aternative candida albicans lifestile

8/8/2019 Aternative Candida Albicans Lifestile

1/24

Alternative Candida albicansLifestyles: Growth onSurfaces

Carol A. Kumamoto and Marcelo D. Vinces

Department 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 on

May 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

biofilm, tissue invasion, invasive growth, thigmotropism, hyphae

Abstract

Candida 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 studied

during liquid growth, but in its natural environment, the gastroin-testinal tract of a mammalian host, the organism associates with

surfaces. Growth on a surface triggers several behaviors, such asbiofilm formation, invasion, and thigmotropism, that are important

for infection. Recent discoveries have identified factors that regulatethese behaviors and revealed the importance of these behaviors for

pathogenesis.

113


2/24

Contents

INTRODUCTION.. . . . . . . . . . . . . . . . 114

SURFACES COLONIZED BYCANDIDA ALBICANS. . . . . . . . . . . 1 1 5

MECHANISMS OF BIOFILMFORMATION AND

DEVELOPMENT.. . . . . . . . . . . . . . 115Biofilm Formation on Implanted

Medical Devices Results in

Drug Refractory Infections . . . . 115Numerous Parameters Influence

Biofilm Formation andStructure . . . . . . . . . . . . . . . . . . . . . . 116

Multiple Mechanisms Contributeto the High Drug Resistance

of Biofilm Cells . . . . . . . . . . . . . . . 116

High Levels of Amino AcidBiosynthesis and Protein

Synthesis in BiofilmC e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7

Defective Biofilm DevelopmentCaused by Mutations that

Alter the Cell Surface and

Compromise Adherence .. . . . . . 118Development of Animal Models

for Biofilms . . . . . . . . . . . . . . . . . . . 118

MECHANISMS OF INVASIVEGROWTH . . . . . . . . . . . . . . . . . . . . . . 119

Tissue Invasion byC. albicans

Hyphae Occurs on Epithelial,Epidermal, and Endothelial

Surfaces DuringCandidiasis . . . . . . . . . . . . . . . . . . . . 119

Protease and PhospholipaseActivities Contribute to

Invasion of Host

Tissue . . . . . . . . . . . . . . . . . . . . . . . . 119Filamentation During Invasive

Growth is Promoted byPhysical Contact .............. 120

Transcription Factors RegulatingInvasive Growth by Embedded

C e l l s . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 0

Signaling Pathways RegulatingInvasive Growth by Embedded

C e l l s . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 3Models for Invasion of Tissue

Surfaces . . . . . . . . . . . . . . . . . . . . . . . 124

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

C ONC LUS IONS .. . . . . . . . . . . . . . . . . . 1 2 6

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 that

time, the incidence of candidiasis has in-creased and C. albicans has become a sig-

nificant nosocomial pathogen. The modern

AIDS 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 immunodeficient hosts aresusceptible to infections ranging from su-

perficial mucosal infections to invasive, life-threatening disease. Candida spp. rank among

the four most common causes of bloodstreaminfections and cardiovascular infections in

U.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 patients

susceptible to candidiasis, increasing the im-portance of C. albicans as a pathogen and

providing impetus for the detailed study ofC. albicansbiology.

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

114 Kumamoto Vinces


3/24

cells are commonly found in association

with surfaces. Therefore, it is important tounderstand how C. albicans cells interact

with surfaces and how the unique aspects ofgrowth on surfaces contribute to C. albicans

pathogenicity. Several distinct C. albicans

behaviors occur on surfaces, including

biofilm formation, invasive growth, andthigmotropism. In this review we describethese behaviors and discuss the mechanisms

that regulate them. Each behavior occursunder specific environmental conditions and

is mediated and controlled by specific fungalproteins.

SURFACES COLONIZED BYCANDIDA ALBICANS

C. albicans is generally found as a commen-sal organism in association with a human or

animal host. In one study, 7.1% of infants were colonized by Candida or other yeasts

on the day of birth and 96% of infants wereorally colonized by approximately one month

of age (94). In adults, gastrointestinal car-riage of Candida spp. is common (78). For

example, fungi were found in 80% of fecal

samples from healthy adults (37). Soll et al.(104) showed that several surfaces in the body

can be colonized, including the vaginal wall,surfaces in the oral cavity, and the anorectal

surface.Individuals carry a particular strain for

long periods, but changes in the colonizing

strain over time can also be detected (78). Asingle individual may carryunrelatedstrains at

different sites (104), and changes in the colo-nizing C. albicansstrain have been observed in

HIV-positive patients (109). Thus, coloniza-tion byC. albicansis not fixed and changes may

occur 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. albicans

cells 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-dimensional

structures 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 findings 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. albicansbehavior andhow these effects con-

tribute to disease.

MECHANISMS OF BIOFILMFORMATION ANDDEVELOPMENT

Biofilm Formation on ImplantedMedical Devices Results in DrugRefractory Infections

A biofilm is a three-dimensional commu-nity of microorganisms embedded in an ex-

opolymeric matrix and attached to a surface.Upon attachment, microorganisms undergo a

change to a sessile (attached) lifestyle. Dental

plaque is a well-known natural example of abacterial biofilm found in humans. C. albicans

also forms a biofilm on dental enamel (61) aswell as on human heart valves, causing endo-

carditis (29).From a human health perspective, biofilms

areimportantbecause they form on implanted

medical devices and result in infections thatare unusually refractory to antimicrobial ther-

apy. Medical device infection contributes toabout half of all nosocomial infections (for

review see Reference 54). Several million vascular and urinary catheters and tens of

thousands of prosthetic heart valves are usedannually in the United States; approximately10% of infections linked to these devices are

due to Candida spp. (54). For example, of theestimated80,000 bloodstream infections asso-

ciatedwith central venouscatheters 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


4/24

Quorum sensing:cell-density-dependentcommunication andcoordination ofmicrobial behavior

via signalingmolecules

rate for device-associated Candida infection is

approximately 30% (54).Cells in a biofilm characteristically exhibit

high resistance to antimicrobial drugs, andtherefore infected devices must usually be re-

moved to cure the infection (54). For somedevices, removal necessitates major surgery

and exposes patients to significant risks. As aresult, the ability of C. albicans to adhere toa medical device and form a biofilm resistant

to antifungal agents represents an importantmedical challenge.

Numerous Parameters InfluenceBiofilm Formation and Structure

Studies have demonstrated that C. albicans

biofilms 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 (filamentous forms of

the organism), and secretion of exopolymericmatrix result in elaboration of the characteris-

tic three-dimensional structure that is typicalof a biofilm. The exopolymeric matrix, com-

posed of carbohydrates, proteins, and other

unidentified components, surrounds the cellsin a mature biofilm (11).

Numerous materials and growth mediasupport the growth of biofilms in the labo-

ratory. The structure of the resulting biofilm,especially the proportions of yeast-form and

hyphal-form cells, is strongly influenced by

parameters such as medium composition,temperature, and the nature of thesubstratum

(59). Mutants unable to form hyphae or yeast-form cells can nevertheless produce a biofilm,

demonstrating that a specific morphology isnot strictly essential for biofilm formation

(10). Because the proportions of the differ-ent morphological forms vary depending onenvironmental conditions, biofilm structure is

highly adaptable.The content of exopolymeric matrix is also

influenced by biofilm incubation conditions.Biofilms incubatedstatically contain relatively

low amounts of matrix, whereas biofilms in-

cubated with shaking produce higher lev-els of matrix (42). This effect of medium

flow is also seen with bacterial biofilms (28).

