Doron Steinberg

Muna Aqawi, Doron Steinberg, Osnat Feuerstein, Michael Friedman, and Sarah Gingichashvili. 2022. “Cannabigerol Effect on Streptococcus mutans Biofilms-A Computational Approach to Confocal Image Analysis.” Frontiers in microbiology, 13, Pp. 880993. Abstract
Biofilms are complex bacterial structures in which bacterial cells thrive as a community. Many bacterial species, including pathogens, form biofilms of high complexity and adaptability to a wide range of environmental conditions. One example of these is Streptococcus mutans, a gram-positive bacterium that has been associated with caries. Cannabigerol, a non-psychoactive cannabinoid, has been shown to affect S. mutans biofilms. In order to better characterize the effect of cannabigerol on biofilms of S. mutans, this paper provides a series of computational assays for biofilm analysis, applied on confocal images of S. mutans biofilms treated with cannabigerol. Confocal images are ubiquitous in biofilm analysis-they are often used to visualize the complex structure and molecular composition of biofilm macrocolonies. In this article, we demonstrate how confocal imaging data can be used to reveal more comprehensive insights into biofilm structure and measure specific anti-biofilm effects. This is accomplished by a series of computational assays, each focusing on a different aspect of biofilm structure.
Ronit Vogt Sionov, Shreya Banerjee, Sergei Bogomolov, Reem Smoum, Raphael Mechoulam, and Doron Steinberg. 2022. “Targeting the Achilles' Heel of Multidrug-Resistant Staphylococcus aureus by the Endocannabinoid Anandamide.” International journal of molecular sciences, 23, 14. Abstract
Antibiotic-resistant Staphylococcus aureus is a major health issue that requires new therapeutic approaches. Accumulating data suggest that it is possible to sensitize these bacteria to antibiotics by combining them with inhibitors targeting efflux pumps, the low-affinity penicillin-binding protein PBP2a, cell wall teichoic acid, or the cell division protein FtsZ. We have previously shown that the endocannabinoid Anandamide (N-arachidonoylethanolamine; AEA) could sensitize drug-resistant S. aureus to a variety of antibiotics, among others, through growth arrest and inhibition of drug efflux. Here, we looked at biochemical alterations caused by AEA. We observed that AEA increased the intracellular drug concentration of a fluorescent penicillin and augmented its binding to membrane proteins with concomitant altered membrane distribution of these proteins. AEA also prevented the secretion of exopolysaccharides (EPS) and reduced the cell wall teichoic acid content, both processes known to require transporter proteins. Notably, AEA was found to inhibit membrane ATPase activity that is necessary for transmembrane transport. AEA did not affect the membrane GTPase activity, and the GTPase cell division protein FtsZ formed the Z-ring of the divisome normally in the presence of AEA. Rather, AEA caused a reduction in murein hydrolase activities involved in daughter cell separation. Altogether, this study shows that AEA affects several biochemical processes that culminate in the sensitization of the drug-resistant bacteria to antibiotics.
Antibiotic resistance has become an increasing challenge in the treatment of various infectious diseases, especially those associated with biofilm formation on biotic and abiotic materials. There is an urgent need for new treatment protocols that can also target biofilm-embedded bacteria. Many secondary metabolites of plants possess anti-bacterial activities, and especially the phytocannabinoids of the Cannabis sativa L. varieties have reached a renaissance and attracted much attention for their anti-microbial and anti-biofilm activities at concentrations below the cytotoxic threshold on normal mammalian cells. Accordingly, many synthetic cannabinoids have been designed with the intention to increase the specificity and selectivity of the compounds. The structurally unrelated endocannabinoids have also been found to have anti-microbial and anti-biofilm activities. Recent data suggest for a mutual communication between the endocannabinoid system and the gut microbiota. The present review focuses on the anti-microbial activities of phytocannabinoids and endocannabinoids integrated with some selected issues of their many physiological and pharmacological activities.
