Page 1 of 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
1
Title page
2 3
Manuscript Type: Technical Note
4 5
Short peptides allow preferential detection of Candida albicans
6
hyphae
7 8 9
Hani E. J. Kaba1, Antonia Pölderl1 and Ursula Bilitewski1* 1
Biological Systems Analysis, Helmholtz Centre for Infection Research (HZI),
Inhoffenstr. 7, D-38124 Braunschweig, Germany.
10 11
* Corresponding author: Ursula Bilitewski
12
Working Group Biological Systems Analysis,
13
Helmholtz Centre for Infection Research (HZI),
14
Inhoffenstr. 7,
15
D-38124 Braunschweig, Germany.
16
[email protected] 17
1 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 15
1
Abstract: Whereas the detection of pathogens via recognition of surface structures
2
by specific antibodies and various types of antibody mimics is frequently described,
3
the applicability of short linear peptides as sensor molecules or diagnostic tools is
4
less well known. We selected peptides which were previously reported to bind to
5
recombinant S. cerevisiae cells, expressing members of the C. albicans ALS cell wall
6
protein family. We slightly modified amino acid sequences to evaluate peptide
7
sequence properties influencing binding to C. albicans cells. Among the selected
8
peptides, decamer peptides with an “AP”-N-terminus were superior to shorter
9
peptides. The new decamer peptide FBP4 stained viable C. albicans cells in their
10
mature hyphal form more efficient than in their yeast form. Moreover, it allowed
11
distinction of C. albicans from other related Candida spp. and could thus be the basis
12
for the development of a useful tool for the diagnosis of invasive candidiasis.
13
2 ACS Paragon Plus Environment
Page 3 of 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
INTRODUCTION
1 2
Candida albicans is an opportunistic fungal pathogen of humans found as
3
commensal in 30 – 70% of healthy individuals.1 This species is responsible for the
4
majority of candidemia, the fourth major cause of nosocomial bloodstream infections
5
with a lethality rate of approximately 40%.2 Other Candida species, which are
6
frequently isolated from infected persons, are C. dubliniensis3 and C. glabrata, which
7
is even more closely related to the baker’s yeast S. cerevisiae than to C. albicans.4
8
C. albicans is present as unicellular yeast (blastospores), but also forms
9
pseudohyphae and switches to the multicellular hyphal form, which is essential for
10
invasive infections.5 Prior to invasion of the host, C. albicans attaches to host cells
11
thanks to a wide repertoire of strong adhesive proteins found in the cell wall. A
12
prominent family of these so-called adhesins is the Agglutinin-Like-Sequence (ALS)
13
family, which includes at least eight glycoprotein encoding genes (ALS1 to ALS7 and
14
ALS9).6 Heterologous expression of the ALS members Als1p and Als5p in non-
15
adherent Saccharomyces cerevisiae was sufficient to allow adhesion to human cells,
16
to extracellular matrix components and to magnetic beads coated with synthetic
17
peptides.7, 8, 9 The peptides, to which binding of C. albicans and of recombinant S.
18
cerevisiae was observed, had the consensus - motif “τφ+”, consisting of an amino
19
acid with a high turn propensity (τ: A, D, G, K, N, P or S), followed by an amino acid
20
with a bulky hydrophobic or an aromatic residue (φ: F, H, I, L, M, T, V, W or Y) and
21
finally a positively charged amino acid (+: either R or K).9
22
Reliable and specific diagnosis of the Candida species which is responsible for an
23
infection is of great clinical importance due to the different resistance profiles of these
24
fungi to various antifungals.10 A number of molecular, culture dependent or indirect
25
methods are currently applied in laboratory diagnostics of Candida infections.11
26
Culture dependent diagnostics is time – consuming, whereas staining of cells by
27
specific binders, such as antibodies, allows rapid diagnosis without further treatment.
28
However, immunoassays for diagnosis of candidiasis are based on the detection
29
either of anti-Candida antibodies circulating in the blood or of mannan, the major
30
polysaccharide from the outer layers of the fungal cell wall. Thus, these assays do
31
not directly detect the pathogen, but determine products resulting from its earlier or
32
actual presence.
