Numerous probiotic microorganisms have repeatedly been shown to produce nanometer-sized structures named extracellular vesicles (EVs). Recently, it has been suggested that similarly to whole microbial cells, EVs produced by probiotics may also demonstrate health benefits to the host, while their application does not involve the risk of infection caused by live microorganisms. In this work, we isolated EVs from two probiotic species originating from different taxonomic domains — yeast Saccharomyces boulardii CNCM I-745 and bacterium Streptococcus salivarius K12. The diameters of S. boulardii EVs were about 142 nm and for S. salivarius EVs about 123 nm. For S. boulardii EVs, 1641 proteins and for S. salivarius EVs, 466 proteins were identified with a liquid chromatography-coupled tandem mass spectrometry and then functionally classified. In both microbial species, metabolic proteins significantly contributed to the cargo of EVs comprising 25% and 26% of all identified vesicular proteins for fungi and bacteria, respectively. Moreover, enzymes associated with cell wall rearrangement, including enzymatically active glucanases, were also identified in EVs. Furthermore, probiotic EVs were shown to influence host cells and stimulate the production of IL-1β and IL-8 by the human monocytic cell line THP-1, and, at the same time, did not cause any remarkable reduction in the survival rate of Galleria mellonella larvae in this invertebrate model commonly used to evaluate microbial EV toxicity. These observations suggest that the EVs produced by the investigated probiotic microorganisms may be promising structures for future use in pro-health applications.

The prevalence of diseases caused by pathogenic fungi of the Candida genus is currently a significant problem, particularly due to the emerging antifungal drug resistance and increasing number of immunocompromised individuals susceptible to opportunistic infections. Recently, it has been shown that fungal extracellular vesicles (EVs) – nanometre-sized structures of cellular origin, equipped with varied cargo enclosed by lipid bilayer – may play a vital role in the response of pathogen cells to antifungal treatment. In this work, we demonstrated that Candida albicans yeast cells grown at the subinhibitory concentrations of three commonly used antifungal drugs – amphotericin B, fluconazole and caspofungin – released a greater number of EVs than fungal cells grown under drug-free conditions. Moreover, these EVs exhibited some variability in size and protein composition, yet they consistently induced the production of the pro-inflammatory cytokine IL-8 by THP-1 macrophage-like cells at levels comparable to control EVs. In studies using the invertebrate model organism Galleria mellonella, EVs released by cells exposed to antifungals did not cause a significant increase in larval mortality, similar to control EVs, although they triggered a remarkably lower activation of phenol oxidase in larval haemolymph. In addition, EVs produced in the presence of caspofungin interacted more noticeably with the membrane of U-937 pro-monocytic cells as indicated by measurements of zeta potential changes. Furthermore, tested EVs contributed to increased tolerance of C. albicans cells to the antifungal drugs. These observations underscore the unmissable role of EVs in the response of pathogen cells to antifungal treatment and highlight the importance of understanding EV functionalities in antifungal resistance.

The microbial cell wall is a dynamic structure responsible for maintaining the shape and integrity of the cell. It is involved in the processes of cell growth, reproduction and division, protection against environmental stress factors, bidirectional selective transport of various molecules, and interactions with other microorganisms inhabiting a particular ecological niche. The cell surface of microorganisms pathogenic to humans is also responsible for constant and direct contact with the host during the initiation and further development of the infection, and proteins exposed on the surface play a key role in this phenomenon. The set of proteins exposed on the cell surface is collectively referred to as the surfaceome. In surfaceome studies, the cell surface shaving technique is increasingly used while maintaining cell integrity, which, in addition to providing insight into the composition of surface proteins, also makes it possible to track the interactions of pathogen molecules with host molecules. This experimental approach will be described in this review with examples of the most relevant groups of identified microbial proteins involved in the host–pathogen interactions.