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Outsmarting the Superbug: developing alternatives to antibiotics
Susan A. McDowell, Ph.D.Department of BiologyBiotechnology ProgramBall State UniversityMuncie, [email protected]
Staphylococcus aureus (staph, MRSA)
Morbidityendocarditis
osteomyelitis
Mortality
most common infectious agent leading to sepsis
1 million sepsis-associated deaths (1999-2005)
Risk factors for virulent S. aureus infections
Hospitalization most common cause of nosocomial cases of pneumonia and surgical wound infection
Invasive procedures hip or knee replacement, catheterization
Immunosuppressionage, HIV
Community exposurecontact sports
Treatment of infectionAntibiotic Mode of Action
Tetracycline Binds to bacterial ribosomes
Quinolones Stop bacterial DNA replication
Penicillin Blocks bacterial cell wall formation
Cephalosporins Block bacterial cell wall formation
Methicillin Blocks bacterial cell wall formation
Vancomycin Blocks bacterial cell wall formation
Mechanisms of antibiotic resistance
Antibiotic
Enzymes that alter antibiotics (chloramphenicol aceyltransferase)
Pumps that transport antibiotics out of the cell
Antibiotic
Enzymes that degrade antibiotics (penicillinase)
Antibiotic
Plasmid with resistance genes
(ampicillin)
Figure adapted from Lisa Melton: http://www.wellcome.ac.uk/doc_WTX026110.html
Chromosomal changes – alter amino acid
sequence/binding site of antibiotic(MRSA – new transpeptidase)
Challenge: Increase in antibiotic resistance
Adapted from Clinical Infectious Diseases, 2006, 42: 389-91
Hea
lthca
re-a
ssoc
iate
d M
RS
A in
fect
ions
(%
)
0
10
20
30
40
50
60
70
1974 1995 2004
Hypothesis
Blocking infection at the level of the host is protective.
How we study host cell invasion
Human umbilical vein endothelial cell (HUVEC)
Host cell
Cell Membrane
Host cell
Cell Membrane
Nucleus
Host cell
Cell MembraneCytoplasm
Nucleus
Host cell
Cell MembraneCytoplasm
Nucleus
Vimentin
Host cell
S. aureus
Cell MembraneCytoplasm
Nucleus Fluorescent secondary antibody binds to protein A of S. aureus
Protein AFc region of IgG
S. aureusATCC#29213
S. aureus
Fibronectin binding protein
Fibronectin
Integrina5b1
Primary mechanism of host invasion
S. aureus
Fibronectin binding protein
Fibronectin
Integrina5b1
S. aureus
Fibronectin binding protein
Fibronectin
Integrina5b1
Our focus – investigate host cell responses that facilitate invasion and could be blocked pharmacologically
Uninfected Infected (1 hr)
The first host cell response we noticed: Actin stress fibers
disassemble during invasion by S. aureus – WHY?
The second host cell response we noticed: Filopodia
formation (a different actin structure) is stimulated during
S. aureus invasion
Stankiewicz, et al., BBRC 391: 2010
Is this assembly of filopodia related to the disassembly of actin?
What we found: When CDC42, a known regulator of actin,
is mutated, host cell invasion is inhibited
HEK CDC42 C507V/V5
Inte
rnal
ized
bac
teria
(%
Con
trol
+/-
SE
M)
*20
40
60
80
100
120
Horn, et al., JPET 326: 2008 *less than control; p < 0.05 Student’s t-test
ML 141 (recently identified inhibitor of CDC41)
**
Derivatives of ML 141
Could a compound that blocks CDC42 activity inhibit invasion?
Could we improve upon that compound?
ML 141
More derivatives of ML 141
Is the inhibition of CDC42 limiting stress fiber disassembly?
Do the derivative compounds work?
RSM 06
Potential Outcomes
We will identify a compound with greater efficacy in inhibition of invasion.
Alternatively, several compounds of similar efficacy as that of ML 141 will be identified in vitro that may demonstrate greater bioavailability in vivo.
Significance
Addressing questions of basic science
how S. aureus is internalized
how infection spreads
Evaluation of the therapeutic benefit of inhibitors
during systemic infection
as a prophylactic for invasive procedures
AcknowledgementsThe N
ational
Institutes
of Health
Der
ron
Bis
hop
Fran Rushing
John
McK
illip
Sharm Knecht
Kelly Gammon
Chr
is V
laho
sJulie Clark (Birdsong)Charron Johnson
Mary Horn
Terri AbrahamCatherine Volk
Laur
a M
icha
el
Parker Siddall
Kelsey HaaningAmy Pierce (Brown)
Jacy WillisRay Kenney
Indiana
Academy o
f
Science
Lilly V
Sarita Tony
Xiaoling Liu
Tiera Liby
Kristin Abel
Amanda Reese
Dan Horan
Sponsore
d Pro
grams
OfficeHonors
College
Tom
Kirc
hhau
sen
Hea
ther
Bru
ns
Janelle Owens
Daniel PetrovichTraci Stankiewicz
Alys Jordan
Nickie MayRobin DeWalt
Ashley Zahrt
Jacob Henry
Shana Ellis
Amber Hampton
Julie Pratt
Jim
Mitc
hell
Larr
y S
klar
Geo
rge
Tego
sM
ark
Hay
nes
College of S
cience
s and H
umanities
Katie Reed
Diana Santana
Lindy Caffo
Rob
Sam
mel
son