Thus, flow of the medium above the sur-face is another variable that influences biofilm

structure.Quorum sensing alsoregulatesbiofilm for-

mation. The quorum-sensing molecule far-nesol is produced continuously by growingC. albicanscells, accumulating to levels corre-

lated with cell number, andacts as an inhibitorof germination, i.e., the yeast-to-hypha tran-

sition (44). Incubation of cells in the presenceof farnesol leads to reduced biofilm formation

(86). Farnesol also has deleterious effects on

mature biofilms (86), suggesting that farnesolregulates biofilm stability as well as biofilm

formation. Similar effects of quorum sensing

on mature bacterial biofilms have been noted(106).

A two-componenthistidinekinase, Chk1p,

plays a role in the response of cells to far-

nesol. chk1 mutant cells are insensitive to theeffects of farnesol on both germination and

biofilm formation (58). Chk1p is a cytoplas-mic protein that probably functions together

with currentlyunknown proteins to detect thefarnesol signal and produce the appropriate

response.

Thus, biofilms vary in themorphology andorganization of their cells and in their con-

tent of extracellular matrix. The plasticity inbiofilm structure in response to medium com-

position, the substratum, flow conditions, andquorum sensing suggests that biofilms located

in 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 ofbiofilms is their high-level resistance to an-

timicrobial drugs (28, 29, 59). Drug resistancereflects a property of individual biofilm cells,

since C. albicanscells released from disrupted

116 Kumamoto Vinces


5/24

biofilms still exhibit substantial levels of resis-

tance (8, 9, 85). In C. albicansbiofilms, resis-tance is probably not due to poor penetration

of drugs into the biofilm structure (1); mu-tants that form structurally defective biofilms

nonetheless exhibit high drug resistance (87).In the early stages of biofilm forma-

tion, changes in gene expression contributeto resistance. Resistance to the antifungalfluconazole can be detected as soon as 2 h

after adherence of C. albicans cells (71). Atthis early time point, resistance is strongly de-

pendent on MDR1, which encodes a flucona-zole efflux facilitator. That is, an mdr1 null

mutant biofilm is 16-fold more sensitive to

fluconazole than is a wild-type biofilm. Dele-tion of CDR1 and CDR2, which encode ho-

mologous drug efflux pumps, also decreases

the resistance of adherent cells (71). Simi-lar results were obtained after 6 h of ad-herent growth, and increased efflux activity

in biofilm cells was detected (74). Thus, the

drug-resistant phenotype of biofilm cells atearly times after adherence results from ex-

pression of drug efflux determinants. Over-expression of MDR1, CDR1, and CDR2 is

also important for drug resistance in drug-resistant clinical strains (83).

In mature biofilms (e.g., after 48 h of incu-

bation) the mechanisms that confer drug re-sistance are different. Mature biofilms formed

from mdr1 cdr1 cdr2 triple null mutantsor the various single and double mutants

are highly resistant to drugs (74, 85). Con-

sistent with these observations, expressionof the drug efflux determinants in mature

biofilms is not high (34, 74) and efflux ac-tivity of mature biofilms is not higher than

the activity in planktonic cells (74). Theseresults argue against a role for the efflux

determinants in drug resistance in maturebiofilms.

Decreased membrane ergosterol content

(74) and altered expression of ergosterolbiosynthetic genes (34) in mature biofilm cells

have been noted. Therefore, increased drugresistance in mature biofilms may be related

to an alteration in membrane sterol content.

Altered membrane composition could resultin changes in membrane properties such as

decreased permeability to drugs, leading to

higher drug resistance. An alternative model has proposed that

most Pseudomonas aeruginosa cells in abiofilm exhibit similar antibiotic resistance as

stationary-phase planktonic cells but that thebiofilm also contains a population of highly

resistant persister cells. The persister cells

are 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 cells

allow recovery of the population after an-tibiotic treatment is discontinued. Although

the existence of persister cells in C. albicans

biofilms has not been tested directly, there is

evidence of cellular heterogeneity in C. al-bicans biofilms (108), and thus this mecha-nism could also contribute to biofilm drug

resistance.In summary, multiple mechanisms con-

tribute to the increased drug resistance ex-

hibited by biofilms. Because drug resistancemechanisms seen in early biofilms differ

from those conferring resistance in maturebiofilms, strategies designed to block early

biofilm formation may differ from strategies

that would be effective for eliminating maturebiofilms.

High Levels of Amino AcidBiosynthesis and Protein Synthesisin Biofilm Cells

To understand the distinctive biofilm lifestyleat the molecular level, microarray studies of

gene expression in C. albicans biofilm cellshave been conducted (34). Results revealed

that, in comparison to postlogarithmic plank-tonic cells that had been grown for thesame length of time, cells in biofilms express

higher 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 biofilms require high-level protein



6/24

biosynthesis, necessitating increased amino

acid biosynthesis.Interestingly, analyses of gene expression

or protein composition in bacterial biofilmcells compared with postlogarithmic plank-

tonic cells grown for the same length of timeshowed some similarities to the results ob-

tained with C. albicans biofilms (98, 111).Among the proteins or transcripts of genespresent at higher concentration in bacterial

biofilm cells were several gene products in-volved in the synthesis of amino acids and

nucleotides or in protein translation. How-ever, when bacterial biofilm cells were com-

pared with planktonic cells that were in the

exponential growth phase, these differencesin gene expression were not observed (98).

These results imply that biofilms contain cells

whose protein synthesis machinery is func-tioning at the level seen in exponential-phasecells, even though the biofilm has been devel-

oping for a long period.

Defective Biofilm DevelopmentCaused by Mutations that

Alter the Cell Surface andCompromise Adherence

Several mutations that reduce biofilm devel-

opment by compromising the first step inbiofilm formation, adherenceto surfaces, have

been studied. One of these mutations affectsEFG1, a major regulator of hyphal develop-

ment (107). Efg1p regulates numerous genes

whoseproducts include many cell surface pro-teins (62, 77, 103). As a result of the cell

surface defect and the defect in productionof hyphae, it is not surprising that efg1 null

mutants are defective in biofilm development(87). On polystyrene, the defect is evident

during the earliest stages of biofilm develop-ment, i.e., adherence and microcolony forma-tion (87). efg1 null mutants are also defective

in adhering to polyurethane central venouscatheter material (65). TheEAP1 gene, which

encodes 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 this

surface protein can mediate attachment topolystyrene (66). Therefore, the failure ofefg1 mutants to initiate biofilm formation onpolystyrene may reflect the absence of Eap1p.

Other mutations affect adherence andbiofilm formation. Mutants lacking ACE2,

which encodes a transcription factor that

regulates expression of chitinase and cellwall proteins, exhibit reduced adherence to

polystyrene and reduced biofilm formation(50). Mutants lacking the NOT4 gene, which

encodes a putative E3 ubiquitin ligase, fail to

attach firmly to a serum-coated plastic surfaceand are defective in biofilm formation (57). As

with the efg1 mutant, the absence of Not4p

or Ace2p may alter the surface properties ofC. albicans, leading to the observed defects inadherence and biofilm development.

Adherence ofCandida glabrata leading to

biofilm formation is mediated in part byEpa6p, a cell surface protein. Expression of

EPA6isactivatedunderconditionsthatleadtobiofilm developmentthrough alterationof the

subtelomeric silencing machinery (48). Takentogether, these results demonstrate that alter-

ation of the cell surface by any one of several

mechanisms results in reduced adherence andreduced biofilm development.

