The study was approved by the Institutional Review Board of Children's Hospital and Research Center at Oakland in accordance with the Declaration of Helsinki. The clinical sample was obtained from the rectal mucosa of a male who had a history of sex with men and presented with severe hemorrhagic proctitis. Briefly, a 26-year-old male presented to a San Francisco Bay area clinic with a complaint of severe rectal pain with blood on defecation. He described a series of encounters with homosexual men and unprotected anal receptive intercourse during the prior month until the onset of rectal pain 5 days prior to the clinic visit. The rectal bleeding had commenced 1 day earlier. The man had a history of gonorrhea in the past but no other known STDs, no known exposure to men with known STDs, and no history of illicit drug use. He was reported to be HIV negative and had no other medical conditions, was not on any over-the-counter or prescription medications, and appeared to be in excellent health. On the physical exam, he was a well-appearing male, afebrile, and normotensive, with no evidence for an ulcerative lesion on the glans or shaft of the penis and no inguinal adenopathy. The anus was inflamed, and on proctoscopy, there was extensive bleeding of the mucosa, with evidence of a purulent discharge. Four swabs from each quadrant of the rectum were obtained and placed in transport medium. Three swabs were sent to the clinical laboratory for standard detection of Neisseria gonorrhoeae by commercial PCR and culture and of C. trachomatis by commercial PCR. The fourth swab was sent to the Chlamydia Research Laboratory at CHORI for in-house C. trachomatis culture.
Reference strains D/UW3, L1/440, L2/434, L2a/TW-396, and L3/404, a clinical L2b strain from Amsterdam (a kind gift from Servaas Morre), and the clinical sample, referred to as L2c, from the above-described case, were analyzed. Each strain was propagated in the human cervical adenocarcinoma cell line HeLa229 using our previously described protocols. The clinical sample was diluted and directly plaque purified using sequential plaque purifications per our referenced protocols. To verify the clonal purity of the plaques, ompA and MLST genes were amplified by PCR and cloned separately using a TOPO TA cloning kit (Invitrogen); 10 clones of each were randomly selected for Sanger sequencing using techniques we have described previously. The confirmed clonal plaques were then individually propagated in tissue culture. A total of two passages were performed for L2c to generate sufficient gDNA for genome sequencing. The EBs for each C. trachomatis isolate were purified from contaminating human cells using DNase treatment followed by gradient ultracentrifugation, and genomic DNA was purified from each isolate using a High Pure PCR template preparation kit (Roche Diagnostics, Indianapolis, IN) as we previously described.
For determination of MOI based on IFUs, duplicate serial 10-fold dilutions of purified EBs were used to infect HeLa cells in 24-well plates. After 24 to 48 h, the cells were fixed in methanol, washed with phosphate-buffered saline (PBS), and stained using the Pathfinder Chlamydia culture confirmation monoclonal antibody (MAb) (Kallestad Diagnostics, Chaska, MN) in accordance with the manufacturer's directions. The number of IFUs per well was divided by the number of cells per well; an average was taken for duplicate wells to arrive at the MOI per strain.
