whitchurch capsig 2018...beta-lactams tested. however, morphological characteristics of each step...
TRANSCRIPT
Pseudomonas*aeruginosa alternate'lifestyles:'opportunities'for'exploitation
Professor'Cynthia'Whitchurch
CAPSIG,'December'12,'2018
@CwhitchD'@ithreeinst
Antibiotic(Resistance(Crisis• Currently*estimated*that*antimicrobial4resistant*infections*kill*50,000*people*each*year*in*Europe*and*the*US.
• 10M*deaths*globally*by*2050
• 20174 WHO*emphasizes*the*need*for*serious*initiatives*to*counteract*the*growth*of*multi4resistant*bacteria*and*spark*R&D.
Combating*infection*by*exploiting*bacterial*lifestyles:
Today&I’ll&discuss&our&attempts&to&understand&and&exploit:1. Biofilm&expansion&along&implanted&medical&devices2. Production&of&public&goods&in&bacterial&biofilms3. Tolerance&of&!@lactam&antibiotics
Pseudomonas*aeruginosa• Gram&negative&rod• Opportunistic&pathogen• Various&chronic&and´&infections• Member&ESKAPE&group&multi>drug&resistant&“superbugs”
• #2&on&WHO&“critical&list”&for&needing&R&D&for&new&antibiotics• High&levels&of&intrinsic&resistance&to&many&classes&of&antibiotics• Highly&impermeant outer&membrane
• Multiple,&inducible&efflux&pumps
• Membrane&vesicles
• Biofilms
• Model&organism&for&study&of&biofilm&development&and&active&biofilm&expansion
Combating*infection*by*exploiting*bacterial*lifestyles:
Today&I’ll&discuss&our&attempts&to&understand&and&exploit:1. Biofilm*expansion*along*implanted*medical*devices2. Tolerance&of&!9lactam&antibiotics
Bacterial)Biofilms• “Biofilms)are)communities)of)bacteria)that)are)generally)stuck)to)a)surface)and)/or)each)other)and)are)encased)in)self8produced)slime”• Normal)mode)of)growth)for)bacteria
• most&bacteria&spend&most&of&their&time&in&biofilm&lifestyle
• Protected)mode)of)growth• Survival&in&hostile&environment• Increased&antibiotic&resistance&&
• 10:10,000x&more&resistant&than&planktonic&bacteria&of&same&species
• Escape&immune&attack• Important• NIH&estimates&over&80%&of&infections&are&biofilms.• Chronic&infections• Medical:device&related&infections&
Pseudomonas)aeruginosa)biofilm on&Foley&catheter
Active'biofilm'expansion• Biofilms)not)always)sessile0 can)actively)expand• Facilitates)rapid)colonisation of)new)territories• Spreads)infection)along)implanted)medical)devices)eg Foley)Catheters• Actively)migrating)biofilms)show)elevated)levels)of)resistance)to)antibiotics• Enables)migration)against)fluid)flow• Complex)self0organised,)multicellular)collective)behaviours
LB0Gelgro @ 370C, Playback speed 300x real0time (4 h)
• Interstitial+biofilm+expansion
Leading(raftsTrail(networkMain(colony
Wild+type+P.#aeruginosa
Glass(slideGellan Gum/AgarGlass(coverslip
Interstitial+biofilm+expansion+model
Twitching(motility=mediated(biofilm(expansion
Interstitial*biofilm*expansion
Twitching)motility)mediated)biofilm)expansion• Powered(by(extension/retraction(of(type(IV(pili
• Polar(location(on(cell• Complex(molecular(machine(• >40(proteins(involved• 100(pN force• Strongest(biological(molecular(motor
Skerker,(J.M.(and(Berg,(H.C.(Proc%Natl.%Acad.%Sci.%USA,(98,(6901N6904((2001).
