Alternative Candida albicans Lifestyles - Growth on Surfaces.pdf

ANRV253-MI59-06 ARI 4 August 2005 11:16

Alternative Candida albicansLifestyles: Growth onSurfacesCarol A. Kumamoto and Marcelo D. VincesDepartment of Molecular Biology and Microbiology, Tufts University, Boston,Massachusetts 02111; email: [email protected], [email protected]

Annu. Rev. Microbiol.2005. 59:11333

First published online as aReview in Advance onMay 2, 2005

The Annual Review ofMicrobiology is online atmicro.annualreviews.org

doi: 10.1146/annurev.micro.59.030804.121034

Copyright c 2005 byAnnual Reviews. All rightsreserved

0066-4227/05/1013-0113$20.00

Key Words

biolm, tissue invasion, invasive growth, thigmotropism, hyphae

AbstractCandida albicans, an opportunistic fungal pathogen, causes a widevariety of human diseases such as oral thrush and disseminated can-didiasis. Many aspects of C. albicans physiology have been studiedduring liquid growth, but in its natural environment, the gastroin-testinal tract of a mammalian host, the organism associates withsurfaces. Growth on a surface triggers several behaviors, such asbiolm formation, invasion, and thigmotropism, that are importantfor infection. Recent discoveries have identied factors that regulatethese behaviors and revealed the importance of these behaviors forpathogenesis.

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Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 114SURFACES COLONIZED BY

CANDIDA ALBICANS . . . . . . . . . . . 115MECHANISMS OF BIOFILM

FORMATION ANDDEVELOPMENT. . . . . . . . . . . . . . . 115Biolm Formation on Implanted

Medical Devices Results inDrug Refractory Infections . . . . 115

Numerous Parameters InuenceBiolm Formation andStructure. . . . . . . . . . . . . . . . . . . . . . 116

Multiple Mechanisms Contributeto the High Drug Resistanceof Biolm Cells . . . . . . . . . . . . . . . 116

High Levels of Amino AcidBiosynthesis and ProteinSynthesis in BiolmCells . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Defective Biolm DevelopmentCaused by Mutations thatAlter the Cell Surface andCompromise Adherence . . . . . . . 118

Development of Animal Modelsfor Biolms . . . . . . . . . . . . . . . . . . . 118

MECHANISMS OF INVASIVEGROWTH . . . . . . . . . . . . . . . . . . . . . . 119Tissue Invasion by C. albicans

Hyphae Occurs on Epithelial,Epidermal, and EndothelialSurfaces DuringCandidiasis . . . . . . . . . . . . . . . . . . . . 119

Protease and PhospholipaseActivities Contribute toInvasion of HostTissue . . . . . . . . . . . . . . . . . . . . . . . . 119

Filamentation During InvasiveGrowth is Promoted byPhysical Contact . . . . . . . . . . . . . . 120

Transcription Factors RegulatingInvasive Growth by EmbeddedCells . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Signaling Pathways RegulatingInvasive Growth by EmbeddedCells . . . . . . . . . . . . . . . . . . . . . . . . . 123

Models for Invasion of TissueSurfaces . . . . . . . . . . . . . . . . . . . . . . . 124

GUIDANCE OF HYPHAE BYTHIGMOTROPISM . . . . . . . . . . . . 125

CONCLUSIONS. . . . . . . . . . . . . . . . . . . 126

Nosocomial: aninfection thatdevelops within ahospital and isproduced by aninfectious organismacquired during thestay of the patient

INTRODUCTION

In the fourth century B.C., growth ofthe opportunistic fungal pathogen Candidaalbicans on the surface of human tissue wasnoted and the oral infection it causes, thrush,was described by Hippocrates. Since thattime, the incidence of candidiasis has in-creased and C. albicans has become a sig-nicant nosocomial pathogen. The modernAIDS epidemic has created a population ofpatients susceptible to candidiasis; oral thrushis one of the most common opportunistic in-fections in AIDS patients (4).

As an opportunist, C. albicans does notusually cause serious disease in immunocom-petent hosts, but immunodecient hosts aresusceptible to infections ranging from su-

percial mucosal infections to invasive, life-threatening disease. Candida spp. rank amongthe four most common causes of bloodstreaminfections and cardiovascular infections inU.S. hospitals (18, 41). In neonatal intensivecare units, Candida spp. are an even more fre-quent cause of bloodstream infections (95).The advances of modern medicine have ledto larger populations of compromised patientssusceptible to candidiasis, increasing the im-portance of C. albicans as a pathogen andproviding impetus for the detailed study ofC. albicans biology.

As is typical for many microorganisms,C. albicans physiology has been frequentlystudied during growth in liquid culture. How-ever, in their natural environment, C. albicans

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cells are commonly found in associationwith surfaces. Therefore, it is important tounderstand how C. albicans cells interactwith surfaces and how the unique aspects ofgrowth on surfaces contribute to C. albicanspathogenicity. Several distinct C. albicansbehaviors occur on surfaces, includingbiolm formation, invasive growth, andthigmotropism. In this review we describethese behaviors and discuss the mechanismsthat regulate them. Each behavior occursunder specic environmental conditions andis mediated and controlled by specic fungalproteins.

SURFACES COLONIZED BYCANDIDA ALBICANS

C. albicans is generally found as a commen-sal organism in association with a human oranimal host. In one study, 7.1% of infantswere colonized by Candida or other yeastson the day of birth and 96% of infants wereorally colonized by approximately one monthof age (94). In adults, gastrointestinal car-riage of Candida spp. is common (78). Forexample, fungi were found in 80% of fecalsamples from healthy adults (37). Soll et al.(104) showed that several surfaces in the bodycan be colonized, including the vaginal wall,surfaces in the oral cavity, and the anorectalsurface.

