Pmp-like proteins Pls1 and Pls2 are secreted into the lumen of the Chlamydia trachomatis inclusion

Abstract

The obligate intracellular pathogen Chlamydia trachomatis secretes effector proteins across the membrane of the pathogen-containing vacuole (inclusion) to modulate host cellular functions. In an immunological screen for secreted chlamydial proteins, we identified CT049 and CT050 as potential inclusion membrane-associated proteins. These acidic, nonglobular proteins are paralogously related to the passenger domain of the polymorphic membrane protein PmpC and, like other Pmp proteins, are highly polymorphic among C. trachomatis ocular and urogenital strains. We generated antibodies to these Pmp-like secreted (Pls) proteins and determined by immunofluorescence microscopy that Pls1 (CT049) and Pls2 (CT050) localized to globular structures within the inclusion lumen and at the inclusion membrane. Fractionation of membranes and cytoplasmic components from infected cells by differential and density gradient centrifugation further indicated that Pls1 and Pls2 associated with membranes distinct from the bulk of bacterial and inclusion membranes. The accumulation of Pls1 and, to a lesser extent, Pls2 in the inclusion lumen was insensitive to the type III secretion inhibitor C1, suggesting that this translocation system is not essential for Pls protein secretion. In contrast, Pls secretion and stability were sensitive to low levels of beta-lactam antibiotics, suggesting that a functional cell wall is required for Pls secretion from the bacterial cell. Finally, we tested the requirement for these proteins in Chlamydia infection by microinjecting anti-Pls1 and anti-Pls2 antibodies into infected cells. Coinjection of anti-Pls1 and -Pls2 antibodies partially inhibited expansion of the inclusion. Because Pls proteins lack classical sec-dependent secretion signals, we propose that Pls proteins are secreted into the inclusion lumen by a novel mechanism to regulate events important for chlamydial replication and inclusion expansion.

FIG. 1.

Genomic arrangement of Pls (Pmp-like secreted) genes. (A) Organization of the genes encoding Pls1 (CT049), Pls2 (CT050), and Pls3 (CT051). Pls2 is paralogously related to Pls1 and Pls3. The percentages of identity (Id) and similarity (Sm) based on BLAST searches are shown. (B) Pls proteins are paralogously related to the passenger domain of the predicted autotransporter polymorphic outer membrane protein PmpC. Regions of similarity between Pls proteins and PmpC were identified by PSI-BLAST. Pls proteins share limited homology to two distinct regions within the passenger domain and to the putative Chlamydia polymorphic middle protein signature domain (ChlamPMP).

FIG. 2.

Pls protein expression in C. trachomatis-infected cells. (A) Rabbits were immunized with purified GST-CT049 (Pls1) or GST-CT050 (Pls2), and specificities of the resulting antisera were determined by screening GFP-tagged CT049 and CT050 expressed in yeast. Expression of recombinant proteins was monitored with anti-GFP antibodies. (B) Pls1 and Pls2 expression during infection was assessed by immunoblot analysis with anti-Pls1 and -Pls2 antisera, using lysates from HeLa cells infected with C. trachomatis L2 for 12 to 48 h. The level of RpoD was monitored to assess the expression of chlamydial proteins, and the host marker GAPDH was used as a loading control. (C) Pls1 and Pls2 antisera (red in merged images) specifically labeled bright punctate structures within the inclusion. HeLa cells were infected with L2 and immunostained with Pls antisera in the presence of excess GST-Pls1 or -Pls2. Note that intra-inclusion bright punctate structures were no longer detected by the antisera in the presence of the corresponding blocking antigen. Bacterial and host DNAs were detected with TOPRO-3 (blue). Closer inspection of stained inclusions reveals background staining of Pls1 (D) and Pls2 (E) with TOPRO-3-positive bacteria, in addition to bright Pls-positive aggregates.

FIG. 3.

Subcellular localization of Pls1 and Pls2 in C. trachomatis-infected cells. HeLa cells were left untreated or infected with L2 at an MOI of ∼1 for 18 or 24 h. Cells were processed for indirect immunofluorescence microscopy with either anti-Pls1 (A) or -Pls2 (B) antibodies (red) and imaged by laser scanning confocal microscopy. Bacterial outer membranes and inclusion membranes were detected with monoclonal antibodies against MOMP and IncA, respectively (green). Note the presence of Pls1 and Pls2 as extrabacterial globular aggregates in the inclusion lumen and in close association with the inclusion membrane. Potential extrusion sites of Pls-positive material are shown by arrowheads.

