Outsmarting the Superbug: developing alternatives to antibiotics Susan A. McDowell, Ph.D. Department of Biology Biotechnology Program Ball State University.

Outsmarting the Superbug: developing alternatives to antibiotics

Susan A. McDowell, Ph.D.Department of BiologyBiotechnology ProgramBall State UniversityMuncie, INsamcdowell@bsu.edu

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

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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