5.5.2.

Specific Aims

Antimicrobial agents indispensably underpin basic and modern medicine. However, the

reduced supply of new antimicrobials coupled with the rapid growth of antimicrobial resistance

(AMR) are serious threats to global health. In the US, each year antibiotic-resistant bacteria and

fungi cause at least and estimated 2,868,700 infections with 35,900 deaths. It is projected that by

2050, 10 million lives annually and a cumulative 100 trillion USD of economic output are jeopardized

by the rise of drug-resistant infection if we could not find a proactive solution now to slow it down.

Therefore, antibiotics development equipped with unexploited mechanism and slow-rate of

antibiotics-resistant development/robust to resistance development is utterly necessary (O’Neill,

2014; CDC, 2019).

Polymyxin E/Colistin has become a last resort antibiotic because of scarcity of novel antibiotics

for treating multidrug resistant bacteria. Colistin is used as a last-line therapy against carbapenem-

resistant Enterobacteriaceae (CRE) to date. Binding of colistin and the lipid A component of

lipopolysaccharide (LPS) cause disruption of the bacterial membrane. However, the clinical efficacy

of colistin is interfered by the emergence of plasmid-encoded (mcr-1) MCR-1 enzyme, a putative 60-

kDa phosphoethanolamine (pEtN) transferase that can transmit colistin resistance trait (Son et al.,

2019; Stojanoski et al., 2016). In order to preserve clinical potency of colistin, including improvement

of its activity and slow down the rate of colistin resistance, colistin-based combination therapy

provides a potential therapeutic option to treat infection caused by the multidrug-resistant bacteria.

Some in vitro and in vivo studies are done to explore the potency of colistin-based combination

(rifampicin, rifabutin, clarithromycin, minocycline, novobiocin, tigecycline) therapy as a potential last

resort therapeutic option (MacNair et al., 2018; Zhou et al., 2020). However, the accurate details

regarding to the mechanism of colistin-based combination therapy against mcr-1-expressing

pathogens/the ability of colistin to potentiate antibiotics against colistin resistant bacteria is still

missing. In order to address this issue and filling the gap of the information related to the modulation

of membrane-binding antibiotics, this proposed study is focused to resolve structural information of

the binding of colistin in combination with other antibiotics (clarithromycin and tigecycline) and

modified lipid A due to the expression of MCR-1 in its native cellular condition using solid-state NMR

technique(ssNMR). In principle, ssNMR enables direct structural studies in cell membrane. However,

poor spectral sensitivity remains a challenge. Integration of high-field dynamic nuclear polarization

(DNP) in generating a state-o-the-art ssNMR can overcome this concern. Specifically, I propose:

1. To test a hypothesis that the colistin resistance mechanism expands beyond reducing the

strength of the electrostatic interaction between colistin and lipid A in the outer membrane

2. To test a hypothesis that fortification of LPS packing provided by mcr-1 could play an important

role in decreasing the uptake and lytic activity of colistin.

3. To assess the binding pattern of colistin, clarithromycin, and tigecycline in modified-lipid A and

the accurate correlation with the synergistic mechanism of this colistin-based combination

therapy in molecular level.

Title

Advanced Solid-State NMR (ssNMR) to Study Binding Mechanism of Colistin-Based Combination

Therapy (Clarithromycin, Tigecycline) in Modified-Lipid A due to The Expression of MCR-1.

Introduction

Colistin/polymyxin E is a last resort antibiotic against multidrug-resistant Gram-negative

bacteria that is classified as polymyxin antibiotics (Grégoire, Aranzana-Climent, Magréault,

Marchand, & Couet, 2017). Even though colistin was neglected in 1970s primarily because of its

nephrotoxicity, clinical use of colistin in the last few years was re-emerged because of the increase

of multidrug resistance (MDR) in Gram-negative bacteria (such as Pseudomonas aeruginosa,

Acinetobacter baumannii, Klebsiella pneumoniae, and carbapenemase-producing

Enterobacteriaceae) and the slow progress of novel antibiotics development (Grégoire et al., 2017).

However, the spread of a novel mobile colistin resistance gene (mcr-1) poses a serious threat for the

sustainability of colistin as a last-line treatment for infections caused by multidrug-resistant Gram-

negative bacteria (Son, Huang, Squire, & Leung, 2019). In order to preserve clinical potency of

colistin, including improvement of its activity and slow down the rate of colistin resistance, colistin-

based combination therapy provides a potential therapeutic option to treat infection caused by the

multidrug-resistant bacteria (MacNair et al., 2018).

