of 20
Materials and Methods
Bacterial strains, plasmids and culture conditions
P. aeruginosa
and
Escherichia coli
were routinely cultured in lysogeny broth (LB; 1% tryptone, 0.5% yeast
extract, 1% sodium chloride) and
S. aureus
in tryptic soy broth (TSB, Becton Dickenson), unless
otherwise indicated, with aeration at 37ºC. Antibiotics were added, when appropriate: car
benicillin at 250
μg/ml for
P. aeruginosa
and gentamycin (Gm) at 30 μg/ml for
P. aeruginosa
and 15 μg/ml for
Escherichia
coli.
All strains used in this study can be found in
Table 1
.
Generating
P. aeruginosa
mutants
Markerless deletion mutations were co
nstructed in PAO1 to inactivate each of the
P. aeruginosa
T6SS
loci. Deletions were introduced using two
-
step allelic exchange, as previously described for
P.
aeruginosa
1
. Briefly, 500 bp upstream and downstream regions of the
tssB
gene from each T6SS loci
(H1
-
H3
-
T6SS) were amplified to construct a contiguous mutant allele by SOE
-
PCR. The deletion allele
was inserted into the donor vector pENTRPEX18Gm by Gateway cloning, transformed into
E. coli
S17.1,
and introduced to
P. aeruginosa
through conjugation usi
ng a mating filter apparatus. Deletion mutants
were first confirmed by PCR and followed by whole genome sequencing and
breseq
analysis to ensure
no secondary mutations arose. All oligonucleotides used to generate
P. aeruginosa
mutants can be found
in
Table
1
.
Macroscopic coculture twitching chemotaxis assay
Motility experiments were performed as previously described
2
4
. Buffered agar plates (10 mM Tris, pH
7.6; 8 mM MgSO4; 1 mM NaPO4, pH 7.6; and 1.5% agar) were poured and allowed to solidify for 1 hour
p
rior to incubation for 16 hours at 37°C and 22% humidity. After solidifying, 4 μL of either growth medium
(TSB) or cell
-
free supernatant derived from an overnight culture of USA300
S. aureus
at OD
600
5.0 and
filter sterilized with a 0.22 μm filter were spo
tted on the surface of the plate and allowed to diffuse for 24
hours at 37°C and 22% humidity to establish a gradient.
P. aeruginosa
PA14 cultures were incubated
overnight in TSB with aeration at 37°C, subcultured 1:100 in TSB the following morning, then
standardized to OD
600
12.0 in 100 μL of 1 mM MOPS buffer supplemented with 8 mM MgSO
4
prior to
inoculating 1 μL on the surface of the plate at five mm from the center of the gradient. Plates were
incubated in a single layer, agar
-
side down, for 24 hours at
37°C with 22% humidity, followed by an
additional 16 hours at room temperature prior to imaging the motility response of
P. aeruginosa.
Images
were captured using a Zeiss stereoscope with Zeiss Axiocam 506 camera and directional motility ratios
were calcu
lated in Fiji before graphing and performing statistical analysis in GraphPad Prism.
BONCAT labeling of
P. aeruginosa
For PSM pulse
-
in and
S. aureus
coculture
proteomics experiments,
P. aeruginosa
PA14
NLL
-
MetRS
cells were grown to OD
600
~2 in defined M14 medium (M9 salts supplemented with 10 mM glucose,
10 mg/ml casamino acids, 1 mM MgSO
4
, 2 μg/ml thiamine (vitamin B1), 2 μg/ml niacin (vitamin B3), 2
μg/ml calcium pantothenate (vitamin B5), 0.1 μg/ml biotin (vita
min B9)). For PSM pulse
-
in, PSM
ɑ1 and
PSMɑ3 (Genscript, 0.2 μg/ml each) were added to
P. aeruginosa
cells; for cocultures,
P. aeruginosa
(PA14
NLL
-
MetRS) and
S. aureus
(USA300_FPR3757) cells were mixed at 1:1 ratio. Labeling was
initiated by the addition o
f 1 mM Anl (Iris
-
Biotech) and cell lysates were harvested immediately after 30
min. Cell pellets were centrifuged at 4°C and washed with ice
-
cold LCMS
-
grade water and frozen at
-
80°C for downstream lysis and chemical enrichment. All samples were lysed by r
esuspension in 1% SDS
in PBS, boiled at 75°C and sonicated with a microtip probe for 15 s at 30% amplification (Qsonica).