3 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 15
1
We had previously shown that antibodies could be generated, which allow the
2
detection of C. albicans via binding to a specific epitope of a protein localized in the
3
cell wall of C. albicans.12 However, antibodies are complex proteins and their
4
production requires immunization of animals and cultivation of mammalian cells.
5
Thus, they are expensive reagents, frequently with a limited stability. That is why
6
there is an interest to develop alternative binders, such as antibody mimetics or even
7
polymeric structures developed as imprints of the analyte.13 We decided to follow an
8
alternative approach and to exploit the endogenous binding capabilities of the
9
fungus. Thus, we investigated whether the adhesive peptides could also be used, in
10
free form, for diagnostic purposes to detect living C. albicans cells. In particular, we
11
were interested in detecting C. albicans in the hyphal morphology, which is strongly
12
related to invasive infections.
13
Starting from peptide sequences reported for binding recombinant S. cerevisiae, we
14
modified the amino acid sequences of the “τφ+”-motif. We identified peptide FBP4,
15
and found it to bind particularly to the surface of C. albicans hyphae and to
16
distinguish C. albicans from the closely related C. dubliniensis as well as from C.
17
glabrata.
18
EXPERIMENTAL SECTION
19
Microbial strains and culture conditions
20
C. albicans SC5314,14 C. dubliniensis DSM1326815 and C. glabrata ATCC200116
21
were used as wild-type strains in this study. Homozygous ALS deletion mutants and
22
re-integrants used in this study were 2373 (∆als5)17, 2407 (∆als5 + ALS5)17, 1467
23
(∆als1)18 and 2151 (∆als1 + ALS1).18 All strains were routinely cultivated in YPD
24
(yeast extract, peptone, dextrose) medium (Sigma-Aldrich Y1375) using Multitron
25
Shakers (Infors-HT, 160 rpm) for the given conditions. Growth of cultures was
26
followed by determination of optical densities of cultures at λ = 600 nm (OD600).
27
Assessment of all OD600 measurements was performed in triplicates (3 x 180 µl) in
28
transparent 96 well plates, using the µQuant microtiter plate reader (Biotek, Bad
29
Friedrichshall, Germany). Alternatively, cells were counted (10 µl of diluted or
30
undiluted culture) using a Neubauer Improved counting chamber (Brand; 0.1 mm
31
depth) under a CKX41/U-RFLT50 microscope (Olympus).
32
Growth of hyphal cultures cannot be followed by determinations of optical densities.
33
For microscopic analysis of cultures, 10 µl of the cell suspension were dropped on 4 ACS Paragon Plus Environment
Page 5 of 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
1
Superfrost glass slides or on the Neubauer Improved counting chamber and
2
analyzed with the CKX41/U-RFLT50 microscope. Pictures of cultures were taken with
3
a ColorView SIS FireWire Camera 3.0 and analyzed with CellA^ 3.0 software
4
(Olympus).
5
Synthetic biotinylated peptides used in this study
6
Peptides used in this study were synthesized by peptides&elephants GmbH,
7
Potsdam, Germany. They are listed in Table 1. All peptides were biotinylated at the
8
C-terminus and thus comprised a C-terminal lysine residue. Peptides were delivered
9
as solids and were stored at – 20 °C until first use. Stock solutions were prepared in
10
PBS (8.0 g · l-1 NaCl, 0.2 g · l-1 KCl, 1.44 g · l-1 Na2HPO4 2 H2O, 0.24 g · l-1 KH2PO4,
11
pH 8.0) supplemented with 1% DMSO at a final concentration of 1 mg · ml-1 .
12
Solutions of all peptides were prepared on the same day and 200 µl “ready to use”
13
aliquots were stored at – 20 °C until use.
14
Table 1. List of free biotinylated peptides (FBPs) used in this work. Peptide
Sequence (N C)
Reference
FBP1
KLRIPSVK-biotin
(9)
FBP2
APRLRFYSLK-biotin
(9)
FBP3
EHAHTPRK-biotin
(9)
FBP4
APKLRIPSVK-biotin
This study
FBP5
RLRFYSLK-biotin
This study
FBP6
APKLRFYSLK-biotin
This study
FBP7
APELDFYSLK-biotin
This study
15
Determination of peptide binding to viable cells
16
Binding of peptides to viable C. albicans cells was determined by indirect
17
fluorescence assays using FITC labeled streptavidin recognizing the C-terminal biotin
18
of the peptides.