Some in vitro and in vivo studies are done to explore the potency of colistin-based combination

(rifampicin, rifabutin, clarithromycin, minocycline, novobiocin, tigecycline) therapy as a potential last

resort therapeutic option. Nevertheless, the accurate details regarding to the mechanism of colistin-

based combination therapy against mcr-1-expressing pathogens/the ability of colistin to potentiate

antibiotics against colistin resistant bacteria is still missing. In order to address this issue and filling

the gap of the information related to the modulation of membrane-binding antibiotics, this proposed

study is focused to resolve structural information of the binding of colistin in combination with other
antibiotics (clarithromycin and tigecycline) and modified lipid A due to the expression of MCR-1 in its

native cellular condition using solid-state NMR technique (ssNMR).

5.5.3 Research Strategy

(a) Significance

Colistin (Polymixin E)

A narrow-spectrum antibiotic, polymyxin E, also known as colistin, is a last resort antibiotic

against multidrug-resistant Gram-negative bacteria that is classified as polymyxin antibiotics; the first

isolation of colistin was conducted by Koyama et al. in 1949 from the broth of Bacillus polymyxain.

Colistin is a linear trilipopeptide linked to a cyclic heptapeptide that is produced by nonribosomal

peptide synthetase systems in Gram-positive bacteria (Figure 1) (Grégoire et al., 2017; Vater, 1996).

During the 1950s, colistin was used in clinical setting in Japan and Europe; in the United

States colistin was used in the form of colistimethate sodium in 1959. Even though colistin was

neglected in 1970s primarily because of its nephrotoxicity, clinical use of colistin in the last few years

was re-emerged because of the increase of multidrug resistance (MDR) in Gram-negative bacteria,

such as Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and

carbapenemase-producing Enterobacteriaceae (Grégoire et al., 2017).

Colistin is a decapeptide compound containing a complex mixture of about 30 different

compounds with two primary components, colistin A and colistin B. Molecular weight of large

molecules, colistin A and colistin B, are 1169 and 1155 g/mol, respectively. They consist of a

hydrophilic cycloheptapeptide ring with three positively charged amine groups, a tail tripeptide moiety

with two positively charged amine groups, and a hydrophobic acyl chain tail (one carbon shorter for

colistin B than for colistin A). As a hydrophilic drug (log P = -2.4), colistin possess an amphipathic

feature because of the presence of both lipophilic and hydrophilic groups. The hydrophobicity of

colistin is corresponded to the fatty acyl moiety. Whereas, its hydrophilicity is due to the presence of

the five ւ-α,γ-diaminobutyric acid (ւ-Dab) residues. The presence of unmasked γ-amino groups of the
five ւ-α,γ-diaminobutyric acid (ւ-Dab) residues in the cyclopeptide ring and tripeptide moiety cause

colistin to display basic properties (acid dissociation constant [pKa] of about 10). Hence, at pH 7.4

colistin is a polycationic compound. In positions 1, 3, 5, 8, and 9, the ւ-Dab molecules have positive

charges. These amino groups are crucial for the electrostatically interaction between colistin and lipid

A portion of lipopolysaccharide (LPS) molecules of Gram-negative bacteria (GNB) which is a key role

in the bactericidal activity of colistin (Bergen et al., 2012; Grégoire et al., 2017; Li, Nation, Milne,

Turnidge, & Coulthard, 2005; M. Rhouma et al., 2015; Mohamed Rhouma, Beaudry, Thériault, &

Letellier, 2016; Shah et al., 2014; T. Velkov, Roberts, Nation, Thompson, & Li, 2013) .

Colistin is delivered either as a prodrug, colistin sulfate when used orally, or as colistin

methanesulfonate (CMS) when used intravenously. While, colistimethate is parenteral and

nebulisation formulations for colistin containing the sodium salt of colistin methanesulfonate (CMS).

Colistin passage through the membrane is restricted by its large molecular weight and its cationic

properties at physiological pH; the primary site of colistin distribution is within the extracellular space

(Grégoire et al., 2017).

LPS, component of the outer membrane (OM) of GNB is the therapeutic target of colistin.

