Cholic Acid-Derived Amphiphile can Combat Gram-Positive Bacteria

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Cholic Acid-Derived Amphiphile can Combat Gram-Positive Bacteria-mediated Infections via Disintegration of Lipid Clusters Sandeep Kumar, Jyoti Thakur, Kavita Yadav, Madhurima Mitra, Sanjay Pal, Arjun Ray, Siddhi Gupta, Nihal Medatwal, Ragini Gupta, Deepakkumar Mishra, Parul Rani, Siladitya Padhi, Priyanka Sharma, Arti Kapil, Aasheesh Srivastava, Ujjaini Dasgupta, U. Deva Priyakumar, Lipi Thukral, and Avinash Bajaj ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00706 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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ACS Biomaterials Science & Engineering

Cholic Acid-Derived Amphiphile can Combat Gram-Positive Bacteria-mediated Infections via Disintegration of Lipid Clusters Sandeep Kumar,1,2 Jyoti Thakur,3 Kavita Yadav,1,2 Madhurima Mitra,1 Sanjay Pal,1,4 Arjun Ray,5,# Siddhi Gupta,1 Nihal Medatwal,1 Ragini Gupta,1 Deepakkumar Mishra,1 Parul Rani,1 Siladitya Padhi,6, Priyanka Sharma,7 Arti Kapil,7 Aasheesh Srivastava,3 U. Deva Priyakumar,6 Ujjaini Dasgupta,8 Lipi Thukral,5 and Avinash Bajaj,1,*

1. Laboratory of Nanotechnology and Chemical Biology, Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone Faridabad-Gurgaon Expressway, Faridabad-121001, Haryana, India. 2. Manipal Academy of Higher Education, Tiger Circle Road, Madhav Nagar, Manipal-576104, Karnataka, India. 3. Department of Chemistry, Indian Institute of Science Education and Research, Bhopal Bypass Road, Bhauri, Bhopal-462066, Madhya Pradesh, India. 4. Kalinga Institute of Industrial Technology, KIIT Road, Patia, Bhubaneswar-751024, Odisha, India. 5. CSIR-Institute of Genomics and Integrative Biology, South Campus, Mathura Road, Opp: Sukhdev Vihar Bus Depot, New Delhi-110025, India. 6. Centre for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Professor CR Rao Road, Gachibowli, Hyderabad - 500032, India 7. Department of Microbiology, All India Institute of Medical Sciences, Sri Aurobindo Marg, Ansari Nagar, New Delhi-110029, India. 8. Amity Institute of Integrative Sciences and Health, Amity University, Amity Education Valley Gurugram, Panchgaon, Manesar, Gurugram-122413, Haryana, India. # Current Address: Center for Computational Biology, Indraprastha Institute of Information Technology, Okhla Industrial Estate, Phase III, New Delhi, 110020, India. Corresponding Author: Avinash Bajaj Email: [email protected] , Ph: 0091-129-2848831

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ABSTRACT Inappropriate and uncontrolled use of antibiotics results in emergence of antibiotic resistance, thereby threatening the present clinical regimens to treat infectious diseases. Therefore, new antimicrobial agents that can prevent the bacteria to develop drug resistance are urgently needed. Selective disruption of bacterial membranes is the most effective strategy for combating microbial infections as accumulation of the genetic mutations cannot allow the emergence of drug resistance against these antimicrobials. In this work, we tested the cholic acid (CA)-derived amphiphiles tethered with different alkyl chains for their ability to combat gram-positive bacterial infections. In-depth biophysical and biomolecular simulation studies suggested that the amphiphile with hexyl chain (6) executes more effective interactions with gram-positive bacterial membranes as compared to other hydrophobic counterparts. Amphiphile 6 is effective against multi-drug resistant gram-positive bacterial strains as well and does not allow the adherence of S. aureus on amphiphile 6 coated catheters implanted in mice. Further, treatment of wound infections with amphiphile 6 clears the bacterial infections. Therefore, current study presents strategic guidelines in design and development of CA-derived membrane-targeting antimicrobials for gram-positive bacterial infections.

Keywords: Antibacterial, Bile Acids, Membrane interactions, MD simulations, Antimicrobials


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INTRODUCTION Infections caused by gram-positive bacteria causes critical medical conditions such as abscesses, endocarditis, osteomyelitis, pneumonia.1 Treatment of these ailments faces extreme challenges due to the emergence of bacterial resistance towards existing antibiotic regimens.2 Most of the commonly used antibiotics target the essential components of bacterial cellular machinery and become ineffective due to different genetic mutations.3 Bacterial membranes are considered as the effective target for antimicrobial agents as physical damage of the membranes can directly kill the bacteria and disrupt other cellular functions of bacteria.4,5 Therefore membrane-targeting bactericidal effect of antimicrobials mediated by electrostatic and hydrophobic interactions cannot allow the bacteria to develop drug resistance as well. Antimicrobial peptides (AMPs) are present in all organisms and act against different microbial infections by performing selective interactions with the microbial membranes.6,7 The appropriate arrangement of different amino acids in α-helices and β-sheets of AMPs provide the muchneeded facial amphiphilic character. AMPs interact selectively with negatively-charged bacterial membranes and remain ineffective towards the zwitterionic host cell membrane.7 A variety of synthetic molecules emulating the features of AMPs,8 short peptides,9 peptide-dendrimer,10 lipopeptides,11,12 amphiphilic cationic small molecules,13 steroidal-derived amphiphiles,14 antibiotic-lipid

conjugates,15

conjugates17,18

and

cationic

arylamide

steroidal

oligomers19

were

antibiotics,16 developed

lipidated as

norspermidine

membrane-targeting

antimicrobials. Development of membrane-targeting antimicrobials can overcome the challenges of AMPs like in vivo toxicity, limited bioavailability and large cost of production 20 Increase in alkyl chain length enhances the antimicrobial activity of membrane-targeting amphiphiles, followed by a reduction in the activity owing to the “cut-off effect” with an increase in chain length.21 Cholic acid (CA) is a naturally occurring bile acid that has three hydroxyl group tethered on steroidal hydrophobic backbone that makes it facial amphiphilic in nature like 3 ACS Paragon Plus Environment


