Expression profile of the nine <i>pmp</i> genes and <i>ompA</i> throughout the development of <i>C. trachomatis.</i>

<p>Reference strain L₂/434 is represented in panel A and E/Bour in panel B. Values represent the mean±SEM based on three independent experiments for time points of 2, 6, 12, 18, 24, 36, and 48 h post infection. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000878#s2" target="_blank">methods</a> for details.</p

Predicted <i>pmpF</i> promoter sequence for reference and clinical strains.

<p>Sequences are for reference strains E/Bour and L₂/434, and clinical strains E/537C-05, E/S-141, E/CS-500-96, and L₂. The predicted transcription promoter for <i>pmpF</i> is located within a 100% conserved region of the <i>pmpG/pmpF</i> IGR, where putative -10 and -35 elements are in blue characters and boxed. Potential A/T spacer region is underlined, and the predicted transcription start site is shown in a larger font below a red asterisk. The putative RBS for <i>pmpF</i> is in orange characters, and the putative RNase E cleavage sites are highlighted in grey. Numbers represent positions relative to the start codon of <i>pmpF</i> (highlighted in yellow). The start codon of <i>pmpG</i> is highlighted in blue.</p

Oligonucleotide primers used for kRT-PCR

a<p>Primers designed based on each <i>pmp</i> sequence of reference strains E/Bour and L₂/434 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000878#pone.0000878-Gomes1" target="_blank">[12]</a>.</p>b<p>Primers designed only for strain L₂.</p>c<p>Primers designed based on the <i>ompA</i> sequence of reference strains E/Bour and L₂/434 (GenBank Accession No. X52557 and M14738, respectively).</p>d<p>Primers designed based on the <i>16SrRNA</i> sequence of reference strains E/Bour and L₂/434 (GenBank Accession No. D85722 and U68443, respectively).</p

Clinical and microbiologic characteristics of female adolescents from whom sera was used for determining the immunoreactivity against rPmpD and rPmpF

a<p>Patients were adolescents 14–19 years of age who had a <i>C. trachomatis</i> infection with only one <i>ompA</i> genotype as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000878#s2" target="_blank">methods</a> or were uninfected;</p>b<p>The diagnosis of cervicitis was based on physical findings consistent with cervicitis as determined by the examining physician; all adolescents infected with <i>C. trachomatis</i> had cervicitis, and none of these patients complained of any symptoms;</p>c<p>A cervical discharge was noted by the examining physician; none of these patients had clinical signs or symptoms consistent with upper genital tract disease.</p

Dot-Blot of serum immunoreactivity against recombinant (r)PmpD and rPmpF.

<p>Sera was obtained from adolescents singly infected and uninfected with a different <i>C. trachomatis</i> clinical strain as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000878#pone.0000878-Gomes3" target="_blank">[17]</a> (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000878#s2" target="_blank">methods</a>). rPmpD and rPmpF concentrations were standardized for use on the blots. Immunoreactivity to each fusion protein for sera from patients infected with strain Ba (n = 3), D (n = 3), E (n = 8), F (n = 5), G (n = 1), Ia (n = 1), J (n = 2) or K (n = 3) are shown. Of note is that immunoreactivity was consistent for sera from patients infected with the same clinical strain except for strain F (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000878#pone-0000878-t002" target="_blank">Table 2</a>); all eight patients infected with strain E were reactive to rPmpD.</p

Expression profile of <i>pmp</i> and <i>ompA</i> genes throughout the development of <i>C. trachomatis</i> clinical strains.

<p>(A) Strain L₂ shares the same <i>ompA</i> genotype as L₂/434; and strains E/537C-05 (B), E/S-141 (C) and E/CS-500-96 (D) share the same <i>ompA</i> genotype as E/Bour. Values represent the mean±SEM based on three independent experiments for time points of 2, 6, 12, 18, 24, 36, and 48 h post infection. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000878#s2" target="_blank">methods</a> for details.</p

Distribution/Location of the putative RNase E cleavage sites within the <i>pmpFE</i> operon coding sequence.