Mark Feldman, Irith Gati, Ronit Vogt Sionov, Sharonit Sahar-Helft, Michael Friedman, and Doron Steinberg. 2022. “Potential Combinatory Effect of Cannabidiol and Triclosan Incorporated into Sustained Release Delivery System against Oral Candidiasis.” Pharmaceutics, 14, 8. Abstract
Candida albicans is a common fungal pathogen. Biofilm formation on various surfaces is an important determinant of C. albicans pathogenicity. Our previous results demonstrated the high potential of cannabidiol (CBD) to affect C. albicans biofilms. Based on these data, we investigated the possibility of incorporating CBD and/or triclosan (an antimicrobial agent that is widely utilized in dentistry) in a sustained-release varnish (SRV) (SRV-CBD, SRV-triclosan) to increase their pharmaceutical potential against C. albicans biofilm, as well as that of the mixture of the agents into SRV (SRV-CBD/triclosan). The study was conducted in a plastic model, on agar, and in an ex vivo tooth model. Our results demonstrated strong antibiofilm activity of SRV-CBD and SRV-triclosan against C. albicans in all tested models. Both formulations were able to inhibit biofilm formation and to remove mature fungal biofilm. In addition, SRV-CBD and SRV-triclosan altered C. albicans morphology. Finally, we observed a dramatic enhancement of antibiofilm activity when combined SRV-CBD/triclosan was applied. In conclusion, we propose that incorporation of CBD or triclosan into SRV is an effective strategy to fight fungal biofilms. Importantly, the data demonstrate that our CBD/triclosan varnish is safe, and is not cytotoxic for normal mammalian cells. Furthermore, we propose that CBD and triclosan being in mixture in SRV exhibit complementary antibiofilm activity, and thus can be explored for further development as a potential treatment against fungal infections.
M. Feldman, R. Sionov, R. Smoum, R. Mechoulam, I. Ginsburg, and D. Steinberg. 2020. “Comparative Evaluation of Combinatory Interaction between Endocannabinoid System Compounds and Poly-L-lysine against Streptococcus mutans Growth and Biofilm Formation.” BioMed Research International, 2020. Publisher's Version Abstract
Endocannabinoid/endocannabinoid-like (EC/EC-like) are natural endogenous compounds which have been found to affect MRSA pathogenicity. Our previous studies showed that EC/EC-like was able to impair staphylococcal biofilm formation and maintenance as well as to alter biofilm-associated virulence factors. In the present study, we investigated the combinatory effect of the selected EC/EC-like with a natural antimicrobial agent, poly-L-lysine, on cariogenic bacteria Streptococcus mutans growth and biofilm formation. Among four tested EC/EC-like, only two, anandamide (AEA) and oleoylethanolamide (OEA), exhibited synergistic combinatory effect with poly-L-lysine against S. mutans. We attribute this distinct effect to differences in the fatty acid chain structure of the selected EC/EC-like compounds. Moreover, AEA exerted a specific antibiofilm mode of action against S. mutans by effecting total inhibition of biofilm formation while still allowing bacteria viability. Finally, we postulate that the presence of EC/EC-like and poly-L-lysine could enhance the permeability and efficacy of each other via hydrophobic and electrostatic interactions with the S. mutans membrane. In conclusion, we assume that a combination of endogenous natural compounds such as EC/EC-like and poly-L-lysine may benefit oral hygiene by preventing dental plaque. © 2020 Mark Feldman et al.
