Vancomycin Derivative Inactivates Carbapenem-Resistant Acinetobacter baumannii and Induces Autophagy

Article abstract of DOI:10.1021/acschembio.0c00091

Vancomycin is a standard drug for the treatment of multidrug-resistant Gram-positive bacterial infections. Albeit, development of resistance (VRE, VRSA) and its inefficacy against persistent infections is a demerit. It is also intrinsically inactive against Gram-negative bacteria. Herein, we report a vancomycin derivative, VanQAmC₁₀, that addresses these challenges. VanQAmC₁₀ was rapidly bactericidal against carbapenem-resistant A. baumannii (6 log₁₀ CFU/mL reduction in 6 h), disrupted A. baumannii biofilms, and eradicated their stationary phase cells. In MRSA infected macrophages, the compound reduced the bacterial burden by 1.3 log₁₀ CFU/mL while vancomycin exhibited a static effect. Further investigation indicated that the compound, unlike vancomycin, promoted the intracellular degradative mechanism, autophagy, in mammalian cells, which may have contributed to its intracellular activity. The findings of the work provide new perspectives on the field of glycopeptide antibiotics.

Full text of DOI:10.1021/acschembio.0c00091

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Letter
Vancomycin Derivative Inactivates Carbapenem-resistant
Acinetobacter baumannii and Induces Autophagy
Paramita Sarkar, Sandip Samaddar, Veena Ammanathan, Venkateswarlu Yarlagadda, Chandradhish
Ghosh, MANJULIKA SHUKLA, GRACE KAUL, Ravi Manjithaya, Sidharth Chopra, and Jayanta Haldar
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.0c00091 • Publication Date (Web): 20 Mar 2020
Downloaded from pubs.acs.org on March 21, 2020
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Vancomycin Derivative Inactivates Carbapenem-resistant
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Acinetobacter baumannii and Induces Autophagy
Paramita Sarkar, Sandip Samaddar, Veena Ammanathan, Venkateswarlu Yarlagadda,
a
a
b
a
a
c
c
b
c
Chandradhish Ghosh, Manjulika Shukla, Grace Kaul, Ravi Manjithaya, Sidharth Chopra,
and Jayanta Haldar a*
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aAntimicrobials Research Laboratory, New Chemistry Unit and School of Advanced Materials,
Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur, Bengaluru
560064, Karnataka, India.
b
Autophagy lab, Molecular Biology & Genetics Unit, JNCASR, Bengaluru
c
CSIR-Central Drug Research Institute, Lucknow 226031, India
Corresponding author: Fax: (+91) 80-2208-2627; E-mail: jayanta@jncasr.ac.in
Abstract: Vancomycin is a standard drug for the treatment of multidrug-resistant Gram-positive
bacterial infections. Albeit, development of resistance (VRE, VRSA) and its inefficacy against
persistent infections is a demerit. It is also intrinsically inactive against Gram-negative bacteria.
Herein, we report a vancomycin derivative, VanQAmC10 that addresses these challenges.
VanQAmC10 was rapidly bactericidal against carbapenem-resistant A. baumannii (6 log10
CFU/mL reduction in six hours), disrupted A. baumannii biofilms and eradicated their stationary
phase cells. In MRSA infected macrophages, the compound reduced the bacterial burden by
1
.3 log CFU/mL while vancomycin exhibited a static effect. Further investigation indicated that
10
the compound, unlike vancomycin, promoted the intracellular degradative mechanism,
autophagy, in mammalian cells which contributed to its intracellular activity. The findings of the
work provide new perspectives to the field of glycopeptide antibiotics.
Keywords: Glycopeptide antibiotics • Antimicrobial resistance • Vancomycin • Acinetobacter
baumannii • biofilm • autophagy
Introduction
The threat of antimicrobial resistance (AMR) has been reiterated several times by World
Health Organization (WHO) and other international healthcare organizations. The upsurge in
1
incidences of multidrug resistance and pan-drug resistance in Gram-negative bacteria (MDR-
2
GNB and PDR-GNB) has become a severe health challenge. Additionally, bacterial
pathogens often evade the host immune system and antibiotic treatment, thereby persisting
within the host. These infections usually result from the ability of bacteria to form biofilms or
1, 3
survive within mammalian cells and are difficult to treat.
This necessitates the need for
development of drugs for the treatment of complicated bacterial infections. Semi-synthetic
approaches are an attractive and clinically successful strategy towards achieving this goal.
