Oral Delivery of Cholic Acid-Derived Amphiphile Helps in Combating Salmonella-Mediated Gut Infection and Inflammation

Article abstract of DOI:10.1021/acs.bioconjchem.8b00880

A major impediment to developing effective antimicrobials against Gram-negative bacteria like Salmonella is the ability of the bacteria to develop resistance against existing antibiotics and the inability of the antimicrobials to clear the intracellular bacteria residing in the gastrointestinal tract. As the 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, a compound with hexyl chain (6) acted as an effective antimicrobial against different Gram-negative bacteria. Apart from its ability to permeate the outer and inner membranes of bacteria; compound 6 can 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 amphiphiles to clear intracellular bacteria and Salmonella-mediated gut infection and inflammation.

Full text of DOI:10.1021/acs.bioconjchem.8b00880

Subscriber access provided by University of Winnipeg Library
Article
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
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
5
1
6
Mishra, Siddhi Gupta, Madhurima Mitra, Vijay Soni, Neha Khare, Priyanka Sharma, Chittur
7
6
8
5
1,*
V. Srikanth, Arti Kapil, Archana Singh, Vinay Kumar Nandicoori, and Avinash Bajaj
1. Laboratory of Nanotechnology and Chemical Biology, Regional Centre for Biotechnology,
NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad-
1
2
3
4
4
21001, Haryana, India.
. Manipal Academy of Higher Education, Manipal-576104, Karnataka, India.
. Department of Chemistry, Indian Institute of Science Education and Research, Bhopal-
62026, Madhya Pradesh, India.
. Kalinga Institute of Industrial Technology, Bhubaneswar-751024, Odisha, India.
5. National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.
6
1
7
. Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-
10029, India.
rd
. Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3 Milestone, Faridabad-
Gurgaon Expressway, Faridabad-121001, Haryana, India.
. CSIR-Institute of Genomics and Integrative Biology, Mathura Road, New Delhi-110025, India.
8
a: authors contributed equally.
Corresponding Authors:
Avinash Bajaj, Email: bajaj@rcb.res.in
1
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 2 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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 Gram-
negative 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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
ACS Paragon Plus Environment

Page 3 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
stimulates acute gastroenteritis. Salmonella has the ability to inhabit the epithelial cells and
2
macrophages to stay and reproduce in the repositories of these cells. Many commonly used
antibiotics like aminoglycosides and glycopeptides cannot get accumulated inside mammalian
3
cells and are therefore, unable to kill the intracellular pathogenic bacteria. The facultative
intracellular nature of microorganisms assists in developing multi-drug resistance (MDR)
4
against antibiotics through sequential accumulation of mutations 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 host-
defense systems.6 Negatively-charged lipopolysaccharide (LPS) at the surface of Gram-
6
negative bacteria provides complex and selective barrier to antimicrobials and its endotoxin
7
nature is responsible for causing severe gut inflammation. 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
9
them a facial amphiphilic character. 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
3
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 4 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
lipopeptides,12 hydrocarbon chain13 or steroidal-derived amphiphiles14 and antibiotic-
amphiphile conjugates15 have been designed mimicking the physicochemical features like
16
facial amphiphilic character of AMPs as effective antimicrobials. 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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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 CA-
derived 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 in-
depth 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.
4
ACS Paragon Plus Environment

Page 5 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
deprotection of Boc residues (Figure 2). We characterized the amphiphiles by H 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.
5
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 6 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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 (MIC ). Structure-activity
99
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 MIC of 4.0 µM. However, further increase in chain
99
6
ACS Paragon Plus Environment

Page 7 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
length diminished the antibacterial activity of amphiphiles (Table 1).21 Our SAR with CA-
derived 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
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
chain lengths. 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
22
concentrations. 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 MIC ) that certified the non-
99
detergent-mediated bactericidal effect of amphiphile (Table 1). We also performed a DLS-
based 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 MIC (4 M) of the compound.
99
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
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 8 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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
IC50
CMC
b
c
d
(µM) (µM) (µM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
S.
S.
E. coli
P.
K.
A.
Flexneri
typhimurium
aeruginosa
pneumoniae
baumannii
1
2
3
4
5
6
7
8
9
128
256
128
32
128
256
128
32
64
256
128
32
256
64
32
16
8
128
256
128
64
16
4
>256
256
128
32
>200
>200
>200
180
83.5
43
82
78
75
20
40
25
62
23
19
90
107
137
385
142
119
31
16
8
16
8
4
4
4
4
4
16
16
4
8
4
4
67
23
32
>256
>256
1
32
16
>256
0.5
8
>256
>256
0.5
68
15
>256
1
>256
2
>256
4
87
10
Polym
yxin B
>200
-e
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
8
ACS Paragon Plus Environment