19
Flow cytometric analysis of germinating yeast cells
20
C. albicans yeast cells with small germ tubes (initial phase of hyphal development;
21
Figure 1A), C. dubliniensis and C. glabrata were analyzed by flow cytometry.
22
Cells from frozen glycerol stocks were resuspended in 1 ml YPD medium (Sigma-
23
Aldrich Y1375). OD₆₀₀ of the resulting cell suspension was determined and an
24
appropriate aliquot was further diluted with 20 ml YPD medium to give a theoretical 5 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 15
1
OD₆₀₀ of 5 x 10-5. Cells were cultivated at 30 °C for 16 – 17.5 h. The OD₆₀₀ was
2
determined and cells were resuspended in 20 ml RPMI medium [8.4 g l-1 RPMI 1640
3
(Sigma-Aldrich R1383), 2 g · l-1 glucose, 0.165 M 3-(N-morpholino) propanesulfonic
4
acid (MOPS)] pH 7.3 at an OD₆₀₀ of 0.24 – 0.3. Cells were cultivated at 37 °C for 3 h.
5
Cells were counted and 5 x 106 cells of each culture were transferred to 2 ml
6
Eppendorf tubes and filled up to 500 µl with PBS. Cells were pelleted [8000 x g, 3
7
min, room temperature (RT)] and the supernatant was removed. Cells were washed
8
with 500 µl PBS and the cell pellet was incubated with 200 µl peptide solution
9
(peptide concentration: 1 mg · ml-1; cell density: 2.5 x 107 cells ml-1) at RT for 30 min
10
with thorough shaking. Cells were pelleted, washed with PBS and subsequently
11
incubated with 200 µl FITC labeled streptavidin (SAV-FITC; 25 µg · ml-1, BD
12
Pharmingen™) at RT for 30 min with thorough shaking and protection from light.
13
Cells were pelleted, washed with PBS and subsequently resuspended in 500 µl PBS
14
containing 0.4% formaldehyde in FACS tubes (Sarstedt) before being analyzed by
15
flow cytometry (BD FACSCanto, Franklin Lakes, USA). As negative controls, peptide-
16
and / or SAV-FITC - solutions were substituted by PBS.
17
Samples were kept on ice and protected from light until they were analyzed in the
18
flow cytometer. For each experiment, 104 events were counted. For data analysis,
19
the FACSDiva 6.0 and FlowJo (TreeStar) software were used. After flow cytometric
20
analysis, fluorescent cells were inspected microscopically (Biozero BZ-8100,
21
Keyence).
22
Flow cytometric analysis of C. albicans SC5314 was performed at least three times
23
for each peptide, using different cultures. A decrease in peptide binding intensity was
24
observed with prolonged peptide storage at – 20 °C. Therefore, only the three first
25
values from each peptide batch were considered for statistical analysis.
26
C. albicans ∆als5 and ∆als5 + ALS5 were analyzed 2 – 3 times, while C. albicans
27
∆als1 and ∆als1+ ALS1, C. dubliniensis and C. glabrata were analyzed only once by
28
flow cytometry.
29
Quantification of fluorescence of cultures containing hyphal and yeast cells
30
Fluorescence intensities of C. albicans hyphae and of C. dubliniensis and C. glabrata
31
were quantified by a fluorescence plate reader (Synergy 4, BioTek Instruments
32
GmbH) or a fluorescence microscope (Biozero BZ-8100, Keyence).
6 ACS Paragon Plus Environment
Page 7 of 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
1
For the generation of short hyphae (early phase of hyphal development; Figure 1 B),
2
cells were first cultivated as described above. Cells from the overnight culture were
3
then resuspended in 20 ml RPMI pH 7.3 at an OD₆₀₀ of 0.1 (C. albicans) and 0.05 (C.
4
dubliniensis and C. glabrata). For the binding assay, 450 µl from each culture were
5
used. Treatment and washing of cells was performed as described above.
