Uncovering the Hidden Antibiotic Potential of Cannabis

Supplementary Information for: Uncovering the hidden antibiotic potential of Cannabis

Maya A. Farha1,2, †, Omar M. El-Halfawy1,2,3, †; Robert T. Gale1,2; Craig R. MacNair1,2; Lindsey A. Carfrae1,2; Xiong Zhang1,2, Nicholas G. Jentsch1,2; Jakob Magolan1,2; Eric D. Brown1,2*

1 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada 2 Michael G. DeGroote Institute of Infectious Disease Research, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada 3 Microbiology and Immunology Department, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt * To whom correspondence should be addressed ([email protected]) † These authors contributed equally

Supplementary Table 1. Antimicrobial activity of cannabinoid analogs against MRSA USA300 MIC Entry Compound Name (Abbreviation) Structure (µg/mL)

1 cannabigerol (CBG) 2

2 cannabidiol (CBD) 2

2 3 cannabinol (CBN)

4 cannabichromene (CBC) 8

5 cannabichromenic acid (CBCA) 2

6 (-)Δ9-tetrahydrocannabinol (THC) 2

2 7 (-)Δ8-tetrahydrocannabinol (-Δ8THC)

8 exo-tetrahydrocannabinol (exo-THC) 2

Δ9-tetrahydrocannabinolic 4 9 (THCAA) acid A

4 10 Δ9-tetrahydrocannabivarin (THCV)

11 cannabigerolic acid (CBGA) 4

12 cannabidivarin (CBDV) 8

13 cannabidiolic acid (CBDA) 16

tetrahydrocannabivarinic 14 (THCVA) 16 acid

15 cannabidivarinic acid (CBDVA) 32

(±) 11-nor-9-carboxy-Δ9- 16 >32 THC

17 (±) 11-hydroxy-Δ9-THC >32

18 cannabicyclol (CBL) >32

Supplementary Table 2. MRSA transposon mutants sensitized to sub-lethal concentrations of CBG.

Gene Function Transposon aroC chorismate synthase SAUSA300_1357 putative endoribonuclease L-PSP SAUSA300_0474 estA tributyrin esterase SAUSA300_2564 - hypothetical protein SAUSA300_0553 - BioY family protein SAUSA300_2233 atpA ATP synthase F1, alpha subunit SAUSA300_2060 conserved hypothetical protein SAUSA300_1780 graS sensor histidine kinase SAUSA300_0646 tcaB teicoplanin resistance associated membrane protein TcaB SAUSA300_2301 - hypotheticalprotein protein SAUSA300_2330 conserved hypothetical protein SAUSA300_0199 accessory secretory protein Asp1 SAUSA300_2587 putative hemolysin III SAUSA300_2129 - hypothetical protein (pyrazinamidase/nicotinamidase pncA) SAUSA300_1899 conserved hypothetical protein SAUSA300_2212 thiE thiamine-phosphate pyrophosphorylase SAUSA300_2047 conserved hypothetical protein SAUSA300_0847 cap5B capsular polysaccharide biosynthesis protein Cap5B SAUSA300_0153 qoxC quinol oxidase, subunit III SAUSA300_0961 ribBA riboflavin biosynthesis protein SAUSA300_1713 sdhA succinate dehydrogenase, flavoprotein subunit SAUSA300_1047 hemB delta-aminolevulinic acid dehydratase SAUSA300_1615 conserved hypothetical protein SAUSA300_1294 - TENA/THI-4 family protein SAUSA300_2050 topB DNA topoisomerase III SAUSA300_2208 - pyruvate ferredoxin oxidoreductase, alpha subunit SAUSA300_1182 - pyridoxal biosynthesis lyase PdxS SAUSA300_0504 sdhB succinate dehydrogenase iron-sulfur subunit SAUSA300_1048 sgtB monofunctional glycosyltransferase SAUSA300_1855 glycosyl transferase, group 1 family protein SAUSA300_0550 putative membrane protein SAUSA300_0917 bioD dethiobiotin synthase SAUSA300_2373 mqo malate:quinone oxidoreductase SAUSA300_2312 cation efflux family protein SAUSA300_2099 putative lipase/esterase SAUSA300_0641 - drug transporter SAUSA300_2451 sucD succinyl-CoA synthetase subunit alpha SAUSA300_1139 lspA lipoprotein signal peptidase SAUSA300_1089 pckA phosphoenolpyruvate carboxykinase SAUSA300_1731 msrA methionine sulfoxide reductase A SAUSA300_1256 - PTS system, galactitol-specific enzyme II, B component SAUSA300_0240 Supplementary Table 3. Bacterial strains and plasmids used in this study.