Individuals carry a particular strain forlong periods, but changes in the colonizingstrain over time can also be detected (78). Asingle individual may carry unrelated strains atdifferent sites (104), and changes in the colo-nizing C. albicans strain have been observed inHIV-positive patients (109). Thus, coloniza-tion by C. albicans is not xed and changes mayoccur during an individuals lifetime.

Within the host, a population of com-mensal organisms is probably bound to mu-cosal surfaces. In mice orally inoculated withC. albicans, binding of yeast-form C. albicanscells to epithelial surfaces in the gastrointesti-nal tract was detected within 3 (82) or 72 h(51) postinoculation. Another yeast species,

Biofilm: acommunity ofmicroorganismsattached to a surface,formingthree-dimensionalstructures containingexopolymeric matrixand cells thatexhibit distinctivephenotypicproperties

Thigmotropism:the directionalresponse of a cell ortissue to touch, orphysical contact witha solid object

Epithelial:pertaining to tissuethat forms the outersurface of the bodyand lines the bodycavities, and to maintubes andpassageways that leadto the exterior, suchas the lining of thegastrointestinal tract

Torulopsis pintolopesii, naturally colonizes mice.This organism is found in layers bound tothe secreting epithelium of the stomach (97).These ndings demonstrate the ability ofcommensal fungal organisms to adhere to tis-sue surfaces within the host.

In summary, growth on a biological sur-face, especially in the mammalian gastroin-testinal tract, is part of the natural lifestyleof C. albicans. We discuss below how the in-teraction of C. albicans with a surface altersC. albicans behavior and how these effects con-tribute to disease.

MECHANISMS OF BIOFILMFORMATION ANDDEVELOPMENT

Biofilm Formation on ImplantedMedical Devices Results in DrugRefractory Infections

A biolm is a three-dimensional commu-nity of microorganisms embedded in an ex-opolymeric matrix and attached to a surface.Upon attachment, microorganisms undergo achange to a sessile (attached) lifestyle. Dentalplaque is a well-known natural example of abacterial biolm found in humans. C. albicansalso forms a biolm on dental enamel (61) aswell as on human heart valves, causing endo-carditis (29).

From a human health perspective, biolmsare important because they form on implantedmedical devices and result in infections thatare unusually refractory to antimicrobial ther-apy. Medical device infection contributes toabout half of all nosocomial infections (forreview see Reference 54). Several millionvascular and urinary catheters and tens ofthousands of prosthetic heart valves are usedannually in the United States; approximately10% of infections linked to these devices aredue to Candida spp. (54). For example, of theestimated 80,000 bloodstream infections asso-ciated with central venous catheters that occurannually in U.S. intensive care units, 11.5%are due to Candida spp. (54). The mortality

www.annualreviews.org C. albicans Growth on Surfaces 115

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Quorum sensing:cell-density-dependentcommunication andcoordination ofmicrobial behaviorvia signalingmolecules

rate for device-associated Candida infection isapproximately 30% (54).

Cells in a biolm characteristically exhibithigh resistance to antimicrobial drugs, andtherefore infected devices must usually be re-moved to cure the infection (54). For somedevices, removal necessitates major surgeryand exposes patients to signicant risks. As aresult, the ability of C. albicans to adhere toa medical device and form a biolm resistantto antifungal agents represents an importantmedical challenge.

Numerous Parameters InfluenceBiofilm Formation and Structure

Studies have demonstrated that C. albicansbiolms form in several stages (21, 29). First,cells, typically yeast form, attach to a surface.Second, cells proliferate on the surface, form-ing microcolonies. Third, growth of cells,production of hyphae (lamentous forms ofthe organism), and secretion of exopolymericmatrix result in elaboration of the characteris-tic three-dimensional structure that is typicalof a biolm. The exopolymeric matrix, com-posed of carbohydrates, proteins, and otherunidentied components, surrounds the cellsin a mature biolm (11).

Numerous materials and growth mediasupport the growth of biolms in the labo-ratory. The structure of the resulting biolm,especially the proportions of yeast-form andhyphal-form cells, is strongly inuenced byparameters such as medium composition,temperature, and the nature of the substratum(59). Mutants unable to form hyphae or yeast-form cells can nevertheless produce a biolm,demonstrating that a specic morphology isnot strictly essential for biolm formation(10). Because the proportions of the differ-ent morphological forms vary depending onenvironmental conditions, biolm structure ishighly adaptable.

The content of exopolymeric matrix is alsoinuenced by biolm incubation conditions.Biolms incubated statically contain relativelylow amounts of matrix, whereas biolms in-

cubated with shaking produce higher lev-els of matrix (42). This effect of mediumow is also seen with bacterial biolms (28).Thus, ow of the medium above the sur-face is another variable that inuences biolmstructure.

Quorum sensing also regulates biolm for-mation. The quorum-sensing molecule far-nesol is produced continuously by growingC. albicans cells, accumulating to levels corre-lated with cell number, and acts as an inhibitorof germination, i.e., the yeast-to-hypha tran-sition (44). Incubation of cells in the presenceof farnesol leads to reduced biolm formation(86). Farnesol also has deleterious effects onmature biolms (86), suggesting that farnesolregulates biolm stability as well as biolmformation. Similar effects of quorum sensingon mature bacterial biolms have been noted(106).

A two-component histidine kinase, Chk1p,plays a role in the response of cells to far-nesol. chk1 mutant cells are insensitive to theeffects of farnesol on both germination andbiolm formation (58). Chk1p is a cytoplas-mic protein that probably functions togetherwith currently unknown proteins to detect thefarnesol signal and produce the appropriateresponse.

Thus, biolms vary in the morphology andorganization of their cells and in their con-tent of extracellular matrix. The plasticity inbiolm structure in response to medium com-position, the substratum, ow conditions, andquorum sensing suggests that biolms locatedin different sites within the host or in associa-tion with different types of devices or surfacesare likely to differ in their properties.