PilU
PilB PilT
PilD(XcpA)
PilC
PilV
PilEPilA
FimU
FimT
PilW
PilX
PilFPilP
PilO PilN FimV
PilM
Outer)Membrane
Inner)Membrane
Periplasm
Cytoplasm
PilQ
PilA
PilY1
PilM
FimX
PilZ
Twitching)motility.mediated)biofilm)expansion
• Complex)collective)behaviour• How)are)the)activities)of)1000’s)of)individual)cells)co<ordinated to)manifest)large<scale)self<organisation ?
Pattern'formation'correlates'to'the'presence'of'phase'bright'trails
Gloag&and&Turnbull&et&al,&PNAS,&2013
30&μm30&μm
What%are%the%phase%bright%trails?
Scratches)in)solidified)media)appear)phase)bright
Expansion)of)P.#aeruginosa biofilm)across)a)scratched)surface)over)2h.)Played)back)at)600x)real)time.Scale)bar)=)100)μm)
Twitching)motility)trails)are)furrows
• Phase'bright'trails'are'furrows'forged'in'substratum'during'migration'that'guide'movements'of'following'cells
Atomic'Force'MicroscopyMichelle'Gee,'Uni Melbourne
Gloag et'al,'Proc Natl Acad Sci USA,'2013Gloag et'al,'Comm Integr Biol,'2013
Raft'head
Raft'trails
Behind'rafts Lattice
Optical'ProfilometryScott'Wade,'Swinburne'Uni Tech
Furrows'confine'cells'into'trails
200nm
680nm
eDNA%in%interstitial%biofilms
P.#aeruginosa expressing)red)fluorescent)protein.)Growth)media)supplemented)with)cell)impermeant eDNA stain)TOTO<I)(green).
eDNA production)during)interstitial)biofilm)expansion
B
Explosive*cell*lysis*releases*eDNA,*produces*membrane*vesicles
Phase&contrast TOTO.1&(eDNA)
Phase&contrastTurnbull&et#al,&2016,&Nature#Comms 7:&Article&No&11220
• Explosive&cell&lysis&occurs&due&to&release&of&tailocins (pyocins)
• The&&endolysin&Lys&Is&essential
‘Tailocins’ are multiprotein particles mor-phologically similar to phage tails [2], andbacteriocin activity has been demon-strated for a subset of them. In the oppor-tunistic human pathogen Pseudomonasaeruginosa, tailocins display a flexible (F)or rigid (R) appearance. These particles,denoted F- and R-type pyocins, respec-tively, have served as models for phage-like bacteriocins [3]. R-type tailocins alsooccur in plant-associated pseudomonadspecies [3,4]. Morphologically similar bac-teriocins have been identified in severalother g-proteobacterial genera, represent-ing species with different lifestyles, such ascarotovoricin of the phytopathogen Pec-tobacterium carotovorum causing soft rot,xenorhabdicin of the entomopathogenicnematode symbiont Xenorhabdus nema-tophila, and maltocin of the opportunisticpathogen Stenotrophomonas maltophilia[5,6]. Bactericidal tailocins are not con-fined to Gram-negative bacteria, as theirgeneral morphology is also conserved in abacteriocin produced by the Gram-posi-tive pathogen Clostridium difficile [7]. Elu-cidation of the tube and sheath structureof the R pyocin contractile tail revealedboth architectural similarities and differ-ences with the nanotubes of phage tails
and the type VI secretion machinery (Fig-ure 1) [8].
Although tailocins bear striking morpho-logical similarity to phage tails, theirgenetic makeup indicates that they shouldnot be considered degenerate pro-phages. Moreover, they have evolvedextensively to fulfill diverse ecologicalroles, as explained below.
Headless but Still FunctionalThe F- and R-type tailocin gene clustersdisplay striking synteny with the genomicregions for tail assembly of phages belong-ing to the families Siphoviridae (e.g.,HK022) and Myoviridae (e.g., P2), respec-tively (http://viralzone.expasy.org) [9].However, in line with their headless tailmorphology, the corresponding genes forphage head assembly and DNA packagingare lacking. On the other hand, the geneclusters encompass cognate regulatorygenes and dedicated lysis cassettes forrelease of the particles. This indicates thatthese tailocins are expressed from well-organized functional units. To assess thetailocin-encoding potential of such geno-mic regions, careful delineation and com-parative analyses of synteny and gene
content are required. Frequently, intactprophages and tailocin clusters with highlysimilar tail regions are present in a particularstrain, sometimes located adjacently.