Development of Animal Modelsfor Biofilms

Although model systems make analysis of the

structure and development of biofilms acces-sible to study, animal models are necessary

to understand the pathogenesis of biofilm-related disease. Two animal models have been

recently described, one utilizing rabbits (100)and one using rats (5). Both systems involveplacement of a central venous catheter fol-

lowed by direct inoculation ofC. albicanscellsinto the lumen of the catheter. The resulting

biofilms arestructurallysimilar to biofilms de-scribed in laboratory model systems, except

for the possible presence of host cells in the

118 Kumamoto Vinces


7/24

biofilm (5). The catheter biofilms also exhibit

drug resistance and express the CDR2 gene,consistent with results obtained in laboratory

model systems (5, 100). In the rat model,biofilm development leads to seeding of the

kidneys with C. albicans, demonstrating dis-seminated disease. These animal models offer

the potential for evaluation of new treatmentsfor biofilm infections based on insights fromlaboratory model systems.

In summary, recent developments haverevealed molecular activities required for

biofilm development. Expression of appropri-ate cell surface molecules for adhesion and

expression of genes needed for high levels

of protein synthesis are critical for successfulbiofilm formation. Mechanisms underlying

antifungal resistance differ in early biofilms

and mature biofilms, indicating that differentstrategies are needed to circumvent this prop-erty of biofilms.

MECHANISMS OF INVASIVEGROWTH

Tissue Invasion byC. albicansHyphae Occurs on Epithelial,Epidermal, and Endothelial

Surfaces During CandidiasisIn superficialcandidiasis, epidermalor epithe-

lial surface invasion by C. albicans hyphae iscommonly observed (79). When fungi colo-

nize an epithelial or epidermal surface, they

adhere to host cells and create depressionsin the surface of the host cells (45, 51, 60,

88). Fungal yeast-form cells also convert tofilamentous hyphae, which penetrate into the

surface.During invasion and traversal of an

endothelial 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 may

then be invaded by hyphal-form cells.In specimens scraped from human oral or

cutaneous lesions, most C. albicans cells are

Endothelial:pertaining to tissuecomposed of a layerof flat squamouscells, such as thoselining blood and

lymphatic vessels andthe heart

found within host cells (20, 70, 73). Access to

the interior of host cells is probably achievedby a combination of enzymatic activities (e.g.,

proteases and phospholipase) and mechani-cal force. Ultrastructural observations reveal

areas 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 result

of their interactions with C. albicans, support-ing the notion that C. albicansuses mechani-

cal force to aid penetration (99). Studies withmodel systems using explanted tissue, animal

infection, or cells in culture confirm these fea-

tures of invasion by C. albicans and demon-stratethat C. albicanshyphae may enter or pass

completely 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 key

component of invasive growth, the process of

penetrating the substratum. Invasion of theepithelial surface allows infecting organisms

to reach thebloodstream,andpenetration anddisruption of endothelial surfaces allow or-

ganisms to escape from the bloodstream andinfect deep tissues. Thus, the invasive behav-

ior ofC. albicanson a biological surface con-

tributes to disease pathogenesis.

Protease and PhospholipaseActivities Contribute toInvasion of Host Tissue

Degradative enzymes secreted by C. albicans

cells 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). Pepstatin

treatment also enhances survival followingintranasal challenge in mice by C. albicans

(31). In addition, several mutants lacking SAP

genes showed decreased invasion (76). Simi-larly, secreted phospholipase activity has been

implicated in tissue invasion. In invading hy-phae, phospholipase activity is concentrated

at the hyphal tip (35, 84); a mutant lacking



8/24

secreted phospholipase B1 exhibits reduced

ability 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 host

cells 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, high

temperature, or microaerophilic conditions

(30, 39). To study invasive hyphal development

specifically, we have used an agar model sys-

tem that mimics some of the features of tissue

invasion.Growth ofC. albicanscells embedded

Figure 1

Invasive growth ofC. albicanswithin host tissue. Sections of fixed tonguetissue from gnotobiotic immunosuppressed piglets orally inoculated with(panel a) wild-type C. albicansor (panel b) efg1 cph1 double null mutantcells. Immunohistochemical analysis with anti-C. albicansantibody isshown. From Reference 91 with permission.

within agarmedium stimulatesthe conversion

of yeast-form cells to filaments that invade themedium (17). Control experiments indicate

that the important cue for hyphal productionis not reduced oxygen levels or gradients of

nutrients but rather physical contact of cellswith agar or other matrixmaterial (17). Unlike

hyphal development stimulated by other cues,invasive filamentation of cells in agar mediumis readily observed in rich medium at low or

high temperature.Studies of immunosuppressed gnotobi-

otic piglets orally inoculated with C. albicans

suggest that contact-dependent filamentation

contributes to invasion of tissue. Theefg1/efg1

cph1/cph1 double mutant strain, which lackstwo transcription factors that regulate fila-

mentation, forms filaments and invades the

tongue of the piglet (91) (Figure 1) or agarmedium (36, 91), but it is defective in fil-amentation under all other conditions (68).

These results suggest that filamentation 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 Regulating

Invasive Growth by Embedded CellsTo date, many genes that differentially affect

filamentous growth depending on whethercells are grown in liquid or on agar medium

have been identified. These genes and their

phenotypes are summarized in Table 1. Tofocus specifically on the molecular mecha-

nisms underlying the filamentous responseto growth within agar medium, the follow-

ing sections summarize the results of stud-ies that make use of the embedded growth

condition (suspending cells within rich agarmedium).

Of the genes known to regulate inva-

sive growth, several are thought to encodetranscription factors (Table 1). The putative

transcription factor CZF1 plays a key role inregulating the response of cells to embed-

ded conditions. Deletion of CZF1 results in

120 Kumamoto Vinces


9/24

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

Gene Filamentation phenotype Putative function Reference(s)

Transcription factors

CPH1 Defective on Spider and SLAD

agar; mild defect within agar

Transcription factor 24, 67

CZF1 Defective within agar Transcription factor 17

EFG1 Hyperfilamentous within agar Transcription factor 36

EFH1 efh1 efg1double mutant

hyperfilamentous within agar

Transcription factor 27

MCM1 Hyperfilamentous when

overexpressed on agar

Transcription factor 93

Signaling components

CEK1 Defective on Lees and Spider agar MAPK 24, 53

CHK1 Defective on serum agar Histidine kinase 117

COS1 Defective on serum and Spider agar Histidine kinase 2, 117

CPP1 Hyperfilamentous and

hyperinvasive on agar

Phosphatase 23

CST20 Defective on Spider agar MAPKKK kinase 24, 53

GAP1 Defective on solid Spider and

SLAD

Amino acid permease 12

GPA2 Defective within agar G-protein -subunit 69a, 72, 96

GPR1 Defective within agar G-protein-coupled receptor 69a, 72

HOG1 Defective in liquid and agar;

hyperinvasive on SLAD agar

MAPK 3

HST7 Defective on Spider and SLAD

agar; hyperfilamentous when

overexpressed within agar

MAPK kinase 24, 53, 96

RAS2 Active allele and mutant defective

within agar

Small G-protein 69a, 96

SLN1 Defective on serum agar Histidine kinase 117

SSK1 Defective on agar; hyperinvasive on

SLAD agar

Two-component response regulator 19

TPK1 Defective on agar PKA catalytic subunit 15

TPK2 Defect in invasive growth of yeast

cells on agar

PKA catalytic subunit 15

Other genes

BMH1 Defective in liquid and within agar;