Initially, colistin binds to lipid A — responsible for the modulation of bacterial permeability— of the

LPS through electrostatic interaction between positively charged ւ-Dab residues of colistin and the

negatively charged phosphate groups of lipid A. Higher affinities of colistin towards LPS (at least

three times higher) compare to those divalent cations result in three-dimensional (3D) structure

alteration on LPS due to the competitive displacement of divalent cations calcium (Ca 2+) and

magnesium (Mg2+) that act as LPS stabilizer. Insertion of hydrophobic terminal fatty acyl chain or the

D-Leu6-L-Leu7 segment into OM cause permeabilization of the OM by the generation of destabilized

areas through which colistin will transit the OM via a self-promoted uptake mechanism. Through

membrane thinning by straddling the interface of hydrophilic head groups and fatty acyl chains,

colistin disrupts the physical integrity of the phospholipid bilayer of the inner membrane (IM).
Eventually, this cause IM lysis, leakage of intracellular content, and cell death (Powers & Hancock,

2003; Mohamed Rhouma et al., 2016; Tony Velkov, Thompson, Nation, & Li, 2010).

Lipid A portion of the LPS is the constituent of the endotoxin of GNB that can be shed by

bacteria during antimicrobial therapy and can be responsible for endotoxic shock. Colistin exhibits an

anti-endotoxin activity because it binds to the lipid A component of LPS. Through this ability, colistin

can prevent endotoxin’s ability to induce shock through the release of cytokines such as tumor

necrosis factor-alpha (TNF-α) and IL-8. Moreover, colistin performs several other mechanisms such

as an inhibition of vital respiratory enzymes (nicotinamide adenine dinucleotide [NADH]-quinone

oxidoreductase) in the bacterial inner membrane, vesicle-vesicle contact pathway, and a hydroxyl

radical death pathway (Deris et al., 2014; Falagas & Kasiakou, 2005; Mohamed Rhouma et al., 2016;

Sentürk, 2005).

Lipid A as a Target of Colistin

LPS, especially lipid A is a therapeutic target of colistin. LPS (Figure 2) contains three

genetically, biologically, and chemically different domains: the more or less acylated and

phosphorylated lipid A being anchored in the bacterial outer membrane, the core oligosaccharide

linked by 3-deoxy-D-manno-oct-ulosonic acid (Kdo) with lipid A, and the so-called O-antigen or O-

specific polysaccharide, with the latter two pointing to the aqueous environment. LPS containing all

three regions are known as smooth (S)-form LPS, while LPS in the absence of the O-antigen are

called rough (R)-form LPS or lipoligosaccharide (LOS). Generally, the lipid A — important for host

receptor recognition— structure is highly conserved, (Alexander & Rietschel, 2001; Steimle,

Autenrieth, & Frick, 2016).

Lipid A (Figure 3) consist of a β(1→6)-linked glucosamine disaccharide backbone which is

mostly phosphorylated at position 1 and 4’ of the saccharides and acylated at positions 2 and 3 of

each monosaccharide portion. Lipid A is anchored in the outer leaflet of the bacterial outer membrane

through electrostatic and mainly hydrophobic interactions. The diglucosamine part of the lipid A is

facing the exterior environment while the lipid A acyl chains point to the hydrophilic interior of the
membrane. Lipid A of E. coli is considered as a representative of all lipid A structures. Its two

glucosamine residues are each substituted by a phosphate residue at positions 1 and 4’.

Esterification with hydroxymiristate (a saturated acyl chain with 14 carbon atoms, as primary acyl

chain) is occurred at the hydroxyl groups at positions 2 and 3 of each monosaccharide. Two

additional secondary acyl chains, laurate (C 12) and myristate (C14), are bound to hydroxymyristates at

positions 2’ and 3’ (Raetz & Whitfield, 2002; Steimle et al., 2016).

Nevertheless, huge variation of lipid A structure are found among the different bacterial

species. Sometimes, within a single species, more than one different lipid A structure can be

observed. In particular, structural differences corelate to the number, position, and length of the

esterified acyl chains, the presence of charged groups on the polar heads as well as the number of

phosphate groups coupled to the disaccharide backbone. Compared to E. coli lipid A, other bacteria

can render distinctively acylated lipid A which is supposed to be mediated by other enzymes than the

ones in E. coli. Among the lipid A of different bacterial species, the number of acyl chains esterified

with the disaccharide backbone is observed to be varied. Moreover, among different bacterial

species, the phosphorylation pattern is also not conserved. Alteration of the net charge of bacterial

surface are enabled by a more or less phosphorylated (negatively charged phosphate groups) lipid A.

Environmental factor such as temperature, pH, important for immune evasion, the presence of

antibiotics and antimicrobial peptides (AMPs). The presence of divalent cations in the surrounding

environment allows the phosphate groups to mediate interactions with neighboring phosphorylated

lipid A structures. These interactions enhance the stability of the bacterial outer membrane and

decrease the permeability of the cell wall (Molinaro et al., 2015; Raetz, Reynolds, Trent, & Bishop,

2007; Raetz & Whitfield, 2002; Steimle et al., 2016).

