Impact of extracellular vesicles of Staphylococcus aureus N305 on the immune response of the host

Impact of extracellular vesicles of Staphylococcus aureus N305 on the immune response of the host. STLOpenday

Extracellular Vesicles and Their Role in <em>Staphylococcus aureus</em> Resistance and Virulence

Staphylococcus aureus is a pathogen of great importance to clinical and veterinary medicine. Recently, there has been a growing interest in S. aureus extracellular vesicles (EVs) in the pathogenesis of this bacterium. Released by living cells into the extracellular milieu, EVs are membranous structures carrying macromolecules such as proteins, nucleic acids, and metabolites. These structures play several physiological roles and are, among others, considered a mechanism of intercellular communication within S. aureus populations but also in trans kingdom interactions. S. aureus EVs were shown to transport important bacterial survival and virulence factors, such as β-lactamases, toxins, and proteins associated with bacterial adherence to host cells, and to trigger the production of cytokines and promote tissue inflammation. In this chapter, we will review the main studies regarding S. aureus EVs, including their composition and roles in host-pathogen interactions, and the possible applications of EVs for vaccines and therapy development against staphylococcal infections

Caractérisation des vésicules extracellulaires dérivées de staphylococcus aureus et leur impact sur la réponse de l'hôte

Bacterial extracellular vesicles (EVs) are nanoparticles carrying macromolecules that can influence host-pathogen interactions. The pathogen Staphylococcus aureus (SA) releases EVs whose characteristics are still largely explored. This thesis’s project provides the first work extensively characterizing the RNA and protein content of profile of SA clinical HG003 strain and its producing cells. We found that EVs comprised all RNA classes including small regulatory RNA. The protein content of EVs was also diverse with various important elements such as virulence factors, transcriptional regulators, and metabolic enzymes. Interestingly, the protein and RNA content of EVs differed from that of its producing cells, suggesting that selective cargo packing exists. The intra- and interspecies role of EVs was also investigated.We found that the addition of HG003 EVs to bacterial cultures improved their growth in restrictive media. In the context of host-pathogen interactions, the cellular response induced by EVs differed from that induced by the living bacteria in both human and bovine models, indicating that EVs could display other physiological functions than those of bacteria which may be important to the infection process. Overall, our data evidence that SA EVs carry important newly discovered elements, and modulate the host response with different intensities, exposure periods, and by different routes from that of live bacteria. This study brings new knowledge about SA EVs potential functional roles in the context of bacterial physiology and staphylococcal infections.Les vésicules extracellulaires (VEs) sont des bio-particules dérivant de la membrane bactérienne et transportant des molécules impliquées dans les interactions avec l’hôte. Le pathogène Staphylococcus aureus (SA) libère des VEs encore largement inexplorées. Cette thèse fournit le premier travail de caractérisation approfondie de la composition en ARN et en protéines des VEs de SA et de ses cellules productrices. Les VEs contiennent toutes les classes d’ARN sous formes intactes ou fragmentées, dont des petits ARN régulateurs. Le contenu en protéines des VEs comprend des facteurs de virulence, des régulateurs transcriptionnels et des enzymes de voies métaboliques variées. Des différences de composition entre les VEs et les cellules productrices ont été détectées, suggérant des processus d’empaquetage sélectif de leur contenu. Au niveau de leurs fonctions biologiques, nous avons montré que l'ajout de VEs à des cultures bactériennes de SA améliorait leur croissance en conditions carencéeDans le cadre des interactions hôte-pathogène, la réponse cellulaire induite par les VEs différait de celle induite par les bactéries vivantes, indiquant que les VEs pourraient avoir d'autres fonctions physiologiques en lien avec l’infection que celles des bactéries. Dans l'ensemble, nous démontrons que les VEs de SA contienent de nouveaux éléments et modulent la réponse de l'hôte avec différentes intensités, temps d'exposition et par des voies différentes de celles des bactéries vivantes. Cette étude apporte des connaissances sur les rôles fonctionnels potentiels des VEs dans le contexte de la physiologie bactérienne et des infections staphylococciques