6
To induce the generation of mature hyphae (Figure 1C), C. albicans cells from frozen
7
glycerol stocks were inoculated in 20 ml RPMI pH 4.0 and incubated at 30 °C for 16.5
8
h. The OD₆₀₀ of the culture was determined and an appropriate aliquot of the cell
9
suspension was resuspended in 50 ml RPMI pH 7.3 to give a theoretical OD600 of
10
0.001. This culture was cultivated at 37 °C for 24 h. C. dubliniensis and C. glabrata
11
were cultivated in a similar way as for mature C. albicans hyphae, however these
12
yeasts did not form hyphae under these conditions. As a control, C. albicans was
13
cultivated in 50 ml ml RPMI pH 4.0 to produce a culture mainly containing the yeast
14
form with only few hyphae (Figure 1D). For the binding assay, 430 µl of each culture
15
were used. Peptide treatment and washing of cells was performed as described
16
above.
17 18 19 20 21
Figure 1. C. albicans cell morphologies. A) Yeast form cells (blastospores) with germ tubes indicating the initial hyphae development (also called “germinating yeast cells”). B) Mixture of yeast form cells and of short hyphae indicating early phases of hyphae development. C) Mature hyphae. D) Yeast form cells.
22 23
Peptide and SAV-FITC treated cell samples were resuspended in 600 µl PBS
24
containing 0.4% formaldehyde. Fluorescence intensities were measured at an
25
excitation wavelength of 488 nm and an emission wavelength of 519 nm in
26
transparent 96 well plates (3 × 180 µl per sample) (monochromators of Synergy 4).
27
Relative fluorescence intensities for C. albicans mature hyphae are the result from
28
two independent cultures (three samples from each culture), while all other data are 7 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
from three samples of a single culture. However, all conclusions were confirmed by
2
different measurement techniques.
Page 8 of 15
3 4
SAFETY CONSIDERATIONS
5
All experiments involving viable Candida species were performed in a bio safety level
6
2 (BSL2) laboratory. Handling of formaldehyde stock solution was performed under
7
the fume hood.
8
RESULTS AND DISCUSSION
9
C. albicans and S. cerevisiae yeast cells expressing Als1p or Als5p had been shown
10
to adhere to peptides in an amino acid sequence dependent manner.9 On the basis
11
of these investigations we chose the adhesive peptides KLRIPSV (basis of FBP1)
12
and APRLRFYSL (basis of FBP2) for the proof of concept of the applicability of
13
peptides as sensor molecules for viable C. albicans. In addition, the non-binding
14
peptide EHAHTPR9 (basis of FBP3) was selected as control peptide.
15
We added a C-terminal lysine residue for biotinylation (Table 1) to allow detection via
16
fluorescently labeled streptavidin (SAV-FITC) and labeled the peptides FBP1, FBP2
17
and FBP3 respectively. In the initial experiments, binding of peptides to C. albicans
18
was analyzed by flow cytometry, which is a powerful method in diagnostic
19
microbiology19, as it allows quantification of the fluorescent staining of single cells.12
20
However, it is not applicable to mature, long hyphae of C. albicans (see Figure 1C),
21
as these would block the systems. To include nevertheless both morphologies, we
22
incubated a culture with germinating C. albicans cells, i.e. a culture containing yeast
23
form cells and cells in the initial phase of the yeast-to-hyphae morphological switch
24
(Figure 1A), with each of the three peptides.
25
The peptide FBP2 showed more efficient binding to C. albicans (36.2 ± 8.0 % of total
26
cell population stained) than FBP1 (4.8 ± 2.7 %), whereas FBP3, the chosen
27
negative control, showed no significant binding (0.9 ± 0.7 %).
28
In general, the peptide length, amino acid composition and sequence affect structure,
29
function and solubility of a given peptide. It had been reported that scrambled or
30
reversed sequences of the τφ+ motif in the peptides abolished cell adhesion and
31
binding of the peptides to the Als5p ligand.9,20,21 8 ACS Paragon Plus Environment
Page 9 of 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
1
To investigate the relevance of the different peptide characteristics, we extended the
2
sequence of FBP1 by additional 2 amino acids at the N-terminus, as FBP2 was a
3
decamer peptide, whereas FBP1 was only an octamer. We added N-terminal alanine
4
and proline residues (AP), as these were the N-terminal amino acids in FBP2,
5
resulting in peptide FBP4. Furthermore, we removed the N-terminal AP present in
6
FBP2 to result in peptide FBP5 (Table 1).