Although vancomycin has been widely used in the clinic for the treatment of MRSA infections,
resistance has been reported in Enterococci and Staphylococci (vancomycin-resistant
4
, 5
Staphylococcus aureus, VRSA and vancomycin-resistant Enterococci, VRE)
. Further, its
inefficacy against Gram-negative bacteria and intracellular Gram-positive bacteria limits broad
clinical usage.6, 7 Efforts have been directed to this end, which involve semi-synthesis and
8
-16
nanoparticle-based approaches through encapsulation of vancomycin.
reported a cationic lipophilic vancomycin derivative that could eradicate Gram-negative
Previously, we had
bacteria both in-vitro and in-vivo. Although the derivative exhibited good activity, its LD was
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28 mg/kg. This warranted an improvement in molecular design to reduce toxicity while
maintaining similar antibacterial activity. Morover, the multifaceted antibacterial activity of a
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semi-synthetic glycopeptide against Gram-negative biofilms and intracellular bacteria remains
underexplored 4, 18. In this repect, we report, the design of a non-toxic vancomycin derivative,
VanQAmC10 (Scheme 1) and its efficacy against acquired resistance in Gram-positive
bacteria and intrinsic resistance in Gram-negative bacteria to vancomycin. Further, the effect
of the compound on biofilms of A. baumannii and the mamallian cellular degradative pathway,
autophagy was investigated.
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Results and Discussion
Design and synthesis. In various biophysical studies and molecular dynamic simulations
against various polymeric and small molecular systems, our group reported that inclusion of
an amide bond between the cationic moiety and the hydrophobic moiety imparts additional
hydrogen bonding capability to the bacterial lipids.
towards bacterial cells over mammalian cells. Therefore, the lipophilic moiety conjugated to
vancomycin here contained an amide group between the cationic centre and lipophilic moiety.
A simple three step synthetic scheme was followed to obtain VanQAmC (Scheme 1). In the
first step, decylamine was conjugated to bromoacetyl bromide to obtain the brominated amide
(1a). Nucleophilic substitution of the bromide in 1a with Boc-protected N,N-dimethyl
propandiamine yielded 1b, which was deprotected to obtain the amphiphilic precursor 1c with
a primary amine group. Then the compound, 1c was reacted with the carboxylic acid group
of vancomycin using N,N,N ′,N ′-tetramethyl-O-(1H-benzotriazol-1-yl)uraniumhexafluoro-
phosphate as a coupling agent to obtain the lipophlic cationic vancomycin derivative,
19, 20
This enhances the selective toxicity
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VanQAmC . The final product was purified through reverse-phase HPLC with around 70%
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yield and was characterized using HPLC, HNMR, CNMR and HR-MS.
Scheme 1. Synthesis of semi-synthetic vancomycin derivative
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In-vitro antibacterial activity. VanQAmC10 exhibited activity comparable to that of
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Table 1. Antibacterial activity of VanQAmC10 against clinical isolates of Gram-negative WHO
priority pathogen A. baumannii
MIC (μM)
PMB
Mero
Levo
Tige
Ceft
VanQAmC10
A. baumannii
NR-13374
NR-13375
NR-13376
NR-13377
NR-13378
NR-13379
NR-13380
NR-13381
NR-13382
NR-13383
NR-13384
NR-13385
NR-17777
NR-17786
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
83.4
>167
167
88.6
177
177
177
22
6.8
6.8
3.4
6.8
6.8
3.4
6.8
6.8
3.4
3.4
6.8
6.8
1.7
3.4
>85.9
>85.9
>85.9
>85.9
>85.9
>85.9
>85.9
>85.9
>85.9
>85.9
>85.9
>85.9
21.5
7.7
7.7
3.9
3.9
3.9
3.9
7.7
7.7
7.7
3.9
7.7
7.7
15.5
3.9
167
>167
>167
10.4
>167
>167
83.4
>167
>167
2.6
11
22
1
22
11
177
88.5
22
10.4
1.3
>85.9
NR-19299
BAA-1605
<0.4
<0.4
167
167
44.3
22
3.4
6.8
>85.9
>85.9
3.9
7.7
ATCC 17978
MTCC 1425
ABR 674
<0.4
ND
<1.3
ND
22
ND
ND
<0.8
ND
21.5
ND
7.7
6.25
10
ND
>64
ND
ND
Levo, levofloxacin; Mero, meropenem; Tige, tigecycline, PMB, Polymixin B, Ceft, ceftaroline;
N.D. Not determined
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Figure 1. Antibacterial activity and mechanism of action of VanQAmC10 (A) MIC against Gram-positive bacteria (B)Time-
kill kinetics against A. baumannii R674; Inhibition of cell wall biosynthesis in A. baumannii upon treatment with test
compounds investigated through (C) Accumulation of UDP-MurNAc-L-Lys-γ-Glu-m-DAP-D-Ala-D-Ala observed by UV