Multiple Mechanisms Contributeto the High Drug Resistanceof Biofilm Cells

One of the most vexing characteristics ofbiolms is their high-level resistance to an-timicrobial drugs (28, 29, 59). Drug resistancereects a property of individual biolm cells,since C. albicans cells released from disrupted

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biolms still exhibit substantial levels of resis-tance (8, 9, 85). In C. albicans biolms, resis-tance is probably not due to poor penetrationof drugs into the biolm structure (1); mu-tants that form structurally defective biolmsnonetheless exhibit high drug resistance (87).

In the early stages of biolm forma-tion, changes in gene expression contributeto resistance. Resistance to the antifungaluconazole can be detected as soon as 2 hafter adherence of C. albicans cells (71). Atthis early time point, resistance is strongly de-pendent on MDR1, which encodes a ucona-zole efux facilitator. That is, an mdr1 nullmutant biolm is 16-fold more sensitive touconazole than is a wild-type biolm. Dele-tion of CDR1 and CDR2, which encode ho-mologous drug efux pumps, also decreasesthe resistance of adherent cells (71). Simi-lar results were obtained after 6 h of ad-herent growth, and increased efux activityin biolm cells was detected (74). Thus, thedrug-resistant phenotype of biolm cells atearly times after adherence results from ex-pression of drug efux determinants. Over-expression of MDR1, CDR1, and CDR2 isalso important for drug resistance in drug-resistant clinical strains (83).

In mature biolms (e.g., after 48 h of incu-bation) the mechanisms that confer drug re-sistance are different. Mature biolms formedfrom mdr1 cdr1 cdr2 triple null mutantsor the various single and double mutantsare highly resistant to drugs (74, 85). Con-sistent with these observations, expressionof the drug efux determinants in maturebiolms is not high (34, 74) and efux ac-tivity of mature biolms is not higher thanthe activity in planktonic cells (74). Theseresults argue against a role for the efuxdeterminants in drug resistance in maturebiolms.

Decreased membrane ergosterol content(74) and altered expression of ergosterolbiosynthetic genes (34) in mature biolm cellshave been noted. Therefore, increased drugresistance in mature biolms may be relatedto an alteration in membrane sterol content.

Altered membrane composition could resultin changes in membrane properties such asdecreased permeability to drugs, leading tohigher drug resistance.

An alternative model has proposed thatmost Pseudomonas aeruginosa cells in abiolm exhibit similar antibiotic resistance asstationary-phase planktonic cells but that thebiolm also contains a population of highlyresistant persister cells. The persister cellsare not mutants but rather wild-type cellswhose physiological state allows them to sur-vive antibiotic treatment (64, 105). By sur-viving antibiotic treatment, the persister cellsallow recovery of the population after an-tibiotic treatment is discontinued. Althoughthe existence of persister cells in C. albicansbiolms has not been tested directly, there isevidence of cellular heterogeneity in C. al-bicans biolms (108), and thus this mecha-nism could also contribute to biolm drugresistance.

In summary, multiple mechanisms con-tribute to the increased drug resistance ex-hibited by biolms. Because drug resistancemechanisms seen in early biolms differfrom those conferring resistance in maturebiolms, strategies designed to block earlybiolm formation may differ from strategiesthat would be effective for eliminating maturebiolms.

High Levels of Amino AcidBiosynthesis and Protein Synthesisin Biofilm Cells

To understand the distinctive biolm lifestyleat the molecular level, microarray studies ofgene expression in C. albicans biolm cellshave been conducted (34). Results revealedthat, in comparison to postlogarithmic plank-tonic cells that had been grown for thesame length of time, cells in biolms expresshigher levels of genes involved in amino acidand nucleotide metabolism, protein synthe-sis, other metabolic functions, and subcellularlocalization. Garcia-Sanchez et al. (34) pro-posed that biolms require high-level protein


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biosynthesis, necessitating increased aminoacid biosynthesis.

Interestingly, analyses of gene expressionor protein composition in bacterial biolmcells compared with postlogarithmic plank-tonic cells grown for the same length of timeshowed some similarities to the results ob-tained with C. albicans biolms (98, 111).Among the proteins or transcripts of genespresent at higher concentration in bacterialbiolm cells were several gene products in-volved in the synthesis of amino acids andnucleotides or in protein translation. How-ever, when bacterial biolm cells were com-pared with planktonic cells that were in theexponential growth phase, these differencesin gene expression were not observed (98).These results imply that biolms contain cellswhose protein synthesis machinery is func-tioning at the level seen in exponential-phasecells, even though the biolm has been devel-oping for a long period.

Defective Biofilm DevelopmentCaused by Mutations thatAlter the Cell Surface andCompromise Adherence

Several mutations that reduce biolm devel-opment by compromising the rst step inbiolm formation, adherence to surfaces, havebeen studied. One of these mutations affectsEFG1, a major regulator of hyphal develop-ment (107). Efg1p regulates numerous geneswhose products include many cell surface pro-teins (62, 77, 103). As a result of the cellsurface defect and the defect in productionof hyphae, it is not surprising that efg1 nullmutants are defective in biolm development(87). On polystyrene, the defect is evidentduring the earliest stages of biolm develop-ment, i.e., adherence and microcolony forma-tion (87). efg1 null mutants are also defectivein adhering to polyurethane central venouscatheter material (65). The EAP1 gene, whichencodes a predicted cell wall, GPI-anchoredprotein, is dependent on Efg1p for expres-sion and may be a critical target of Efg1p.

Expression of EAP1 in nonadherent Saccha-romyces cerevisiae cells resulted in increased ad-herence to polystyrene, suggesting that thissurface protein can mediate attachment topolystyrene (66). Therefore, the failure ofefg1 mutants to initiate biolm formation onpolystyrene may reect the absence of Eap1p.