Bacterial Strain Description Reference

S. aureus USA300 USA300 LAC isolated in 2002 from a skin and soft tissue Laboratory infection of an inmate in the Los Angeles County Jail in stock* California, USA.; hypervirulent community-associated MRSA; cured of antibiotic resistance plasmid; also known as JE2; parent of the NTML.

Nebraska Transposon Mutant Library Screening Array; NTML 1920 S. aureus subsp. aureus USA300 JE2, transposon (Tn) NARSA* mutants arrayed in five 384-well microtiter plates. EryR

B. subtilis 168 BGSC** B. subtilis CRISPRi essential gene knockdown strain CRISPRi collection collection 1

P. aeruginosa Clinical isolate PAO1 2

E. coli lacZ+Y-I-, E. coli with constitutive expression of β- ML35 galactosidase but lacking the lactose permease 3 ML35 with periplasmic β-lactamase ML35pBR322 This study K-12 BW25113 Lab stock

A. baumannii ATCC19606 A. baumannii lacking lipooligosaccharides (LOS) ATCC ATCC19606-LOS- 4

Bacterial Plasmid Description Reference pBR322 Promoterless bioluminescent reporter plasmid encoding 5 luxABCDE, AmpR CmR

* Provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution by BEI Resources, NIAID, NIH. ** Bacillus Genetic Stock Center Amp, ampicillin; Ery, erythromycin; Cm, chloramphenicol; Spec, spectinomycin; Strep, streptomycin Strains and plasmids constructed in this study are available from the authors upon request.

CBN CBG CBD 3

2.5 3

0 0

2.0 6

0 6

6 D

D 2

/ /

1.5 V

o o

1.0

0 1

A A A 0.5

0.0 0 0 5 1 2 4 8 5 1 2 4 8 5 1 2 4 8 25 16 25 0. 16 25 0. 16 125 0. 125 0. 125 0. 0625 0. 0625 0. 0625 0. 03125 0. 03125 0. 03125 0. 0. 0. 0. 0. Concentration, mg/ml Concentration, mg/ml Concentration, mg/ml CBC CBCA D 9-THC 3

2.0 2.5

0 0

6 2.0

0 0

6 6

D 2 1.5

/ /

V 1.5

C 1.0

f o

1.0

o o

0 1

6 6

A 0.5 A A 0.5 0 0.0 0.0 5 1 2 4 8 25 0. 16 5 1 2 4 8 5 1 2 4 8 125 0. 25 0. 16 25 0. 16 0625 0. 125 0. 125 0. 03125 0. 0625 0. 0625 0. 0. 03125 0. 03125 0. 0. 0. Concentration, mg/ml Concentration, mg/ml Concentration, mg/ml D 9-THCA-A D 8-THC Exo-THC 3

2.5 2.0

0 0

2.0 6 0

6 1.5

6 D

D 2

/ O

/ /

1.5 V

V C

C 1.0

o o

1.0

0 1

6 6

6 0.5

A A

A 0.5

0.0 0.0 0 5 1 2 4 8 5 1 2 4 8 5 1 2 4 8 25 16 25 16 25 0. 16 125 0. 125 0. 125 0. 0625 0. 0625 0. 0625 0. 0. 03125 0. 03125 0. 03125 0. 0. 0. 0. 0. Concentration, mg/ml Concentration, mg/ml Concentration, mg/ml THCV CBGA CBDV

2.0 1.5 3

6 6

1.5 6

D D

1.0 D 2

V C

C 1.0

0 0.5

0 1

0 6

6 0.5

A A

0.0 0.0 0 5 1 2 4 8 5 1 2 4 8 25 16 25 0. 16 125 0. 5 1 2 4 8 125 0. 0625 0. 25 0. 16 0625 0. 03125 0. 125 0. 03125 0. 0. 0625 0. 0. 0. 03125 0. 0. Concentration, mg/ml Concentration, mg/ml Concentration, mg/ml CBDA THCVA CBDVA

