Oral Delivery of Cholic Acid-Derived Amphiphile Helps in Combating

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Oral Delivery of Cholic Acid-Derived Amphiphile Helps in Combating Salmonella-mediated Gut Infection and Inflammation Kavita Yadav, Prabhu S Yavvari, Sanjay Pal, Sandeep Kumar, Deepakkumar Mishra, Siddhi Gupta, Madhurima Mitra, Vijay Soni, Neha Khare, Priyanka Sharma, Chittur V Srikanth, Arti Kapil, Archana Singh, Vinay K. Nandicoori, and Avinash Bajaj Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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Bioconjugate Chemistry

Oral Delivery of Cholic Acid-Derived Amphiphile Helps in Combating Salmonella-mediated Gut Infection and Inflammation Kavita Yadav,1,2,a Prabhu Srinivas Yavvari,3,a Sanjay Pal,1,4 Sandeep Kumar,1,2 Deepakkumar Mishra,1 Siddhi Gupta,1 Madhurima Mitra,1 Vijay Soni,5 Neha Khare,1 Priyanka Sharma,6 Chittur V. Srikanth,7 Arti Kapil,6 Archana Singh,8 Vinay Kumar Nandicoori,5 and Avinash Bajaj1,* 1. Laboratory of Nanotechnology and Chemical Biology, Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad121001, Haryana, India. 2. Manipal Academy of Higher Education, Manipal-576104, Karnataka, India. 3. Department of Chemistry, Indian Institute of Science Education and Research, Bhopal462026, Madhya Pradesh, India. 4. Kalinga Institute of Industrial Technology, Bhubaneswar-751024, Odisha, India. 5. National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India. 6. Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi110029, India. 7. Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone, FaridabadGurgaon Expressway, Faridabad-121001, Haryana, India. 8. CSIR-Institute of Genomics and Integrative Biology, Mathura Road, New Delhi-110025, India. a: authors contributed equally. Corresponding Authors: Avinash Bajaj, Email: [email protected]

ACS Paragon Plus Environment

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract: A major impediment to develop effective antimicrobials against Gram-negative bacteria like Salmonella is the ability of the bacteria to develop resistance against existing antibiotics and inability of the antimicrobials to clear the intracellular bacteria residing in the gastrointestinal tract. As critical balance of charge and hydrophobicity is required for effective membrane-targeting antimicrobials without causing any toxicity to mammalian cells, herein we report the synthesis and antibacterial properties of Cholic Acid-derived amphiphiles conjugated with alkyl chains of varied hydrophobicity. Relative to other hydrophobic counterparts, compound with hexyl chain (6) acted as an effective antimicrobial against different Gramnegative bacteria. Apart from its ability to permeate the outer and inner membranes of bacteria; compound 6 can to cross the cellular and lysosomal barriers of epithelial cells and macrophages and kill the facultative intracellular bacteria without disrupting the mammalian cell membranes. Oral delivery of compound 6 was able to clear the Salmonella-mediated gut infection and inflammation, and was able to combat persistent, stationary and multi-drug resistant clinical strains. Therefore, our study reveals the ability of Cholic Acid-derived amphiphile to clear intracellular bacteria and Salmonella-mediated gut infection and inflammation.


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Introduction. Gut infections caused by Salmonella (a Gram-negative bacteria) alone affect millions of patients annually where Salmonella enterica serovar Typhi (S. typhi) induces enteric fever (Typhoid) and other serotypes like Salmonella enterica serovar Typhimurium (STM) stimulates acute gastroenteritis.1 Salmonella has the ability to inhabit the epithelial cells and macrophages to stay and reproduce in the repositories of these cells.2 Many commonly used antibiotics like aminoglycosides and glycopeptides cannot get accumulated inside mammalian cells and are therefore, unable to kill the intracellular pathogenic bacteria.3 The facultative intracellular nature of microorganisms assists in developing multi-drug resistance (MDR) against antibiotics through sequential accumulation of mutations4 or through activation of proteases against naturally present antimicrobial peptides (AMPs).5 Combating infections caused by Gram-negative bacteria is highly challenging due to intricate molecular structure of their membranes, emergence of MDR and their ability to impair hostdefense systems.6 Negatively-charged lipopolysaccharide (LPS) at the surface of Gramnegative bacteria provides complex and selective barrier to antimicrobials6 and its endotoxin nature is responsible for causing severe gut inflammation.7 Therefore, synthetic small molecular amphiphiles targeting the bacterial membranes present a valuable strategy as it is difficult for the bacteria to develop drug resistance against membrane-targeting antimicrobials.8 An ideal antimicrobial needs to be stable in the hostile environment of gastrointestinal tract (GIT) as well and should have the ability to kill intracellular bacteria. Naturally occurring AMPs adopt -helical or -sheet-based secondary conformations providing them a facial amphiphilic character.9 Clear segregation of cationic and hydrophobic residues on these AMPs helps in their interactions with bacterial membranes followed by its permeabilization and disruptions.10 Several small molecules based on peptides,11


