Transcription of TP0126, Treponema pallidum Putative OmpW Homolog, Is Regulated by the Length of a Homopolymeric Guanosine Repeat

An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum, the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis

Unique Features of MgpB Including C-terminal Homology to MgpC, Periodic Repeats, and Overrepresented Amino Acids are Illustrated using <i>in silico</i> Analyses.

<p>(A) The distal C-termini of MgpB and MgpC are homologous, share periodic lysine repeats (every fourth or seventh amino acid; orange underline) and are proline-rich (blue). Remaining lysine residues not included within the periodic repeat are in orange/black. BLAST alignment of the predicted transmembrane domains (gray) and beyond for aa 1,352–1,443 and 940–1,053 of MgpB and MgpC, respectively, demonstrate the cytoplasmic domain of MgpB has significant homology to aa 940–1,032 of MgpC with an e-value of 1e-21. (B) Schematic diagram of MgpB showing amino acid repeat sequences and periodicity including (boxes): (I) asparagine repeats at aa 71 (green, underlined), (II) alternating asparagine repeats at aa 733 (green, underlined), (III) alternating serine repeats at aa 778 (blue, underlined), (IV) “NQLL” motif found twice at aa 727 and 908 (bold, dashed underline in boxes II and IV), (V) serine repeats at aa 989 (blue, underlined), (VI) “LVNL” sequence found at aa 1,233 and 1,288 (double underline), and (VII) lysine residues repeated every fourth amino acid in the threonine/proline-rich distal conserved C-terminus, aa 1,431 (orange, underlined; also shown in panel B). Sequences in boxes I, IV, VI, and VII were 100% conserved in all strains while sequences containing alternating asparagine and serine repeats varied, indicated by numbered sequences within boxes II, III, and V. Sequence 1 corresponds to G37 and the number of variants expressing each unique sequence is shown to the right in brackets (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.s006" target="_blank">S2 Fig</a> for a complete alignment). Shown below is an illustration of the overrepresented amino acids within overlapping ~240 aa segments of MgpB, determined using the SAPS <i>B</i>. <i>subtilis</i> reference set; overrepresented residues (99% quantile point) are indicated by “++”. The complete SAPS analysis including underrepresented amino acids and comparisons to other reference sets (<i>E</i>. <i>coli</i> and a random sample of proteins) can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.s004" target="_blank">S4 Table</a>.</p

MgpB Contains a Single C-terminal Transmembrane Domain.

<p>(A) Schematic depiction of MgpB showing the location of the N-terminal segment absent in the mature protein (aa 1–58, red), variable regions B, EF, and G (yellow), and the position of the previously described transmembrane domains M1–M5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.ref039" target="_blank">39</a>] (gray and red, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.s001" target="_blank">S1 Table</a> for coordinates). The recombinant peptides spanning MgpB used for the production of rabbit antibodies are indicated with black bars. The N-, D1-, and D2-domains described by Opitz and Jacobs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.ref034" target="_blank">34</a>] spanning aa 200–384, 769–954, and 1,123–1,360, respectively, are shown in blue with the precise binding locations of attachment inhibiting monoclonal antibodies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.ref034" target="_blank">34</a>] indicated by lines with circles. (B) Transmembrane domains in MgpB predicted by the TMHMM program. The N-terminal signal peptide and a single C-terminal transmembrane domain (both red) are identified with high probability, predicting a topology in which the majority of the protein is extracellular (pink) with a short cytoplasmic domain (blue), a prediction corroborated by the majority of algorithms used. (C) The N-terminal transmembrane helix identified in panel B includes the predicted MgpB signal peptide. Sequencing of mature MgpB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.ref039" target="_blank">39</a>] indicates that the N-terminus begins at amino acid 59 (solid underline following red arrow). Using the Gram-positive network, all computational programs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#sec002" target="_blank">Materials & Methods</a>) identify a signal peptide spanning amino acids 1–30 (bold) with the predicted cleavage site occurring between the amino acids VGG-YF; results using the Gram-negative network give a shorter signal peptide covering amino acids 1–26 with the cleavage site between VIT-GV (dotted underline).</p

Amino Acid Diversity and Predicted B-cell Epitopes are Distributed throughout the MgpB Adhesin.

