Chlamydial strains, host cell lines, and chlamydial infections
Chlamydia trachomatis strain 434/Bu (serovar L2) and C. caviae strain GPIC elementary bodies were purified from infected monolayers by passage over a 30% Hypaque density gradient. Purified chlamydiae were stored in 0.25 M sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid (SPG) at -80°C prior to use . HeLa and ChoK1 cells (ATCC) were cultured in Minimal Essential Medium supplemented with 10% fetal bovine serum (Gibco) and 10 μgml-1 gentamicin and grown at 37°C in 5% CO2. Elementary bodies diluted in SPG were added to the cells at multiplicities of infection (MOI) as indicated. Infected cells were centrifuged at 500 g for 1 h at room temperature (RT), after which the inocula were removed, the cells were washed with Hank's Balanced Salt Solution (Gibco) and medium was added.
Cells to be used for microscopic analysis were cultured on sterile glass coverslips at 50 to 80% confluency. After 30–35 h, infected cells were fixed in 100% methanol for 10 min and washed with phosphate-buffered saline (PBS). Cells were then subjected to fluorescent antibody staining or stored at 4°C.
Vaccinia virus recombinants
Full length C. caviae incA and a truncated incA fragment (nucleotides 1–459) were amplified using Pwo polymerase (Boehringer). Terminal oligonucleotide primers (Table 1; see additional file 1) were designed to yield restriction enzyme sites for cloning into plasmid pRB21, a shuttle vector that allows simple selection of VV recombinants . Alanine substitutions of serine or threonine residues in IncA were produced using a three-primer technique . All amplification products were confirmed by DNA sequencing. Recombinant Vaccinia virus diluted in PBS were inoculated onto HeLa cells and incubated at room temperature for 1 h prior to addition of MEM-10 and incubation at 37°C . Production of IncA and the identification of different IncA isoforms were monitored by immunoblot analysis of VV-infected HeLa cell lysates collected 16 hours post-infection .
Construction of plasmids used in transfection experiments
Plasmids pcDNA3.1(+) and pcDNA4/HisMax C (Invitrogen) were used as vectors in all experiments (Fig. 1). Plasmid pcDNA4/HisMax C vector encodes a polyhistidine tag that was fused to the amino terminus of each recombinant polypeptide. Intact or truncated coding regions of C. caviae incA, or incC, and C. trachomatis incA, were amplified using primers indicated in Table 1. Restriction sites for Eco RI and Eco RV were introduced into the forward and reverse primers, respectively, for each amplification product to be cloned. Reactions were carried out using Pwo polymerase and chlamydial genomic DNA as template. Plasmids from positive clones were purified (Qiagen) and confirmed by sequence analysis.
Antibodies and Immunofluorescence Microscopy
Monoclonal antibody A57B9, directed at a genus common epitope on chlamydial GroEL (HSP60), was used to label chlamydial developmental forms . Polyclonal anti-IncA was produced as described . Monoclonal antibodies to C. trachomatis IncA (mAb 3H7) or to C. caviae IncA (mAb 17F12) have been previously described [4, 22]. Monoclonal antibody to polyhistidine was purchased from Clontech.
Fixed monolayers were incubated in 2% bovine serum albumin in PBS (BSA-PBS), as a blocking reagent, for 20 min. Primary antibodies diluted in BSA-PBS were then incubated on cells for 1 h. Cells were washed three times with PBS and then incubated in the dark with the appropriate secondary antibodies for 1 h. Secondary antibodies were purchased from Southern Biotechnology Associates. The cells were washed three times in PBS and coverslips were inverted onto 3 μl of Vectashield (Vectors Laboratories) containing the DNA-specific fluorochrome 4', 6'-diamino-2-phenylindole (DAPI; Sigma; 2 μgml-1). Labeled monolayers were examined at 1000× magnification using a Leica fluorescence microscope and images were collected with a SPOT digital camera system (Diagnostic Instruments). Images were processed in Photoshop 5.0 (Adobe Software) and Canvas 6 (Deneba Software).
Transfections and transfection/infection experiments
HeLa or ChoK1 cells grown on sterile glass coverslips were transfected with the indicated plasmid constructs using Lipofectamine 2000 (Gibco) according to manufacturer's instructions. All quantitative infection/transfection analyses were conducted in cells with MOI of 1. Transfected cells were incubated 24 h post transfection and then either fixed with methanol or infected with chlamydiae. Infected cells were incubated 30 h prior to methanol fixation, unless indicated. The efficiency of infection of transfected cells was determined by labeling inclusions with anti-polyhistidine (to detect the product of the transgene) and anti-HSP60 (to detect the chlamydiae). Following labeling, coverslips were removed and the infection rates determined by counting infected and transfected cells, and total numbers of transfected cells, under 1000× magnification.
In experiments where HeLa cells were doubly transfected with plasmids expressing incA and incC, a pcDNA3.1(+) construct encoding IncA in the absence of the 6-his tag was used in combination with pcDNA4/HisMax C encoding IncC. Cells were transfected as described and examined by fluorescence microscopy, using anti-IncA and anti-6-His reagents to detect IncA and IncC, respectively.
Standard deviations were calculated for each combination of plasmid and chlamydial strain from the average of two or three wells in the same experiment. The number of transfected cells that were infected by chlamydiae in a particular experiment was evaluated in at least three independent experiments for each plasmid construct tested. A total of at least 300 individual transfected cells were counted for each tested plasmid construct. In each case, the significance of the differences between means were evaluated using both the Student's T test and the Kruskall-Wallis test, as calculated using the Instat software program (GraphPad Software). Reported P values represent calculations based on the Student's T test.