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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