5 8 8

0 0

4 0

0 0

6 6 6

6 6

O /

3 /

V V V

C 4

f f

2 o

0 0

6 2 6

6 2

A A 1 A 0 0 0 5 1 2 4 8 5 1 2 4 8 5 1 2 4 8 25 0. 16 25 0. 16 25 0. 16 125 0. 125 0. 125 0. 0625 0. 0625 0. 0625 0. 03125 0. 03125 0. 03125 0. 0. 0. 0. Concentration, mg/ml Concentration, mg/ml Concentration, mg/ml

+/- 11-Nor-9-Carboxy D9-THC +/- 11-OH D9-THC CBL

3 3 1.5

0 0

D D

D 2 2 1.0

/ /

o o

0 0.5 0

0 1 1

A A

0 0 0.0 5 1 2 4 8 5 1 2 4 8 5 1 2 4 8 25 0. 16 25 0. 16 25 0. 16 125 0. 125 0. 125 0. 0625 0. 0625 0. 0625 0. 03125 0. 03125 0. 03125 0. 0. 0. 0. Concentration, mg/ml Concentration, mg/ml Concentration, mg/ml Supplementary Fig. 1. Effect of cannabinoids on biofilm formation of MRSA USA300. Shown is the effect of increasing concentrations of the cannabinoids on MRSA biofilm formation. The average A600nm measurements of crystal violet stained biofilms normalized by the OD600 of planktonic cells are shown with error bars representing S.E.M. (n = 4). CBG CBD CBN 10 9 10 9 10 9 10 8 10 8 10 8 10 7 10 7 10 7 10 6 10 6 10 6

10 5 10 5 10 5

CFU/ml CFU/ml CFU/ml 10 4 10 4 10 4 10 3 10 3 10 3 10 2 10 2 10 2 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Concentration, g/ml Concentration, g/ml Concentration, g/ml

9 8 CBCA (-)  -THC (-)  -THC 10 9 10 9 10 9 10 8 10 8 10 8 10 7 10 7 10 7 10 6 10 6 10 6

10 5 10 5 10 5

CFU/ml CFU/ml CFU/ml 10 4 10 4 10 4 10 3 10 3 10 3 10 2 10 2 10 2 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Concentration, g/ml Concentration, g/ml Concentration, g/ml

9 Exo-THC  -THCA-A THCV 10 9 10 9 10 9 10 8 10 8 10 8 10 7 10 7 10 7 10 6 10 6 10 6

10 5 10 5 10 5

CFU/ml

CFU/ml CFU/ml 10 4 10 4 10 4 10 3 10 3 10 3 10 2 10 2 10 2 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Concentration, g/ml Concentration, g/ml Concentration, g/ml CBGA CBDV CBDA 10 9 10 9 10 9 10 8 10 8 10 8 10 7 10 7 10 7 10 6 10 6 10 6

10 5 10 5 10 5

CFU/ml CFU/ml CFU/ml 10 4 10 4 10 4 10 3 10 3 10 3 10 2 10 2 10 2 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Concentration, g/ml Concentration, g/ml Concentration, g/ml

9 THCVA CBDVA () 11-Nor-9-carboxy- -THC 10 9 10 9 10 9 10 8 10 8 10 8 10 7 10 7 10 7 10 6 10 6 10 6

10 5 10 5 10 5

CFU/ml

CFU/ml CFU/ml 10 4 10 4 10 4 10 3 10 3 10 3 10 2 10 2 10 2 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 Concentration, g/ml Concentration, g/ml Concentration, g/ml

9 () 11-Hydroxy- -THC CBL 10 9 10 9 10 8 10 8 10 7 10 7 10 6 10 6

10 5 10 5

CFU/ml CFU/ml 10 4 10 4 10 3 10 3 10 2 10 2 0 5 10 15 20 0 5 10 15 20 Concentration, g/ml Concentration, g/ml

Supplementary Fig. 2. Killing of S. aureus USA300 persisters by various cannabinoids detected after 1 hour of treatment shown as mean ±S.E.M. a CBG 0.25 m g/ml CBG 0.5 m g/ml CBG 1 m g/ml

