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Editorial papers

Mikol. Lek. 2004, 11 (2): 139-144

ISSN 1232-986X

Candida secreted aspartyl proteinases –

expression and function during infection

Candida wydzielaj¹ce proteinazy aspartylowe – ekspresja i funkcja podczas zaka¿enia

Julian R. Naglik, Elina Tsichlaki, Stephen J. Challacombe

Department of Oral Medicine, Pathology & Immunology, GKT Dental Institute, Kings College London (Guy’s Campus), London, UK

The opportunistic fungal pathogen Candida albicans possesses a repertoire of virulence attributes in order to colonise, infect, and evade host defence

mechanisms. In particular, the secreted aspartic proteinases (Saps), encoded by a SAP gene family with ten members, appear to play a central role in

C. albicans pathogenicity as demonstrated primarily through gene expression studies, the using SAP-deficient mutants, and proteinase inhibitors. In

recent years we have been interested in determining whether SAP gene expression patterns observed in vitro and in animal models are representative of

those expressed in the context of human disease. The in vivo analysis of SAP gene expression profiles in over 130 oral and vaginal clinical C. albicans

samples confirmed the differential expression of the SAP family in humans and correlated the expression of specific SAP genes with active disease

and anatomical location (J. Inf. Dis., 2003, 188, 469-479). To extend these qualitative findings, we have recently established real-time RT-PCR analy-

sis of the SAP genes to determine the quantitative levels of each SAP mRNA transcript present in the different infection situations. In preliminary expe-

riments, we have first assessed the quantitative SAP1-10 expression profiles in C. albicans cells during growth in YPD and YCB/BSA (Sap2-inducing

medium). All the SAP genes could be detected at most time points in both YPD and YCB/BSA and SAP9 was consistently the most highly expressed

gene. In YPD, no significant changes in SAP1-10 expression were observed throughout the time course; however, the predicted upregulation of SAP2

expression in YCB/BSA was clearly evident by 6 h, with a peak induction at 24 h at which point SAP2 was induced to levels 20-fold that of ACT1. The

real time RT-PCR assay is presently being used to assess the quantitative expression of SAP1-10 during oral and vaginal infections in humans to

discover which SAP genes are the most highly expressed and which are up- or down-regulated during mucosal Candida infection.

Key words: Candida, pathogenicity, secreted aspartyl proteinase, quantitative gene expression, real-time RT-PCR

Oportunistyczny patogen grzybiczy, jakim jest Candida albicans, ma wiele w³aœciwoœci chorobotwórczych umo¿liwiaj¹cych kolonizacjê, zara¿enie

i unikniêcie mechanizmów obronnych gospodarza. W szczególnoœci wydzielane proteazy aspartylowe (Saps), kodowane przez rodzinê 10 genów SAP,

wydaj¹ siê odgrywaæ g³ówn¹ rolê w patogennoœci C. albicans, co zosta³o potwierdzone dziêki badaniom nad ekspresj¹ genów, badaniom z zastosowa-

niem mutantów pozbawionych SAP oraz przy u¿yciu inhibitorów SAP. W ostatnich latach skoncentrowano zainteresowania nad zagadnieniem, czy

wzór ekspresji genów SAP obserwowany in vitro oraz w badaniach na zwierzêtach odnosi siê równie¿ do ludzi. Analiza in vivo profilu ekspresji genów

SAP w ponad 130 próbkach C. albicans pobranych od ludzi z b³ony œ³uzowej jamy ustnej lub pochwy potwierdzi³a ró¿n¹ ekspresjê genów z rodziny