one allele defective only in liquid

14-3-3 protein 92

FAB1 Defective on Spider and serum agar Phosphatidylinositol 3-phosphate

5-kinase

7

PLD1 Defective on Spider agar;

hyperfilamentous on

cornmeal/Tween agar

Phosphatidylcholine-specific

phospholipase D1

47

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

protein

69



10/24

MAPK:mitogen-activatedprotein kinase

PKA: protein kinaseA

reduced invasive filamentation. Ectopic CZF1

expression results in accelerated filamenta-tion (17) (Figure 2). Czf1p does not affect

morphogenesis under other conditions, sug-gesting that a unique pathway is stimulated in

embedded cells.Mutation ofCPH1, the C. albicanshomo-

logue of the S. cerevisiae STE12 transcriptionfactor, results in defective filamentous growthin certain media (24, 62, 67) and mildly de-

fective filamentous growth within agar (36).The czf1 cph1 double null mutant is more de-

fective than either single mutant, indicatingthat the two genes have partially overlapping

functions (17).EFG1 encodes a basic helix-loop-helix

transcription factor that is required for hy-

phal development under most laboratory con-

ditions (68, 107). However, C. albicans efg1mutant cells are more filamentous than wild-type cells when embedded (36) (Figure 2a,b).

Thus, underembedded conditions,EFG1 acts

as a negative regulator of filamentous growth.The relatedEFH1 geneisalsobelievedtoplay

a negative role in regulating filamentation inthe embedded condition, perhaps in conjunc-

tion with Efg1p (27). The effects of the efg1

mutation are epistatic to the effects of the czf1

mutation and partially epistatic to the effects

of the cph1 mutation (36), and therefore, theefg1 cph1 double null mutant filaments duringgrowth within agar medium, as noted above.

CZF1 affects filamentation 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 filamentation 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 and

Efg1p each define two distinct signalingpathways that regulate filamentous growth

under 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 in

which these cascades regulate morphogene-sis (Figure 3). Under the standard condi-

tions used to promote filamentous growth,e.g., the use of inducers such as serum, neutral

pH, microaerophilicconditions, temperature,

Figure 2

Invasive growth ofC. albicanswithin 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. albicansmaltase gene.

122 Kumamoto Vinces


11/24

Figure 3

The standard and embedded programs for regulation of filamentation. 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 filamentationonly 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 figure 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 performs

a backup, positive function in the standardprogram. During embedded growth, filamen-

tation is promoted by contact with agar orother matrix material. Filamentation under

embedded conditions is controlled by the em-

bedded program, in which Efg1p acts as a

negative regulator and Czf1p functions as anantagonist of Efg1p repressor activity. Thefunction of Cph1p partially overlaps that of

Czf1p 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. albicansgenes GPR1, GPA2, and RAS2

regulate filamentous growth under embed-

ded conditions (69a, 72, 96). GPR1, whichencodes a G-protein-coupled receptor, andGPA2, which encodes a G-protein -subunit

homologue, probably function in transmis-sion of signals. gpr1 and gpa2 mutants are

defective in filamentous growth under vari-ous conditions, particularly during embedded

growth (69a, 72, 96). Recent studies suggestthat GPR1 and GPA2 act in the cAMP-PKA

pathway (69a, 72). These studies demonstrate

suppression of the gpa2 defect by additionof exogenous cAMP. In addition, the gpa2



12/24

mutation is rescued by overexpression ofTPK1, a component of the cAMP-PKA path-

way, but not by overexpression ofHST7, a

member of the MAPK pathway (69a).Under standard inducing conditions, the

small G-protein-encoding RAS1 gene posi-tively regulates filamentous growth and is be-

lieved to act in both the cAMP and MAPKpathways (62a) (Figure 3). Deletion ofRAS1

causes a defect in filamentation under em-

bedded conditions, and this defect can besuppressed by addition of exogenous cAMP,

suggesting a positive role for both RAS1 andcAMP in regulating filamentous growth in

this condition (69a). Although under stan-

dard conditions exogenous cAMP acceler-ates filamentous growth, the influence of

cAMP on filamentation of embedded cells is

unclear.Takentogether, the datasuggest thatunder

embedded conditions the MAPK pathway ac-

tivates a positive regulator, Cph1p, (53) and

promotes filamentous growth, whereas thecAMP-dependent pathway, including RAS1,GPR1, and GPA2, acts on a negative regula-tor, Efg1p, (14) that suppresses filamentous

growth. 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 roles

in filamentation in liquid and agar media (15).The tpk1 mutant is defective for filamentous

growth on agar media and is only slightly

affected in liquid, and the tpk2 mutant isonly partially defective on agar but is strongly

blocked for filamentation in liquid. The basisfor the differential effects ofTPK1 and TPK2

is currently unclear.Other genes that affect morphogenesis

on agar medium include SLN1, COS1, andCHK1, which encode two-component histi-dine kinases, SSK1, which encodes a response

regulator, 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 specifically affect filamentation on agar

medium have been described, but the mech-

anisms by which they act are incompletelyunderstood. These genes are summarized in

Table 1.

Models for Invasion of TissueSurfaces

Studies of the efg1 and/or the efg1 cph1 dou-ble null mutant in different animal models of

infection reveal two mechanisms for penetra-tion of tissue surfaces. One mechanism occurs

on mucosal surfaces and involves Efg1p-

dependent adherence and proliferation on thesurface, followed by Efg1p-independent pen-

etration of the surface. A second mechanism

occurs on endothelial surfaces and involvesuptake of fungi by endocytosis, followed byintracellular, Efg1p-dependent hyphal devel-

opment that allows fungal penetration of the

surface.The extensive surface growth of wild-type

C. albicans in pseudomembranous oral can-didiasis in the immunosuppressed gnotobiotic

piglet is dependent on Efg1p and is not ob-

served with the efg1 cph1 double null mutant(6). In addition,defects in adhesion ofefg1 null

mutant or efg1 cph1 double null mutant cellsto oral epithelial cells and Caco-2 cells have

been observed (26, 112).In several systems, the overall extent of in-

vasion is reduced in the absence of Efg1p,

probably reflecting 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 ofEFG1 function. This model is based

on the observations that efg1 null mutants arehyperfilamentous and hyperinvasive in agar(36) (Figure 2a,b) and that EFG1 transcrip-

tion is downregulated during filamentation inliquid medium (107, 110).

Therefore, because Czf1p is a negativeregulator of Efg1p (36), Czf1p may act as a

switch that inactivates Efg1p and promotes

124 Kumamoto Vinces


13/24

the conversion from surface growth to inva-

sive growth. Activation of the Czf1p switchoccurs as a response to contact with agar or

other matrix material. Czf1p antagonism ofEfg1p is not the only mechanism that pro-

motes invasive filamentation, since there arealso high-temperature-activated mechanisms

that 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 such

as endothelial cells are stimulated to takeup C. albicans cells by endocytosis, and thus

a 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 efg1

mutant cells are internalized by macrophages

(68) or neutrophils (56). Defective exit by ger-mination and defective endocytosis probably

contribute to the failure ofefg1 mutants to in-vade and damage endothelial cell monolayers

(81).In summary, tissue invasion requires

degradative activities secreted by the invad-

ing C. albicanshyphae and mechanical forcesexerted by the hyphae. The mechanisms

that regulate invasion are adapted to thenature of the surface to which C. albicanscells

are bound. The mechanism for invasion of

mucosal surfaces resembles the embeddedprogram for regulation of filamentation. That

is, following Efg1p-dependent proliferationon the surface, Efg1p is downregulated and

contact-dependent hyphal development en-sues. A second mechanism involving entry of

fungi into host cells via endocytosis followedby intracellular, Efg1p-dependent filamenta-tion and exit from the host cells is important

on endothelial surfaces. In this situation,control of hyphal development resembles the

standard program for regulation of filamen-tation. The cues that promote filamentation

on 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 for

filamentation contributes to the remarkableversatility ofC. albicansas a pathogen.