Different Trails of TailDomesticationGenomic tailocin clusters of the R- and F-type are abundantly present in thegenomes of P. aeruginosa strains, eitherindividually or as R-to-F fused regions simi-lar to the pyocin R2–F2 gene organization inP. aeruginosa PAO1 [3]. Two additionaltailocins resembling different Myoviridae(pro)phages have been identified in Pseu-domonas fluorescens and in Pseudomo-nas syringae [3,4]. Phylogenetic analysis ofstructural components shared betweenphages and the abundant R-type tailocinsreveals a distinct clustering with differentphage genera, as illustrated for their sheathproteins in Figure 2A. Whereas the R2-typepyocin appears to be related to a pseudo-monad phage (PS17), a probable ancestryshared with nonpseudomonad phages ofdifferent genera is inferred for other pseu-domonad tailocins (e.g., Vibrio parahaemo-lyticus phage VP882, Hapunalikevirusgenus; Shigella flexneri phage SfV, Mulike-virus genus). This strongly argues against a
(A) (B)
Extended sheath
Contracted sheath
Tail fiber
Baseplate
Tube
SpikeBaseplate hub
Figure 1. Structure of a Bactericidal Tailocin. (A) The structure consists of a rigid tube (orange subunits), contractible sheath (blue subunits), lipopolysaccharide(LPS)-targeting tail fibers (red) attached to the baseplate (gray), and spike (black) connected via the baseplate hub (pale yellow) to the central tube. (B) Tail-tubearchitecture of pyocin R2 in extended conformation (EMDB-6270, PDB 3J9Q) with top view of a transverse section showing a hexameric disc (marked with a box in panelA). Two sheath protomers (cyan and blue) and two tube protomers (yellow and orange) are shown in surface representation; the other subunits (cartoon) are shown ingray (sheath) and white (tube).
588 Trends in Microbiology, October 2015, Vol. 23, No. 10
OMX&3D.SIM
eDNA facilitates,interstitial,biofilm,expansion
Gloag et#al#&#Whitchurch,.2013, PNAS,#110:11541611546
0
50
100
150 ***
- +
Inte
rstit
ial b
iofil
m a
rea
(mm2 )
Glass.slideGellan Gum/Agar
Glass.coverslip
eDNA:• maintains.cell.alignment• coherence.with.neighbours• increases.frequency.of.individual.movements
6 DNaseI +.DNaseI
Twitching)motility.mediated)biofilm)expansion
• Self&organised via0physical0means:• Furrows0forged0in0substratum0by0aggregates0confine0following0cells0into0“highways”0that0guide0cell0movements0(“Stigmergy”)
• eDNA is0required0to:0• facilitate0cell0movements0(! frequency0tfp binding)• control0traffic0flow0throughout0furrow0network0(alignment,0coherence)• assemble0“bulldozer”0aggregates0to0forge0interconnected0furrows• Furrows0confine/concentrate0eDNA
A
with0attached0tfp
with0unattached0tfp
eDNA
velocity+0DNase I& DNase I
Gloag et0al,0Proc Natl Acad Sci USA,02013Gloag et0al,0Comm Integr Biol,02013Gloag et0al,0Scientifica,02015
Exploiting*trail*following*tendencies
• Can%we%exploit%this%new%understanding%of%basic%biology%%(biophysics)%to%develop%innovative%(non;antibiotic)%approaches%to%infection%control?• Our%observations%show%that%furrows%guide%bacterial%movement• Can%we%slow%rate%of%biofilm%expansion%along%medical%devices%by%introducing%furrows?
Catheter'Associated.Urinary.Tract.Infections.(CAUTIs).