spectroscopy at 260 nm; (D) Mass spectrum of UDP-MurNAc-PP (structure inset) identified at m/z 1194.334 (E) SEM
micrographs of A. baumannii treated with VanQAmC10 against exponentially growing bacteria (Scale bar, 1 μm). ‘*‘ indicates
<
50 cfu/mL
vancomycin against vancomycin-sensitive bacteria (MIC 0.4 μM against MRSA, 0.5 μM
against E. faecium). Against vancomycin-resistant bacteria, a 100-330 fold enhancement in
activity as compared to vancomycin was observed. It showed a minimum inhibitory
concentration (MIC) of 0.1 μM and 0.2 μM against VISA and VRSA repectively; 2.2 μM and 4
μM against VRE VanA and VanB phenotypes respectively. Vancomycin showed a MIC of 250
μM (VRE VanA) and 750 μΜ (VRE VanB) (Figure 1A). The activity of the compound was also
tested against various Gram-negative pathogens such as E. coli, K. pneumoniae, P.
aeruginosa and A. baumannii (Table 1 and Table S1). While the compound showed some
activity against E. coli and P. aeruginosa, the highlight of the work was its potency against A.
baumannii (Table 1). A. baumannii is notorious due to its unprecedented ability to acquire
resistance to most antibiotics which led to the World Health Organisation categorising it as the
number one critical priority pathogen for drug-discovery. Given the challenge presented by
infections caused by this pathogen, this finding is promising towards extending the use of
vancomycin against this pathogen.21 VanQAmC10 showed an MIC in the range 3.9-15.5 μM
against various multidrug-resistant clinical isolates of A. baumannii. The bacteria tested were
resistant to the currently used antibiotics, levofloxacin, meropenem, ceftaroline and only
susceptible to polymyxin B and tigecycline. Its bactericidal activity was rapid; 4 log CFU/mL
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reduction was observed within 2 h at around 3 x MIC (28 μM) , while complete killing (6 log10
CFU/mL reduction) was observed in 6 h (Figure 1B). At 7 μM (just below MIC), the compound
initially showed a 2.5 log CFU/mL reduction at 4 h and then subsequent growth upto 24 h.
1
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The incorporation of the lipophilic cationic moiety permitted interaction with the negatively
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charged bacterial membrane and resulted in depolarization and permeabilization of both the
outer and inner membranes of the bacteria (Figure S1). The outer membrane permeabilization
caused by the lipophilic cationic vancomycin derivative permit the entry of the compound to
the cell wall region and thereby inhibit cell wall biosynthesis (Figure 1C and 1D). The
membrane disruptive mode of action was further supported by the SEM images of VanQAmC10
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Figure 2. Activity of VanQAmC10 against biofilms and stationary phase of A. baumannii (A) Confocal laser scanning images
of biofilms of multi-drug resistant strain when mature biofilms were left untreated, treated with vancomycin, VanQAmC10
,
colistin at a concentration of 50 μM each (scale bar, 5 μm). (B) Viability of bacterial cells in biofilms post-treatment with
test compounds (C) Viability of stationary phase cells 2 h post-treatment with test compound.‘*‘ indicates <50 cfu/mL
treated cells where cell debris and lysis is visible (Figure 1E).
Disruption of biofilms of carbapanem-resistant A. baumannii. A. baumannii is notorious
for biofilm related infections.22 Most of the conventional antibiotics are rendered ineffective
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due to the permeability barrier created by the extracellular polymeric matrix (EPS). Therefore,
the activity of VanQAmC against A. baumannii biofilms was of immense interest. Confocal
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laser scanning microscopy showed that VanQAmC treated biofilms were disrupted and their
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thickness reduced to 2.2 μm at 50 μM while untreated and vancomycin treated biofilms were
1 μm and 8.8 μm thick (Figure 2A). This reduction was comparable to that of colistin taken
1
at the same concentration. The conjugation of the cationic lipophilic moiety onto vancomycin
facilitated interaction with the negatively charged components of the EPS resulting in
disintegration of the biofilms. To further investigate biofilm eradication, the viability of bacteria
within these disrupted biofilms upon treatment at various concentrations was determined.