GUIDANCE OF HYPHAE BYTHIGMOTROPISM

The direction of C. albicans hyphal growth

can be determined by the topography ofthe substratum. This behavior, known as

thigmotropism, allows hyphae to be guidedby ridges in the substratum (113). In stud-

ies of this behavior, hyphae penetrate the

pores of nucleopore membranes and, afterpenetration, follow the face of the mem-

brane. During penetration, hyphae grow

both away from and toward the agar un-derneath the membrane, implying that theorientation of hyphal growth is not due

to chemotropism but to thigmotropism (38,

101). Helical growth of hyphae occurs whencells are grown on firm surfaces, such as cel-

lophane (102). These thigmotropic responsesto surface features are reminiscent of the

behavior of plant-pathogenic fungi on leaf

surfaces.Thigmotropism plays a major role in the

location of infectable sites on plants by phy-topathogenic fungi. For example, Uromycesappendiculatus, the causative agent of beanrust, produces hyphae that are guided by

the topography of the bean leaf surface to

grow toward stomata, where they differen-tiate into appressoria, specialized structures

needed 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 pathogens

Puccinia hordeiandMagnaporthe grisea also use

thigmotropicbehavior on thesurface of plantsto locate openings in the epidermal layer of

plant tissue and initiate invasive growth (43,89, 116). Hence, thigmotropism plays an im-

portant role in guidance of hyphal growth,



14/24

MS:mechanosensitive

in regulation of development, and in disease

progression. 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 a

general feature of fungal hyphae that mustforage for nutrients on surfaces and withinmaterials.

The thigmotropic response ofC. albicans

may be an adaptation for penetrating tissue.

Studies of cultured intestinal enterocytesinfected with C. albicans (115) and of biop-

sies of oral candidiasis taken from AIDS

patients (90) demonstrated orientation of hy-phae along ridges or grooves and penetration

into 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 organisms

from archeans, E. coli, and yeast to higher eu-karyotes (13, 49, 55, 118) and are believed

to 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 the

opening of the channels. C. albicanspossesses

MS channel activity. Reorientation of hyphalgrowth in response to ridges is attenuated by

treatment with an inhibitor of MS channels,Gd3+, which suggests that MS channels are

responsible for sensing substrate topography

(113).

To summarize, thigmotropic behavior al-

lows C. albicans to respond with greatprecision to topographical features of the

surface. This behavior may contribute totissue invasion by directing penetrating

hyphae toward more vulnerable regions ofthe tissue. Further studies will illuminate the

details 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. albicanscells form biofilms with char-acteristic three-dimensional structures and

high antifungal resistance. This response to

contact with a surface is significant for hu-man health because of the large numbers

of implanted medical devices used in mod-ern medicine. On tissue surfaces, C. al-

bicans initially adheres and grows on thesurface and subsequently produces invasive

filaments that penetrate the surface. Thisbehavior allows organisms to escape from

their normal niche in the gastrointestinal

tract and reach the bloodstream, settingthe stage for disseminated, life-threatening

infection. 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 develop

methods for interrupting the progression to

disease.

SUMMARY POINTS

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

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

3. Biofilm formation, a behavioral response ofC. albicanscells growing on a surface, is

associated with medical device infection.

4. The morphology and organization ofC. albicansbiofilms are influenced by numerous

parameters, and thus variability may be observed in biofilms located at different siteswithin the host.

126 Kumamoto Vinces


15/24

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

6. Secreted degradative enzymes and the hyphal form of growth play key roles during

tissue invasion byC. 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, biofilmdevelopment, colonization, and invasion.

ACKNOWLEDGMENTS

We are grateful to Perry Riggle, Julia Kohler, and Linc Sonenshein for careful reading of the

manuscript and for helpful comments. Our research on this project was supported by grantAI38591 from the National Institute of Allergy and Infectious Diseases.

LITERATURE CITED1. Al-Fattani MA, Douglas LJ. 2004. Penetration ofCandida biofilms by antifungal agents.

Antimicrob. Agents Chemother. 48:329197

2. Alex LA, Korch C, Selitrennikoff CP, Simon MI. 1998. COS1, a two-component histidinekinase that is involved in hyphal development in the opportunistic pathogen Candida

albicans. Proc. Natl. Acad. Sci. USA 95:7069733. Alonso-Monge R, Navarro-Garcia F, Molero G, Diez-Orejas R, Gustin M, et al. 1999.

Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence ofCandida albicans. J. Bacteriol. 181:305868

4. Ampel NM. 1996. Emerging disease issues and fungal pathogens associated with HIV

infection. Emerg. Infect. Dis. 2:10916

5. Andes D, Nett J, Oschel P, Albrecht R, Marchillo K, Pitula A. 2004. Development andcharacterization of an in vivo central venous catheter Candida albicans biofilm model.

Infect. Immun. 72:6023316. Andrutis KA,Riggle PJ,Kumamoto CA, Tzipori S. 2000.Intestinal lesions associated with

disseminated candidiasis in an experimental animal model.J. Clin. Microbiol. 38:2317237. Augsten M, Hubner C, Nguyen M, Kunkel W, Hartl A, Eck R. 2002. Defective hyphal

induction of a Candida albicansphosphatidylinositol 3-phosphate 5-kinase null mutant onsolid media does not lead to decreased virulence. Infect. Immun. 70:446270

This careful studyof the resistance ofbiofilm cells

demonstrates thatresuspended cellsfrom a biofilmexhibit enhancedantifungalresistance.

8. Baillie GS, Douglas LJ. 1998. Effect of growth rate on resistance ofCandida albicans

biofilms to antifungal agents. Antimicrob. Agents Chemother. 42:19005

9. Baillie GS, Douglas LJ. 1998. Iron-limited biofilms ofCandida albicansand their suscep-

tibility to amphotericin B. Antimicrob. Agents Chemother. 42:21464910. Baillie GS, Douglas LJ. 1999. Role of dimorphism in the development ofCandida albicans

biofilms. J. Med. Microbiol. 48:6717911. Baillie GS, Douglas LJ. 2000. Matrix polymers ofCandida biofilms and their possible role

in biofilm resistance to antifungal agents. J. Antimicrob. Chemother. 46:39740312. Biswas S, Roy M, Datta A. 2003. N-acetylglucosamine-inducible CaGAP1 encodes a

general amino acid permease which co-ordinates external nitrogen source response and

morphogenesis in Candida albicans. Microbiology 149:2597608



16/24

13. Blount P. 2003. Molecular mechanisms of mechanosensation: big lessons from small cells.Neuron 37:73134

14. Bockmuhl DP, Ernst JF. 2001. A potential phosphorylation site for an A-type kinase

in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans.Genetics157:152330

15. Bockmuhl DP, Krishnamurthy S, Gerads M, Sonneborn A, Ernst JF. 2001. Distinct andredundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis

and growth ofCandida albicans. Mol. Microbiol. 42:12435716. Borg M,RuchelR. 1988. Expression of extracellular acid proteinase by proteolytic Candida

spp. during experimental infection of oral mucosa. Infect. Immun. 56:62631

The authorsdemonstrate thatgrowth within agarpromotesfilamentation andthatCZF1 is animportantregulator of thisresponse.