Other mutations affect adherence andbiolm formation. Mutants lacking ACE2,which encodes a transcription factor thatregulates expression of chitinase and cellwall proteins, exhibit reduced adherence topolystyrene and reduced biolm formation(50). Mutants lacking the NOT4 gene, whichencodes a putative E3 ubiquitin ligase, fail toattach rmly to a serum-coated plastic surfaceand are defective in biolm formation (57). Aswith the efg1 mutant, the absence of Not4por Ace2p may alter the surface properties ofC. albicans, leading to the observed defects inadherence and biolm development.

Adherence of Candida glabrata leading tobiolm formation is mediated in part byEpa6p, a cell surface protein. Expression ofEPA6 is activated under conditions that lead tobiolm development through alteration of thesubtelomeric silencing machinery (48). Takentogether, these results demonstrate that alter-ation of the cell surface by any one of severalmechanisms results in reduced adherence andreduced biolm development.

Development of Animal Modelsfor Biofilms

Although model systems make analysis of thestructure and development of biolms acces-sible to study, animal models are necessaryto understand the pathogenesis of biolm-related disease. Two animal models have beenrecently described, one utilizing rabbits (100)and one using rats (5). Both systems involveplacement of a central venous catheter fol-lowed by direct inoculation of C. albicans cellsinto the lumen of the catheter. The resultingbiolms are structurally similar to biolms de-scribed in laboratory model systems, exceptfor the possible presence of host cells in the

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biolm (5). The catheter biolms also exhibitdrug resistance and express the CDR2 gene,consistent with results obtained in laboratorymodel systems (5, 100). In the rat model,biolm development leads to seeding of thekidneys with C. albicans, demonstrating dis-seminated disease. These animal models offerthe potential for evaluation of new treatmentsfor biolm infections based on insights fromlaboratory model systems.

In summary, recent developments haverevealed molecular activities required forbiolm development. Expression of appropri-ate cell surface molecules for adhesion andexpression of genes needed for high levelsof protein synthesis are critical for successfulbiolm formation. Mechanisms underlyingantifungal resistance differ in early biolmsand mature biolms, indicating that differentstrategies are needed to circumvent this prop-erty of biolms.

MECHANISMS OF INVASIVEGROWTH

Tissue Invasion by C. albicansHyphae Occurs on Epithelial,Epidermal, and EndothelialSurfaces During Candidiasis

In supercial candidiasis, epidermal or epithe-lial surface invasion by C. albicans hyphae iscommonly observed (79). When fungi colo-nize an epithelial or epidermal surface, theyadhere to host cells and create depressionsin the surface of the host cells (45, 51, 60,88). Fungal yeast-form cells also convert tolamentous hyphae, which penetrate into thesurface.

During invasion and traversal of anendothelial surface, the initial entry of yeast-form cells into endothelial cells is by an endo-cytotic process (22). Damage to the endothe-lial surface (33, 52) results in exposure of theunderlying basement membrane, which maythen be invaded by hyphal-form cells.

In specimens scraped from human oral orcutaneous lesions, most C. albicans cells are

Endothelial:pertaining to tissuecomposed of a layerof at squamouscells, such as thoselining blood andlymphatic vessels andthe heart

found within host cells (20, 70, 73). Access tothe interior of host cells is probably achievedby a combination of enzymatic activities (e.g.,proteases and phospholipase) and mechani-cal force. Ultrastructural observations revealareas of clearing around penetrating hyphae,supporting a role for lytic enzymes during in-vasion (73, 99, 109). In addition, in samplescollected from cutaneous candidiasis cases,the corneocytes appear deformed as a resultof their interactions with C. albicans, support-ing the notion that C. albicans uses mechani-cal force to aid penetration (99). Studies withmodel systems using explanted tissue, animalinfection, or cells in culture conrm these fea-tures of invasion by C. albicans and demon-strate that C. albicans hyphae may enter or passcompletely through host epithelial or epider-mal cells with minimal damage to the host cell(16, 32, 46, 88, 114).

In summary, hyphal penetration is a keycomponent of invasive growth, the process ofpenetrating the substratum. Invasion of theepithelial surface allows infecting organismsto reach the bloodstream, and penetration anddisruption of endothelial surfaces allow or-ganisms to escape from the bloodstream andinfect deep tissues. Thus, the invasive behav-ior of C. albicans on a biological surface con-tributes to disease pathogenesis.

Protease and PhospholipaseActivities Contribute toInvasion of Host Tissue

Degradative enzymes secreted by C. albicanscells are important for tissue invasion. Inhibi-tion of the secreted aspartyl protease (SAP)class of enzymes with the inhibitor pep-statin decreases tissue invasion (76). Pepstatintreatment also enhances survival followingintranasal challenge in mice by C. albicans(31). In addition, several mutants lacking SAPgenes showed decreased invasion (76). Simi-larly, secreted phospholipase activity has beenimplicated in tissue invasion. In invading hy-phae, phospholipase activity is concentratedat the hyphal tip (35, 84); a mutant lacking


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secreted phospholipase B1 exhibits reducedability to penetrate cell monolayers (63) ortissue (75). These data strongly implicate se-creted proteases and phospholipases in suc-cessful invasion.

Filamentation During InvasiveGrowth is Promoted by PhysicalContact

As discussed above, hyphae that are capa-ble of exerting mechanical force on hostcells are characteristically seen in specimenstaken from infected tissue and probably con-tribute to invasion. Several experimental reg-imens stimulate hyphal growth in the labora-tory including the use of special media, hightemperature, or microaerophilic conditions(30, 39).