HeLa cell monolayers grown in minimal essential medium (MEM) containing 10% fetal bovine serum (UCSF Cell Culture Facility, San Francisco, CA) and 1 microg/ml gentamicin (MP Biomedicals, Solon, OH) at a confluence of 80% on 12-mm coverslips (Fisher Scientific, Pittsburgh, PA) in 24-well plates were infected with either reference strains L1/440, L2/434, L2a/TW-396, L3/404, and D/UW3, clinical strain L2b from Amsterdam, or the clinical strain L2c from this study in sucrose-phosphate-glutamine (219 mmol/liter sucrose, 3.82 mmol/liter KH2PO4, 8.59 mmol/liter Na2HPO4, 4.26 mmol/liter glutamic acid, 10 microg/ml gentamicin [MP Biomedicals], 100 microg/ml vancomycin [Acros Organics, Morris Plains, NJ], and 25 U/ml nystatin [MP Biomedicals] in distilled water, pH 7.4) at an MOI of 1, unless indicated, for 2 h on an orbital shaker at room temperature. The inocula were aspirated, and the infected monolayers were cultured in a humidified incubator at 37 C with 5% CO2 in Dulbecco's modified MEM (Cellgro, Manassas, VA) with GlutaMAX-1 (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum (UCSF Cell Culture Facility), 0.45% glucose solution (Cellgro), 20 mM HEPES (UCSF Cell Culture Facility), 0.08% NaHCO3, and 1 microg/ml cycloheximide. At 12, 18, 24, 36, and 48 h (48 h for D/UW3 only) postinfection, the coverslips were fixed with methanol for 10 min, rinsed in PBS, and incubated for 30 min with anti-C. trachomatis specific lipopolysaccharide (LPS) MAb (Virostat, Portland, ME) and anti-IncA MAb 3H7 (gift from Daniel D. Rockey) or polyclonal anti-IncA (gift from Ted Hackstadt). The secondary antibodies were Cy-3 conjugated IgG (Jackson ImmunoResearch, West Grove, PA) for LPS and fluorescein isothiocyanate (FITC)-labeled IgG (Jackson ImmunoResearch) or Alexa 488 (Invitrogen) for IncA and chlamydial heat shock protein 60. DAPI (4',6-diamidino-2-phenylindole dihydrochloride) (Vector Laboratories, Burlingame, CA) was used to stain DNA. The inclusions formed by the reference LGV and D/UW3 strains and the clinical L2c strain were visualized on a Zeiss 510 confocal microscope. Light microscopy was used to examine the D/UW3, LGV, and clinical L2c strains at 36 h, but no inclusions were observed for any LGV strains at this time point.
We examined incA expression using our previously described techniques, with slight modifications. HeLa cells were infected at 0, 2, 12, 24, and 48 h with D/UW3, L2/434, and L2c at an MOI of 5. Total RNA was extracted using an RNeasy minikit (Qiagen, Valencia, CA) per the manufacturer's instructions; on-column DNase (Qiagen) treatment was performed to remove contaminating DNA. cDNA was generated from 2 microg of total RNA using TaqMan reverse transcriptase (RT) reagents and random hexamers (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed in replicate using SYBR green chemistry, reagents, primers (see Table S1 in the supplemental material), thermocycling, and standard curves as we previously described. 16S rRNA was used for normalization. Negative controls and the standard curves for each gene were used as previously described. Analysis of the dissociation curves was used to verify the specificity of the amplified products. The conversion of the mean threshold cycle values determined the relative amounts of target and control gene from the respective standard curve. Three independent experiments were performed.
Growth curves for strains D/UW3, L2/434, and L2c were generated using quantitative PCR as we previously described. Briefly, each strain at an MOI of 1 was grown in HeLa cells and harvested at 0, 12, 18, 24, 36, and 48 h, and total RNA was extracted and reverse transcribed as described above. Each quantitative PCR consisted of 1x SYBR green master mix (Applied Biosystems), 1 microl of cDNA, and 5 pmol of each primer (16S rRNA and glyceraldehyde-3-phosphate dehydrogenase [GAPDH] as described in reference 56) in a total reaction volume of 25 microl run on an ABI 7900 (Applied Biosystems) using our thermocycling profile as previously described; standard curves were generated, and negative controls for each primer pair were included in each run as we previously described. GAPDH was used for normalization. Melting curves were used to verify the specificity of the reactions and the absence of primer dimmers. Samples were amplified in triplicate where the mean was used for analysis. Three independent experiments were performed.
Reference strains D/UW3 and L2/434 and the clinical L2c strain from this study were analyzed in a cytotoxicity assay adapted from the method of Belland et al. (42). HeLa cells were grown to 80% confluence as described above in 24-well plates. The monolayers were treated with 1 ml DEAE-dextran (45 microg/ml) in Hanks' balanced salt solution (HBSS) for 15 min at 37 C. The cells were infected at an MOI of 100 (~5 x 107 IFU) with each strain and mock infected in duplicate at room temperature for 4 h on an orbital shaker. The inocula were removed, and the cells were washed with HBSS and incubated for an additional 4 h in growth medium as described above except for the addition of 10 microg/ml gentamicin (MP Biomedicals), 25 microg/ml vancomycin (Fisher) to prevent any nonchlamydial bacterial growth. The monolayers were visualized under light microscopy at x400 magnification. The cells were scored for rounding, detachment, and lysis compared to the levels for uninfected control cells by the following metric: (-), same as cell control; 3+, 100% of cells affected; 2+, 75% of cells affected; and 1+, 25% of cells affected.