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AMPs,.22-24 CA has been derivatized with many charged and polar chemical moieties and have been tested for antimicrobial activities.25 Membrane-targeting antimicrobials need a fine balance of combination of charge moieties and hydrophobic scaffolds for performing selective membrane disruptions of bacteria over mammalian cells. Different molecular mechanisms of bacterial membranes disruptions like toroidal pore, carpet or barrel stave have been proposed. We have shown the ability of CA-derived amphiphiles in combating gut infections mediated by gramnegative bacteria.24 We also demonstrated that hydrophobicity of the side chain on CA-derived amphiphiles helps in maintaining the rigidity of the amphiphile and assists in mediating specific interactions with lipopolysaccharide layer present in the membranes of gram-negative bacteria.26 Herein, we present the structure activity relationship (SAR) screening of CA-derived amphiphiles against gram-positive bacteria followed by extensive biophysical and biochemical assays to probe the interactions of these antimicrobials with gram-positive bacterial membranes. The antibacterial activity of the most effective amphiphile was further explored against clinically isolated bacteria and murine wound and catheter infections models. Our results showed that amphiphile 6 mediates specific interactions with bacterial lipids and is able to combat the grampositive bacteria-mediated infections. MATERIAL AND METHODS Materials. 3,3’-diethylthiadicarbocyanine (DiSC2) (173754), propidium iodide, polymyxin B (81334), levofloxacin (28266), ciprofloxacin (17850), neomycin (72133), bovine serum albumin (BSA) (A2153) and dimyristoylphosphatidyl glycerol (DMPG) (P6412) were purchased from Sigma-Aldrich (St. Louis, USA). Luria-Bertani (LB) medium, Mueller Hinton (MH) broth (M391) and

tryptone

soy

broth

(TSB)

(M011)

were

purchased

from

HiMedia.

Dipalmitoylphosphatidylethanolamine (DPPE) (850705) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). 1,6-Diphenyl 1,3,5-hexatriene (DPH) (AC117300010) was purchased from Acros Organics (Fisher Scientific, USA). HPA sensor chip was obtained from GE Healthcare 4 ACS Paragon Plus Environment

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(Bio-Sciences AB, Uppsala, Sweden). LC-MS grade solvents Methanol (#34699), Acetonitrile (#34967), Formic acid (#56302-1L-GL) are obtained from Honeywell (Germany). Staphylococcus aureus (MTCC-737), Staphylococcus oralis (MTCC-2696), Staphylococcus pneumoniae (MTCC-1936) and Bacillus subtilis (MTCC-441) were obtained from Microbial Tissue and Cell Culture (MTCC), Institute of Microbial Technology, Chandigarh, India and were cultured in MH broth and MH agar media. Bioluminescent S. aureus strain (Xen 36) was purchased from Perkin-Elmer (Santa Clara, CA, USA). Clinical strains were obtained from All India Institute of Medical Sciences, New Delhi. Ethics statement. All animal experiments were performed after due ethical approval from Institutional Animal Ethics Committee of Regional Centre for Biotechnology (RCB/IAEC/2016/001 and RCB/IAEC/2016/10 and RCB/IAEC/2017/015). Clinical strains were obtained from All India Institute of Medical Sciences, New Delhi after due ethical approval from Institute Ethics Committee of All India Institute of Medical Sciences (IEC/NP-433/09/10.2015) and Regional Centre for Biotechnology (RCB-IEC-H-7). Antibacterial assay. The antibacterial activity of amphiphiles was expressed as minimum inhibitory concentration (MIC99), the concentration at which 99% bacterial death was observed.27 Log phase cultures of all strains (grown for 3-4 h in LB broth) having 105 CFU/mL of bacteria were treated with different concentrations of amphiphiles, highest being 256 µM in 96-well plates. Culture broth alone was taken as sterility control and polymyxin B was used as positive control. The plates were then incubated for 18h at 37ºC and MIC99 values were determined by measuring the OD at 600 nm. MIC99 values are reported as averaged values of at least three independent biological experiments performed in quadruplicates. Antimicrobial susceptibility of clinical isolates of S. aureus were done as per CLSI (Clinical Laboratory Standards Institute) guidelines and the isolates were categorized as susceptible (S), intermediate (I) and resistant (R) according to the CLSI inhibition zone diameter interpretive standards (Table S4).28,29 5 ACS Paragon Plus Environment


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Time kill assay. Log phase culture of S. aureus (105 CFU/mL) was obtained and resuspended in fresh MH broth. Cells were then treated with amphiphile 6 at 1X and 4X MIC99 values for different time points (0-360 min), serially diluted in broth and plated over MH agar plates. Growth cultures with no treatment were taken as negative control. The starting inoculum or the untreated cells were plated after dilution and recorded as the time zero. After incubating the agar plates for 18 h at 37 ºC, colonies were counted and data was analyzed. Log10(CFU/mL) values of CFU data are reported as Mean ± SEM of four replicates. Anisotropy experiments. DPH-doped model gram-positive membranes were prepared by traditional

thin-film

method.