To study invasive hyphal developmentspecically, we have used an agar model sys-tem that mimics some of the features of tissueinvasion. Growth of C. albicans cells embedded

Figure 1Invasive growth of C. albicans within host tissue. Sections of xed tonguetissue from gnotobiotic immunosuppressed piglets orally inoculated with(panel a) wild-type C. albicans or (panel b) efg1 cph1 double null mutantcells. Immunohistochemical analysis with anti-C. albicans antibody isshown. From Reference 91 with permission.

within agar medium stimulates the conversionof yeast-form cells to laments that invade themedium (17). Control experiments indicatethat the important cue for hyphal productionis not reduced oxygen levels or gradients ofnutrients but rather physical contact of cellswith agar or other matrix material (17). Unlikehyphal development stimulated by other cues,invasive lamentation of cells in agar mediumis readily observed in rich medium at low orhigh temperature.

Studies of immunosuppressed gnotobi-otic piglets orally inoculated with C. albicanssuggest that contact-dependent lamentationcontributes to invasion of tissue. The efg1/efg1cph1/cph1 double mutant strain, which lackstwo transcription factors that regulate la-mentation, forms laments and invades thetongue of the piglet (91) (Figure 1) or agarmedium (36, 91), but it is defective in l-amentation under all other conditions (68).These results suggest that lamentation in re-sponse to contact with a surface contributesto invasion of epithelial tissue and is de-pendent on factors other than Efg1p andCph1p.

Transcription Factors RegulatingInvasive Growth by Embedded Cells

To date, many genes that differentially affectlamentous growth depending on whethercells are grown in liquid or on agar mediumhave been identied. These genes and theirphenotypes are summarized in Table 1. Tofocus specically on the molecular mecha-nisms underlying the lamentous responseto growth within agar medium, the follow-ing sections summarize the results of stud-ies that make use of the embedded growthcondition (suspending cells within rich agarmedium).

Of the genes known to regulate inva-sive growth, several are thought to encodetranscription factors (Table 1). The putativetranscription factor CZF1 plays a key role inregulating the response of cells to embed-ded conditions. Deletion of CZF1 results in

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Table 1 C. albicans genes influencing filamentous growth within or on agar media

Gene Filamentation phenotype Putative function Reference(s)Transcription factorsCPH1 Defective on Spider and SLAD

agar; mild defect within agarTranscription factor 24, 67

CZF1 Defective within agar Transcription factor 17EFG1 Hyperlamentous within agar Transcription factor 36EFH1 efh1 efg1 double mutant

hyperlamentous within agarTranscription factor 27

MCM1 Hyperlamentous whenoverexpressed on agar

Transcription factor 93

Signaling componentsCEK1 Defective on Lees and Spider agar MAPK 24, 53CHK1 Defective on serum agar Histidine kinase 117COS1 Defective on serum and Spider agar Histidine kinase 2, 117CPP1 Hyperlamentous and

hyperinvasive on agarPhosphatase 23

CST20 Defective on Spider agar MAPKKK kinase 24, 53GAP1 Defective on solid Spider and

SLADAmino acid permease 12

GPA2 Defective within agar G-protein -subunit 69a, 72, 96GPR1 Defective within agar G-protein-coupled receptor 69a, 72HOG1 Defective in liquid and agar;

hyperinvasive on SLAD agarMAPK 3

HST7 Defective on Spider and SLADagar; hyperlamentous whenoverexpressed within agar

MAPK kinase 24, 53, 96

RAS2 Active allele and mutant defectivewithin agar

Small G-protein 69a, 96

SLN1 Defective on serum agar Histidine kinase 117SSK1 Defective on agar; hyperinvasive on

SLAD agarTwo-component response regulator 19

TPK1 Defective on agar PKA catalytic subunit 15TPK2 Defect in invasive growth of yeast

cells on agarPKA catalytic subunit 15

Other genesBMH1 Defective in liquid and within agar;

one allele defective only in liquid14-3-3 protein 92

FAB1 Defective on Spider and serum agar Phosphatidylinositol 3-phosphate5-kinase

7

PLD1 Defective on Spider agar;hyperlamentous oncornmeal/Tween agar

Phosphatidylcholine-specicphospholipase D1

47

RBR1 Defective on acidic M-199 soft agar pH-regulated putative cell wallprotein

69


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MAPK:mitogen-activatedprotein kinase

PKA: protein kinaseA

reduced invasive lamentation. Ectopic CZF1expression results in accelerated lamenta-tion (17) (Figure 2). Czf1p does not affectmorphogenesis under other conditions, sug-gesting that a unique pathway is stimulated inembedded cells.

Mutation of CPH1, the C. albicans homo-logue of the S. cerevisiae STE12 transcriptionfactor, results in defective lamentous growthin certain media (24, 62, 67) and mildly de-fective lamentous growth within agar (36).The czf1 cph1 double null mutant is more de-fective than either single mutant, indicatingthat the two genes have partially overlappingfunctions (17).

EFG1 encodes a basic helix-loop-helixtranscription factor that is required for hy-phal development under most laboratory con-ditions (68, 107). However, C. albicans efg1mutant cells are more lamentous than wild-type cells when embedded (36) (Figure 2a,b).Thus, under embedded conditions, EFG1 actsas a negative regulator of lamentous growth.The related EFH1 gene is also believed to playa negative role in regulating lamentation inthe embedded condition, perhaps in conjunc-tion with Efg1p (27). The effects of the efg1

mutation are epistatic to the effects of the czf1mutation and partially epistatic to the effectsof the cph1 mutation (36), and therefore, theefg1 cph1 double null mutant laments duringgrowth within agar medium, as noted above.

CZF1 affects lamentation during growthin agar (17). The effects of a CZF1 mutation,however, are not observed in the absence ofEFG1 (36), which suggests that CZF1 pro-motes lamentation by antagonizing EFG1-mediated repression. Yeast two-hybrid datasuggest that this effect may involve phys-ical interaction between Czf1p and Efg1p(36).

