Proteolytic Prevotella: Catalysts of Complement Dysregulation Linking the Vaginal Microbiome and Preterm Birth

Abstract

Preterm birth (PTB) occurs in 10% of pregnancies worldwide, often resulting in brain injury, long term disability, and requiring neonatal intensive care. Despite its high prevalence, the underlying causes of PTB are poorly understood. Bacterial vaginosis (BV) has been associated with a 2-fold increase in PTB risk; select BV-associated bacteria including proteolytic Prevotella species have been directly linked to preterm delivery. Oral Prevotella spp. secrete a well-characterized cysteine protease that targets and cleaves key complement regulator C3 to produce the pro-inflammatory anaphylatoxin C3a. Complement dysregulation has been implicated in PTB but linkages between BV bacteria and changes in cervicovaginal complement signalling have not been explored. Here, we are the first to describe a cysteine-protease mediated C3-cleavage mechanism from BV- and PTB-associated Prevotella bivia in vitro. Anti-C3c Western blot analysis depicted a conserved cleavage profile from cell-free bacterial supernatants producing bands corresponding to the release of C3a (109 kDa a’-chain) as well as an uncharacterized band at 35 kDa; this phenotype was inhibited upon the addition of cysteine protease inhibitor E-64. N-terminomic analysis of cleavage products revealed cuts C-terminal to the anaphylatoxin domain (R748) and CUB1 domains (M990) that were lost with protease inhibitor treatment and corresponded to the 109 and 35 kDa products observed by western blot. Production of C3a was specifically quantified using a C3a ELISA and was significantly different from controls (ANOVA, p = 0.0001). Bioinformatic analysis of the P. bivia genome revealed three candidate C10 proteases as likely targets for further characterization, secretion of these proteases was confirmed using cell-shaving proteomics and molecular modelling was used to identify the most likely candidate conferring C3 cleavage. We also explored the relationships between cervicovaginal Prevotella spp., complement activation and PTB in a cohort of high risk pregnancies. Baseline vaginal samples collected >72 hours prior to delivery from patients with no known antimicrobial exposure were selected for multiplex immunological profiling of complement proteins, PTB-associated cytokines and matrix metalloproteases (N = 66) and a subset was profiled for vaginal microbiome composition using 16S rNA gene amplicon sequencing (n = 28). Approximately half of the cohort delivered preterm (47%) with a median time to delivery of 4.8 weeks, compared to 8.7 weeks for term patients (Mann-Whitney U-test, p=0.0001); no other clinical features differed significantly across patients who delivered preterm vs. term. Presence and abundance of Prevotella spp. was significantly associated with preterm delivery (Mann-Whitney U-test, p = 0.0033; Spearman Correlation, p = 0.0063, ρ = -0.5036) and robust PCA revealed that differences in variation between samples was driven by activation of C3 and C5. C3 activation was negatively correlated with gestational age at birth (Spearman Correlation, p = 0.0441, ρ = -0.2486), and C5 activation was positively correlated with cervical dilation at sampling (Spearman Correlation, p = 0.0120, ρ = 0.3122). Taken together, this data reveals a novel mechanism for immune modulation by BV-associated bacterium Prevotella bivia with implications for preterm delivery, particularly by way of C3- and C5-mediated cervicovaginal inflammation. With further biochemical characterization, the cysteine proteases described here provide potential targets for prophylactic treatments that could reduce the burden of PTB on families and children.