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The aims of the present study were to determine whether Allium sativum (garlic) extract has any effect on the morphology transformation of Candida albicans, and to investigate whether it could alter the gene expression level of SIR2, a morphogenetic control gene and SAP4, a gene encoding secreted aspartyl proteinase. Candida albicans cells were incubated with a range of concentrations of fresh garlic extract, and the morphology was monitored via light microscopy. Garlic extract treatment caused the transition of yeast form to hyphal form to be obviated. The expression of SIR2 was down-regulated from 1.2- to 2.5-fold with increasing concentration of the garlic extract, as determined from relative quantitative reverse transcription-polymerase chain reaction. There was no difference in the SAP4 expression in control vs treated cultures. Garlic and its bioactive components have the ability to suppress hyphae production and to affect the expression level of SIR2 gene. Hyphal production is an essential virulence determinant of C. albicans for invasive infections, therefore garlic and its constituents can be effective not only against colonizing C. albicans strains present in mucosal infections, but also virulent strains causing systemic or invasive candidiasis.
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ORIGINAL ARTICLE
Inhibition of hyphae formation and SIR2 expression in
Candida albicans treated with fresh Allium sativum (garlic)
extract
C.F. Low
1
, P.P. Chong
1,2
, P.V.C. Yong
1
, C.S.Y. Lim
1
, Z. Ahmad
1
and F. Othman
3
1 Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra Malaysia, Serdang, Selangor, Malaysia
2 Institute of Bioscience, University Putra Malaysia, Lebuh Silikon, Serdang, Selangor, Malaysia
3 Department of Human Anatomy, Faculty of Medicine and Health Sciences, University Putra Malaysia, Serdang, Selangor, Malaysia
Introduction
The dimorphic yeast, Candida albicans, is a commensal
organism and is commonly found in oral cavity, gastro-
intestinal tract, female’s genital tract and occasionally on
the surface of skin and mucous membrane. Candida sp.is
the most common cause of opportunistic mycoses and
the infections of Candida sp.are generally referred to as
candidiasis, which can be further classified into superficial
candidiasis, mucocutaneous candidiasis and systemic or
invasive candidiasis. Superficial candidiasis is relatively
common as compared with systemic candidiasis, which is
a more serious infection and can prove fatal. Superficial
infections of C. albicans could worsen into invasive infec-
tions and disseminate to other body sites. Virulent factors
that contribute to candidiasis are mainly undefined and
under investigation. The virulent factors include secreted
protease (Borg-von Zepelin et al. 1998), phospholipase
(Ghannoum 2000) and the ability to change morphology
from budding yeast cells into hyphal or filamentous form,
which is observable in C. albicans (Scherer and Magee
1990). The ability of C. albicans to change its morphology
from yeast-like cells into hyphal and even mycelial cells
and thus, adhere to the host cells and subsequently pene-
trate host tissues remains an important virulent determi-
nant for invasive infection.
Allium sativum, which is also commonly known as gar-
lic, is a species from the family of Alliaceae that has been
Keywords
Allium sativum, Candida albicans, SIR2 gene.
Correspondence
Pei Pei Chong, Department of Biomedical
Science, Faculty of Medicine and Health
Sciences, University Putra Malaysia, 43400
Serdang, Selangor, Malaysia. E-mail:
cpp@medic.upm.edu.my
2008 0793: received 9 May 2008, revised 10
July 2008 and accepted 18 July 2008
doi:10.1111/j.1365-2672.2008.03912.x
Abstract
Aims: The aims of the present study were to determine whether Allium sativum
(garlic) extract has any effect on the morphology transformation of Candida
albicans, and to investigate whether it could alter the gene expression level of
SIR2, a morphogenetic control gene and SAP4, a gene encoding secreted aspar-
tyl proteinase.
Methods and Results: Candida albicans cells were incubated with a range of
concentrations of fresh garlic extract, and the morphology was monitored via
light microscopy. Garlic extract treatment caused the transition of yeast form
to hyphal form to be obviated. The expression of SIR2 was down-regulated
from 1Æ2- to 2Æ5-fold with increasing concentration of the garlic extract, as
determined from relative quantitative reverse transcription-polymerase chain
reaction. There was no difference in the SAP4 expression in control vs treated
cultures.
Conclusions: Garlic and its bioactive components have the ability to suppress
hyphae production and to affect the expression level of SIR2 gene.