Amphipathic nature of lipid A contributes to its propensity to aggregate and limited solubility in

organic solvents and poor solubility in aqueous media (Raetz & Whitfield, 2002). At the anomeric

phosphate under acidic conditions, purified lipid A is unstable in solution being susceptible to

hydrolysis. Methylation, dephosphorylation or diacylation are chemical modification that presumably

enhance stability of lipid A (Ribeiro, Zhou, & Raetz, 1999). Regarding to lipid A NMR analysis, there

were some solvents that are used previously such as deuterated dimethyl sulfoxide (d 6DMSO),

deuterated chloroform (CDCl3) or 2:3:1 (v/v) deuterated chloroform-deuterated methanol-deuterium

oxide (CDCl3-CD3OD-D2O). Furthermore, it has been recommended that addition of deuterated

ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) to a sample enhance

signal resolution significantly. Sharper signals and improve resolution can also be achieved by

increasing sample temperature to 60-80℃ (Schweda & Richards, 2003).

Colistin Resistance in Gram-negative Bacteria

Resistance of Gram-negative bacteria towards colistin can be occurred through intrinsic

resistance, acquired resistance, and transferable resistance. Intrinsic resistance is triggered naturally

by the functional expression of certain chromosomal genes. Mutations in two-component regulatory

systems involved in the lipopolysaccharide synthesis pathway (such as PhoP/PhoQ in Klebsiella

pneumoniae and Pseudomonas aeruginosa and PmrA/PmrB in Salmonella enterica, Acinetobacter

baumannii, and Pseudomonas aeruginosa). Plasmid-borne genes mediated (such as mcr-1, mcr-2,

mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, mcr-8, and mcr-9) transferable resistance to colistin in Gram-

negative pathogens (Carroll et al., 2019; Xu, Wei, et al., 2018).

Use of colistin in veterinary medicine, primarily intended for the prevention and treatment of

Enterobacteriaceae infections has been lasted for decades. Estimation of the global demand for

colistin in agriculture is about 11942 tons per year by the 2015, increasing to 16500 tones by the year

2021, at an average annual growth rate of 4.75%. Colistin use for veterinary medicine and as a feed

additive was re-evaluated due to the discovery of the mobilized colistin-resistance gene (mcr-1) —

gene encoding for a phosphoethanolamine transferase — in Southern China, in 2015. Therefore, in

2017, China strictly banned the use of colistin as a growth promoter for livestock production.

Epidemiological studies showed that mcr-1 is harbored in no less than ten diversified species of the

Enterobacteriaceae such as Escherichia coli, Klebsiella pneumoniae, Salmonella, Enterobacter

aerogenes, Enterobacter cloacae, Cronobacter sakazakii, Shigella sonnei, Kluyvera spp., Citrobacter
spp., and Raoultella ornithinolytica. In the absence of selection process, mcr-1 plasmids will be

maintained in Enterobacteriaceae population and transferred to the human population (Liu et al.,

2016; Sun, Zhang, Liu, & Feng, 2018).

The Current Situation of mcr-1-positive Enterobacteriaceae in Humans

Prevalence study regarding to the carriage of mcr-1-positive Enterobacteriaceae was first

observed by Liu and coworkers in China; it found that of 1% inpatients (16/1322) were carriage of

mcr-1-positive Enterobacteriaceae (MCRPE). Later, sporadic human cases with mcr-1-positive has

been recorded in over 40 countries across five continents (Liu et al., 2016; Sun et al., 2018).

A retrospective cross-sectional assessment of prevalence of MCRPE conducted by Wang and

coworkers in 2017 discovered that from 17498 isolates associated with infection, mcr-1 was detected

in 76 (1%) of 5332 E. coli isolates, 13 (<1%) of 348 Klebsiella pneumoniae, one (<1%) of 890

Enterobacter cloacae and one (1%) of 162 Enterobacter aerogenes. Most mcr-1-positive E.

coli (MCRPEC) found in this study were commonly not susceptible to ciprofloxacin, cefotaxime, and

cefepime but carbapenem. This result suggested that MCRPEC could recruit other resistant genes

and become multidrug resistant pathogen (Wang et al., 2017). In 2016, Vasquez and coworkers

reported the fourth isolate in the US containing the mcr-1 gene that is potentially travel-associated

mcr-1 acquisition (Vasquez et al., 2016). The first case of mcr-1 in E.coli (ST117 lineage) in The US

hospital setting was reported by Macesic and coworkers (Macesic et al., 2017). Farzana and

coworkers reported the first case of a mcr-1 positive E. coli in normal flora from a patient admitted in

