From cell surface adhesion to From cell surface adhesion to final architecture of final architecture of XylellaXylella

fastidiosafastidiosa biofilms: investigation biofilms: investigation with advanced microscopywith advanced microscopy

Mônica Mônica Mônica Mônica A.CottaA.CottaA.CottaA.CottaUniversidadeUniversidade EstadualEstadual de Campinas (UNICAMPde Campinas (UNICAMP))

InstitutoInstituto de de FísicaFísica GlebGleb WataghinWataghin, Campinas, Campinas--SP, BrazilSP, Brazil

InP AuInPInPInAs

Materials ScienceMaterials Science

Semiconductor nanowires

L.H.G.Tizei

D.S.Oliveira et al. Nano Letters (2013)

T.Chiaramonte et al., Nano Lett (2011)

L.H.G.Tizei et al., Phys.Rev.Lett. (2011)

D.S.Oliveira et al. Nano Letters (2013)

Eshelby twist

Scanning Probe MicroscopyScanning Probe Microscopy

A.C.Narvaez et al.,

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P.Sahoo et al., J.Phys.Chem.C (2011)

Electrical modesElectrical modesElectrical modesElectrical modesCCCC----AFMAFMAFMAFMKPFMKPFMKPFMKPFM

A.C.Narvaez et al., Nanotechnol. (2009)

J.Phys.Chem.C (2011)

BDA C

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Dibucaine (DBC)

G.S.Lorite et al., Biophysical Chemistry (2009)

XylellaXylella fastidiosafastidiosa

IFGW/LNB:IFGW/LNB:IFGW/LNB:IFGW/LNB:Prof. Mônica A.CottaAlberto L.D.Moreau (PhD) Gabriela S.Lorite (M.Sc)Eng. João H. Clerici

IAC/CCIAC/CCIAC/CCIAC/CC::::Dr.Alessandra A.de SouzaCarol Rodrigues

Materials Science & MicroscopyMany years ago…. (~2008)

Eng. João H. Clerici

Univ. Ulm, Germany:Univ. Ulm, Germany:Univ. Ulm, Germany:Univ. Ulm, Germany:Prof. Boris MizaikoffDr. Christine Kranz

Dr. Richard Janissen (PD)

IFGW/LNB:IFGW/LNB:IFGW/LNB:IFGW/LNB:Prof. Mônica A.CottaDr. Richard Janissen (now TU Delft)Dr. Prasana SahooAlberto L.D.Moreau (now ITFSP)Gabriela S.Lorite (now Univ.Oulu)Duber M.MurilloEng. João H. Clerici

IAC/CCIAC/CCIAC/CCIAC/CC::::Dr.Alessandra A.de SouzaCarol RodriguesDr.Juarez P.TomazBarbara Niza

XylellaXylella fastidiosafastidiosa -- nownow

Prof. Hernandes F. de CarvalhoProf. Carlos L.Cesar

Profa. Marcia L.A.TemperiniMarcelo Nobrega

XylellaXylellaXylellaXylella fastidiosafastidiosafastidiosafastidiosa

Gram negative bacteriasmall slender rods,~200–300 nm in diameter and 0.9–3.5μm in lengthSlow growth rate (~6-24hs) and harmless

Several economically important diseases in citrus plantscitrus variegated chlorosisannual losses over USD 300million to citrus industry

First phytopathogenic bacterium with a complete genome sequenceNature 406406406406, 151 (2000)

Pathogenicity results from biofilm formation inside the plant xylem vessels

Blockage and consequent water stress plant starves!

http://www.cnr.berkeley.edu/xylella/

Bacterial Bacterial BiofilmsBiofilms

Biofilm Formation in 5 steps: Biofilm Formation in 5 steps: Biofilm Formation in 5 steps: Biofilm Formation in 5 steps:

(1)(1)(1)(1) Pioneer bacterial cells reversible attachment to surface Pioneer bacterial cells reversible attachment to surface Pioneer bacterial cells reversible attachment to surface Pioneer bacterial cells reversible attachment to surface

(2)(2)(2)(2) Irreversible attachement (influence of EPS Irreversible attachement (influence of EPS Irreversible attachement (influence of EPS Irreversible attachement (influence of EPS ---- extracellular polymeric substanceextracellular polymeric substanceextracellular polymeric substanceextracellular polymeric substance))))

(3) (3) (3) (3) CellCellCellCell----Cell adhesion and proliferation Cell adhesion and proliferation Cell adhesion and proliferation Cell adhesion and proliferation

(4) (4) (4) (4) Mature biofilmMature biofilmMature biofilmMature biofilm

K. Sauer, Genome Biology 4444, 219 (2003)

(5) Detachment (5) Detachment (5) Detachment (5) Detachment

SPM SPM SPM SPM Large sizesBiological sample reproducibility issues

Fractal analysisFractal analysisFractal analysisFractal analysisJAP (2009)JAP (2009)JAP (2009)JAP (2009)

N.Killiny et al, Appl.Environ.Microbiol. 75757575, 521 (2009)