Significance and Impact of the Study: Hyphal production is an essential viru-
lence determinant of C. albicans for invasive infections, therefore garlic and its
constituents can be effective not only against colonizing C. albicans strains
present in mucosal infections, but also virulent strains causing systemic or
invasive candidiasis.
Journal of Applied Microbiology ISSN 1364-5072
ª2008 The Authors
Journal compilation ª2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 2169–2177 2169
widely used for medicinal purposes throughout recorded
history. An individual component of garlic that was iden-
tified to inhibit the growth of fungi through the inhibi-
tion of succinate dehydrogenase was allicin. Other
findings on the effect of garlic extract as an anti-Candida
agent included observed pits formation on surface of
C. albicans cells (Lemar et al. 2002).
Allicin, one of the active components of freshly crushed
garlic homogenates, has demonstrated potent anti-bacte-
rial and anti-fungal properties (Ankri and Mirelman
1999). Allicin in its pure form was found to exhibit: (i)
anti-bacterial activity against a wide range of gram-
negative and -positive bacteria, including multidrug-
resistant enterotoxicogenic strains of Escherichia coli;
(ii) anti-fungal activity, particularly against C. albicans;
(iii) anti-parasitic activity, including some major human
intestinal protozoan parasites such as Entamoeba histo-
lytica and Giardia lamblia; and (iv) anti-viral activity.
Through studies using electron microscopy, Lemar et al.
(2002) showed that fresh garlic extract is more efficient in
anti-Candida effect than dried garlic powder; and that
garlic and its bioactive components destroy the Candida
cell membrane integrity, resulting in the escape of much
of the cytoplasm, formation of pits and eventual cellular
collapse.
The ability of Candida sp. to adhere to the host tissue,
produce secretory aspartyl proteases and phospholipase
enzyme, and transform from yeast to hyphal phase are
among the major determinants of its pathogenicity.
Beginning as a single budding yeast cell, C. albicans have
the capacity to generate different phenotypic variants in
several generations of growth, forming heterogeneous
populations that possess the phenotype that is needed for
survival and well suited for sudden environmental
changes. Candida albicans is capable of growing in a bud-
ding yeast form (blastospore), or in filamentous forms
ranging from pseudohyphae to true hyphae (Sudbery
et al. 2004). The phenomenon whereby C. albicans is able
to undergo different types of morphological changes has
been termed as ‘phenotypic switching’. A small
proportion of clinical strains of C. albicans undergo
white-opaque switching. The silent information regulatory
gene (SIR2) was identified as one of the candidate genes
that play a role in phenotypic switching in C. albicans
(Pe
´rez-Martı
´net al. 1999).
The secreted aspartyl proteinases (SAP) from C. albi-
cans are encoded by a multi-gene family with at least nine
members (SAP1 to SAP9). SAP are recognized as viru-
lence factors of Candida species and the proteolytic
activity of SAP has been associated with tissue invasion
(Ru
¨chel et al. 1991). Among the large group of SAP pro-
teins, the SAP4, SAP5 and SAP6 form a distinctive
subgroup (DeBernardis et al. 1995) that was identified
during hyphae formation at neutral pH (Hube et al.
1994). As blood and salivary pH are approximately neu-
tral, and hyphae formation is important during tissue
invasion (Edwards 2000), therefore, SAP4, SAP5 and
SAP6 proteins were presumed to be important in dissemi-
nated candidiasis.
There is a dearth of studies on the effect of garlic
extract on C. albicans yeast and hyphal morphology and
also the factors involved in regulating the switching phe-
nomenon. Hence, this study was undertaken to observe
the effects of garlic extract on C. albicans morphology,
and on the gene expression of SAP4 and SIR2 upon expo-
sure to fungistatic dose of garlic.
Materials and Methods
Allium sativum (garlic) extract preparation
Garlic extract was prepared freshly before each test
according to Lemar et al. (2002). A total weight of 1 g
fresh garlic was ground in 10 ml of distilled water to pre-
pare a stock solution of 100 mg ml
)1
(w v). The extract
was allowed to stand for 30 min in room temperature
and then centrifuged at 5000 rev min
)1
for 10 min. The
supernatant fluid was passed through a sterile 0Æ22-lm
filter (Millipore, UK). The required concentration was
prepared by serial dilution of the stock solutions.
