Release of a Potent Polymorphonuclear Leukocyte Chemoattractant Is Regulated by White-Opaque Switching in Candida albicans

Abstract

Previous studies employing transmembrane assays suggested that Candida albicans and related species, as well as Saccharomyces cerevisiae, release chemoattractants for human polymorphonuclear leukocytes (PMNs). Because transmembrane assays do not definitively distinguish between chemokinesis and chemotaxis, single-cell chemotaxis assays were used to confirm these findings and test whether mating-type or white-opaque switching affects the release of attractant. Our results demonstrate that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata release bona fide chemoattractants for PMNs. S. cerevisiae, however, releases a chemokinetic factor but not a chemoattractant. Characterization of the C. albicans chemoattractant revealed that it is a peptide of approximately 1 kDa. Whereas the mating type of C. albicans did not affect the release of chemoattractant, switching did. White-phase cells released chemoattractant, but opaque-phase cells did not. Since the opaque phase of C. albicans represents the mating-competent phenotype, it may be that opaque-phase cells selectively suppress the release of chemoattractant to facilitate mating.

Polymorphonuclear leukocytes (PMNs) represent the first line of defense in combating microbial infections. By sensing chemoattractants, PMNs migrate toward sites of infection. In vitro, PMNs orient and chemotax up spatial gradients of formyl methyl peptides (11, 53-55), which are released by bacteria. These peptides bind to a receptor on the surface of PMNs (26, 31). The receptor-mediated signal is transduced through regulatory cascades that control the actin cytoskeleton in order to polarize and direct the movement of the PMNs toward the site of infection (6, 18, 52). The spatial, and possibly temporal, patterns of receptor occupancy dictate orientation and subsequent chemotaxis (11, 12).

The process of PMN chemotaxis may play a role in controlling the levels of Candida spp. carriage in the commensal state, as well as in clearing yeast from sites of infection (10, 34). The attraction of PMNs to sites of infection may also contribute to the inflammatory response associated with vaginitis and other forms of mucosal candidiasis. Immunocompromised individuals are far more prone to mucosal candidiasis and candidemias (3), supporting the suggestion that the cellular immune response plays a role in suppressing such infections. The attraction of PMNs to sites of fungal infection may be mediated by an attractant either released by the colonizing yeast cells or released by human cells activated by the yeast. Interestingly, however, in animal models of vaginitis and in human cases of vaginitis the frequent absence of PMNs has been noted (10). The absence of PMNs at the site of a fungal infection may be due either to unresponsive PMNs or to the failure by the colonizing yeast to release a chemoattractant. The release of PMN chemoattractants by colonizing yeast and the responsiveness of PMNs to such chemoattractants, therefore, warrant careful scrutiny as they relate to yeast infections.

It has been demonstrated, by using a transmembrane assay to assess chemotaxis, that Candida albicans and related species release a chemotactic or chemokinetic signal that functions through the fMLP receptor (5, 7, 8). In the transmembrane, or “Boyden Chamber,” assay (4, 57), cells are placed on one side of a filter separating two reservoirs. The reservoir on the side of the membrane supporting the PMNs contains buffer lacking chemoattractant, whereas the reservoir on the other side of the membrane contains buffer with chemoattractant. Presumably, the spatial gradient of attractant generated across the membrane stimulates orientation and directed movement, leading to an increase in PMNs on the reverse surface of the membrane facing the reservoir with attractant. However, attractants can stimulate random movement (chemokinesis), which also results in an increase in the number of cells on the surface of the membrane facing the reservoir with attractant. Although “checkerboard” assays have been developed to help distinguish between chemotaxis and chemokinesis (57), they do not definitively achieve this goal (32, 50, 55, 57). In particular, transmembrane assays measure the final number of cells that penetrate or traverse the membrane rather than the oriented behavior of individual cells. Realizing the need to directly assess chemotaxis at the single cell level, Zigmond (53, 56) pioneered the use of a chamber composed of a bridge that supports cells, bordered on either side by two wells: one containing buffer alone and the other containing buffer plus chemoattractant. When the wells are filled with their respective solutions, a transient spatial gradient of chemoattractant develops across the bridge. The speed at which the gradient develops and then flattens is directly proportional to molecular weight. By plotting the time of maximum response as a function of molecular weight for known chemoattractants and chemokinetic factors, one can generate a reference plot that can be used to estimate the approximate molecular weight of an unidentified attractant from the time it takes for the latter to elicit a maximum response (35, 36).

