Relative abundances of <i>L</i>. <i>crispatus</i> proteins in cervicovaginal lavages of women with positive <i>L</i>. <i>crispatus</i> 16S rDNA microarray results (n = 13) among two vaginal pH categories.

<p>Homologous proteins from different strains are numbered (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150767#pone.0150767.t001" target="_blank">Table 1</a>). Box plots represent median (black line), first and third quartiles (box) and range within 1.5 times the interquartile range from the box (whiskers). Outliers are plotted as points. *p-value<0.05. Abbreviations: GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ENO: enolase; ALDO: fructose-bisphosphate aldolase; GPI: glucose-6-phosphate isomerase; PulA: pullulanase type I; CDP: cell division protein; HL: hydrolase; SD: YSIRK signal domain/LPXTG anchor domain surface protein; HP: hypothetical protein.</p

Relative abundances of <i>L</i>. <i>iners</i> proteins in cervicovaginal lavages of women with positive <i>L</i>. <i>iners</i> 16S rDNA microarray results (n = 31) among three cervicovaginal microbiota groups.

<p>GAPDH_1, GAPDH_2, ALDO, GPI, and DPS were significantly decreased in women with moderate and severe dysbiosis, compared to women with a <i>L</i>. <i>iners</i>-dominant cervicovaginal microbiota. Box plots represent median (black line), first and third quartiles (box) and range within 1.5 times the interquartile range from the box (whiskers). Outliers are plotted as points. *p-value<0.05; ** p-value<0.01; ***p-value<0.001. Abbreviations: GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ALDO: fructose-bisphosphate aldolase; GPI: glucose-6-phosphate isomerase; DPS: DNA starvation/stationary phase protection protein; EFtu: elongation factor Tu; PK: pyruvate kinase.</p

Cervicovaginal <i>Lactobacillus iners</i> and <i>L</i>. <i>crispatus</i> proteins identified by mass spectrometry.

<p>Cervicovaginal <i>Lactobacillus iners</i> and <i>L</i>. <i>crispatus</i> proteins identified by mass spectrometry.</p

Relative abundances of <i>L</i>. <i>iners</i> proteins in cervicovaginal lavages of women with positive <i>L</i>. <i>iners</i> 16S rDNA microarray results (n = 31) among three vaginal pH categories.

<p>GAPDH_1, GAPDH_2, ALDO, GPI, and DPS were significantly decreased in women with a vaginal pH ≥5, compared to women with a vaginal pH between 4 and 5. Box plots represent median (black line), first and third quartiles (box) and range within 1.5 times the interquartile range from the box (whiskers). Outliers are plotted as points. *p-value<0.05; ** p-value<0.01; ***p-value<0.001. Abbreviations: GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ALDO: fructose-bisphosphate aldolase; GPI: glucose-6-phosphate isomerase; DPS: DNA starvation/stationary phase protection protein; EFtu: elongation factor Tu; PK: pyruvate kinase.</p

Relative abundances of <i>L</i>. <i>crispatus</i> proteins in cervicovaginal lavages of women with positive <i>L</i>. <i>crispatus</i> 16S rDNA microarray results (n = 13) among three cervicovaginal microbiota groups.

<p>Homologous proteins from different strains are numbered (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150767#pone.0150767.t001" target="_blank">Table 1</a>). Box plots represent median (black line), first and third quartiles (box) and range within 1.5 times the interquartile range from the box (whiskers). Outliers are plotted as points. *p-value<0.05. Abbreviations: GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ENO: enolase; ALDO: fructose-bisphosphate aldolase; GPI: glucose-6-phosphate isomerase; PulA: pullulanase type I; CDP: cell division protein; HL: hydrolase; SD: YSIRK signal domain/LPXTG anchor domain surface protein; HP: hypothetical protein.</p

SpaCBA pili are glycosylated with mannose and fucose.

<p><b>(A) SpaCBA pili are glycosylated on <i>L</i>. <i>rhamnosus</i> GG cells—</b>Cell wall-associated proteins of <i>L</i>. <i>rhamnosus</i> GG wild type (1), the pilus-deficient Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357, 2) and the Δ<i>welE</i>::Tc^R mutant on which the pili are overexposed (CMPG5351, 3), were probed with mannose- and fucose-specific lectins (HHA and AAL resp.). Pili content was visualized by probing with SpaC antiserum (SpaC, black arrow and HMW: high molecular weight pili). Interference of the Msp1 glycoprotein was ruled out (open arrow). Blots and gels were performed in triplicate. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(B) Purified pili are glycosylated—</b>SDS-PAGE separated pili (pool A) were stained with PAS glycostain and Sypro^® to visualize their protein content. Pili content was shown by probing of Western blotted samples with SpaC antiserum (HMW: high molecular weight pili). Purified Msp1 (open arrow) was used as a positive control. Representative gels are shown, experiment was carried out in triplicate. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(C) SpaCBA pili bind mannose-specific lectins—</b>Purified pili fractions (PRM and pili pool B) were subjected to PAS glycostaining and both Sypro^® and Silver stain to visualize their protein content. Absence of 75 kDa signals on PAS and lectin blots rule out the interference Msp1 (cf. open arrow). Western blotted samples were probed with SpaC antiserum (HMW: high molecular weight pili) and the mannose-specific lectins HHA and GNA, visualizing the pili content of the samples and the presence of mannose, respectively. Representative gels and blots of in triplicate-repeated experiment. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(D&E) Mannose- and fucose-specific lectins bind SpaCBA pili—</b>Binding of lectins to plate-coated pili was measured to ELISA. Wells coated with coating buffer served as a negative control. Mannan and Lewis X were coated as a positive control for the mannose-specific HHA (<i>Hippeastrum</i> hybrid, D) and fucose-specific AAL (<i>Aleuria aurantia</i>, E) lectins, respectively. Error bars represent standard deviations of three independent experiments (paired t-test, p < 0.05)</p

Probing of fucose and mannose residues on <i>L</i>. <i>rhamnosus</i> GG pili using AFM.

<p>Fig 1A and 1C depict the adhesion forces and Fig 1B and 1D the rupture length histograms (n = 1024) obtained in buffer, from the interaction between <i>L</i>. <i>rhamnosus</i> GG wild type and fucose- and mannose-binding lectin probes (AAL and HHA resp.). In Fig 1E and 1F the force data for the interaction of a pili-deficient Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357) with the two lectin probes are displayed. Insets show representative retraction force curves.</p

Immunogold labeling reveals colocalization of SpaA and fucose on SpaCBA pili.

<p>Immunoelectron microscopy double labeling of <i>L</i>. <i>rhamnosus</i> GG cells (A and B) and the Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357) (C) with SpaA antiserum and the fucose-specific <i>Aleuria aurantia</i> lectin (AAL). Detection of SpaA and AAL was done using 5 nm (white arrows) and 10 nm gold particles (black arrows) respectively. The scale bar represents 500 nm. Original overall pictures are shown as insets of A and B.</p