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Outsmarting the Superbug: developing alternatives to antibiotics

Susan A. McDowell, Ph.D.Department of BiologyBiotechnology ProgramBall State UniversityMuncie, INsamcdowell@bsu.edu1Staphylococcus aureus (staph, MRSA)Morbidityendocarditisosteomyelitis

Mortality most common infectious agent leading to sepsis1 million sepsis-associated deaths (1999-2005)

2Risk 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

3Treatment of infectionAntibiotic

Mode of Action

TetracyclineBinds to bacterial ribosomes

QuinolonesStop bacterial DNA replication

Penicillin Blocks bacterial cell wall formationCephalosporins Block bacterial cell wall formation MethicillinBlocks bacterial cell wall formation VancomycinBlocks bacterial cell wall formation 4Mechanisms of antibiotic resistance

AntibioticEnzymes that alter antibiotics (chloramphenicol aceyltransferase)Pumps that transport antibiotics out of the cell

AntibioticEnzymes that degrade antibiotics (penicillinase)AntibioticPlasmid with resistance genes(ampicillin)Figure adapted from Lisa Melton: http://www.wellcome.ac.uk/doc_WTX026110.htmlChromosomal changes alter amino acid sequence/binding site of antibiotic(MRSA new transpeptidase)5Challenge: Increase in antibiotic resistanceAdapted from Clinical Infectious Diseases, 2006, 42: 389-91

6HypothesisBlocking infection at the level of the host is protective.

7How we study host cell invasion

Human umbilical vein endothelial cell (HUVEC)8Host cell

Cell Membrane9Host cell

Cell MembraneNucleus10Host cell

Cell MembraneCytoplasmNucleus11Host cell

Cell MembraneCytoplasmNucleusVimentin 12Host cell

S. aureusCell MembraneCytoplasmNucleusFluorescent secondary antibody binds to protein A of S. aureusProtein AFc region of IgGS. aureusATCC#2921313

S. aureusFibronectin binding proteinFibronectin Integrina5b1Primary mechanism of host invasion14

S. aureusFibronectin binding proteinFibronectin Integrina5b115

S. aureusFibronectin binding proteinFibronectin Integrina5b116

Our focus investigate host cell responses that facilitate invasion and could be blocked pharmacologically

17UninfectedInfected (1 hr)The first host cell response we noticed: Actin stress fibers disassemble during invasion by S. aureus WHY?

18The 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/V5Internalized bacteria (% Control +/- SEM)*20406080100120Horn, et al., JPET 326: 2008 *less than control; p < 0.05 Students t-test ML 141 (recently identified inhibitor of CDC41)

**Derivatives of ML 141Could a compound that blocks CDC42 activity inhibit invasion?

Could we improve upon that compound?

ML 141

More derivatives of ML 141Is the inhibition of CDC42 limiting stress fiber disassembly?

Do the derivative compounds work?

RSM 06

Potential OutcomesWe 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.SignificanceAddressing questions of basic science how S. aureus is internalizedhow infection spreads

Evaluation of the therapeutic benefit of inhibitorsduring systemic infectionas a prophylactic for invasive procedures

AcknowledgementsThe National Institutes of Health Derron BishopFran RushingJohn McKillipSharm KnechtKelly GammonChris VlahosJulie Clark (Birdsong)Charron JohnsonMary HornTerri AbrahamCatherine VolkLaura MichaelParker SiddallKelsey HaaningAmy Pierce (Brown)Jacy WillisRay KenneyIndiana Academy of ScienceLilly VSarita TonyXiaoling LiuTiera LibyKristin AbelAmanda ReeseDan HoranSponsored Programs OfficeHonors CollegeTom Kirchhausen Heather BrunsJanelle OwensDaniel PetrovichTraci StankiewiczAlys JordanNickie MayRobin DeWaltAshley ZahrtJacob HenryShana EllisAmber HamptonJulie PrattJim MitchellLarry SklarGeorge TegosMark HaynesCollege of Sciences and HumanitiesKatie ReedDiana SantanaLindy CaffoRob Sammelson27