Unique Insights in the Cervicovaginal Lactobacillus iners and L. crispatus Proteomes and Their Associations with Microbiota Dysbiosis

Abstract

Background: A Lactobacillus-dominated cervicovaginal microbiota (VMB) protects women from adverse reproductive health outcomes, but the role of L. iners in the VMB is poorly understood. Our aim was to explore the association between the cervicovaginal L. iners and L. crispatus proteomes and VMB composition.

Methods: The vaginal proteomes of 50 Rwandan women at high HIV risk, grouped into four VMB groups (based on 16S rDNA microarray results), were investigated by mass spectrometry using cervicovaginal lavage (CVL) samples. Only samples with positive 16S results for L. iners and/or L. crispatus within each group were included in subsequent comparative protein analyses: Lactobacillus crispatus-dominated VMB cluster (with 16S-proven L. iners (ni) = 0, and with 16S-proven L. crispatus (nc) = 5), L. iners-dominated VMB cluster (ni = 11, nc = 4), moderate dysbiosis (ni = 12, nc = 2); and severe dysbiosis (ni = 8, nc = 2). The relative abundances of proteins that were considered specific for L. iners and L. crispatus were compared among VMB groups.

Results: Forty Lactobacillus proteins were identified of which 7 were specific for L. iners and 11 for L. crispatus. The relative abundances of L. iners DNA starvation/stationary phase protection protein (DPS), and the glycolysis enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate isomerase (GPI), were significantly decreased in women with L. iners-containing dysbiosis compared to women with a L. iners-dominated VMB, independent of vaginal pH and L. iners abundance. Furthermore, L. iners DPS, GAPDH, GPI, and fructose-bisphosphate aldolase (ALDO) were significantly negatively associated with vaginal pH. Glycolysis enzymes of L. crispatus showed a similar negative, but nonsignificant, trend related to dysbiosis.

Conclusions: Most identified Lactobacillus proteins had conserved intracellular functions, but their high abundance in CVL supernatant might imply an additional extracellular (moonlighting) role. Our findings suggest that these proteins can be important in maintaining a Lactobacillus-dominated VMB. Functional studies are needed to investigate their roles in vaginal bacterial communities and whether they can be used to prevent vaginal dysbiosis.

Fig 1. Relative abundances of L. iners proteins in cervicovaginal lavages of women with positive L. iners 16S rDNA microarray results (n = 31) among three cervicovaginal microbiota groups.

GAPDH_1, GAPDH_2, ALDO, GPI, and DPS were significantly decreased in women with moderate and severe dysbiosis, compared to women with a L. iners-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.

Fig 2. Relative abundances of L. iners proteins in cervicovaginal lavages of women with positive L. iners 16S rDNA microarray results (n = 31) among three vaginal pH categories.

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.

Fig 3. Relative abundances of L. crispatus proteins in cervicovaginal lavages of women with positive L. crispatus 16S rDNA microarray results (n = 13) among three cervicovaginal microbiota groups.

Homologous proteins from different strains are numbered (Table 1). 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.

Fig 4. Relative abundances of L. crispatus proteins in cervicovaginal lavages of women with positive L. crispatus 16S rDNA microarray results (n = 13) among two vaginal pH categories.

Homologous proteins from different strains are numbered (Table 1). 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.