Page 9 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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 (DiSC (5)) usually gets accumulated into lipid membranes of
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
24
hyper polarized cells and its fluorescence gets quenched. Substantial increase in DiSC (5)
2
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 MIC and only 1h of treatment
99
at 4X MIC was sufficient to clear the bacterial load (Figure 3D).
99
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
26
on compound 6 treatment (Figure 3E). 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).
9
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 10 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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 DiSC (5) over time on compound 6
2
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
1
0
ACS Paragon Plus Environment

Page 11 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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 MIC ) at different time show that the compound 6 is able to disrupt the
99
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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.
1
1
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 12 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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
1
2
ACS Paragon Plus Environment

Page 13 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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
29
normal gastro-intestinal emptying time and tested for antibacterial activity. We observed that
compound 6 showed MIC of 8 M after its incubation in SGF confirming its gastric stability.
99
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
1
3
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 14 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
th
for STM confirmed no bacterial load by 4 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).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Invasion of Salmonella into intestinal epithelium induces acute inflammation triggered by LPS-
31
Toll like receptor (TLR) interactions. 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
5
G, 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).
1
4
ACS Paragon Plus Environment

Page 15 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 16 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Remarkable activity against multi-drug resistant (MDR) clinical strains. Uncontrolled use
of antimicrobials is known to generate drug resistant among bacterial strains due to
33
accumulation of mutations in key targets of these antimicrobials. 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 MIC of
99
compound 6. Both the bacteria were unable to generate drug resistance against compound 6
1
6
ACS Paragon Plus Environment

Page 17 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
till 40 passages, and we observed MIC of 4 M for compound 6 at first and last passage for
99
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
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
resistant STM as well with same efficacy (MIC = 4 M). Similarly, E. coli did not generate
99
any drug resistance against compound 6 as developed by levofloxacin and Polymyxin B
(Figure 6D).
34
To test the efficacy of compound 6 against persistent bacteria, 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 MIC for 2h killed these bacteria where
99
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
12
in MIC of Polymyxin B at ~10 CFU/mL for STM and E. coli. These results clearly confirmed
99
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
1
7
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 18 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
that compound 6 was bactericidal against all the clinical strains including multi-drug resistant
strains of E. coli, P. aeruginosa and K. pneumoniae with MIC of 1-8 M (Table 2). Compound
99
6
was also effective against Ciprofloxacin resistant S. typhi strains with MIC of 1-2 M.
99
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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
5
in MIC of compound 6 at higher CFU of STM (E) and E. coli. (F) over MIC at 10 CFU/mL.
99
99
1
8
ACS Paragon Plus Environment

Page 19 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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.
S. typhi
E. coli
A. baumannii
K. pneumoniae
P. aeruginosa
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
1
6011/10
7375/10
742/09
R
R
R
R
R
R
R
R
R
R
S
S
R
S
S
S
S
S
S
S
S
2
2
2
2
2
2
2
2
1
2
1284
6/78
6/23
9/203
6/33
8/341
6/40
9/44
6/38
6/27
I
R
R
R
R
R
R
R
R
R
R
I
4
4
4
4
4
4
4
8
4
4
4/307
1/303
6/303
3/216
5/405
5/321
4/202
5/412
6/312
9/229
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
4
8
4
4
4
4
4
4
4
4
4/230
8/407
8/307
7/47
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I
8
4
4
4
4
4
8
4
4
8
4/316
6/243
1/231
1/318
3/313
6/264
17/288
7/227
20/261
7/233
S
S
S
R
S
R
R
R
S
S
S
S
S
R
R
R
R
R
S
S
S
S
S
R
S
R
R
R
S
S
8
8
1
S
S
S
S
S
S
S
S
S
R
R
S
R
R
R
R
R
R
R
R
S
R
R
R
R
R
R
7
8
7300/01
13521/2
111608/08
15878/08
4274/15
2353
8-16
8
12823
12744
6/47
4-8
8
7408
4
7399
4
2
6052
7/9272
R
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.
1
9
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 20 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental Section
General: Cholic acid, Boc-Glycine, n-alkyl alcohols, Dicyclohexylcarbodiimide (DCC), Pyrene,
Anhydrous dichloromethane, Chloroform (HPLC grade, ≥99.9%), MTT (3-(4,5-dimethylthiazol-
2
-yl)-2,5-diphenyltetrazolium bromide), N-phenyl-naphthyl amine (NPN), Paraformaldehyde,
DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride), 3',3'- diethylthiadicarbocyanine
DiSC (5), Propidium Iodide (PI), polyclonal rabbit anti-LAMP1 (Cat. No. L1418), Hoechst
2
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
2
0
ACS Paragon Plus Environment