1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine

(DPPE,

4.151mg), 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG, 1.377 mg) and DPH (18.6 µg) were dissolved in a mixture of chloroform and methanol (2:1). Organic solvents were removed by rotary motion under a stream of dry nitrogen and any residual trace of solvent was evaporated under vacuum for 6 h to obtain a thin lipid film. This lipid film was hydrated in Milli-Q water (8 mL) for another 6 h. Lipid emulsion was processed through four freeze-thaw cycles at 60 °C to 4 °C with intermittent vortexing resulting in multi-lamellar vesicles (MLVs). These MLVs were sonicated using 35 kHz Bandelin sonicator (Berlin, Germany) at 60 °C for 45 min to get small unilamellar vesicles. DPH-doped vesicles (200 µL) were incubated with amphiphiles at 10 % w/w ratio for 6 h and change in anisotropy was measured in 96-well plate using SpectraMax M5 multimode microplate reader (Molecular Devices, Sunnyvale, CA, USA) in end point mode using λex at 350 nm and λem of 452 nm. Steady state fluorescence anisotropy (rs) was then calculated using equation I. (I ∥ ― GI ⊥ )

𝑟𝑠 = (I ∥ + 2GI ⊥ ) ……………(I) where Ill and I┴ are emission intensities in parallel and perpendicular directions to plane of polarized excitation. G is an instrument-specific factor measured to correct instrument polarization as described previosuly.30 6 ACS Paragon Plus Environment

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Surface plasmon Resonance experiments. SPR experiments were performed using BiacoreTM T200 (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden). Model gram-positive membranes were prepared by traditional thin-film method as described above using DPPE (4.151mg) and DMPG (1.377 mg) lipids in 3:1 (w/w) ratio. Liposomes were coated on HPA sensor chip. Following the immobilization of liposomes, BSA was injected (75 µL, 0.1 mg/mL in PBS) to cover the nonspecific binding sites. Binding of amphiphile with the lipid monolayer were then studied at a flow rate of 30 µL/min with both contact and dissociation time of 30 seconds. A series of sensorgrams were collected at five different concentrations using two state model as per literature protocol.25,26 Sensorgram for each binding event was fitted by two-state model and analyzed by curve-fitting using numerical integration analysis given by the BIAevaluation software. Computational Methods Lipid

bilayers.

The

mixed

membrane

of

zwitterionic

1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was built by using Membrane Builder in CHARMM-GUI, consisting of a total of 150 POPE and 50 POPG molecules. Subsequently, a production run was performed for 342 ns for membrane stability.31-33 Molecules preparation and force field parameters. The three-dimensional structures of amphiphiles were constructed followed by obtaining necessary force field parameters that are compatible with the CHARMM force fields used for lipids in this study. Initially, we generated force field parameters for the parent structure that is common to all amphiphiles, and amphiphile 1, 6 and 9 were obtained from the CHARMM36 all-atom general force field (cgenff) based on analogy.34 This was followed by obtaining parameters for each of the substituents and by comparing the types of each bond, angle, etc. with the ones in cgenff. The analogy between the atom types and those already existed in cgenff was enough to use these parameters directly.



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Structure preparation. Three independent systems, containing one copy of the molecule along with the prepared heterogeneous membrane were prepared. The molecules were placed ~1 nm distance away from the heterogeneous membrane at the start of the simulation. The three systems of molecule and membrane will be referred as amphiphile 1, 6 and 9 that contained 15223,15214 and 15194 TIP3P water molecules for solvation, respectively. Each of the systems contained 47 Na ions to neutralize the system.35 Molecular Dynamics Simulations Protocol. We utilised GROMACS 4.6.1 to perform the MD simulations and forcefield CHARMM 36 was applied to both lipids and proteins.36-38 The van der Waals cutoff was 1.2 nm and real space cutoff was 0.9 nm. The long-range electrostatic interactions were treated with pme (particle mesh Ewald method) with a grid spacing of 0.12 nm. 39 Periodic boundary conditions were used and the bond lengths were restrained with LINCS algorithm, and a time step of 2 fs for numerical integration of the equations of motion was used. To begin, the starting structure, including membrane and amphiphiles were energy minimised using steepest descent algorithm with emtol of 1000 kJ/mol. Next, we performed equilibration run of 100 ps in NVT ensemble Nose-Hoover at 303.15 K with a coupling constant of 1.0 ps where the amphiphiles and lipid molecules were separately coupled. The pressure coupling was performed with Parrinello-Rahman thermostat for 100 ps semi-isotropically. The pressure was maintained at 1 bar with a coupling constant of 2.0 ps. For analysing the trajectories, the coordinates were saved every 20 ps.40 The production simulation was carried out at 303.15 K and time for each trajectory was 0.7 microseconds.


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Membrane thickness. The analysis of membrane thickness was calculated using previously developed tools.41 Briefly, the thickness was calculated as the distance between two phosphate bonds (PO4) in the membrane headgroup region. We leveraged starting and final time-points during the course of the simulations to calculate the thickness. This resulted in distance-dependent XY membrane thickness profiles in which the protein translational motion of the protein was removed (i.e., the position of the center of mass of the protein was constant). Clustering. For the lipid clustering, we used our previously developed in-house python-based method for visualizing the clustering of different lipids.42 The program considers the phosphate of every individual lipid in the membrane for mapping onto the hexagonal grid-based matrix. The data was normalized for consistency and the color shade of the cell is a function of the concentration of lipids in a single hexagonal cell (lightest = 1/per cell, medium = 2/per cell, dark = >3/per cell). The final plot was generated using MatPlotLib, a plotting library for the Python programming language.43 Membrane permeabilization studies. S. aureus cells in mid log phase were harvested, washed and resuspended in phosphate buffered saline (PBS) at ~106 CFU/mL. The bacterial suspension was incubated with 3,3’-diethylthiacarbocyanine (DiSC2(5)) dye (10 μM) for 80 min. Fluorescence of the bacterial suspension was measured every 10 min using ex of 637 nm and em of 670 nm to monitor the stabilization in the fluorescence. Once saturation was reached, the test amphiphile 6 and Polymyxin B were added as per 1X and 2X MIC99 values of amphiphile 6 and incubated for 30 min at 37 °C. After incubation, cells (200 µL) were added to black 96-well plate and change in fluorescence was measured for 20 min. The change in membrane potential was measured as a dose-dependent increase in fluorescence and kinetics data was plotted as a function of time.44 Flow cytometry analysis. Bacterial suspension (105 CFU/mL) was centrifuged at 5000 rpm for 10 min at 4 oC. Cell pellet was washed and re-suspended in PBS (1 mL). Cells (105 CFU/mL) were then treated with different concentrations of amphiphile 6 at 37 ºC for 30 and 60 min. After stipulated time periods, propidium iodide (PI) (15 µM) was added to the treated suspensions and 9 ACS Paragon Plus Environment