17. Brown DH Jr, Giusani AD, Chen X, Kumamoto CA. 1999. Filamentous growth of

Candida albicansin response to physical environmental cues, and its regulation by

the unique CZF1 gene. Mol. Microbiol. 34:65162

18. Calderone RA. 2002. Introduction and historical perspectives. In Candida and Candidiasis,

ed. RA Calderone, pp. 313. Washington, DC: ASM Press

19. Calera JA, Zhao XJ, Calderone R. 2000. Defective hyphal development and avirulencecausedbyadeletionoftheSSK1 response regulator genein Candida albicans.Infect. Immun.

68:51825

20. Cawson RA, Rajasingham KC. 1972. Ultrastructural features of the invasive phase ofCandida albicans. Br. J. Dermatol. 87:43543

This articleincludes a studyof the stagesof biofilmdevelopment andthe architecture ofthe mature biofilm.

21. Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA.

2001. Biofilm formation by the fungal pathogen Candida albicans: development,

architecture, and drug resistance. J. Bacteriol. 183:538594

22. Clemons KV, Calich VL, Burger E, Filler SG, Grazziutti M, et al. 2000. Pathogenesis I:

interactions of host cells and fungi. Med. Mycol. 38(Suppl. 1):9911123. Csank C, Makris C, Meloche S, Schroppel K, Rollinghoff M, et al. 1997. Derepressed

hyphal growthand reduced virulencein a VH1family-relatedprotein phosphatase mutant

of the human pathogen Candida albicans. Mol. Biol. Cell8:25395124. Csank C, Schroppel K, Leberer E, Harcus D, Mohamed O, et al. 1998. Roles of the Can-

dida albicansmitogen-activated protein kinase homolog, Cek1p, in hyphal developmentand systemic candidiasis. Infect. Immun. 66:271321

25. Davis D. 2003. Adaptation to environmental pH in Candida albicans and its relation topathogenesis. Curr. Genet. 44:17

26. Dieterich C, Schandar M, Noll M, Johannes FJ, Brunner H, et al. 2002. In vitro re-

constructed human epithelia reveal contributions ofCandida albicans EFG1 and CPH1 toadhesion and invasion. Microbiology 148:497506

27. Doedt T, Krishnamurthy S, BockmuhlDP, Tebarth B, Stempel C, et al.2004.APSESpro-teins regulate morphogenesis and metabolism in Candida albicans. Mol. Biol. Cell15:3167

8028. Donlan RM, Costerton JW. 2002. Biofilms: survival mechanisms of clinically relevant

microorganisms. Clin. Microbiol. Rev. 15:1679329. Douglas LJ. 2003. Candida biofilms and their role in infection. Trends Microbiol. 11:303630. Ernst JF. 2000. Transcription factors in Candida albicans: environmental control of mor-

phogenesis. Microbiology 146(Pt. 8):17637431. Fallon K, Bausch K, Noonan J, Huguenel E, Tamburini P. 1997. Role of aspartic proteases

in disseminated Candida albicansinfection in mice. Infect. Immun. 65:5515632. Farrell SM, Hawkins DF,RyderTA. 1983.Scanningelectronmicroscope study ofCandida

albicansinvasion of cultured human cervical epithelial cells. Sabouraudia 21:25154

128 Kumamoto Vinces


17/24


18/24

53. Kohler JR, Fink GR. 1996. Candida albicansstrains heterozygous and homozygous for mu-

tations in mitogen-activated protein kinase signaling components have defects in hyphaldevelopment. Proc. Natl. Acad. Sci. USA 93:1322328

54. Kojic EM, Darouiche RO. 2004. Candida infections of medical devices. Clin. Microbiol.Rev. 17:25567

55. Koprowski P, Kubalski A. 2001. Bacterial ion channels and their eukaryotic homologues.Bioessays23:114858

56. Korting HC, Hube B, Oberbauer S, Januschke E, Hamm G, et al. 2003. Reduced expres-sion of the hyphal-independent Candida albicansproteinase genes SAP1 and SAP3 in theefg1 mutant is associated with attenuated virulence during infection of oral epithelium. J.

Med. Microbiol. 52:6233257. Krueger KE, Ghosh AK, Krom BP, Cihlar RL. 2004. Deletion of theNOT4 gene impairs

hyphal development and pathogenicity in Candida albicans. Microbiology 150:2294058. Kruppa M, Krom BP, Chauhan N, Bambach AV, Cihlar RL, Calderone RA. 2004. The

two-component signal transduction protein Chk1p regulates quorum sensing in Candida

albicans. Eukaryot. Cell3:10626559. Kumamoto CA. 2002. Candida biofilms. Curr. Opin. Microbiol. 5:6081160. Kvaal C, Lachke SA, Srikantha T, Daniels K, McCoy J, Soll DR. 1999. Misexpression of

the opaque-phase-specificgenePEP1 (SAP1)inthewhitephaseofCandida albicansconfers

increased virulence in a mouse model of cutaneous infection. Infect. Immun. 67:66526261. Lamfon H, Porter SR, McCullough M, Pratten J. 2003. Formation ofCandida albicans

biofilms on non-shedding oral surfaces. Eur. J. Oral Sci. 111:4657162. Lane S, Birse C, Zhou S, Matson R, Liu H. 2001. DNA array studies demonstrate con-

vergent regulation of virulence factors by Cph1, Cph2, and Efg1 in Candida albicans. J.

Biol. Chem. 276:489889662a. Leberer E, Harcus D, Dignard D, Johnson L, Ushinsky S, et al. 2001. Ras links cellu-

lar morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling

pathways in the pathogenic fungus Candida albicans. Mol. Microbiol. 42:6738763. Leidich SD, Ibrahim AS, Fu Y, Koul A, Jessup C, et al. 1998. Cloning and disruption of

caPLB1, a phospholipase B gene involved in the pathogenicity ofCandida albicans. J. Biol.

Chem. 273:260788664. Lewis K. 2001. Riddle of biofilm resistance. Antimicrob. Agents Chemother. 45:999100765. Lewis RE, Lo HJ, Raad II, Kontoyiannis DP. 2002. Lack of catheter infection by the

efg1/efg1 cph1/cph1 double-null mutant, a Candida albicansstrain that is defective in fila-mentous growth. Antimicrob. Agents Chemother. 46:115355

66. Li F, Palecek SP. 2003.EAP1, a Candida albicansgeneinvolved in binding human epithelialcells. Eukaryot. Cell2:126673

67. Liu H, Kohler J, Fink GR. 1994. Suppression of hyphal formation in Candida albicansbymutation of a STE12 homolog. Science 266:172326. Erratum. 1995. Science 267(5194):17

This important

study demonstratesthat a mutantlacking both Efg1pand Cph1p isnonfilamentousunder almost allconditions and isavirulent.