7
Incubation of cells with the extended peptides FBP2 and FBP4 resulted in a
8
significantly higher ratio of stained cells (36.2 ± 8.0 % and 43.3 ± 9.0 % of total cell
9
population) compared to the shorter versions FBP1 and FBP5 (4.8 ± 2.7 % and 11.0
10
± 4.0 %). These results indicated an optimal length of 10 amino acids.
11
We next studied the influence of the amino acid sequence on the basis of the
12
sequences of FBP2 and FBP4, without changing the peptide length. Replacement of
13
“R” in the third position in FBP2 by “K” as in the third position in FBP4 resulted in
14
FBP6. Keeping the C-terminal part of FBP2 and replacing the N-terminal amino acids
15
3 and 5 by unrelated amino acids delivered FBP7. These changes of the amino acid
16
sequences resulted in significantly reduced binding efficiency (15.9 ± 4.8 and 2.8 ±
17
2.2% respectively). These results indicated that among the chosen peptides, FBP4
18
and FBP2 showed best binding properties.
19
To determine the relevance of ALS – proteins for peptide binding, we incubated the
20
deletion mutants ∆als5 and ∆als1 of C. albicans with the peptides FBP2 and FBP4.
21
As shown in Table 2, we observed a reduced staining efficiency of both strains by
22
FBP2 and FBP4 showing that both peptides do not bind to a single ALS – protein.
23
This is also supported by the finding that there was still significant residual binding of
24
both peptides (up to 32 % of the population) in the single gene deletion mutants.
25
Table 2. Flow cytometric analysis of peptide binding to ALS mutants.
stained ratio of cell population [%] ∆als5 Peptide FBP2 19.2 ± 13.6 FBP4 32.2 ± 19.5 ∆als1 Peptide FBP2 6,4 FBP4 15,4
∆als5 + ALS5 36.1 ± 12.7 51.9 ± 19.6 ∆als1 + ALS1 15.3 34.6
26
9 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
The results obtained by flow cytometry were confirmed by microscopic analysis of
2
FBP4 treated cultures, to allow analysis of peptide binding to the different
3
morphological forms of C. albicans (Figure 2A - C).
Page 10 of 15
4 5 6 7 8 9 10
Figure 2. Analysis of peptide binding to fungal cells by fluorescence microscopy. Cultivation and treatment of cells was performed as described in the experimental section. (A) C. albicans (initial hyphal development) (60x; exposure time 1.8 s). (B) C. albicans mature hyphae (30x; exposure time 1.3 s). (C) C. albicans yeast (30x; exposure time 1.3 s). (D) C. dubliniensis (60x; exposure time 1.3 s). (E) C. glabrata (60x; exposure time 1.3 s).
11
The photographs highlight that binding of the peptide occurred mainly at the germ
12
tubes (Figure 2A) and the hyphal walls (Figure 2B), whereas the mother cells (Figure 10 ACS Paragon Plus Environment
Page 11 of 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
1
2A) and yeast cells (Figure 2C) were only weakly stained so that they are hardly
2
visible under the fluorescence microscope. Variable lengths of the germ tubes could
3
be the reason for varying fluorescence intensities of cells within a cellular population.
4
As hyphal cells cannot be analyzed by flow cytometry due to the multicellular nature
5
of mycelia, we quantified fluorescence intensities of cell populations with a
6
fluorescence microtiter plate reader. These quantitative data also indicated the
7
preference of FBP4 to bind to hyphae of C. albicans compared to yeasts (Table 3).
8 9 10 11
Table 3. Relative fluorescence intensities (RFI) determined with a fluorescence microtiter plate reader resulting from FBP4 binding to fungal cells obtained from different cultivation conditions. The background values resulted from treatment with SAV-FITC only.