Sample preparation for mass spectrometry
For chemical enrichment of BONCAT
-
labeled proteins from lysates, 2.5 mg of lysates were us
ed for each
sample. Lysates were first reduced by dithiothreitol (DTT), alkylated with chloroacetamide and reacted
with
aza
-
dibenzocyclooctyne
(DBCO) agarose beads (Click Chemistry Tools) via copper
-
free click
chemistry. The reaction was incubated in dark
at room temperature overnight, followed by rigorous resin
washing with i) 40 ml 0.8% (wt/vol) SDS/PBS; ii) 40 ml 8 M urea in tris hydrochloride (pH = 8.0) and iii)
40 ml 20% (vol/vol) acetonitrile/H
2
O to remove nonspecific protein binding. Peptides were de
tached from
DBCO agarose with on
-
bead digestion with 0.1
g trypsin and 0.05
g endoproteinase LysC at 37°C
overnight. Supernatant was then collected for further detergent removal (HiPPR column, Thermo Fisher
Scientific), and peptides were desalted with
C
18
StageTips.
LC
-
MS analyses of desalted samples
Peptides were subjected to LC
-
MS/MS analysis on an EASY
-
nLC 1200 (Thermo Fisher, San Jose, CA)
coupled to a Q Exactive HF Orbitrap mass spectrometer (Thermo Fisher, Bremen, Germany) equipped
with a Nanospr
ay Flex ion source. Samples (2 μL out of 10 μL total) were directly loaded onto an Aurora
25 cm x 75 μm ID, 1.6 μm C18 column (Ion Opticks, Victoria, Australia) heated to 50°C. The peptides
were separated with a 60 min gradient at a flow rate of 350 nL/min
as follows: 2
6% Solvent B (3.5 min),
6
-
25% B (42 min), 25
-
40% B (14.5 min), 40
-
98% B (1 min), and held at 98% B (14 min). Solvent A
consisted of 97.8% H2O, 2% ACN, and 0.2% formic acid and solvent B consisted of 19.8% H2O, 80 %
ACN, and 0.2% formic acid.
The Q Exactive HF was operated in data dependent mode with full scan
resolution set to 60,000 at m/z 200 in profile mode, full scan target set to 3 × 10
6
, and a maximum injection
time of 15 ms. Full scan mass range was set to 375−1500 m/z. Data dependent
MS2 scans were collected
for charge state 2
-
5 precursors in centroid mode using a loop count of 12, AGC target of 1 × 10
5
, an
intensity threshold of 1 × 10
5
,
and a maximum injection time of 45 ms. Isolation width was set at 1.2 m/z,
scan range was 200
-
20
00 m/z, and a fixed first mass of 100 was used. Normalized collision energy was
set at 28. Peptide match was set to off, and isotope exclusion was on. Dynamic exclusion was set to
exclude after 1 time for 45 sec. All mass spectrometry raw data used for thi
s study have been deposited
to the
California Institute of Technology Research Data Repository for public access under DOI
10.22002/gbjeb
-
6kg53
.
Proteomics data processing and analysis
Proteomics data analysis was performed in Proteome
Discoverer 2.5 (Thermo Scientific) using the
SequestHT search algorithm and a nonredundant Uniprot
P. aeruginosa
FASTA file plus common
contaminants (CRAPome). Sequest search parameters were as follows: fully tryptic peptides with 6
-
144
residues and no mor
e than 2 missed cleavages, precursor mass tolerance of 35 ppm and fragment mass
tolerance of 0.05 Da, and a maximum of 3 equal modifications. Dynamic modifications were as follows:
Cysteine carbamidomethylation, Methionine oxidation, Asparagine and Glutam
ine deamidation, protein
N
-
terminal acetylation, protein N
-
terminal Met
-
loss, and protein N
-
terminal Met
-
loss plus acetylation.
Percolator FDRs were set at 0.01 (strict) and 0.05 (relaxed). The consensus level peptide and PSM FDR
filters were also set at
0.01 (strict) and 0.05 (relaxed). Strict parsimony principle was set to true. Protein
quantification was also performed in Proteome Discoverer by summed abundances of the precursor
intensities of all high confidence unique and razor peptides.
