Species-selective killing of bacteria by antimicrobial peptide-PNAs
Madhav Mondhe1, Ashley Chessher1, Shan Goh2, Liam Good2 and James E. M. Stach1
School of Biology, Newcastle University, Newcastle upon Tyne, United Kingdom1, Department of Pathology
and Infectious Diseases, Royal Veterinary College, University of London, London, United Kingdom2
Supporting Information
Figs S1 and S2
Tables S1-S6
Figure S1. Venn diagram of the fifteen possible species combinations (lettered A-O) in a theoretical bacterial
community composed of the four species used in this study. The key on the right indicates the antimicrobial
agent that would result in the bactericide of the desired species target(s). Peptide-PNAs are in bold. Only the
peptide-PNAs designed in this study are capable of species-specific bactericide for individual (C and D) and
mixed (M) Gram-negative species. * Peptide-PNA (Ec1000) was non-specific, but a number of E. coli- specific
PNAs were designed (Table S4) that have yet to be evaluated.
Figure S2. The predicted structures of the E. coli PNA transporter protein SbmA (pink) and YgaD (blue) of B.
subtilis. Structures were predicted using the I-TASSER platform [1]. YgaD was identified by homology
searching using HHPred [2] with SbmA as the input sequence. Protein sequences were aligned and rendered by
PyMOL [3]
Table S1. Antibiotic susceptibility of strains used in this study
Target
Cell wall
synthesis
Class
Penicillins
Ribosome 50S
B. subtilis
E. coli
K. pneumoniae
S. Typhimurium
S
S
R
S
Amoxicillin
25
S
S
R
S
Cefalexin
30
S
S
S
S
Cefuroxime
30
S
S
S
S
Cefotaxime
30
S
S
S
S
Glycopeptide
Vancomycin
30
S
R
R
R
Carbapenem
Imipenem
10
S
S
S
S
Aminoglycosides
Amikacin
30
S
S
S
S
Streptomycin
30
S
R
S
S
Neomycin
30
S
S
S
S
Gentamicin
30
S
S
R
S
Erythromycin
15
S
R
R
R
Azithromycin
15
S
R
R
R
Lincomycin
15
S
R
R
R
Clindamycin
10
S
R
R
R
Nalidixic acid
30
S
S
S
S
Pipemidic acid
20
S
S
S
S
Fleroxacin
5
S
S
S
S
Oflaxacin
5
S
S
S
S
5
S
S
R
S
Macrolides
Quinolones
Fluoroquinolones
Tetrahydrofolic
acid synthesis
Sensitive (S)/Resistant (R)
10
Lincosamides
Nucleic acid
synthesis
Amount per disc (μg)
Methicillin
Cephalosporins
Ribosome 30S
Antibiotic
Trimethoprim
Table S2. S. Typhimurium-specific PNAs
Gene
PNA sequence
Tm
Orthologue
essential in E.
coli
acrB
adhE
adk
avrA
bcr
cheA
clpB
clpX
csdA
cstA
dcoC
ddl
dfp
dnaK
fic
ftsZa
gcd
glmM
glnA
glyS
hisI
hisM
hrpA
hybB
icdA
katG
lpxA
lysS
maf
metG
miaE
mig-3
mrp
murB
murG
mviN
napH
nrfC
pabB
pdxK
phoR
polA
prfB
pstS
pyrF
recC
GGCATGTCTT
GCCATAATGC
CGCATTACGA
ATCATCTTTA
GTCACATCGA
CTCACGCTAT
CGCATAACTC
GTCATGAGTC
TTCATGATAT
TTCATAGTTG
TTCATAATAA
GCCATGTTGC
CTCATCATGT
CCCATCTAAA
CTCATAGTGC
AACATAATCT
GCCATAAAAA
CTCATAGCGT
GACATACTTG
GACATGGCCG
AACATATCCT
ATCACAAATC
AGCATCAGTG
GATCGTCTCC
TCCATTCACC
CTCATATCTC
ATCACGAATC
GACATGTTGA
GGCATATTTT
GTCATAGTGG
GTCATACTAT
TACATGGTGT
TTCATCCCGA
GTCATGGCTG
CTCATCGAGA
TGCATGGCTG
GCCATCTTTC
CTCATGGCTG
ATCATTCCTG
CCCATAAAAT
AGCACGCGTC
ACCATAATGT
GTTATTCACC
TTCATAATGT
GTCATGACCA
AACATAATTA
55.8
56.4
59.2
40.5
58.6
51.1
53.2
55.1
42.8
46.3
42.5
59.3
46.8
50.1
51.2
46.1
57.4
53.8
50.5
68.2
46.7
