LIFE-AND-DEATH battles rage in Robert Wheeler’s lab at theUniversity of Maine.

The combatants — zebrafish and Candida albicans — fightto the bitter end in glass-bottom microplates.

Similar perilous battles are being fought inside humans. TheC. albicans fungus is a leading cause of hospital-acquired infec-tion that annually kills several thousand patients nationwide.

During the staged scuffles in Wheeler’s lab in Hitchner Hall, anesthetizedzebrafish are injected with Candida and placed in a gelatinous material calledagarose.

A laser microscope captures and magnifies the struggles inside the zebrafishblood vessels in real time in high-definition color detail.

The microplate clashes provide the assistant professor of microbiology withthe ability to view how immune cells fight the microbe, identify genes involvedin virulence, test new drugs and learn how gene perturbations affect host-pathogen interaction.

“We’re using zebrafish to ask really specific questions that cannot beanswered another way,” Wheeler says. “These questions have been inaccessiblefor a long time. We hope to be able to better utilize existing therapies and beable to develop better therapies.”

In March 2012, Wheeler received a three-year, more than $421,600 grantfrom the National Institutes of Health to ask and answer these questions in theproject: “Genetics & Visualization of Innate Host Response to Candida albicansInfection In Vivo.”

The goal is that the resulting answers will save human lives.The grant is the most-recent funding Wheeler has received during his 13-

year quest to unravel the mysteries of Candida.

MILLIONS OF C. albicans live peacefully in digestive tracts of people withhealthy immune systems. Despite being the culprit of pesky vaginal infectionsin adults and oral infections in babies, for the most part, “the organism hasevolved to coexist rather than constantly attack,” Wheeler says. “It’s part of ournatural microflora.”

A UMaine biomedical lab looks foranswers in the transformation ofa peaceful yeast to fatal fungus

Battle linesBy Beth Staples

umainetoday.umaine.edu 2524 UMaineToday Spring 2013

umainetoday.umaine.edu 2726 UMaineToday Spring 2013

But when a person’s immune system iscompromised — as occurs with organtransplant patients and people with cancerand human immuno deficiency virus —Candida albicans transforms from peacefulyeast to an invasive, potentially fatalfungus that infects vital organs.

Candida’s Jekyll-to-Hyde conversionproves deadly for about one-third ofpeople afflicted with bloodstream in -fections of the pathogen. It’s clear, saysWheeler, that better diagnostics and thera-pies are needed.

Wheeler also uses mice to studyimmunity to C. albicans. The research isbeneficial, he says, but limited because thelive mammals aren’t see-through and don’tfit under a microscope.

But transparent tropical fish larvae

measuring a few millimeters fit the bill.And the ability to conduct experiments invivo — “within the living” zebrafish —have been and continue to be elucidating.

Zebrafish also have backbones, sharemany of the same genes as people, andhave the ability to respond to infectionsand vaccinations in ways similar tohumans, Wheeler says.

For the life-and-death battles inmicroplates, Wheeler uses engineeredzebrafish with green fluorescent immunesystem cells and fluorescent red C. albicansfungus. A laser scanning confocal micro-scope captures layered, 3D images of theskirmishes blow-by-blow, in real time.

On Wheeler’s iPad, a battle that lastsfor hours is condensed into a time-lapsedmovie that can be viewed in minutes. The

movie of green zebrafish immune cellsgobbling up red C. albicans resembles aPac-Man arcade game.

The Pac-Man reference is one exampleof how Wheeler explains the fungal host-microbial pathogen interaction in waysthat make sense to nonscientists.

He also compares sugar layers of thefungus cell wall using everyday objects andterminology, including M&M candiesand GORE-TEX.

ZEBRAFISH AND fungi are familiar foeswith apparent elaborate knowledge of eachother’s respective arsenals.

“For each mode of host immunity, thechallenger has designed a defense, which,in turn, leads the host to devise a newavenue of attack,” according to Wheeler.