SAP, a ekspresja okreœlonych genów SAP korelowa³a z aktywnoœci¹ choroby i anatomiczn¹ lokalizacj¹ (J. Inf. Dis., 2003, 188, 469-479). W celu roz-

szerzenia tych jakoœciowych wyników, nasza grupa ostatnio opracowa³a metodê oceny genów SAP za pomoc¹ real-time RT-PCR w celu oceny iloœcio-

wej poziomu transkryptu mRNA ka¿dego genu SAP obecnego w ró¿nych stanach klinicznych. We wstêpnej analizie oceniono iloœciowo ekspresjê 10

genów SAP w komórkach C. albicans podczas wzrostu na pod³o¿u YPD i YCB/BSA (pod³o¿e stymuluj¹ce Sap2). Wszystkie geny SAP mog³y byæ wy-

kryte w wiêkszoœci przedzia³ach czasowych zarówno na pod³o¿u YPD i YCB/BSA, a SAP9 by³ genem, którego ekspresja by³a najwy¿sza. Na pod³o¿u

YPD nie obserwowano istotnych ró¿nic w ekspresji genów SAP1-10 podczas ca³ego badania, natomiast przewidziana wzmo¿ona ekspresja SAP2 na

pod³o¿u YCB/BSA by³a stwierdzana w 6 godz., ze szczytem w 24 godz., gdzie ekspresja SAP2 wzros³a do 20 razy w porównaniu ze stwierdzan¹

w przypadku ACT1. Obecnie metoda real-time RT-PCR jest u¿ywana do iloœciowej oceny genów SAP1-10 w czasie infekcji jamy ustnej i pochwy u lu-

dzi, celem okreœlenia, które geny SAP s¹ najbardziej aktywne oraz, które s¹ hamowane, a które aktywowane podczas infekcji b³on œluzowych wywo³a-

nej C. albicans.

S³owa kluczowe: Candida, patogennoœæ, wydzielane proteinazy aspartylowe, iloœciowa ocena ekspresji genów, real-time RT-PCR

139

Streszczenie

Abstract

Introduction

Candida albicans is the most common fungal pathogen

of humans and is a significant problem of growing clinical im-

portance. The majority of patients, notably immunosuppres-

sed individuals with human immunodeficiency virus (HIV)

infection, experience some form of superficial mucosal can-

didiasis (oral or vaginal) and many suffer from recurrent in-

fections. Candida species usually reside as commensal

organisms as part of the normal microflora and determining

exactly how the transformation from ‘commensal-to-patho-

gen’ takes place and how it can be prevented is a continuing

challenge for medical mycologists.

Most pathogens including Candida species have deve-

loped an effective battery of putative virulence factors and

specific strategies to assist in their ability to colonise host

tissues, cause disease, and overcome host defences. For

C. albicans, the virulence attributes most widely studied in

recent years are hyphal formation, surface recognition mo-

lecules, phenotypic switching, and extracellular hydrolytic

enzyme production. The extracellular hydrolytic enzymes,

specifically the secreted aspartyl proteinases (Sap), are one

of very few gene products that have been shown to directly

contribute to C. albicans virulence.

Secreted aspartyl proteinases of C. albicans

While little is known about the extracellular proteinases

of most dimorphic human pathogenic fungi, the proteolytic

system of C. albicans is well described. Many pathogenic

Candida species are known to possess SAP genes inclu-

ding C. dubliniensis (1), C. tropicalis (2) and C. parapsilo-

sis (3, 4). However, the C. albicans Saps, encoded by ten

SAP genes, are by far the best characterised proteinases

secreted by Candida (reviewed in) (5-7). Biochemically, the

Saps are characterised by (a) having an optimum pH activity

in the acid range and (b) their inhibition by pepstatin A, a he-

xapeptide from Streptomyces (8). Three distinct groups are

clustered within the proteinase family; Sap1-3 are ~67%

identical and Sap4-6 ~89% identical, with Sap7 being the

most divergent with only 20-27% sequence homology to

the other Saps (4). Sap9 and Sap10 are unlike the other

proteinases as they both have C-terminal consensus sequ-

ences typical for glycosylphosphotidylinositol (GPI) proteins

(9). The reader is guided to the following review articles to

obtain more details on various aspects of SAP research: di-

scovery and characterization of the SAP gene family; struc-

ture, processing, activation and regulation; purification, acti-

vity and enzymatic properties; in vitro SAP gene expression

in culture medium, and role in pathogenesis and virulence

(5-7, 10-17).