Figure 1 Anti-fungal activity of different concentrations of Allium
sativum extract on Candida albicans. Discs contained from the top in
clockwise direction; 100 (top-most), 10, 1 and 0Æ1mgml
)1
of A. sati-
vum extract. The disc in the middle of each plate is a negative control
disc that was soaked in sterile distilled water.
Garlic suppresses CaSIR2 expression C.F. Low et al.
2170 Journal compilation ª2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 2169–2177
ª2008 The Authors
Preparation of paper disc impregnated with Allium
sativum extract
Different concentrations of fresh A. sativum extract that
ranged from 0Æ1 to 100 mg ml
)1
through a serial
10-fold dilution was prepared from the stock solution
(100 mg ml
)1
) using sterile distilled water. Twenty
microlitre of each diluted solution of the extract was
pipetted onto separate paper discs. Negative control
disc was prepared using 20 ll of sterile distilled water.
The discs were allowed to stand at room temperature
for 1 h before use.
Susceptibility testing (disc diffusion method) of Candida
albicans
Candida albicans (strain 14053) was purchased from the
American Type Culture Collection (ATCC). The fungal
cells were grown on YEPD agar at 37C and passaged
at least twice to ensure purity and viability. Inocula
were prepared by picking five colonies of about 1 mm
in diameter from 24-h-old cultures of Candida sp. The
colonies were suspended in 5 ml of phosphate-buffered
saline (PBS). The resulting suspension was vortexed for
15 s and the cell density was adjusted to 0Æ5 McFarland
units. The inocula were centrifuged to pellet the cells
and the PBS buffer was substituted with the same vol-
ume of YEPD broth. This procedure yielded a yeast
stock suspension of 1 ·10
6
to 5 ·10
6
cells ml
)1
. The
inocula were spread on YEPD agar using a sterile cot-
ton swab. The culture plates were allowed to stand at
room temperature for 15 min to dry. The plant extract
disc was applied on the agar and allowed to stand at
room temperature for 15 min to allow diffusion and
then, incubated at 37C for 24 h. The diameter of the
inhibition zone was measured.
Treatment of Candida albicans with varying concentra-
tions of garlic extract for gene expression analysis
Separately, each flask of cells (1 ·10
6
to
5·10
6
cells ml
)1
) in YEPD broth was treated with
freshly prepared garlic extract at final concentrations of
20, 40, 60, 80 and 100 mg ml
)1
(w v) and incubated at
37C for 24 h. Total RNA was extracted using RNeasy
mini kits (Qiagen, Germany) according to the manufac-
turer’s operating instructions, with slight modifications
that entail the use of 50U zymolyase (ICN Chemicals,
USA) to lyse the yeast cell wall. DNA digestion proto-
col was performed to remove DNA contamination
using DNase (Promega, UK). Samples were then puri-
fied using an RNA cleanup protocol through the use of
RNeasy column chromatography.
Semi-quantitative reverse transcription-polymerase chain
reaction (RT-PCR) for comparing SIR2 and SAP4 gene
expression levels
Single-stranded cDNA was synthesized from the extracted
RNA using MMLV reverse transcriptase and random hex-
amer oligonucleotides (Promega). Candida albicans SIR2
gene was amplified from the cDNA using CaSIR2 for-
ward primer (5¢-ACGAGCAGGATTGAAACTGGAA-3¢)
and CaSIR2 reverse primer (5¢-CCAAAATGGATTGGT-
GCTTGTT-3¢). CaSAP4 gene was amplified using CaSAP4
forward primer (5¢-ATGTTCTTGCAAAATATCTTGAG-
TG-3¢) and CaSAP4 reverse primer (5¢-CTAATTAATG-
CCAACAATGTTAGAC-3¢). The primer sequences were
designed with the aid of Primer3 software. In addition,
actin gene was also amplified as an internal control (house-
keeping gene). The actin gene was amplified using CaACT
forward primer (5¢-ACCGAAGCTCCAATGAATCCAAAA-
TCC-3¢) and CaACT reverse primer (5¢-GTTTGGTCAA-
TACCAGCAGCTTCCAAA-3¢). Actin was used as a
housekeeping gene to normalize the inconsistency of RNA
concentrations during total RNA extraction protocol. For
every SIR2 gene amplification, a negative control without
MMLV enzyme was subjected to the same cycling condi-
tions; to ensure that all PCR products obtained originated
from cDNA and not from genomic DNA contamination.
