Candida wydzielające proteinazy ...

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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 (Guys Campus), London, UK
Abstract
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
Streszczenie
Oportunistyczny patogen grzybiczy, jakim jest Candida albicans, ma wiele w³aciwoci chorobotwórczych umo¿liwiaj¹cych kolonizacjê, zara¿enie
i unikniêcie mechanizmów obronnych gospodarza. W szczególnoci wydzielane proteazy aspartylowe (Saps), kodowane przez rodzinê 10 genów SAP,
wydaj¹ siê odgrywaæ g³ówn¹ rolê w patogennoci 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 ³uzowej jamy ustnej lub pochwy potwierdzi³a ró¿n¹ ekspresjê genów z rodziny
SAP, a ekspresja okrelonych genów SAP korelowa³a z aktywnoci¹ choroby i anatomiczn¹ lokalizacj¹ (J. Inf. Dis., 2003, 188, 469-479). W celu roz-
szerzenia tych jakociowych wyników, nasza grupa ostatnio opracowa³a metodê oceny genów SAP za pomoc¹ real-time RT-PCR w celu oceny ilocio-
wej poziomu transkryptu mRNA ka¿dego genu SAP obecnego w ró¿nych stanach klinicznych. We wstêpnej analizie oceniono ilociowo 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 wiêkszoci 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 ilociowej oceny genów SAP1-10 w czasie infekcji jamy ustnej i pochwy u lu-
dzi, celem okrelenia, które geny SAP s¹ najbardziej aktywne oraz, które s¹ hamowane, a które aktywowane podczas infekcji b³on luzowych wywo³a-
nej C. albicans.
S³owa kluczowe: Candida, patogennoæ, wydzielane proteinazy aspartylowe, ilociowa ocena ekspresji genów, real-time RT-PCR
139
Julian R. Naglik, Elina Tsichlaki, Stephen J. Challacombe
Mikol. Lek. 2004, 11 (2)
Introduction
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.
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).
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
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
140
Candida secreted aspartyl proteinases expression and function during infection
A
B
Oral Infected
Oral Carriage
Vaginal Infected
Vaginal Carriage
%
100
%
100
80
80
60
60
40
40
20
20
0
0
SAP1 SAP2 SAP3 SAP4 SAP5 SAP6 SAP7 SAP8
SAP1 SAP2 SAP3 SAP4 SAP5 SAP6 SAP7 SAP8
Gene
Gene
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 Fishers 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)
CAF 2-1 YPD
CAF 2-1 YCB/BSA
50
40
30
20
10
0
0 h
1 h
3 h
6 h
12 h
24 h
30 h
Fig. 2. Growth curve of C. albicans CAF2-1 cells
in YPD and YCB/BSA over 30 h
Culture time
ACT 1
SAP 2
SAP 9
20
10
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
0
-10
-20
1 h
3 h
6 h
12 h
24 h
Culture time
30 h
141
Julian R. Naglik, Elina Tsichlaki, Stephen J. Challacombe
Mikol. Lek. 2004, 11 (2)
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
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.
142
SAP4 was not detected until 30 h growth
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).
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 Guys and St Thomass Charita-
ble Foundation.
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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
143
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