Raw prote
in quantification data exported from ProteomeDiscoverer 2.5 was imported into R and analyzed
using the romics analysis package (
https://jeffsocal.github.io/romics
). On
ce imported the data is filtered
for common protein contaminants and normalized between runs using Breiman and Cutler's Random
Forests for Classification and Regression (
https://cran.r
-
p
roject.org/web/packages/randomForest
)
method selected based on best performance in lowering sample replicate variation while maintaining
quantitative dynamic range for label free analysis. Protein expression differences b
etween samples were
evaluated in the R romics package using the limma algorithm for differential expression
(
https://bioinf.wehi.edu.au/limma/
). Differential expression
volcano plots were generated using Prism
(GraphPad). For PSM pulse
-
in proteomics analysis, principal component analysis revealed an outlier for
PSM
-
treated samples (data not shown). Differential expression analysis performed before (log
2
fold
change and ra
w P
-
values) and after (log
2
fold change and Benjamini Hochberg FDR adjusted P
-
values)
removal of outlier yielded consistent observations, and the final volcano plots shown represent data
analysis post removal of the outlier. Protein set enrichment analysis
for significantly up
-
regulated and
down
-
regulated
P. aeruginosa
proteins was performed using STRING v11 database (Search Tool for
Retrieval of Interacting Genes/Proteins) by uploading protein accession codes (PAO1 UniProt) to the
server (
https://string
-
db.org/
), and results visualized using python libraries matplotlib and networx.
Fluorescence microscopy
To visualize the ClpV1 foci,
P. aeruginosa
PAO1 ClpV1
-
GFPmut3 was grown in mi
nimal medium (M8
salts supplemented with 0.2% glucose and 1.2% tryptone, M8T) with aeration at 37°C. Cells were sub
-
cultured in fresh M8T, grown to mid
-
log phase (OD
600
of ~0.3
-
0.6), standardized to OD
600
of 0.3, pelleted,
and resuspended in M8T
(vehicle) or 8 μg/ml PSMs (4 μg/ml α1 and 4 μg/ml α3). PSMs were sonicated
for 5 min prior to use. 0.5 μL of cells were inoculated onto a 4
-
chamber glass
-
bottom 35 mm dish (Cellvis,
Cat. no. D35C4
-
30
-
1.5
-
N) before placing an agarose pad on top. Agarose pad
s were made by pipetting
550 μL of M8T with 2% molten agarose (Lonza, Cat. no. 50081) into each quadrant of a 4
-
chamber glass
-
bottom dish and drying uncovered for ~1 hr 15 min at room temperature, followed by ~1 hr covered with
a lid at room temperature, t
hen ~1 hr 15 min at 37°C before transferring the pads onto the inoculated
glass
-
bottom dish. For polymyxin B, since significant cellular toxicity was observed at all concentrations
examined, which also increased ClpV1
-
GFP puncta formation when added at T
0
,
a final concentration of
4 μg/ml of polymyxin B was added on top of the agarose pad ~2 hrs after the cells were inoculated, and
then allowed to diffuse for ~40 min. The imaging was performed with an inverted Nikon Ti2 A1R
Galvanometer Scanning Confocal Mi
croscope, using a 100x Plan Apo oil objective (1.45 NA). Images
were acquired between 2
3 hrs after the cells were inoculated onto the dish. A 488 nm laser was used
to excite GFPmut3. Images were saved and analyzed using the Nikon NIS
-
Elements AR softwar
e and
the representative images are shown in the Maximum Intensity projection. The ClpV1
-
GFPmut3 puncta
were manually quantified and divided by the total cellular area per field of view (FOV). The mean
fluorescence intensity (F.I.) was quantified by calcul
ating the average of the mean F.I. of the total cellular
area per FOV. A total of three biological replicates with four technical replicates (FOVs) per condition,
per biological replicate, were analyzed.
For determination of inner membrane permeability to propidium iodide,
P. aeruginosa
cells harboring
P
tac
-
gfp
on a plasmid were grown in M8T with 250 μg/ml carbenicillin to mid
-
log phase and prepared as
described above except
0.5 μM propidium iodide (PI) wa
s added to the agarose pads and polymyxin was
added directly to the cells at T
0
. Phase contrast and epifluorescent images were acquired with an Andor
Sona camera. Fluorescent images were acquired every 20 minutes with TxRed images taken at 18 ms
exposure a
nd 20% Sola fluorescent light and GFP imaged at 2 ms exposure and 20% Sola fluorescent
light. The rate of PI uptake was determined
by dividing the total fluorescence intensity of PI by the total
fluorescence intensity of
GFP
in each FOV
. A total of three b
iological replicates with two technical
replicates (FOVs) per condition, per biological replicate, were analyzed.