0
0·05
0·1
0·15
0·2
0·25
0·3
0 5 10 15 20 25 30
Incubation time (h)
Optical density
0
0·5
1
1·5
2
2·5
Figure 2 Inhibitory effect of Allium sativum extract on growth profile
of Candida albicans. The optical density values for untreated
C. albicans cells ( ), 0 mg ml
)1
garlic extract follow the scale of
the y-axis on the right; whereas the optical density values for the
treated cells ( , 20; , 40; , 60; , 80 and
, 100 mg ml
)1
) follow the y-axis scale on left.
C.F. Low et al. Garlic suppresses CaSIR2 expression
ª2008 The Authors
Journal compilation ª2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 2169–2177 2171
Amplification of SIR2 and actin genes was carried out for
30 PCR cycles. Finally, gel electrophoresis of PCR products
were carried out using 1Æ5% (w v) agarose gel, and the
results were visualized using a CCD imager machine
(AlphaImager). Quantitation of the PCR products was per-
formed by using AlphaImager software by comparing the
intensity of the target genes’ bands with the intensities of
bands displayed by known molecular weight markers.
(c) Incubation with 80 mg ml–1 A. sativum extract at 0, 6 and 24 h
0 h 24 h 6 h
(i) (ii) (iii)
(b) Incubation with 20 mg ml–1 A. sativum extract at 0, 6 and 24 h
0 h 6 h 24 h
(i) (ii) (iii)
(a) Without A. sativum extract treatment at 0, 6 and 24 h
0 h 6 h 24 h
(i) (ii) (iii)
Figure 3 Microscopic analysis of Candida albicans hyphae production. Magnification ·20. (a) Without treatment with Allium sativum extract at 0,
6 and 24 h; (b) incubation with 20 mg ml
)1
of A. sativum extract at 0, 6 and 24 h; (c) incubation with 80 mg ml
)1
of A. sativum extract at 0, 6
and 24 h.
Garlic suppresses CaSIR2 expression C.F. Low et al.
2172 Journal compilation ª2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 2169–2177
ª2008 The Authors
Results
Growth and morphology of cells in varying garlic extract
concentrations
The disc diffusion susceptibility assay showed that
20 mg ml
)1
and above of garlic extract was able to reduce
the growth of C. albicans, but was not sufficient to be
fungicidal. In contrast, 100 mg ml
)1
of garlic extract was
able to produce a prominent zone of inhibition (Fig. 1).
The growth rates of C. albicans exposed to garlic extract
were diminished, with an extended lag phase, and
decreased cell counts in the exponential phase (Fig. 2).
Incubation of C. albicans in culture medium supple-
mented with fresh garlic extract resulted in inhibition of
hyphae production. As shown in Fig. 3a, in the absence
of garlic extract, at 0 h prior to adding sterile water for
control, the cells existed in yeast form; then gradually
produced germ tubes and pseudohyphae at 6 h. Extensive
hyphae production was observable by 24 h. In contrast,
20 mg ml
)1
of garlic extract had not induced any true
hypha production, and very little germ tubes were visible
(a) SIR2 gene
(b) SAP4 gene
Garlic
conc: 0 20 40 60 80 100
mg ml–1
Figure 4 Gel electrophoresis of reverse transcription polymerase chain reaction products of (a) SIR2 and (b) SAP4 genes from Candida albicans
ATCC strain after treatment with fresh garlic extract.
C.F. Low et al. Garlic suppresses CaSIR2 expression
ª2008 The Authors
Journal compilation ª2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 2169–2177 2173
even after 24 h of incubation (Fig. 3b). Cells incubated
with 80 mg ml
)1
of garlic extract had no microscopically
observable hyphae production after 24 h of incubation,
and no budding of cells (Fig. 3c). Incubation with
100 mg ml
)1
of garlic extract resulted in severe suppres-
sion of growth and very few viable cells.