68. Lo H-J, Kohler JR, DiDomenico B, Loebenberg D, Cacciapuoti A, Fink GR. 1997.

Nonfilamentous C. albicansmutants are avirulent. Cell90:93949

69. LotzH,SohnK,BrunnerH,Muhlschlegel FA, Rupp S. 2004.RBR1, a novel pH-regulatedcellwallgeneofCandida albicans,isrepressedbyRIM101 andactivatedbyNRG1.Eukaryot.Cell3:77684

69a. Maidan MM, De Rop L, Serneels J, Exler S, Rupp S, et al. 2005. The G protein-coupled

receptor Gpr1 and the G protein Gpa2 act through the cAMP-PKA pathway to inducemorphogenesis in Candida albicans. Mol. Biol. Cell(Epub ahead of print)

70. Marrie TJ, Costerton JW. 1981. The ultrastructure ofCandida albicans infections. Can.J. Microbiol. 27:115664

130 Kumamoto Vinces


19/24

71. Mateus C, Crow SA Jr, Ahearn DG. 2004. Adherence of Candida albicans to silicone

induces immediate enhanced tolerance to fluconazole. Antimicrob. Agents Chemother. 48:335866

72. Miwa T, Tagaki Y, Shinozaki M, Yun C-W, Schell WA, et al. 2004. Gpr1, a puta-tive G-protein-coupled receptor, regulates morphogenesis and hypha formation in the

pathogenic fungus Candida albicans. Eukaryot. Cell3:9193173. Montes LF, Wilborn WH. 1968. Ultrastructural features of host-parasite relationship in

oral candidiasis. J. Bacteriol. 96:13495674. Mukherjee PK, Chandra J, Kuhn DM, Ghannoum MA. 2003. Mechanism of fluconazoleresistance in Candida albicansbiofilms: phase-specific role of efflux pumps and membrane

sterols. Infect. Immun. 71:43334075. Mukherjee PK, Seshan KR, Leidich SD, Chandra J, Cole GT, Ghannoum MA. 2001.

Reintroduction of the PLB1 gene into Candida albicansrestores virulence in vivo. Micro-

biology 147:258597

76. Naglik JR, Challacombe SJ, Hube B. 2003. Candida albicanssecreted aspartyl proteinases

in virulence and pathogenesis. Microbiol. Mol. Biol. Rev. 67:4002877. Nantel A, Dignard D, Bachewich C, Harcus D, Marcil A, et al. 2002. Transcription

profiling of Candida albicans cells undergoing the yeast-to-hyphal transition. Mol. Biol.

Cell13:34526578. Odds FC. 1987. Candida infections: an overview. Crit. Rev. Microbiol. 15:1579. Odds FC. 1994. Pathogenesis ofCandida infections. J. Am. Acad. Dermatol. 31:S25

80. Perera TH, Gregory DW, Marshall D, Gow NA. 1997. Contact-sensing by hyphae of

dermatophytic and saprophytic fungi. J. Med. Vet. Mycol. 35:2899381. Phan QT, Belanger PH, Filler SG. 2000. Role of hyphal formation in interactions of

Candida albicanswith endothelial cells. Infect. Immun. 68:34859082. Pope LM, Cole GT. 1981. SEM studies of adherence ofCandida albicansto the gastroin-

testinal tract of infant mice. Scan Electron Microsc. Pt. 3:738083. Prasad R, Panwar SL, Smriti. 2002. Drug resistance in yeasts: an emerging scenario. Adv.

Microb. Physiol. 46:155201

84. Pugh D, Cawson RA. 1977. The cytochemical localization of phospholipase in Candidaalbicansinfecting the chick chorio-allantoic membrane. Sabouraudia 15:2935

85. Ramage G, Bachmann S, Patterson TF, Wickes BL, Lopez-Ribot JL. 2002. Investigationof multidrug efflux pumps in relation to fluconazole resistance in Candida albicansbiofilms.

J. Antimicrob. Chemother. 49:97380

86. Ramage G, Saville SP, Wickes BL, Lopez-Ribot JL. 2002. Inhibition of Candida albi-cansbiofilm formation by farnesol, a quorum-sensing molecule. Appl. Environ. Microbiol.

68:54596387. Ramage G, VandeWalleK, Lopez-Ribot JL, Wickes BL. 2002. The filamentation pathway

controlled by the Efg1 regulator protein is required for normal biofilm formation anddevelopment in Candida albicans. FEMS Microbiol. Lett. 214:95100

88. Ray TL, Payne CD. 1988. Scanning electron microscopy of epidermal adherence andcavitation in murinecandidiasis: a role forCandida acidproteinase.Infect. Immun. 56:194249

89. Read ND, Kellock LJ, Collins TJ, Gundlach AM. 1997. Role of topography sensing forinfection-structure differentiation in cereal rust fungi. Planta 202:16370

90. Reichart PA, Philipsen HP, Schmidt-Westhausen A, Samaranayake LP. 1995. Pseu-domembranous oral candidiasis in HIV infection: ultrastructural findings. J. Oral Pathol.

Med. 24:27681



20/24

91. Riggle PJ, Andrutis KA, Chen X, Tzipori SR, Kumamoto CA. 1999. Invasive lesions

containing filamentous forms produced by a Candida albicansmutant that is defective infilamentous growth in culture. Infect. Immun. 67:364952

92. Roberts RL, Mosch HU, Fink GR. 1997. 14-3-3 proteins are essential for RAS/MAPKcascade signaling during pseudohyphal development in S. cerevisiae. Cell89:105565

93. Rottmann M, Dieter S, Brunner H, Rupp S. 2003. A screen in Saccharomyces cerevisiae

identified CaMCM1, an essential gene in Candida albicanscrucial for morphogenesis.Mol.

Microbiol. 47:9435994. Russell C, Lay KM. 1973. Natural history ofCandida species and yeasts in the oral cavitiesof infants. Arch. Oral Biol. 18:95762

95. Saiman L, Ludington E, Pfaller M, Rangel-Frausto S, Wiblin RT, et al. 2000. Risk factors

for candidemia in Neonatal Intensive Care Unit patients. The National Epidemiology ofMycosis Survey study group. Pediatr. Infect. Dis. J. 19:31924

96. Sanchez-Martinez C, Perez-Martin J. 2002. Gpa2, a G-protein alpha subunit requiredfor hyphal development in Candida albicans. Eukaryot. Cell1:86574

97. Savage DC, Dubos RJ. 1967. Localization of indigenous yeast in the murine stomach. J.

Bacteriol. 94:18111698. Schembri MA, Kjaergaard K, Klemm P. 2003. Global gene expression in Escherichia coli

biofilms. Mol. Microbiol. 48:25367

This articledemonstrates thedeformation ofhuman corneocytesbyC. albicanshyphae duringhyphal penetration.

99. Scherwitz C. 1982. Ultrastructure of human cutaneous candidosis. J. Invest. Der-

matol. 78:2005100. Schinabeck MK, Long LA, Hossain MA, Chandra J, Mukherjee PK, et al. 2004. Rabbit

model of Candida albicans biofilm infection: liposomal amphotericin B antifungal lock

therapy. Antimicrob. Agents Chemother. 48:172732101. Sherwood J, Gow NA, Gooday GW, Gregory DW, Marshall D. 1992. Contact sensing

in Candida albicans: a possible aid to epithelial penetration. J. Med. Vet. Mycol. 30:46169102. Sherwood-Higham J, Zhu WY, Devine CA, GoodayGW, Gow NA, Gregory DW. 1994.