Adhesiveness and life cycleAdhesiveness and life cycle

S. Chatterjee et al, Ann.Rev.Phytopathol. 46464646, 243(2008)

K.L.Newman et al., PNAS 101101101101, 1737 (2004)

Reversible adhesion:Reversible adhesion:Reversible adhesion:Reversible adhesion:Electrostatic natureElectrostatic natureElectrostatic natureElectrostatic nature

Initial adhesion stagesInitial adhesion stages

K. Sauer, Genome Biology 4444, 219 (2003)

Irreversible adhesion: Irreversible adhesion: Irreversible adhesion: Irreversible adhesion: Chemical natureChemical natureChemical natureChemical nature

SPM & IR SPM & IR –– Glass surfacesGlass surfaces

Live Bacteria grown inside AFM liquid cell at 28C

ATRATRATRATR---- IRIRIRIR

Conditioning film due to Conditioning film due to Conditioning film due to Conditioning film due to culture medium:culture medium:culture medium:culture medium:changes in changes in changes in changes in hydrophobicityhydrophobicityhydrophobicityhydrophobicity

G.S.Lorite et al., Journal of Colloids and Interface Science 359 (2011) 289–295

Dry samples (cells around BF, 7 days)

XylellaXylella fastidiosafastidiosa BiofilmsBiofilms

1800 1600 1400 1200 10000.00

0.04

0.08

0.12

0.16

0.20

1578.6

995.2

1043.2

1077.2

1111.91300.9

1408.9

1460.0

1549.8

1620.1

Abs

orpt

ion

Inte

nsity

(u.

a.)

Experiment A Experiment B PW conditioning film

1656.8

0 500 1000 1500 2000 2500 3000 3500

0.00

0.01

0.02

0.03

0.04

0.05

Hei

ght P

eaks

Abs

ropt

ion

Time (min)

Experiment B(b)

ATRATRATRATR---- IRIRIRIR

polysaccharide groups (C–O–P, P=Osymmetric stretch, C–C and C–O stretch)

polyphosphate groups

Wavenumber (cm -1)

G.S.LoriteG.S.LoriteG.S.LoriteG.S.Lorite et al.et al.et al.et al.Colloid and Surfaces B: Colloid and Surfaces B: Colloid and Surfaces B: Colloid and Surfaces B: BiointerfacesBiointerfacesBiointerfacesBiointerfaces (2013)(2013)(2013)(2013)

Different surfacesDifferent surfaces

G.S.Lorite et al., PLOS One (2013)

Different surfacesDifferent surfaces

- What happens after incubation in culture media (PW)?

- How homogeneous are these surfaces (roughness and electrically)?

celullose (EC, CA): zeta potential ~ -10 to -50 mV

SPM analysisSPM analysisSi EC CA

Surface potentialAfter PW incubation

G.S.Lorite et al., PLOS One (2013)

Force spectroscopyForce spectroscopy

Tip functionalization XadA1 adhesinePBS buffer

control

Surface

Protein

offset

control

AC

AC

Force spectroscopyForce spectroscopy

G.S.Lorite et al., PLOS One (2013)

Force spectroscopyForce spectroscopy

G.S.Lorite et al., PLOS One (2013)

Specific binding to CA surfaces less likely than for Si and ECSpecific binding to CA surfaces less likely than for Si and ECSpecific binding to CA surfaces less likely than for Si and ECSpecific binding to CA surfaces less likely than for Si and EC

SP + force spectroscopy: different shapes of biofilmsSP + force spectroscopy: different shapes of biofilmsSP + force spectroscopy: different shapes of biofilmsSP + force spectroscopy: different shapes of biofilms

Intriguing features

Bacterial Poles

Textures

G.Lorite et al., J.Coll.Inter.Sci. (2011)

XylellaXylellaXylellaXylella fastidiosafastidiosafastidiosafastidiosa "mutant” cells "mutant” cells "mutant” cells "mutant” cells ---- with Green Fluorescent Protein (GFP)with Green Fluorescent Protein (GFP)with Green Fluorescent Protein (GFP)with Green Fluorescent Protein (GFP)

WFM, SPM (LNB) & SDCLSM, INCT on Biophotonics

XylellaXylellaXylellaXylella fastidiosafastidiosafastidiosafastidiosa “mutant” cells“mutant” cells“mutant” cells“mutant” cells

“Follow” Xylella fastidiosa life cycle“Follow” Xylella fastidiosa life cycleValidate model for single microrganism

shared traits with human bacteriamore general understanding

ConclusionsConclusionsQuantitativeQuantitativeQuantitativeQuantitative measurements of important biological parameters:

- Roughness

- Hidrophobicity

- Electrostatic character

- Surface modification

hydrophilic surfaces

- Surface modification

for different materials on which Xyllela fastidiosa can growBiology: complete life cycle evolution of Xylella fastidiosa

Biofilm structure that maximizes nutrient flow and adhesion to different surfaces

issues on mechanical stabilityadaptation to host/vector transmission mechanism

Important:Important:Important:Important: physicists, biologists, chemists working in same environment!!physicists, biologists, chemists working in same environment!!physicists, biologists, chemists working in same environment!!physicists, biologists, chemists working in same environment!!

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