<p>(A) Amino acid diversity within the MgpB adhesin is concentrated within and around variable regions B, EF, and G. All available <i>mgpB</i> sequences were translated <i>in silico</i> and aligned to create a hypothetical consensus sequence; the percentage of sequences matching the consensus at each residue is shown in gray, while the percentage of sequences with variant amino acids is noted with colored bars as indicated in the key above and as follows: (1) blue, second most common residue (meaning the most common variant differing from the consensus), (2) orange, third most common residue, (3) green, fourth most common, (4) pink, fifth most common, and (5) aqua, sixth most common residue. Deletions (as compared to the consensus) are indicated by dark gray bars descending from the top of the graph. As an example, at the first amino acid, 100% of the sequences encode methionine (represented by a solid gray line), whereas five different amino acids were detected at position 117 [noted in gray (the consensus), blue, orange, green, and pink (four variants)]. The strain typing region and serine repeats are designated in orange and purple, respectively, while dashed red lines indicate the semi-conserved regions in EF that contain a high number of synonymous mutations. The location of the MgpB conserved and variable regions are noted below the x-axis in gray and black, respectively, as is the location of the C-terminal putative transmembrane domain (TM) and the experimentally determined start of the mature protein (red arrow). (B) The location of predicted linear B-cell epitopes, as determined using the BepiPred 1.0b Server. High (>0.9) and low (<0.9) scoring epitopes are indicated in red and gray, respectively. (C) The location of predicted conformational epitopes, as determined using the CBTOPE algorithm. Residues with scores above 4.0 indicate conformational epitopes and are shaded red (those below the 4.0 cutoff are in gray). Vertical shading indicates the following: strain typing region (orange), amino acid diversity including and surrounding the three variable regions (yellow), serine repeats (purple), and the C-terminal transmembrane domain (red).</p

The Distribution of Synonymous and Non-Synonymous Mutations Differ within Defined Regions of the <i>mgpB</i> Expression Site.

<p>Nucleotide (DS_nt, in black) and amino acid diversity scores (DS_aa, in red) for overlapping windows of 12 nucleotides or 4 amino acids, respectively, are shown on the y-axis with MgpB amino acid position on the x-axis. As detailed in Materials & Methods, a DS = 0 indicates 100% sequence conservation while a DS = 1 indicates 100% variability. Synonymous mutations are apparent in regions where black peaks are visible indicating that the DS_nt is greater than DS_aa (for example, aa 880–920 of region EF). Below the x-axis, the total number of sequences analyzed per region (<i>N</i>), the average % of non-synonymous and synonymous mutations, and the average DS values for each region are listed.</p

The Majority of MgpB-Reactive Antibodies Bind to the Surface of Intact <i>M</i>. <i>genitalium</i> Cells.

<p>Binding of region-specific antibodies to MgpB was measured using intact (PBS-treated, “No Triton” dark gray) and Triton X-100 permeabilized (“+ Triton”, light gray) <i>M</i>. <i>genitalium</i>. The increased reactivity with treated compared to untreated cells was significant for the conserved N-terminus (rMgpB-1; *, p = 0.024), variable region EF (rMgpB-EF; **, p < 0.001), and the putative cytoplasmic domain (rMgpB-4b; **, p = 0.0004); conversely there was a significant decrease in antibody binding in treated compared to untreated cells for the second conserved region (**, p = 0.007 and p = 0.0061 for rMgpB-2a and -2b, respectively). The assay was repeated in triplicate with results of a typical experiment shown above with each condition tested in quadruplicate; error bars indicate standard deviation.</p

Summary of Antibody Accessibility, Hemadsorption Inhibition, Amino Acid Variation and Immunogenicity of MgpB.

<p>^<i>a</i> ND—not determined</p><p>^<i>b</i> “X” indicates any amino acid</p><p>Summary of Antibody Accessibility, Hemadsorption Inhibition, Amino Acid Variation and Immunogenicity of MgpB.</p

Analysis of the <i>Mycoplasma genitalium</i> MgpB Adhesin to Predict Membrane Topology, Investigate Antibody Accessibility, Characterize Amino Acid Diversity, and Identify Functional and Immunogenic Epitopes

