Outer membrane vesicles are the powerful cell-to-cell communication vehicles that allow bacteria to monitor extracellular milieu

Existence of cell-to-cell communication strategies among bacteria (i.e., defined as inter-species, intra-species, and inter-kingdom crosstalk) reveals how genius they are (1,2). The common and well-studied language for bacterial communication is quorum sensing (QS), an informative signaling pathway within a bacterial population (3-5). Specifically, bacteria secrete signaling modules under QS regulation, known as autoinducers (AIs), to share quorum information and regulate group behaviours. QS modulates the expression of bacterial pathogenicity factors involved in the infection process, as a similar regulation could occur in the physiology of host cells. Detailed mechanisms are extensively reviewed (6,7).

Bacterial conversation enables them to feel the surrounding stimuli and coordinate their gene expression accordingly. This cooperation in sensing and responding to environmental changes could result in arrays of behaviour, ranging from symbiosis to virulence, biofilm formation, stress adaptation and natural product production, leading bacteria to live like multicellular organisms (8-10). Furthermore, bacteria need to manage competition for ecological niches and resources that favour their survival and genetic persistence. This strategy underlies mechanisms of complex interactions that occur among bacteria as well as bacteria-hosts.

While the potency of QS has gained the interest of researchers for a long time, only recently QS-regulated pathways have been reported to be involved in membrane vesicles (MVs) or outer membrane vesicles (OMVs) biogenesis in Gram-positive and Gram-negative bacteria, respectively (11). OMVs have been recognized as an alternative vehicle for cell-to-cell signaling in bacteria. This spherical membranous structure can package cargoes such as virulence factors, biologically active proteins, DNA, RNA, as well as signaling molecules, playing an important role in bacterial adaptation and survival (2,12). Despite tremendous progress in the OMVs field (e.g., involvement in disease-related processes and delivery mechanisms into host cells), there is still some uncertainty in the biogenesis of OMVs and their precise sorting mechanisms within bacterial communities.

So far, there is enough evidence connecting QS and OMVs, indicating that either QS molecules could serve as OMVs cargo or they are able to control the release of OMVs in several nosocomial pathogens (i.e., Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli) (13). The latter mechanism could act either by stimulating OMVs secretion, a phenomenon evidenced in P. aeruginosa, Vibrio harveyi, Stenotrophomonas maltophilia, as well as in plant pathogens such as Xanthomonas oryzae and Xanthomonas campestris or vice versa by inhibiting its release as reported in Xylella fastidiosa (14-18).

For instance, through a QS-regulated process, OMVs released by the environmental pathogen Chromobacterium violaceum (C. violaceum) deliver the antimicrobial compound violacein to compete with other bacteria, exerting its toxicity in vivo even over far distances (19). As a QS metabolite produced by various bacterial species, the OMV cargo violacein contributes to a broad range of antimicrobial activities (20). Batista et al. showed that violacein of C. violaceum is not only toxic against Gram-positive bacteria, but also it induces OMV biogenesis for its own delivery and contributes to biofilm formation (19). Another example of bacterial usage of OMVs related to cope with nutritional challenges (i.e., iron-limited conditions) was reported by Lin et al. (21). They found that the type VI secretion system H3-dependent effector for iron uptake TseF is packed into OMVs. This finding suggested that TseF is incorporated into OMVs and interacts with the iron-binding P. aeruginosa quinolone signal (PQS); this interaction facilitates the delivery of the metal ion to its receptors on cell surface receptors (FptA or OprF) (21). Of note, for most bacterial pathogens, sensing, sequestering and uptaking efficiently environmental iron is critical to enable colonization and pathogenicity, and for this reason, this process involves multi-component complexes (22).

In a recent issue of Science of The Total Environment, Zhao et al. reported the isolation and purification of PQS-containing OMVs (PQS-OMVs) produced by P. aeruginosa and evaluated the effects of this signaling molecules on the formation and structure of P. aeruginosa biofilm (23). Their study indicated that P. aeruginosa biofilm capacity is regulated by OMV-mediated PQS that promotes the increase of bacterial biomass, leading to biofilm formation. Subsequently, the authors quantified in detail the extracellular polysaccharides (PS) and proteins, the two main matrix components of biofilms. Their analyses revealed that proteins and PS have a synergistic effect, although the expression of proteins regulated by OMV-mediated PQS played the dominant role with respect to PS in the formation of P. aeruginosa biofilms. Next, in a dual-species biofilm formed by P. aeruginosa and S. aureus, the research proved that OMV-mediated PQS exerted inhibitory effects on S. aureus growth, leading to a decrease in extracellular polymeric substances produced by S. aureus (23). These two major opportunistic pathogens, commonly co-isolated from cystic fibrosis patients, are often found growing together in biofilms in lungs and wounds (24). It should be noted that bacteria in diverse conditions (i.e., lab growth, hospital setting and during infection) behave differently as evidenced by phenotype changes as well as in exporting QS molecules and stimulating OMV biogenesis (25-29). For example, the traffic of PQS between the inner and outer membranes defines the yield of OMVs production. Florez et al. showed that OMVs production depends on PQS export rates rather than a defect in its production; therefore, the accumulation of PQS in the inner membrane resulted in a poor OMVs production due to early saturation of the export pathway (30).