The transcription factors Cph1p andEfg1p each dene two distinct signalingpathways that regulate lamentous growthunder many conditions (30). Cph1p is regu-lated by a MAPK cascade, and Efg1p is reg-ulated by the cAMP/PKA signaling cascade.The studies described above reveal the exis-tence of at least two different programs inwhich these cascades regulate morphogene-sis (Figure 3). Under the standard condi-tions used to promote lamentous growth,e.g., the use of inducers such as serum, neutralpH, microaerophilic conditions, temperature,

Figure 2Invasive growth of C. albicans within agar medium. Colonies were grown on the surface of agar medium,surface cells were washed off, and the remaining cells that were embedded within the agar werephotographed from the side. White arrowhead shows the top of the agar. Panel a, WT cells; panel b, efg1null mutant cells; panel c, czf1 null mutant cells; and panel d, cells ectopically expressing CZF1 from thepromoter of the C. albicans maltase gene.

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Figure 3The standard and embedded programs for regulation of lamentation. Filamentation stimulated bystandard inducing conditions (e.g., serum, 37C, neutral pH, starvation, microaerophilic growth) or byembedded growth requires many of the same signaling components and transcription factors. Notabledifferences include (a) the role of Efg1p as a positive regulator in the standard program and as a negativeregulator in the embedded program, and (b) a Czf1p-dependent pathway that promotes lamentationonly in the embedded program. Bold arrows indicate the relatively greater importance of theEfg1-mediated pathways in both programs. Dashed lines indicate unclear relationships. Blunt arrowsindicate negative effects. Transcription factors are depicted as boxes. For simplicity, many factors withuncertain relationships to these pathways have been left out of the gure but are discussed in greaterdetail in the text.

and nutritional signals (30, 39), hyphal de-velopment by the standard program requiresEfg1p as a positive regulator. Cph1p performsa backup, positive function in the standardprogram. During embedded growth, lamen-tation is promoted by contact with agar orother matrix material. Filamentation underembedded conditions is controlled by the em-bedded program, in which Efg1p acts as anegative regulator and Czf1p functions as anantagonist of Efg1p repressor activity. Thefunction of Cph1p partially overlaps that ofCzf1p in the embedded program. The po-tential contributions of other transcriptionalregulators of morphogenesis (25) to regula-tion in the embedded condition are currentlyunknown.

Signaling Pathways RegulatingInvasive Growth by Embedded Cells

The C. albicans genes GPR1, GPA2, and RAS2regulate lamentous growth under embed-ded conditions (69a, 72, 96). GPR1, whichencodes a G-protein-coupled receptor, andGPA2, which encodes a G-protein -subunithomologue, probably function in transmis-sion of signals. gpr1 and gpa2 mutants aredefective in lamentous growth under vari-ous conditions, particularly during embeddedgrowth (69a, 72, 96). Recent studies suggestthat GPR1 and GPA2 act in the cAMP-PKApathway (69a, 72). These studies demonstratesuppression of the gpa2 defect by additionof exogenous cAMP. In addition, the gpa2


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mutation is rescued by overexpression ofTPK1, a component of the cAMP-PKA path-way, but not by overexpression of HST7, amember of the MAPK pathway (69a).

Under standard inducing conditions, thesmall G-protein-encoding RAS1 gene posi-tively regulates lamentous growth and is be-lieved to act in both the cAMP and MAPKpathways (62a) (Figure 3). Deletion of RAS1causes a defect in lamentation under em-bedded conditions, and this defect can besuppressed by addition of exogenous cAMP,suggesting a positive role for both RAS1 andcAMP in regulating lamentous growth inthis condition (69a). Although under stan-dard conditions exogenous cAMP acceler-ates lamentous growth, the inuence ofcAMP on lamentation of embedded cells isunclear.

Taken together, the data suggest that underembedded conditions the MAPK pathway ac-tivates a positive regulator, Cph1p, (53) andpromotes lamentous growth, whereas thecAMP-dependent pathway, including RAS1,GPR1, and GPA2, acts on a negative regula-tor, Efg1p, (14) that suppresses lamentousgrowth. The signaling components that lieupstream of Czf1p are not yet known.

The two genes encoding isoforms of cat-alytic subunits of PKA, a component of thecAMP signaling pathway, have distinct rolesin lamentation in liquid and agar media (15).The tpk1 mutant is defective for lamentousgrowth on agar media and is only slightlyaffected in liquid, and the tpk2 mutant isonly partially defective on agar but is stronglyblocked for lamentation in liquid. The basisfor the differential effects of TPK1 and TPK2is currently unclear.

Other genes that affect morphogenesison agar medium include SLN1, COS1, andCHK1, which encode two-component histi-dine kinases, SSK1, which encodes a responseregulator, and HOG1, which encodes an os-moregulating MAPK gene (2, 3, 19, 117).These genes may not act within the MAPKor cAMP pathways but rather within indepen-dent pathways.

The activities of numerous other genesthat specically affect lamentation on agarmedium have been described, but the mech-anisms by which they act are incompletelyunderstood. These genes are summarized inTable 1.

Models for Invasion of TissueSurfaces

Studies of the efg1 and/or the efg1 cph1 dou-ble null mutant in different animal models ofinfection reveal two mechanisms for penetra-tion of tissue surfaces. One mechanism occurson mucosal surfaces and involves Efg1p-dependent adherence and proliferation on thesurface, followed by Efg1p-independent pen-etration of the surface. A second mechanismoccurs on endothelial surfaces and involvesuptake of fungi by endocytosis, followed byintracellular, Efg1p-dependent hyphal devel-opment that allows fungal penetration of thesurface.

The extensive surface growth of wild-typeC. albicans in pseudomembranous oral can-didiasis in the immunosuppressed gnotobioticpiglet is dependent on Efg1p and is not ob-served with the efg1 cph1 double null mutant(6). In addition, defects in adhesion of efg1 nullmutant or efg1 cph1 double null mutant cellsto oral epithelial cells and Caco-2 cells havebeen observed (26, 112).