FIG. 4.

The accumulation of Pls proteins in the inclusion lumen is sensitive to Amp. (A) HeLa cells were infected with L2 for 16 h and either left untreated or treated with 10 μg/ml Amp or 70 μM of the TTS inhibitor C1 for an additional 8 h. At 24 h postinfection, cells were processed for immunofluorescence microscopy, and Pls1 or Pls2 was detected with specific antisera. In addition, bacteria were detected with anti-Omp2 and anti-LPS antibodies. Inclusion membranes were detected with anti-IncA antibodies. Note the accumulation of Pls1 and Pls2 in the cytoplasm of enlarged RBs and the lack of Pls-positive intra-inclusion globular structures in Amp-treated cells. (B) Pls1 and Pls2 transiently localize to the surfaces of enlarged RB outer membranes after recovery from Amp treatment. HeLa cells were infected in duplicate with L2 for 16 h and treated with 10 μg/ml Amp for 8 h. One set of cells was fixed, and the other was allowed to recover from Amp for an additional 24 h (Amp + Rec.). Pls1 and -2 and MOMP were detected by indirect immunofluorescence, as described above. Note the reduced levels of Pls1 and Pls2 expression in Amp-treated cells (middle panels) and the association of Pls1 and Pls2 at the surfaces of enlarged RBs (arrowheads) in cells recovering from Amp treatment. Host and bacterial DNAs were stained with TOPRO-3 (blue).

FIG. 5.

Pls1 and Pls2 are differentially processed in Amp-treated cells. HeLa cells were infected with L2 at an MOI of ∼1 and harvested at 30 h postinfection. Prior to cell harvesting, parallel samples were treated with either 10 μg/ml Amp or 70 μM C1 for 0, 2, 6, or 10 h. Total protein lysates were generated and subjected to immunoblot analysis with anti-Pls1, -Pls2, -Omp2, -IncA, and -RpoD antibodies. Tubulin levels are shown as a loading control. Note the loss of Pls1 and Pls2 during prolonged Amp treatment, despite the accumulation of other bacterial markers.

FIG. 6.

Pls1 and Pls2 cofractionate with membranes. HeLa cells infected with L2 at an MOI of ∼1 were harvested at 48 h postinfection. The PNS was ultracentrifuged to generate cytosol-enriched HSS and HSP. Total membranes and intact bacteria partitioned with the HSP. Membranes in the HSP were further separated by density gradient centrifugation, and samples were collected from the top (T), middle (M), and bottom (B) fractions. The bottom fraction consisted mostly of intact bacteria. PNS and membrane fractions were solubilized in SDS sample buffer, boiled under reducing conditions, and subjected to immunoblot analysis. Note the partitioning of low-MW processed forms of Pls1 and Pls2 with cytosolic fractions and the association of larger processed Pls forms with middle membrane fractions. Other bacterial membrane proteins (MOMP and Omp2) remained at the bottom of the gradient.

FIG. 7.

Microinjection of Pls1 and Pls2 antisera inhibits inclusion expansion. HeLa cells were infected with L2 at an MOI of ∼1. Infected cells were then microinjected with Pls1 and Pls2 preimmune sera or Pls1 and Pls2 sera at 4 h postinfection. At 30 h, cells were fixed, and microinjected cells were detected with fluorophore-conjugated anti-rabbit IgG and Hoechst 33258. The sizes of ∼170 inclusions in microinjected cells from each experiment were measured in pixels, and the distributions of inclusion size were analyzed. The rectangular box corresponds to data points between the 25th and 75th percentiles of the total distribution. The horizontal line denotes the median. The individual dots correspond to data points above the 95th and below the 5th percentile of the total distribution. A two-tailed t test was performed for each data set. The average size of inclusions from cells microinjected with a combination of Pls1 and Pls2 antisera was 35.2% smaller than that for cells injected with preimmune sera. Parts A and B represent results from two independent experiments.