Owing to the ability of OMVs to distribute widely PQS in an aqueous environment, it is reasonable to think that they can enable long distance transport of this essential element, thus allowing trans-feeding of bacteria at distant sites of the host during infection. In addition, the effective biofilm dispersion is dependent on the production of PQS-induced OMVs, which likely act as delivery vehicles for matrix-degrading enzymes. Accordingly, OMVs represent promising bacterial-derived molecules that could operate as an alternative for antibiotics for the treatment or inhibition of biofilm-forming species.

The studies connecting QS to OMVs in P. aeruginosa opened up new perspectives on other clinically relevant nosocomial pathogens such as multidrug-resistant (MDR) Acinetobacter spp (28,29). Previous studies have shown that Acinetobacter spp. can successfully release OMVs carrying plasmids containing resistance genes (i.e., bla_OXA-24) to recipient cells through horizontal gene transfer (31,32). To this point, Chatterjee et al. reported that A. baumannii strain ST 1462 releases OMVs capable to transfer an intact plasmid harbouring a carbapenem resistance gene (bla_NDM-1) during in vitro growth (33). Notably, these OMVs carrying bla_NDM-1 were found in an active form allowing high frequency of transformation and transmittable abilities not only intra-species (A. baumannii) but also inter-species (E. coli) (33). Carbapenem-resistant A. baumannii, i.e., extensively drug-resistant or pandrug-resistant isolates, are responsible for substantial life-threating hospital-acquired infections in patients with severe underlying diseases, mainly in intensive care units, often related to invasive procedures or indwelling devices (10,34,35). A recent study reported by Huang et al. showed an increase in the production of OMVs under antibiotic stress (36). Under stimulation by different antibiotics, A. baumannii releases OMVs at different levels of efficiency; compared to other tested antibiotics, levofloxacin was the strongest OMVs inducer both in particle number, protein level and particle diameter. Moreover, the stress induced by levofloxacin led to the encapsulation of large amounts of intracellular components into OMVs by activating efflux pumps proteins; AdeB, AdeA and AcrB were identified as the most expressed proteins. Both in vitro and in vivo experiments showed that bacteria are able to pack pumped antibiotics into OMVs; this excellent drug-resistance ability becomes a strategy to kill other bacteria (36). Moreover, the orally administration of the antibiotic-loaded OMVs could kill pathogenic bacteria in the intestine as demonstrated by the effective killing of enterotoxigenic E. coli in a mouse model of intestinal infection (36).

More recently, Dhurve et al. reported that A. baumannii DS002 releases OMVs carrying proteins associated with cell wall/membrane biogenesis, inorganic ion transport and metabolism through QS signalling (37). The OMVs cargo content underlies the prominent role of OMVs in cell physiology, signaling activities, transport functions and pathogenesis, as well as in the defence mechanisms against host immunity (37). Interestingly, OMVs were selectively enriched in TonB-dependent transporters (TonRs), outer membrane proteins that capture and transport iron chelated by siderophores into A. baumannii DS002 cells. Thus, the OMV-associated TonRs appeared to play a critical role in the survival of A. baumannii in certain conditions such as nutrient-limiting polymicrobial environments (37). Despite only some pathogenicity properties in A. baumannii have been shown to be under QS control such as surface-associated motility and biofilm formation (38), the potential linkage between OMVs production mediated by QS remains to be investigated. In our opinion, OMVs biogenesis in A. baumannii could be induced through QS signaling and their role warrants extensive examinations.

Conclusion and future perspective

Overall, Zhao et al. provided new insights into the role of OMV-mediated PQS on biofilm formation, structure and composition of EPS in P. aeruginosa as well as interspecific inhibitory effects of PQS in the context of microbial community. We learned that bacterial pathogens during their growth and metabolism excrete OMVs with biologically active proteins as well as transmissible DNA sequences associated with diverse functions. The multiple advantages of OMVs as drug delivery carriers, biofilm inhibitor or anti-bacteria adhesion will broaden effective/alternative therapeutic approaches. Due to the increased rates of antibiotic resistance as well as biofilm-associated infections, OMVs delivery could potentially direct us to explore innovative tools (i.e., development of artificial OMVs or the engineering of the natural ones) for clinical application in order to better control intestinal, pulmonary or even systemic infections caused by MDR pathogens. The intracellular trafficking of OMVs can be studied in detail using advanced in vitro models such as human tissue-derived organoids.

Acknowledgments

Funding: This research was funded by the Italian Ministry of Health (Ricerca Corrente and 5 × 1000). The research was also supported by Bandi Ateneo Sapienza, grant number RP120172B7FF9E6F to C.A. The Italian Ministry of Health and Bandi Ateneo Sapienza had no role in paper preparation and decision of publishing.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editor (Chenyu Zhang) for the series “Extracellular RNAs and Human Health” published in ExRNA. The article has undergone external peer review.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://exrna.amegroups.com/article/view/10.21037/exrna-22-18/coif). The series “Extracellular RNAs and Human Health” was commissioned by the editorial office without any funding or sponsorship. CA serves as an unpaid editorial board member of ExRNA from December 2021 to November 2023. This research was funded by the Italian Ministry of Health (Ricerca Corrente and 5 × 1000). The research was also supported by Bandi Ateneo Sapienza, grant number RP120172B7FF9E6F to CA. The Italian Ministry of Health and Bandi Ateneo Sapienza had no role in paper preparation and decision of publishing. Salary of MS was funded by the Italian Ministry of Health (starting grant SG-2018-12365432). This funder had no role in study design, decision to publish, and preparation of the manuscript. Salary of DS was funded by POR Lazio FSE 2014-202O and Sapienza Ateneo funding. These funders had no role in study design, decision to publish, and preparation of the manuscript. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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