Periodic Reporting for period 1 - MINERVA (Role of Metabolic INteractions in the Eco-evolutionary dynamics of bacteRial communities in the VAgina)
Reporting period: 2021-04-01 to 2023-03-31
More than one out of four women in the world suffers from bacterial vaginosis. Their vaginal secretions become more abundant and have a strong fishy odor. Women with bacterial vaginosis show increased risks of getting most sexually transmissible infections and are more prone to complications during pregnancy. We know that bacterial vaginosis is linked to a modification of the vaginal microbiome, the ensemble of bacteria, fungi and viruses that inhabit the vagina. The vaginal microbiome is different in women suffering from bacterial vaginosis: i) certain bacterial species are preferentially present in the case of bacterial vaginosis; ii) it contains a higher diversity of bacterial species, and iii) it displays increased levels of a particular metabolic activity related to specific sugar molecules, the sialic acids. We know little, however, about the relative contribution of each of these bacterial species to the disease or to the changes in metabolic activity. We know even less about the interactions between bacteria from the same species and between different species, and about how these interactions are related to vaginal health and disease. In addition, bacterial species associated with bacterial vaginosis can also be found in the vaginal microbiome of healthy women, blurring our understanding of the development of bacterial vaginosis.
The MINERVA project addressed these questions with the ultimate aim of understanding the role of the bacterial metabolism of special sugars in the transition from vaginal health to disease. We used clinical isolates of three bacterial species of the vaginal microbiome and evaluated their genetic diversity and their metabolic profiles.
The MINERVA project addressed these questions with the ultimate aim of understanding the role of the bacterial metabolism of special sugars in the transition from vaginal health to disease. We used clinical isolates of three bacterial species of the vaginal microbiome and evaluated their genetic diversity and their metabolic profiles.
To investigate the role of sialic acid metabolism in bacterial vaginosis, we explored both the genetic and phenotypic diversity among clinical isolates of each bacterial species: Gardnerella spp, Prevotella bivia and Atopobium vaginae. We have collected vaginal clinical samples, created an isolate collection of 88 Gardnerella spp., 65 P. bivia and seven A. vaginae, and developed a growth protocol on solid surfaces. This growth assay allows both reliable monoculture growth of all our isolates as well as community experiments with co-cultures of isolates, so that we can test community-level interactions.
We first assessed genotypic and phenotypic diversity among our Gardnerella spp. and P. bivia isolates. We sequenced a conserved gene to evaluate genetic relationships among isolates, and we targeted three different sialidase genes, related to sialic acid metabolism. We further performed biochemical assays to quantify bacterial sialidase enzyme activity. For A. vaginae, we could not find any sialidase genes, and only one isolate displayed a very low sialidase activity. For P. bivia, the genetic diversity was relatively low and all isolates possessed a putative sialidase gene, but the actually expressed sialidase activity varied greatly between isolates. For Gardnerella spp. we found great genotypic variation in terms of whole-genome relatedness and of patterns of presence/absence of sialidase genes. When crossing the genetic data with the actual sialidase activity production, we conclude that the presence of the nanH3 sialidase gene was predictive of expressed sialidase activity in Gardnerella spp, and we observed a very large sialidase phenotypic diversity among isolates, both qualitatively and quantitatively. No pattern of genotypic nor phenotypic diversity, however, was linked to a clinical diagnosis of BV.
We then evaluated the sialidase production patterns at the community-level. For this we performed bacterial co-cultures, so that we could evaluate the direction and strength of intra- and interspecific interactions. The experimental system we implemented enabled us to measure expressed sialidase activity for 82 co-cultures formed by a selection of Gardnerella spp., P. bivia and A. vaginae isolates, in a spatially structured environment. All isolates were categorized based on their expressed sialidase activity in monoculture (strong-, low-, or non-producer), and we assessed intra and inter-specific interactions through combinations of two or three isolates of different taxa and sialidase categories. Our results support the hypothesis that both intra- and interspecific interactions likely play a key role in sialic acid-related bacterial functions. Indeed, sialidase activity in cocultures can strongly diverge from expectations based on monoculture data with numerous cases suggesting the presence of synergistic interactions amongst bacterial vaginosis-associated bacteria. Synergistic interactions where also found for interspecific co-cultures and were strongest in co-cultures of Gardnerella spp. and A. vaginae isolates with co-cultures expressing substantially higher sialidase activity levels than the monocultures of both partner isolates added together.
The first scientific article of this project (corresponding to WP1 and 2) is currently in the final phase of preparation and another one will follow (for WP4). The results have further been presented at the institute seminars, to our international scientific collaborators (mainly in South Africa) and to the medical collaborators at the hospital, and at the international conference of the French Society of Microbiology.