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incubated for another 15 min at 37 ºC in dark. Unbound PI was removed by washing cells with PBS (1 mL). PI positive cells (%) were quantified by flow cytometry by using the appropriate laser (488 nm) for PI on BD FACSVerse flow cytometer (Biosciences Corp., NJ, USA). Polymyxin B was taken as the positive control. Fluorescence for PI positive cells was acquired using a logarithmic scale for 10,000 events per sample and quadrants were adjusted as per unstained S. aureus cells. Data was presented as percentage of PI positive cells with respect to untreated cells.45 Amphiphile resistance propensity of S. aureus. The MIC99 of 6 was determined against S. aureus as described earlier in antibacterial assays. For consecutive experiment (following day), the bacterial dilution was prepared by using the bacterial suspension from the samples treated at ½X MIC99 concentration of the amphiphile 6. MIC99 data was recorded by measuring the O.D. value at 600 nm using SpectraMax M5 multimode microplate reader (Molecular Devices, Sunnyvale, CA, USA) by taking the average of quadruplicate O.D. values for each concentration. The process was continuously repeated for 35 passages. Levofloxacin and Polymyxin B were used as controls. The fold change of MIC99 for amphiphile 6 and the two control antibiotics were plotted against the number of days. Mutant Concentration Prevention Assay. S. aureus was grown for overnight in Mueller Hinton broth. Bacterial culture (100 μL of 105-1012 CFU/mL) was inoculated into 96-well plate containing 100 μL of amphiphiles, which were 2-fold diluted from a highest concentration of 256 µM and incubated at 37 ºC for 16-18 h. Polymyxin B was taken as positive control. MIC99 data was recorded by measuring the O.D. value at 600 nm using SpectraMax M5 multimode microplate reader (Molecular Devices, Sunnyvale, CA, USA). MIC99 was determined by taking the average of quadruplicate O.D. values for each concentration. Wound infection studies.47 Female BALB/c mice (6-8 weeks) were prepared by shaving dorsal right-side belly. Next day, mice were anesthetized by injecting 100 µL of ketamine-xylazine 10 ACS Paragon Plus Environment

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solution (ketamine 100 mg/kg and xylazine 12.5 mg/kg) intraperitoneally. Target area was swabbed by povidone-iodine solution and cleaned with 70% ethanol. Wound was created by making a round cut having diameter of ~1 cm and S. aureus (Xen 36) log phase culture in saline (30 L of 106 CFU/mice) was inoculated over it. After 16 h of infection, mice were randomized into three groups, group 1 mice were treated with saline, group 2 mice were treated with neomycin (20 mg/kg) and group 3 mice were treated with amphiphile 6 (20 mg/kg).46 Treatment was continued thrice a day for three days. Mice were subjected for imaging under In Vivo Imaging System (IVIS) Spectrum (PerkinElmer, Santa Clara, CA, USA) on day 4 and bacterial load was confirmed in terms of photon intensity. After imaging, infected skin was harvested and homogenized in saline (1 mL) and different dilutions were plated over LB agar plate supplemented with kanamycin (200 ug/mL). Plates were incubated at 37 ºC for 24 h and colonies were counted. Coating of catheters and quantification of amphiphile load on catheters. Sterilized catheter sections (~ 0.5 cm) were dipped into amphiphile 6 solution (10 mg/mL in dichloromethane) for 15 times and dried after each dip to get a uniform coating. Quantitation of the amphiphile loaded on catheters was done by LCMS/MS using Linear Ion Trap Quadrupole (QTRAP 4500, SCIEX, USA) with Turbo VTM source and electrospray ionization (ESI) probe coupled to a high pressure UHPLC (ExionLCTM AC, SCIEX, USA). Liquid chromatographic separation was done using a Kinetex® C8 (100 A) column, 50 mm x 2.1 mm and 1.7 um particle size) (Phenomenex, India), at 55°C, using a binary mobile phase with Solvent A : Solvent B (60:40, v/v) used in isocratic flow; Solvent A: 0.1% formic acid in methanol and Solvent B 0.1% formic acid in acetonitrile. Injection volume was set at 5 uL and total run time was optimized to 5 min with an isocratic flow rate of 0.3 mL/min and LC retention time was noted. For quantitation of amphiphile, Multiple Reaction Monitoring (MRM) to Enhanced Product Ion (EPI) scan in the positive ion mode was done using Q1 precursor ion (664.7 Da) and Q3 11 ACS Paragon Plus Environment