Dhaka Medical College Hospital. (Refath et al., 2019)

Velasco and coworkers reported the first isolates of E. coli harbored a plasmid-borne colistin

resistance gene (mcr-1) from 123 drug-resistant isolates collected from January-June 2018 at a

tertiary hospital in Manila, Philippines. Two E. coli clinical isolates were found to be mcr-1 positive

where one isolate carried 12 antibiotic resistance genes in addition to mcr-1, including the ESBL

blaCTX-M-55 and the other E. coli isolate carried 6 antibiotic resistance genes in addition to mcr-1
(Velasco et al., 2020). Prevalence study in Vietnam showed that 36.8% of the isolates (57 colistin-

resistant isolates were obtained from 98 residents) harbored chromosomal mcr-1. Moreover, 63.2%

and 1.8% of the isolates carried mcr-1 on the plasmid and the plasmid/chromosome, respectively

(Yamaguchi et al., 2020). A retrospective screening and analysis to study mcr-1 and blaNDM

distribution and the antibiotics resistance in regard to polymyxins (polymyxin B and colistin) and

carbapenems of positive strains of 12858 Gram-negative bacteria isolated from wildlife, patients,

livestock, poultry and environment in 14 provinces of China from 2010 to 2019 conducted by Fan and

coworkers. It found that a total of 70 strains of 10 species carried the mcr-1 gene with the positive

rates of patients, livestock and poultry, and environmental strains were 0.62% (36/5828), 4.07%

(29/712), 5.43% (5/92), respectively. All mcr-1 carriers were not resistant to carbapenems, among

which, 66 strains were resistant and 4 were sensitive to polymyxins. While, the strains harbored

blaNDM gene had different level of resistance to carbapenems and were sensitive to polymyxins. This

study indicated that the emergence and rise in polymyxins and carbapenems-resistant bacteria was

primarily associated with the selective pressure of antibiotics. Therefore, use of polymyxins and

carbapenems for disease prevention and control in the clinical setting, poultry, livestock, and

environment should be strictly regulated to prevent further intra- and interspecies dissemination of

resistant genes and to manage the spread of a broad-spectrum drug resistant or multidrug resistant

bacteria (Fan et al., 2020). A study conducted by Huang and coworkers found that specific E. coli

harboring mcr-1 and blaNDM (MCR-CREC) were rapidly selected as a result of increased use of colistin

in clinical settings. In particular, the increase of the minimum inhibitory concentration (MIC) of MCR-

CREC to colistin from <2mg/L in 80% strains to 2mg/L in 100% strains were observed (Huang et al.,

2020).

MCR-1

The protein encoded by mcr-1 is a putative 60.1-kDa (541 amino acid) cytoplasmic

transmembrane protein known as MCR-1, phosphoethanolamine (pEtN) transferase which is

observed in Gram-negative bacteria. The proposed MCR-1 catalytic mechanism is that in the
beginning MCR-1 binds the substrate phosphatidylethanolamine (PE) and donates pEtN to generate

an MCR-1/pEtN complex. Next, the complex attaches to the most abundant lipid A species in E. coli,

Kdo2-lipid A and transfers pEtN to either the 1’ or 4’-phosphate position of lipid A to neutralize the

surrounding negative charges (Figure 3). Removal of negative charge on lipid A results in elimination

of the electrostatic interaction that is required for the initial binding of polymyxins (polymyxin B or E) to

lipid A (Ma, Zhu, Yu, Ahmad, & Zhang, 2016; Son et al., 2019; Wei et al., 2018; Xu, Lin, Cui, Srinivas,

& Feng, 2018).

MCR-1 consist of two distinct domains, an N-terminal transmembrane domain assembled by

five putative α-helices, and a soluble C-terminal α/β/α sandwich domain that faces the periplasmic

space. As a member of the pEtN transferase family, MCR-1 shares ~40% sequence identity with its

structural homologues, NmEptA and CjEptC. The shape of the soluble periplasmic domain of MCR-1

is hemispherical and adopts an α/β/α topology consisting a centrally located seven-stranded β-sheet

decorated with eight main α-helices. The soluble domain of MCR-1 features some β-α-β-α motifs and

three disulfide linkages between C281/C291, C356/C364, and C414/C422. The catalytic domain of

MCR-1 containing α/β/α represent the characteristic of alkaline phosphatase superfamily (Figure 5).

