The spread of antimicrobial resistance continues to be a priority health concern worldwide, necessitating exploration of alternative therapies. Cannabis sativa has long been known to contain antibacterial cannabinoids, but their potential to address antibiotic resistance has only been superficially investigated. Here, we show that cannabinoids exhibit antibacterial activity against MRSA, inhibit its ability to form biofilms and eradicate pre-formed biofilms and stationary phase cells persistent to antibiotics. We show that the mechanism of action of cannabigerol is through targeting the cytoplasmic membrane of Gram-positive bacteria and demonstrate in vivo efficacy of cannabigerol in a murine systemic infection model caused by MRSA. We also show that cannabinoids are effective against Gram-negative organisms whose outer membrane is permeabilized, where cannabigerol acts on the inner membrane. Finally, we demonstrate that cannabinoids work in combination with polymyxin B against multi-drug resistant Gram-negative pathogens, revealing the broad-spectrum therapeutic potential for cannabinoids..

SummaryThe PhoPR two-component signal transduction system controls one of the major responses to phosphate limitation in Bacillus subtilis. When activated it directs expression of phosphate scavenging enzymes, lowers synthesis of the phosphate-rich wall teichoic acid (WTA) and initiates synthesis of teichuronic acid, a non-phosphate containing replacement anionic polymer. Despite extensive knowledge of this response, the signal to which PhoR responds has not been identified. Here we report that one of the main functions of the PhoPR two-component system in B. subtilis is to monitor WTA metabolism. PhoR autokinase activity is controlled by the level of an intermediate in WTA synthesis that is sensed through the intracellular PAS domain. The pool of this intermediate generated by WTA synthesis in cells growing under phosphate-replete conditions is sufficient to inhibit PhoR autokinase activity. However WTA synthesis is lowered upon phosphate limitation by the combined effects of PhoP∼P-mediated activation of tuaA-H transcription and repression of tagAB. These transcriptional changes combine to lower the level of the inhibitory WTA metabolite thereby increasing PhoR autokinase activity. This amplifies the PHO response with full induction being achieved ∼ 90 min after the onset of phosphate limitation.

In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as Staphylococcus aureus, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that β-O-GlcNAcylation of S. aureus wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure of S. aureus TarS, the enzyme responsible for polyribitol phosphate β-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase.

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to β-lactam antibiotics. The membrane-associated TagA glycosyltransferase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T . italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme’s lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to β-lactam antibiotics. The membrane-associated TagA glycosyltranserase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme's lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.