VanQAmC reduced the number of viable bacteria in a concentration dependent manner. At
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0 μM, it reduced cell viability by 1.8 log CFU/mL. At higher concentrations of 25 μM and 50
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μM, 2.8 log CFU/mL and 3.8 log CFU/mL reduction was obtained respectively. Colistin on
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the other hand could completely eliminate the viable cells (6 log10 CFU/mL reduction).
Tobramycin was found to be ineffective while vancomycin caused a 1 log CFU/mL reduction
1
0
of viability of bacteria within the biofilm at 50 μM (Figure 2B).
Eradication of stationary phase cells of A. baumannii. Biofilms consist of bacteria in
various metabolic phases; the reduction in viable cells in biofilms indicated activity against
stationary phase bacteria as well. While vancomycin did not show any significant reduction in
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viability (0.6 log CFU/mL), VanQAmC reduced cell viability by 3 log CFU/mL and 5.5 log
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CFU/mL at 5 μM and 10 μM respectively within 2 h (Figure 2C). Since the integrity of the
membrane is crucial for bacterial survival irrespective of its metabolic state, the membrane-
disruptive nature of the compound resulted in this rapid bactericidal activity (Figure S2). The
currently used antibiotic, tobramycin showed a lower activity with a 2.7 log CFU/mL reduction,
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while colistin completely killed the stationary-phase bacteria (Figure 2C). However, the
advantage of VanQAmC10 offered over colistin was that it did not induce any observable
resistance in A. baumannii (Figure S3). Upto 19 passages, the compound showed no increase
in MIC, while colistin induced resistance from the sixth passage itself. The membrane-
disrupting, non-specific targeting by the vancomycin derivative may have contributed to this
lack of propensity to induce resistance.
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Figure 3. (A) Intracellular killing kinetics of VanQAmC10 against MRSA (B) Schematic of the tandem mRFP-GFP-LC3 assay
(C) Representative microscopic images of merged GRP and RFP channels of RFP–GFP–LC3 expressing HeLa cells treated
with indicated compounds at 100 μM for 2 hours (scale bar 10 μm); (D) Comparison of number of puncta per cell of
autolysosomes (red) and autophagosomes (black) in treated and untreated cells; (E) Intracellular activity of compounds
against intracellular pathogen Salmonella typhimurium.
Toxicity. The in vitro and in vivo toxicity of the compound was then tested. The HC of the
50
compound was found to be greater than 1000 μM as opposed to 100 μM for a vancomycin
derivative of comparable activity lacking an amide between the cationic centre and lipophilic
17
moiety (Figure S4). The LD in mice when administered intravenously was found to be 70
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mg/kg as opposed to 28 mg/kg for the derivative lacking the amide spacer. The H-bonding
ability of the amide moiety has been reported to result in better interaction with the polar head
groups of the bacterial membrane than with the mammalian cell membrane. This increased
interaction due to H-bonding ability with the bacterial membrane makes up for the lower chain
length of the hydrophobic moiety which results in reduced interaction with the lipid membranes.
Both the factors together probably contributed to the enhancement in the selectivity of the
compound towards bacteria.