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product ion (439.3 Da) during MRM scan. Purified amphiphile 6 was reconstituted in methanol and centrifuged at 12000 rpm for 10 min to remove any debris and the supernatant was used. Amphiphile 6 was serially diluted in methanol solvent for making different concentrations. Linear regression line were plotted with correlation coefficient values of at least 0.94 using Analyst 1.6.3 (SCIEX, USA) with quantitation wizard. Six concentrations were taken for calibration curve with regression value of 0.94. Samples were extracted from the catheters two times and diluted in methanol for estimation on LC MS/MS. Samples were run with 3 technical replicates and concentrations corresponding to peak area were quantified using the calibration curve and averaged. In vitro catheter infection assay. Log phase culture of S. aureus (50 L) was taken in 1 mL TSB/well in 12 well plate. Catheters (three/well) were incubated in this S. aureus culture for three days and media (1 mL) was replenished in every 24 h. Catheters were taken out at day 4 and vortexed in 1 mL of TSB medium for 20 min. The vortexed suspensions were serially diluted and plated over LB agar plates supplemented with chloramphenicol (25 µg/mL). Colonies were counter after 24 h of incubation at 37 ºC and expressed as log10. In vivo catheter infection studies. Catheter infection model was developed by subcutaneous implantation of coated and uncoated catheters followed by bacterial inoculation. Female BALB/c mice (6-8 weeks old) were shaved near the area of interest 24 h before infection. Mice were anesthetized on day 0 by 100 µL of ketamine-xylazine solution (ketamine 100 mg/Kg and xylazine 12.5 mg/Kg). The shaved area was swabbed by applying the povidone-iodine solution and cleaned with 70% ethanol. Catheters were inserted subcutaneously on the right dorsal side of mice using forceps and scissors after making a small incision (~1 cm). Log phase culture of S. aureus (Xen 36) (30 µL of 106 CFU/mL per mice) was resuspended in saline and inoculated over catheters and skin was sutured with silk thread. Mice were randomized in three groups (n = 5/group) where group 1 mice had control uncoated catheters, mice in group 2 had DCM-coated 12 ACS Paragon Plus Environment

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and mice in group 3 had amphiphile 6-coated catheters. Bioluminescence imaging was performed on day 7 using In Vivo Imaging System (IVIS) Spectrum (Perkin-Elmer, Santa Clara, CA, USA). Bacterial load was measured by photon intensity and average radiance were plotted. Catheter sections were taken in 1 mL saline and vortexed for 20 min to obtain the bacterial suspension that were serially diluted and plated over kanamycin (200 µg/mL) plates. The plates were kept at 37 ºC and colonies were counted next day. Both wound infection and catheter infection data analysis was performed in Graph Pad prism 7 and statistical analysis was performed by unpaired two-tailed Student’s t-test.



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RESULT AND DISCUSSION Amphiphile 6 is most effective against gram-positive bacteria. We used nine CA-derived amphiphiles where C-24 carboxylic acid of CA was modified with alkyl chains of different length (methyl to dodecyl) and three glycine residues were tethered at three hydroxyl termini of CA (Figure 1A).24 Antibacterial activities of these amphiphiles were tested against different grampositive bacterial strains, S. aureus, B. subtilis, S. pneumoniae and S. oralis using broth dilution assay to determine the MIC99. We observed a decrease in MIC99 of the amphiphiles with increase in alkyl chain length till hexyl chain and further increase in alkyl chain length increases the MIC99 (Figure 1B). Amphiphile 6 with hexyl chain is the most active amphiphile with MIC99 of 4.0 μM. Poor antimicrobial activity with longer alkyl chain lengths can been attributed to amphiphile’s sparing solubility or formation of micellar aggregates.24 As the critical micellar concentration (CMC) of amphiphile 6 is ~31 μM (higher than MIC99.), amphiphile 6 executes non-detergentmediated bactericidal effect against gram-positive bacteria (Figure 1B).24 Hemolytic assays against human red blood cells and cytotoxicity assays against lung epithelial cells (A549) confirm that amphiphile 6 is ~10 fold more selective for gram-positive bacteria over RBCs and ~6-fold less toxic against mammalian cells (Figure 1B).24 Cholic acid (CA) has very poor antibacterial activity and has MIC99 of 20 mM.48 Therefore, conjugation of the hydrophobic tails at carboxyl terminal and glycine units at its hydroxyl terminals helps in providing much needed electrostatic and hydrophobic interactions with bacterial membranes for antibacterial activity. We tested the antibacterial activity of amphiphile 6 after its incubation in aqueous solution for 24 and 48 h and observed that amphiphile retains its antibacterial activity.


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Figure 1. A) General structure of cholic acid-derived amphiphiles with different hydrophobic alkyl chains. B) Antibacterial activities of cholic acid-derived amphiphiles against different grampositive laboratory strains (B. subtilis, S. aureus, S. pneumoniae, S. oralis) and their toxicities against human red blood cells and mammalian cells. IC50, HC50 and CMC values were reproduced from reference 24, Copyright © 2019 American Chemical Society.



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Amphiphile 6 induces maximum membrane-perturbations. We selected amphiphile 1 with methyl chain (MIC99 = 64.0 µM), amphiphile 6 with hexyl chain (MIC99 = 4.0 µM) and amphiphile 9 with dodecyl chain (MIC99 > 256.0 µM) with distinct antibacterial activity against S. aureus and compared the interactions of these amphiphiles with model gram-positive bacterial membranes to decipher the enhanced activity of amphiphile 6 over amphiphiles 1 and 9 (Figure 2A). In a rigid environment,