Here, we used a chamber designed based on that of Zigmond (53) to confirm prior observations based on transmembrane filter assays that Candida species release bona fide PMN chemoattractants (5, 7, 8). We also used this single-cell assay to test whether mating-type (14, 22) or white-opaque (38) switching affects the release of chemoattractant by Candida albicans. Our results demonstrate that all five tested pathogenic Candida spp. (C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata) release a bona fide chemokinetic and chemotactic factor(s), whereas three unrelated strains of Saccharomyces cerevisiae release a chemokinetic, but not a chemotactic, factor. In C. albicans, strains heterozygous for the mating type (MTL) locus (a/α) and strains homozygous for the MTL locus (a/a or α/α) in the white-phase phenotype, release a chemoattractant. However, strains homozygous for the MTL locus (a/a or α/α) that have switched to the opaque-phase phenotype do not release a chemoattractant. Since the opaque-phase phenotype of C. albicans represents the mating-competent phenotype of this species (23, 28), the possibility that opaque-phase cells suppress the release of a chemoattractant to facilitate mating is examined.

MATERIALS AND METHODS

Isolation of PMNs.

A total of 10 ml of blood was collected in a heparinized vacutainer (Becton Dickinson, Franklin Lakes, N.J.) from healthy young adult donors. After 5 min of incubation at 37°C, 5 ml was layered onto 5 ml of Polymorphoprep (Axis-Shield, Oslo, Norway) in a 15-ml Falcon centrifuge tube (Becton Dickinson). Centrifugation for 30 to 35 min at 450 × g generated a distinct PMN-enriched fraction relatively free of mononuclear cells and erythrocytes. The PMN layer was removed and mixed with an equal volume of Hanks buffered salt solution (Gibco-BRL, Gaithersburg, Md.) containing 0.01 M HEPES (pH 7.4) (H-HBSS) to restore normal osmolarity. The PMN suspension was washed three times in H-HBSS by centrifugation at 400 × g for 10 min. The final pellet was resuspended at a final cell concentration of 1.5 × 10⁶ cells per ml in H-HBSS. The final PMN preparations contained between 1 and 3% mononuclear cells and erythrocytes.

Maintenance of yeast cultures.

The following yeast strains were used in this study: C. albicans strain 3153A (a/α) (37), C. albicans strain WO-1 (α/α) (38), C. albicans strain P37005 (a/a) (22), C. albicans strain 19F (α/α) (22), C. albicans strain L26 (a/a) (22), C. albicans strain 12C (a/a) (22), C. albicans strain P57072 (α/α) (22), C. dubliniensis strain P30 (17), C. tropicalis strain T1 (16), C. parapsilosis strain 313 (the present study), C. glabrata strains 1480.47 (25) and 35B11 (20), and S. cerevisiae strains MGD407 (2), and LP388 and LA761. Strains LP388 and LA761, obtained from vaginal S. cerevisiae infections, were generous gifts from J. D. Sobel, Wayne State University. Both formed asci on sporulation medium and were, therefore, diploid. Strain MGD407 (MATa/α ura3-52 leu2-3,112) was a generous gift from Brian Rymond, University of Kentucky. Cells maintained on nutrient agar were inoculated into the appropriate liquid nutrient medium and grown to mid-log or early stationary phase. C. albicans strains were grown in modified Lee medium (1). All others were grown in YPD medium (2% glucose, 2% Bacto Peptone, 1% yeast extract).

Conditioned buffer.

A total of 5 ml of cell culture was pelleted, and the cells washed three times in 5 ml of H-HBSS. Cells were then resuspended in 5 ml of H-HBSS, followed by incubation on a rotating shaker for 3 h. Cells were then pelleted, and the supernatant was passed first through a 0.8-μm-pore-size Acrodisc syringe filter (Pall Corp., Ann Arbor, Mich.) and then through a 0.22-μm-pore-size Millipore syringe filter (Millipore Corp., Bedford, Mass.).

Chemotaxis assay.

The quartz chamber used to analyze single-cell chemotaxis with bright-field optics has been described in detail by Shutt et al. (35) (Fig. 1A). To initiate a chemotaxis experiment, 200 μl of a suspension of ca. 2 × 10⁶ PMNs per ml was distributed onto a 24-by-30-mm Thermanox coverslip (Nunc, Inc., Naperville, Ill.). After 5 min at 37°C, the coverslip was inverted and clamped to the top of the inverted chamber. The chamber was turned upside down, and source and sink wells were filled with test solution and H-HBSS, respectively. The second coverslip was then placed on the bottom and clamped. The chamber was positioned on an inverted microscope (Axiovert 100; Zeiss Corp.) equipped with a long-distance working lens and condenser.

FIG. 1.

Diagrams of the chemotaxis chamber (A) and spatial gradient generated across the bridge (B). After assembly of the chamber, PMNs (cells) are supported by the glass coverslip clamped to the chamber. The sink well contains buffer alone, whereas the source well contains test solution. When fMLP is placed in the source well, it generates a spatial gradient across the bridge. Because the bridge is made of quartz, high-magnification bright-field optics with a long working distance lens and condenser allows single cell analysis. Images are frame-grabbed at 4-s intervals for motion analysis.

Analysis of cell behavior.