Intracellular activity and autophagy. Intracellular survival of S. aureus is a critical contributor
2
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for persistence and vancomycin is known to be intracellularly ineffective. On treatment with
VanQAmC , mouse macrophages infected with MRSA resulted in a lower intracellular
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bacterial burden. The untreated intracellular bacterial burden increased by 0.5 log CFU/mL
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and 1.6 log CFU/mL in 6 h and 12 h (Figure 3A). Upon treatment with vancomycin at 60 μM,
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around 0.5 log CFU/mL reduction in the intracellular bacterial burden was observed upto
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12 h. VanQAmC (at 40 μM) exhibited better intracellular activity than the parent drug. 1 log
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CFU/mL and 1.3 log10 CFU/mL reduction in bacterial titre was observed in 6 h and 12 h
respectively. The morphology of the RAW 264.7 cells post incubation with VanQAmC10
remained unaffected (Figure S5). Compounds that induce autophagy have been reported to
reduce intracellular survival of pathogens.24 The 1.3 log reduction of intracellular pathogen
prompted us to explore if VanQAmC10 exhibits such a property. Autophagy is a cellular
degradative pathway that is crucial for defense against pathogen invasions. The process
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involves the fusion of the lysosome with the phagosome. An upregulation in the process is
associated with the clearance of intracellular pathogens. A tandem mRFP-GFP-LC3 assay
was performed to study the effect of VanQAmC on autophagy (Figure 3B). The induction of
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autophagy was monitored by quantitating the autophagic vesicles by tagging the LC3 protein,
which is a biomarker for autophagosomes and autolysosomes. HeLa cells were transfected with
a tandem red fluorescence protein-green fluorescence protein-LC3 expressing plasmid (mRFP-
GFP-LC3). GFP signal is sensitive to the acidic and proteolytic conditions of the lysosome lumen
26
and hence, its fluorescence is quenched, while that of RFP is stable. The combination of an
acid-sensitive GFP with an acid-insensitive RFP allows the visualization of change from
autophagosomes (neutral pH) to autolysosomes (with an acidic pH). Upon induction of
autophagy, the GFP-RFP labelled LC3 proteins are employed in the formation of
autophagosomes. These are positive for both GFP and RFP and hence, appear yellow. Fusion
with the lysosome results in a drop of pH leading to quenching of GFP fluorescence, making the
autophagolysosomes appear red.27 Both vancomycin and VanQAmC10 showed a higher
number of autophagosomes as compared to the untreated cells (Figure 3C and 3D). However,
interestingly, VanQAmC10 promoted the fusion of autophagosomes with lysosomes. A two-
fold increase in the number of red puncta as compared to vancomycin-treated and untreated
cases was observed. Vancomycin on the other hand showed no increase which is in
accordance with a literature report that vancomycin blocks autophagy. S. aureus is known to
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prevent phagosomal maturation during infection in order to create a niche for replication.
The upregulation of autophagy may have therefore contributed to the reduction of intracellular
29
bacteria. . This was further supported by the two-fold reduction of intracellular Salmonella
typhimurium exhibited by VanQAmC , although it was inactive against the exponentially
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growing bacteria (MIC >200 μM, Figure 3E). Vancomycin, as expected did not show any
significant reduction. This finding indicates the potential for exploring the role of host immune
system as a combinatorial mechanism with antibiotic effect to enhance the efficacy in battling
intracellular pathogens.
Conclusion
With the severity of AMR on the rise, strategies to fight them are necessary. Bacterial
infections are further complicated by the inherent ability of bacteria to form biofilms and reside
within macrophages which protect them from the antimicrobial action of the host immune
system and external agents. This is the first report of a vancomycin derivative effective against
multiple aspects of bacterial infections. VanQAmC could eradicate biofilms and associated
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cells of carbapenem resistant A. baumannii and does not induce resistance. Another important
finding is the ability of the compound to promote autophagy, which is a contributing factor to
its ability to clear intracellular pathogens. This multi-faceted antibacterial activity of the
compound offer a new perspective to the field of new glycopeptide antibiotics.
Experimental Section
Synthetic procedures, characterization data and experimental details are included in the
supporting information.
Supporting Information
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Supporting informatiuon contains data on evaluation of mechanism of action against
exponential phase bacteria, graph of percentage hemolysis evaluation, resistance study
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against A. baumannii, representative images of cells treated with VanQAmC . Synthetic
procedures, characterization, protocols for microbiological assays are also included.
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We thank C. N. R. Rao, F. R. S. (JNCASR) for his constant support. We gratefully
acknowledge BIRAC SRISTI PMU 2016/002 and JNCASR for the funding. J.H. acknowledges
Sheikh Saqr Career Award Fellowship. S. S. is grateful to BIRAC-GYTI for post-doctoral
fellowship. We thank R. G. Prakash (In-house animal facility) for in-vivo study. We
acknowledge the NMR facilities at NCBS, India, and Technology Platform Services, C-CAMP,
India.
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A vancomycin derivative that disrupts biofilms of the critical priority pathogen Acinetobacter baumannii and
kills intracellular MRSA. The three distinct mechanisms of action exerted by the compound against bacteria
such as inhibition of cell wall biosynthesis, infiltration of membrane and induction of autophagy
demonstrates its potential as a new antibacterial agent.
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