Discovery of Linear Low-Cationic Peptides to Target Methicillin-Resistant Staphylococcus aureus in Vivo

Article abstract of DOI:10.1021/acsinfecdis.8b00230

The development and rapid spread of multidrug resistant (MDR) bacteria cause severe public crises. New antibacterial compounds are urgently needed to treat bacterial infections. By circumventing the disadvantages of cationic peptides here, we engineered a short, linear, low-cationic peptide bacaucin-1a, which exhibited remarkable antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Bacaucin-1a was efficient in the prevention of MRSA associated infections in both in vitro and in vivo models with a unique mode of action. The discovery of low-cationic antibiotic candidates will extend our antibiotic pipeline in the fight against antibiotic resistant bacteria.

Full text of DOI:10.1021/acsinfecdis.8b00230

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Article
Discovery of linear low-cationic peptides to target
methicillin-resistant Staphylococcus aureus in vivo
Yuan Liu, Meirong Song, Shuangyang Ding, and Kui Zhu
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00230 • Publication Date (Web): 29 Oct 2018
Downloaded from http://pubs.acs.org on October 29, 2018
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Discovery of linear low-cationic peptides to target methicillin-resistant
Staphylococcus aureus in vivo
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Yuan Liu, Meirong Song, Shuangyang Ding, Kui Zhu*,†,‡,§
†
‡
‡,§
†Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of
Veterinary Medicine, China Agricultural University, No.2 Yuanmingyuan West Road, Haidian,
Beijing, China 100193
‡National Center for Veterinary Drug Safety Evaluation, College of Veterinary Medicine, China
Agricultural University, No.2 Yuanmingyuan West Road, Haidian, Beijing, China 100193
§Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety and Beijing
Laboratory for Food Quality and Safety, China Agricultural University, No.2 Yuanmingyuan
West Road, Haidian, Beijing, China 100193
*
Correspondence author: zhukcau@gmail.com (K.Z.).
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The development and rapid spread of multi-drug resistant (MDR) bacteria causes a severe public
crisis. New antibacterial compounds are urgently needed to treat bacterial infections. By
circumventing the disadvantages of cationic peptides, here we engineered a short, linear, low
cationic peptide bacaucin-1a, which exhibited remarkable antibacterial activity against
methicillin-resistant Staphylococcus aureus (MRSA). Bacaucin-1a was efficient to prevent
MRSA associated infections in both in vitro and in vivo models with unique mode of action. The
discovery of low cationic antibiotic candidates will extend our antibiotic pipeline for fighting
antibiotic resistant bacteria.
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Keywords: antibacterial, antibiotic, MRSA, peptides
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The global spread of MDR bacteria poses a severe threat to worsen public health.1-3 To address
this challenge, an efficient strategy is to develop novel antibiotics to directly combat pathogens
with different modes of action.4-9 Antimicrobial peptides and their analogs are potentially
effective candidates against pathogens.10-13 Such peptide-derivatives including lipopeptides,
glycopeptides, lantibiotics, short peptides and other variants,14,15 are produced by diverse
organisms to compete with pathogenic microbes, to facilitate signal transduction, or to serve as
physiological cues modulating population dynamics. Compared to these natural analogs, some of
their derivatives play a crucial role in treating bacteria associated infections in the clinic. For
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example, glycopeptides such as vancomycin, and lipopeptides such as polymyxins are
currently used as the last resort antibiotics against Gram-positive and Gram-negative bacteria,
respectively. However, bacterial pathogens have evolved resistance rapidly due to the overuse
and misuse of these antibiotics. For instance, bacteria can modify the negatively charged lipid A
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on the surface with a positively charged ethanolamine to resist cationic polymyxins. Thus, there
is a great need to develop new peptide-based antibiotic leads to restrict the emergence of
antibiotic resistant bacterial pathogens.
Nowadays, synthetic short peptides with antibacterial activities have attracted more attentions.