The location of the active site of MCR-1 is within the soluble domain and in the center on the highly

conserved T285 (act as a catalytic nucleophile in substrate modification) residue that is observed in

all pEtN transferases. The active site contains a zinc ion that is important for function; MCR-1 is a

zinc metalloprotein, validated by all of the published crystal structure containing at least one zinc ion

in common. The active site possesses a distinct pattern of hydrophobic and charged residues. A

distinct electronegative potential to attract and bind the -NH 3+ moiety of the substrate pEtN is provided

by the interaction between a small cavity adjacent to the most conserved zinc site and T285.

Transmembrane domain of MCR-1 is essential for the correct binding and orientation of the lipid

substrates, PE and lipid (Hinchliffe et al., 2017; Ma et al., 2016; Stojanoski et al., 2016).

(b) Innovation

Colistin-Based Combination Therapy

 It is found that combination of colistin and the antibiotics (exhibiting activity against Gram-

positive and Gram-negative bacteria) such as rifampin, rifabutin, clarithromycin, minocycline, and

novobiocin enable these antibiotics to achieve the highest therapeutic potential as combinatorial

partners of colistin. It observed the reduction in MIC (minimum inhibitory concentration) below the

corresponding Gram-positive clinical breakpoint for all Enterobacteriaceae tested. Moreover,

susceptibility to colistin combination treatment (with clarithromycin, novobiocin, and rifampicin) was

broadly conserved across beyond laboratory-generated mcr-1 strains of Enterobacteriaceae; colistin

potentiation was strongly related to antibiotic class. Particularly, broad-spectrum antibiotics such as

fluoroquinolone and beta-lactam classes showed a limited reduction in MIC and no impact on clinical

breakpoints. Whereas, rifamycin and macrolide class, as well as minocycline and novobiocin,

exhibited the highest degree of synergism in combination with colistin to fight against mcr-1-positive

Enterobacteriaceae. Observation found that mcr-1 confers a high degree of resistance to the

bactericidal and lytic activity of colistin but provides minimal protection to its outer membrane

perturbation (MacNair et al., 2018).

The success of colistin-based combination treatment in inhibiting the growth of an mcr-1-

positive strain is merely determined by the antimicrobial activity of the antibiotic partner. As a result,

resistance to the antibiotic partner would be likely responsible for the reduction in efficacy in this

colistin-based combination treatment, rather than to the potentiation ability of colistin. It is showed by

the result of the spontaneous E. coli mutant expressing mcr-1 produced in the presence of rifampicin

and colistin were later resistant to the same combination. Nonetheless, changing rifampicin with

either novobiocin or clarithromycin at therapeutically relevant levels kept the bacteria susceptibility to

the colistin-based combination treatment. Hence, colistin combination therapy possess the unique

benefit that, despite the chance for the resistance development, treatment efficacy could be re-

established through substitution of the antibiotic partner. This study found that colistin and

clarithromycin combination shows efficacy against mcr-1-positive Klebsiella pneumoniae in murine

and bacteremia infection models at clinically relevant doses (MacNair et al., 2018; Rasko &

Sperandio, 2010).

Another study to explore the potency of colistin-based combination therapy to treat infection

against Gram-negative bacteria was conducted by Zhou and coworkers (2020). Particularly,

combination of colistin and tigecycline — the first of glycycline class that has a wide spectrum of

activity — was investigated at the clinically achievable concentrations in vitro and in murine thigh

infection model against carbapenem-resistant E. coli harboring blaNDM-5 and mcr-1. The result showed

that clinically achievable concentration of colistin and tigecycline generated a synergistic activity in

both in vitro and in vivo studies. Their different mechanism of action at separate bacterial targets

likely underpin the potentiation effect of this combination. Tigecycline is required to enter the bacterial

cells in order to exert its effect on the cytoplasm via binding to the ribosomal complex (Bauer, Berens,

Projan, & Hillen, 2004). Generally, there are two strategies that facilitate tigecycline uptake across the

bacterial cell wall and cytoplasmic membrane, passive diffusion and an energy-dependent active

transport system (Chopra & Roberts, 2001). The outer membrane surrounds the cell wall of Gram-

negative bacteria and tigecycline is transferred through membranes via porin channels in the absence

of colistin (Roberts, 2003). It is presumed that tigecycline passive accumulation is due to the outer-

membrane disruption and instable regions in cytoplasmic membrane caused by the colistin activity

(MacNair et al., 2018). Result from the intracellular tigecycline accumulation assays displayed that the

presence of colistin did promote tigecycline uptake (Zhou et al., 2020).

