A component of the innate immune response prevents ... · A component of the innate immune response prevents bacterial biofilm development Singh, P ... immune response prevents biofilm

A component of the innate immuneresponse prevents bacterial biofilmdevelopment

Singh, P. K., Parsek, M. R., Greenberg, E. P., and Welsh, M. J.Nature 417:552-5

A Little on BiofilmsA Little on Biofilms Biofilms are communitiesBiofilms are communities

for bacteriafor bacteria

Once established difficultOnce established difficultto removeto remove

Safe haven for bacteriaSafe haven for bacteriafrom from biocidalbiocidal compounds compounds

Link betweenLink betweenenvironmental and clinicalenvironmental and clinical

Donlan and Costerton Clin. Microbiol. Rev. 15:167-93.

Study DesignStudy Design Authors proposed that some factor of the innateAuthors proposed that some factor of the innate

immune response prevents biofilm formationimmune response prevents biofilm formation

LactoferrinLactoferrin - - chelatorchelator of iron, present in bodily of iron, present in bodilysecretionssecretions

GFP expressing GFP expressing P. P. aeruginosaaeruginosa used used

Flow cell chambers for biofilm developmentFlow cell chambers for biofilm development

+/- +/- lactoferrinlactoferrin, , conalbuminconalbumin, or , or deferoxaminedeferoxamine

ConfocalConfocal microscopy with time lapse microscopy with time lapse

Checked biofilm resistance to Checked biofilm resistance to biocidalbiocidalcompounds (compounds (tobramycintobramycin and H and H22OO22))

A Little on Flow CellA Little on Flow Cell

Monitor bacterial attachment in a systemMonitor bacterial attachment in a system

Control rate of flowControl rate of flow

Integrated with microscopy techniquesIntegrated with microscopy techniques

ScopeSlide

FreshMedia

SpentMedia

Iron...Who Cares?Iron...Who Cares? Bacteria, animals, and plants need ironBacteria, animals, and plants need iron

Activity of some enzymes require ironActivity of some enzymes require iron ex: ex: NorRNorR in in E. coliE. coli ( (YgaAYgaA in in S. S. typhimuriumtyphimurium))

Activated by NO in anaerobic environmentActivated by NO in anaerobic environment

NO produced by macrophage (NO produced by macrophage (iNOSiNOS))

D'Autreaux, Tucker, Dixon, & Spiro. Nature 437:769-72.

Figure 1 - Growth CurveFigure 1 - Growth Curve

Used 20 _g/mL

Figure 2 - Figure 2 - ConfocalConfocalMicroscopyMicroscopy LactoferrinLactoferrin prevented prevented

formation of biofilmformation of biofilm

What if other bacteriaWhat if other bacteriaare present?are present?

4 hrs

7 days

24 hrs

3 days

Figure 3 - Bacteria BehavingFigure 3 - Bacteria BehavingBadlyBadly Squatters -Squatters -

localized in 15 localized in 15 __mmcircle until divisioncircle until division

Ramblers -Ramblers -remained attached,remained attached,but moved outsidebut moved outsidecirclecircle

Flyers - detachedFlyers - detachedfrom surfacefrom surface

Time Lapse MicroscopyTime Lapse Microscopy

Monitored bacterial behavior w/ or w/oMonitored bacterial behavior w/ or w/olactoferrinlactoferrin

Control video (no Control video (no lactoferrinlactoferrin))

Experimental video (with Experimental video (with lactoferrinlactoferrin))

Figure 4 - Twitching MotilityFigure 4 - Twitching Motility Specialized motilitySpecialized motility

mediated by type 4mediated by type 4pilipili Increase in type 4Increase in type 4

genes or proteins?genes or proteins?

40 have been40 have beenidentified in identified in P.P.aeruginosaaeruginosa ((J. S. Mattick,Annual Rev. Microbiol 56:289-314))

Twitching motilityTwitching motilitystimulated by ironstimulated by ironchelatorschelators and may and mayprevent biofilmprevent biofilmformationformation

Fe unsat. lactoferrin

No lactoferrin

lacto. (mutant)

No lacto. (mutant)

Fe sat. lactoferrin

Figure 5 - Biofilm ProtectionFigure 5 - Biofilm Protection Susceptibility to Susceptibility to tobramycintobramycin and H and H22OO22

Presence or absence of iron Presence or absence of iron chelatorchelatorconalbuminconalbumin

Questions/Comments/ConcernsQuestions/Comments/Concerns