Such peptides are easily generated through the process of solid-phase peptide synthesis, and are
readily accessible for further chemical modifications. However, despite several synthetic peptide
leads have entered clinical trials,19,20 three crucial obstacles facing to develop new generation of
synthetic peptide-based antibiotics are cost-ineffective for chemical synthesis, poor protease
stability and nonspecific toxicity.21,22 Compared to the complicated natural structures,
antibacterial short peptides can dramatically reduce the production cost. Several attempts have
achieved promising results for treating bacterial infections in both in vitro and in vivo models
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such as MP196 (a synthetic hexapeptide, strongly enriched in tryptophan and arginine residues) ,
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SNAPPs (structurally nanoengineered antimicrobial peptide polymers) and CS-g-K (a cationic
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peptidopolysaccharide). Such short antibacterial peptides and the derivatives always contain
multiple positive charged amino acid residues that are ideal for binding to the negatively charged
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bacterial surface. Nevertheless, these peptides with intrinsic cationic-rich properties are also
easily resulted in off-target toxicity under physiological conditions,26,27 due to the nonspecific
electrostatic interaction between the positively charged groups in the peptides and the abundant
negatively charged biomacromolecules on cell surfaces. Therefore, we hypothesized that the
short and linear peptide analogs with low cationicity would be more potential, especially in terms
of stability and safety.
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It is advantageous to look back into the history of previously known but undeveloped
compounds for discovering new antibiotics. The family of cyclic noncationic lipopeptides from
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Bacillus species are great examples, which includes surfactin, lichenysins, iturin and other
classes. They all contain multiple negatively charged amino acid residues in lieu of positive ones
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in the chiral peptide sequences, suggesting that they share different modes of action. Taken
together, these examples further indicated that the potential of low cationic compounds can be
used to combat MDR bacteria. Inspired by these positive evidence and our unflagging effort in
developing surfactin-inspired antibiotics to combat MDR bacterial pathogens, we herein reported
a short, linear, low-cationic heptapeptide antibiotic composed of all natural amino acid residues
termed bacaucin-1a, which exhibited specific antibacterial activity against Staphylococcus
aureus, especially for methicillin-resistant Staphylococcus aureus (MRSA) in both in vitro and in
vivo.
Results and discussion
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Screening linear low-cationic peptides. Bacaucin-1, H-ELLSRVD-OH, was chosen as a
model to achieve our goal in this study. We first used the L-alanine (Ala, A) screening method to
investigate the role of each amino acid residue in antibacterial activity against S. aureus ATCC
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9213 (Figure 1A). The replacement of Ala, the smallest chiral amino acid, will keep the basic
structure of original peptide and indicate the function of side chains in amino acid residues.
Interestingly, we found that the sole substitution of L-arginine (Arg, R) at position 5 totally lost
antibacterial activity (Figure S1). To further elucidate the structure-activity relationship (SAR) of
the side chains (R groups), we synthesized 39 L-type derivatives of bacaucin-1 based on their
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polarities and electronic properties (Table S1). All these compounds were tested for antibacterial
activity against S. aureus ATCC 29213. We found that the sole substitution of hydrophilic and
negatively charged L-glutamic acid (Glu, E) by hydrophobic A at position 1 shows the best
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antibacterial activity with minimum inhibitory concentration (MIC) of 2 µg mL (Figure 1B).
Hence, this derivative of bacaucin-1 was named as bacaucin-1a, with negatively charged
potential surface and measured zeta potential of -6.08 ± 0.19 mv (Figure 1C). Altogether, it
suggest that bacaucin-1a is a low-cationic peptide with antibacterial activity against S. aureus.
Additionally, compared to the all L-type one, the derivatives of bacaucin-1a with all D-type or
partial D-type amino acid residues show a 4-fold to 32-fold reduced antibacterial activity (Table
S3).
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Guanidine group is crucial for antibacterial activity in bacaucin-1a. Compared to the other
analogs of R groups at position 5 (Figure 1B), we found both the length of side chains and the
positively charged guanidine group are crucial for antibacterial activity. To elaborate the function
of guanidine group, we used 1,2-cyclohexanedione to block the guanidine group of arginine and
get the derivatization of cyclohexanedione-bacaucin-1a conjugate (CBC). The purity of the
conjugate product was 97.05% based on ultra-performance liquid chromatography-tandem mass
(UPLC-MS/MS) analysis (Figure 2A). As expected, CBC completely lost its antibacterial
activity (Figure 2B), which confirmed the crucial role of the guanidine group in its antibacterial
activity. Additionally, significant reduced activity (more than 64-fold) was observed when
“
C=NH” (arginine) was replaced by “C=O” (citrulline) (Figure 1B), suggesting that the imino in
the guanidine group plays a crucial role in the antibacterial activity of bacaucin-1a.