Relative quantitative RT-PCR of SIR2 and SAP4
The C. albicans ATCC isolate subjected to different con-
centrations of garlic extract was harvested for RNA isola-
tion after 24 h of incubation. The RNA extracted was
reverse transcribed to cDNA and subjected to PCR using
SIR2 gene-specific primers. The RNA extraction and RT-
PCR were performed in triplicates for greater reliability of
the results. The results showed that actin housekeeping
gene was expressed at every concentration of the garlic
extract. Figure 4 exhibits the representative gel electro-
phoresis results of the RT-PCR. The slight inconsistent
level of actin expression between different samples is
because of different starting amounts of RNA used and
also the inefficiency of cDNA synthesis. However, this
was compensated by the normalization in our calcula-
tions. Tables 1 and 2 describe the contents of each lane in
the gels in Fig. 4a,b, respectively, and also display concen-
tration of each PCR product as well as the normalized
SIR2 actin and SAP4 actin ratios. The chart in Fig. 5
shows a general trend of reduction in SIR2 expression in
cells exposed to fresh garlic extracts compared with cells
that had not been treated with garlic extract. The SIR2
mRNA was down-regulated 1Æ6-, 1Æ2-, 1Æ2-, 2Æ3- and 2Æ5-
fold, respectively at garlic extract concentrations of 20,
40, 60, 80 and 100 mg ml
)1
, respectively. In contrast, the
garlic extract apparently had no significant effect on the
SAP4 mRNA expression level as the SAP4 actin ratio
remained fairly constant under the conditions tested. The
normalized SAP4 gene expression level for cells incubated
with garlic extract showed no prominent increase or
decrease in fold changes compared with cells grown in
the absence of garlic.
The authenticity of the PCR products was verified via
DNA sequencing using an outsourced commercial
sequencing service provider (BioSynTec Sdn Bhd, Malay-
sia). The sequences were analysed via nucleotide Blast
against the nonredundant (nr) database in GenBank. The
sequence similarity analysis confirmed that the identity of
the PCR products were SAP4 and SIR2 genes of C. albi-
cans, respectively.
Table 1 Concentration of reverse transcription polymerase chain reaction (RT-PCR) products of SIR2 and actin genes for each sample in the gel
electrophoresis results in Fig. 4a
Lane
DNA
sample
Garlic extract
concentration
(mg ml
)1)
RT-PCR
primers
Concentration
of PCR products
(ng ll
)1
)
Normalized
ratio (SIR2 actin)
Fold change
relative
to control
M 100 bp DNA ladder
S1 CA ATCC§ 0 SIR2 11Æ95 0Æ78 1
A1 CA ATCC 0 Actin 15Æ34
N* CA ATCC 0 SIR2
S2 CA ATCC 20 SIR2 20Æ87 0Æ48 1Æ6
A2 CA ATCC 20 Actin 43Æ37
N* CA ATCC 20 SIR2
S3 CA ATCC 40 SIR2 18Æ21 0Æ62 1Æ2
A3 CA ATCC 40 Actin 29Æ15
N* CA ATCC 40 SIR2
S4 CA ATCC 60 SIR2 23Æ44 0Æ66 1Æ2
A4 CA ATCC 60 Actin 35Æ53
N* CA ATCC 60 SIR2
S5 CA ATCC 80 SIR2 25Æ01 0Æ35 2Æ3
A5 CA ATCC 80 Actin 72Æ38
N* CA ATCC 80 SIR2
S6 CA ATCC 100 SIR2 20Æ65 0Æ31 2Æ5
A6 CA ATCC 100 Actin 65Æ89
N* CA ATCC 100 SIR2
M 100 bp DNA ladder (Fermentas)
*Without MMLV reverse transcriptase.
Values are obtained from the mean of triplicate experiments.
Fold change is calculated by dividing the normalized ratio of the untreated control (0 mg ml
)1
) to that of each sample.
§Candida albicans ATCC.
Garlic suppresses CaSIR2 expression C.F. Low et al.
2174 Journal compilation ª2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 2169–2177
ª2008 The Authors
Discussion
Medicinal, insecticidal, anti-bacterial and anti-fungal
properties have been ascribed to garlic. In addition, inter-
est in garlic as an anti-fungal agent has been renewed
recently. Garlic extract was demonstrated to be fungicidal
against pathogenic yeasts especially C. albicans (Ghan-
noum 1990). In the present study, the fungicidal concen-
tration of the aqueous garlic extract against C. albicans
was higher than those reported by other authors previ-
ously. An early study had reported the minimal inhibitory
concentration (MIC) of aqueous garlic extract to be lower
than 2 mg ml
)1
(Ghannoum 1988). Lemar et al. (2002)
reported that 10 mg ml
)1
or greater fresh garlic extract
was required for fungicidal action. In our study, we
observed that at least 100 mg ml
)1
of fresh garlic extract
was needed to produce a fungicidal effect in disc diffusion
assay. This discrepancy between our study and previous
studies could be attributed to the different types or varie-
ties of garlic found in the different geographical locations.
