Tiny Can Be Big: The Contribution of Microbial Extracellular Vesicles to Host-Pathogen Interactions

Abstract

The human vagina hosts a microbiome formed by dense numbers of diverse bacterial species which can be categorised based on their species diversity. Despite the variation, the low-diversity communities (i.e. a dominant single species of Lactobacillus) is understood to represent the microbiota of healthy premenopausal women. The diversified community, on the other hand, is formed mostly by anaerobic microorganisms with several species that are associated with bacterial vaginosis (BV). BV is a polymicrobial condition that develops due to the imbalance of the microbiome and is linked to a higher risk of acquiring other genital infections, including the infection caused by the parasite Trichomonas vaginalis. This flagellated protozoan is a causative agent of trichomoniasis, one of the world’s most common non-viral sexually transmitted infections. The co-existence of these pro- and eukaryotic microorganisms (i.e. bacteria and protozoan) in the vagina, as revealed by metagenomics, suggests a functional relationship between vaginal microbiomes and this pathogen. Indeed, previous research from our group has shown that microbiome-parasite interactions directly modulate pathogenesis. Therefore, in this thesis, I envisaged that these microorganisms should engage in some kind of communication between themselves and with the host. Extracellular vesicles (EVs) are now a recognised mechanism for cell-to-cell communication. They serve as a vehicle for the delivery of bioactive molecules. Once taken up by the target cells, EVs can elicit functional responses and promote phenotypic changes affecting physiological or pathological status. This study proposed that EVs might be an important component of the intercellular and interkingdom communication among the triad parasite-bacteria-human. Here, I have explored whether these microorganisms produce EVs, examined their molecular cargoes and evaluated how the delivery of microbial EVs may alter host and pathogen responses. T. vaginalis has been previously shown to produce EVs with protein and RNA cargoes. However, the RNA cargo was left uncharacterised. Here, we demonstrated that this RNA cargo is indeed encapsulated into these vesicles and is preferentially composed of small size RNAs. Importantly, these molecules are efficiently delivered to human cells. By employing the deep sequencing and bioinformatic analysis, we identified biotypes of RNAs in T. vaginalis EVs and showed preferential packaging of tRNA fragments into these vesicles. Aiming to understand how EVs from the vaginal microbiome may influence host-parasite behaviour, two bacterial species of the vagina were selected for characterisation of their EVs - Lactobacillus gasseri and Gardnerella vaginalis - representatives of a healthy or dysbiotic microbiome, respectively. Both bacteria were shown to release EVs during their normal growth. The application of the purification step increased our confidence in their cargo. By applying an advanced proteomics approach, SWATH, we demonstrated the specific enrichment of L. gasseri EVs for bacteriocins and other antimicrobial compounds while showing that G. vaginalis EVs were enriched for virulence factors and potential toxins. To our knowledge, this was the first study to look into the EVs proteome of two bacteria belonging to the opposite status of the same microbiome simultaneously. Finally, I investigated the contribution of bacterial EVs to the modulation of host response and parasite behaviour. L. gasseri EVs reduced pathogen T. vaginalis cytoadhesion, motility and caused the formation of large parasite aggregates. G. vaginalis EVs, on the other hand, were shown to promote the cytoadhesion, motility and growth of T. vaginalis. Both types of bacterial EVs were capable of inducing an early innate immune response from the human cells. My research revealed that EVs from these vaginal bacteria contribute significantly to host-parasite interactions. Strikingly, their cargoes and effects on host and pathogen match with the expected ecological niche of these bacteria within the vaginal biome. This study shows that microbial EVs largely contribute to the host response and pathogen behaviour, highlighting that this knowledge can be explored towards new antimicrobial interventions in the future. It shows that EVs have the potential to be exploited in novel therapeutics approaches aiming to prevent and combat infections while simultaneously preserving or restoring a healthy microbiome.