Putative functions of the C. albicans Sap proteins

during infection

The virulence attributes utilised by C. albicans to co-

lonise and infect host tissues will vary depending on the

type of infection, the stage and site of infection, and the

nature of the host response. Thus, C. albicans must be

highly adapted to an existence on and within the host. Gi-

ven that the Saps are the only described extracellular

proteinases of C. albicans, which must thus rely on this

one enzyme family for protein acquisition, this strongly al-

ludes to the versatile proteolytic nature of the Sap family.

One basic function for the C. albicans Saps is likely to

include digestion of host proteins to provide nitrogen for

the cells. This is catered for by the distinct differences in

pH optima of the proteinases, with Sap1-3 (yeast-associa-

ted) having optimum activity at pH 3-5 and Sap4-6 (hyphal-

associated) at pH 5-7 (18). Based on current data, Sap ac-

tivity ranges from pH 2 to 7, a property that may prove vital

to C. albicans survival and infection within the host and

thus to its success as an opportunistic pathogen.

The Saps may have also evolved to fulfil more direct

virulence functions. Based predominantly on Sap2 data,

the Saps are likely to contribute to host tissue adhesion

and invasion by degrading host structural and intercellular

proteins, or by destroying cells and degrading proteins of

the immune and host cascade system to avoid or resist

antimicrobial attack (reviewed in) (5-7). Extracellular ma-

trix and host surface proteins such as fibronectin, lami-

nin, and mucin, are efficiently degraded by Sap2 (16,

19, 20), which may promote C. albicans adherence to

mucosal sites. Several host defence proteins such the

proteinase inhibitor a-macroglobulin, enzymes of the re-

spiratory burst of macrophages, salivary lactoferrin, and

almost all immunoglobulins, including secretory IgA, can

also be hydrolysed by Sap2 (16, 21), signifying a functio-

nal role for Sap in immune evasion. In addition, Saps may

activate the proinflammatory cytokine interleukin 1b from

its precursor, suggesting a role for Saps in the induction

and maintenance of the inflammatory response in vivo

(22). Furthermore, the proteinases can inactivate the epi-

dermal cysteine proteinase inhibitor cystatin A (23), clea-

ve human big endothelin 1 (a vasoconstrictive peptide)

(24), and activate host protein precursors such as blood

clotting factors (25), but these have no obvious advanta-

ges for the fungus. The data indicate that C. albicans

proteinases are likely to have multiple targets and func-

tions in vivo, which are required for nutrient acquisition,

adherence, penetration and invasion, and immune eva-

sion or escape. The substrate specificities of the other

members of the Sap family have not yet been investigated

in detail and so the full range of targets and functions of

this versatile C. albicans gene family in vivo is yet to be

elucidated.

Expression of the C. albicans Sap proteins during infection

The contribution of the Saps to C. albicans pathogene-

sis has been demonstrated in multiple ways, but primarily

through gene expression studies and the use of SAP-defi-

cient mutants and proteinase inhibitors. These studies establi-

shed that different SAP genes appear to be crucial for both

mucosal (Sap1-3) and systemic infections (Sap4-6), and

are involved in C. albicans adherence, tissue damage, and

evasion of host immune responses. However, it should be

noted that Sap involvement in C. albicans pathogenicity is

complex, which is highlighted by the fact that Sap produc-

tion is associated with a number of other putative virulence

attributes of C. albicans including hyphal formation, adhe-

sion, and phenotypic switching. For full details on the use of

SAP-deficient mutants and proteinase inhibitors in deciphe-

ring the role of Saps in C. albicans pathogenesis, the rea-

der is guided to the following review article (5). Below, the

140

Julian R. Naglik, Elina Tsichlaki, Stephen J. Challacombe

Mikol. Lek. 2004, 11 (2)