Stability and safety assessment of bacaucin-1a. We next sought to evaluate the protease
resistance and serum stability of bacaucin-1a, and found that bacaucin-1a is resistant to
proteinase K, trypsin, pepsin, papain and 50% fetal bovine serum, superior to bacaucin-1 (Table
S4). To further elaborate the mechanism of bacaucin-1a resistant to trypsin, we evaluated the
resistance of bacaucin-1a and its substitutions to trypsin by UPLC-MS/MS method (Figure 2C).
The substitutions of either L-serine (Ser, S) or L-valine (Val, V) dramatically increased the
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percentage of hydrolyzed product under trypsin treatment (Figure 2D), suggesting that the
neighboring serine and/or valine residues around arginine might contribute to the resistance.
Furthermore, bacaucin-1a maintained antibacterial activity after heating at 80 °C for 1 h and
exhibited high stability under physiological conditions with pH ranging from 6 to 8 (Figure S2).
Lastly, we evaluated the toxicity of bacaucin-1a on red blood cells and mammalian cells. No
hemolysis and no cytotoxicity were observed at the concentration of bacaucin-1a above 50-fold
of MIC value (Figure S3). Taken together, these findings indicate that low cationic bacaucin-1a
as a lead structure is promising for the development of new antibacterial agent.
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Antibacterial spectrum of bacaucin-1a. Bacaucin-1a exhibited high specificity to S. aureus,
MRSA and MDR-SA isolates, instead of other bacterial species (Table S5). To further confirm
the antibacterial activity of bacaucin-1a, 100 clinical MRSA isolates from both human and
animal origins were tested. Compared to daptomycin which exhibits the MIC values ranged from
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0.25 μg mL to 2 μg mL , the MIC values of bacaucin-1a ranged from 2 μg mL to 4 μg mL
against all tested isolates (Figure 3A, Table S6). We defined the coefficient of variance, which
was the ratio of MICmax to MICmin of an antibiotic. The value of coefficient of bacaucin-1a was
much lower than daptomycin, suggesting that the antibacterial activity of bacaucin-1a was more
stable for diverse MRSA isolates (Figure 3B). To evaluate whether bacaucin-1a potentiates the
activity of other antibiotics, five antibiotics from different classes were tested in the combination
with bacaucin-1a. Bacaucin-1a shown synergy with ampicillin and rifampicin especially for
rifampicin, but not for the other tested antibiotics (Figure S4). To assess whether this synergy
was strain-specific, we determined six other rifampicin-resistant isolates, including four MRSA
isolates, and one of each Enterococcus faecalis and Escherichia coli. Results showed that the
synergy was species specific (Figure S5), which indicates that the potentiation of rifampicin in S.
aureus might be associated with the mode of action of bacaucin-1a.
Mode of action of bacaucin-1a. To study the underlying mechanism, we first focused on the
configuration of bacaucin-1a based on dynamic light scattering and nuclear magnetic resonance
(NMR) analysis. No significant intensity changes was observed in the 26-hour continuous
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measurement (Figure S6A), indicating that no intermolecular self-assembly of bacaucin-1a in
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aqueous solution. Besides, no intramolecular interaction of bacaucin-1a was observed through H
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and C NMR analysis (Figure S6B, C), suggesting that bacaucin-1a belongs to the family of
extended/random-coil peptides which lack secondary structure in solution.