the

absorption

transition

moments

of

the

fluorescence

probe

like

diphenylhexatriene (DPH) oriented along the electric vector of the polarized incident light are preferentially excited and the extent of polarization of fluorescence emission is described in terms of the anisotropy (rs). In contrast, fluorescence probes in solution or in fluidic environment can freely rotate and does not induce any polarization of the excited light.49 Therefore, anisotropy measurements provide the changes in the environment of the biological membranes on their interactions with drugs and antimicrobials.50 DPH-doped model gram-positive bacterial vesicles using DPPE and DMPG lipids were prepared and incubated with the amphiphiles. Change in membrane rigidity in terms of DPH anisotropy was measured. Amphiphile 6 showed ~1.5-fold decrease in rigidity of bacterial membranes as compared to amphiphiles 1 and 9 (Figure 2B), thereby confirming the ability of amphiphile 6 to disrupt the membranes more effectively over amphiphile 1 and 9. We used the surface plasmon resonance (SPR) to calculate the binding affinities of 1 and 6 with model gram-positive lipid monolayers as amphiphile 9 being insoluble in water.51 Amphiphile 6 showed significantly higher binding response and strong irreversible interactions with membranes as compared to poor and reversible binding of amphiphile 1 (Figure 2C, 2D). The binding constants (K1) for amphiphile 1 and 6 were comparable suggesting similar electrostatic interactions with negatively-charged phospholipid head groups (Figure 2E). Comparison of K1 and K2 suggested that overall higher affinity (KA) of amphiphile 6. This increase in affinity for amphiphiles 6 during the second step arises from insertion of the amphiphile into the hydrophobic 16 ACS Paragon Plus Environment

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core of the membrane.52 Slower (~130 fold) dissociation for amphiphile 6 resulted in its 100-fold greater overall binding constant (KA) as compared to amphiphile 1 (Figure 2E). These results suggest that tethering of hexyl chain helps in stronger association of amphiphile 6 with bacterial membranes.

Figure 2. A) SAR showing the antibacterial activities of amphiphiles against S. aureus. (# presents > 256 M). B) Effect of the amphiphile 1, 6 and 9 on membrane rigidity of model grampositive bacterial membranes. Data is presented as mean ± SD of three replicates and statistical analysis was performed by unpaired two-tailed Student’s t-test. C-D) Surface plasmon resonance sensorgrams for amphiphile 1 (C) and 6 (D) on their interactions with model grampositive bacterial membranes. E) Binding constants calculated from binding sensorgrams for amphiphile 1 and 6. Data is presented as mean ± SD of three replicates. 17 ACS Paragon Plus Environment


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Amphiphile 6 induces disintegration of lipid clusters in bacterial membranes. We performed individual simulations of amphiphiles 1, 6 and 9 in bacterial membrane environment to probe the amphiphile-membrane interactions at atomistic-level (see experimental section for details). We monitored the precise mobility of the three amphiphiles with respect to the various constituent chemical groups, including cholic acid backbone, amino ester group and hydrophobic tail (Figure 3A). The root means square fluctuation (mobility/flexibility) as a function of each atom in all molecules is shown in Figure 3B. Amphiphile 9 showed maximum mobility across all the three groups. In contrast, amphiphile 1 and amphiphile 6 are less flexible at the backbone region. To find out the effect of these amphiphiles on membrane, we measured the membrane thickness for starting and final structures (Figure 3C). Amphiphile 6 membrane interactions showed thinnest membranes as compared to other two amphiphiles. Representative snapshots of time evolution structures of amphiphile 6 displayed membrane insertion across bacterial membranes (Figure 3D). The drastic reduction in membrane size along with fine balance of flexibility across different chemical groups strongly suggests an underlying molecular mechanism of amphiphile 6 to disrupt the membrane. Next, we sought out to determine the mechanistic insights underlying membrane perturbations of amphiphile 6. We used an in-house developed algorithm to compute lipid clustering within heterogeneous gram-positive bacterial membranes containing POPE and POPG lipids (see experimental section for details). The hexagon-based plot, as shown in Figure 3E, captures area based (grid) lipid frequencies and the high intensity of the color shows maximum number of lipids in a given area. We found a clear distinguishing lipid clustering pattern by end of the simulation, whereby the lipid clusters observed at the beginning of the simulation were separated. These findings suggest an interesting role of dynamic lipid-amphiphile interactions and diverse lipid composition in gram-positive membrane during membrane disruption process. Above two panels of Figure 3E are of the starting complex and the below two panels are of the end complex. As 18 ACS Paragon Plus Environment

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the amphiphile disrupts the membrane, we see thinning of the lipid clusters and therefore, above two panels have more darker cells (having more lipid clusters) than in the below panels (less lipid clusters).

Figure 3. A) Molecular structure of cholic acid-derived amphiphile where each atom number is labelled. B) Root mean square fluctuation of amphiphile 1 (black), 6 (red) and 9 (green) are 19 ACS Paragon Plus Environment


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plotted as a function of each atom. C) The top and below graphs represent the first and last structure-based thickness calculation from the MD simulations. D) Representative snapshots of amphiphile 6 simulations during the course of time evolution show the membrane association followed by insertion. E) Lipid clustering analysis plotted as hexagon-based plots for starting (above two boxes) and final structures (below two boxes). Each hexagon represents frequency of lipids present in that particular grid cell, with higher color intensity showing higher lipid number. The POPG and POPE lipids are colored in purple and red, respectively.