1.50 1.50 1.50

o o o

i i i

t t

1.25 1.25 t 1.25

a a a

r r r

h h h

t t t

w w

1.00 1.00 w 1.00

o o o

r r r

g g g

d d d

e e

0.75 0.75 e 0.75

z z z

i i i

l l l

a a a

m m m

r r

0.50 0.50 r 0.50

o o o

n n n

- - -

M M

0.25 0.25 M 0.25

Q Q Q

I I I 0.00 0.00 0.00 0 500 1000 1500 2000 0 500 1000 1500 2000 0 500 1000 1500 2000 NTM L clones NTM L clones NTM L clones

b t Enrichment by cellular localization

- p

m bioD, tenA, hemB, aroC, pckA r

m Cytosol

bioY, atpA, msa, lspA, sdhB, qoxC, ltaA, r

O Plasma membrane

a G

l graS, tcaA, sgtB u

l -4 -3 -2 -1 0

l 10 10 10 10 10 e

C p-value c

Enrichment by biological process s

exts racel polysaccharide biosyn

e capB

c o

r Response to antibiotics graS, tcaA

a Electron transport chain

c qoxC, sdhB, sdhA

g o

l TCA cycle mqo1, sucD, sdhB

i b

cellular respiration

- qoxC, sdhB, sdhA m r vitamin biosynthesis

e bioD, tenA, thiE, pdxS, ribA

O metabolic processes tenA, thiE, bioD, pncA, pdxS, ribA, atpA, G estA, Mqo1, sucD, sdhB 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 p-value

Supplementary Fig. 3. Chemical genomic studies using the Nebraska Transposon Mutant Library. a, Chemical genomic screen of the transposon library at various sub-lethal concentrations of CBG. b, Enrichment by cellular localization, whereby transposon mutants were classified based on gene ontology (GO). Enrichment was based on functional overrepresentation of the mutants resulting in sensitivity to CBG, using a Fisher’s exact test to calculate p-value. c, Enrichment by biological processes, whereby transposon mutants were classified based on gene ontology (GO). Enrichment was based on functional overrepresentation of the mutants resulting in sensitivity to CBG, using a Fisher’s exact test to calculate p-value.

)

L 2

m /

g 1

µ (

0.5 G

B 0.3 C 0.1

0 0 1.6 3.1 6.3 12.5 25 50 100 Sodium bicarbonate (mM)

Supplementary Fig. 4. Combination of CBG with sodium bicarbonate against MRSA USA300. The extent of inhibition is shown as a heat plot, such that the darkest blue color represents full bacterial growth.

)

0.25 ) L

L 0.25

m /

0.125 / g

g 0.125

µ (

(

0.0625

B 0.0625

n i 0.0312 i

x 0.0312

y y

0.0156 m

0.0156

l o

o 0

P P 0 0 0.25 0.5 1 2 4 8 0 0.25 0.5 1 2 4 8

CBC (µg/mL) CBD (µg/mL)

) ) L

0.25 L 0.25

m m

/ /

0.125 g 0.125

µ µ

( (

0.0625 B

n n i

i 0.0312 x

0.0312 x y

y m

0.0156 m 0.0156

y y l

o o

0 P 0 P 0 0.25 0.5 1 2 4 8 0 0.25 0.5 1 2 4 8 CBN (µg/mL) THC (µg/mL)

Supplementary Fig. 5. Checkerboard analysis of CBC, CBD, CBN and THC in combination with polymyxin B against E. coli (K-12 BW25113). The extent of inhibition is shown as a heat plot, such that the darkest blue color represents full bacterial growth.

0.12

0.09 Control CBG 2 g/ml PmB 0.125 g/ml

492 0.06

A PmB + CBG

0.03

0.00 0 10 20 30 Time, min

Supplementary Fig. 6. CBG is not active against the OM of E. coli, as measured by hydrolysis (absorbance at 492 nm) of nitrocefin upon permeation across the OM of an E. coli expressing a periplasmic β-lactamase.

1 H NMR (700 MHz, CDCl3)

13 C NMR (176 MHz, CDCl3)

Supplementary Fig. 7. Spectral data for CBG synthesis. This spectral data is consistent with previously reported literature. Choi, Young Hae, et al. Phytochemical Analysis: An International Journal of Plant Chemical and Biochemical Techniques 15.6 (2004): 345-354. References

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