Fields of cells were digitized at 15 frames per min onto the hard disk of a Macintosh computer (Apple Computer, Cupertino, Calif.) equipped with a framegrabber board (Data Translation, Inc., Marlboro, Mass.) and 2D-DIAS software (39, 43, 45). Perimeters were outlined and converted to beta-spline replacement images. The centroid of each perimeter image was computed. Both perimeter and centroid tracks were generated. Cell centroid tracks were used to measure motility parameters, including instantaneous velocity, directional change, and chemotactic index (CI), whereas the replacement images were used to compute morphology parameters, including maximum length, maximum width, and roundness, according to formulas previously described (9, 11, 39, 47a, 48, 51). To measure the frequency of lateral pseudopod formation, difference pictures were generated at 4-s intervals by placing the image at frame n over the image at frame n − 1. Regions in the n image not overlapping the n − 1 image were considered expansion zones. A lateral pseudopod was considered to be an expansion zone formed from the main axis of translocation at an angle ≥ 30° that attained a minimum of 5% total cell area and contained nonparticulate cytoplasm. The axis of translocation was determined by drawing a vector from the centroid of the cell in frame n−1 to that in frame n. The CI was computed as the net distance moved directly toward the source of chemoattractant divided by the total distance moved in that same time period. The percent positive chemotaxis was computed as the proportion of PMNs exhibiting a positive CI over the period of analysis. Only cells translocating at rates of 3 μm per min or greater were analyzed. This ranged between 75 and 85% of PMNs in a given experiment. Significance was tested by the Student t test. A P value of >0.05 was considered insignificant.

RESULTS

Chemotaxis assay.

PMNs from a healthy young donor were dispersed on the bridge of a chamber and covered with a coverslip (Fig. 1A). The well on one side of the bridge was filled with buffer alone (the “sink”), whereas the well on the other side was filled with buffer plus test solution (“source”) (Fig. 1A). Cell behavior was then videorecorded. In the case of the chemoattractant fMLP, a gradient developed between 5 and 25 min (Fig. 1B) that caused PMNs to orient in and chemotax up the spatial gradient. In Fig. 2A, representative perimeter tracks are presented of PMNs on the bridge of a chamber in which both wells were filled with buffer lacking chemoattractant. PMNs moved in all directions, since there was no spatial gradient of chemoattractant in which they could orient. In Fig. 2B, representative perimeter tracks are presented of PMNs on the bridge of a chamber in which the well on the left hand side of the bridge was filled with buffer lacking chemoattractant, while the well on the right-hand side was filled with buffer containing 10⁻⁷ M fMLP. After 5 min of incubation, the majority of cells oriented in the direction of the gradient and moved in the direction of increasing fMLP concentration, as indicated by the arrow at the bottom of the panel. The paths of the representative cells in Fig. 2B were relatively persistent in the direction of increasing fMLP concentration. In the process of chemotaxis, PMNs suppressed lateral pseudopod formation. In buffer the average frequency of lateral pseudopod formation was 5.80 per 10 min, whereas in a spatial gradient of fMLP, the frequency was 0.64 per 10 min, a ninefold decrease in frequency (P < 0.01) (Table 1.) The paths of PMNs in buffer (Fig. 2A) included more sharp turns than PMNs in a spatial gradient of fMLP (Fig. 2B). This was reflected by a mean directional change parameter of 37.1° ± 2.1° per 4 s for cells in buffer compared to 24.2° ± 7.0° per 4 s for PMNs in a spatial gradient of fMLP (P < 0.01) (Table 1). Cells also became more elongate in buffer. This was reflected by an increase in maximum length and a decrease in roundness (Table 1).

FIG. 2.

Perimeter tracks of PMNs responding to no chemotactic gradient (A) and a chemotactic gradient of fMLP (B) in the gradient chamber described in Fig. 1. Perimeters were smoothed and plotted at 4-s intervals. The dark gray area is the last in each track. In panel A buffer alone was placed in the sink and source wells, whereas in panel B buffer alone was placed in the sink well and buffer plus 10⁻⁷ M fMLP was placed in the source well. Tracks begin after 5 min of incubation in the chamber. The fMLP gradient is represented by an arrow (pointing toward source) in panel B.

TABLE 1.

Five infectious yeasts (C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata), but not S. cerevisiae, release a potent chemoattractant for human PMNs