The above-described experiments were repeated using 1 microg/ml of doxycycline to pretreat cells before infection. In addition, the assay was repeated using L2/434 and a higher passage number (passage no. 20) for L2c. A total of two independent experiments were performed for each of these studies.
To detect the toxin protein, a Western blot analysis was performed as we have previously described, with modifications. Briefly, L2/434, D/UW3, and L2c were independently grown in HeLa cells as described above at an MOI of 100. Purified EBs from each isolate were solubilized in Laemmli buffer, run on NuPAGE Novex 4 to 12% bis-Tris precast gels (Invitrogen) with a BenchMark prestained protein ladder (Invitrogen), and electrophoretically transferred onto a BioTrace polyvinylidene difluoride (PVDF) membrane (Pall Life Science, Port Washington, NY) at 120 V for 2 h in 1x NuPAGE transfer buffer (Invitrogen) with 10% methanol. The membrane was incubated with 5% skim milk in 1x Tris-buffered saline (TBS) and 0.1% Tween 20, washed with 1x TBS-0.1% Tween 20, and reacted overnight with a polyclonal antiserum against recombinant CT166 (gift from Harlan Caldwell) at a 1:500 dilution. The membrane was washed, and secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase [HRP]; Bio-Rad, Hercules, CA) was applied at a 1:1,000 dilution, incubated at room temperature, and washed in 1x TBS prior to detection using the SuperSignal West Pico chemiluminescent substrate (enhanced chemiluminescence [ECL] detection system; Thermo Scientific, Rockford, IL) and CL-XPosure Film (Thermo Scientific).
The purified genomic DNA from the clinical case was shotgun sequenced using a combination of the 454/Roche GS-FLX Titanium and GS Junior instruments (454 Life Sciences, Branford, CT). The 809,874 reads were generated with an average read length of 404 nt. Preliminary analysis suggested an unusually high proportion of contamination of DNA from the HeLa cell culture. Therefore, 618,844 reads of human origin were removed (identified by mapping to the hs19 golden path release of the genome []) using Newbler gsMapper 2.5 software.
We assembled a random subset of 50,000 of the remaining reads de novo using the 454 gsAssembler software program, version 2.0.01.14, with default parameters. The order of the contigs in the final genome sequence was determined using a combination of the possible connections between contigs suggested by overlapping reads and information from mapping contigs against the L2/434 reference genome. After determination of the correct path through the contig graph, the contigs were assembled to form the final sequence. To accomplish this, the ends of adjoining contigs were matched using BLAST against the database of reads. Reads found in both contigs were assembled into a consensus sequence that bridged the two contigs to form a single, larger contig. The majority of gaps were small enough to be spanned by a single read. The plasmid was a single contig with reads overlapping the beginning and end, indicating the expected circular redundancy.
For verification of the sequences, we aligned the original reads to the consensus chromosomal and plasmid templates using gsMapper 2.3; 91.6% of the 191,030 human screened reads (average length, 436 nt) mapped to the chromosome, and 6.8% mapped to the plasmid. The remaining unmapped reads were found not to assemble de novo into any contigs consisting of more than 2 reads, suggesting that these were mostly contamination that had not matched the human reference sequence. The mapped assemblies were inspected to remove a small number of errors. The final assembly, with an average redundancy of coverage of approximately 75-fold, was used for annotation. The identity of each base in the final consensus sequence was determined by majority vote. The gsMapper software revealed two possible structural variants in a minority of sequence reads. The first was a deletion between coordinates 133542 and 155549 present in 4 sequence reads. The second was a deletion between bases 849581 and 849942 present in 19 sequence reads.
The L2c consensus chromosome and plasmid sequences were annotated automatically using the Integrative Services for Genomics Analysis pipeline. Promoter 2.0 prediction (), Bacterial PROMoter prediction (), and the Berkeley Drosophila Genome Project neural network () were used to identify the ftsK putative promoter region, including the initiation site, -10 and -35 promoter elements, and ribosome binding sites. The default parameters were used for prediction scores for significance.