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lipopeptides,12 hydrocarbon chain13 or steroidal-derived amphiphiles14 and antibioticamphiphile conjugates15 have been designed mimicking the physicochemical features like facial amphiphilic character of AMPs as effective antimicrobials.16 Cholic Acid (CA) provides an ideal scaffold for antimicrobials due to its inherent facial amphiphilic nature where three hydroxyl groups are appended on its concave side with a hydrophobic backbone on convex side of the molecule.17,18 Cationic steroid antibiotics based on CA scaffold adopt facial amphiphilic character like AMPs.19, 20 A critical balance of charge and hydrophobicity is essential for membrane-targeting antimicrobials to execute distinct membrane disruptions. Antimicrobial activity of amphiphiles in general increases with alkyl chain length but does not follow a linear relationship due to “cutoff effect”, thereafter, their activity gets diminished with an increase in chain length.21 Numerous studies have reported the impact of hydrophobicity on antimicrobial activity but the ability of these amphiphiles to kill intracellular bacteria and gut has never been attempted.11,13 Therefore, we decided to study the effect of hydrophobicity on antibacterial activity of CAderived amphiphiles against Gram-negative bacteria and combating gut infections as endogenous and biocompatible nature of CA make it suitable candidate for oral delivery. In this manuscript, we present the synthesis of CA-derived amphiphiles and their antimicrobial activities against different Gram-negative bacteria. We then probed the amphiphiles by indepth mechanistic studies and tested the ability of the most potent amphiphile to kill intracellular bacteria. We then tested the oral formulation of the most effective amphiphile against Salmonella-mediated gut infections and inflammation in mice model. Finally, we tested the ability of the bacteria to develop resistance and activity of the amphiphile against multi-drug resistant clinical strains.


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Results and Discussion: CA is a suitable scaffold for synthesis of antimicrobial amphiphiles. We modified the CA scaffold at C-24 carboxylic acid with different hydrophobic alkyl chains and introduced cationic charge in the form of three glycine residues at hydroxyl termini to provide a net facial amphiphilic character to the molecule (Figure 1). We used three simple steps to synthesize these amphiphiles: a) esterification of C-24 carboxylic acid of CA with different alkyl alcohols, b) conjugation of three Boc-protected glycine units at three hydroxyl termini of CA and c) deprotection of Boc residues (Figure 2). We characterized the amphiphiles by 1H NMR and mass spectra studies.

Figure 1. Molecular structures of Cholic Acid (CA)-derived amphiphiles (1-9) with different hydrophobic alkyl chains having glycine units attached to three hydroxyl termini of CA.


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Figure 2. Synthesis of CA-derived amphiphiles where carboxyl terminal at C24 was derivatized with different alcohols using ester bond and three hydroxyl groups at C3, C7, and C12 were conjugated with glycine to introduce charged groups.

Compound 6 is most active amphiphile. We tested the antibacterial activities of these amphiphiles against different Gram-negative bacterial strains including Shigella flexneri, STM, Escherichia coli (E. coli), Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii using broth dilution assay and presented the activities as minimum inhibitory concentration required for 99% killing of the bacteria (MIC99). Structure-activity relationship (SAR) studies revealed the surge in antibacterial activity of amphiphiles with increase in alkyl chain length with a “cut-off effect” at hexyl chain. In particular, compound 6 with hexyl chain was most active with MIC99 of 4.0 µM. However, further increase in chain


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length diminished the antibacterial activity of amphiphiles (Table 1).21 Our SAR with CAderived amphiphiles witnessed the cut-off effect at hexyl chain as inclusion of CA scaffold provided additional hydrophobicity to the amphiphile and reduced the cut-off effect of higher chain lengths.21 Antimicrobial inactivity with longer alkyl chain lengths has been attributed to amphiphile’s limited solubility or propensity to form micellar aggregates. Detergent-like mechanism involves the solubilization of targeted bacterial membranes into mixed micelles that is followed by many detergents like n-Octyl-β-D-Glucopyranoside.22 In contrast, AMPs and membrane-targeting amphiphiles, below their critical miceller concentration, cause toroidal pores in bacterial membranes and induce leakage of intracellular contents due to asymmetric expansion, chain disordering and membrane thinning at concentrations.22 To overrule the detergent-mediated bactericidal effect of these amphiphiles, we determined critical micelle concentration (CMC) of amphiphiles using pyrene fluorescence assay. Intuitively, there was a decrease in CMC of amphiphiles with increase in chain length. Compound 6 had a CMC of ~31 µM (~8-fold more than its MIC99) that certified the nondetergent-mediated bactericidal effect of amphiphile (Table 1). We also performed a DLSbased size analysis of most effective compound 6 and found that it formed self-assembled nano-sized aggregates at 50 M that was >12-fold higher than MIC99 (4 M) of the compound. Further, we then assessed the selectivity of the amphiphiles for bacterial membranes over mammalian cell membranes using hemolytic and cytotoxicity assays. Hemolytic assay witnessed that compound 6 is ~10 fold more selective for Gram-negative bacteria over RBCs (Table 1). Subsequently toxicity of amphiphiles against lung epithelial cells (A549) suggested that compound 6 is ~6-fold less toxic against mammalian cells (Table 1).