50.4
59.9
54.2
50.5
39.4
53.7
57.4
47.9
56.7
43.0
55.1
55.8
60.2
56.7
63.0
48.7
56.6
47.2
49.5
67.5
50.7
46.5
44.5
58.2
45.5
no
no
yes
n/a
no
no
no
no
yes
no
n/a
no (ddlB)
yes
no
no
yes
no
no
no
yes
no
no
no
no
no
no
yes
no
n/a
yes
n/a
n/a
no
yes
yes
no (murJ)b
no
no
no
no
no
no
no
no
no
no
No. of
mismatches
with E. coli
orthologue
Off-target essential gene
0
0
1
n/a
1
1
0
1
2
0
n/a
2
2
0
1
2
1
0
1
1
0
2
6
0
0
4
1
1
n/a
1
n/a
n/a
0
2
1
3
2
0
5
2
4
0
8
0
0
4
no
no
no
no
no
no
no
no
no
no
yjgMc
no
c
lpdA , rimMc
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
rho
no
no
no
nrfCc
no
no
no
murBc,d
no
no
no
no
no
no
c
hflK , STM2913c, STM4259c
no
recF
rep
rfaB
rfaL
rfbK
rfbV
rhsE
rnb
rnt
rplC
rpmJ
rpoD
ruvB
sbcB
sinI
smvA
sopA
sspB
STM0268
STM0345
STM0438
STM0557
STM0858
STM1003
STM1008
STM1097
STM1128
STM1129
STM1133
STM1552
STM1556
STM1559
STM1560
STM1637
STM2273
STM2274
STM2406
STM2619
STM2633
STM2844
STM2913
STM3012
STM4262
tdk
thrS
topB
trmD
trxA
ulaA
uppS
vacB
wcaD
GACATATATT
CGCATGGGAA
TTCATATATA
AGCATCTTTT
TTCATTAGTC
AGCATATGAT
TGCACGTTCT
AACATATTCG
GACATCTGAG
ATCATTGTAT
TGCATTTCAT
TCCATAAGAC
TCCAGCACCG
GTCACTTGAT
TGCATTCAGA
AACTCTCCCT
TTCATTAGAA
TCCATACAGG
TCCATGAATA
TGCATTTCGG
TCCACAGATA
CTCATTCTTA
TTCATTATTA
AACATAATGA
TCCATGATTT
TTCATGCTTC
ATCATAGTTC
GACATCCAGT
ATCATTTATC
TTCACAACTT
CTCATAATGG
ATCATGATGC
CTCATTTGTC
GCCATAATAT
TTCATAATTT
CTCATTTTGC
ATCATGACCT
GCCATGCTCA
TCCATGATTT
AACACAAATT
GTCATGACCT
ATCATGCTAC
TCCATAAGTA
GCCATTGGTC
GGCATGTTAT
CGCATCAGGT
AACACAAGCC
CTCATATATA
TCCATAAGCG
AACATAACGC
GACATTGAGG
ACTCCTCCAG
42.4
69.4
36.1
47.2
43.5
51.8
56.1
50.0
56.8
43.0
47.9
52.3
64.8
50.9
56.5
50.5
47.4
56.0
50.4
57.6
53.9
39.5
37.1
52.7
46.8
46.8
44.7
57.2
38.7
48.1
49.3
53.4
44.0
47.4
39.0
45.4
51.6
60.1
46.8
53.3
54.5
49.3
49.0
58.7
53.6
62.6
62.4
36.1
57.9
58.2
61.1
55.0
no
no
no
no
no (cpsG)
n/a
no
no
no
yes
no
no
no
no
n/a
n/a
n/a
no
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
no
no
no
yes
no
no
no (ispU)
no (rnr)
no
5
3
2
1
5
n/a
8
3
1
1
3
0
0
4
n/a
n/a
n/a
1
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
3
1
4
5
0
1
1
3
0
no
no
trxAc
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
STM2633c
yfiFc
no
no
no
no
tnaBc,d
no
no
no
rplSc,d, STM0857c
no
STM2913c
no
STM1008c
no
c
hlfK , pyrFc, STM2406c
no
no
no
no
no
no
rfaBc
no
no
no
no
yafD
yaiZ
ybeX
ybjO
ycaL
yccJ
ychK
ychM
yciG
ydcN
ydgP
ydiN
yeaZ
yebW
yecS
yeeF
yfaW
yfgE
yhcK
yjcD
yjjY
yliB
yneI
yoaE
ytfE
ytfP
a PNA
CGCATCGCAT
CGCATAACGA
TCATGGCGTC
CCCAATGAAT
TTCATACTAA
GGCATTCGAA
CTCATCGTAC
ATCACATGTG
GCCATATTAT
TCCATTTTCC
AGCATGGTTT
GACATTTGTT
CGCATGAGGT
AACATTACAT
TGCATTCGCG
GTCATTCTCC
TCCATTTTAA
GACATAAAAT
TCCATTCATG
GACATAGAAA
GTCATGTTAC
GTCATGTTGT
GTCATGTTCT
TCCATGACAA
GCCATAGCCG
CGCATTGCTA
60.8
61.9
60.6
54.3
43.0
63.0
49.1
53.4
44.7
44.6
57.1
49.1
65.3
47.4
61.8
47.4
43.5
51.3
48.3
57.1
48.9
52.0
47.3
56.6
64.2
56.0
no
no
no
no
no
no
no (rssA)
no
no
no
no (rsxG)
no
no
no
no
no
no (rhmD)
no (hda)
no (nanR)
no
no
no (gsiB)
no (sad)
no
no
no
6
6
0
5
3
2
3
5
1
4
4
3
3
1
4
4
6
7
4
2
0
3
5
0
1
0
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
used in this study.