• 40%%of%all%nosocomial%infections
• Largest%reservoirs%of%multi6drug%resistant%‘super6bugs’%in%healthcare%facilities%• Risk%of%CAUTIs%increases%3610%%each%day%of%catheterisation
• Within%30%days%100%%of%patients%will%develop%bacteriuria
• Complications:%pyelonephritis,%bacteremia
• Current%approach:%remove%Foley%catheters%within%3%days%to%prevent%bladder%infections
• Colonisation of%catheter%surface%within%urethra%is%asymptomatic• 2/3%of%CAUTI’s%are%due%to%interstitial%biofilms
Intraluminal%biofilmInterstitial%biofilm Intraluminal%biofilm
Tissue Interstitial%biofilm
Engineered(furrows(impede(biofilm(expansion(by(P.#aeruginosa
InoculumPDMS
Growth1mediaSlide
Furrows Smooth
Gloag et1al,1Frontiers.Microbiol,12017
Inoculation1pointFurrows
5 10 20 50 1000
100
200
300
400
500
Furrow width (µm)
Mig
ratio
n ra
te (µ
m/h
)
FurrowsSmooth
** *
**
Narrow&furrows&inhibit&biofilm&expansion&by&other&CAUTI&pathogens&by&disrupting&collective&behaviours
5 5 5 10 20 50 100
0
20
40
60
80
Furrow width (µm)
Per
cent
age
redu
ctio
n (%
)
P. aeruginosaPr. vulgaris20 µm IF
10 µm IF
15 µm IF
Gloag et(al,(Frontiers)Microbiol,(2017
• 405(–fold(decrease(in(expansion(rate
• Can(potentially(extend(time(before(catheter(removal((from(3(days(to(10015(days)
• No(antibiotics!
Combating*infection*by*exploiting*bacterial*lifestyles
Today&I’ll&discuss&my&team’s&attempts&to&understand&and&exploit:1. Biofilm&expansion&along&implanted&medical&devices2. Tolerance*of*!7lactam*antibiotics
• !!lactams(are(the(most(widely(used(class(of(antibiotics(world!wide• Includes(penicillins,(carbapenems,(monobactams,(cephalosporins• "nhibit(peptidoglycan(synthesis(by(inhibiting(transpeptidase(activity(of(PBPs• Induce(filamentation and(rapid(lysis in(many(bacterial(species
!!lactam'antibiotics
To evaluate the generality of the observed progression forother beta-lactams, we carried out similar experiments for cefsu-lodin, a beta-lactam with a different profile from cephalexin andampicillin. Cefsulodin targets PBP1a and PBP1b in E. coli, and itis generally considered to be elongation specific (Jacoby andYoung, 1991). We first examined cells grown in liquid cultureunder cefsulodin treatment. Bulges still formed at the midcellsite; however, YFP and membrane stain images of these cellsdid not show membrane gaps near the bulging site (Figure 3A).Selective images of a representative cell show that, unlike ceph-alexin and ampicillin, cefsulodin does not block septation; therepresentative cell underwent two rounds of division before lysis.Bulges formed at nascent poles on septated filaments, and lysis
occurred separately in daughter cells (Figure 3B; Movie S2). Weconclude that there is a common pathway to cell lysis under allbeta-lactams tested. However, morphological characteristicsof each step vary for different classes of beta-lactams basedon their specific cellar targets.
Three Modes of Bulge Formation within Isogenic CellPopulationsDo all E. coli cells follow the same morphological dynamics asshown in representative cells? To answer this question, werecorded and analyzed the morphological dynamics of !200wild-type cells under cephalexin treatment, focusing on bulgeformation. We found that cells separated into three distinct
0 10 20 30 40 50 60 700
2
4
6
8
time (min)
L and
D ( µ
m)
69 69.5 70 70.5 71 71.5 72 72.50
2
4
6
8
time (min)
L and
D ( µ
m)
right before lysis (t4)
lysis begins(t5)
drug added (t0)
filamentation(t1)
bulge formation begins (t2)
bulge formation ends (t3)
A
0
1
2
t
D
filamentationbulging
lysist0
t1
t2
t3t4t5
cell length Lbulge depth D
τBL = t4-t2τB = t3 t2
B
C
ttttttttt
-
Figure 2. Live-Cell Microscopy with Automated Imaging Analysis Reveals Bulge Formation as an Intermediate Step toward Lysis(A) Selected images of a representative E. coli cell at different stages of cephalexin treatment (yellow, cell contour; red, cell length; cyan, bulge depth). The time of
these snapshots (t0!t5) is indicated in gray dots in (B).