Characterization of Staphylococcus aureus extracellular vesicles and their impact on the host immune response

Les vésicules extracellulaires (VEs) sont des bio-particules dérivant de la membrane bactérienne et transportant des molécules impliquées dans les interactions avec l’hôte. Le pathogène Staphylococcus aureus (SA) libère des VEs encore largement inexplorées. Cette thèse fournit le premier travail de caractérisation approfondie de la composition en ARN et en protéines des VEs de SA et de ses cellules productrices. Les VEs contiennent toutes les classes d’ARN sous formes intactes ou fragmentées, dont des petits ARN régulateurs. Le contenu en protéines des VEs comprend des facteurs de virulence, des régulateurs transcriptionnels et des enzymes de voies métaboliques variées. Des différences de composition entre les VEs et les cellules productrices ont été détectées, suggérant des processus d’empaquetage sélectif de leur contenu. Au niveau de leurs fonctions biologiques, nous avons montré que l'ajout de VEs à des cultures bactériennes de SA améliorait leur croissance en conditions carencéeDans le cadre des interactions hôte-pathogène, la réponse cellulaire induite par les VEs différait de celle induite par les bactéries vivantes, indiquant que les VEs pourraient avoir d'autres fonctions physiologiques en lien avec l’infection que celles des bactéries. Dans l'ensemble, nous démontrons que les VEs de SA contienent de nouveaux éléments et modulent la réponse de l'hôte avec différentes intensités, temps d'exposition et par des voies différentes de celles des bactéries vivantes. Cette étude apporte des connaissances sur les rôles fonctionnels potentiels des VEs dans le contexte de la physiologie bactérienne et des infections staphylococciques.Bacterial extracellular vesicles (EVs) are nanoparticles carrying macromolecules that can influence host-pathogen interactions. The pathogen Staphylococcus aureus (SA) releases EVs whose characteristics are still largely explored. This thesis’s project provides the first work extensively characterizing the RNA and protein content of profile of SA clinical HG003 strain and its producing cells. We found that EVs comprised all RNA classes including small regulatory RNA. The protein content of EVs was also diverse with various important elements such as virulence factors, transcriptional regulators, and metabolic enzymes. Interestingly, the protein and RNA content of EVs differed from that of its producing cells, suggesting that selective cargo packing exists. The intra- and interspecies role of EVs was also investigated.We found that the addition of HG003 EVs to bacterial cultures improved their growth in restrictive media. In the context of host-pathogen interactions, the cellular response induced by EVs differed from that induced by the living bacteria in both human and bovine models, indicating that EVs could display other physiological functions than those of bacteria which may be important to the infection process. Overall, our data evidence that SA EVs carry important newly discovered elements, and modulate the host response with different intensities, exposure periods, and by different routes from that of live bacteria. This study brings new knowledge about SA EVs potential functional roles in the context of bacterial physiology and staphylococcal infections

Effect of vancomycin on the production of extracellular vesicles in Staphylococcus aureus

Extracellular vesicles (EVs) are nano-sized biological particles (30 to 150 nm) produced by any living cell that are actively secreted into the surrounding environment. They carry biologically active components involved in intercellular signaling and communication, and contribute to important physiological processes. Depending on their composition, they participate, among other things, in pathogenesis, modulation of the immune response and exchange of material. Secretory factors are important components of pathogenesis in Staphylococcus aureus, a major pathogen for human and animal health. The role of EVs in infections caused by S. aureus is poorly understood yet although. The human strain S. aureus HG003 derived from NCTC8325, widely used as a model in antibiotic resistance studies, has a regulatory sRNA involved in virulence and adaptation to antibiotics. However, the role of S. aureus HG003 EVs in antibiotic resistance has not been investigated yet. In this work, we characterized HG003 EVs, in terms of their protein and RNA content. The RNA-seq approach was used to characterize the RNA content of EVs from S. aureus HG003 prepared from early and late stationary growth phases in the presence and absence of vancomycin, an antibiotic frequently used in S. aureus treatment. Preliminary results showed that there is a significant increase in EVs diameter from late stationary growth phase of HG003 compared to early stationary phase, whereas no significant difference was observed in absence and presence of vancomycin. The transcriptome and proteome analyses of HG003 EVs and eukaryotic RNA-seq are ongoing. This work will improve our knowledge on the role of EVs in S. aureus pathogenesis, and in its adaptation of antibiotic treatment

