Cannabidiol Is a Novel Modulator of Bacterial Membrane Vesicles

ORIGINAL RESEARCH published: 10 September 2019 doi: 10.3389/fcimb.2019.00324 Cannabidiol Is a Novel Modulator of Bacterial Membrane Vesicles Uchini S. Kosgodage 1 , Paul Matewele 1 , Brigitte Awamaria 1 , Igor Kraev 2 , Purva Warde 3 , Giulia Mastroianni 4 , Alistair V. Nunn 5 , Geoffrey W. Guy 6 , Jimmy D. Bell 5 , Jameel M. Inal 3 and Sigrun Lange 7* 1 Cellular and Molecular Immunology Research Centre, School of Human Sciences, London Metropolitan University, London, United Kingdom, 2 School of Life, Health and Chemical Sciences, The Open University, Milton Keynes, United Kingdom, 3 Bioscience Research Group, Extracellular Vesicle Research Unit, School of Life and Medical Sciences, University of Hertfordshire, Hatfield, United Kingdom, 4 School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom, 5 Research Centre for Optimal Health, School of Life Sciences, University of Westminster, London, United Kingdom, 6 GW Pharmaceuticals Research, Cambridge, United Kingdom, 7 Tissue Architecture and Regeneration Research Group, School of Life Sciences, University of Westminster, London, United Kingdom Edited by: Jyl S. Matson, University of Toledo, United States Reviewed by: Medicharla Venkata Jagannadham, Centre for Cellular Molecular Biology (CCMB), India Bo Peng, Sun Yat-sen University, China *Correspondence: Sigrun Lange Specialty section: This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 04 April 2019 Accepted: 28 August 2019 Published: 10 September 2019 Citation: Kosgodage US, Matewele P, Awamaria B, Kraev I, Warde P, Mastroianni G, Nunn AV, Guy GW, Bell JD, Inal JM and Lange S (2019) Cannabidiol Is a Novel Modulator of Bacterial Membrane Vesicles. Front. Cell. Infect. Microbiol. 9:324. doi: 10.3389/fcimb.2019.00324 Membrane vesicles (MVs) released from bacteria participate in cell communication and host-pathogen interactions. Roles for MVs in antibiotic resistance are gaining increased attention and in this study we investigated if known anti-bacterial effects of cannabidiol (CBD), a phytocannabinoid from Cannabis sativa, could be in part attributed to effects on bacterial MV profile and MV release. We found that CBD is a strong inhibitor of MV release from Gram-negative bacteria (E. coli VCS257), while inhibitory effect on MV release from Gram-positive bacteria (S. aureus subsp. aureus Rosenbach) was negligible. When used in combination with selected antibiotics, CBD significantly increased the bactericidal action of several antibiotics in the Gram-negative bacteria. In addition, CBD increased antibiotic effects of kanamycin in the Gram-positive bacteria, without affecting MV release. CBD furthermore changed protein profiles of MVs released from E. coli after 1 h CBD treatment. Our findings indicate that CBD may pose as a putative adjuvant agent for tailored co-application with selected antibiotics, depending on bacterial species, to increase antibiotic activity, including via MV inhibition, and help reduce antibiotic resistance. Keywords: bacterial membrane vesicles (MVs), cannabidiol (CBD), antibiotic resistance, gram-negative, gram-positive, E. coli VCS257, S. aureus subsp. aureus Rosenbach INTRODUCTION Outer membrane vesicles (OMVs) and membrane vesicles (MVs) are released from Gram-negative and Gram-positive bacteria and participate in inter-bacterial communication, including via transfer of cargo molecules (Dorward and Garon, 1990; Li et al., 1998; Fulsundar et al., 2014; Jan, 2017; Toyofuku et al., 2019). MVs are released in greater abundance from Gram-negative, than Gram-positive bacteria and their production seems crucial for bacterial survival and forms part of the stress response (McBroom and Kuehn, 2007; Macdonald and Kuehn, 2013; Jan, 2017). Gram-negative bacteria generate, in addition to common one-bilayer vesicles (OMV), also double-bilayer vesicles (O-IMVs), and in some stress conditions other types of MVs (Pérez-Cruz et al., 2016) and therefore we will use the umbrella term “membrane vesicles” (MVs) hereafter. MVs are important in biofilm formation and dissemination of toxins in the host (Wang et al., 2015; Cooke et al., 2019). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 September 2019 | Volume 9 | Article 324 Kosgodage et al. CBD—A Novel Modulator of Bacterial MVs E. coli and S. aureus cultures were maintained by plating on Mueller-Hinton agar plates and weekly sub-culturing was performed according to previously established methods (Iqbal et al., 2013). Before MV isolation, all bacterial growth medium (LB broth) was pre-treated before use by ultracentrifugation at 100,000 g for 24 h to ensure minimum contamination with extracellular vesicles (EVs) from the medium (Kosgodage et al., 2017). For MV isolation, bacteria were grown in EV-free medium (as described above) for 24 h at 37◦ C, the culture medium was collected and centrifuged once at 400 g for 10 min for removal of cells, followed by centrifugation at 4,000 g for 1 h at 4◦ C to remove cell debris. The resultant supernatant was then centrifuged for 1 h at 100,000 g at 4◦ C and the isolated MV pellet was resuspended in Dulbecco’s phosphate buffered saline (DPBS; ultracentrifuged and sterile filtered using a 0.22 µm filter) and centrifuged again at 100,000 g for 1 h at 4◦ C. The resulting MV pellet was sterile filtered (0.45 µm) once and then resuspended in sterile filtered DPBS. The quantitative yield of vesicles was ∼6.5 × 109 MVs per liter of culture. The isolated MV pellets were then either used immediately, or stored at −80◦ C for further experiments. MVs participate in host-pathogen interactions (Gurung et al., 2011; Koeppen et al., 2016; Bitto et al., 2017, 2018; Codemo et al., 2018; Turner et al., 2018; Cecil et al., 2019) and may also be involved in antibiotic resistance, for instance by protecting biofilms from antibiotics via increased vesiculation (Manning and Kuehn, 2011). Furthermore, MVs from Porphyromonas gingivalis have been linked to metabolic remodeling in the host (Fleetwood et al., 2017), while MVs from Neisseria gonorrhoeae have been shown to target host mitochondria and to induce macrophage death (Deo et al., 2018). Besides roles for cellular and bacterial communication, the use of MVs as nano-carriers for various compounds, including for antibiotic and vaccine delivery, has also raised considerable interest in the research community (Gnopo et al., 2017; Rüter et al., 2018; Tan et al., 2018; Wang et al., 2018). The regulation of bacterial MV biogenesis and release may therefore be of great importance, both in relation to interbacterial communication, including biofilm formation, their host interactions as commensals, as well as in host-pathogen interactions and in antibiotic resistance. Cannabidiol (CBD) is a phytocannabinoid from Cannabis sativa with anti-inflammatory (Martin-Moreno et al., 2011 (...truncated)