Test solutiona	No. of cells	Mean instantaneous velocity (μm/min) ± SD	Chemokinetic stimulation (%)	Mean directional change (degrees/4 s) ± SD	Mean maximum length (μm) ± SD	Mean roundness (%) ± SD	Frequency of lateral pseudopod formation/10 min	CI	% Positive chemotaxis
Buffer	26	10.2 ± 3.4		37.1 ± 2.1	15.0 ± 3.4	72 ± 4	5.80	+0.03b	47b
fMLP	25	14.3 ± 4.6	42	24.2 ± 7.0	18.3 ± 2.2	64 ± 10	0.64	+0.46	87
C. albicans (3153A)	20	15.7 ± 8.6	56	23.2 ± 8.3	19.9 ± 2.2	64 ± 4	0.76	+0.33	92
C. dubliniensis (P30)	88	15.3 ± 2.3	52	27.2 ± 2.5	20.0 ± 2.0	64 ± 4	0.75	+0.53	92
C. tropicalis (T1)	21	19.3 ± 4.9	91	23.2 ± 6.5	16.5 ± 1.6	66 ± 8	1.00	+0.60	96
C. parapsilosis (313)	23	15.6 ± 3.6	55	31.9 ± 3.1	22.1 ± 2.2	62 ± 8	0.77	+0.40	87
C. glabrata (1480.47)	20	13.7 ± 2.4	36	33.7 ± 1.0	19.9 ± 2.2	63 ± 6	0.66	+0.58	92
C. glabrata (35B11)	21	16.0 ± 3.2	57	26.8 ± 6.8	24.7 ± 5.0	48 ± 11	0.59	+0.39	94
S. cerevisiae (LA761)	30	15.6 ± 3.3	53	37.6 ± 2.0	15.0 ± 1.7	87 ± 6	6.66	−0.02	55
S. cerevisiae (MGD467)	19	14.9 ± 1.5	46	32.2 ± 6.0	13.0 ± 3.0	86 ± 5	7.26	−0.05	47
S. cerevisiae (LP388)	16	14.2 ± 3.1	41	35.5 ± 4.9	14.8 ± 2.1	88 ± 3	8.00	+0.09	52

^aBuffer, 10⁻⁷ M fMLP or buffer conditioned by noted yeast species (strain).

^bOne arbitrary direction was taken, even though buffer was in both troughs of the chamber. The parameters represent those expected in the absence of a gradient (i.e., no chemotaxis). The CI was close to 0.00, and the percent positive chemotaxis was close to 50%, both measures reflecting no chemotaxis.

When cells were placed on the bridge of a spatial gradient chamber with buffer in both wells and one well was arbitrarily picked as the source, the computed CI was +0.03, which is very close to 0.00, the value for random movement (i.e., no chemotaxis) (Table 1). However, when 10⁻⁷ M fMLP was placed in one well and buffer in the other, the computed CI was +0.46, reflecting a strong positive chemotactic response. In the nonchemotactic configuration (i.e., buffer in both wells), 47% of PMNs moved toward the arbitrary source and 53% toward the arbitrary sink, reflecting random directionality (Table 1). In the chemotaxis configuration, 87% of PMNs moved toward the true source, whereas only 13% moved toward the well containing buffer alone (Table 1). When both wells of the chamber were filled with buffer, PMNs translocated with a mean instantaneous velocity of 10.2 ± 3.4 μm/min, but in spatial gradients of fMLP, PMNs translocated with a mean instantaneous velocity of 14.3 ± 4.6 μm/min (P < 0.01) (Table 1). The 42% increase in velocity is considered to represent a chemokinetic response to fMLP. The parameters presented in Table 1 for PMNs moving in buffer and a gradient of fMLP were those for PMNs of a single healthy donor but were highly representative of PMNs of three additional healthy donors (data not shown).

If a test solution contains a chemoattractant as potent as fMLP, one would expect PMNs to orient in and chemotax up a gradient of that attractant in a manner similar to that in a gradient of fMLP (Fig. 2B and Table 1), but if a test solution contains no chemoattractant, one would expect PMNs to behave in a manner similar to that in buffer (Fig. 2A and Table 1). If a test solution contains a chemokinetic factor, then one would expect an increase in velocity similar to that in an fMLP gradient. The possibility exists that a test solution may contain either chemotactic or chemokinetic activity or both activities.

Release of chemoattractants by Candida spp.

Several studies demonstrated that C. albicans released one or more factors that stimulate cell migration across a membrane in a transfilter assay (5, 7, 8). To test whether C. albicans and related species release a bona fide PMN chemoattractant, cells from each Candida species were incubated in buffer for 3 h and centrifuged, and the supernatant (conditioned buffer) was filtered and tested in the source well of the chemotaxis chamber (Fig. 1A). C. albicans strain 3153A, which is a/α and thus constitutively in the white phase (22), released a potent PMN chemoattractant. Conditioned buffer from this strain, when used to generate a spatial gradient, stimulated individual PMNs to orient and crawl in a directed fashion toward the source trough (Fig. 3A). PMNs responded to this gradient, with an average CI of +0.33 and a percent positive chemotaxis of 92% (Table 1). Perimeter tracks of representative PMNs in spatial gradients of the C. albicans attractant(s) (Fig. 3A) were qualitatively similar to those in gradients of fMLP (Fig. 2B). The instantaneous velocities in the two gradients were 15.7 ± 8.6 and 14.3 ± 4.6 μm/min, respectively (P = 0.21) (Table 1). PMNs chemotaxing in a spatial gradient of C. albicans attractant exhibited an increase in mean instantaneous velocity over that in buffer of 56%, a decrease in mean directional change, an increase in length, a decrease in roundness, and an eightfold decrease in the average frequency of lateral pseudopod formation (Table 1). All of these differences had P values of <0.01. The parameters of PMNs in a spatial gradient of C. albicans attractant were similar to those of PMNs in a spatial gradient of fMLP (Table 1). The P values were all >0.05. These results demonstrate unequivocally that C. albicans releases a bona fide chemotaxis factor.