Candida albicans appears to be a major fungal patho-
gen of humans and 55% of blood stream infections due
to yeast are caused by C. albicans (Pfaller et al. 2001).
The frequencies of C. albicans in candidal infections in
North America and Europe are c. 50% (Messer et al.
2006). This opportunistic yeast pathogen switches
between several general phenotypes distinguishable by col-
ony as well as cellular morphology spontaneously and
reversibly at a high frequency (Klar et al. 2001). Hyphal
form is associated with pathogenicity as it could promote
tissue penetration during the early stage of the infection,
whereas the yeast form is associated with the dissemina-
tion of the infection in the bloodstream. The family of
SIR genes appears to play an important role in silencing
the phenotypic switching of the species. Disruption of
two copies of SIR2 gene in C. albicans resulted in higher
frequency of production of variant colony morphologies
(Pe
´rez-Martı
´net al. 1999). SIR2 has been implied to
influence the morphological switching of C. albicans cells
from budding yeast form to hyphal forms (Pe
´rez-Martı
´n
et al. 1999). The upregulation of SIR2 expression deter-
mined through real-time PCR during transformation
from yeast form to hyphal form also suggests its involve-
ment in morphology switching in addition to its proven
role in colony variant phenotypic switching (C.S. Lim,
H.F. Seow, R. Rosli and P.P. Chong, personal communi-
cation). On the other hand, SAP4 gene was implicated as
a virulence gene as it is expressed in the hyphal tips dur-
ing hyphae formation (Chen et al. 2002). Inhibition of
the pathogenic and virulence genes expression could
Table 2 Concentration of reverse transcription polymerase chain reaction (RT-PCR) products of SAP4 and actin genes for each sample in the gel
electrophoresis results in Fig. 4b
Lane
DNA sample
garlic extract
concentration
RT-PCR
primers
Concentration
of PCR products
(ng ll
)1
)
Normalized
ratio
(SAP4 actin)
Fold change
relative
to control
M 1 kb DNA ladder
S1 CA ATCC (0 mg ml
)1
) SAP4 1Æ82 0Æ11 1
S1N CA ATCC (0 mg ml
)1
)* SAP4 –
A1 CA ATCC (0 mg ml
)1
) Actin 16Æ26
S2 CA ATCC (20 mg ml
)1
) SAP4 2Æ13 0Æ12 0Æ92
S2N CA ATCC (20 mg ml
)1
)* SAP4
A2 CA ATCC (20 mg ml
)1
) Actin 16Æ98
S3 CA ATCC (40 mg ml
)1
) SAP4 1Æ01 0Æ06 0Æ55
S3N CA ATCC (40 mg ml
)1
)* SAP4
A3 CA ATCC (40 mg ml
)1
) Actin 15Æ60
S4 CA ATCC (60 mg ml
)1
) SAP4 1Æ84 0Æ11 1
S4N CA ATCC (60 mg ml
)1
)* SAP4
A4 CA ATCC (60 mg ml
)1
) Actin 16Æ90
S5 CA ATCC (80 mg ml
)1
) SAP4 1Æ12 0Æ07 0Æ64
S5N CA ATCC (80 mg ml
)1
)* SAP4
A5 CA ATCC (80 mg ml
)1
) Actin 15Æ34
S6 CA ATCC (100 mg ml
)1
) SAP4 2Æ44 0Æ12 1Æ1
S6N CA ATCC (100 mg ml
)1
)* SAP4
A6 CA ATCC (100 mg ml
)1
) Actin 19Æ99
M 1 kb DNA ladder
*Without MMLV reverse transcriptase.
Values are obtained from the mean of triplicate experiments.
Fold change is calculated by dividing the normalized ratio of the untreated control (0 mg ml
)1
) to that of each sample.
C.F. Low et al. Garlic suppresses CaSIR2 expression
ª2008 The Authors
Journal compilation ª2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 2169–2177 2175
therefore logically suppress the ability of the species in
infecting the host.