Page 21 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
on aluminium sheet coated with Silica gel 60 GF 254 (Merck, Germany) and stained with
1
ethanolic solution of phosphomolybdic acid. H NMR spectra were recorded in CDCl and
3
DMSO-d using a Bruker Avance 400 MHz spectrometer. Chemical shifts (δ) are reported in
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ppm with tetramethyl silane (TMS) as the internal standard. Mass spectrometry analysis were
performed with an micrOTOF-Q II 10330 system (Bruker).
36
Antibacterial assay. Antibacterial efficacy of the molecules is reported as minimum inhibitory
concentration (MIC ) that is the lowest concentration of antimicrobial agent inhibiting 99%
99
growth of bacteria. Bacterial strains were freshly inoculated in MH broth, incubated for 6h and
5
re-suspended further in broth to achieve 10 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
o
then incubated at 37 C for overnight. MIC data was recorded by measuring the absorption at
99
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
5
12
different CFUs (10 -10 CFU/mL) of bacteria were used to determine the MIC .
99
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
5
at 9000 rpm for 2 min. and washed with PBS. Bacterial suspensions (10 CFU/mL) were then
o
treated with 2X MIC of compound 6 and incubated at 37 C for 2h. Ampicillin (8 M) was taken
99
as a control. Bacterial suspension without any treatment served as negative control. After
2
1
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 22 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
treatment, bacterial suspensions were serially diluted and plated on MH agar plates. Viable
o
colonies were counted after 24h of incubation at 37 C and bacterial CFU were represented as
mean ± SD of three replicates.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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
5
suspension (10 CFU/mL) was then treated with compound 6 (8 M) and Ampicillin (8 M) at
3
7oC for 2h. Treated bacterial suspension was then serially diluted in MH broth and plated on
o
MH agar plates. Viable colonies were counted after 24h of incubation at 37 C and bacteria
CFU were represented as mean ± SD of three replicates.
6
Time kill kinetics was performed by incubating 10 ꢀCFU/mL of mid-log-phase bacteria with
1
X and 4X MIC of amphiphile. Aliquots were taken after 0, 15, 30, 60, 120, 240, and 360
99
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 MIC were further used for antibacterial
99
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
40
41
membrane depolarization assay, and propidium iodide assay, and Transmission Electron
42
Microscopy studies were performed as per reported protocol.
2
2
ACS Paragon Plus Environment

Page 23 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
43
Atomic force microscopy. 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
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0 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.
28
5
In vitro Infection Assay. HeLa or RAW 264.7 cells (2 X 10 ) 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
1
0 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
2
3
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 24 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
5 min at room temperature followed by washing with PBS. Cells were then incubated with
.1% saponin and 1% bovine serum albumin (PBS buffer) for 1h and then stained with
0
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 anti-
rabbit 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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
concentrations of amphiphile solutions were recorded using  of 334 nm. The I and I395
ex
375
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.
45
To test the stability of compound 6 in simulated gastric fluid (SGF), 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
2
4
ACS Paragon Plus Environment

Page 25 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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
28
For infection studies, 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
6
fasted for 4h and then infected intragastrically with STM (~10 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
2
5
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 26 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was performed using 20 μL reaction volume with final primer concentration of 10 nM and c-
DNA (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.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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
46
methods. All the strains were tested for antimicrobial susceptibility to the drugs recommended
47
by CLSI (Clinical Laboratory Standards Institute) for that particular organism. 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
8
containing 1.5 x 10 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
2
6
ACS Paragon Plus Environment