<div><p><i>Mycoplasma genitalium</i> is a sexually transmitted pathogen and is associated with reproductive tract disease that can be chronic in nature despite the induction of a strong antibody response. Persistent infection exacerbates the likelihood of transmission, increases the risk of ascension to the upper tract, and suggests that <i>M</i>. <i>genitalium</i> may possess immune evasion mechanism(s). Antibodies from infected patients predominantly target the MgpB adhesin, which is encoded by a gene that recombines with homologous donor sequences, thereby generating sequence variation within and among strains. We have previously characterized <i>mgpB</i> heterogeneity over the course of persistent infection and have correlated the induction of variant-specific antibodies with the loss of that particular variant from the infected host. In the current study, we examined the membrane topology, antibody accessibility, distribution of amino acid diversity, and the location of functional and antigenic epitopes within the MgpB adhesin. Our results indicate that MgpB contains a single transmembrane domain, that the majority of the protein is surface exposed and antibody accessible, and that the attachment domain is located within the extracellular C-terminus. Not unexpectedly, amino acid diversity was concentrated within and around the three previously defined variable regions (B, EF, and G) of MgpB; while nonsynonymous mutations were twice as frequent as synonymous mutations in regions B and G, region EF had equal numbers of nonsynonymous and synonymous mutations. Interestingly, antibodies produced during persistent infection reacted predominantly with the conserved C-terminus and variable region B. In contrast, infection-induced antibodies reacted poorly with the N-terminus, variable regions EF and G, and intervening conserved regions despite the presence of predicted B cell epitopes. Overall, this study provides an important foundation to define how different segments of the MgpB adhesin contribute to functionality, variability, and immunogenicity during persistent <i>M</i>. <i>genitalium</i> infection.</p></div

Antibodies Elicited During Persistent Experimental <i>M</i>. <i>genitalium</i> Infection of Non-Human Primates Recognize Variable Region B and the Conserved C-terminus.

<p>ELISA reactivity of primate serum collected at weekly time points over the course of infection measured against the recombinant MgpB proteins spanning the conserved (rMgpB-1, -2a, -2b, -4a, and -4b) and variable regions (rMgpB-B,-EF, and-G), as indicated in the legends to the right. ODs shown represent values after subtracting the readings from no antigen control wells. (A) In primate A01220 (cervical and salpingeal pocket infection), antibody reactivity to recombinant proteins spanning the variable rMgpB-B, as well as the proximal and distal conserved C-terminus (rMgpB-4a and -4b), increased during infection, peaking at week 2 post-inoculation. (B) Primate J00106 (cervical infection only) exhibited a similar pattern of reactivity against rMgpB-B and -4a, with the antibody response peaking around 8 weeks post-infection. Data above represents the average of two independent experiments, with each condition tested in triplicate.</p

Attachment domains within MgpB are Located within the Proximal Conserved C-terminus and Share Homology with the Attachment Domain of the <i>M</i>. <i>pneumoniae</i> P1 Protein.

<p>(A) The results of the quantitative hemadsorption inhibition assay showing that antibodies reacting with the proximal conserved C-terminus (α-rMgpB-4a) diluted 1:640 inhibit hemadsorption by approximately 40% (**, p < 0.0001). At dilutions with comparable reactivity to MgpB (1:20, α-rMgpB-2b and-EF; 1:40, α-rMgpB-2a and-G; 1:80, α-rMgpB-1 and -4b; 1:320 α-rMgpB-B), antibodies reacting with other regions of MgpB have no statistical impact on the binding of red blood cells with results similar to untreated control wells not pretreated with antibodies. (B) Hemadsorption inhibition experiments demonstrating the inhibitory effect of α-rMgpB-4a antibodies was dose dependent; two-fold dilutions of this antibody ranging between 1:20 and 1:1,280 significantly decrease hemadsorption (**, p < 0.0001; *, p = 0.007) with lower dilutions completely inhibiting the binding of red blood cells with results comparable to the no bacteria control wells. In panels A and C, error bars represent standard deviation. (C) Alignment of the proximal and distal conserved C-termini of MgpB (aa 1,184–1,444) with the C-terminus of the homologous P1 adhesin (aa 1,337–1,627) of <i>M</i>. <i>pneumoniae</i>, for which the attachment domain has also been defined. Within P1, the location of peptides 7 and 8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.ref048" target="_blank">48</a>] are noted with purple and green lines, respectively, and the binding epitope for the attachment inhibiting monoclonal antibody [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138244#pone.0138244.ref049" target="_blank">49</a>] (designated PepD) is underlined in red. Amino acids identically conserved between MgpB and P1 are highlighted in gray; additionally, predicted transmembrane domains predicted by TMHMM are boxed and the location of cytoplasmic proline (white/blue) and lysine (orange/black) residues are indicated. The arrows beneath the P1 sequence at the distal conserved C-termini note the location of a simple tandem repeat of unknown significance.</p