7


Table 1. Antibacterial activities of CA-derived amphiphiles (1-9) in comparison with Polymyxin B against different Gram-negative laboratory strains, and their activities against human red blood cells and mammalian cells along with their critical micelle concentration. MIC99 (µM)a

HC50 (µM)b

IC50 (µM)c

CMC (µM)d

S. Flexneri

S. typhimurium

E. coli

P. aeruginosa

K. pneumoniae

A. baumannii

1

128

128

64

256

128

>256

>200

82

107

2

256

256

256

64

256

256

>200

78

137

3

128

128

128

32

128

128

>200

75

385

4

32

32

32

16

64

32

180

20

142

5

16

8

16

8

16

8

83.5

40

119

6

4

4

4

4

4

4

43

25

31

7

16

16

4

8

4

4

67

62

23

8

32

>256

32

16

8

>256

68

23

15

9

>256

>256

>256

>256

>256

>256

87

19

10

Polym yxin B

1

1

2

0.5

4

0.5

>200

90

-e

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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a: MIC99 (Concentration at which 99% bacterial killing is observed) was measured using broth dilution assay. b: Hemolytic activities of compounds against human red blood cells (RBCs). HC50 is the concentration at which 50% RBC lysis was observed. c: Cytotoxic activities of compounds against mammalian lung epithelial (A549) cells is reported as concentration at which 50% cell death was observed. d: Critical micelle concentrations of compounds was measured by Pyrene fluorescence assay. e: not determined.

Compound 6 can permeabilize and disrupt the bacterial membranes. To find the mechanism of antimicrobial action of compound 6, we assessed the ability of compound 6 to permeabilize the outer and inner bacterial membranes. N-phenyl naphthylamine (NPN) being a lipophilic dye has weak fluorescence in aqueous environment and its fluorescence increases on penetration in disrupted hydrophobic cell membrane.23 Profound increase in NPN


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fluorescence on compound 6 treatment of bacteria confirmed its ability to permeabilize the outer bacterial membranes (Figure 3A). Membrane-potential sensitive cationic dye, 3,3’diethylthiadicarbocyanine iodide (DiSC2(5)) usually gets accumulated into lipid membranes of hyper polarized cells and its fluorescence gets quenched.24 Substantial increase in DiSC2(5) fluorescence on compound 6 treatment established the depolarization of inner bacterial membranes of STM (Figure 3B). Release of intracellular contents from bacteria on membrane perturbations were confirmed by dose- and time-dependent accumulation of membrane impermeable dyes like propidium iodide (PI) inside the bacteria (Figure 3C).25 We then performed in vitro time-kill assay against STM that revealed bactericidal nature of compound 6 as no viable colonies were visible after 6h of treatment at 1X MIC99 and only 1h of treatment at 4X MIC99 was sufficient to clear the bacterial load (Figure 3D). Further, we investigated the impact of compound 6 on membrane disruptions using Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM). Micrographs from AFM studies depicted the formation of grooves and lesions appearing on the bacterial surface on compound 6 treatment (Figure 3E).26 Complete disintegration of the bacteria with massive amount of membranous residues and debris around the damaged cells established the leakage of cytoplasmic fluid from the bacteria on compound 6 treatment (Figure 3E). Micrographs from TEM of untreated STM revealed an intact outer and inner membrane with normal intracellular contents (Figure 3F). In contrast, we observed bacterial cells with a clear cytoplasmic zone on compound 6 treatment suggesting the possible leakage of intracellular contents.27 Number of bacterial cells with clear cytoplasmic zone increased after 12h of treatment with disruptions in the bacterial membranes (Figure 3F).


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Figure 3. A) Time dependent increase in fluorescence of N-phenyl-1-naphthylamine (NPN) on treatment of compound 6 with STM confirm the ability of 6 to perturb outer bacterial membranes. B) Increase in fluorescence intensity of DiSC2(5) over time on compound 6 treatment show the ability of 6 to disrupt inner bacterial membranes. C) Dose and time dependent increase in uptake of propidium iodide (PI) by STM cells in terms of % PI positive cells validate the membrane disruptions after treatment with compound 6. D) In vitro time kill


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assay in terms of CFU on treatment of STM confirm bactericidal effect of 6. E-F) Representative Atomic Force (G) and Transmission Electron (F) micrographs of STM after treatment with compound 6 (1X MIC99) at different time show that the compound 6 is able to disrupt the bacterial membrane and induce leakage of cellular contents.

Compound 6 kills intracellular bacteria. To assess the ability of compound 6 to kill intracellular STM without disrupting mammalian cell membranes, we treated the infected mammalian cells with compound 6 for 4h. CFU analysis confirmed its ability to clear the intracellular bacteria in infected epithelial (HeLa) (Figure 4A) and macrophage (RAW 264.7) cells (Figure 4B) with >90% mammalian cell viability. Fluorescence micrographs of untreated and compound 6-treated mCherry-STM infected cells validated the reduced intracellular bacterial load on compound 6 treatment in HeLa (Figure 4C) and RAW 264.7 cells (Figure 4D). Salmonella containing vacuoles (SCVs), natural habitat of Salmonella in mammalian cells, provide another intracellular protection against antimicrobials apart from outer cellular membrane.28 We therefore co-stained SCVs of untreated and compound 6 treated STM infected HeLa cells with LAMP1 antibody, and witnessed a significant reduction in the number of SCVs on compound 6 treatment (Figure 4E). These observations confirmed the ability of the compound 6 to cross mammalian cellular and intracellular barriers to kill the bacteria.