Impact of extracellular vesicles derived from Staphylococcus aureus Newbould 305 on innate immune response of mice mammary gland

Mastitis, an inflammation of the mammary gland, is the most prevalent production disease in dairy herds worldwide. It affects animal health and milk quality and causing huge economic losses. Staphylococcus aureus is one of the most important etiological agents of mastitis, and treatment against S. aureus-induced mastitis is still ineffective. Although S. aureus is extensively studied, the mechanisms involved in its pathogenicity are not yet fully understood. Since secretory factors are important components in S. aureus pathogenesis, the role of extracellular vesicles (EVs) in infections is an emerging field of research in S. aureus. EVs are indeed nano-sized biological particles ranging from 30 to 150 nm and that are actively secreted into the surrounding environment by any living cell. They play a crucial role in intercellular communication and vectorization of bioactive molecules, and can modulate the metabolism and physiology of target cells, participating in pathogenesis, modulation of the immune response and exchange of material. Various virulence factors have been identified in S. aureus EVs. Additionally, EVs secreted by S. aureus N305 strain, a bovine mastitis isolate, induced the expression of IL-8 in vitro, and promoted tissue inflammation, deterioration, and local cytokine production, in vivo, suggesting a role of EVs in mastitis pathogenesis. The objective of this work is to investigate the involvement of S. aureus EVs in the host-pathogen interactions at the cellular and molecular levels, with special attention to the modulation of cellular and immune response. For that, an RNA-seq approach was chosen to identify eukaryotic genes and signaling pathways targeted and triggered by S. aureus N305 EVs in vitro, which is ongoing. Afterwards, the expression of relevant genes activated in vitro will be measured in mammary gland tissues exposed to EVs to confirm the mechanisms of action of EVs in vivo. This work will improve our knowledge on the interactions of EVs with host cells, bringing information about S. aureus mastitis pathogenesis. Additionally, it will also provide molecular and functional data for the use of EVs as biotechnological agents, e.g. for the development of vaccines or drugs for mastitis prevention

Impact of extracellular vesicles derived from Staphylococcus aureus Newbould 305 on innate immune response of mice mammary gland

Mastitis, an inflammation of the mammary gland, is the most prevalent production disease in dairy herds worldwide. It affects animal health and milk quality and causing huge economic losses. Staphylococcus aureus is one of the most important etiological agents of mastitis, and treatment against S. aureus-induced mastitis is still ineffective. Although S. aureus is extensively studied, the mechanisms involved in its pathogenicity are not yet fully understood. Since secretory factors are important components in S. aureus pathogenesis, the role of extracellular vesicles (EVs) in infections is an emerging field of research in S. aureus. EVs are indeed nano-sized biological particles ranging from 30 to 150 nm and that are actively secreted into the surrounding environment by any living cell. They play a crucial role in intercellular communication and vectorization of bioactive molecules, and can modulate the metabolism and physiology of target cells, participating in pathogenesis, modulation of the immune response and exchange of material. Various virulence factors have been identified in S. aureus EVs. Additionally, EVs secreted by S. aureus N305 strain, a bovine mastitis isolate, induced the expression of IL-8 in vitro, and promoted tissue inflammation, deterioration, and local cytokine production, in vivo, suggesting a role of EVs in mastitis pathogenesis. The objective of this work is to investigate the involvement of S. aureus EVs in the host-pathogen interactions at the cellular and molecular levels, with special attention to the modulation of cellular and immune response. For that, an RNA-seq approach was chosen to identify eukaryotic genes and signaling pathways targeted and triggered by S. aureus N305 EVs in vitro, which is ongoing. Afterwards, the expression of relevant genes activated in vitro will be measured in mammary gland tissues exposed to EVs to confirm the mechanisms of action of EVs in vivo. This work will improve our knowledge on the interactions of EVs with host cells, bringing information about S. aureus mastitis pathogenesis. Additionally, it will also provide molecular and functional data for the use of EVs as biotechnological agents, e.g. for the development of vaccines or drugs for mastitis prevention