In several systems, the overall extent of in-vasion is reduced in the absence of Efg1p,probably reecting defective adherence (26,40, 56, 114). However, invasion of host ep-ithelium by C. albicans can occur in the ab-sence of Efg1p (91) (Figure 1b). In fact,invasive growth probably requires downregu-lation of EFG1 function. This model is basedon the observations that efg1 null mutants arehyperlamentous and hyperinvasive in agar(36) (Figure 2a,b) and that EFG1 transcrip-tion is downregulated during lamentation inliquid medium (107, 110).

Therefore, because Czf1p is a negativeregulator of Efg1p (36), Czf1p may act as aswitch that inactivates Efg1p and promotes

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the conversion from surface growth to inva-sive growth. Activation of the Czf1p switchoccurs as a response to contact with agar orother matrix material. Czf1p antagonism ofEfg1p is not the only mechanism that pro-motes invasive lamentation, since there arealso high-temperature-activated mechanismsthat do not require Czf1p (17). The result ofEfg1p and Czf1p activities is the characteris-tic invasion of epithelial surfaces seen in pseu-domembranous oral candidiasis.

In contrast, other host cell types suchas endothelial cells are stimulated to takeup C. albicans cells by endocytosis, and thusa second mechanism for invasion is promi-nent on these surfaces. In this situation,invasion is initiated by endocytosis of yeast-form cells. Within host cells, the fungi un-dergo Efg1p-dependent formation of germtubes and exit from the host cell. Germina-tion within host cells is defective when efg1mutant cells are internalized by macrophages(68) or neutrophils (56). Defective exit by ger-mination and defective endocytosis probablycontribute to the failure of efg1 mutants to in-vade and damage endothelial cell monolayers(81).

In summary, tissue invasion requiresdegradative activities secreted by the invad-ing C. albicans hyphae and mechanical forcesexerted by the hyphae. The mechanismsthat regulate invasion are adapted to thenature of the surface to which C. albicans cellsare bound. The mechanism for invasion ofmucosal surfaces resembles the embeddedprogram for regulation of lamentation. Thatis, following Efg1p-dependent proliferationon the surface, Efg1p is downregulated andcontact-dependent hyphal development en-sues. A second mechanism involving entry offungi into host cells via endocytosis followedby intracellular, Efg1p-dependent lamenta-tion and exit from the host cells is importanton endothelial surfaces. In this situation,control of hyphal development resembles thestandard program for regulation of lamen-tation. The cues that promote lamentationon the two types of surfaces are different and

the molecules involved in controlling hyphalmorphogenesis are used in different ways.The existence of the two mechanisms forlamentation contributes to the remarkableversatility of C. albicans as a pathogen.

GUIDANCE OF HYPHAE BYTHIGMOTROPISM

The direction of C. albicans hyphal growthcan be determined by the topography ofthe substratum. This behavior, known asthigmotropism, allows hyphae to be guidedby ridges in the substratum (113). In stud-ies of this behavior, hyphae penetrate thepores of nucleopore membranes and, afterpenetration, follow the face of the mem-brane. During penetration, hyphae growboth away from and toward the agar un-derneath the membrane, implying that theorientation of hyphal growth is not dueto chemotropism but to thigmotropism (38,101). Helical growth of hyphae occurs whencells are grown on rm surfaces, such as cel-lophane (102). These thigmotropic responsesto surface features are reminiscent of thebehavior of plant-pathogenic fungi on leafsurfaces.

Thigmotropism plays a major role in thelocation of infectable sites on plants by phy-topathogenic fungi. For example, Uromycesappendiculatus, the causative agent of beanrust, produces hyphae that are guided bythe topography of the bean leaf surface togrow toward stomata, where they differen-tiate into appressoria, specialized structuresneeded for invasion of the leaf. Induction ofappressorium formation is caused by archi-tectural features of the stomatal guard cellsand can be mimicked in the laboratory us-ing polystyrene membranes bearing ridges ofthe appropriate height. The plant pathogensPuccinia hordei and Magnaporthe grisea also usethigmotropic behavior on the surface of plantsto locate openings in the epidermal layer ofplant tissue and initiate invasive growth (43,89, 116). Hence, thigmotropism plays an im-portant role in guidance of hyphal growth,


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MS:mechanosensitive

in regulation of development, and in diseaseprogression.

Thigmotropism has also been demon-strated in dermatophytes and in saprophytessuch as Mucor mucedo and Neurospora crassa(80). Thus, thigmotropic behavior of hyphaeis not restricted to pathogenic fungi; it is ageneral feature of fungal hyphae that mustforage for nutrients on surfaces and withinmaterials.

The thigmotropic response of C. albicansmay be an adaptation for penetrating tissue.Studies of cultured intestinal enterocytesinfected with C. albicans (115) and of biop-sies of oral candidiasis taken from AIDSpatients (90) demonstrated orientation of hy-phae along ridges or grooves and penetrationinto the regions between enterocytes or cor-neocytes. The authors suggested that thig-motropism may allow hyphae to locate theintercellular regions.

MS ion channels are found in organismsfrom archeans, E. coli, and yeast to higher eu-karyotes (13, 49, 55, 118) and are believedto play roles in processes such as osmosensa-tion, topographic sensing, touch, and hearing.MS channels are activated by stretch forceson the plasma membrane, resulting in theopening of the channels. C. albicans possessesMS channel activity. Reorientation of hyphalgrowth in response to ridges is attenuated bytreatment with an inhibitor of MS channels,Gd3+, which suggests that MS channels areresponsible for sensing substrate topography(113).

To summarize, thigmotropic behavior al-lows C. albicans to respond with greatprecision to topographical features of thesurface. This behavior may contribute totissue invasion by directing penetratinghyphae toward more vulnerable regions ofthe tissue. Further studies will illuminate thedetails of the molecular mechanisms usedby C. albicans to sense the features of itsenvironment.

