Synteny of a locus containing cruzipain genes in <i>T. cruzi</i>, <i>T. dionisii</i> and <i>T. b. brucei</i>.

<p>Segments from the chromosome 6 of <i>T. cruzi</i> CL Brener non-Esmeraldo-like and Esmeraldo-like haplotypes, corresponding to TcIII and TcII, respectively, showing 3 to 4 cruzipain gene copies (entire or partial) flanked by orthologous genes marked with different colors according to the legend. Data from the draft assembly of <i>T. cruzi</i> G, M6241 cl6 and <i>T. dionisii</i> allowed to place one, three or two cruzipain gene copies, respectively, within the same syntenic region (figure do not reflect their actual position on chromosomes). Syntenic region from the chromosome 6 of <i>T. b. brucei</i> comprising 11 copies in tandem of brucipain genes was included in the alignment. The shades of vertical gray bars indicate the variable degrees of divergence between sequences according to the legend. The accession codes of all contigs/scaffolds and GenBank accession numbers (in bold) are presented below the corresponding sequences.</p

Alignment of predicted amino acid sequences from entire cruzipain of <i>T. cruzi</i> (TcI, TcII, TcIII, TcVI and Tcbat) and homologues from <i>T. cruzi</i>-like (<i>T. c. marinkellei</i> and <i>T. dionisii</i>), <i>T. rangeli</i> and <i>T. b. brucei</i>.

<p>Pre, pro, catalytic domain and C-terminal extension amino acid sequences of cruzipain genes from <i>T. cruzi</i> Sylvio X10.6 and G (TcI), TCC1994 (Tcbat), Y and Esmeraldo cl3 (TcII), M6241 cl6 (TcIII), CL Brener (TcVI) Non-Esmeraldo-like (TcIII) and Esmeraldo-like (TcII) haplotypes and homologues from <i>T. c. marinkellei</i> (344), <i>T. dionisii</i> (211), <i>T. rangeli</i> (LDG and AM80) and <i>T. b. brucei</i> (TREU 927). The CATL family signatures of pro-domain motifs ERFININ (ERFN) and GNFD (GTFD) are indicated in bold and underlined, the subsites S1, S2 and S2′ are in bold, and the conserved Trp181 are indicated by (*).The glutamine [Q] of the oxyanion hole, cysteine [C], histidine [H] and asparagine [N] of catalytic triad in the catalytic domain, and 8 cysteines in the C-terminal extension are indicated by arrow heads.</p

Cluster analysis of mid-vaginal samples positive for “<i>Ca</i>. M. girerdii”.

<p>Relative abundance of microbial taxa in mid-vaginal bacterial communities of “<i>Ca.</i> M. girerdii” positive women is shown. The dendrogram was generated using Ward’s method with Manhattan distance. This analysis includes only mid-vaginal samples that exhibited at least 0.1% “<i>Ca</i>. M. girerdii” by 16S rDNA profiling. Clinical diagnosis is indicated in the first bar, and presence of <i>T. vaginalis</i> by RT-PCR is indicated in the second bar (orange designates a negative result and pink designates a positive result). The three samples dominated by <i>L. crispatus</i> and the three samples with the highest prevalence of <i>L. iners</i> were negative for <i>T. vaginalis</i>.</p

Polymorphism and network analyses of catalytic domain sequences of cruzipain genes from <i>T. cruzi</i> isolates of TcI-VI and Tcbat.

<p>(A) Polymorphic nucleotide sites on catalytic domains of cruzipain encoding genes; (B) Network based on polymorphic nucleotides constructed with the Neighbour-Net algorithm excluding all conserved sites and with Uncorrected p-distance. The numbers in nodes correspond to bootstrap values from 100 replicates. CLBrener1-5 are sequences from TriTrypDB: Tc00.1047053509429.320, Tc00.1047053507537.20, Tc00.1047053507603.270, Tc00.1047053507603.260 and Tc00.1047053507537.10; *GenBank accession numbers of all sequences included in these analyses are listed on Table1. Major types of sequences from <i>T. cruzi</i> isolates of different DTUs are indicated by different colors according to the legend.</p

Representation of “<i>Ca.</i> M. girerdii” genomes.