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The method of screening spontaneous mutants has been successfully applied to reveal potential
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targets of novel antibiotics. For example, telomycin is found to bind to bacterial phospholipid
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cardiolipin, and humimycins specifically kills MRSA by inhibiting lipid II flippase. Based on
this approach, we evolved the wild-type S. aureus ATCC 29213 in the presence of sublethal
levels of bacaucin-1a to obtain the mutants that resist to bacaucin-1a. After 90 days of continuous
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induction, we got the mutants that survive at 64 μg mL bacaucin-1a (Figure S7A, B), which is
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2 folds of MIC (2 μg mL ). However, the total protein expression in mutants shown the same
patterns to that in the parent strain (Figure S8). Therefore, we further sequenced the whole-
genomes of the mutants to gain a deep insight into the mode of action of bacaucin-1a. We
identified four single-nucleotide polymorphisms (SNPs) not present in the wild-type one (Table
S7). Each resistant isolate contained SNPs in GMP synthase and RNA polymerase sigma factor
sigB (Figure 3C, S7C), confirmed by our optimized PCR tests and genetic sequencing analysis
(Table S8). While, the other two SNPs were excluded as the potential targets of bacaucin-1a, due
to the false-positive mutation by second generation sequencing. In S. aureus, GMP synthase
participates in the de novo biosynthesis of purine nucleotides and glutamate metabolism.34
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Besides, RNA polymerase sigma factor sigB, by reversibly binding to the RNA polymerase, is
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a master regulator for the stress response, such as temperature tolerance and osmotic stress.
Combining the fact that the synergy of bacaucin-1a and rifampicin targeting RNA polymerases
Figure S4, S5), it suggests that GMP synthase and RNA polymerase sigma factor sigB may be
(
the intracellular targets of bacaucin-1a. However, it remains unclear that whether these two
intracellular targets are responsible for the high selectivity of bacaucin-1a against S. aureus.
To further investigate how bacaucin-1a reaches the intracellular targets, we evaluate the
membrane integrity of S. aureus treated by bacaucin-1a. We first observed the collapsed and
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lysed bacterial cells induced by bacaucin-1a by scanning electron microscope (SEM) (Figure
S9). Meanwhile, the integrity of bacterial membrane was severely disrupted through
LIVE/DEAD cell assays (Figure S10A). Additionally, increased membrane permeability was
shown by the uptake of propidium iodide (PI) in dose and time dependent manners (Figure
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S10B, C), which was consistent with the increased Ca efflux (Figure S10D). These results
suggest that bacaucin-1a may reach the cytosol of S. aureus through membrane disruption.
We next exploited the intracellular secondary responses of S. aureus treated by bacaucin-1a.
Recent studies have shown that the stressful conditions may trigger an apoptotic-like death
(
ALD) mediated by recA and lexA genes or the mazEF pathway in bacteria.39,40 Interestingly, we
found that bacaucin-1a induced ten-fold increase of the expression of recA and lexA genes based
on mRNA expression analysis, but not for the mazEF gene (Figure 3D). These results indicate
that bacaucin-1a triggers an apoptotic-like death (ALD) pathway to induce programmed cell
death in S. aureus. Meanwhile, dissipation of membrane potential and increased level of total
reactive oxygen species (ROS) were observed as well (Figure S10E, F). Taken together, these
results suggest that bacaucin-1a kills S. aureus through multiple strategies (Figure 3E).
Bacaucin-1a exhibits therapeutic efficacy in cellular and animal models. Given the
attractive antibacterial activity of bacaucin-1a against MRSA in vitro and its unique mode of
action, we further investigated its potential as a therapeutic agent in in vitro and in vivo models.
Bacaucin-1a could effectively reduce the number of intracellular S. aureus in infected Vero cells
(Figure 4A, B). Bacaucin-1a, similar to vancomycin, dramatically increased the survival rate and
alleviated the pathologic changes of mice in the peritonitis-sepsis model (Figure 4C, D, E and
S11). The infected mice without treatment were all died within 12 h. Collectively, these findings
demonstrate the ability of bacaucin-1a against S. aureus associated infections at both cellular and
in vivo levels.
Conclusions
In conclusion, our approach demonstrate that an alternative lead can be used to treat S. aureus
associated infections. Unlike the classic cationic antimicrobial peptides, we rationally engineered
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a short, linear, low cationic heptapeptide bacaucin-1a with remarkable antibacterial activity based
on systematic structure-activity relationship study. Bacaucin-1a specifically kills MRSA in both
in vitro and in vivo models. Our approach can be extended to other natural products to construct
promising antibacterial agents for fighting antibiotic resistant bacteria.
