Promysalin is a salicylate-containing antimicrobial with a cell-membrane-disrupting mechanism of action on Gram-positive bacteria

Promysalin was previously described as a narrow spectrum molecule with a unique species-specific activity against Pseudomonas aeruginosa. Here we demonstrate that promysalin is active against Gram-positive and Gram-negative bacteria using a microdilution assay. Promysalin acts on Gram-positive bacteria with a mechanism of action involving cell membrane damage with leakage of intracellular components. The evaluation of MICs and MBCs on 11 promysalin analogs, synthesized utilizing diverted total synthesis, allowed the identification of the structural moieties potentially involved in cell membrane interaction and damage. The mechanism of action of promysalin against Gram-negative bacteria is still not clarified, even if a synergistic effect with the bisguanidine chlorhexidine on cell membrane disruption has been observed.


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General procedure A: acylation of ethyl L-pyroglutamate.
NEt 3 (2 eq.), followed by acid chloride (1.2 eq.) were added dropwise to a stirred solution of ethyl Lpyroglutamate (1 eq.) in toluene (0.5 M) at 0 °C under N 2 atmosphere. The mixture was stirred at 80 °C for 3 h and cooled to room temperature. Sat. NaHCO 3 was added and the organic layer was separated.
The aqueous layer was extracted with EtOAc (× 2). The combined organic extracts were washed with brine, dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The product was purified using flash column chromatography in 0-30% EtOAc/hexane.

General procedure B: reductive elimination.
To a stirred solution of acylated pyroglutamate (1 eq.) in dry toluene (0.2 M) was added Superhydride ® (lithium triethylborohydride) (1.2 eq., 1M in THF) at -78 °C under N 2 atmosphere. The mixture was stirred at -78 °C for 1h, then DMAP (0.1 eq.) and DIPEA (5.7 eq.) were added, followed by very slow addition of TFAA (1.2 eq.). The reaction mixture was gradually warmed to room temperature and stirred for 3 h. Water (× 10) was added and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (× 2); the combined organic extracts were washed with brine, dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The residue was purified using flash column chromatography in 0-50 % ethyl acetate: hexane.

General procedure C: hydrolysis of ethyl ester.
To a solution of ethyl ester (1 eq.) was added dropwise a solution of LiOH (1.5 eq.) in water (EtOH : H 2 O 2:1, 0.08 M) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 5 h.
EtOH was removed in vacuo, the aqueous layer was washed with 40 % ethyl acetate in diethyl ether (× 2), cooled to 0 °C and acidified using 5% citric acid. The product was extracted using 5% CH 3 OH : CH 2 Cl 2 (× 3). The combined organic extracts were washed with brine, dried over anhydrous Na 2 SO 4 and concentrated in vacuo to afford carboxylic acid.

General procedure D: Yamagouchi esterification.
NEt 3 (3 eq.) followed by 2,4,6-trichlorobenzoyl chloride (2 eq.) were added dropwise to a stirred solution of acid (1eq.) in THF (0.03 M) at 0 °C under N 2 atmosphere. The mixture was warmed to room temperature and stirred for 2h. THF was removed in vacuo and the residue was dissolved in toluene (0.03M). DMAP (3 eq.) followed by alcohol (0.8 eq.) in toluene were added at 0 °C under N 2 atmosphere. The resulting suspension was stirred overnight at room temperature. EtOAc (× 15) was added, the organic layer was washed with sat. NH 4 Cl, brine, dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The product was purified by flash column chromatography 0-70 % EtOAc: hexane.

General procedure E: MEM deprotection.
To a stirred solution of MEM ether (1 eq.) in CH 2 Cl 2 (0.15 M) was added TiCl 4 (2 eq., 1M in CH 2 Cl 2 ) at S 6 ammonia (20 times) was added. The aqueous layer was extracted with ethyl acetate (×2), and the combined organic extracts were washed with brine and dried over anhydrous Na 2 SO 4 . The solvent was removed in vacuo. The product was purified using preparative TLC.

