Biogenesis of Outer Membrane Vesicles Concentrates the Unsaturated Fatty Acid of Phosphatidylinositol in Capnocytophaga ochracea.

Divya Naradasu,Satoshi Takenawa,Shruti Sharma,Masanori Toyofuku,Akihiro Okamoto,Takamitsu Soma,Waheed Miran,Nobuhiko Nomura

doi:10.3389/fmicb.2021.682685

Abstract

Bacterial outer membrane vesicles (OMVs) are spherical lipid bilayer nanostructures released by bacteria that facilitate oral biofilm formation via cellular aggregation and intercellular communication. Recent studies have revealed that Capnocytophaga ochracea is one of the dominant members of oral biofilms; however, their potential for OMV production has yet to be investigated. This study demonstrated the biogenesis of OMVs in C. ochracea associated with the concentration of unsaturated fatty acids of phosphatidylinositol (PI) and characterized the size and protein profile of OMVs produced at growth phases. Transmission electron microscopy showed isolated spherical structures from cells stained with heavy metals, indicating the production of OMVs with a size ranging from 25 to 100 nm. Lipidome analysis revealed the presence of phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, and PI as the main lipids. Some unsaturated fatty acids of PI were present specifically in OMV and little in the outer membrane, suggesting that OMVs are generated from a specific region of the membrane through blebbing rather than a random process such as cell lysis. Furthermore, the lack of similar PI accumulation in the OMV of Porphyromonas gingivalis suggests that C. ochracea has a different biogenesis mechanism. The blebbing mechanism was further supported by higher OMV production occurring at the exponential phase in comparison to the stationary phase, where cell lysis is more likely to occur. Further, comparative protein profile of OMVs isolated under different growth phases may indicate that the OMV cargo does not largely vary with growth phases. The present study provides a basis for further understanding the roles of C. ochracea OMVs in oral biofilms as well as systemic diseases that C. ochracea involves.

Highlights

Among the adapted modes for survival, the production of outer membrane vesicles (OMVs) by bacteria, Gram-negative bacteria, plays a prominent role in interactions between themselves and the host (Jan, 2017; Lynch and Alegado, 2017)
Late exponential growth phase cells were used for the observation of OMVs, where thin sections of cells were subjected to transmission electron microscopy (TEM) analysis
The results revealed that stained particles had diameters mostly ranging between 25 and 100 nm, and their membranes showed the same structure and staining profile as the outer cell membrane (Figure 1), indicating the release of OMVs in C. ochracea

Summary

Introduction

Among the adapted modes for survival, the production of outer membrane vesicles (OMVs) by bacteria, Gram-negative bacteria, plays a prominent role in interactions between themselves and the host (Jan, 2017; Lynch and Alegado, 2017). OMVs have gained recognition for their role in bacterial virulence. They are known to play similar or even more invasive roles as their parent cells in mediating adherence, host cell damage, modulation of host immune responses, and biofilm formation. The subgingival plaque is home to multispecies biofilms, and its development is strongly associated with the onset of chronic periodontitis (Kolenbrander et al, 2010). The increased concentrations of the gram-negative, anaerobic, and proteolytic bacteria such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are strongly associated with symptoms of chronic periodontitis, and all three species secrete OMVs (Mohanty et al, 2019). OMVs, through their contributions to aggregation, communication, nutrient acquisition, and defense, play a key role in enhancing biofilm formation (Wang et al, 2015). The relationship between biofilms and OMVs by pathogens such as Helicobacter pylori, Francisella, Pseudomonas aeruginosa, Vibrio cholerae, and Pseudomonas putida from diverse niches have been well validated (Yonezawa et al, 2011; van Hoek, 2013; Murphy et al, 2014)

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