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Experimental section
Materials. All bacterial strains used in this study are listed in Table S2. Unless otherwise
noted, strains were grown in brain heart infusion (BHI) broth or on BHI agar plates at 37 °C.
Vero cells were grown in Modified Eagle’s Medium (MEM, Gibco) supplemented with 1% heat
inactivated fetal bovine serum (FBS, Invitrogen), 1% (w/v) penicillin-streptomycin and 1% (w/v)
sodium pyruvate (Sigma-Aldrich). Ampicillin, colistin, ciprofloxacin, rifampicin, tetracycline,
vancomycin and daptomycin were obtained from China Institute of Veterinary Drug Control.
Peptide synthesis. All derivatives of bacaucin-1 and bacaucin-1a were synthesized by GL
Biochem (Shanghai, China). All the peptides (C to N terminals) were synthesized from natural
amino acids using an Fmoc (9-fluorenylmethoxycarbonyl) strategy-based solid phase peptide
synthesis method. All the detailed information of these derivatives can be found in Table S1.
Zeta potential measurement: The zeta-potential of bacaucin-1a was measured as bellow.
Bacaucin-1a was first dissolved in distilled water to reach a final concentration of 128 µg/mL.
After incubation at room temperature for 30 min, 1 mL of the solution was taken out for the
surface potential measurement using Malvern Zetasizer (Nano-ZS). All the measurement was
carried out at 25 °C.
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Antibacterial tests. Minimum inhibitory concentrations (MICs) of bacaucin-1, its
derivatives and other antibiotics were determined by broth micro-dilution according to the CLSI
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015 guideline. Daptomycin was tested in the cation-adjusted Muller-Hinton broth with
supplemented to 50 µg/mL calcium (Hope Bio Technology), while the others were tested in
cation-adjusted Muller-Hinton broth (Land Bridge Technology). MIC values were defined as the
lowest concentrations of antibiotics with no visible growth of bacteria after incubation at 37 °C
for 16-20 h.
Stability of bacaucin-1a. The proteolytic resistance of bacaucin-1a and its derivatives were
evaluated by treated with four kinds of proteinases (Sangon Biotech) including proteinase K,
trypsin and papain which were dissolved in PBS (pH 7.0), while pepsin was dissolved in PBS
(pH 2.0) at 37 °C for 1 h. To determine the stability of bacaucin-1a in the presence of serum,
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bacaucin-1a was pre-treated with 50% fetal bovine serum at 37 °C for 12 h and the residual
antimicrobial activity was determined. To determine the thermostability and pH stability,
bacaucin-1a was treated in the temperature range from 20 °C to 121 °C for 1 h and in the pH
range from 2.0 to 12.0 at 37 °C for 1 h, respectively. Subsequently, all samples were readjusted to
pH 7.0 to determine the residual antimicrobial activity against S. aureus ATCC 29213.
Safety assessment. Hemolytic activity of the derivatives were determined according to a
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previous publication. Briefly, sheep blood cells were prepared from fresh sterile defibrinated
sheep blood (Land Bridge Technology) and treated with various concentrations of derivatives at
3
7 °C for 1 h, using PBS and 0.2% Triton X-100 as negative and positive controls, respectively.
The absorption of released hemoglobin was measured at 576 nm. Cytotoxicity on mammalian
cells was performed on Vero cells by water-soluble tetrazolium salt-1 (WST-1, Roche) assay.43
Cell membrane tests. Membrane permeability and dissipated membrane potential of S.
aureus ATCC 29213 induced by bacaucin-1a were tested by 10 nmol/L propidium iodide (PI)
and 3, 3-dipropylthiadicarbocyanine iodide (DiSC (5)) (Aladdin). The concentration of
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intracellular Ca was measured by fluorescent probe Fluo-3AM (Beyotime) after S. aureus
ATCC 29213 treated by bacaucin-1a for 1 h. The total ROS of S. aureus ATCC 29213 in the
presence or absence of bacaucin-1a was probed with 2’,7’-dichlorofluor-escein diacetate (DCFH-
DA). Rosup was used as the positive control for ROS production.