FIG. 3.

Perimeter tracks of PMNs challenged with potential gradients of chemoattractant generated from buffer conditioned by the noted species in the source wells of the chamber. Perimeters were smoothed and plotted at 4-s intervals. The dark gray area is the last in each track. The gradient of potential attractant is represented by an arrow at the bottom of each panel. Note that chemotaxis (directed movement up each gradient) is induced by buffer conditioned by every tested species but S. cerevisiae.

Similar experiments revealed that the species C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata also released one or more bona fide chemotactic and chemokinetic factors. The conditioned buffers of these species resulted in CIs ranging between +0.39 and +0.60, and percent positive chemotaxis measures between 87 and 96% (Table 1). Perimeter tracks of representative PMNs revealed orientation and directed movement up spatial gradients generated by the conditioned buffer of C. dubliniensis (Fig. 3B), C. tropicalis (Fig. 3C), C. parapsilosis (Fig. 3D) and C. glabrata (Fig. 3E). The conditioned buffer from all four species also caused decreases in the frequency of lateral pseudopod formation, decreases in the directional change parameter, an increase in maximum length, a decrease in roundness, and chemokinetic stimulation ranging between 36 and 91% (P < 0.01 for all parameters compared to buffer) (Table 1).

The only exception was S. cerevisiae. Although the buffers conditioned by three tested strains of S. cerevisiae, two natural isolates from vaginitis patients (LP388 and LA761) and one laboratory strain (MGD407), induced increases in instantaneous velocity (P < 0.01 compared to buffer), which are considered chemokinetic responses, none stimulated chemotaxis (Table 1). In each case, directional change was high, maximum length was low and roundness high, as in buffer (Table 1). The CI of PMNs in all three cases, however, was close to 0.00, the value for true random movement (Table 1). The percent positive chemotaxis in all three cases ranged between 47 and 55%, close to the 50% value representing true directional randomness. Perimeter tracks of representative PMNs revealed random movement (Fig. 3F).

Switching regulates chemoattractant in C. albicans.

In C. albicans, zygosity at the mating-type locus (MTL) regulates the capacity to undergo white-opaque switching, and switching in turn regulates the capacity to mate (22, 23, 28, 42, 46). Although cells heterozygous at the MTL locus (a/α) are unable to switch, cells homozygous at the MTL locus (a/a or α/α) are able to switch (22, 28). To test whether MTL zygosity, mating type, and switching affect the production of chemoattractant by C. albicans, five strains homozygous at the MTL locus, three a/a and two α/α, were tested in the white and opaque phases for the production of PMN chemoattractant (Table 2). Buffer conditioned by white-phase cells of each of the five strains stimulated chemotaxis and chemokinesis. The CIs of PMNs responding to spatial gradients of chemoattractant released from white-phase cells of the five strains ranged from +0.27 to +0.50, and the percent positive chemotaxis values ranged from 82 to 92% (Table 2). The majority of perimeter tracks of PMNs in gradients generated by conditioned buffer from all four strains revealed directed movement up each gradient (Fig. 4A, C, E, and G). The percent chemokinetic stimulation over control cells ranged from 25 to 65% (P < 0.01 in all cases compared to buffer) (Table 2). The spatial gradients of chemoattractant released by white-phase cells of the five strains caused a decrease in mean directional change and a dramatic decrease in the frequency of lateral pseudopod formation, a result similar to the changes in spatial gradients of fMLP (P < 0.01 compared to buffer) (Table 2).

TABLE 2.

Opaque-phase cells of C. albicans do not produce a PMN chemoattractant

Source trough of chambera			No. of cells	Mean instantaneous velocity (μm/min) ± SD	Chemokinetic stimulation (%)	Mean directional change (degrees/4 s)	Frequency of lateral pseudopod formation/10 min	CI	% Positive chemotaxis
Strain	MTL genotype	Phase
-	(Buffer)	NA	26	10.2 ± 3.4		37.0 ± 2.1	5.8	+0.03b	47b
-	fMLP	NA	25	14.3 ± 4.6	42	24.2 ± 7.0	0.7	+0.46	87
WO-1	α/α	Wh	22	15.8 ± 3.0	55	29.0 ± 4.7	0.7	+0.27	82
		Op	19	12.2 ± 1.0	21	34.0 ± 5.1	7.1	−0.07	52
137005	a/a	Wh	16	12.5 ± 1.8	25	28.7 ± 5.6	0.6	+0.49	86
		Op	19	11.7 ± 2.2	14	39.9 ± 2.3	7.9	−0.01	48
19F	α/α	Wh	21	15.6 ± 1.9	53	22.4 ± 6.9	0.6	+0.50	88
		Op	21	12.5 ± 4.4	23	35.4 ± 9.9	7.1	+0.03	47
L26	a/a	Wh	13	15.5 ± 3.1	52	31.2 ± 3.1	0.6	+0.50	90
		Op	17	15.0 ± 4.2	49	38.5 ± 4.6	5.7	−0.09	50
12C	a/a	Wh	16	16.8 ± 4.9	65	30.2 ± 8.1	0.7	+0.49	92
		Op	15	14.4 ± 3.1	41	35.6 ± 1.9	6.8	−0.09	54