Difference in annotation of start codon location between E. coli K12 and S. Typhimurium. The start
codon of E. coli was used.
c Off-target gene reported to be essential in S. Typhimurium with 9 or 10 bp matches with the PNA
d Off-target gene reported to be essential in E. coli with 9 or 10 bp matches with the PNA
b
Table S3. S. Typhimurium-specific PNAs designed using essential genes from E. coli
Genea
accC
accD
asd
bcsB (yhjN)
birA
cca
cdsA
coaD
cysS
def
dxs
entD
era
fabA
fabZ
fmt
folA
folC
folE
folK
ftsK
ftsL
ftsQ
ftsX
groS (groES)
hemC
hemD
hemG
hemH
hemL
PNA sequence
Tm
AACATGTTCG
CTCATTGGTG
TTCATAGCGT
TTCATCGCAT
TTCATGCAAT
TTCACGCCAC
AGCAAAAGCA
TGCATGACAG
AACATCGAAG
GACATAATTA
CTCATCGCAA
AGCATCGTTT
CTCATTCCAA
ACCATGTTTT
GTCAAAATAC
GACACGTTGG
ATCATTAATT
TTCATTGTTT
GGCATTAATC
GTCATACGGT
CTCAAAAAAG
ATCATGCGTT
GACATATCAG
TTCATTGGCC
CTCATTGATA
GTCATTATCA
CTCATGCGGG
TTCACGTGTT
CGCATTGCCG
CTCATGGAAA
55.8
53.0
53.2
50.6
50.6
58.9
66.3
60.9
60.2
46.7
56.3
54.8
48.6
48.3
51.5
64.5
40.2
42.2
52.0
57.5
55.0
54.5
51.6
54.5
44.6
45.8
64.9
53.0
63.8
56.8
No. of
mismatches
with E. coli
orthologue
1
2
4
1
1
1
1
2
1
2
4
5
1
1
1
1
3
3
2
1
1
1
1
2
1
6
1
1
1
2
Off-target essential gene
no
no
no
ispBe
mufSe
no
no
no
no
no
ispBe
no
no
dere
no
no
no
e
folC , pheTd,e, rplCd,e
no
no
no
no
no
lpxKe
no
no
no
no
no
no
holA
holB
infC
ispA
ispE
ispF
ispH
ligA
lnt
lolB
lolC (ycfU)
lptA (yhbN)
lptG (yjgQ)
lpxB
map
minD
minE
mraY
mreC
mreD
mukF
murAb
murE
murF
murI
nadD
nadE
nrdA
nrdB
pgsA
pheT
plsC
pyrG
pth
rho
ribA
ribC (ribE)
ribD
ribE (ribH)
rimN (yrdC)
rplU
rpmA
rpsB
rpsN
rseP (yaeL)
secD
ssb
tadC (yfhC)
tdcF (yjgF)
tilS
thrS
tmk
ATCATTAGTT
TTCATGCCGC
TTAATACTTT
TCCATTTACT
ATCATTTCAC
CGCATTATGC
TGCATGTTTA
TCCATATCAC
GCCATTTTTA
GTCATAGTGA
TAATGTACAT
TTCATAAGAG
TGCATTATAC
GCCATTAACG
GCCATGAATT
GCCATAAAAA
GCCATAATTT
AACATGAACC
TTCATAGGCT
ACCATTACCC
CTCATAGGTT
TCCATTATTG
GCCACCTGTT
ATCATGCGAT
GCCATAAACT
TTCATATCAC
GTCATTTAAC
TTCATGTGGT
GCCATTGAAG
TGCATAGTGA
TTCATTGTTT
AGCATAACAA
GTCATGCGGA
GCCACGTTTT
TTCATAATGG
TGCATGTATT
AACATATCTT
TGCATGGCTT
TTCATATTCT
TTCACTTGTT
TACATAAAAA
GCCATTTAAT
GCCATGATTT
TTCATTGATT
AGCATAAAAC
AACACGGCAG
GCCATAATTG
GGCATTATAC
CTCATGATTT
GTCATGATCC
GGCATGTTAT
CCCATTTTTT
43.4
58.9
37.7
42.7
50.7
54.2
50.3
46.5
46.5
54.6
45.5
50.4
45.8
57.3
55.2
57.4
48.0
57.9
51.6
52.1
48.6
43.5
57.9
55.0
53.9
43.1
46.0
54.0
59.7
57.6
42.2
57.5
66.0
55.9
49.0
49.0
43.4
58.6
37.8
45.4
49.7
47.9
52.5
43.5
56.1
69.1
52.1
50.4
43.8
52.5
53.6
41.8
1
3
1
4
6
1
2
1
4
1
1
2
2
1
1
1
1
2
2
5
2
4
1
1
1
1
1
3
4
1
3
1
1
2
1
2
3