Outer membrane permeability
P. aeruginosa
cells were grown in M14 at 37
°
C with aeration to early stationary phase and standardized
to OD
600
= 1.0. Cells were then washed in fresh M14 and incubated with 20
μ
M 1
-
N
-
Phenylnaphthylamine
(NPN) and polymyxin B or synthetic
PSM
ɑ1 and PSMɑ3
for 15 minutes. 100
μ
L from each condition were
added to a black, clear
-
bottom 96 well plate and NPN fluorescen
ce read (excitation: 350 nm, emission:
420 nm) on a Tecan Infinite M200 plate reader. Relative NPN fluorescence was calculated by dividing
fluorescence of treated cells by fluorescence of untreated cells.
P. aeruginosa
growth rate
P. aeruginosa
cell
s were grown in
M14
to mid
-
log phase, washed in fresh
M14,
standardized to OD
600
=
0.
05,
and added to a clear
-
bottom 96
-
well plate with medium alone, polymyxin B, or
synthetic
PSM
ɑ1
and PSMɑ3. The plate was covered with a gas permeable membrane and OD
600
m
easurements were
taken at 10 min intervals
on a Tecan infinite M200 plate reader
. The plate was incubated at 37°C and
was shaken for 1 s before each measurement.
Mammalian cell culture
The human CF bronchial epithelial cell line CFBE41o
-
(obtained from J. P. Clancy, Cincinnati Children’s
Hospital) originated from a person with CF that was homozygous for the ΔF508 mutation in CFTR.
CFBE41o
-
cells were routinely cultured in minimal essenti
al media (MEM) supplemented with 10% fetal
bovine serum (FBS), 2 mM L
-
glutamine, 5 U/ml penicillin
-
5 mg/ml streptomycin, and 0.5 mg/ml plasmocin
at 37°C with 5% CO2. CFBE41o
-
cells were seeded at near confluency on Transwell filters (Costar). After
conflue
ncy, CFBE41o
-
cells were differentiated at an air
-
liquid interface (ALI) for 1
-
2 weeks before use
in experiments. The identity and purity of the CFBE41o
-
cells were verified by short tandem repeat
profiling (University of Arizona Genetics Core). Cells were
tested quarterly for mycoplasma using a
Southern Biotech mycoplasma detection kit.
Coinfection of CFBE cells
ALI
-
differentiated CFBE41o
-
cells were moved to antibiotic
-
free media by washing with MEM (Gibco)
supplemented with 2 mM L
-
glutamine and replacin
g basolateral media with MEM supplemented with
10% FBS and 2 mM L
-
glutamine.
P. aeruginosa
PAO1 and
S. aureus
USA100 were pre
-
washed in MEM
supplemented with 2 mM L
-
glutamine and inoculated at a 1:1 ratio at an MOI of approximately 250 onto
the apical side
of polarized CFBE41o
-
cells. After 2 hours of attachment, non
-
attached bacteria were
removed, and apical media was adjusted to 0.4% L
-
arginine for a total of 6 hours.
RNA sequencing
RNA was isolated from
P. aeruginosa
PAO1 and
S. aureus
USA100 mono
-
or 1
:1
coculture
following 6
-
hour infection on polarized CFBE41o
-
cells. RNA was collected by phenol:chloroform extraction using
RNA
-
Bee (AMS Biotechnology) and zirconia/silica beads in a BeadBeater (BioSpec Products) from two
independent, biological replicate
s. RNA was precipitated with isopropanol and linear acrylamide, and
RNA pellets were washed by ethanol precipitation. RNA was treated with Turbo DNase (Ambion) and
purified by RNA Clean and Concentrator (Zymo Research). DNA removal was confirmed by 260/280
and
260/230 ratios and by PCR for the
P. aeruginosa
rplU
gene. RNA integrity was determined by agarose
gel electrophoresis and visualization of 5S, 16S, 18S, 23S, and 28S bands.