We first assessed genotypic and phenotypic diversity among our Gardnerella spp. and P. bivia isolates. We sequenced a conserved gene to evaluate genetic relationships among isolates, and we targeted three different sialidase genes, related to sialic acid metabolism. We further performed biochemical assays to quantify bacterial sialidase enzyme activity. For A. vaginae, we could not find any sialidase genes, and only one isolate displayed a very low sialidase activity. For P. bivia, the genetic diversity was relatively low and all isolates possessed a putative sialidase gene, but the actually expressed sialidase activity varied greatly between isolates. For Gardnerella spp. we found great genotypic variation in terms of whole-genome relatedness and of patterns of presence/absence of sialidase genes. When crossing the genetic data with the actual sialidase activity production, we conclude that the presence of the nanH3 sialidase gene was predictive of expressed sialidase activity in Gardnerella spp, and we observed a very large sialidase phenotypic diversity among isolates, both qualitatively and quantitatively. No pattern of genotypic nor phenotypic diversity, however, was linked to a clinical diagnosis of BV.
We then evaluated the sialidase production patterns at the community-level. For this we performed bacterial co-cultures, so that we could evaluate the direction and strength of intra- and interspecific interactions. The experimental system we implemented enabled us to measure expressed sialidase activity for 82 co-cultures formed by a selection of Gardnerella spp., P. bivia and A. vaginae isolates, in a spatially structured environment. All isolates were categorized based on their expressed sialidase activity in monoculture (strong-, low-, or non-producer), and we assessed intra and inter-specific interactions through combinations of two or three isolates of different taxa and sialidase categories. Our results support the hypothesis that both intra- and interspecific interactions likely play a key role in sialic acid-related bacterial functions. Indeed, sialidase activity in cocultures can strongly diverge from expectations based on monoculture data with numerous cases suggesting the presence of synergistic interactions amongst bacterial vaginosis-associated bacteria. Synergistic interactions where also found for interspecific co-cultures and were strongest in co-cultures of Gardnerella spp. and A. vaginae isolates with co-cultures expressing substantially higher sialidase activity levels than the monocultures of both partner isolates added together.
The first scientific article of this project (corresponding to WP1 and 2) is currently in the final phase of preparation and another one will follow (for WP4). The results have further been presented at the institute seminars, to our international scientific collaborators (mainly in South Africa) and to the medical collaborators at the hospital, and at the international conference of the French Society of Microbiology.
The role of G. vaginalis as a possible single-cause etiological factor for BV remains highly debated. The presence of G. vaginalis in vaginal samples from both BV-positive and BV-negative women has raised interest on the one hand in assessing the diversity among Gardnerella isolates, and on the other hand on the importance of the concomitant presence of other bacterial species in the vaginal microbiome.
With the MINERVA project, we provided an overview of Gardnerella spp. diversity, in particular in terms of sialidase activity, whose detection is strongly associated to BV diagnostic. We found large phenotypic diversity across various Gardnerella clades and no correlation between BV diagnostic and taxonomy, sialidase gene detection or expressed sialidase activity. With the aim of shifting the focus to the bacterial community scale, we further explored to genetic and phenotypic diversity of two other bacterial species commonly found in association with Gardnerella spp. in BV-associated vaginal samples. All tested P. bivia isolates displayed sialidase activity but with highly variable levels, and none of the few tested A. vaginae isolates produced detectable levels of sialidase activity under our experimental conditions. Confirming the importance of community context and intra- and interspecific interactions for metabolic activity, we found that sialidase activity in cocultures can strongly diverge from expectations based on monoculture data with numerous cases suggesting the presence of synergistic interactions amongst BV-associated bacteria.
Our project contributed to the vaginal microbiome field through three main aspects: (i) it provides an experimental protocol for metabolic assessment in mono- and co-cultures under spatially explicit conditions, (ii) it improved the knowledge on Gardnerella spp. diversity and its link to BV diagnostic, and (iii) it brought experimental insights and support for the role of interactions in microbiome metabolism.
With the MINERVA project, we provided an overview of Gardnerella spp. diversity, in particular in terms of sialidase activity, whose detection is strongly associated to BV diagnostic. We found large phenotypic diversity across various Gardnerella clades and no correlation between BV diagnostic and taxonomy, sialidase gene detection or expressed sialidase activity. With the aim of shifting the focus to the bacterial community scale, we further explored to genetic and phenotypic diversity of two other bacterial species commonly found in association with Gardnerella spp. in BV-associated vaginal samples. All tested P. bivia isolates displayed sialidase activity but with highly variable levels, and none of the few tested A. vaginae isolates produced detectable levels of sialidase activity under our experimental conditions. Confirming the importance of community context and intra- and interspecific interactions for metabolic activity, we found that sialidase activity in cocultures can strongly diverge from expectations based on monoculture data with numerous cases suggesting the presence of synergistic interactions amongst BV-associated bacteria.
Our project contributed to the vaginal microbiome field through three main aspects: (i) it provides an experimental protocol for metabolic assessment in mono- and co-cultures under spatially explicit conditions, (ii) it improved the knowledge on Gardnerella spp. diversity and its link to BV diagnostic, and (iii) it brought experimental insights and support for the role of interactions in microbiome metabolism.