141

Candida secreted aspartyl proteinases – expression and function during infection

Fig. 1. Detection of C. albicans SAP1-8 mRNA expression from oral (A) and vaginal (B) clinical samples. The total percentage of subjects expressing each SAP gene is illu-

strated in each group: oral infected patients (n=40), vaginal infected patients (n=40), asymptomatic oral Candida carriers (n=29), and asymptomatic vaginal Candi-

da carriers (n=28). All the SAP genes were expressed, with SAP2 and SAP5 being the most common during both infection and carriage. The Fisher’s exact test in-

dicates that when compared with the carrier group, SAP1, SAP3, SAP4, SAP7, and SAP8 expression is correlated with oral disease, whereas SAP1, SAP3, SAP6,

SAP7, and SAP8 expression is correlated with vaginal disease (shown by asterix). (Original data published in J. Infect. Dis., 2003, 188, 469-479)

Fig. 2. Growth curve of C. albicans CAF2-1 cells

in YPD and YCB/BSA over 30 h

Fig. 3. Quantitative expression of SAP2 and SAP9

in YCB/BSA over 30 h using real time Taq-

Man RT-PCR. SAP9 was consistently the

most highly expressed SAP gene throug-

hout the 30 h time course with mRNA le-

vels similar to that of ACT1. SAP2 expres-

sion was noticeably induced by 6 h with

a peak induction at 24 h, at which point

SAP2 was induced to levels 20-fold that of

ACT1. This data supports the view that

SAP2 is the main gene up-regulated during

growth in protein-containing media

OD 600 nm

Fold mRNA expression

50

CAF 2-1 YPD

CAF 2-1 YCB/BSA

40

30

0

0 h

Culture time

1 h

3 h

6 h

12 h

24 h

30 h

10

20

ACT 1

SAP 2

SAP 9

20

10

0

-10

-20

1 h

3 h

6 h

12 h

24 h

30 h

Culture time

A

%

100

80

40

20

0

SAP1 SAP2 SAP3 SAP4 SAP5 SAP6 SAP7 SAP8

60

Percent of subjects

%

100

80

40

20

0

60

Percent of subjects

Oral Infected

Gene

SAP1 SAP2 SAP3 SAP4 SAP5 SAP6 SAP7 SAP8

Gene

Oral Carriage

Vaginal Infected

Vaginal Carriage

B

142

Julian R. Naglik, Elina Tsichlaki, Stephen J. Challacombe

Mikol. Lek. 2004, 11 (2)

expression of the C. albicans Sap proteins during the infec-

tive process is addressed.

C. albicans SAP gene expression during mucosal

and systemic infections has been studied in vitro and in

vivo in animal models and patient samples mainly by use

of reverse transcriptase PCR (RT-PCR). In our own stu-

dies, we have analysed SAP1-8 expression in over 130

subjects with oral and vaginal C. albicans infection or

asymptomatic carriage. SAP2 and SAP5 were the most

common genes expressed during both infection and car-

riage (26, 27). SAP1, SAP3, SAP4, SAP7, and SAP8

expression correlated with oral disease (fig. 1A), whereas

SAP1, SAP3, SAP6, SAP7, and SAP8 expression corre-

lated with vaginal disease (fig. 1B). Furthermore, SAP1,

SAP3 and SAP8 were preferentially expressed in vaginal

rather than oral infections (not shown). The results indica-

ted that not only are certain C. albicans SAP genes pre-

ferentially expressed in the oral cavity and vaginal lumen

of humans, but that individual proteinases are more frequ-

ently expressed during active infection compared with

carriage.

Using C. albicans-infected oral (28) and vaginal (29)