^aBuffer was conditioned by cells of the strains and phases noted. -, No strain; NA, not applicable; Wh, white; Op, opaque.

^bSee Table 1, footnote b.

FIG. 4.

Perimeter tracks of PMNs challenged with potential gradients of chemoattractant in buffer conditioned by white-phase and opaque-phase cells of each tested homozygous (a/a or α/α) strain of C. albicans. Strains WO-1, 157072, and 19F are α/α, whereas 137005 is a/a. Perimeters were smoothed and plotted at 4-s intervals. The dark gray image is the last in each track. The gradient of potential attractant is represented by an arrow at the bottom of each panel. Note that chemotaxis was induced by buffer conditioned by white-phase, but not opaque-phase, cells of each strain of C. albicans.

In marked contrast, conditioned buffer from opaque phase cells of all five strains did not stimulate chemotaxis. The CIs were all close to 0.00, ranging between −0.09 and +0.03 (Table 2). The percent positive chemotaxis values were all close to 50% (the value for random movement), ranging between 47 and 54% (Table 2). Perimeter tracks revealed randomly directed translocation in each case (Fig. 4B, D, F, and H).

Although buffer conditioned by opaque-phase cells did not stimulate chemotaxis, it did stimulate chemokinesis, ranging from 14 to 49% stimulation (P < 0.01 in all cases compared to buffer) (Table 2). For all but one strain, chemokinetic stimulation by buffer conditioned by opaque-phase cells was lower than that by buffer conditioned by white-phase cells (P < 0.01, except for L26, where P = 0.53) (Table 2). However, buffer conditioned by opaque-phase cells did not cause a decrease in directional change or a decrease in the frequency of lateral pseudopod formation (Table 2), changes associated with chemotactic stimulation (44). These results support the conclusion that whereas white-phase cells of either a/a or α/α strains release a PMN chemoattractant, opaque-phase cells of the same strains do not.

The experiments comparing white- and opaque-phase cells from two homozygous strains, one a/a (L26) and one α/α (WO-1), were repeated with PMNs from a second healthy donor. The results were highly similar to those obtained with PMNs from the primary donor. For both strains, spatial gradients generated from buffer conditioned by white-phase cells induced chemokinesis (instantaneous velocities of 16.62 ± 2.0 and 14.9 ± 1.2 μm/min, respectively) and chemotaxis (CIs of +0.30 and +0.41, respectively) and suppressed lateral pseudopod formation, whereas opaque- phase cells stimulated chemokinesis (instantaneous velocities of 14.0 ± 2.9 and 13.4 ± 3.7 μm/min, respectively) but did not stimulate chemotaxis (CIs of −0.02 and −0.01, respectively) or suppress lateral pseudopod formation. These results demonstrate that the lack of response by PMNs to opaque-phase cell-conditioned buffer is not a peculiarity of the PMNs of the major healthy donor.

The chemoattractant of C. albicans is a peptide.

To test whether the chemoattractant released by C. albicans was a peptide, we tested whether it was heat labile and sensitive to proteinase. Boiling and treatment with proteinase K removed chemotactic, but not chemokinetic, activity from buffer conditioned by white-phase cells (Table 3). These results suggest that the chemotactic factor released by white-phase C. albicans cells is a protein.

TABLE 3.

The chemoattractant appears to be a protein synthesized by white-phase, but not by opaque-phase, cells

Treatment	Preparationa	Strain	Phase(s)b	No. of cells	Mean instantaneous velocity (μm/min) ± SD	Chemokinetic stimulation (%)	Frequency of lateral pseudopod formation/10 min	CI	% Positive chemotaxis
Buffer	None			26	10.2 ± 3.4		5.8	+0.03c	47c
Boiling	Control	L26	Wh	13	15.5 ± 3.1	53	0.6	+0.50	90
	Test		Wh	16	20.0 ± 3.3	96	5.7	−0.01	49
	Test		Op	19	14.7 ± 2.1	44	6.0	+0.02	55
Proteinase K	Control	WO-1	Wh	22	15.8 ± 3.0	55	0.7	+0.27	82
	Test		Wh	20	14.4 ± 2.7	48	7.3	−0.07	55
Phase mixing	Control	137005	Wh		12.5 ± 1.8	25	0.6	+0.49	86
	Test		Wh+Op	19	9.9 ± 2.2	0	0.8	+0.22	90
Phase mixing	Control	19F	Wh	21	15.6 ± 1.9	53	0.6	+0.50	88
	Test		Wh+Op	15	11.2 ± 2.3	10	0.75	+0.30	89
Pepstatin	Control	WO-1	Wh	22	15.8 ± 3.0	55	0.7	+0.27	82
	Test		Wh	21	16.0 ± 3.2	57	0.6	+0.39	94
	Test		Op	20	19.4 ± 4.4	91	6.1	+0.04	52

^aControl was performed in the absence of treatment.