Screening bacaucin-1a-resistant mutants. Resistant S. aureus mutants were obtained
through continuous passage culture. 1 to 10,000 diluted S. aureus ATCC 29213 overnight culture
were inoculated in fresh MHB containing 0.25 × MICs, 0.5 × MICs, 1 × MICs and 2 × MICs and
4
× MICs of bacaucin-1a . The bacterial cultures were then incubated at 37 °C under continuous
shaking at 200 rpm for 24 h.
Subsequently, the cultures from the second highest concentrations with visible growth (OD600
≥
2) were 1 to 100 diluted into a series of MHB media, until the presence of bacaucin-1a resistant
mutants. Lastly, the culture that could grow at high MIC levels was passaged on antibiotic free
BHI plates. Meanwhile, the correspondence MICs were determined as described above.
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Whole genome sequencing (WGS) and SNPs (single-nucleotide polymorphisms)
identification. Total DNA from S. aureus ATCC 29213 and bacaucin-1a resistant mutants (16 ×
MIC and 32 × MIC) were extracted using the Wizard Genomic DNA Purification kit (Promega).
Then, the libraries were constructed using the NEXT Ultra DNA Library Prep kit (New England
Biolabs) and 150 bp paired-end reads were sequenced by Illumina Hiseq 2500 (Annoroad).
Lastly, the draft assembly of the sequences were generated using CLC Genomics Workbench 8
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All MiSeq reads were mapped to the reference genome of S. aureus ATCC 29213 (RefSeq
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assembly accession: GCF_001879295.1) using BWA for identification of the variants. Single-
nucleotide polymorphisms (SNPs) were found in the bacaucin-1a resistant strains.
Bacteria-cell coculture. MRSA T144 were first stained by pHrodo Red (Life Technologies)
for 45 min. Subsequently, Vero cells were infected with stained MRSA T144 cells at an MOI of
100, and co-culture with various bacaucin-1a concentrations for 5 hours at 37°C in 5% CO2.
After 50 µg/mL vancomycin treatment and PBS (phosphate buffered saline) washing, Vero cells
were then fixed (4% paraformaldehyde), permeabilized (0.1% Triton X-100) and stained with
actinGreen (Life Technologies) followed by Hoechst 33342 (Beyotime) staining. Lastly,
fluorescent images were obtained by a confocal laser scanning microscope (Leica TCS SP8).
Mouse peritonitis-sepsis model. BALB/c female mice (n = 6 per group) were infected with
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.5 mL of MRSA T144 suspension (7.5 × 10 CFU per mouse) via intraperitoneal injection. At
one hour post-infection, all mice were treated with bacaucin-1a at single intraperitoneal doses of
, 2, 10, 20 and 40 mg/kg. Meanwhile, the other two groups were treated with vancomycin at
doses of 2 and 20 mg/kg as positive controls. Survival rates of treated mice were recorded during
8 h.
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Ethics statement. Mice were maintained in strict accordance with the regulations for the
Administration of Affairs Concerning Experimental Animals approved by the State Council of
People’s Republic of China (11-14-1988). The animal study protocols were reviewed and
approved by the Institutional Animal Care and Use Committee (IACUC) of the China
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Agricultural University and were performed in accordance with the relevant guidelines and
regulations (ID: SKLAB-B-2010-003). The laboratory animal usage license number is SYXK-
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016-0008, certified by Beijing Association for Science and Technology. All animals were
performed with 6-8 week old female BALB/c mice, purchased from the Vital River Laboratory
Beijing, China) and housed under specified pathogen-free condition for one week.
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Associated content
In supporting information section, Figure S1 describes the antibacterial activity of L-alanine
substitutions of bacaucin-1. Figure S2 and S3 provide the stability and safety evaluation of
bacaucin-1a. Figure S4 and S5 show the synergistic activity of bacaucin-1a and other antibiotics.
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Figure S6 displays the H NMR and C NMR spectrum. Figure S7 and S8 describe the screening
of bacaucin-1a resistant mutants and SDS-PAGE analysis of total protein expression. Figure S9
and S10 show the morphological changes and membrane dysfunctions of S. aureus by bacaucin-
1
a. Figure S11 provides the histopathological changes of mice organs in control, infected and
treated groups. Table S1 and S2 show the chemical information of derivatives and bacterial
strains used in this study. Table S3 and S4 describe the effect of conformation of amino acid and
proteinase and serum on the antibacterial activity of bacaucin-1a. Table S5 and S6 show the
antibacterial spectrum and activity of bacaucin-1a. Table S7 and S8 describe the SNPs in
bacaucin-1a resistant mutants and primers used in this study.