Amphiphile 6 is bactericidal against S. aureus. We performed time dependent killing assay where S. aureus cells were treated with amphiphile 6 for different time points and CFUs were counted. We did not observe any bacterial colony within 2h of treatment of amphiphile 6 at 4X MIC99 (Figure 4A). To explore the mechanism of antimicrobial action of amphiphile 6, we studied its ability to depolarize the gram-positive bacterial membranes by membrane-potential sensitive fluorescent dye, 3',3'-diethylthiadicarbocyanine or DiSC2 (5) that usually gets accumulated into lipid membranes of hyperpolarized cells.53 On amphiphile 6 treatment at 2X MIC99, we observed a multi-fold increase in DiSC2 (5) fluorescence suggesting the depolarization of cytoplasmic membrane of gram-positive (S. aureus) bacteria (Figure 4B). Propidium iodide (PI) is a membrane impermeable fluorescent dye and can permeate only through the disrupted cell membranes and shows an increase in fluorescence upon intercalating with DNA. We observed a dose and time dependent accumulation of PI in bacteria on treatment with amphiphile 6 that confirmed the membrane-mediated bactericidal effect (Figure 4C). Next, we tested the ability of amphiphile 6-coated catheters to prevent the bacterial growth during in vitro conditions. We coated the sterilized catheter pieces (∼0.5 cm) with amphiphile 6 by dipping the catheters in a solution of amphiphile 6 in dichloromethane (DCM). DCM was later evaporated resulting in amphiphile 6-coated catheters. Quantification amphiphile 6 loaded on catheters by 20 ACS Paragon Plus Environment

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mass spectrometry showed that there was 36.08  2.62 µg of amphiphile on each catheter. These coated catheters were then incubated in S. aureus suspension. Uncoated and DCM-coated catheters were used as a control. After 3 days, CFU analysis showed >4-log reduction of adhered bacteria on amphiphile 6-coated catheters as compared to uncoated and DCM-coated catheters (Figure 4D). Therefore, these results confirm the bactericidal effect of amphiphile 6 on grampositive bacteria and their ability to prevent the adherence of bacteria on catheters.

Figure 4. A) Time kill assay against S. aureus show the bactericidal nature of amphiphile 6. Data is presented as Mean  SEM of three biological replicates and statistical analysis was performed using two-way ANOVA. B) DiSC2(5) based membrane permeabilization assay of amphiphile 6 treatment on S. aureus confirm the depolarization of membranes. C) Time and dose-dependent



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increase in percentage of propidium iodide (PI) positive S. aureus cells on amphiphile 6 treatment. Data is presented as mean ± SEM of three replicates and statistical analysis was performed by unpaired two-tailed Student’s t-test. D) CFU analysis of bacteria (S. aureus) shows the ability of amphiphile 6-coated catheters to prevent the adherence of S. aureus on 6-coated catheters as compared to uncoated and DCM-coated catheters. Data is presented as mean ± SD and statistical analysis was performed by unpaired two-tailed Student’s t-test.

Amphiphile 6 is effective against S. aureus-mediated wound infections. We used murine topical wound infection model to test the antibacterial efficacy of amphiphile 6 using bioluminescent S. aureus (Xen 36) strain which is an auto bioluminescent strain and does not require any exogenous substrate.54 We created wounds on dorsal side of female BALB/c mice and infected them with S. aureus as described in experimental section. After 16 h of infection, mice were randomized into three groups (4 mice/group) where one group of mice were treated with saline (untreated control). Recent study have shown that addition of neomycin to mupirocin (most commonly used antibiotic for skin infections) treatment helps in combating the S. aureus infections.54 Therefore, we have used the neomycin as a positive control for our animal studies and mice in group 2 were treated with neomycin (20 mg/kg) for three days.46 MIC99 of neomycin against S. aureus is 4 M. Group 3 mice were treated with amphiphile 6 (20 mg/kg) three times a day and treatments were continued for three days (Figure 5A). We performed bioluminescent imaging on day 4 by subjecting the mice to in vivo imaging system. Bioluminescence images witnessed a significant reduction in bioluminescent intensity from amphiphile 6-treated mice (Figure 5B) and bioluminescence quantification confirmed a ~1-log change in average radiance in amphiphile 6 treated mice as compared to untreated mice (Figure 5C). CFU analysis showed > 2-log decrease of bacterial load in amphiphile 6 treated mice in comparison to untreated mice thereby confirming the ability of amphiphile 6 to combat the wound infection (Figure 5D). 22 ACS Paragon Plus Environment

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Figure 5. A) Schematic representation of a wound infection model depicting different treatment groups and experimental plan. B) Bioluminescent images of untreated, neomycin and amphiphile 6 treated groups on the 4th day of infection show reduced bioluminescent intensity in both neomycin and amphiphile 6 treated mice as compared to untreated control. C) Quantification of the average radiance of three groups show a significant reduction bioluminescence in amphiphile 6 treated mice. D) CFU analysis of S. aureus infected wounds after 3 days of treatment show > 2-log decrease in bacterial count upon amphiphile 6 treatment compared to untreated. Data is presented as mean ± SD and statistical analysis was performed by unpaired two-tailed Student’s t-test. 23 ACS Paragon Plus Environment


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Amphiphile 6 prevents S. aureus growth on coated catheters. Substratum like heart valves, catheters or other indwelling devices allow the colonization of bacteria on their surface and can cause systemic infections.55 Therefore, we used the catheter infection model to test the ability of amphiphile 6 coatings to prevent biofilm formation. Uncoated, DCM-coated and amphiphile 6coated catheter pieces (∼0.5 cm, 1 catheter/mice, 5 mice/group) were inserted subcutaneously in three different groups of mice in an incision made on the dorsal side of mice. These incision sites were then infected with bioluminescent S. aureus (Xen 36) and observed for 7 days after skin suturing (Figure 6A). Mice in group 1 received the uncoated catheters, mice in group 2 received DCM coated catheters and group 3 mice received the amphiphile 6 coated catheters. Bioluminescence imaging performed on day 7 revealed a significant reduction in bioluminescence from mice with amphiphile 6-coated catheters as compared to mice with uncoated and DCMcoated catheters (Figure 6B). Quantification of bioluminescence intensity revealed ~ 1.5 log reduction in the bioluminescence as compared to mice with uncoated and DCM-coated catheters (Figure 6C), thereby suggesting the ability of the amphiphile 6 to prevent the adherence of the bacteria. Next, we removed the catheters and performed CFU assay to quantify the bacterial load on the catheters. CFU analysis confirmed a >4 log reduction in bacterial load on amphiphile 6coated catheters as compared to uncoated and DCM-coated catheters (Figure 6D), establishing the ability of amphiphile 6 coating in preventing S. aureus growth on catheters in murine models.