RNA
-
seq library preparation and sequencing were performed by the Health Sci
ences Sequencing Core
at Children’s Hospital of Pittsburgh (Pittsburgh, PA). RNA concentration and integrity was confirmed by
fluorometric quantification (Qubit) and Tapestation analysis (Agilent). RNA was rRNA
-
depleted using
Ribo
-
Zero Epidemiology, and se
quencing libraries were prepared using Truseq stranded Total RNA Kit
(Illumina). Single end sequencing was performed on a NextSeq 500. Approximately 75 million 75 bp
reads were obtained for each sample. Reads were processed and mapped to the
P. aeruginosa
PAO1
genome and differential expression analysis was performed in CLC Genomics Workbench. Statistically
significant changes were considered as p≤0.05. The
P. aeruginosa
and
S. aureus
co
-
infection
transcriptome sequencing (RNA
-
seq) data is part of a large
r study on the response of
P.
aeruginosa
to co
-
infection
. This data has been deposited in the NCBI Sequence Read Archive
(BioProject #PRJNA865724).
Bacterial competition assay
For
in vitro
coculture
competition,
P. aeruginosa
(PA14) and
S. aureus
(USA300
WT and Δ
psm
ɑ1
-
4
δATG
-
ATT) overnight cultures were diluted to OD
600
= 0.1 in fresh M14 medium, mixed at 1:1 ratio by
species and grown for 24 hr. PSM
ɑ1 and PSMɑ3 (Genscript, 5 μg/ml each) were added to the medium
at time of inoculation of
coculture
. Harvest
ed
coculture
s were serially diluted and plated on
Pseudomonas
isolation agar (PIA) for selective isolation of
P. aeruginosa
and Mannitol Salt Agar for selective isolation
of
S. aureus
.
For bacterial competition associated with CFBE41o
-
cells,
P. aeruginos
a
(PAO1) was grown overnight in
LB
and
S. aureus
(USA100)
was grown overnight in TSB at 37 ̊C. Overnight cultures were washed with
phosphate buffered saline (PBS). Strains were OD
-
normalized and mixed at a 1:1 ratio. As before,
differentiated and polarized
CFBE41o
-
cells were infected at an MOI ~250 with the 1:1 bacterial mixture.
After 1 hour of attachment to the airway cells, planktonic bacteria were removed, and the competition
assay was run for a total of 6 hours. Following competition, 0.1% Triton
-
X was
added to the apical
compartment to remove attached cells and enumerate bacteria. For each assay, the initial 1:1 inoculum
(input) and 6
-
hour time point (output) were serially diluted in PBS and spotted on PIA to count
P.
aeruginosa
and on tryptic soy agar supplemented with 10 mg/L colistin sulfate (Sigma) and 15 mg/L
nalidixic acid (Sigma) to count
S. aureus
. Colony forming units (CFUs) were determined for both species,
and competitive index was calculated as: [(WT
output
/competitor
o
utput
)/(WT
input
/competitor
input
)].
Extended Data Figures
Supplementary Figure 1. PSM peptide sequences, structure and genome location.
a.
Amino acid sequences for all 7 known peptides in the PSM family of
Staphylococcus aureus
with
deposited Protein Data Bank (PDB) structures shown. f=N
-
formylation.
b.
Regulatory operons and gene
locations for PSMs in
S. aureus
(annotations based on strain U
SA300 FPR3757). Adapted from
5
. The
psm
ɑ1
-
4
mutant used previously
11
, was generated via deletion of the entire
psm
α
operon
(
psm
ɑ1
-
ɑ4
).
To generate the double
psm
αδ
mutant, since
psmδ
is encoded within the coding region of RNAIII,
psmδ
was inactivated through mutation of the start codon from ATG to ATT
11
.
Supplementary Figure 2. BONCAT labeling and enrichment of nascent
P. aeruginosa
proteome.
a.
General scheme of a BONCAT experime
nt.
