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&acute&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

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625

0

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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