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Figure 4. A-C) Intracellular bacterial load in terms of CFU/mL and % cell viability of infected HeLa (A) and RAW 264.7 (B) cells after 4h treatment of STM infected cells at different doses of compound 6. Compound 6 was able to clear the intracellular load without causing any toxicity to mammalian cells. (# presents no colonies were observed). C-D) Representative confocal micrographs of mCherry-labelled STM infected HeLa (C) and RAW 264.7 (D)


cells on

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compound 6 (10 M) treatment as compared to untreated cells show significant decrease in intracellular bacteria. E) Representative confocal micrographs of mCherry-STM infected HeLa cells stained with LAMP1 antibody (Green) revealed a significant reduction in number of Salmonella containing vacuoles (SCVs) on 6 treatment.

Compound 6 can clear Salmonella induced gut infection, and combat inflammation: To test the efficacy of compound 6 in combating STM-induced gut infection and inflammation, we first assessed its stability of compound 6 in simulated gastric fluid (SGF). We incubated the compound 6 formulation (prepared in carboxymethyl cellulose) in SGF for 2h simulating the normal gastro-intestinal emptying time29 and tested for antibacterial activity. We observed that compound 6 showed MIC99 of 8 M after its incubation in SGF confirming its gastric stability. Further, we determined the dose-dependent biocompatibility of compound 6 formulation on oral delivery in BALB/c mice. There was no mortality and change in body weight of mice on using 5, 10 and 20 mg/kg of the compound 6 for five days with a frequency of 3 doses per day (Figure S1A). H&E sections of liver witnessed a normal histology with intact hepatocytes like control mice (Figure S1B) and spleen sections confirmed the intact morphology of tissue with normal plasmacytes (Figure S1B). To test the ability of compound 6 to combat in vivo infections, we used streptomycin-pretreated STM infection model in BALB/c mice as this model resembles many aspects of human infection causing edema, massive infiltration of neutrophils and epithelial ulceration.30 Mice were pre-treated with streptomycin to clear the gut microbiota and infected with STM using oral gavage for 24h to establish the infection. We randomized the mice into three groups (untreated, compound 6 treated, and Ciprofloxacin treated) for treatment schedules that was three times a day for four days (Figure 5A). CFU analysis of fecal samples on different days


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for STM confirmed no bacterial load by 4th day (Figure 5B) and compound 6 treatment cleared the STM infection from the spleen as well (Figure 5C). For performing non-invasive imaging of STM infection, we infected the streptomycin pre-treated mice with bioluminescent Salmonella Typhimurium-Xen33 and followed the similar treatment regimes. Bioluminescence imaging confirmed the established STM infection in untreated mice and a significant reduction in bioluminescence on compound 6 treatment validated the clearance of STM infection as observed in CFU analysis (Figure 5D). Invasion of Salmonella into intestinal epithelium induces acute inflammation triggered by LPSToll like receptor (TLR) interactions.31 STM-induced inflammation in general causes multi-fold increase in expression of pro- and anti-inflammatory cytokines through Type III secretory system along with shortening of colon and swelling of spleen.32 Compound 6 treatment significantly diminished the inflammation at the colon site as we observed normal colon length as compared to shortened colon in case of infected mice (Figure 5E, 5F). Similarly, compound 6 treatment caused normal spleen as compared to inflamed spleen of infected mice (Figure 5G, 5H). Gene expression by real time PCR denoted a significant reduction in expression of pro-inflammatory genes like TNF- and anti-inflammatory genes like IL-10 on compound 6 treatment that confirm the ability of compound 6 to combat Salmonella-induced gut inflammation (Figure 5I).


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Figure 5. A) Schematic plan of the study to test the ability of compound 6 to combat Salmonella infection in streptomycin pre-treated STM infection model in BALB/c mice. B) Quantification of bacterial burden in terms of CFU/g of fecal samples on different days of treatment with compound 6 and Ciprofloxacin confirm the bactericidal nature of 6 in clearing gut bacteria. Data presented is mean ± SD of four mice. C) Quantification of bacterial burden in terms of CFU/g of spleen of STM infected mice on compound 6 treatment shows its ability to clear the infections from spleen. Data presented is mean ± SD of four mice. D) Bioluminescent images of untreated, compound 6 and Ciprofloxacin treated BALB/c mice infected with bioluminescent Salmonella typhimurium-Xen33 confirm significant reduction in bacterial load on compound 6 treatment. E-H) Representative images and change in length of colon (E, F) and spleen (G, H) of STM infected mice after compound 6 treatment show that colon and spleen maintain their normal length after compound 6 treatment. Data presented is mean ± SD of four mice. I) Relative quantification of inflammatory genes by qRT-PCT from colon tissues on different treatments show significant reduction in levels of pro-inflammatory cytokines on compound 6 treatment. Data presented is mean ± SD of three mice done in duplicates. Remarkable activity against multi-drug resistant (MDR) clinical strains. Uncontrolled use of antimicrobials is known to generate drug resistant among bacterial strains due to accumulation of mutations in key targets of these antimicrobials.33 Emergence of MDR against wide-spectrum of antibiotics affects the therapeutic outcome thereby complicating the treatment regimens. Therefore, we assessed the ability of STM and E. coli to develop drug resistance against compound 6 upon serial passaging of bacteria in presence of sub MIC99 of compound 6. Both the bacteria were unable to generate drug resistance against compound 6