There are some questions still remained regarding to these studies. The observed

susceptibility of mcr-1-expressing bacteria to outer membrane disruption (MacNair et al., 2018) is not

supported by the classic focus of the proposed mechanism for mcr-1 and other polymyxin resistance

in eliminating the electrostatic interaction between colistin and lipid A (Ma et al., 2016; Son et al.,

2019; Xu, Lin, et al., 2018). However, it is important to note that the pEtN modification of mcr-1

causes only limited decrease to the formal charge of the phosphate ester of lipid A (from -1.5 to -1)

(Nikaido, 2003). Therefore, the hypothesis regarding to the colistin resistance mechanism expands
beyond reducing the strength of the electrostatic interaction between colistin and lipid A in the outer

membrane (MacNair et al., 2018).

The addition of cationic groups like pEtN can change outer-membrane architecture via an

increase in repulsion between neighboring LPS molecules that bolsters membrane packing is one

potentially underappreciated aspect of mcr-1 that presumably involves in its ability to resist the

growth-inhibitory activity of colistin (Nikaido, 2003; T. Velkov et al., 2013). The high intrinsic polymyxin

resistance found in N. meningitides has been speculated having a relationship with this phenomenon

(Nikaido, 2003). In particular, phosphoethanolamine transferase LptA is the primary component of

polymyxin resistance in N. meningitides; this enzyme possess a closely related structure with MCR-1

(Liu et al., 2016; Tzeng et al., 2005). Bolstered LPS packing provided by mcr-1 could contribute in

decreasing the uptake and lytic activities of colistin (MacNair et al., 2018).

The accurate details of the mechanism of colistin in affecting the energy-dependent transport

of tigecycline is still vague. Moreover, it is still unknown whether the mechanism of colistin in

increasing accumulation of tigecycline in bacterial cells is specific to certain antibiotic or applied to

broader range of antibiotics.

Despite of the absence of accurate details regarding to the mechanism of colistin-based

combination therapy against mcr-1-expressing pathogens/the ability of colistin to potentiate antibiotics

against colistin resistant bacteria, imposing colistin resistant bacteria to colistin-based combination

therapy not only provides a potential last resort therapeutic option but also may help to unravel the

Achilles heel of the resistant pathogens.

(c) Approach

Protein expression and purification

MCR-1 expression and purification are carried out based on the previously established

protocols (Wei et al., 2018). The full-length of the plasmid-mediated transferable colistin resistance

gene (mcr-1) gene is generated. Referring to the result of secondary structure predictions, the

catalytic domain of MCR-1 contains 326 aa, from P216 to R541. Overexpression of the catalytic
domain uses the plasmid pET-28(+) vector between the NcoI and XhoI restriction sites. Subsequent

confirmation of the gene insertion is achieved by sequencing. The plasmid is then transformed into

the E. coli strain BL21 (DE3) pLysS. Luria broth (LB) (containing 50mg/L kanamycin and 34mg/L

chloramphenicol) is used for selection of the transformants. A single colony is selected and cultured

in LB with the antibiotics listed before at 37℃. 0.25mM isopropyl-β-D-thiogalactopyranoside (IPTG) is

added to the culture to induce protein expression when optical density (OD) 600 reach 0.6. Incubation is

continued at 25℃ for 20 h. The cells are harvested by centrifugation and then resuspended in PBS

buffer with 1mM phenylmethanesulfonyl fluoride at 4℃. The cells are lysed, and the lysate was

clarified by centrifugation at 16,000 g to remove insoluble debris. Then, a 0.4-μm filter membrane is

used to filter the supernatant. The flow-through is loaded in a Ni-nitrilotriacetic acid (NTA) column,

equilibrated with lysis (PBS) buffer. The beads are washed with 10 mM imidazole in lysis buffer and

the bound protein is eluted by 150 mM imidazole in lysis buffer. The eluate is subjected for further

purification on a Superdex 200 Increase 10/300 GL gel-filtration column (GE Healthcare Life

Sciences, Little Chalfont, United Kingdom) equilibrated with 10 mM Tris-HCl (pH 8.0).

Extraction, purification, and identification of LPS-lipid A

LPS-lipid A extraction is carried out according to the established protocols (Hankins, Madsen,

Needham, Brodbelt, & Trent, 2013). Bacterial cultures grown on LBA plates with a proper level of

colistin and clarithromycin are stripped and washed with washing buffer containing 30 mM Tris-HCl.

In order to disrupt the cell wall, lysozyme is added to the bacterial cells (suspended in a buffer of 30

mM Tris-HCl and 20% sucrose). The lysates are dissolved in 3 mM EDTA and further sonicated.

