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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
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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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Contents ARI 8 August 2005 15:56
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
Ann
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Contents ARI 8 August 2005 15:56
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/
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