Download The contribution of electrostatic forces to the process of adherence

FEMS Microbiology Letters 120 (1994) 257-262
© 1994 Federation of E u r o p e a n Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
257
F E M S L E 06063
The contribution of electrostatic forces
to the process of adherence of Candida albicans
yeast cells to substrates
S t e p h e n A. K l o t z *
Department of Medicine, Veterans Affairs Medical Center, Kansas City, MO 64128, USA
(Received 2 February 1994; revision received 12 April 1994; accepted 16 April 1994)
Abstract: The contribution of electrostatic interactions to the adherence process of Candida albicans and other Candida species
was investigated by mixing cationic or anionic exchange resins possessing free - C O 0 - or -NH~ groups, respectively, on their
surface. The adherence process of yeast cells to the anionic exchange resin is a saturable event that is essentially complete by 60
min. There is no measurable interaction of yeast cells with the cationic exchange resin. All clinical isolates of C. albicans, C.
pseudotropicalis, one isolate each of C. tropicalis and Torulopsis glabrata possessed electrostatic charge as defined by this method,
whereas two clincal isolates of C. parapsilosis, C. krusei and one isolate of C. tropicalis did not have measurable electrostatic
surface charge. The adherence process to the exchange resin with the free -NH4" group was d e p e n d e n t upon the pH of the
suspending m e d i u m and varied from one isolate to another. Fixing yeast cells, or alternatively, endothelial cells, in such a m a n n e r
as to change the surface charge of both and then measuring adherence of yeast cells to the target cells was an event that was not
controlled by electrostatic interactions as they are defined herein. It appears that whatever contribution electrostatic charges make
to the adherence process, that at best, it is a minor contribution.
Key words: Electrostatic force; Adherence; Candida albicans
Introduction
The process of adherence of the fungus, Candida albicans, to cells, to extracellular matrix, or
perhaps to inanimate objects such as plastic
catheter surfaces is believed to be important in
the pathogenesis of all forms of disease involving
this commensal yeast [1]. Receptor (adhesin)ligand interactions such as those involving a fi-
* Corresponding author. Fax: (816) 861 4700 ext. 3821
SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 2 0 9 - A
bronectin adhesin [2] or a iC3b adhesin [3] interacting with subendothelial extracellular matrix or
endothelial cells, respectively, are currently receiving considerable attention. These specific interactions are modelled loosely after the 'lock
and key' concept applied to enzymes and their
substrates and imply a high degree of specificity
of interaction between the pathogen and the host
target tissue. However, it is also apparent that the
adherence process is not explainable by one single specific event and that several different specific interactions may be occurring [4] or that
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'non-specific' forces or events may be contributing to the adherence process. For example, it has
long been recognized that hydophobicity contributes to adhesion or adherence of C. albicans
in vitro [5] and perhaps in vivo [6]. Although
recognized earlier in work on Candida adherence
[5], the role of electrostatic forces in the process
of adherence of this fungus to substrates has
never been investigated systematically.
Previous work by us has established that altering the electrostatic charges on the surface of C.
albicans will affect the adherence of yeast cells to
underlying substrates. For example, treating yeast
cells with formaldehyde increases the net negative surface charge on the microorganism [5] and
markedly reduces the number of yeast cells which
adhere to the surface of ex vivo porcine endothelium [7]. In addition formaldehyde-treated yeast
cells demonstrate a 49% reduction in adherence
to a plastic surface, whereas carbodiimide-treated
yeast cells which are more positively charged
demonstrate a 72% increase in adherence to a
plastic surface [5]. Thus, in vitro it appears that
electrostatic forces in concert with other forces,
both non-specific such as hydrophobicity and specific such as r e c e p t o r - l i g a n d interactions, contribute to the adherence process. However, the
magnitude a n d / o r the importance of electrostatic interactions in the adherence process remains unknown. In early studies of adherence it
was appreciated that the presence of calcium
a p p e a r e d to be important to the adherence process. For example, Skerl et al. [8] demonstrated
that significantly more yeast cells adhered to immobilized fibronectin in the presence of ionized
calcium that did so in the absence of this divalent
cation. Since ionized calcium is a charged particle
it was possible that the contribution of this metal
to the adherence process was purely electrostatic
in character.
Materials and Methods
Fungi
Candida species were maintained on Sabouraud dextrose agar and subcultured monthly. For
assay purposes, a loopful of microorganisms was
placed in Sabouraud dextrose broth (or in some
experiments, brain heart infusion medium) and
grown for 20 h at room temperature with shaking.