Page 27 of 35
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
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.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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
2
7
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 28 of 35
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Supporting Information Available. The Supporting Information is available free of charge
on the ACS Publications website at www.pubs.acs.org
Figure S1, Synthesis of amphiphiles, tables S1-S2.
References
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1. Als, D., Radhakrishnan, A., Arora, P., Gaffey, M. F., Campisi, S., Velummailum, R., Zareef,
F., Bhutta, Z. (2018) A. Global trends in typhoidal Salmonellosis: a systematic review. Am. J.
Trop. Med. Hyg. 99, 10-19.
2. Coburn, B., Grassl, G. A., Finlay, B. B. (2007) Salmonella, the host and disease: a brief
review. Immunol. Cell Biol. 85, 112-118.
3. Brezden, A., Mohamed, M. F., Nepal, M., Harwood, J. S., Kuriakose, J., Seleem, M. N.,
Chmielewski, J. (2016) Dual targeting of intracellular pathogenic bacteria with a cleavable
conjugate of kanamycin and an antibacterial, cell penetrating peptide. J. Am. Chem. Soc. 138,
10945-10949.
4
. Threlfall, F. J. (2002) Antimicrobial drug resistance in Salmonella: problems and perspectives
in food- and water-borne infection, FEMS Microbiol. Rev. 26, 141-148.
. Groisman, E. A., Parra-Lopez, C., Salcedo, M., Lipps, C. J., Heffron, F. (1992) Resistance to
host antimicrobial peptides is necessary for Salmonella virulence. Proc. Nat. Acad. Sci. U.S.A
5
89, 11939-11943.
6. Nikaido, H. (1994) Prevention of drug access to bacterial targets: permeability barriers and
active efflux. Science 264, 382–388.
. Rosenfeld, Y., Shai, Y. (2006) Lipopolysaccharide (Endotoxin)-host defense antibacterial
7
peptides interactions: Role in bacterial resistance and prevention of sepsis. Bochim. Biophys.
Acta Biomembr. 1758, 1513-1522.
2
8
ACS Paragon Plus Environment

Page 29 of 35
Bioconjugate Chemistry
1
2
3
8
. Hurdle, J. G., O’Neill, A. J., Chopra, I., Lee, R. E. (2011) Targeting bacterial membrane
function: an underexploited mechanism for treating persistent infections. Nat. Rev. Microbiol. 9,
62-75.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9. Mahlapuu, M., Håkansson, J., Ringstad, L., Björn, C. (2016) Antimicrobial peptides: an
emerging category of therapeutic agents. Front Cell Infect. Microbiol. 6, 194.
10. Nguyen, L. T., Haney, E. F., Vogel, H. J. (2011) The expanding scope of antimicrobial
peptide structures and their modes of action. Trends Biotechnol. 29, 464-472.
11. Konai, M. M., Haldar, J., (2017) Fatty acid comprising lysine conjugates: Anti-MRSA agents
that display in vivo efficacy by disrupting biofilms with no resistance development. Bioconjugate
Chem. 28, 1194-1204.
12. Kimura, R., Shibata, M., Koeda, S., Miyagawa, A., Yamamura, H., Mizuno, T. (2018)
Development of new antimicrobial agents from cationic PG-surfactants containing oligo-lys
peptides. Bioconjugate Chem. 29, 4072-4082.
13. Hoque, J., Konai, M. M., Sequeira, S. S., Samaddar, S., Haldar, J. (2016) Antibacterial and
antibiofilm activity of cationic small molecules with spatial positioning of hydrophobicity: an in
vitro and in vivo evaluation. J. Med. Chem. 59, 10750–10762.
1
4. Singla, P., Dalal, P., Kaur, M., Arya, G., Nimesh, S., Singh, R., Salunke, D. B. (2018) J. Med.
Chem. 61, 1026576–10275.
5. Bera, S., Zhanel, G. G., Schweizer, F. (2008) Design, synthesis, and antibacterial activities
1
of neomycin-lipid conjugates: polycationic lipids with potent gram-positive activity. J. Med.
Chem. 51, 6160-6164.
2
9
ACS Paragon Plus Environment