P. aeruginosa
cells constitutively expressing NLL
-
MetRS
were treated with 1 mM azidonorleucine (Anl) to initiate protein labeling. Newly synthesized proteins (blue
circles) were selectively tagged with azide functional groups on Anl residues and chemic
ally distinct from
the pre
-
existing cellular proteome (grey circles). Labeled proteins were enriched via copper
-
free azide
-
alkyne click chemistry onto DBCO
-
alkyne agarose beads. Enriched proteins were digested and analyzed
by LC
-
MS/MS or visualized via in
-
gel fluorescence following copper
-
catalyzed azide
-
alkyne click
chemistry onto TAMRA
-
alkyne fluorophore (
b
). Anl
-
labeled proteins in each sample were detected and
visualized by TAMRA fluorescence (552 nm ex/ 578 nm em) gel via copper
-
catalyzed click chemist
ry as
previously described
6
. Briefly, lysates were quantified by the Pierce
TM
BCA Protein Assay kit (Thermo
Fisher Scientific), and samples containing 100
g protein were incubated with 2.5
M alkyne
-
TAMRA
(Click Chemistry Tools), 100
M CuSO
4
, 500
M
tris(3
-
hydroxypropyltriazolylmethyl)amine (THPTA
ligand; Click Chemistry tools) and 5 mM aminoguanidine hydrochloride. The click reaction was initiated
by addition of 5 mM sodium ascorbate and allowed to proceed for 30 min in the dark at room temperature,
followed by methanol/chloroform precipitation. Precipitated proteins in each sample were then washed
twice with methanol, resuspended in PBS and SDS loading buffer, and visualized via SDS
-
PAGE
electrophoresis (NuPAGE Novex 4
-
to12% bis
-
Tris gels; Thermo Fis
her Scientific). Imaging was done on
a Typhoon gel imager (GE Healthcare) for fluorescence or coomassie (Instant
-
Blue) signal intensities.
c.
Chemical compounds used for BONCAT in
-
gel fluorescence and enrichment in this study.
Supplementary Figure 3. Enrichment analysis of proteins differentially regulated in response to
PSM pulse
-
in and
S. aureus
coculture
.
Gene set enrichment analysis (GSEA)
7
was performed
for
PSM pulse
-
in (a) and
S. aureus
coculture
(b)
using the fgsea packa
ge for R
(https://bioconductor.org/packages/release/bioc/html/fgsea/html).
GSEA compares a rank
-
ordered list of genetic and/or proteomic data to curated annotations. Briefly,
proteins with quantified differential expressions were provided as a rank
-
ordered
list by log
2
-
transformed
fold changes with annotations pulled from the
Pseudomonas
Genome Project (pseudocap.gaf) and
downloaded UniProtKB entry annotations for
P. aeruginosa
. Analysis revealed significantly enriched
Gene Ontology (GO) terms for cellular
components, molecular functions and biological processes
with
indicated false discovery rate (FDR) adjusted
P
values. The number of permutations was set to 10,000
for
P
value calculation. For complete lists of GSEA output, see
Supplementary Table 2
and
3
.
Supplementary Figure 4.
P. aeruginosa
proteome ranked by average raw abundances (T6SS core
components highlighted).
a.
P. aeruginosa
nascent protein synthesis in response to PSM pulse
-
in and
S. aureus
coculture
during
Anl
labeling period is ranked by individual protein raw abundance as indicated by Label
-
Free
Quantification (LFQ) values.
b.
P. aeruginosa
nascent protein synthesis in control (
-
PSM and
monoculture) samples.
Supplementary Figure 5. PSMs induce py
overdine biosynthesis in
P. aeruginosa
.
P. aeruginosa
overnight cultures (PA14 P’
pvdG
-
mScarlet)
were
diluted to O.D. (600 nm)
of 0.1 in fresh
M14 medium. Aliquots (100 μL
) of culture were added to 96
-
well clear, flat
-
bottom polystyrene plates with
or without 10 μg/ml PSMs (5 μg/ml PSM
ɑ1 and PSMɑ3 each), followed by incubation at 37°C with
continuous orbital shaking at 1200 rpm using a microplate reader (VarioScan). Fluores
cence intensity
(mScarlet: 565 nm ex/ 600 nm em; pyoverdine: 405 nm ex and 460 nm em) and optical density (UV
absorption at 500 nm) measurements were taken every 30 min for 18 h. Measurements for triplicate wells
were averaged, normalized to growth and plo
tted for raw fluorescence intensity units (
a
) with statistical
quantification of end time points (
b
). Data shown represent the mean and standard deviation of at least
three independent replicates. Statistical significance was determined by multiple
t
-
test
followed by
multiple comparisons test for False Discovery Rate adjusted
P
-
value
.