<p>A circular representation of the “<i>Ca.</i> M. girerdii” reference genome (strain VCU_M1) assembled from metagenomic sequences from a mid-vaginal sample. Position 1 is set to the start of the <i>dnaA</i> gene. Outermost circles (1–3) show the alignment (97% or greater identity) of contigs of three different strains from metagenomic assemblies from mid-vaginal samples containing high proportions of “<i>Ca</i>. M. girerdii”. Circle 4 (red) represents the reference strain (VCU_M1). Circles 5 (dark red) and 6 (blue) represent the predicted coding sequences in the forward and reverse orientations respectively. Circle 7 (black) shows the GC content, and circle 8 shows GC skew (pink (-), green (+)).</p

Phylogenetic tree of 16SrRNA shows uncultured “<i>Ca.</i> M. girerdii” clusters most closely with other uncultivated organisms in the Pneumoniae Group.

<p>The maximum likelihood tree was inferred by RAxML 7.2.7 using the gamma-distributed heterogeneity rate categories with 1,000 bootstraps. The 16S rRNA gene alignments were manually inspected. The Hominis Group is shaded in blue, the Pneumoniae Group in green, the Hemoplasma Group in gray and the Spiroplasma Group in purple. The 16S rRNA sequence of “<i>Ca</i>. M. girerdii” VCU_M1, “<i>Ca</i>. M. girerdii” VCU_PA1, “<i>Ca</i>. M. girerdii” VCU JB1 and “<i>Ca</i>. M. girerdii” VCU_G1 were identical. “<i>Ca</i>. M. girerdii” groups most closely with “Mnola”, which shows 100% identity in 16S rRNA sequence, uncultivated organisms from the oral sample of a low birth weight infant (HG764209, HG764210, and HG764212) and uncultivated species from rumen and termite gut in the Pneumoniae Group. A partial 16S rRNA sequence from the vaginal sample of a woman who delivered full term (JX871253) also exhibits 99% identity with “<i>Ca</i>. M. girerdii”, but was not included in the analysis due to its length.</p

Network genealogies of predicted amino acid sequences from all domains of genes encoding cruzipain in <i>T. cruzi</i> and homologues in other trypanosome species.

<p>Networks produced using the Neighbour-Net algorithm in SplitsTree v4.11.3, excluding all conserved sites and with Uncorrected p-distance. Networks were produced using entire sequences (A), pre- and pro-domains (B) or restricted to catalytic domains (C) of cruzipain encoding genes from the different trypanosomes are indicated by different symbols and colors according to the legend. Numbers in nodes correspond to support values estimated by performing 100 bootstrap replicates using the same parameter optimized for network inferences.</p

Unique strategies of “<i>Ca</i>. M. girerdii”.

<p>Putative transporters, enzymes involved in carbohydrate metabolism and virulence factors are represented in red for “<i>Ca</i>. M. girerdii”. Comparisons with other genital mycoplasmas are indicated with color-coded boxes: <i>M. genitalium</i> (MG, blue) <i>U. parvum</i> (UP, green) and <i>M. hominis</i> (MH, purple). Arrows indicate direction of transport. Light gray arrows represent metabolic strategies unique to other genital mycoplasma. Metabolic reconstruction was performed using ASGARD and careful inspection of manual annotations.</p

Expanded “<i>Ca</i>. M. girerdii” BspA-like protein family.

<p>The diverse family of 26 BspA-like protein family members from the “<i>Ca</i>. M. girerdii” strain VCU_M1 is depicted in panel (A). Predicted transmembrane domains and TpLRR domains are represented. An alignment of TpLRR domains from <i>Tannerella forsythia</i> BspA (AAC82625.1, bases 382–1347), “<i>Ca</i>. M. girerdii” strain VCU_M1 MGM1_3780 (bases 449–782) and <i>T.vaginalis</i> BspA-like TVAG_495790 (XP_001327783.1, bases 112–1077) is shown in panel (B).</p

Phylogenetic Tree based on inferred amino acid sequences confirms placement of “<i>Ca.</i> M. girerdii” in the Pneumoniae group.

<p>“<i>Ca</i>. M. girerdii” is located within the Pneumoniae group, denoted in green, in a subclade along with the <i>Ureaplasma</i> species, <i>M. iowae</i> and <i>M. penetrans</i>. The tree was inferred using amino acid sequences of 57 orthologs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110943#pone.0110943.s008" target="_blank">Tables S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110943#pone.0110943.s009" target="_blank">S5</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110943#pone.0110943.s010" target="_blank">S6</a>). Numbers at nodes correspond to the support values from 1,000 bootstrap replicates.</p