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Figure 6. A) Schematic representation of the catheter infection model depicting different treatment groups. B) Bioluminescent images of uncoated, DCM coated and 6-coated catheters on 7th day of infection show a reduction in bioluminescent intensity in 6-coated catheters as compared to uncoated control. C) Quantification of the average radiance of three groups suggest a significant reduction in bacterial load on 6-coated catheters. D) CFU analysis of S. aureus after 7 days of catheter infection show more than 4-log change in the bacterial load on 6-coated catheters compared to the uncoated catheters. Data is presented as mean ± SD and statistical analysis was performed by unpaired two-tailed Student’s t-test.



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Significant activity against multi-drug resistant (MDR) clinical strains. Use of antibacterial agents can confer multiple drug resistance (MDR) in pathogenic bacteria through activation of multiple resistance genes.56,57 Such strains may act as a major source of hospital-acquired infections. Therefore, we monitored the ability of S. aureus to develop drug resistance against amphiphile 6 upon serial passaging of bacteria in presence of sub MIC99 of amphiphile 6. S. aureus could not develop drug resistance against amphiphile 6 till 35 passages as the amphiphile 6 maintained a steady value of MIC99 (4 µM) at first and at last passage. In contrast, the exposure of levofloxacin resulted in >1000-fold and polymyxin B induced ~30-fold increase in MIC99 after 35 passages (Figure 7A). Mutant Prevention Concentration (MPC) assay showed only 2-fold increase in MIC99 at 1012 CFU/mL of S. aureus whereas Polymyxin B showed an 8-fold increase in MIC99 at ~1012 CFU/mL (Figure 7B). These observations suggest the ability of the amphiphile 6 to be effective against persister cells as well.58 We explored the activity of amphiphile 6 against ten different S. aureus clinical strains obtained from All India Institute of Medical Sciences, New Delhi. We characterized all the strains as per clinical guidelines and categorized them into susceptible (S), intermediate (I), and resistant (R) towards different antimicrobials according to CLSI (Clinical and Laboratory Standards Institute) guidelines as mentioned in experimental section. Our antimicrobial screening revealed that amphiphile 6 was bactericidal against all the clinical multi-drug resistant S. aureus strains with MIC99 of 4 µM (Figure 7C).


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Figure 7. A) Development of drug resistance by S. aureus in terms of fold change in MIC99 over MIC99 at first passage for amphiphile 6 and its comparison with levofloxacin and polymyxin B. B) MIC99 of amphiphile 6 at different CFUs of S. aureus. C) Antibacterial activities of amphiphile 6 and drug resistance profiles of different clinical strains of S. aureus.



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CONCLUSIONS In summary, we performed SAR of cholic acid-derived facial amphiphiles with varied hydrophobicity against gram-positive bacteria and showed that amphiphile 6 with hexyl chain was most active against Gram-positive bacteria and induce bactericidal effect. Biophysical studies validated that amphiphile 6 performed stronger interactions than other amphiphiles with gram-positive bacterial membranes and MD simulation studies showed that amphiphile 6 induces de-clustering of lipid aggregates in simulated membranes. Amphiphile 6 effectively cleared the wound infection in mice model and prevented the S. aureus growth on subcutaneously implanted catheters. Our findings conclude that an optimum balance between charge and hydrophobicity in amphiphile 6 enables it for most suitable interaction with grampositive bacterial membrane, leading to an efficient killing. Therefore, this study promises for designing cholic acid derived amphiphiles for future therapeutics of gram-positive infections. Acknowledgements We are grateful to RCB, IGIB, IISER and AIIMS for intramural funding. Work in A.B. laboratory is

supported

by

the

following

grants:

BT/PR12297/MED/29/895/2014

(DBT)

and

BT/PR17525/MED/29/1021/2016 (DBT). S.P. thank UGC, and K.Y. and S.K. thank RCB for research fellowships. Financial support from the DBT-RA Program in Biotechnology and Life Sciences is gratefully acknowledged. Small animal facility of Regional Centre for Biotechnology is

supported

BT/PR5480/INF/22/158/2012

(DBT).

A.S.

is

supported

by

BT/PR17525/MED/29/1021/2016 (DBT). We thank Amity Lipidomics Research Facility at Amity University Haryana funded by DST-FIST grant (SR/FST/LSI-664/2016). L.T. thanks support from CSIR intramural funding for this project under MLP 1805 grant.

We thank CSIR-4PI for

supercomputing resources. We also thank the support of DBT e-Library Consortium (DeLCON) for providing access to e-resources.


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Table of Contents Graphic

Cholic Acid-Derived Amphiphile can Combat Gram-Positive Bacteria-mediated Infections via Disintegration of Lipid Clusters Sandeep Kumar, Jyoti Thakur, Kavita Yadav, Madhurima Mitra, Sanjay Pal, Arjun Ray, Siddhi Gupta, Nihal Medatwal, Ragini Gupta, Deepakkumar Mishra, Parul Rani, Siladitya Padhi, Priyanka Sharma, Arti Kapil, Aasheesh Srivastava, U. Deva Priyakumar, Ujjaini Dasgupta, Lipi Thukral, and Avinash Bajaj


Cholic Acid-Derived Amphiphile can Combat Gram-Positive Bacteria

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