(B and C) Measurement of cell length (L) and bulge depth (D) of a representative E. coli cell throughout beta-lactam treatment (B) and during bulging and lysis
(C, zoom-in view of the box in B). Both bulge-formation time (tB) and bulge lifetime (tBL, zoom-in inset in C) are defined based on bulge-depth measurements.
Adaptive time resolutions were adopted to characterize processes with different kinetics: 1 min/frame for the first 30 min after adding drug, 12.5 s/frame for
filamentation and bulge stagnation, and!125 ms/frame for bulge formation and final lysis. Notice the simultaneous cell-length shrinkage during the abrupt bulge
formation and the second increase in bulge depth right before lysis.
Scale bar represents 2 mm. See also Figure S2 and Movie S1.
Molecular Cell
Cell-Shape Dynamics under Beta-Lactam Treatment
Molecular Cell 48, 705–712, December 14, 2012 ª2012 Elsevier Inc. 707
Yao(et(al,(Molecular(Cell,(2013
P.#aeruginosa#meropenem'MIC'vs'MBC
32 16 8 4 2 1 0.5 0.250.1
25
0.062
5
0.031
25
0.015
625
0
5000
10000
15000
20000
Concentration (µg/mL)
Fluo
resc
ence
MBC (Resazurin)
32 16 8 4 2 1 0.5 0.250.1
25
0.062
5
0.031
25
0.015
625
0.0
0.2
0.4
0.6
0.8
Concentration (µg/mL)
OD
605
MIC (OD)
• P.#aeruginosa#can%tolerate%very%high%concentrations%of%!2lactams• Bacteriostatic%not%bactericidal%• eg PA14%meropenem%MIC%=0.521%vs%MBC%=%32%µg/mL
• Mechanism%of%!2lactam%tolerance%by%P.#aeruginosa#is%unclear
Resazurin%redox%dye%(metabolic%activity)–reduced%to%resorufin (fluorescent)Proportional%to%aerobic%respiration
Tolerance)of)! lactam)antibiotics)by)P.#aeruginosa
• All$classes$of$!+lactam$antibiotics$(penicillins,$carbapenems,$cephalosporins,$monobactams)$induce$en#masse#transition$to$spherical$cell$morphotype• Up$100%$of$population$transitions$to$viable$spherical$cells$within$$hrs
5x)MIC)Meropenem0)h 1)h
4)h 24)h
5$µm
Rods
Transitioning
Spheres
LiveDead
Monahan$et#al#&$Whitchurch,$2014,$AAC#58:1956+62$
0)h 2)h 24)h
1$µm
!!lactam'induced'spherical'cells'lack'a'functional'cell'wall
• Loss%of%rod%cell%shape%suggests%cell%wall%is%defective
Bacillary%cell
!7lactam%induced%spherical%cell
History(repeating….• Rapid induction,of,spherical,cells,of,P.#aeruginosa by,!5lactams,is,not,a,new,observation.• 1969,• “sphere5with5two5horns” 2(h 24(h
1,µm
Spherical*cell*transition*is*reversible
• Spherical+cells+revert+to+rods+when+transferred+to+antibiotic5free+medium• Rods+grow+and+divide+normally
0*h 2*h
6*h 24*h
Are$!%lactam%induced$spherical$cells$L%forms?• L#forms)are)bacterial)cells)with)absent)or)defective)cell)walls)that)can)grow%and%proliferate through)processes)not)involving)normal)cell)division)machinery
• L#forms)first)described)in)1930’s)by)Emmy)Kleineberger# Lister)institute• Literature)over)several)decades)of)early)20th centuries)indicates:
• All)“wild”)bacterial)species)are)likely)to)have)capacity)to)exist)as)L#forms• Bacterial)cells)can)readily%transition%between)normal)and)L#forms
• The)biological)role)of)L#forms)is)unknown• Are)L#forms)an)artefact of)the)lab)or)do)they)have)a)biological)function?