^bWh, white; Op, opaque; Wh+Op, white- and opaque-phase cells were mixed 50:50.

^cSee Table 1, footnote b.

To estimate the approximate size of the chemoattractant, we used a bioassay used previously to estimate the sizes of the two T-cell chemoattractants released by human immunodeficiency virus-infected T-cell syncytia (35, 36). This assay is based on Einstein's diffusion equation, which predicts that the time at which a spatial gradient achieves the steepness necessary to elicit a chemotactic response and the time it takes for the concentration necessary to elicit a chemokinetic response, are directly proportional to their molecular weight. Therefore, a reference plot can be generated in which the time it takes each known factor to elicit a maximum response is plotted as a function of the molecular weight of each respective factor (Fig. 5). The molecular weight of an unknown chemoattractant can then be estimated from its position along the plot based on time of maximum response, which can be estimated by plotting percent positive chemotaxis as a function of incubation time in the spatial gradient chamber (Fig. 6). In the case of the chemotactic factor released by C. albicans, the maximum response (peak of the plot) was attained at approximately 7 min (Fig. 6), which translates into a molecular mass of 10³ Da (Fig. 5).

FIG. 5.

Estimating the molecular mass of the chemoattractant release by C. albicans. Chemokinetic and chemotactic agents with known molecular masses were individually placed in the source well, buffer was placed in the sink well of the chamber in Fig. 1, and responding cells were placed at the bridge. Different fields of cells were continuously videorecorded through a 150-min period. Cells were then analyzed by computer-assisted methods for chemotaxis and/or chemokinesis. The maximum response time was the time of maximum chemotactic or chemokinetic response. The response times are the average of three experiments. The standard deviations of all knowns were less than 20% of the mean. The maximum response time was plotted as a function of molecular mass. Known molecules (small filled circles) and responding cells included the following: cyclic AMP (cAMP; 328 Da), Dictyostelium discoideum amoebae; fMLP (438 Da), human PMNs; RANTES (8 kDa), IL-16 (14 kDa), and gp120 (120 kDa); and peripheral blood T cells. The maximum response time of the attractant released by C. albicans was 7 min (peak of % positive chemotaxis plotted as a function of time in Fig. 6). The position along the reference plot at 7 min (large filled circle) was used to estimate molecular mass along the vertical axis (∼10³ Da). See references 35 and 36 for details of this bioassay.

FIG. 6.

The percent positive chemotaxis in the chamber in Fig. 1 was plotted as a function of time for PMNs responding to a gradient of chemoattractant released by C. albicans (3153A) cells, which were in the white phase. Measurements were made for each 5-min interval and were the means of three separate experiments. The standard deviations of all measurements were less than 15% of the mean. The peak was at approximately 7 min.

The absence of chemoattractant in opaque phase cell cultures is not due to proteinases.

The results described above suggest that the chemoattractant released by C. albicans is a low-molecular-weight protein. The white-opaque transition regulates a number of phase-specific genes (21, 29, 30, 41, 47), including two that encode proteinases, SAP1 and SAP2 (13, 30, 49). The possibility that the absence of chemoattractant in buffer conditioned by opaque phase cells may be the result of degradation due to secreted aspartyl proteinase activity was therefore considered. To test this possibility, white- and opaque-phase cells were mixed 50:50, and the mixture used to condition buffer. If buffer conditioned by opaque-phase cells lacks chemoattractant as a result of secreted proteinase degradation, then buffer conditioned by the mixture of white- and opaque-phase cells should also have no activity. Buffer conditioned by 50:50 mixtures of white- and opaque-phase cells of two strains (137005 and 19F) stimulated both chemotaxis and chemokinesis, resulted in a decrease in directional change, and suppressed lateral pseudopod formation, like buffer conditioned by white-phase cells alone (Table 3). Buffer conditioned by a mixture of 90% opaque- and 10% white-phase cells also stimulated chemotaxis (data not shown). In addition, we tested whether pepstatin, which inhibits the activity of the secreted aspartyl proteinases released by opaque-phase cells (29), resulted in the accumulation of chemoattractant when it was added to opaque-phase cells during the conditioning of buffer. It did not (Table 3), supporting the conclusion that opaque-phase cells simply do not release a chemotactic factor.