Acknowledgements
The authors thank Prof. Wenhan Lin (Peking University) for detailed NMR analysis and Dr.
Zhuojun Dai (Duke University) and Jianqi Wang (Chinese University of Hong Kong) for
dynamic light scattering measurement. This work was supported by the National Key Research
and Development Program of China (2017YFC1600305, 2018YFD0500506), National Natural
Science Foundation of China (31772796), Beijing Dairy Industry Innovation Team and Beijing
Municipal Science and Technology Project (D171100002017004).
Notes
The authors declare no competing financial interest.
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Figure 1 Structure-activity relationship (SAR) of bacaucin-1a.
A) Flowchart of the screening progression of low-cationic peptide antibiotic in this study. (B)
(
Antibacterial activity of bacaucin-1a derivatives. Each amino acid of bacaucin-1a was substituted
for another one based on their polarities and electronic properties. The R groups (side chains) of
each amino acid residue are shown. The amino acid compositions of bacaucin-1a were displayed
in red column. Minimum inhibitory concentrations (MICs) are the lowest concentration of a
synthesized peptide that inhibits the growth of S. aureus ATCC 29213 by broth micro-dilution
method. All the peptides consists of L-type amino acids. See also the Supporting information
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Table S1. (C) Chemical structure, surface potential and zeta potential of bacaucin-1a.
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Figure 2 Active site and trypsin resistance of bacaucin-1a.
(
(
A) Synthesis of 1,2-cyclohexanedione-bacaucin-1a conjugate (CBC) in borate buffered saline
BBS, pH = 8.5). The purity of CBC was determined by UPLC-MS/MS. (B) Antibacterial
activity of bacaucin-1a and CBC against S. aureus ATCC 29213. Bacaucin-1a lost its
antibacterial activity after guanidine group of arginine was conjugated. ***P < 0.001, determined
by Student’s t test. (C) Analysis of trypsin resistance in bacaucin-1a by UPLC-MS/MS. The
retention times and corresponding pear areas of parent sequence and hydrolyzed product were
shown. Percent (ALLSRVD, %) = 3560452 / (3560452 + 241453) = 93.65%, related to the first
columns in Figure 2D. (D) Percentage of bacaucin-1a to the combination of bacaucin-1a and
hydrolyzed product (ALLSR).
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Figure 3 Mode of action of bacaucin-1a.
A) Antibacterial activity of bacaucin-1a against 100 clinical MRSA isolates from both human
(
and animal origins. (B) Bacaucin-1a displayed more stable antibacterial activity than daptomycin.
Coefficient of variance represents the ratio of MICmax to MIC . The smaller value of coefficient
min
of variance, the more stable antibacterial activity. **P < 0.01, determined by Student’s t test. (C)
The SNPs of GMP synthase and RNA polymerase SigB in bacaucin-1a resistant mutants. (D)
Bacaucin-1a induced programmed cell death in S. aureus via the apoptotic-like death (ALD)
pathway. N.S., not significant, **P < 0.01, ***P < 0.001, determined by non-parametric one-way
ANOVA. (E) Schematic representation of mode of action of bacaucin-1a against S. aureus.
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Figure 4 Bacaucin-1a was efficient in preventing infections in both cellular and in vivo
models.
(A) Fluorescent micrographs of Vero cells infected with MRSA T144 in the presence of
bacaucin-1a. DNA, F-actin and MRSA T144 were stained by hoechst, actin-green 488 and
pHrodo red succinimidyl (NHS) ester, respectively. Scale bars, 25 µm. (B) Decreased numbers of
the intracellular bacteria treated by bacaucin-1a. Data are represented as mean ± SD. ***P <
0
.001, determined by non-parametric one-way ANOVA. (C) Scheme of the experimental
protocol for the mouse peritonitis-sepsis model. (D and E) Survival rates of the mice treated with
bacaucin-1a (D) and vancomycin (E) infected by MRSA T144 (n = 6 per group).
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