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till 40 passages, and we observed MIC99 of 4 M for compound 6 at first and last passage for STM (Figure 6A) and E. coli (Figure 6B). In contrast, exposure of Ciprofloxacin and Polymyxin B allowed the STM to develop resistance by >2000 fold for ciprofloxacin and >30 fold for Polymyxin B after 40 passages (Figure 6A). Compound 6 was able to kill these ciprofloxacin resistant STM as well with same efficacy (MIC99 = 4 M). Similarly, E. coli did not generate any drug resistance against compound 6 as developed by levofloxacin and Polymyxin B (Figure 6D). To test the efficacy of compound 6 against persistent bacteria,34 we treated the STM and E. coli cultures with high dose of Ampicillin (100 μg/mL) for 3h at 37°C to get the persister cells. Treatment of STM persisters with compound 6 at 2X MIC99 for 2h killed these bacteria where Ampicillin was found ineffective (Figure 6C). Similarly, treatment of STM bacteria from their stationary phase with compound 6 for 2h cleared all the bacteria (Figure 6C). Compound 6 was also able to clear persistent and stationary E. coli as well with same efficacy (Figure 6D). We also witnessed that compound 6 was consistent in its antibacterial activity up to ~1012 CFU/mL of STM (Figure 6E) and E. coli (Figure 6F) whereas there was a ~4-8-fold increase in MIC99 of Polymyxin B at ~1012 CFU/mL for STM and E. coli. These results clearly confirmed the ability of compound 6 to kill even the least susceptible cells.35 We then assessed the activity of compound 6 against 50 different clinical strains (10 each for S. typhi, E. coli, P. aeruginosa, K. pneumoniae and A. baumannii) obtained from All India Institute of Medical Sciences, New Delhi. We characterized all the strains as per clinical guidelines and categorized their responses towards different antimicrobials into susceptible (S), intermediate (I), and resistant (R) according to CLSI (Clinical and Laboratory Standards Institute) guidelines as mentioned in supporting information. Our antimicrobial assays revealed


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that compound 6 was bactericidal against all the clinical strains including multi-drug resistant strains of E. coli, P. aeruginosa and K. pneumoniae with MIC99 of 1-8 M (Table 2). Compound 6 was also effective against Ciprofloxacin resistant S. typhi strains with MIC99 of 1-2 M.

Figure 6. A, B) Development of drug resistance by STM (A) and E. coli (B) in terms of fold change in MIC99 over MIC99 at first passage for compound 6 and its comparison with Ciprofloxacin, Levofloxacin and Polymyxin B. C, D) Antibacterial activity of compound 6 and Ampicillin against persistent and stationary STM (C) and E. coli (D) bacteria. E, F) Fold change in MIC99 of compound 6 at higher CFU of STM (E) and E. coli. (F) over MIC99 at 105 CFU/mL.


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Table 2. Antimicrobial activity of compound 6 against different clinical strains of S. typhi, E. coli, A. baumannii, K. pneumoniae and P. aeruginosa; and susceptibility profiles of these strains against different drugs as per CLSI guidelines.

Strain

Ciprofloxacin

Cefotaxime

Amikacin

Compound 6

Strain

Ciprofloxacin

Cefotaxime

Amikacin

Compound 6

Strain

Ciprofloxacin

Cefotaxime

Amikacin

Compound 6

Strain

Ciprofloxacin

Cefotaxime

Amikacin

Compound 6

P. aeruginosa

Compound 6

K. pneumoniae

Amoxicillin

A. baumannii

Cefexime

E. coli

Ciprofloxacin

S. typhi

Strain

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


16011/10

R

S

S

2

1284

I

R

I

4

4/307

R

R

R

4

4/230

R

R

R

8

4/316

S

S

S

8

17375/10

R

S

R

2

6/78

R

R

R

4

1/303

R

R

R

8

8/407

R

R

R

4

6/243

S

S

S

8

7742/09

R

S

S

2

6/23

R

R

R

4

6/303

R

R

R

4

8/307

R

R

R

4

1/231

S

S

S

8

7300/01

R

S

S

2

9/203

S

R

S

4

3/216

R

R

R

4

7/47

R

R

R

4

1/318

R

R

R

8-16

13521/2

R

S

S

2

6/33

R

R

R

4

5/405

R

R

R

4

12823

R

R

R

4

3/313

S

R

S

8

111608/08

R

S

S

2

8/341

R

R

R

4

5/321

R

R

R

4

12744

R

R

R

4

6/264

R

R

R

4-8

15878/08

R

S

S

2

6/40

R

R

R

4

4/202

R

R

R

4

6/47

R

R

R

8

17/288

R

R

R

8

4274/15

R

S

S

2

9/44

R

R

R

8

5/412

R

R

R

4

7408

R

R

R

4

7/227

R

R

R

4

2353

R

S

S

1

6/38

R

R

R

4

6/312

R

R

R

4

7399

R

R

I

4

20/261

S

S

S

4

26052 R S S 2 6/27 R R R 4 9/229 R R R 4 7/9272 R R R 8 7/233 S S S 8 Sensitivity of strains (R: resistant, S: sensitive, I: intermediate) was determined as mentioned in experimental section. Activity of Compound 6 was presented as MIC99 in µM (Concentration at which 99% bacterial killing is observed) and was measured using broth dilution assay.