RHE, Schaller et al. discovered that the SAP1-3 subfami-

ly were expressed in the initial stages of infection. Fur-

thermore, Sap1-3 contributed to tissue damage in both

models, as determined by use of SAP1-3 deficient mu-

tants (29, 30), which demonstrated a role for Sap1-3 in

establishing C. albicans infections at human mucosal

surfaces. In a murine model of oropharyngeal candidia-

sis, SAP1-6 and SAP9 transcripts were detected continu-

ously throughout the course of infection with SAP5 and

SAP9 being most highly expressed (31). Also, during ga-

stric candidiasis in immunocompetent and defined immu-

nodeficient gnotobiotic mice all ten SAP genes could be

detected in both mouse models, suggesting that the ab-

sence of certain components of the host defence system

did not appear to alter SAP gene expression during ga-

stric candidiasis (32). Furthermore, in a mouse model of

gastrointestinal infection, SAP4 and SAP6 were constitu-

tively expressed, whereas SAP2, SAP3 and SAP5 mRNA

were only occasionally detected (33). In addition, in an

intraperitoneal infection model, SAP1, SAP2, SAP4,

SAP5, SAP6, and SAP9 were discovered to be the most

commonly expressed proteinase genes within the first 72 h

(34). Lastly, using in vivo expression technology (IVET) in

a mouse model of oesophageal candidiasis, transcription

of SAP5 and SAP6 predominated indicating a possible

role for the SAP4-6 subfamily in mouse mucosal infec-

tions (35).

The expression studies described above have been

key in contributing to our understanding of which SAP ge-

nes might be associated with different mucosal and sys-

temic Candida infections. Although a number of these

studies have revealed differential expression of the SAP

genes, all the SAP genes assayed can often be detected.

This is perhaps due to the low threshold level of mRNA

detection in the different studies, which generally use va-

riations of sensitive RT-PCR-based protocols to detect

SAP gene expression. Consequently, the studies provide

little knowledge of the precise transcript levels of each

SAP gene present in the different infection situations. To

address this, we have recently established real-time qu-

antitative analysis based on the TaqMan system to provi-

de us with more useful information with regard to which

SAP genes are up- and down regulated during different

stages and types of infection.

Quantitative expression of C. albicans SAP1-10 genes in vitro

Gene-specific primer and probes amplifying target se-

quences of <150 bp have been designed to analyse the

quantitative expression of the C. albicans SAP1-10 ge-

nes using real-time RT-PCR. The efficiency and sensitivity

of each primer/probe set was determined using serial

ten-fold dilutions of genomic DNA and RNA isolated from

C. albicans NCPF 3156 grown in YCB/BSA medium (Yeast

carbon base, 1% bovine serum albumin). The primer/pro-

be sets were equally efficient with regard to detecting the

serial dilutions, but the ACT1 and SAP1 primer/probe

sets were slightly less efficient at amplifying their respecti-

ve amplicons (not shown). Thus, a slight correction factor

needed to be incorporated into the data analysis to acco-

unt for this minor discrepancy.

Table I: Fold mRNA expression of SAP1-10 relative to ACT1 in C. albicans CAF2-1 cells grown in YPD for 30 h

Fold mRNA expression

a

Gene

1 h

3 h

6 h

12 h

24 h

30 h

SAP1

–491

–221

–278

–71

–61

–24

SAP2

–374

–447

–512

–110

–171

–17

SAP3

–2469

–5461

–1846

–416

–503

–66

SAP4

b

–36

SAP5

–10226

–37510

–13170

–1629

–422

–41

SAP6

–31324

–14067

–18306

–2684

–1380

–163

SAP7

–140

–187

–341

–1101

–363

–41

SAP8

–1646

–3421

–4656

–982

–820

–91

SAP9

–4.53

–4.84

–4.61

–1.02

1.01

9.25

SAP10

–355

–440

–139

–35

–11

–15

ACT1

1

1

1

1

1

1

a

Values are average of triplicate readings. All but two values are negative, indicating that gene expression is less than that of ACT1

b

SAP4 was not detected until 30 h growth

143

Candida secreted aspartyl proteinases – expression and function during infection

In preliminary experiments the quantitative expression of

the SAP1-10 gene family was determined in C. albicans

CAF2-1 cells during 30 h growth in YPD (1% yeast extract,

2% peptone, 2% dextrose) and YCB/BSA (Sap2-inducing

medium). It has previously been established using Northern

blotting that the predominant gene expressed by C. albi-

cans in the yeast phase in protein-containing medium

(YCB/BSA) is SAP2 (36, 37). Thus, the aims were two fold;

first to confirm that the SAP1-10 primer/probe sets were

able to detect the predicted changes in SAP2 expression,

and secondly to determine the changes in the expression

levels of the other SAP genes under these same growth

conditions.