DISCUSSION

Human PMNs serve as the first line of defense in clearing microorganisms from sites of infection. PMNs are recruited from the vascular system through signals emanating from the site of infection. This can be accomplished in two ways. First, a signal from the microorganism can induce professional host signaling cells in their microenvironment to release chemoattractants that attract PMNs to the site of infection. Alternatively, the microorganism can directly release chemoattractants that attract PMNs. Recently, it was suggested that whereas chemotactic signals that attract PMNs over short distances may be in the form of spatial gradients, signals that attract PMNs over long distances may be in the form of waves of attractant relayed outwardly by host cells (11, 44). PMNs have been demonstrated to read both forms (spatial gradients and waves) of chemotactic signal (11).

It has long been known that individual PMNs undergo positive chemotaxis in vitro in spatial gradients of formyl-methyl-peptides (53, 54, 56), which are released naturally by bacteria. This chemotactic response has been demonstrated to be mediated through a specific fMLP receptor (26, 31) and to be a true chemotactic response through studies in which single-cell behavior was analyzed (53). Single-cell assays represent the only method for definitively distinguishing between chemotactic and chemokinetic stimulation (32, 50, 55, 57). The results from transmembrane assays suggested that a number of Candida species, as well as S. cerevisiae, released chemotactic factors (5, 7, 8). An elegant study by Edens et al. (8) demonstrated that these factors mediate a response through the fMLP receptor. In the present study, by using a single-cell chemotaxis assay, we confirmed the majority of these results, demonstrating that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata all release a bona fide PMN chemoattractant. We have demonstrated that as in the case of fMLP (11), these chemoattractants suppress the frequency of lateral pseudopod formation and turning. These attractants also induce a chemokinetic response. However, until the Candida spp. attractant is purified and tested, we cannot be certain that chemotactic and chemokinetic stimulation are due to a single factor. Our results, however, do not support the conclusion that S. cerevisiae releases a chemotactic factor. Our results do demonstrate that S. cerevisiae releases a chemokinetic factor, which may explain the positive results obtained with transmembrane assays (8).

Our characterization studies indicate, as did previous studies (8), that the C. albicans chemoattractant is a small protein. By passing conditioned medium through filters with known pore size, Edens et al. (8) estimated the molecular mass of the attractant to be between 0.5 and 1.0 kDa. Using a bioassay, we have similarly estimated the molecular mass to be ∼1.0 kDa. The attractant is sensitive to heat denaturation and proteinase K digestion, suggesting that it is a low-molecular-mass polypeptide, like fMLP. This is consistent with previous transmembrane studies demonstrating that the C. albicans chemoattractant mediates its response through the fMLP receptor (8). Experiments are now under way to identify the C. albicans chemoattractant and to test whether it alone mediates both the chemotactic and the chemokinetic responses. The possibility that there may be separate chemotactic and chemokinetic factors emerges from three observations. First, whereas buffer conditioned by opaque-phase cells is devoid of a chemotactic factor, it does consistently contain chemokinetic activity, albeit at lower levels than buffer conditioned by white-phase cells. Second, although buffer conditioned by mixtures of white- and opaque-phase cells contains high levels of chemoattractant activity, it exhibits reduced levels of chemokinetic activity. Finally, although proteinase K and high temperature remove chemotactic activity from conditioned buffer, these treatments do not remove chemokinetic activity.

The most intriguing result of our study, however, is the discovery that opaque-phase cells of C. albicans do not release a chemotactic agent. They do appear to release a chemokinetic agent, although the chemokinetic activity of buffer conditioned by opaque-phase cells, as noted, was lower than that of buffer conditioned by white-phase cells for all five tested MTL-homozygous strains. The absence of a chemoattractant represents one more unique phenotypic characteristic of opaque-phase cells (40, 41). Given the newly discovered role white-opaque switching plays in C. albicans mating (22, 23, 24, 28), the absence of attractant must be considered within this context, as well as pathogenesis. Although 97% of natural isolates of C. albicans are a/α, only 3% are a/a or α/α (22). a/α strains cannot undergo white-opaque switching. However, C. albicans can undergo the white-opaque transition after it has undergone homozygosis at the MTL locus to either a/a or α/α. The opaque-phase phenotype in turn represents the mating-competent phenotype of C. albicans (22, 28). Hence, the absence of a secreted chemoattractant may either be fortuitous and irrelevant to mating or it may play a role in the mating strategy. Possibly, the absence of a PMN chemoattractant makes opaque-phase cells less vulnerable to clearing by white blood cells, hence facilitating mating. Although mating occurs inside the host at low frequency (15, 27), it occurs at extremely high frequencies on skin (19), presumably because internal body temperature (37°C) causes conversion of the opaque-phase cell to the white-phase phenotype (33, 38, 41, 47). The temperature of skin (32°C) supports the opaque-phase phenotype. Although none of these observations provide a complete explanation for the selective absence of a chemoattractant in opaque-phase cell cultures, they do suggest that an answer may be found in the emerging relationships between MTL homozygosis, switching, mating, and pathogenesis (42, 46).