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Conclusions. In summary, we have synthesized a series of Cholic Acid-derived facial amphiphiles with alkyl chains of different length. SAR studies against Gram-negative bacteria concluded that compound 6 with hexyl chain was most active and can disrupt the bacterial membranes. Treatment of the infected epithelial and macrophage cells with compound 6 validated the ability of the amphiphile to kill intracellular bacteria without any toxicity. Gastric stability of compound 6 allowed targeting the Salmonella residing in the GIT and effective interactions with LPS help in combating Salmonella-induced gut inflammation. Therefore, this study opens new avenues for design and exploring of Cholic Acid-derived amphiphiles for combating gut infections. Experimental Section General: Cholic acid, Boc-Glycine, n-alkyl alcohols, Dicyclohexylcarbodiimide (DCC), Pyrene, Anhydrous dichloromethane, Chloroform (HPLC grade, ≥99.9%), MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide), N-phenyl-naphthyl amine (NPN), Paraformaldehyde, DAPI

(4′,6-Diamidino-2-phenylindole

dihydrochloride),

3',3'-

diethylthiadicarbocyanine

DiSC2(5), Propidium Iodide (PI), polyclonal rabbit anti-LAMP1 (Cat. No. L1418), Hoechst 33258, Streptomycin, Polymyxin B, Levofloxacin, Ciprofloxacin, and MacConkey MUG agar were purchased from Sigma-Aldrich. RNA-II Kit from MN, Germany, i-Script cDNA Synthesis Kit from Bio-Rad, USA and SYBR Green from Takara was purchased. Prolong gold anti-fade reagent was obtained from Molecular Probes, Life Technologies. Mueller Hinton (MH) broth and agar were purchased from HiMedia. Dimethylaminopyridine (DMAP), 4M HCl in dioxane and chromatography solvents like petroleum ether, ethyl acetate, dichloromethane and methanol were purchased from SDFC Ltd. India. Thin layer chromatography was carried out


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on aluminium sheet coated with Silica gel 60 GF 254 (Merck, Germany) and stained with ethanolic solution of phosphomolybdic acid. 1H NMR spectra were recorded in CDCl3 and DMSO-d6 using a Bruker Avance 400 MHz spectrometer. Chemical shifts (δ) are reported in ppm with tetramethyl silane (TMS) as the internal standard. Mass spectrometry analysis were performed with an micrOTOF-Q II 10330 system (Bruker). Antibacterial assay.36 Antibacterial efficacy of the molecules is reported as minimum inhibitory concentration (MIC99) that is the lowest concentration of antimicrobial agent inhibiting 99% growth of bacteria. Bacterial strains were freshly inoculated in MH broth, incubated for 6h and re-suspended further in broth to achieve 105 CFU/mL. Bacterial culture (100 μL) was then inoculated into each well of a 96-well plate containing 100μL of compounds that were 2-fold diluted from a highest concentration of 512 µM (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 µM). Media (200 μL) without any bacteria and bacterial solution (200 μL) without any treatment was used as control. Polymyxin B and Ciprofloxacin were used as positive controls. Bacterial culture was then incubated at 37oC for overnight. MIC99 data was recorded by measuring the absorption at 600 nm using a Spectramax M5 microplate reader (Molecular Devices Corp; Sunnyvale, CA, USA). MIC99 value was determined in quadruplicates for each concentration at least three independent times. For mutant prevention concentration assay, bacterial suspension with different CFUs (105-1012 CFU/mL) of bacteria were used to determine the MIC99. For antibacterial activity against stationary phase bacteria, mid-log phase culture was allowed to grow for 16-18h for bacteria to reach stationary phase. Bacterial suspension was centrifuged at 9000 rpm for 2 min. and washed with PBS. Bacterial suspensions (105 CFU/mL) were then treated with 2X MIC99 of compound 6 and incubated at 37oC for 2h. Ampicillin (8 M) was taken as a control. Bacterial suspension without any treatment served as negative control. After


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treatment, bacterial suspensions were serially diluted and plated on MH agar plates. Viable colonies were counted after 24h of incubation at 37oC and bacterial CFU were represented as mean ± SD of three replicates. To obtain the persister bacterial cells, stationary phase bacteria were treated with Ampicillin (100 μg/mL) for 3h at 37°C to generate the persister cells. Culture was then centrifuged at 6000 rpm for 5 min. and washed 2-3 times to remove complete Ampicillin. Bacterial suspension (105 CFU/mL) was then treated with compound 6 (8 M) and Ampicillin (8 M) at 37oC for 2h. Treated bacterial suspension was then serially diluted in MH broth and plated on MH agar plates. Viable colonies were counted after 24h of incubation at 37oC and bacteria CFU were represented as mean ± SD of three replicates. Time kill kinetics was performed by incubating 106 CFU/mL of mid-log-phase bacteria with 1X and 4X MIC99 of amphiphile. Aliquots were taken after 0, 15, 30, 60, 120, 240, and 360 min, subjected to serial 10-fold dilutions in MH broth and plated. Viable colonies were counted and data is presented as mean ± SD of four replicates. To study the ability of bacteria to develop drug resistance, MIC99 value of amphiphile, Ciprofloxacin, Levofloxacin and Polymyxin B was determined against bacteria as described above in antibacterial assay. Treated bacteria at 1/2X MIC99 were further used for antibacterial assay to determine MIC99 of amphiphile and other drugs. Fold change in MIC99 for each amphiphile and control antibiotics was plotted against the number of days. Hemolytic assay,37 cytotoxicity assay,38 outer membrane permeabilization assay,39 inner membrane depolarization assay,40 and propidium iodide assay,41 and Transmission Electron Microscopy42 studies were performed as per reported protocol.