To verify the regions of diversity (L2-D hybrids), single amplicons of these regions (the tarp gene, incA, hctB, and toxin locus genes; the IGR upstream of ftsK) that were identified by the SAS program Q-plotGenome (see below) and genomic analyses (see below) as divergent from L2/434 were amplified, cloned using a TOPO TA cloning kit (Invitrogen), and Sanger sequenced using primers (see Table S1 in the supplemental material) designed to amplify and sequence the full length of each as previously described. Ten clones were sequenced for each. The sequences were compared with those of the following available reference strains (GenBank accession numbers are given in parentheses): A/2497 (EU121607), B/HAR36 (EU121608), C/TW3 (EU121609), D/UW3 (AE001273), L2/434/Bu (AM884176), and L2b/UCH-1/proctitis (AM884177) for the tarp gene; A/HAR13 (CP000051), B/Jali20/0T (FM872308), C/TW3 (EU121596), D/UW3 (AE001273), L2/434/Bu (AM884176), and L2b/UCH-1/proctitis (AM884177) for hctB; A/HAR13 (CP000051), B/TW5 (DQ064209), B/TZ1A828/OT (FM872307), Ba/Apache-2 (DQ064210), C/TW3 (DQ064211), D/UW3, E/Bour (DQ064214), F/IC-CAL3 (DQ064215), G/UW57 (EU247624), H/UW4 (EU247625), i/UW12 (DQ064218), Ia/870 (DQ064219), J/UW36 (DQ064220), K/UW31 (DQ064221), L1/440 (DQ064222), L2/434/Bu, L2b/UCH-1/proctitis, and L3/404 (DQ064224) for incA; C/TW3 (AY647994), D/UW3, H/UW4 (AY647999), and L2/434 for the cytotoxin locus; and A/HAR13, B/Jali20/0T, D/UW3, L2/434, and L2b/UCH-1/proctitis for the IGR upstream of ftsK.
To detect recombination, we developed a program called Q-plotGenome that was written as a set of macros in the SAS software 9.2 language (SAS Institute, Inc., Cary, NC) to compare the genome sequences of L2c with those of reference strains L2/434 and D/UW3. The core of the program is similar to that of SimPlot except that it covers the entire 1-Mb genome instead of a discrete sliding window of limited size; it samples a series of fixed-length subsequences (windows) from one genome and then compares them to windows from the other genome. Q-plotGenome is, thus, a tool for comparing DNA sequences in the range of 1 Mb plus and for displaying results graphically. The description of Q-plotGenome is detailed in Text S1 in the supplemental material.
To identify statistically significant recombination breakpoints or regions, SimPlot software 3.5.1 () was utilized as we have previously described. The parameters included a window size of 200 base pairs (bp), a step of 20 bp, neighbor-joining trees calculated using the Kimura-2-parameter distance model (60), and 1,000 bootstrap replicates to determine confidence for each branch (Fig. 7).
For a global analysis, we used a version of the BLAST score ratio approach to identify recombinants. We aligned the databases of both (i) raw L2c sequences and (ii) 100-nt windows along the L2c chromosome sequence separated by 50 nt against the L2/434 genome as a reference and the other complete genomes listed in the previous section. Matches with a BLAST score ratio of <0.95 were plotted using Circos software (Fig. 5).
For analysis of specific regions, nucleotide sequences were aligned by ClustalW 1.8, MUSCLE, or MAUVE. Gaps were removed using GBLOCKS with default parameters. Phylogenic inference and tree plotting were performed using the MEGA 3.1 package described previously or PHYLIP programs DNApars, Seqboot, and Consensus. Neighbor-joining trees were calculated using the Kimura 2-parameter model that assumes constant nucleotide frequencies and their rates of substitution among sites (60) and 1,000 bootstrap replicates.
For detection of strain D-specific SNPs within the L2c data, we first identified SNPs between the D/UW3 and L2/434 genomes using the show-snps tool of the MUMmer package. We then mapped L2c against L2/434 using gsMapper 2.3 and extracted the number of "D"-like and "L2"-like nucleotides at each variant position from the 454AllDiffs.txt output using a custom script. Similar analyses were performed for D(s)/2923.
The complete chromosome and plasmid sequences were submitted to the NCBI GenBank database (accession no. CP002024). The NCBI genome project identification number is 47581. The raw data from the 191,030 human screened L2c reads were deposited in the NCBI short read archive (sra; accession no. SRP002231). The sequence of the L2c toxin gene from the initial plaque purification was submitted to GenBank (accession no. 2981755).