Extracellular)vesiculationBacillus,)E.)coli
Intracellular)vesicles)released)by))lysis)of)“mother”)cell#Listeria,)Enterococcus
!!lactam!induced,spherical,cells,are,L!forms
• P.#aeruginosa#has%the%capacity%to%rapidly%and%reversibly%transition%enmasse#to%L4form%lifestyle%in%response%to%exposure%to%!4lactam%antibiotics• Enables%survival%and%development%of%elevated%resistance%to%!4lactams• Loss%of%cell%wall%armour?%Exploitable%‘Achilles’%heel’%vulnerability?%
!4lactam
A"new"way"to"kill"bacteria:"exploiting"the"!5lactam"tolerance"response?
– !5lactam"induced"L5forms"lack"intact"cell"wall• Greater(access(of(antibiotics(to(sites(of(action
• Loss(of(function(of(multi4drug(efflux(pumps(
• Increased(susceptibility(to(antibiotics?
Exploiting*P.#aeruginosa L,forms
• Hypothesis:– Antimicrobial.peptides.may.efficiently.kill.L7forms.by.interacting.with.exposed.regions.of.cytoplasmic.membrane• Antimicrobial.peptides.tested:.
• Nisin (Lactobacillus+lactisbacteriocin)7 food.preservative
• LL737.(Cathelicidin,.human.defensin)
• Mellitin (bee.venom)
Antimicrobial,peptides,kill,P.#aeruginosa L3forms
Untreated LL337,alone Nisin,alone
128$µg/mL 128$µg/mLMonahan$et#al#&$Whitchurch,$2014,$AAC#58:1956<62
AMPs%induce%rapid%lysis of%L4forms
– 99%$killing$within$1$h$for$LL237
Playback$at$10x$real$timeStarts$13$min$after$peptide$addition
Scale$bar$3$µmMonahan$et#al#&$Whitchurch,$2014,$AAC#58:1956262
Summary' !'lactam+induced+CWD+cells+(L'forms)• P.#aeruginosa#undergoes*rapid*and*reversible*en#masse#transition*to*L3form*lifestyle*upon*exposure*to*!3lactam*antibiotics*• Does*not*require*specialised growth*conditions3
• osmoprotectants not*required,*occurs*in*sputum*&*serum• May*provide*mechanism*for*tolerance*of*!3lactam*antibiotics*in#vivo• May*be*exploitable*to*develop*novel*antibiotics
• Overcome*the*outer3membrane*barrier*of*Gram*negative*pathogens
Summary
• Bacteria)have)the)capacity)to)adopt)a)number)of)different)lifestyles)that)enable)tolerance)of)stressful)environments• Alternate)lifestyles)enable)survival)until:
• antibiotic-levels-fall-below-MIC-(pharmacodynamics/pharmacokinetics))• or)resistance-is-acquired through)elevated)expression)of)intrinsic)resistance)genes))(!?lactamases,)efflux)pumps),)mutation)or)horizontal)gene)transfer.
• Understanding)bacterial)lifestyles)may)provide)novel)opportunities)to)exploit)to)combat)infection
Planktonic) Biofilms
CWD)cells
AcknowledgementsThe0ithree institute,0UTS
Lynne%TurnbullErin%GloagLeigh%MonahanIbrahim%AwadhDavid%MaxGiulia%BallerinJames%LazenbySarah%OsvathRosalia Cavaliere
Microbial0Imaging0Facility,0UTS
Collaborators:UTSNicola%PettyIan%CharlesMatt%WandMilos%Toth
Uni of0Melbourne@ ChemistryMichelle%Gee
Swinburne0Uni of0TechnologyScott%Wade