<p>The sequence is for reference strains E/Bour and L₂/434 and for clinical strains E/537C-05, E/S-141, E/CS-500-96 and L₂. Black vertical lines represent all RNase E cleavage sites conserved among all strains under study; green vertical lines show the ones only conserved among the four “E” strains; orange vertical lines represent those specific solely for both L₂ strains. Numbers represent nucleotide positions relative to the start codon of <i>pmpF.</i></p

Phase variation mediated by variable homopolymeric tracts.

<p>Panel A. The graph shows the evolution throughout passaging of the percentage of sequence reads with different ‘A’ counts in the homopolymeric tract upstream from CT533/<i>lpxC</i> for the strain E/CS1025/11. The poly(A) tract corresponds to poly(T) in the annotated leading strand. Panel B. Schematic view of the putative promoter region of CT533/<i>lpxC</i>. The predicted transcription start site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref126" target="_blank">126</a>] is labeled by +1. The variable poly(A) tract (in bold) falls between the predicted -35 and -10 hexamers (underlined). BLAST analyses revealed the existence of variable number of ‘A’ counts in <i>C</i>. <i>trachomatis</i> genomes, and also that the nucleotide indicated with an arrow is deleted exclusively in all LGV strains. Panel C. The graph shows the percentage of sequence reads with different ‘G’ counts in the variable homopolymeric tract of CT166 found in the initial populations of the epithelial-genital strains. “G” counts of nine correspond to an “ON” protein. Panel D. Schematic view of the four positions (numbers 1 to 4) relative to a gene at which contingency loci (e.g., homopolymeric tracts) can cause phase variation (adapted from van der Woude MW and Bäumler AJ, Clin Microbiol Rev <b>17</b>:581–611, 2004 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.ref152" target="_blank">152</a>]). Whereas positions 1 and 2 are associated with transcription initiation and position 4 with translation (ON/OFF), the mechanism regarding the position 3 is not completely disclosed. We found heterogeneity in length within homopolymeric tracts located in positions 2 (for CT533/<i>lpxC</i>) (blue), 3 (for CT043/<i>slc1</i> and the operon CT134-CT135), and 4 (for CT166—cytotoxin) (red).</p

Impact of <i>in vitro</i> passaging on the <i>C</i>. <i>trachomatis</i> growth kinetics.

<p>Panels A and B. Comparison of the growth rates and doubling times between ancestral (grey) and evolved populations (black). The percentage values above the bars correspond to the growth rate increment of the evolved population relatively to the ancestral. Panel C. Comparison of the one-step growth curve between D/CS637/11 CT135-positive and CT135-negative strains. Cells grown in the same conditions were infected at a MOI of 1, and cell scrapings were collected over time after infection for analysis of inclusion-forming units (IFUs). The black line represents the evolved CT135-negative D/CS637/11 strain, whereas the grey line represents the ancestor CT135-positive strain. The shaded area indicates the time points chosen for RNA-seq differential expression comparative analyses.</p

Mutational scenario throughout experimental evolution.

<p>Panel A. Chromosomal location of the genomic alterations observed during the <i>in vitro</i> passaging. The chromosomal position of each mutation (scale adjusted and given by the locus name) and the type of mutation event (inactivating events represented in red) are shown for each strain (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.s005" target="_blank">S3 Table</a> for details). Inactivating SNPs or indels refer to events leading to protein truncation (regardless the length of the resulting protein). For the strain D/CS637/11, the CT135 inactivating event involved the entire gene deletion between direct repeats (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133420#pone.0133420.g003" target="_blank">Fig 3</a>). <b>Panel B</b>. Dynamics of the emergence and spread of mutations and their frequency in the evolving bacterial populations. For each time-point (passages 5–7, 10, 20, 30, 50 and 100), circular graphs show the frequency of the mutations in the bacterial population, where each color represents a different mutated locus. The number of bacterial generations was estimated taking into account the minimum and maximum values of the mean doubling time of the strains analyzed at each time-point, and assuming a conservative approach by considering 15 hours of exponential phase <i>per</i> bacterial life-cycle (i.e, <i>per</i> passage). Loci designations are based on genome annotation of the D/UW3 strain (GenBank accession number NC_000117).</p