This yields yeast cells at the early stationary phase
of growth. In some experiments the fungi were
cultured for 12 h which yields fungi in the early
logarithmic phase of growth. The fungi in each
instance were washed by centrifugation and suspended in Hank's balanced salt solution containing 2 mM calcium and 2 m M magnesium (buffer).
Electrostatic charge determination
Following the wash of yeast cells in buffer, 0.5
g of either anionic or cationic exchange resin
beads of 1 0 0 - 2 0 0 / x m mesh (Bio-Rad, Richmond,
CA) were added to a 15-ml acid-washed screwtopped test tube along with 5 ml of buffer containing 1 x l0 s yeast cells m l - I at p H 7.35. A
sample of the suspension was removed from the
test tube, allowed to settle for 10 min and then
the absorbance of the 'supernatant' was determined at 530 nm. This value was denoted as E 0.
The test tubes were then gently rocked for 60 rain
to insure proper mixing, a sample removed, allowed to settle for 10 min, and the absorbance of
the 'supernatant' determined. This value was
termed E60. The value attached to the degree of
electrostatic interactions among the yeast cells
was then determined by the formula 1 - E 6 o / E o.
The greater the value, 1 - E6o/Eo, the greater
the electrostatic interaction. This value, 1 E6o/Eo, is termed ' b o u n d ' yeast cells for the sake
of simplicity. When no resin beads were added
there was no significant difference in the beginning and ending absorbance values.
Miscellaneous methods
Bovine aortic endothelial cells were grown to
confluence on plastic coverslips in the bottom of
24-well tissue culture trays by previously described methods [9]. Some wells containing endothelial cells were treated with formaldehyde or
glutaraldehyde, washed and yeast cells added to
each well to determine the amount of adherence.
Following incubation for 1 h the coverslips were
washed by immersion, fixed and stained with
hematoxylin and silver. The number of yeast cells
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adhering to 100 endothelial cells was then determined microscopically [9].
Results
Yeast cells were mixed with either cationic or
anionic exchange resin and a m e a s u r e m e n t of
electrostatic charge on the surface of yeast cells
recorded. Fig. 1 shows that the interaction of
yeast cells with the charged beads is a saturable
phenomenon. Only the anionic resin containing
free -NH~- groups and therefore positively
charged, allowed interaction with yeast cells.
Cationic resin beads containing free - C O O groups had no measurable interaction with yeast
cells. A scanning electron p h o t o m i c r o g r a p h
demonstrating the b i n d i n g / a d h e r e n c e of the
yeast cells to anionic resin beads is shown in
Fig. 2.
A number of different Candida species yeast
cells were then mixed with anionic exchange resin
in order to measure electrostatic interactions of
the cells. All of these were clinical isolates and as
can be seen in Table 1 there was a broad range of
interaction of yeast cells with the resin beads. A
0.25 -
0.15
rn
-
0.10-
0.05-
0.00
o
2'o
,'o
6'o
8'0
,~0
Incubation Time (minutes)
Fig. 1. Measurement of electrostatic charge on the surface of
Candida albicans yeast cells as determined by the interaction
with anionic exchange resin at pH 7.35 and room temperature. B equals the value of 1 - E 6 o / E o. Other isolates had
similar saturation kinetics, r = 0.8470 for the points shown.
Error bars represent 2 standard deviations. Dotted line is best
fit sigmoidal curve.
Table 1
Survey of the electrostatic charge on the surface of clinical
isolates of Candida species yeast cells
Species
Isolate
Bound (1 - E 6 o / E o)
Candida albicans
1
2
3
4
5
1
0.094
0.100
0.515
0.800
0.303
0.265
Candida tropicalis
Candida parapsilosis
Candida krusei
Candida pseudotropicalis
Torulopsis glabrata
2
0.000
1
0.000
2
0.000
1
2
1
2
1
0.000
0.000
0.340
0.280
0.700
Yeast cells were incubated with anionic exchange resin for 1 h
at room temperature, pH 7.35.
C. albicans isolate had the greatest measurable
charge of all the isolates and all C. albicans
isolates had some measurable charge, whereas
two isolates each of C. parapsilosis and C. krusei
did not have any measurable electrostatic charge.