Several researchers have investigated the anti-fungal
mode of action of garlic on fungi. Garlic has been
reported to affect the lipid constituents of the outer sur-
face of C. albicans (Ghannoum 1988). Ajoene, a derivative
of ethanolic garlic extract, was found to inhibit the phos-
phatidylcholine (PC) synthesis in the cytosolic membrane
(San-Blas et al. 1997) and block the morphogenic trans-
formation (San-Blas et al. 1993) of the dimorphic fungus,
Paracoccidioides brasiliensis. This correlates well with our
study, which shows that high concentrations of garlic
extract could suppress hyphae formation. Another com-
pound of interest in garlic is allyl alcohol (AA), a meta-
bolic product that accumulates after trituration of garlic
cloves. It was reported that AA and garlic extract expo-
sure can induce oxidative stress in C. albicans cells
(Lemar et al. 2005). In addition, a recent study showed
that diallyl disulfide (DADS), a garlic constituent, could
induce cell death in Candida through oxidative stress by
depleting glutathione (Lemar et al. 2007).
As SAP4 has been regarded as a virulence factor of C.
albicans and is closely associated with hyphal tip advance-
ment, we had anticipated that garlic would have an effect
on its expression concurrent with the suppression of
hyphae formation. However, in our RT-PCR analysis we
were unable to observe any distinct pattern of decrease in
SAP4 expression. Although there were initial decreases in
SAP4 expression at garlic concentrations of 40 and
80 mg ml
)1
, the level of expression at 100 mg ml
)1
of
garlic concentration was not significantly different from
that of the untreated cultures. Thus, the hyphal suppres-
sion by garlic appears to be unrelated to SAP genes. The
effect of fresh garlic extract on the C. albicans SIR2 gene
expression has been observed in this study, whereby the
expression of SIR2 gene in C. albicans was apparently
reduced with increasing concentrations of garlic extract.
In the present investigation, garlic extract was also dem-
onstrated to suppress hyphae formation in C. albicans.
These effects of garlic suggest that it could be used for
prophylaxis against Candida infections, particularly in
immunocompromised and immunosuppressed patients
who are prone to opportunistic yeast infections.
Acknowledgements
The authors are grateful to the University Putra Malaysia
for the Research University Grant Scheme (RUGS), which
provided the financial support for this project.
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Relative quantitation of SIR2 and SAP4 genes
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0
0·1
0·2
0·3
0·4
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0·7
0·8
0·9
0 20 40 60 80 100
Garlic extract concentration (mg ml–1)
Ratio of
gene of interest/actin
Figure 5 Relative quantitation of SIR2 and SAP4 gene expressions
(normalized to house-keeping gene, actin) in Candida albicans ATCC
strain after 24 h of treatment with different concentrations of fresh
garlic extract. The first bar, 0 mg ml
)1
, is the untreated control. ( ),
SIR2; ( ), SAP4.
Garlic suppresses CaSIR2 expression C.F. Low et al.
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... Then the plates were allowed to stand in an upright position for 15 min to allow for the proper diffusion of the extracts into the medium before incubation. The plates were incubated at room temperature (28 ± 2 °C) for about 7 d [21] . The experiment was performed in triplicates to minimize the error ratio. ...
... The experiment was performed in triplicates to minimize the error ratio. At the end of incubation period, the inhibition zones around each well was measured for 3, 7 d intervals after the inoculation to an accuracy of 0.1 mm and the effect was calculated as a mean of triplicate tests to evaluate the antifungal activity [21,22] . ...
... The petri plates with 10 mL of each solution including; fungicide 50% (WP) Captan, DMSO-water (50%, v/v), methanol-water (80%, v/v), sterilized distilled water, and n-hexane were mixed with 90 mL of sterilized PDA media and maintained as controls. Then the plates were incubated at room temperature (28 ± 2 °C) for about 7 d [21] . At the end of the incubation period, the percentage inhibition of mycelial growth was calculated using the equation below [23] . ...
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... Then the plates were allowed to stand in an upright position for 15 min to allow for the proper diffusion of the extracts into the medium before incubation. The plates were incubated at room temperature (28 ± 2 °C) for about 7 d [21] . The experiment was performed in triplicates to minimize the error ratio. ...
