Sepsis causes millions of deaths per year worldwide and is a current global health priority declared by the WHO. Sepsis-related deaths are a result of dysregulated inflammatory immune responses indicating the need to develop strategies to target inflammation. An important mediator of inflammation is extracellular adenosine triphosphate (ATP) that is released by inflamed host cells and tissues, and also by bacteria in a strain-specific and growth-dependent manner. Here, we investigated the mechanisms by which bacteria release ATP. Using genetic mutant strains of Escherichia coli (E. coli), we demonstrate that ATP release is dependent on ATP synthase within the inner bacterial membrane. In addition, impaired integrity of the outer bacterial membrane notably contributes to ATP release and is associated with bacterial death. In a mouse model of abdominal sepsis, local effects of bacterial ATP were analyzed using a transformed E. coli bearing an arabinose-inducible periplasmic apyrase hydrolyzing ATP to be released. Abrogating bacterial ATP release shows that bacterial ATP suppresses local immune responses, resulting in reduced neutrophil counts and impaired survival. In addition, bacterial ATP has systemic effects via its transport in outer membrane vesicles (OMV). ATP-loaded OMV are quickly distributed throughout the body and upregulated expression of genes activating degranulation in neutrophils, potentially contributing to the exacerbation of sepsis severity. This study reveals mechanisms of bacterial ATP release and its local and systemic roles in sepsis pathogenesis.
Sepsis is a severe condition often caused by the body’s immune system overreacting to bacterial infections. This can lead to excessive inflammation which damages organs and requires urgent medical care. With sepsis claiming millions of lives each year, new and improved ways to treat this condition are urgently needed. One potential strategy for treating sepsis is to target the underlying mechanisms controlling inflammation. Inflamed and dying cells release molecules called ATP (the energy carrier of all living cells), which strongly influence the immune system, including during sepsis. In the early stages of an infection, ATP acts as a danger signal warning the body that something is wrong. However, over time, it can worsen infections by disturbing the immune response. Similar to human cells, bacteria release their own ATP, which can have different impacts depending on the type of bacteria and where they are located in the body. However, it is not well understood how bacterial ATP influences severe infections like sepsis. To investigate this question, Spari et al analysed how ATP is released from Escherichia coli, a type of bacteria that causes severe infections. This revealed that the bacteria secrete ATP directly in to their environment and via small membrane-bound structures called vesicles. Spari et al. then probed a mouse model of abdominal sepsis which had been infected with E. coli that release either normal or low levels of ATP. They found that the ATP released from E. coli impaired the mice’s survival and lowered the number of neutrophils (immune cells which are important for defending against bacteria) at the site of the infection. The ATP secreted via vesicles also altered the role of neutrophils but in more distant regions, and it is possible that these changes may be contributing to the severity of sepsis. These findings provide a better understanding of how ATP released from bacteria impacts the immune system during sepsis. While further investigation is needed, these findings may offer new therapeutic targets for treating sepsis.

Bacteroides spp. are prominent gut commensals that are believed to modulate the intestinal environment, in part, by producing outer membrane vesicles (OMVs). Bacteroides OMVs have been ascribed many functions in vitro, but the genetic underpinnings behind OMV biogenesis and regulation are unclear. Understanding the mechanism of OMV biogenesis is required to determine the importance of Bacteroides OMVs in vivo. Here, we describe our methodology for screening Bacteroides thetaiotaomicron VPI-5482 to identify genes required for OMV biogenesis and regulation in a high-throughput format. This protocol is easily adaptable and can potentially be employed to further our knowledge of OMV biogenesis in other bacteria.

Shigellosis is one of the leading causes of diarrheal disease in low- and middle-income countries, particularly in young children, and is more often associated with antimicrobial resistance. Therefore, a preventive vaccine against shigellosis is an urgent medical need. We have proposed Generalised Modules for Membrane Antigens (GMMA) as an innovative delivery system for Shigella sonnei O-antigen, and an Alhydrogel formulation (1790GAHB) has been extensively tested in preclinical and clinical studies. Alhydrogel has been used as an adsorbent agent with the main purpose of reducing potential GMMA systemic reactogenicity. However, the immunogenicity and systemic reactogenicity of this GMMA-based vaccine formulated with or without Alhydrogel have never been compared. In this work, we investigated the potential adjuvant effect of aluminium salt-based adjuvants (Alhydrogel and AS37) on S. sonnei GMMA immunogenicity in mice and rabbits, and we found that S. sonnei GMMA alone resulted to be strongly immunogenic. The addition of neither Alhydrogel nor AS37 improved the magnitude or the functionality of vaccine-elicited antibodies. Interestingly, rabbits injected with either S. sonnei GMMA adsorbed on Alhydrogel or S. sonnei GMMA alone showed a limited and transient body temperature increase, returning to baseline values within 24 h after each vaccination. Overall, immunisation with unadsorbed GMMA did not raise any concern for animal health. We believe that these data support the clinical testing of GMMA formulated without Alhydrogel, which would allow for further simplification of GMMA-based vaccine manufacturing.