1
1
3
1
3
1
4
2
1
4
6c
5
2
1
1
no
no
no
no
no
no
no
ydfBe
no
no
no
no
tadCd,e
no
no
no
ssbe
no
prfAe
no
dnaNe
hemKd, prmCe, valSe
no
rnce
no
no
hutHd, STM0932d
no
no
lolBe
folCe, rplCd,e
coaDe
folKe
hisSd,e
pstSd, STM1556d
no
no
rpsSe
no
no
no
no
d,e
murD , STM1008e, STM2633e
groSe
no
no
minEe
lptGe
kdsBd,e, fabGd,e, yjeAe
no
no
no
tnaB (mtr)
trpS
ubiA
ubiB
waaU (rfaK)
wzyE (wecF)
ydiL
yeaZ
yejM
yhbV
yhhQ
yigP
GCCATTCAGC
GTCATTTTTT
TCCATCTTTA
GTCATTTGGC
ATCATAATAG
CTCATTCGGC
ATCATTTTTT
CGCATGAGGT
ACCATACCTG
TTCATAGCTC
GTCATATTGT
GGCATTCCAC
58.5
40.9
42.6
55.2
43.7
55.5
38.1
65.3
53.4
46.8
46.0
58.7
4
1
2
1
6
2
4
3
3
2
1
3
no
STM4259d, ydiLe
no
no
no
no
d
d,e
narY , ompN , STM4259d, trpSe
no
no
murBd,e
no
no
hemEd,
narYd,
S. Typhimurium gene name in parenthesis
PNA used in this study
c Orthologues in E. coli and S. Typhimurium have different annotations of the start codon location. If
the E. coli start codon is applied to both, there are 2 mismatches.
d Off-target gene reported to be essential in S. Typhimurium with 9 or 10 bp matches with the PNA
e Off-target gene reported to be essential in E. coli with 9 or 10 bp matches with the PNA
a
b
Table S4. E. coli-specific PNAs
Gene
PNA sequence
Tm
Orthologue in S.
Typhimurium
alsK
bamA
bamD
can
chpS
cohE
dicA
fbaA
ispU
mazE
racR
secM
yagG
yceQ
ydfB
yefM
TGCATGAGGC
GCCATCGTTA
GTCATGACGT
TTCATGGAGG
CGCATCTGCA
TTCATAACGA
TCCATAGTTA
GACATGTCTC
ATCACGCGTT
ATCATAACCC
AGCATTGCTT
CTCACGTTAT
GTCATCAGAA
GACACTCTGC
TCCATATCAC
TTGACATGCG
65.6
56.0
58.6
60.5
60.4
52.6
46.3
52.4
59.3
49.7
54.2
47.7
56.3
57.1
46.5
59.9
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
Off-target
essential gene
E. coli
no
no
yidC
hemL
no
lolD
no
rpmB
no
no
no
rimN
no
no
no
no
No. of cells (1 x 104)
Mixed culture
B. subtilis
E. coli
K. pneumoniae S. enterica
B. subtilis - K. pneumoniae
B. subtilis - S. Typimurium
E. coli - S. Typimurium
E. coli - K. pneumoniae
K. pneumoniae - S. Typimurium
B. subtilis - K. pneumoniae - S.
Typimurium
6
6
-
6
4
-
0.6
2.7
4
0.7
1
3
4.7
-
1.3
0.7
Table S5. Number of cells used for inoculation of mixed culture.
Table S6. Oligonucleotides used in this study
Oligo name Target
ECpdf-F
def
ECpdf-R
SEpdf-F
def
BSpdf-R
actcattaacccggaattgct
caggcggtcgagtttttcta
def
KPpdf-R
BSpdf-F
cgagacgatgtacgcagaag
ggctttcagacgatccagtt
SEpdf-R
KPpdf-F
Oligo sequence* (5’ -> 3’)
ctggagaaagatggcgaaac
aatgcgctgttgtttgagtg
def
ggaaacacctgcggaaacc
ctgcacggctcttgctaaaa
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3.
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