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Atomic force microscopy.43 Amphiphile treated bacteria were applied on fresh surface of clear glass slides (1 mm thick) and allowed to dry overnight before imaging. The images were obtained in tapping mode in air using WITec alpha300RS with scanning range of 100 x 100 x 20 micron in X, Y and Z respectively. All images were scanned with a speed of 0.3 Hz/line speed with a resolution of 512 x 512 pixels. A silicon noncontact cantilever from Nanosensors with a resonance frequency of 180 kHz and 42 N/m spring constant was used. Every scan resulted in a topography and a phase image that were acquired simultaneously. Data was processed using WITec Project software. In vitro Infection Assay.28 HeLa or RAW 264.7 cells (2 X 105) were seeded in a 6 well plate for 24h. Cells were then washed with 1X DPBS and supplemented with fresh media containing 10% FBS without any antibiotics. Cells were then infected with STM for 1h with multiplicity of infection (MOI) as 1:40. Extracellular bacteria were washed off with gentamicin (100 µg/mL). Infected cells were treated with different concentrations of amphiphile (4, 8 and 10 µM) or ciprofloxacin (4 µM) for 4h. Cells were then lysed using water, serially diluted in sterile PBS and plated onto MH agar plates for overnight, and CFUs were calculated. Cell viability after infection and post treatment were assessed using MTT assay in a separate set of experiments. For confocal microscopy, cells adhered on cover slips were fixed with 4% paraformaldehyde for 5 min at room temperature, subsequently washed with PBS and nucleus counter stain was done with DAPI. Cells were then washed and coverslip was mounted using prolong gold anti-fade reagent. Confocal laser scanning microscopic imaging was finally performed to visualize the infected bacteria using Leica Confocal SP5 microscope. To stain the Salmonella containing vacuoles; mCherry-STM infected HeLa cells on amphiphile treatment were washed twice with PBS and fixed in 2.5% paraformaldehyde for


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15 min at room temperature followed by washing with PBS. Cells were then incubated with 0.1% saponin and 1% bovine serum albumin (PBS buffer) for 1h and then stained with polyclonal rabbit anti-LAMP1 antibody for 1h at room temperature. After 3 washings with PBS containing 0.1% saponin and 1% BSA, cells were incubated with Cy5-conjugated goat antirabbit immunoglobulin G for 1h. Cells were washed, incubated in Hoechst 33258 stain for 5 min. and observed using Leica confocal SP5 microscope using a 63X oil objective. Pyrene fluorescence assay.44 Fluorescence spectra of pyrene (0.3 g/mL) with different concentrations of amphiphile solutions were recorded using ex of 334 nm. The I375 and I395 were measured at the wavelengths corresponding to the first and third vibrionic bands using Hitachi F7000 Fluorescence Spectrophotometer (Hitachi High Technologies, USA). Ratio of I395 and I375 were plotted against concentration of amphiphiles. Intersection of best fit lines was considered as CMC of the amphiphile. Animal experiments: All animal experiments were performed after animal ethical clearance from Institute Animal Ethical Committee of National Institute of Immunology and Regional Centre for Biotechnology. Oral formulations of compound 6 were prepared in polysorbate-80 and carboxymethyl cellulose. Compound 6 (2.0 mg) was suspended in polysorbate-80 (50 µL) and suspension was vortexed for 10 min followed by an addition of ethanol (50 µL). Suspension was further vortexed for 5 min to get the clear solution that was further diluted by the addition of 0.5% carboxymethyl cellulose (900 µL); and mixed by vortexing. To test the stability of compound 6 in simulated gastric fluid (SGF),45 SGF (with pH 1.2) was prepared using NaCl (0.2% w/v), pepsin (0.32% w/v) and HCl (0.7% v/v). Compound 6 (5.833 mM in 1 mL PBS) was then incubated with 9 mL of SGF. After 2 h, an aliquot (1 mL) from SGF incubation mixture was taken and pH was neutralized using saturated sodium bicarbonate