Chlamydia trachomatis is an obligate intracellular pathogen responsible for the most prevalent bacterial sexually transmitted disease globally. The high prevalence of chlamydial infections underscores the urgent need for licensed and effective vaccines to prevent transmission in populations. Bacterial outer membrane vesicles (OMVs) have emerged as promising mucosal vaccine carriers due to their inherent adjuvant properties and the ability to display heterologous antigens. In this proof-of-concept study, we evaluated the immunogenicity of Salmonella OMVs decorated with C. trachomatis MOMP-derived CTH522 or HtrA antigens in mice. Following a prime-boost intranasal vaccination approach, two OMV-based C. trachomatis vaccines elicited significant humoral responses specific to the antigens in both systemic and vaginal compartments. Furthermore, we demonstrated strong antigen-specific IFN-γ and IL17a responses in splenocytes and cervical lymph node cells of vaccinated mice, indicating CD4+ Th1 and Th17 biased immune responses. Notably, the OMV-CTH522 vaccine also induced the production of spleen-derived CD8+ T cells expressing IFN-γ. In conclusion, these results highlight the potential of OMV-based C. trachomatis vaccines for successful use in future challenge studies and demonstrate the suitability of our modular OMV platform for intranasal vaccine applications.

Bacterial extracellular vesicles (BEVs) contribute to stress responses, quorum sensing, biofilm formation and interspecies and interkingdom communication. However, the factors that regulate their release and heterogeneity are not well understood. We set out to investigate these factors in the common gut commensal Bacteroides thetaiotaomicron by studying BEV release throughout their growth cycle. Utilising a range of methods, we demonstrate that vesicles released at different stages of growth have significantly different composition, with early vesicles enriched in specifically released outer membrane vesicles (OMVs) containing a larger proportion of lipoproteins, while late phase BEVs primarily contain lytic vesicles with enrichment of cytoplasmic proteins. Furthermore, we demonstrate that lipoproteins containing a negatively charged signal peptide are preferentially incorporated in OMVs. We use this observation to predict all Bacteroides thetaiotaomicron OMV enriched lipoproteins and analyse their function. Overall, our findings highlight the need to understand media composition and BEV release dynamics prior to functional characterisation and define the theoretical functional capacity of Bacteroides thetaiotaomicron OMVs.

Investigations into the biological role and composition of bacterial extracellular vesicles have grown in popularity in recent years. Vesicles perform a variety of functions during interactions with eukaryotic host cells, ranging from antibiotic resistance to immune modulation. It is necessary to isolate vesicles in order to understand their biological functions. Here we describe a polymer-based precipitation method allowing high-yield isolation of extracellular vesicles and their cargo RNA from the Gram-positive bacterium Staphylococcus aureus.

Norovirus infection is influenced by the presence of commensal bacteria, and both human and murine norovirus (MNV) bind to these bacteria. These virus-bacterial interactions, as well as MNV infection, promote the increased production of bacterial extracellular vesicles (bEVs). However, no correlation has been made between specific bacterial groups, their vesicles, and their impact on norovirus infection. The current study evaluated the impact of select bacterial compositions of murine microbiomes using antibiotic (ABX) cocktails on MNV infection and bEV production. The goal of this research was to determine if increases in bEVs following MNV infection in mice were associated with changes in specific bacterial populations. Bacterial taxa were found to be differentially abundant in both ABX-treated and untreated mice, with the greatest change in bacterial taxa seen in mice treated with a broad-spectrum ABX cocktail. Specifically, Lachnospiraeae were found to be differentially abundant between a variety of treatment factors, including MNV infection. Overall, these results demonstrate that MNV infection can alter the abundance of bacterial taxa within the microbiota, as well as their production of extracellular vesicles, and that the use of selective antibiotic treatments can allow the detection of viral impacts on the microbiome that might otherwise be masked.

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1α and IL-1β. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

Tenacibaculum dicentrarchi is the second most important pathogen in Chilean salmon farming. This microorganism causes severe skin lesions on the body surface of farmed fish. The bacterium can also adhere to surfaces and form biofilm, survive in fish skin mucus, and possess different systems for iron acquisition. However, the virulence mechanisms are still not fully elucidated. Outer membrane vesicles (OMV) are nanostructures released by pathogenic Gram-negative bacteria during growth, but none has been described yet for T. dicentrarchi. In this study, we provide the first reported evidence of the fish pathogen T. dicentrarchi producing and releasing OMV from 24 h after incubation, increasing thereafter until 120 h. Analyses were conducted with T. dicentrarchi TdCh05, QCR29, and the type strain CECT 7612T . The OMV sizes, determined via scanning electron microscopy, ranged from 82.25 nm to 396.88 nm as per the strain and incubation time point (i.e., 24 to 120 h). SDS-PAGE revealed that the number of protein bands evidenced a drastically downward trend among the T. dicentrarchi strains. In turn, the OMV shared five proteins (i.e., 22.2, 31.9, 47.7, 56.3, and 107.1 kDa), but no protein pattern was identical. A heterogeneous amount of protein, RNA, and DNA were obtained, depending on the time at which OMV were extracted. Purified OMV were biologically active and induced a cytotoxic effect in macrophage-enriched cell cultures from rainbow trout (Oncorhynchus mykiss) head kidneys. This is the first step towards understanding the role that OMV could play in the pathogenesis of T. dicentrarchi.

OBJECTIVE: Bacteria can package protein cargo into nanosized membrane blebs that are shed from the bacterial membrane and released into the environment. Here, we report that a type of pathogenic bacteria called enterohemorrhagic Escherichia coli O157 (EHEC) uses their membrane blebs (outer membrane vesicles) to package components of their type 3 secretion system and send them into host cells, where they can manipulate host signaling pathways including those involved in infection response, such as immunity. Usually, EHEC use a needle-like apparatus to inject these components into host cells, but packaging them into membrane blebs that get taken up by host cells is another way of delivery that can bypass the need for a functioning injection system.