For instance, innate immune cellsrecognize surface molecules of the deadlyfungus, including sugar β-glucan. Whenthe immune cells recognize the Candidacell wall, the immune system goes on highalert and responds to eliminate the threat.

In some instances, the Candida funguscovers up the β-glucan with a thickprotective cell layer, thereby blocking theimmune system’s response. Wheelercompares this dense sugar coating toGORE-TEX clothing. Just as GORE-TEX doesn’t let water penetrate, this outerlayer prevents immune molecules fromtouching the β-glucan.

Some pharmaceutical drugs makeCandida more recognizable. In addition tokilling fungi, one antifungal drug has aside effect of uncovering the β-glucan,Wheeler says.

“If we’re able to expose the β-glucan,the immune system goes crazy,” says RemiGratacap, a postdoctoral research fellowfrom Grenoble, France. “You see almost athreshold where the immune system isable to cope and if you go just past that,suddenly (β-glucan) can’t cope anymore.”

The UMaine group, says Wheeler, isalso trying to better understand howCandida gets from one place to another inthe body. Since Candida cannot moveindependently, Wheeler seeks to discoverif the pathogen is carried in immune cells.

Candida, Wheeler says, can changeshapes, from bunches of yeast to long fila-ments. Both shapes serve it well: Candidatravels easily in the blood in yeast formand penetrates tissues best as a filament.

Gratacap says that Wheeler, a propo-nent of re-examining long-standing scien-tific concepts accepted as true, is“ridiculously clever.”

WHEELER COMES naturally by it. Hispaternal grandfather is John ArchibaldWheeler, a theoretical physicist whocollaborated with Albert Einstein and isheralded for his contributions to gravita-tion and quantum mechanics. The formerPrinceton professor, who coined the termsblack hole and wormhole, died in 2008.

Robert Wheeler earned degrees fromHarvard and Stanford universities and didhis postdoctoral work at Whitehead Insti-tute, a nonprofit research institution inCambridge, Mass., where biomedicalresearchers seek to improve human health.

Wheeler, a sought-after expert in thefield, is slated to lecture this semester inGermany and France.

Wheeler lauds UMaine colleaguesCarol Kim, professor of microbiology anddirector of the Graduate School ofBiomedical Sciences and Engineering, andClarissa Henry, associate professor ofbiological sciences, for their help, collegial-ity and excellent research with zebrafish.The Zebrafish Research Facility, run byMark Nilan, is only a few steps fromWheeler’s lab.

Battle lines

The switch to drug-containing media causesCandida filaments (green fluorescence) tolose its GORE-TEX outer coat and expose theimmune-stimulating sugar β-glucan (redreactivity). The surface-label (purple) marksold growth without drugs, where β-glucan ismasked.

“It’s hard to overstate how instrumen-tal the well-run facility has been,” Wheelersays. “I really don’t know if I could havedone this work anywhere else.”

His research group has already made asignificant breakthrough discovery regard-ing C. albicans.

After receiving grants in 2008 and2009 from the Maine Agricultural andForest Experiment Station and theNational Institutes of Health, he andstudents Kimberly Brothers and ZacharyNewman started viewing interactionsbetween fungi and immune cells. Theyshowed for the first time that NADPHoxidase is required for regulation of C.albicans filamentation in vivo.

These observations, first made byBrothers, implied the deadly fungus mightspend more time inside zebrafish phago-cytes — immune cells that ingest microor-ganisms, other cells and foreign particles— than researchers had believed.

This, Gratacap says, demonstrates thatexamining these microscale battles indifferent hosts can lead to striking insights. �

“These questions have been inaccessible for a long time.We hope to be able to better utilize existing therapiesand be able to develop better therapies.”

Robert Wheeler

Researchers in the Wheeler lab include, leftto right, Sarah Barker, a postdoctoralresearch fellow from Yorkshire, England;Xiaojie Ji, first-year biomedical sciencedoctoral student from China; and RemiGratacap, a postdoctoral research fellowfrom Grenoble, France.