Nanotechnology and microbiology: basic science and ...chtm.unm.edu/incbnigert/Presentations/Nanotech Symposia_Rebeil.pdf · Nanotechnology and microbiology: basic science and applications

Nanotechnology and microbiology: basic science and applications that can

impact cell biology

Roberto Rebeil1st Annual Symposium Integrating Nanotechnology

With Cell Biology and Neuroscience08/15/07

Focus of research is on host-pathogen interactions to understand and combat disease

Animal cellBacterial cell

Virus

Illnesses spread by insects represent a significant portion of emergent and reemergent diseases

Temporal-geographical spread of WNV in the US through mosquitoes

Plague (Drug resistance)

Tularemia

Lyme disease

Relapsing fever

Typhus

West Nile virus

Rocky Mountain Spotted Fever

Dengue

Yellow fever

Malaria (Drug resistant)

Y. pestis is a bacterial pathogen of significant historical and current interest

• Justinian Plague (541-544)• Black Death (1347-1351)• Oriental Plague (1855-1900)

• Multiple recent smaller epidemics• Classified as a reemergent pathogen• Classified as a biowarfare/bioterrorism agent

Y. pestis is maintained in nature by flea- rodent enzootic cycles

40-60%Lethality

99%Lethality

Both the virulence and geographical distribution of Y. pestis are linked to the flea vector

Various organ systems:liver, spleen, lung

Bloodstream: bacteria/ml?ID50 = 1 X 108 bacteria/ml

21 oC

37 oC

Y. pestis virulence factors: anti-phagocytic and anti-inflammatory

Cornelis et al., 2002

F1 pseudocapsule V-antigen

Atomic force microscopy can help to visualize bacterial nanostructures

Yersinia pestis 21 ºC Yersinia pestis 37 ºC

Jonas et al. 2007

Novel microscopy techniques like HSI-MVCR help to visualize true host-pathogen interactions

E. coli pAsRed2 Y. pestis pAsRed2

Y. pestis pathogenesis is dependent on both modified lipid A and YOPS

21 °C

37 °CTN

F-α

(pg/

mL)

X 1

0

0

200

400

600

800

1000

1200

Neg. control 1.0 pg 10.0 pg 100.0 pg 1.0 ng 10 ng

LPS concentration

Y. pestis LPS 37oCY. pestis LPS 21oCUntreatedE. coli LPS (10 ng)

O

O

O

OHN

O

OH

O

O

O

O

OH

HNO

OH

O

P

P

O

O

OH

HOO

OH

OH

OOOO

O

O

O

O

O

O

HNO

O

OH

O

O

O

O

OH

HNO

OH

O

P

P

O

O

OH

HOO

O

OH

OH

O

O

O

O

OH

HNO

OH

OH

O

O

O

O

OH

HNO

OH

O

P

P

O

O

OH

HOO

OH

OH

Rebeil et al., 2004

TIRF microscopy permits the study of receptor- ligand interactions on the cell surface at the

nanoscale level

RAW 264.7 mouse macrophage

= fluorescently labeled LPS

Y. pestis transmission by fleas is by a unique mechanism

Novel application of X-ray photoelectron spectroscopy (XPS)

3Na 1sN Auger

O Auger

2

Fe 2p

1

Si 2pSi 2sP 2pP 2s O 2s

1

23

Element At. % Mass %

C 73 66O 19 23N 6 7Si 1 2P 0.4 1Fe 0.3 1Na 0.1 0.2

C 1s

N 1s

O 1s

XPS shows that significant changes occur to the flea-bloodmeal after digestion

C/N

YE1YE2

YE Hem

in1YE H

emin2 LBBHI

FBS1 HS MBFBM 1h

rFBM 2h

r

0123456789

10111213

YE1YE2YE Hemin1YE Hemin2LBBHIFBS1HSMBFBM 1hrFBM 2hr

O/N

YE1YE2

YE Hemin1

YE Hemin2 LB BHI

FBS1 HS MBFBM 1h

rFBM 2h

r

0

1

2

3

4YE1YE2YE Hemin1YE Hemin2LBBHIFBS1HSMBFBM 1hrFBM 2hr

1 hr 2 wks

Time of Flight- Secondary Ion Beam spectroscopy will provide additional information

720 730 740 750 760 770 7800

50

100

150

Boxc

ar b

inni

ng

Integral: 22606 \\S825939\CURRENT ANALYSIS\REBEIL AU-MICA SAMPLES 9-06\DPPC-20SEP2006-0

766745725 776747727723717 779743 774753751 771761729 733722715 737 763 773749720 755 765759

735

757

750 800 850 900 950 1000 10500

500

1000

1500

2000

2500

Boxc

ar b

inni

ng

Integral: 71472 \\S825939\CURRENT ANALYSIS\REBEIL AU-MICA SAMPLES 9-06\DOPC-20SEP2006-

825801745 772 817

787983

809

Free Iron plays a crucial role in Y. pestis gene regulation: XPS can help determine the state of

iron in the flea

3Na 1sN Auger

O Auger

2

Fe 2p

1

Si 2pSi 2sP 2pP 2s O 2s

1

23

Element At. % Mass %

C 73 66O 19 23N 6 7Si 1 2P 0.4 1Fe 0.3 1Na 0.1 0.2

C 1s

N 1s

O 1s

Y. pestis flea mass

Lab generated mass

XPS analysis reveals different Iron species in Y. pestis clumps depending on growth conditions

1

23

45

61

23

456

Fe2p 3/2Fe2p 1/2

1

2

34

5

12

345

Fe2p 3/2Fe2p 1/2

Applications to cell biology and pathogenesis: host-pathogen, cell-cell, neuron-synapse.

Note: Great technology requires great biology

Aknowledgements

My Lab• Bryan Carson• Kevin Crown• Bryce Ricken

Rocky Mountain Labs• Joe Hinnebusch• Clayton Jarret

Sandians• Allan Burns• Jeri Timlin• Tony Ohlhausen• Bill Wallace• Luke Brewer• David Haaland• Howland Jones• Mike Sinclair