Supplementary Figure 6. PolymyxinB inhibits
P. aeruginosa
growth and induces T6SS firing.
a.
P. aeruginosa
growth over time monitored by OD
600
under indicated treatment conditions.
b.
Representative fluorescence microscopy images of
P. aeruginosa
ClpV1
-
GFPmut3 treated with
polymyxin B.
Supplementary Figure 7
.
Diverse c
-
di
-
GMP sensing and regulatory enzym
es
8
mediate
P.
aeruginosa
interspecies response to PSMs and
S. aureus.
Domain structures of c
-
di
-
GMP
sensing/regulatory enzymes detected to be significantly up
-
regulated in
response to PSMs pulse
-
in (
a
) and
S. aureus
coculture
(
b
). Red color indicates sil
ent domains.
GGDEF/AGDEF/GGEEF: diguanylate cyclase (DGC) domain; EAL/EVL: phosphodiesterase (PDE)
domain; PAS, PAC, REC are other sensory and/or regulatory domains frequently associated with DGC
and PDE domains.
For references, see
Aragon et al., 2015
9
, J
ones et al., 2014
10
, Cai et al., 2020
11
,
Okegbe et al., 2017
12
, McLaughlin et al., 2012
13
, Basu Roy & Sauer, 2014
14
, Li et al., 2013
15
, Bellini et
al., 2017
16
.
Supplementary Figure 8. Functional enrichment analysis of
P. aeruginosa
genes differentially
regulated during co
-
infection with
S. aureus
.
Up
-
regulated (fold change > 2,
P
<0.05) and down
-
regulated (fold change <
-
2,
P
< 0.05) genes from RNA
sequencing exp
eriment were analyzed for functional classification using the PANTHER (Protein ANalysis
THrough Evolutionary Relationships) Classification System (
http://pantherdb.org/about.jsp
). Selected
functional categories that showed statistically significant enrichment (FDR adjusted P
-
value <0.05) are
plotted. Population total (PT) refer to all protein/genes quantified in each experiment; population hits (PH)
refer to all protein/gene
s annotated as indicated categories in each experiment; list hits (LH) refer to
protein/genes annotated as indicated functional categories in subset lists;
list total (LT) refer to total
number of proteins/genes in each sublist.
Percentage expected = PH/PT
; Percentage hits = LH/LT.
Table 1. Strains and primers used in this study.
Name
Genotype or sequence
Source
Notes
P. aeruginosa
strains
UCBPP
-
PA14
WT
Laboratory collection
PA14 NLL
-
MetRS
attTn7::mini
-
Tn7T
-
Gm
R
P
trc
:
nll
-
Ec
metRS
Babin
et
al
., 2017
17
PA14
P’pvdG
-
mScarlet
P’pvdG
-
mScarlet
(pSB175)
Zarrella
et al
., 2022
1
8
PAO1
WT
Laboratory collection
PAO1 ClpV1
-
GFPmut3
Obtained from Joseph
Mougous (UW)
PAO1 ΔH1
-
T6SS
tssB1
(∆
PA0083
)
This study
PAO1 ΔH2
-
T6SS
hsiB2
(∆
PA1657
)
This study
PAO1 ΔH3
-
T6SS
hsiB3
(∆
PA2365
)
This study
S. aureus
strains
USA100
WT
Laboratory collection
USA300 LAC
WT
Laboratory collection
USA300 LAC
Δ
psmα
1
-
4
Δ
psmα1
4
Sayed
et al
., 2015
1
9
USA300 LAC
Δ
psmαδ
Δ
psmα1
4 δ
ATG
-
ATT
Sayed
et al
., 2015
1
9
Primers
PA0083
UpF01
-
attB1
GGG GAC AAG TTT GTA CAA
AAA AGC AGG CTA CGT CGC
CGA ACG GGT CGG CTC
This study
For
PAO1
tssB1
PA0083
UpR01
GGA ATC CTC TTA CGC CTG
CGG TCC CAT CTT GTT TCT CCC
TCG CG
This study
For
PAO1
tssB1
PA0083
DownF01
CCG CAG GCG TAA GAG GAT
TCC
This study
For
PAO1
tssB1
PA0083
DownR01
-
attB2
GGG GAC CAC TTT GTA CAA
GAA AGC TGG GTA GCA GCC
ATA GGG CTC GCC G
This study
For
PAO1
tssB1