <i>c-myc</i> levels were standardized on <i>GAPDH</i>. Experiments were performed three times in duplicate. (B) Immunoblots of whole cell lysates of <i>Cp. psittaci</i> infected (middle column), infected and chloramphenicol-treated (right) and control (left) cells probed with anti-LPS, anti-c-Myc and anti-β-actin antibodies. Bar diagrams below the immunoblots represent a quantification of c-Myc protein related to β-actin protein concentration. Experiments were performed three times.</p

Full-length IncA of <i>Cp. psittaci</i> and G3BP1 proteins interact <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Purified GST, GST-IncA (aa 1-338) and GST-IncA (aa 121-383) or (C) GST and GST-G3BP1 (aa1-466) stained with Coomassie. (B) Results of GST pull-down experiments performed with IncA full-length and truncation clone fused to GST and [³⁵S]-methionine-labeled full-length G3BP1 or (E) with Hep-2 cell lysate instead of labeled G3BP1. (D) Results of GST pull-down experiment performed with full-length G3BP1 fused to GST and [³⁵S]-methionine-labeled IncA (aa 121-383). GST served as control. IVT: 10 µl of the <i>in vitro</i> translation product. Input: 25% (75 µg protein) of Hep-2 cell lysate used for the interaction assay. (F) Whole cell lysate of HEK293 cells transfected with a His-tagged codon-adapted IncA mammalian expression construct was subjected to co-immunoprecipitation experiments using anti-His antibody. Co-immunoprecipitated endogenous G3BP1 was detected with a mouse anti-G3BP1 monoclonal antibody. Controls were beads alone (c) or pre-immune serum (lgG). Input: 10% (100 µg) of the protein used for immunoprecipitation.</p

G3BP1 is accumulated around the chlamydial inclusion in <i>Cp. psittaci</i> infected Hep-2 cells.

<p>Hep-2 cells were infected with <i>Cp. psittaci</i> at a MOI of 3, and at 48 h postinfection the cells were fixed and stained with anti-chlamydial LPS (red), anti-G3BP1 (green) antibodies and DAPI (blue, nuclei), and viewed under a fluorescence (1–3) or a confocal laser-scanning microscope (4–6). Non-infected cells served as control (1–3). The anti-G3BP1 (1 and 4) and anti-LPS (5) or DAPI (2) signals were merged (3 and 6). A fraction of endogenous G3BP1 is concentrated around chlamydial inclusions (6). Bar = 20 µm.</p