24

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solution (70 µL). From this, a stock solution of 32 µM of compound 6 was prepared and used for testing the antibacterial efficacy keeping DMSO stock solution of compound 6 as the positive control. For dose tolerated studies, BALB/c mice were randomized into five groups (n = 4 mice per group); and different doses of compound 6 formulation (0, 5, 10 and 20 mg/kg) were given using oral gavage three times a day for four days. Change in body weight of mice were monitored. After four days, organs were harvested and H&E staining was performed as per published protocol.45 For infection studies,28 BALB/c mice were orally administered with streptomycin (3.75 mg/mice) orally in saline solution after 4 hours of fasting. After 24h of antibiotic treatment; mice were fasted for 4h and then infected intragastrically with STM (~106 CFU/mice) in PBS (200 µL) by gavage. Mice were then randomized into three groups a) control untreated, b) Ciprofloxacin, and c) compound 6 treated mice (4 mice/group). Group b mice were treated with Ciprofloxacin (20 mg/kg) and group c mice were treated with compound 6 suspension (20 mg/kg) thrice a day for four consecutive days. Fecal pellets from each group were collected every day before dosing and plated on MacConkey MUG agar plates with antibiotic (Streptomycin at 50 g/mL) selection marker followed by incubation for 2-3 days. After completion of treatment, mice were sacrificed ,and colon and spleen were photographed and length was measured. Bacterial load in the spleen was then quantified from tissue homogenates. Colon tissues were stored in tissue protectant for quantification of inflammatory markers. Real time PCR studies. Total RNA from mouse colon samples was isolated using the Nucleo Spin RNA-II Kit according to the manufacturer's protocol. One microgram of each total RNA sample was used to synthesize c-DNA using the i-Script cDNA Synthesis Kit. Real-time PCR


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was performed using 20 μL reaction volume with final primer concentration of 10 nM and cDNA (corresponding 40 ng RNA) per well (diluted with nuclease free water) in a 96-well plate by using SYBR Green according to the manufacturer's instruction in the Bio-Rad CFX 96™ Real Time Detection System. All reactions were normalized to the housekeeping gene B2M for mouse samples. Primer sequences used are mentioned in Table S1. Drug resistance profiling of clinical strains. Antibacterial activity against clinical strains were determined after Institute Ethical Clearance from All India Institute of Medical Sciences, New Delhi and Regional Centre for Biotechnology, Faridabad. Clinical isolates of E. coli, P. aeruginosa, A. baumannii, S. typhi, and K. pneumoniae (10 each) were selected for antimicrobial susceptibility testing. The strain identification was done by standard biochemical methods.46 All the strains were tested for antimicrobial susceptibility to the drugs recommended by CLSI (Clinical Laboratory Standards Institute) for that particular organism.47 Antimicrobial susceptibility was determined by Kirby-Bauer disk diffusion method using Amoxicillin (10 µg), Ciprofloxacin (5 µg), Cefixime (5 µg), Amikacin (30 µg) and Cefotaxime (30 µg) disks procured from HiMedia Laboratories limited, Mumbai, India.48 Briefly, 3-5 colonies from an 18- 24h old culture were added to a tube containing 4-5 mL of 0.85% normal saline and vortexed to form a suspension. The suspension was adjusted to match the turbidity of 0.5 McFarland turbidity standards that is equivalent to a suspension containing 1.5 x 108 CFU/mL. This bacterial suspension was inoculated on Mueller Hinton agar medium (BD Difco, Sparks, MD, USA). The inoculated plates were allowed to stand for 5-10 min to allow excess moisture to be absorbed and antibiotic disks were then applied using sterile forceps. After 18-20h of incubation, the plates were examined and inhibition zone diameters were measured to the nearest whole millimeter using a ruler. All the results were analyzed as


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per CLSI 2017 guidelines. The isolates were categorized as Susceptible (S), Intermediate (I) and Resistant (R) according to the CLSI inhibition zone diameter interpretive standards as mentioned in Table S2. Statistical analysis. All graphs were drawn in Graph Pad Prism 7. Statistical significance among the treated groups is calculated either by unpaired Student’s t-test or one-way ANOVA (Dunnett test) or two-way ANOVA. Conflicting interests: Authors declare no conflicting interests. Ethics statement. All animal experiments were performed after due ethical approval from Institutional Animal Ethics Committee of National Institute of Immunology (IAEC#388/15), and Regional Centre for Biotechnology (RCB/IAEC/2016/001). All experiments with human blood samples and clinical bacterial isolates were performed 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). Acknowledgements: The support from RCB, NII and AIIMs-New Delhi core funds and Department of Biotechnology (DBT), India are greatly acknowledged. A.B. is supported by BT/PR12297/MED/29/895/2014 and BT/PR17525/MED/29/1021/2016 from Department of Biotechnology, Govt. of India. K.Y. and S.K. thank RCB, P.S.Y. and S.P. thank UGC for research fellowships. We thank Dr. Somanath Kundu for helping in confocal microscopy experiments. We are thankful to Advanced Instrumentation Research Facility, Jawaharlal Nehru University, Delhi, India for AFM studies. Small animal facility at Regional Centre for Biotechnology is funded by DBT (BT/PR5480/INF/22/158/2012). We thank Dr. Beth McCormick for providing Salmonella strain. We are also grateful to support of DBT e-Library Consortium (DeLCON) for providing access to e-resources


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46. Mackie, T., Collee, J., McCartney, J. (2007) Mackie and McCartney practical medical microbiology. New Delhi (India): Elsevier. 47. Clinical and Laboratory Standards Institute. (2012) Performance standards for antimicrobial susceptibility testing, Twenty-second informational supplement. CLSI document M100-22. Wayne, PA: Clinical and Laboratory Standards Institute. 48. Clinical and Laboratory Standards Institute (2015) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard. CLSI Document M07A10, 10th ed. Wayne, PA: Clinical and Laboratory Standards Institute.


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TOC Graphic


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Oral Delivery of Cholic Acid-Derived Amphiphile Helps in Combating

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