Table I shows the SAP expression levels in YPD througho-

ut the 30 h time course. All the SAP genes were detec-

ted at all time points, except SAP4, which was detected

only at the final 30 h time point. SAP9 was consistently

the most highly expressed gene relative to actin at all ti-

me points. In general, no significant changes in SAP1-10

expression was observed for the first 6 h growth in YPD,

but at the later time points most of the SAP genes appea-

red to be expressed at higher levels relative to actin,

especially the 30 h time point. However, at 30 h C. albi-

cans CAF2-1 was at stationery phase (fig. 2) and so this

apparent increase in expression in all ten SAP genes mi-

ght be due to lower levels of ACT1 mRNA being present,

rather than a specific upregulation of the SAP genes. Ne-

vertheless, it is interesting to note that SAP9 was expres-

sed 9-fold more than ACT1 during stationery phase (tab.

1).

When CAF2-1 was grown in YCBBSA, the SAP genes

were generally expressed at higher levels (relative to

ACT1). SAP9 again was consistently the most highly

expressed SAP gene throughout the 30 h time course

with mRNA levels similar to that of ACT1 (fig. 3). SAP2

expression was relatively low in the preculture (not

shown) and at the early 1 h time point, being expressed

70-fold less than ACT1 (fig. 3). However, SAP2 mRNA

was noticeably induced by 6 h with a peak induction at

24 h, at which point SAP2 was induced to levels 20-fold

that of ACT1. This upregulation of SAP2 mRNA coinci-

ded with the onset of exponential C. albicans growth in

this culture medium (fig. 2). By 48 h, SAP2 mRNA levels

had decreased, but were still 3-fold higher than ACT1

(fig. 3). Our quantitative expression data supports previo-

us studies using Northern analysis demonstrating that

SAP2 is the main gene up-regulated during growth in pro-

tein-containing media.

The high expression of SAP9 in both YPD and YCB/BSA

has not previously been reported, but since the SAP9 mRNA

levels do not seem to change significantly throughout the

time course in either culture medium, this indicates that

Sap9 production is independent of the presence of protein.

Sap9 possesses C-terminal consensus sequences typical

for GPI proteins and probably has functions different to that of

SAP1-8, and so the consistent high expression of SAP9 in

both YPD and YCB/BSA might be a reflection of this feature.

The function of SAP9 in C. albicans growth, survival and/or

pathogenesis remains to be elucidated.

Our data have confirmed the effectiveness of real-time

RT-PCR in determining the quantitative expression levels

of all ten members of the C. albicans SAP gene family

and in detecting the up- and down- regulation of SAP mRNA

in cells grown under different culture conditions. We aim

to extend these preliminary data to assess the quantitati-

ve expression of SAP1-10 genes in oral and vaginal RHE

models and patient samples in order to give us a better

idea of which SAP genes are most highly expressed and

which are up- and down-regulated during C. albicans in-

fections in vivo.

Acknowledgements

This work was financially supported by the Guy’s and St Thomas’s Charita-

ble Foundation.

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Part of this work has been presented at the EURESCO Conference on Fun-

gal Virulence Factors and Disease held in September 2001, Seefeld, Au-

stria, and at the 6

th

ASM Conference on Candida and Candidiasis held in Ja-

nuary 2002, Tampa, Florida.

Praca by³a czêœciowo prezentowana podczas konferencji EURESCO na te-

mat chorób grzybiczych i grzybiczych czynników chorobotwórczych we

wrzeœniu 2001 (Seefeld, Austria) oraz na 6 Konferencji ASM na temat Can-

dida i kandydozy w styczniu 2002 (Tampa, Florida).

Address for correspondence:

Dr Julian R. Naglik

Department of Oral Medicine, Pathology & Immunology

Kings College London (Guy’s Campus)

Floor 28, Guy’s Tower

London SE1 9RT

United Kingdom

Phone: [phone]

Fax: [phone]

Received: 31-03-2004

Approved: 12-05-2004