... The experiment was performed in triplicates to minimize the error ratio. At the end of incubation period, the inhibition zones around each well was measured for 3, 7 d intervals after the inoculation to an accuracy of 0.1 mm and the effect was calculated as a mean of triplicate tests to evaluate the antifungal activity [21,22] . ...
... The petri plates with 10 mL of each solution including; fungicide 50% (WP) Captan, DMSO-water (50%, v/v), methanol-water (80%, v/v), sterilized distilled water, and n-hexane were mixed with 90 mL of sterilized PDA media and maintained as controls. Then the plates were incubated at room temperature (28 ± 2 °C) for about 7 d [21] . At the end of the incubation period, the percentage inhibition of mycelial growth was calculated using the equation below [23] . ...
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... The mechanism of anti-biofilm may be in connection with the Ras/cAMP pathway [129]. One study confirmed that a compound from garlic extract, allicin (25, Figure 10), was also able to suppress hyphae formation in C. albicans [57]. ...
... connection with the Ras/cAMP pathway [129]. One study confirmed that a compound from garlic extract, allicin (25, Figure 10), was also able to suppress hyphae formation in C. albicans [57]. ...
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... A decrease in SIR2 gene expression was observed during exposure to fresh garlic extract containing allicin; the relative quantification of SIR2 gene expression in the control group untreated with the tested substance was approximately 0.8 (ratio of the gene of interest/actin). After treatment with allicin at doses of 20, 40, 60, 80, and 100 mg/mL, it decreased to approximately 0.5, 0.6, 0.65, 0.35, and 0.3, respectively [41]. Another virulence factor of C. albicans is the ECE1 gene (endothelin converting enzyme 1 gene), which encodes a peptide toxin, candidalysin, which, by destabilizing the cell membrane of vaginal cells, facilitates infection and is responsible for the immunopathogenesis of fungal VVC [42]. ...
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... In addition, garlic extract has been found to inhibit C. albicans growth by forming pits on the surface [101]. Low et al. showed that treatment with garlic extract prevented yeast cell transition to hyphae and SIR2 expression was downregulated when the garlic extract concentration was increased, indicating that garlic and its bioactive components suppressed C. albicans hyphae production and affected SIR2 gene expression [103]. The compound ajoene, derived from ethanolic garlic extract, inhibits the synthesis of phosphatidylcholine in cytosol and prevents the morphogenic transformation of fungi [104]. ...
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Western diets are rapidly spreading due to globalization, causing an increase in obesity and diseases of civilization. These Western diets are associated with changes in the gut microbiota related to intestinal inflammation. This review discusses the adverse effects of Western diets, which are high in fat and sugar and low in vegetable fiber, on the gut microbiota. This leads to gut dysbiosis and overgrowth of Candida albicans, which is a major cause of fungal infection worldwide. In addition to an unhealthy Western diet, other factors related to disease development and gut dysbiosis include smoking, excessive alcohol consumption, lack of physical activity, prolonged use of antibiotics, and chronic psychological stress. This review suggests that a diversified diet containing vegetable fiber, omega-3 polyunsaturated fatty acids, vitamins D and E, as well as micronutrients associated with probiotic or prebiotic supplements can improve the biodiversity of the microbiota, lead to short-chain fatty acid production, and reduce the abundance of fungal species in the gut. The review also discusses a variety of foods and plants that are effective against fungal overgrowth and gut dysbiosis in traditional medicine. Overall, healthy diets and lifestyle factors contribute to human well-being and increase the biodiversity of the gut microbiota, which positively modulates the brain and central nervous system.
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The opportunistic fungal pathogen Candida albicans produces secretory aspartyl proteinases, which are believed to be virulence factors in infection. We have studied the in vitro expression of seven known members of the SAP gene family in a range of strains and serotypes by Northern analysis. SAP1 and SAP3 were regulated during phenotypic switching between the white and opaque forms of the organism. The SAP2 mRNA, which was the dominant transcript in the yeast form, was found to be autoinduced by peptide products of Sap2 activity and to be repressed by amino acids. The expression of the closely related SAP4-SAP6 genes was observed only at neutral pH during serum-induced yeast to hyphal transition. No SAP7 mRNA was detected under any of the conditions or in any of the strains tested. Our data suggest that the various members of the SAP gene family may have distinct roles in the colonization and invasion of the host.