M. Aqawi, R. Gallily, R.V. Sionov, B. Zaks, M. Friedman, and D. Steinberg. 2020. “Cannabigerol Prevents Quorum Sensing and Biofilm Formation of Vibrio harveyi.” Frontiers in Microbiology, 11. Publisher's Version Abstract
Cannabigerol (CBG) is a non-psychoactive cannabinoid naturally present in trace amounts in the Cannabis plant. So far, CBG has been shown to exert diverse activities in eukaryotes. However, much less is known about its effects on prokaryotes. In this study, we investigated the potential role of CBG as an anti-biofilm and anti-quorum sensing agent against Vibrio harveyi. Quorum sensing (QS) is a cell-to-cell communication system among bacteria that involves small signaling molecules called autoinducers, enabling bacteria to sense the surrounding environment. The autoinducers cause alterations in gene expression and induce bioluminescence, pigment production, motility and biofilm formation. The effect of CBG was tested on V. harveyi grown under planktonic and biofilm conditions. CBG reduced the QS-regulated bioluminescence and biofilm formation of V. harveyi at concentrations not affecting the planktonic bacterial growth. CBG also reduced the motility of V. harveyi in a dose-dependent manner. We further observed that CBG increased LuxO expression and activity, with a concomitant 80% downregulation of the LuxR gene. Exogenous addition of autoinducers could not overcome the QS-inhibitory effect of CBG, suggesting that CBG interferes with the transmission of the autoinducer signals. In conclusion, our study shows that CBG is a potential anti-biofilm agent via inhibition of the QS cascade. © Copyright © 2020 Aqawi, Gallily, Sionov, Zaks, Friedman and Steinberg.
R.V. Sionov, M. Feldman, R. Smoum, R. Mechoulam, and D. Steinberg. 2020. “Anandamide prevents the adhesion of filamentous Candida albicans to cervical epithelial cells.” Scientific Reports, 10, 1. Publisher's Version Abstract
Candidiasis is a fungal infection caused by Candida species that have formed a biofilm on epithelial linings of the body. The most frequently affected areas include the vagina, oral cavity and the intestine. In severe cases, the fungi penetrate the epithelium and cause systemic infections. One approach to combat candidiasis is to prevent the adhesion of the fungal hyphae to the epithelium. Here we demonstrate that the endocannabinoid anandamide (AEA) and the endocannabinoid-like N-arachidonoyl serine (AraS) strongly prevent the adherence of C. albicans hyphae to cervical epithelial cells, while the endocannabinoid 2-arachidonoylglycerol (2-AG) has only a minor inhibitory effect. In addition, we observed that both AEA and AraS prevent the yeast-hypha transition and perturb hyphal growth. Real-time PCR analysis showed that AEA represses the expression of the HWP1 and ALS3 adhesins involved in Candida adhesion to epithelial cells and the HGC1, RAS1, EFG1 and ZAP1 regulators of hyphal morphogenesis and cell adherence. On the other hand, AEA increased the expression of NRG1, a transcriptional repressor of filamentous growth. Altogether, our data show that AEA and AraS have potential anti-fungal activities by inhibiting hyphal growth and preventing hyphal adherence to epithelial cells. © 2020, The Author(s).}, funding_text 1=his study was partially supported by Agriculture Ministry of Israel. We are grateful to Dr. Yael Feinstein-Rotkopf for operating the Nikon spinning scan microscope at our Interdepartment Core Research Facility. We thank Muna Aqawi and Sarah Gingichashvili for their support.