CONCLUSIONS

Contact with surfaces elicits unique physio-logical responses in C. albicans. On solid sur-faces, C. albicans cells form biolms with char-acteristic three-dimensional structures andhigh antifungal resistance. This response tocontact with a surface is signicant for hu-man health because of the large numbersof implanted medical devices used in mod-ern medicine. On tissue surfaces, C. al-bicans initially adheres and grows on thesurface and subsequently produces invasivelaments that penetrate the surface. Thisbehavior allows organisms to escape fromtheir normal niche in the gastrointestinaltract and reach the bloodstream, settingthe stage for disseminated, life-threateninginfection. Understanding the unique physiol-ogy of C. albicans on surfaces and the adap-tations of the organism that favor infection,researchers will be better equipped to developmethods for interrupting the progression todisease.

SUMMARY POINTS

1. Growth on surfaces is a natural part of the C. albicans lifestyle.

2. The unique physiology of cells growing on a surface makes an important contributionto pathogenesis.

3. Biolm formation, a behavioral response of C. albicans cells growing on a surface, isassociated with medical device infection.

4. The morphology and organization of C. albicans biolms are inuenced by numerousparameters, and thus variability may be observed in biolms located at different siteswithin the host.

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5. Multiple mechanisms contribute to the high resistance of biolms to antifungal drugs.

6. Secreted degradative enzymes and the hyphal form of growth play key roles duringtissue invasion by C. albicans.

7. Mechanisms that promote invasion of epithelial surfaces differ from mechanisms usedfor invasion of endothelial surfaces.

8. EFG1 regulates several aspects of growth on surfaces, including adhesion, biolmdevelopment, colonization, and invasion.

ACKNOWLEDGMENTS

We are grateful to Perry Riggle, Julia Kohler, and Linc Sonenshein for careful reading of themanuscript and for helpful comments. Our research on this project was supported by grantAI38591 from the National Institute of Allergy and Infectious Diseases.

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Contents ARI 8 August 2005 15:56

Annual Review ofMicrobiology

Volume 59, 2005

Contents

FrontispieceGeorges N. Cohen xiv

Looking BackGeorges N. Cohen 1

Signaling in the Arbuscular Mycorrhizal SymbiosisMaria J. Harrison 19

Interplay Between DNA Replication and Recombination inProkaryotesKenneth N. Kreuzer 43

Yersinia Outer Proteins: Role in Modulation of Host Cell SignalingResponses and PathogenesisGloria I. Viboud and James B. Bliska 69

Diversity and Evolution of Protein TranslocationMechthild Pohlschrder, Enno Hartmann, Nicholas J. Hand, Kieran Dilks,

and Alex Haddad 91

Alternative Candida albicans Lifestyles: Growth on SurfacesCarol A. Kumamoto and Marcelo D. Vinces 113

Yeast Evolution and Comparative GenomicsGianni Liti and Edward J. Louis 135

Biology of Bacteriocyte-Associated Endosymbionts of PlantSap-Sucking InsectsPaul Baumann 155

Genome Trees and the Nature of Genome EvolutionBerend Snel, Martijn A. Huynen, and Bas E. Dutilh 191

Cellular Functions, Mechanism of Action, and Regulation of FtsHProteaseKoreaki Ito and Yoshinori Akiyama 211

vii

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Mating in Candida albicans and the Search for a Sexual CycleR.J. Bennett and A.D. Johnson 233

Applications of Autouorescent Proteins for In Situ Studies inMicrobial EcologyEstibaliz Larrainzar, Fergal OGara, and John P. Morrissey 257

The Genetics of the Persistent Infection and Demyelinating DiseaseCaused by Theilers VirusMichel Brahic, Jean-Franois Bureau, and Thomas Michiels 279

Intracellular Compartmentation in PlanctomycetesJohn A. Fuerst 299

Biogenesis of Inner Membrane Proteins in Escherichia coliJoen Luirink, Gunnar von Heijne, Edith Houben,

and Jan-Willem de Gier 329

Genome-Wide Responses to DNA-Damaging AgentsRebecca C. Fry, Thomas J. Begley, and Leona D. Samson 357

The Rcs Phosphorelay: A Complex Signal Transduction SystemNadim Majdalani and Susan Gottesman 379

Translational Regulation of GCN4 and the General Amino AcidControl of YeastAlan G. Hinnebusch 407

Biogenesis, Architecture, and Function of Bacterial Type IV SecretionSystemsPeter J. Christie, Krishnamohan Atmakuri, Vidhya Krishnamoorthy,

Simon Jakubowski, and Eric Cascales 451

Regulation of Bacterial Gene Expression by RiboswitchesWade C. Winkler and Ronald R. Breaker 487

Opportunities for Genetic Investigation Afforded by Acinetobacterbaylyi, A Nutritionally Versatile Bacterial Species that is HighlyCompetent for Natural TransformationDavid M. Young, Donna Parke, and L. Nicholas Ornston 519

The Origins of New Pandemic Viruses: The Acquisition of New HostRanges by Canine Parvovirus and Inuenza A VirusesColin R. Parrish and Yoshihiro Kawaoka 553

Vaccine-Derived Polioviruses and the Endgame Strategy for GlobalPolio EradicationOlen M. Kew, Roland W. Sutter, Esther M. de Gourville, Walter R. Dowdle,

and Mark A. Pallansch 587

viii Contents

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INDEXES

Subject Index 637

Cumulative Index of Contributing Authors, Volumes 5559 675

Cumulative Index of Chapter Titles, Volumes 5559 678

ERRATA

An online log of corrections to Annual Review of Microbiology chaptersmay be found at http://micro.annualreviews.org/

Contents ix

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