Then, the crude LPS is precipitated following the removal of cell debris by 1 hour (1h) of

centrifugation at 16,000 rpm. Resuspension of crude LPS is conducted in 30 mM Tris-HCl and 0.2%

SDS and subjected to successive treatments of DNase I (25μg/ml), RNase A (100μg/ml) at 37℃ for 1

h and proteinase K at 37℃ for 1 h. Termination of the is carried out by incubation at 100℃ for 1 h.

The purified LPS-lipid A is obtained after the removal of SDS contaminants by two rounds of

treatment with acidified ethanol and 95% ethanol. The purified lipid A species in LUG loading buffer
[250 mM Tris-HCl (pH 6.8), 10% (vol/vol) SDS, 1% bromophenol blue, 10% (vol/vol) glycerol, 5%

(vol/vol) 2-mercaptoethanol] are visualized with SDS-PAGE (10%) combined with silver staining.

Solid-State NMR Sample Preparation

Hydration was conducted to the dry lipid films containing the lipid A/phospholipids mixture

(Phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocoline (C18:1, DOPC); 1,2-dimyristoleoyl-sn-3-

phosphocoline (C14:1, DMoPC); and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (C18:1, DOPG)) by

vortexing with each 1.5 mL of a colistin-clarithromycin and colistin-tigecycline solution (15 mM Tris-

HCl, 25 mM NaCl, pH 7.0), separately. Liposome is collected by centrifugation (20,000 x g) and load

into Bruker 1.3 or 3.2 mm zirconia rotors (Medeiros-Silva et al., 2018).

Native membrane vesicles (MVs) preparation is conducted by first transferring 10 mL of an E.

coli overnight culture grown in Luria broth (LB) at 37℃ with orbital shaking at 250 rpm into ~200 ml

fresh LB medium. Upon reaching optical density (OD) 600 0.6, 10 aliquots of 20 ml are centrifuged for 5

minutes at 4000 g. The supernatant is discarded, and the cell pellets are washed in PIPES buffer (50

mM PIPES pH 7.4, 150 mM NaCl). The bacteria are resuspended with the AMUPol, dissolved in

PIPES buffer containing 500 mM of trehalose solution for 5 min at 37℃, and then they are spun for 5

minutes at 14,000 rpm. The cell pellets are snap-frozen using liquid N 2 and packed into a 3.2-mm

sapphire rotor with silica plug between the zirconia spinning cap (Sani, Zhu, Hofferek, & Separovic,

2019).

DNP/solid-state NMR

DNP/solid-state NMR measurements are carried out using on a 9.4 T wide-bore Bruker

Avance-III NMR Spectrometer (Wissembourg, Germany) equipped with a 3.2-mm low-temperature

triple-resonance MAS probe. The temperature and spinning frequency of all experiments are

maintained at 110 K and 8kHz, respectively. Optimization of the spectrometer is conducted in order to

ensure that the microwave irradiation of the gyrotron occurred at the maximum DNP enhancement of

nitroxide (polarizing agent that is used is AMUPol) at 263.334GHz. NMRPipe

(https://www.ibbr.umd.edu/nmrpipe/install.html) is used to process all spectra and Gnuplot (open

source; http://www.gnuplot.info/) is used for plotting purpose (Sani et al., 2019).

13
C-detected cross-polarization saturation recovery experiments are used to determine recycle

delays. In this experiment, recycle delays are set at 1.3 x 1HT1 as described by Sani and coworkers.

The 13C signal intensities were fitted with a single exponential decay in Topspin 3.5 (Bruker) (Sani et

al., 2019).

13
C solid-state NMR
13
C cross-polarization MAS (CPMAS) experiments are conducted with 108 kHz proton

excitation pulse and 100 kHz SPINAL decoupling. The FIDs are zero filled to 4 k points and 10 Hz
13
line broadening is applied. Internal silicon plug C signal set at 0 ppm is used to reference the

spectra (Sani et al., 2019).

15
N solid-state NMR
15
N CPMAS are carried out with 108 kHz proton excitation pulse and 100 kHz SPINAL

decoupling. The FIDs are zero filled to 4 k points and 75 Hz line broadening is applied (Sani et al.,

2019).
Figure 1. Chemical structure of Colistin (Mohamed Rhouma et al., 2016)
 Figure 3. Structure of E. coli lipid A (Steimle et al., 2016)

Figure 2. General structure of lipopolysaccharides of Gram-negative bacteria (Steimle et al., 2016)

Figure 4. Molecular-structure modeling and electrostatic potential on the surface of full-length MCR-1

(Wei et al., 2018)

Figure 5. Structure of the catalytic domain of MCR-1 phosphoethanolamine transferase (Stojanoski et

al., 2016)
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