Helical growth of hyphae ofCandida albicans. J. Med. Vet. Mycol. 32:43745103. Sohn K, Urban C, Brunner H, Rupp S. 2003. EFG1 is a major regulator of cell wall

dynamics in Candida albicansas revealed by DNA microarrays. Mol. Microbiol. 47:89102104. Soll DR, Galask R, Schmid J, Hanna C, Mac K, Morrow B. 1991. Genetic dissimilarity

of commensal strains ofCandida spp. carried in different anatomical locations of the samehealthy women. J. Clin. Microbiol. 29:170210

105. Spoering AL, Lewis K. 2001. Biofilms and planktonic cells ofPseudomonas aeruginosa have

similar resistance to killing by antimicrobials. J. Bacteriol. 183:674651106. Stanley NR, Lazazzera BA. 2004. Environmental signals and regulatory pathways that

influence biofilm formation. Mol. Microbiol. 52:91724

The authorsidentifyEFG1 as akey regulator ofmorphogenesis in

C. albicans.

107. Stoldt VR, Sonnenborn A, Leuker CE, Ernst JF. 1997. Efg1p, an essential regu-

lator of morphogenesis of the human pathogenCandida albicansis a member of a

conserved class of bHLH proteins regulating morphogenetic processes in fungi.

EMBO J. 16:198291

108. Suci PA, Tyler BJ. 2003. A method for discrimination of subpopulations ofCandida albi-cansbiofilm cells that exhibit relative levels of phenotypic resistance to chlorhexidine. J.

Microbiol. Methods53:31325109. Sweet SP. 1997. Selection and pathogenicity of Candida albicans in HIV infection. Oral

Dis. 3(Suppl. 1):S8895110. Tebarth B, Doedt T, Krishnamurthy S, Weide M, Monterola F, et al. 2003. Adaptation

of the Efg1p morphogenetic pathway in Candida albicansby negative autoregulation and

PKA-dependent repression of the EFG1 gene. J. Mol. Biol. 329:94962

132 Kumamoto Vinces


21/24

111. Vilain S, Cosette P, Zimmerlin I, Dupont JP, Junter GA, Jouenne T. 2004. Biofilm

proteome: homogeneity or versatility? J. Proteome Res. 3:13236112. Villar CC, Kashleva H, Dongari-Bagtzoglou A. 2004. Role ofCandida albicanspolymor-

phism in interactions with oral epithelial cells. Oral Microbiol. Immunol. 19:26269

The authorsdemonstrate thatinhibition of MS

channels inhibitsthigmotropism inC. albicans.

113. Watts HJ, Very A-A, Perera THS, Davies JM, Gow NAR. 1998. Thigmotropism and

stretch-activated channels in the pathogenic fungus Candida albicans. Microbiology

144:68995

114. Weide MR, Ernst JF. 1999. Caco-2 monolayer as a model for transepithelial migrationof the fungal pathogen Candida albicans. Mycoses42(Suppl. 2):6167115. Wiesner SM, Bendel CM, Hess DJ, Erlandsen SL, Wells CL. 2002. Adherence of yeast

and filamentous forms ofCandida albicansto cultured enterocytes. Crit. Care Med. 30:67783

116. Xiao JZ, Watanabe T, Kamakura T, Oshima A, Yamaguchi I. 1994. Studies on the cellu-lar differentiation ofMagnaporthe grisea. Physiological aspects of substratum surfaces in

relation to appressorium formation. Physiol. Mol. Plant Pathol. 44:22736

117. Yamada-Okabe T, Mio T, Ono N, Kashima Y, Matsui M, et al. 1999. Roles of threehistidine kinase genes in hyphal development and virulence of the pathogenic fungusCandida albicans. J. Bacteriol. 181:724347

118. Zhou XL, Kung C. 1992. A mechanosensitive ion channel in Schizosaccharomyces pombe.EMBO J. 11:286975



22/24

Annual Revie

Microbiology

Volume 59, 20

Contents

Frontispiece

Georges N. Cohen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p xiv

Looking Back

Georges N. Cohen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1

Signaling in the Arbuscular Mycorrhizal Symbiosis

Maria J. Harrison p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 19

Interplay Between DNA Replication and Recombination in

Prokaryotes

Kenneth N. Kreuzer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p43

Yersinia Outer Proteins: Role in Modulation of Host Cell Signaling

Responses and Pathogenesis

Gloria I. Viboud and James B. Bliska p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p69

Diversity and Evolution of Protein Translocation

Mechthild Pohlschrder, Enno Hartmann, Nicholas J. Hand, Kieran Dilks,

and Alex Haddad p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 91

Alternative Candida albicansLifestyles: Growth on Surfaces

Carol A. Kumamoto and Marcelo D. Vinces p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 113

Yeast Evolution and Comparative Genomics

Gianni Liti and Edward J. Louis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 135

Biology of Bacteriocyte-Associated Endosymbionts of Plant

Sap-Sucking Insects

Paul Baumannp p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

155

Genome Trees and the Nature of Genome Evolution

Berend Snel, Martijn A. Huynen, and Bas E. Dutilh p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 191

Cellular Functions, Mechanism of Action, and Regulation of FtsH

Protease

Koreaki Ito and Yoshinori Akiyama p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 211

vii


23/24

Mating in Candida albicansand the Search for a Sexual Cycle

R.J. Bennett and A.D. Johnson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 233

Applications of Autofluorescent Proteins for In Situ Studies in

Microbial Ecology

Estibaliz Larrainzar, Fergal OGara, and John P. Morrissey p p p p p p p p p p p p p p p p p p p p p p p p p p p p 257

The Genetics of the Persistent Infection and Demyelinating Disease

Caused by Theilers VirusMichel Brahic, Jean-Franois Bureau, and Thomas Michiels p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 279

Intracellular Compartmentation in Planctomycetes

John A. Fuerst p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 299

Biogenesis of Inner Membrane Proteins in Escherichia coli

Joen Luirink, Gunnar von Heijne, Edith Houben,

and Jan-Willem de Gier p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 329

Genome-Wide Responses to DNA-Damaging Agents

Rebecca C. Fry, Thomas J. Begley, and Leona D. Samson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 357

The Rcs Phosphorelay: A Complex Signal Transduction System

Nadim Majdalani and Susan Gottesman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 379

Translational Regulation ofGCN4 and the General Amino Acid

Control of Yeast

Alan G. Hinnebusch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 407

Biogenesis, Architecture, and Function of Bacterial Type IV Secretion

Systems

Peter J. Christie, Krishnamohan Atmakuri, Vidhya Krishnamoorthy,

Simon Jakubowski, and Eric Cascalesp p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p

451

Regulation of Bacterial Gene Expression by Riboswitches

Wade C. Winkler and Ronald R. Breaker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 487

Opportunities for Genetic Investigation Afforded byAcinetobacter

baylyi, A Nutritionally Versatile Bacterial Species that is Highly

Competent for Natural Transformation

David M. Young, Donna Parke, and L. Nicholas Ornston p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 519

The Origins of New Pandemic Viruses: The Acquisition of New Host

Ranges by Canine Parvovirus and Influenza A VirusesColin R. Parrish and Yoshihiro Kawaoka p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 553

Vaccine-Derived Polioviruses and the Endgame Strategy for Global

Polio Eradication

Olen M. Kew, Roland W. Sutter, Esther M. de Gourville, Walter R. Dowdle,

and Mark A. Pallansch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 587

viii Contents


24/24

INDEXES

Subject Index p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 637

Cumulative Index of Contributing Authors, Volumes 5559 p p p p p p p p p p p p p p p p p p p p p p p p p p p 675

Cumulative Index of Chapter Titles, Volumes 5559 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 678

ERRATA

An online log of corrections to Annual Review of Microbiology chapters

may be found at http://micro.annualreviews.org/

aternative candida albicans lifestile

Documents