Cultivation Treatment RFI leading to Early hyphal Candida albicans SC5314 development FBP4 2094 ± 137 Background 587 ± 26 Candida dubliniensis DSM13268 FBP4 1431 ± 94 Background 630 ± 15 Candida glabrata ATCC2001 FBP4 804 ± 25 Background 577 ± 47 Mature Candida albicans SC5314 hyphae FBP4 2596 ± 274 Background 884 ± 41 Yeast Candida albicans SC5314 FBP4 683 ± 27 Background 315 ± 9 Candida dubliniensis DSM13268 FBP4 798 ± 14 Background 491 ± 26 Candida glabrata ATCC2001 FBP4 758 ± 27 Background 338 ± 23 12 13
We then tested the applicability of peptide FBP4 to the related fungi C. dubliniensis
14
and C. glabrata. While C. glabrata tends to remain in the yeast morphology under
15
most cultivating conditions,4 C. dubliniensis forms less hyphae in vivo and fails to 11 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 12 of 15
1
form hyphae under laboratory conditions inducing hyphal development in C.
2
albicans.22 This correlates to our observation that C. dubliniensis and C. glabrata did
3
not develop hyphal or pseudohyphal morphologies like C. albicans under the chosen
4
cultivation conditions. As hyphal development in C. albicans and C. dubliniensis
5
requires different cultivation conditions, which also influence the transcriptional
6
profiles of the strains,22 we decided to use the same cultivation conditions for all
7
strains and focus our studies on the different morphologies of C. albicans. Flow
8
cytometric analysis showed that FBP4 only weakly stained C. dubliniensis (6.2 %)
9
and C. glabrata cells (2.2 %) compared to C. albicans. This was confirmed by
10
fluorescence microscopy (Figure 2 D, E) as well as by the quantitative data obtained
11
by a fluorescence microtiter plate reader (Table 4). The quantitative data showed that
12
binding of FBP4 to C. dubliniensis was slightly stronger than to C. glabrata. This
13
could be due to the presence of ALS orthologues in C. dubliniensis in contrast to C.
14
glabrata.23, 24
15 16
CONCLUSIONS
17
We could show that 1) suspended C. albicans cells could be detected by incubation
18
with labeled peptides without the need for pre-treatment of the cells, confirming
19
previous observations.21 2) The binding efficiency was dependent on the length and
20
the sequence of the peptide indicating the specificity of the interaction of FBP2 and
21
FBP4 with C. albicans. The significant reduction of peptide binding to ∆als – deletion
22
mutants also indicates the specificity of the binding. 3) In particular, peptide FBP4
23
preferentially bound to the surfaces of hyphal cells and binding of FBP4 to cells with
24
this morphology was significantly more efficient than to yeast form cells. 4) Binding of
25
FBP4 to C. dubliniensis as well as to C. glabrata was significantly weaker compared
26
to C. albicans under all tested conditions.
27
Peptides as specific recognition components of pathogens offer the significant
28
advantage of being easily available through chemical synthesis, and being cheaper
29
and more robust than antibodies. Detection can be based on standard analytical
30
formats, such as flow cytometry or fluorescence microscopy.
31 12 ACS Paragon Plus Environment
Page 13 of 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
Analytical Chemistry
ACKNOWLEDGEMENTS
2
The authors thank Sonja Kunstmann and Heiko Kima for their contribution to
3
experiments during their lab course studies, Dr. Lothar Gröbe (EXIM, HZI,
4
Braunschweig, Germany) for his support in flow cytometry and Dr. Lois L. Hoyer
5
(University of Illinois, Urbana, Illinois, USA) for providing ALS mutant strains. This
6
work was financially supported by the Federal Ministry of Education and Research of
7
Germany (BMBF) through the project “The Lab in a Hankie — Impulse Centre for
8
Integrated Bioanalysis”, grant no. 03IS2201 A-N.