General procedure F: TBDPS deprotection.
TBAF (3 eq. 1M in THF) was added dropwise to a stirred solution of silyl ether (1 eq.) in THF (0.2 M) at 0 °C. The reaction mixture was stirred at room temperature for 1h. Sat. NH 4 Cl was added. The aqueous layer was extracted with ethyl acetate (× 2). The combined organic extracts were washed with brine, dried over anhydrous Na 2 SO 4 and concentrated in vacuo. The product was purified using preparative TLC.
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putida RW10S1 was verified. For agar well diffusion assay, 10 7 CFU/ml were inoculated in melted M17 or MRS or TSB agar media (15 g/l). After the medium solidification, a well of 1 cm of diameter was created using a sterile tip and loaded with a 50 µl of DMSO of promysalin at different concentration.
The plates were then incubated at the appropriate temperature for 18 h, and the presence or the absence of an inhibition halo around the wells were verified.
S 58 P S P S S P S P A B C Figure S1. Agar diffusion assay carried out using P. putida RW10S1 (promysalin producers), and P. stutzeri LMG 2333 (promysalin sensitive) as reference strains 4 spotted in TSB agar. Promysalin production and activity was tested against the M17 soft agar overlay containing Streptococcus thermophilus DSM 20617T (A), and the MRS soft agar overlay containing Pediococcus acidilactici PAC1.0 (B) and the TSB soft agar overlay containing Pesudomonas stutzeri LMG 2333 (C). P, and S in the figure represent the growth of the promysalin-producer P. putida RW10S1, and the promysalin-sensitive strain respectively in TSB agar.
A B P P P P Figure S2. Agar diffusion assay carried out using P. putida RW10S1 (promysalin producers) as reference strains 4 spotted in TSB agar. Promysalin production and activity was tested against the TSB soft agar overlay containing  To evaluate whether membrane damage was linked to cell leakage of intracellular components, microbial cells grown for 18 h in the appropriate medium in Petri dishes were collected and diluted in sterile filtered (0.2 µm) phosphate-buffered saline (PBS) (NaCl 8 g/L; KCl 0.2 g/L; Na 2 HPO 4 1,44 g/L; KH 2 PO 4 0.24 g/L; pH 7.4) to a final concentration of 10 8 events per mL. The cell suspension was diluted to 10 6 events/mL and then exposed to promysalin (100 µg/mL) or its derivative analogues (100 µg/mL), chlorhexidine (100 µg/mL) (Sigma-Aldrich) or benzalkonium chloride (100 µg/mL) (Sigma-Aldrich) at 37 °C. The cell suspension was also exposed to a DMSO control. At the time requested, a sample was collected and subjected to SYBR Green I/PI double staining and analysis by flow cytometry and, when necessary, to a standard plate count in the appropriate medium. In flow cytometry, particles/cells that pass through the beam will scatter light, which is detected as forward scatter (FSC) and side scatter (SSC). FSC correlates with cell size, cell shape and cell aggregates, whereas SSC depends on the density of the particles/cells (i.e., the number of cytoplasmic granules and membrane size). In this manner, cell populations can often be distinguished based on differences in their size and density. Cell suspensions were subjected to dual nucleic acid staining with cell permeant SYBR Green I (1X) and cell impermeant propidium iodide (PI) (5 µg/mL) (Sigma-Aldrich, Milan, Italy). SYBR Green I permeates the membrane of total cells and stains nucleic acids with green fluorescence. After incubation at room temperature for 15 min, the labeled cell suspensions were diluted to approximately 10 6 events per mL, and analyzed by flow cytometry. Cell suspensions that were prepared as described above were analyzed using a flow cytometer with the following threshold settings: FSC 5,000, SSC 4,000, and 20,000 total events collected. All parameters were collected as logarithmic signals, and a 488-nm laser was used to measure the FSC values. The rate of events in the flow was generally lower than 2,000 events/s. The obtained data were analyzed using BD AccuriTM C6 software 1.0 (BD Biosciences, Milan, Italy). Cell-membrane damage was carried out by applying double staining with SYBR Green I and PI. The SYBR Green I fluorescence intensity of stained cells was recovered in the Milan, Italy). The cFSE-labeled cell suspension was then exposed to promysalin (100 µg/mL) or its derivative analogues (100 µg/mL), chlorhexidine (100 µg/mL) (Sigma-Aldrich) or benzalkonium chloride (100 µg/mL) (Sigma-Aldrich) at 37 °C. As a control, the cFSE-labeled cell suspension was also exposed to a volume of DMSO solvent equal to that used for promysalin and its derivative analogues. C D Figure S5. The effect of promysalin and gramicidin on Streptococcus thermophilus DSM 20617 T cell membrane integrity. Flow cytometry density diagrams show the cFSE vs PI fluorescence of cells exposed to promysalin or gramicidin (100 µg/mL and 100 mM, respectively). A) Cells before exposure to antimicrobials. B) Cells after 60 min of exposure to DMSO. C) Cells after 60 min of exposure to promysalin. D) Cells after 60 min of exposure to gramicidin. Viable cells are gated in G1. Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.
T0 DMSO chlorexidine A B C Figure S6. The effect of DMSO and chlorhexidine on Streptococcus thermophilus DSM 20617 T cell membrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to DMSO (a volume equal to that used for promysalin and its derivative analogs), and chlorhexidine (100 µg/mL). A) Cells before exposure to antimicrobials. B) Cells after 60 min of exposure to DMSO. C) Cells after 60 min of exposure to promysalin. Viable cells are gated in G1. Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.