S. Ron-Doitch, Y. Soroka, M. Frusic-Zlotkin, D. Barasch, D. Steinberg, and R. Kohen. 2020. “Saturated and aromatic aldehydes originating from skin and cutaneous bacteria activate the Nrf2-keap1 pathway in human keratinocytes.” Experimental Dermatology. Publisher's Version Abstract
Skin homeostasis is constantly challenged by environmental factors, affecting its delicate redox balance. The skin is also home to a wide variety of bacterial species, including Staphylococci. The cutaneous redox state is governed by the Nrf2-keap1 pathway, which is responsible for the induction of phase II cytoprotective enzymes, thus sustaining a healthy oxidative state. As part of normal metabolism, both bacteria and cutaneous tissue emit copious amounts of volatile organic compounds (VOCs), one subgroup of which are aldehydes. α,β-unsaturated aldehydes are known activators of Nrf2-keap1 pathway by direct oxidation of the keap1 protein. However, we did not encounter reports of Nrf2 activation by saturated or aromatic aldehydes, neither bacteria nor skin-derived. We hypothesized that non–α,β-unsaturated aldehydes derived from skin or cutaneous bacteria may act as Nrf2-keap1 pathway activators and therefore afford protection against environmental insults. The saturated aldehydes nonanal and decanal (known skin metabolites) and the aromatic aldehyde benzaldehyde (known skin and Staphylococcus epidermidis metabolite) were shown to induce the Nrf2-keap1 pathway in human keratinocytes. We also identified a newly described aromatic aldehyde, 3-furaldehyde (3-FA), emitted from S. aureus and S. epidermidis cultures, which also activated the pathway. Moreover, Nrf2-keap1 induction led to a significant protection against UVB-induced apoptosis. The mechanism involved in this activation has been partially elucidated. This work emphasizes the importance of cutaneous bacteria, as well as healthy skin lipid peroxidation processes in the maintenance and regulation of the cellular antioxidant response, namely with regard to coping with environmental stressors. © 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Infections caused by antibiotic-resistant strains of Staphylococcus aureus have reached epidemic proportions globally. Our previous study showed antimicrobial effects of anandamide (AEA) and arachidonoyl serine (AraS) against methicillin (MET)-resistant S. aureus (MRSA) strains, proposing the therapeutic potential of these endocannabinoid/endocannabinoid-like (EC/EC-like) agents for the treatment of MRSA. Here, we investigated the potential synergism of combinations of AEA and AraS with different types of antibiotics against MRSA grown under planktonic growth or biofilm formation. The most effective combinations under planktonic conditions were mixtures of AEA and ampicillin (AMP), and of AraS and gentamicin (GEN). The combination with the highest synergy in the biofilm formation against all tested bacterial strains was AEA and MET. Moreover, the combination of AraS and MET synergistically caused default of biofilm formation. Slime production of MRSA was also dramatically impaired by AEA or AraS combined with MET. Our data suggest the novel potential activity of combinations of EC/EC-like agents and antibiotics in the prevention of MRSA biofilm formation. Copyright: © 2020 Feldman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
L. Friedman, R. Smoum, M. Feldman, R. Mechoulam, and D. Steinberg. 2019. “Does the Endocannabinoid Anandamide Affect Bacterial Quorum Sensing, Vitality, and Motility?” Cannabis and Cannabinoid Research, 4, 2, Pp. 102-109. Publisher's Version Abstract
Introduction: The endocannabinoid anandamide (AEA) is a neurotransmitter produced and released "on demand." Numerous studies have been conducted on AEA and on the endocannabinoid system (ECS), but none of them have investigated their effect on prokaryotes. Quorum sensing (QS) is a process of bacteria-bacteria communication. In this cross-Talk, the bacteria secrete and recognize signal molecules termed autoinducers (AIs). It has been shown that the QS system regulates expression of many physiological and virulence factors of bacteria. Materials and Methods: QS was measured using the bioluminescence property of the bacterium Vibrio harveyi. The effect of AEA on QS-related gene expression was measured using real-Time polymerase chain reaction. 0.18% agar plates were used for surface movement assay. Results: No dose response of AEA could be determined up to 100 μg/mL on bacterial growth either wild-Type (WT) V. harveyi or mutant strains. However on addition of AEA, QS was reduced significantly for WT and other V. harveyi strains mutated at different locations of the QS cascade (BB152; HAI-1 synthase mutant, BB886; Sensor-2-, BB170; Sensor-1-, MM30; AI-2). Genes related to the QS pathway, such as luxS, showed significant reduction in expression in the presence of AEA. Motility tests showed that continuous exposure to AEA reduced V. harveyi ability to spread on a soft agar surface, but pre-exposure to AEA did not have any effect on its motility. Conclusions: This study presents the first evidence that the endocannabinoid AEA affects specific functions of a prokaryotic organism (e.g., QS and motility). Our results present novel, not yet been observed biological functions of the ECS, namely as a possible line of defense against bacteria. © Copyright 2019, Mary Ann Liebert, Inc., publishers 2019.