9 10
REFERENCES
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
(1) Gow, N. A.; van de Veerdonk, F. L.; Brown, A. J.; Netea, M. G. Nat. Rev. Microbiol. 2012, 10, 112–122. (2) Pfaller, M. A.; Diekema, D. J. Clin. Microbiol. Rev. 2007, 20, 133–163. (3) Sullivan, D. J.; Westerneng, T. J.; Haynes, K. A.; Bennett, D. E.; Coleman, D. C. Microbiology, 1995, 141, 1507–1521. (4) Fidel Jr., P. L.; Vazquez, J. A.; Sobel, J. D. Clin. Microbiol. Rev. 1999, 12, 80–96. (5) Sudbery, P. E. Nat. Rev. Microbiol. 2011, 9, 737–748. (6) Hoyer, L. L.; Green, C. B.; OH; S; ZHAO, X. Med. Mycol. 2008, 46, 1–15. (7) Fu Y.; Rieg G.; Fonzi W. A.; Belanger P. H.; Edwards Jr. J. E.; Filler S. G. Infect. Immun. 1998, 66, 1783–1786. (8) Gaur N. K.; Klotz S. A. Infect. Immun. 1997, 15, 5289–5294. (9) Klotz S. A.; Gaur N. K.; Lake D. F.; Chan V.; Rauceo J.; Lipke P. N. Infect. Immu. 2004, 72, 2029–2034. (10) Kragelund, C.; Kieffer-Kristensen, L.; Reibel, J.; Bennett, E. P. Clin. Oral Investig. 2013, 17, 957–965. (11) Ellepola A. N.; Morrison C. J. J. Microbiol. 2005, 43, 65–84. (12) Kaba, H. E. J.; Maier, N.; Schliebe-Ohler, N.; Mayer, Y.; Müller, P. P.; van den Heuvel, J.; Schuchhardt, J.; Hanack, K.; Bilitewski, U. J. Microbiol. Methods 2015, 108, 61–69. (13) Bowen, J.L.; Manesiotis, P; Allender, C.J. Mol. Imprinting 2013, 35-40. (14) Gillum, A. M.; Tsay, E. Y.; Kirsch, D. R. Mol. Gen. Genet. 1984, 198, 179–182. (15) Leinberger, D. M.; Schumacher, U.; Autenrieth, I. B.; Bachmann, T. T. J. Clin. Microbiol. 2005, 43, 4943–4953. (16) Csank, C.; Haynes, K. FEMS Microbiol. 2000, Lett. 189, 115–120. (17) Zhao, X.; OH, S.; Hoyer L. L. Medical Mycology 2007, 45, 429–434. (18) Zhao, X.; OH, S.; Cheng, G.; Green, C. B.; Nuessen, J. A.; Yeater, K.; Leng, R. P.; Brown, A. J. P.; Hoyer, L. L. Microbiology 2004, 150, 2415–2428. (19) Álvarez-Barrientos, A. ; Arroyo, J.; Canton, R.; Nombela, C. ; Sanchez-Perez, M. Clin. Microbiol. Rev. 2000, 13, 167–195. (20) Ahmad, M. F.; Yadav, B.; Kumar, P.; Puri A.; Mazumder, M.; Ali, A.; Gourinath, S.; Muthuswami, R.; Komath, S. S. PLoS ONE 2012, DOI:10.1371/journal.pone.0035305. 13 ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1 2 3 4 5 6 7 8
Page 14 of 15
(21) Garcia-Sherman, M. C.; Lysak, N.; Filonenko, A.; Richards H.; Sobonya, R. E.; Klotz, S. A.; Lipke, P. N. PLoS ONE 2014, DOI:10.1371/journal.pone.0086067. (22) O’Connor, L; Caplice, N; Coleman, D. C.; Sullivan, D. J.; Moran, G. P. Eukaryot. Cell 2010, 9, 1383–1397. (23) Hoyer, L. L.; Fundyga, R.; Hecht, J. E.; Kapteyn, J. C.; Klis, F. M.; Arnold, J. Genetics 2001, 157, 1555–1567. (24) Fu Y.; Phan Q. T..; Lou G.; Solis, N. V.; Liu Y. ; Cormack, B. P.; Edwards Jr. J. E.; Ibrahim, A. S.; Filler S. G. Infect. Immun. 2013, 81, 2528–2535.
9 10
14 ACS Paragon Plus Environment
Page 15 of 15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
Analytical Chemistry
For TOC only
2
15 ACS Paragon Plus Environment