Two isolates of C. albicans were then selected
and the degree of electrostatic interaction of yeast
cells with both cationic and anionic resin beads
determined over a broad range of pH. T h e r e was
no measurable interaction of C. albicans with the
cationic exchange resin. The electrostatic charge
of yeast cells as determined by the interaction of
cells with anionic exchange resin varied unpredictably with the p H of the suspending medium.
C. albicans yeast cells cultured in brain heart
infusion medium adhered to the anionic exchange resin about three times more avidly than
did yeast cells cultured in Sabouraud dextrose
broth. Also, stationary phase yeast cells adhered
to the exchange resin in significantly greater
numbers that did yeast cells in the logrithmic
phase of growth. Adding 5 - 2 0 % fetal calf serum
to the suspending buffer entirely obliterated any
binding of yeast cells to anionic exchange resin.
Previous work has established that formaldehyde-treated yeast cells do not adhere as well as
viable untreated yeast cells to ex vivo porcine
endothelium [7]. If formaldehyde treatment in-
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creases the net negative charge on the surface of
cells as has been claimed [5] then treating the
endothelium with formaldehyde should reduce
the adherence of viable untreated yeast cells to
this tissue as well. As is shown in Table 2 such is
not the case. On the contrary, treating the endothelium with formaldehyde actually increased
the adherence of C. albicans yeast ceils, whereas,
Fig. 2. Scanning electronphotomicrograph of Candida albicans yeast cells adhering to the surface of anionic exchange resin beads.
Bar on the lower part of the figure equals 1 /xm.
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Table 2
T h e adherence of Candida albicans yeast cells to bovien
endothelial cells in tissue culture following the electrostatci
alteration of the surface of yeast cells or the endothelial cells
n ~
Control (viable yeast cells
plus normal endothelium)
Endothelium treated with:
1.5% formaldehyde
2% glutaraldehyde
Yeast cells treated with:
1.5% formaldehyde
Yeast
cells/100
endothelial
cells ( _+SD b)
P
3
6 7 + 16
7
4
105 + 16
70 + 18
< 0.05
NS
3
31 + 11
< 0.05
A d h e r e n c e assay was performed at p H 7.4, 37°C in 95%
a i r / 5 % CO2.
a n = n u m b e r of coverslips.
b DS, standard deviation.
c NS, not significant.
treatment with glutaraldehyde seemingly does not
affect the adherence process. Therefore in this
somewhat more complex interaction of living yeast
cell and living or fixed endothelial cell, rather
than the exchange resin as previously worked
with, electrostatic interactions a p p e a r to be overwhelmed in the adherence process or are purely
secondary to other forms of interactions.
Discussion
This work establishes that measurable electrostatic charges exist on the surface of Candida
species isolates and that these charges may play
some minor role in the adherence process of the
yeast cell to a substratum. For example, the interaction of the yeast cells with the anionic resin
exchange beads and not with the cationic exchange beads supports the concept of electrostatic interactions in the process since the beads
differ only in their net surface charge. However,
the electrostatic interactions are clearly dependent upon the p H of the milieu in which these
experiments are conducted. The electrostatic
charge varies greatly with the p H and no two
isolates are the same (data not shown). This
observation is in agreement with Persi et al. [10]
who found that C. albicans yeast cell adherence
to vaginal epithelial cells was highly dependent
upon the p H and was different for two clinical
isolates. However, more recent work did not
demonstrate an effect of p H on the adherence
process of C. albicans to acrylic [11].
Ionized calcium has been shown by this laboratory and others to be important in the adherence
process of the fungus to substratum [12]. This ion
(calcium) and other cations are a prominent part
of the D L V O colloid theory of interaction of a
small particle (such as a microbe) with a negatively charged substratum [13]. In this theory the
cationic metals are treated solely as charged particles. However, recent work by us would indicate
that the favorable effect that calcium has upon
yeast cell adherence to a substratum is due more
to its conformation or configuration than to its
charge. For example, in the adherence of C.
albicans yeast cells to immobilized fibronectin,
calcium clearly was the one cation which enhanced adherence. Terbium, a trivalent earth
metal which is thought to be isomorphic with
calcium enhances adherence of yeast cells to fibronectin to about half that of calcium. Magnesium, only when added to calcium, enhanced
adherence. The presence of other cations did not
affect the adherence of yeast cells. Therefore, the
contribution of calcium to the adherence process
appears to depend a great deal upon the shape of
this particular cation rather than solely upon its
positive charge.
It thus appears that electrostatic interactions,
although present in the process of adherence of
yeast cells to some substrata, is a minor force
which makes only a modest, at best, contribution
to adherence.
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