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SYBR Green I fluorescence -A PI fluorescence -A A B Figure S7. The effect of DMSO and promysalin on Lactobacillus paracasei DSM 5622 T cell membrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to DMSO (a volume equal to that used for promysalin) and promysalin (100 µg/mL). A) Cells incubated 60 min at 37 °C in presence of DMSO. B) Cells after 60 min of exposure to promysalin. Viable cells are gated in G1. Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.

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SYBR Green I fluorescence -A PI fluorescence -A A B Figure S8. The effect of DMSO and promysalin on Staphylococcus aureus ATCC 25923 cell membrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to DMSO (a volume equal to that used for promysalin) and promysalin (100 µg/mL). A) Cells incubated 60 min at 37 °C in presence of DMSO. B) Cells after 60 min of exposure to promysalin. Viable cells are gated in G1. Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.

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PI fluorescence -A A B C SYBR Green I fluorescence -A Figure S9. The effect of DMSO, promysalin and chlorhexidine on Bacillus subtilis DSM 347 cell membrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to DMSO (a volume equal to that used for promysalin) and promysalin (100 µg/mL). A) Cells incubated 60 min at 37 °C in presence of DMSO. B) Cells after 60 min of exposure to promysalin. C) Cells after 60 min of exposure to chlorhexidine. Viable cells are gated in G1. Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.

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SYBR Green I fluorescence -A PI fluorescence -A A B C Figure S10. The effect of promysalin and chlorhexidine on Pseudomonas stutzeri LMG 2333 cell-membrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to promysalin or chlorhexidine (100 µg/mL) for 1h at 37 °C. A) Cells exposed to DMSO as control; B) Cells exposed to promysalin; C) Cells exposed to chlorhexidine. Viable cells are gated in G1, Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.

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SYBR Green I fluorescence -A PI fluorescence -A A B C Figure S11. The effect of promysalin and chlorhexidine on Pseudomonas aeruginosa ATCC 10145 cellmembrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to promysalin or chlorhexidine (100 µg/mL) for 1h at 37 °C. A) Cells exposed to DMSO as control; B) Cells exposed to promysalin; C) Cells exposed to chlorhexidine. Viable cells are gated in G1, Dead cells with damaged membranes are gated in G3.  Figure S12. The effect of promysalin and chlorhexidine on Escherichia coli ATCC 25922 cell-membrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to promysalin or chlorhexidine (100 and 200 µg/mL, respectively). A) and B) Cells after 20 min of exposure to antibacterials. C) and D) Cells after 1.5 h-exposure to antimicrobials. Viable cells are gated in G1, and viable cells with slightly damaged cell membranes are gated in G2. Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.

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SYBR Green I fluorescence -A PI fluorescence -A A B C Figure S13. The effect of promysalin and chlorhexidine on Acetobacter aceti MIM2000/28 cell-membrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to promysalin or chlorhexidine (100 µg/mL) for 1h at 37 °C. A) Cells exposed to DMSO as control; B) Cells exposed to promysalin; D) Cells exposed to chlorhexidine. Viable cells are gated in G1, Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.

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SYBR Green I fluorescence -A PI fluorescence -A promysalin, 60 min chlorhexidine 60 min A B C Figure S14. The effect of promysalin and chlorhexidine on Saccharomyces cerevisiae BC1 cell membrane integrity. Flow cytometry density diagrams show SYBR Green I vs PI fluorescence of cells exposed to promysalin and chlorhexidine (100 µg/mL). A) Cells before exposure to antimicrobials. B) Cells after 60 min of exposure to promysalin. C) Cells after 60 min of exposure to chlorhexidine. Viable cells are gated in G1. Dead cells with damaged membranes are gated in G3. The transition of cell populations from gate G1 to gate G3 is correlated to cell membrane damage.     Figure S18. Growth of Saccharomyces cerevisiae BC1 in the absence and presence of different concentrations of chlorhexidine. Chlorhexidine concentrations (µg/ml) are indicated. The MIC for chlorhexidine was 8 µg/ml. In presence of chlorhexidine 1, 2 and 4 µg/ml, the growth curves of S. cerevisae were not significantly different from the growth in absence of the biocide (control).