Effect of vancomycin on the production of extracellular vesicles in Staphylococcus aureus

Extracellular vesicles (EVs) are nano-sized biological particles (30 to 150 nm) produced by any living cell that are actively secreted into the surrounding environment. They carry biologically active components involved in intercellular signaling and communication, and contribute to important physiological processes. Depending on their composition, they participate, among other things, in pathogenesis, modulation of the immune response and exchange of material. Secretory factors are important components of pathogenesis in Staphylococcus aureus, a major pathogen for human and animal health. The role of EVs in infections caused by S. aureus is poorly understood yet although. The human strain S. aureus HG003 derived from NCTC8325, widely used as a model in antibiotic resistance studies, has a regulatory sRNA involved in virulence and adaptation to antibiotics. However, the role of S. aureus HG003 EVs in antibiotic resistance has not been investigated yet. In this work, we characterized HG003 EVs, in terms of their protein and RNA content. The RNA-seq approach was used to characterize the RNA content of EVs from S. aureus HG003 prepared from early and late stationary growth phases in the presence and absence of vancomycin, an antibiotic frequently used in S. aureus treatment. Preliminary results showed that there is a significant increase in EVs diameter from late stationary growth phase of HG003 compared to early stationary phase, whereas no significant difference was observed in absence and presence of vancomycin. The transcriptome and proteome analyses of HG003 EVs and eukaryotic RNA-seq are ongoing. This work will improve our knowledge on the role of EVs in S. aureus pathogenesis, and in its adaptation of antibiotic treatment

Value of the Galleria mellonella model for exploring the Staphylococcus aureus extracellular vesicles impact on host-pathogen interactions

International audienceIntroduction and objectivesExtracellular vesicles (EVs) are nanometric particles secreted from the membrane into the surrounding environment. They play a crucial role in intercellular communication through their ability to transport bioactive molecules. Various functions have been attributed to EVs produced by Staphylococcus aureus: biofilm formation, cytotoxicity, immunomodulation... These functions suggest their involvement in host-pathogen interactions and pathogenesis. However, in vivo data remain scarce to date. The aim of this work was to test the larval Galleria mellonella model to assess the impact of EVs in vivo.Materials and methodsDerivated HG003 reference strain-EVs were purified by steric exclusion chromatography. Different quantities of EVs were injected into the larval circulatory system to determine the minimum lethal dose. Impact of EVs, alive bacteria and PBS buffer was compared at different times after injection, both on aspects of mortality, macroscopy/microscopy, and expression of factors of larval innate immunity (e.g., opsonins, anti-microbial peptides...) by RT-qPCR from 2 distinct environments, (i) hemolymph (HL; larval circulatory system) and (ii) fat body (FB). All experiments were performed in triplicate.Results, discussion and conclusionPreliminary data showed that only a significant quantity of EVs (1010 EVs) were responsible for early larval mortality. Microscopy data allowed us to visualize S. aureus bacteria as well as clusters of EVs, each condition exhibiting a specific immune response and cellular remodeling. The larval immune response proved to be specific to the condition tested (bacteria vs. EVs), and depended on post-infection time and localization (HL vs. FB). Compared to bacteria, EVs induced an early activation of the majority of tested-innate factors in the HL, whereas a rather late activation in the FB. In addition, certain immune players were specifically induced by EVs. The G. mellonella model therefore appears suitable for assessing the contribution of S. aureus EVs to pathogenesis