CELLULAR MECHANISMS OF PHAGOCYTOSIS OF CANDIDA BY M U RI N E MACROPHAGES

Rita K,Sposzta, Rosangela R Da Silva, L,~szl6 Mar6di, and Siamon Gordon

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

II. Pathogenicity of C alb icans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

III. Anticandidal Function of Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 IV. Cell Biology of Interaction between Maerophages and

C. a lb icans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 A. Binding and Entry Mechanisms of C. a lb icans Yeasts . . . . . . . . . . . . . . . . . . 320 B. Maturation of Candida Phagosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

C. Germ Tube Formation and Candida Escape from

Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 D. Entry Mechanisms of Filamentous Form of C. a lb icans . . . . . . . . . . . . . . . . 236

V. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

Advances in Cell and Molecular Biology of Membranes and Organelles Volume 6, pages 317-331. Copyright �9 1999 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0610-6

317

318 R. KAPOSZTA, R. P. Da SILVA, L. MARODI, and S. GORDON

I. I N T R O D U C T I O N

The clinical importance of Candida albicans infection has been increasing for the last decades, due paradoxically to the development of medical care. Despite sig- nificant efforts to develop reliable diagnostic methods and effective antifungal agents, disseminated candidiasis remains a diagnostic and therapeutic challenge (Beck-Sagu6 et al., 1993; Wenzel, 1995). Better understanding of host defence against Candida should lead to the development of more effective therapeutic strategies. Mononuclear phagocytes play an important role in combating candidal infection (Balish et al., 1993; Mar6di, 1997). Macrophages (M~) can ingest both serum-opsonised and unopsonised Candida, and kill the intracellular yeasts mostly by oxygen-dependent mechanisms. However, antifungal capacity of immunologically nonactivated MO is limited, and treatment of these cells with interferon (IFN)-y results in significant increase of Candida killing (Mar6di et al, 1993). Little is known about the molecular mechanisms of Candida binding to MO, and the cell biology of internalization of this fungus. The complex biology of fungal growth and dimorphism of Candida make it even more difficult to char- acterize. On the other hand, the ability of Candida to regulate its cellular morphol- ogy in response to environmental conditions and bind to MO directly without opsonins makes this pathogen a useful microbiological tool for the cell biologist to study antimicrobial mechanisms in MO. Furthermore, development of nonfila- mentous C. albicans mutants should help us to better understand the details of M~-Candida interaction.

Phagocytosis of C. albicans by immunologically nonactivated mouse peritoneal M~ is relatively rapid and involves early recruitment and fusion of late endosomes and lysosomes with the Candida-phagosomes (K~iposzta et al., 1999). Formation of phagolysosomes favors the growth of filamentous fungus, which is invasive and destroys the cellular membranes. We discuss here the interaction of MO with Can- dida, particularly in relation to the cell biology of this process.

II. P A T H O G E N I C I T Y OF C. ALBICANS

C. albicans is part of the normal microbial flora on body surfaces in humans. It grows as single ellipsoidal yeasts (blastoconidia or blastospores) under most laboratory culture conditions, but different environmental signals can induce development of pseudohyphae or hyphae (mycelial/filamentous forms) as shown schematically in Figure 1. These forms are long branched chains of cells, which remain attached after multiplication. In the hyphae, the cells are highly elongated and separated by perpendicular septal walls, while the pseudohyphae consist of yeastlike cells with constrictions at the interface of elongated blastoconidia. Germ tubes appear at the initial stage of sprouting, the blastoconidia-hyphae conversion (Shepherd et al., 1985). These forms are

Cellular Mechanisms of Phagocytosis of Candida by Murine Macrophages 319

7 A

B

12

Figure 1. Different morphological forms of C. albicans. (A) Budding blastoconidia. (B) Pseudohypha, a chain of elongated blastoconidia. (C) Hypha.

often seen in the same mycelium, which suggests a close interrelationship between them, and all can be observed in the infected tissues. The virulence of the different forms depends largely on the culture conditions used to iso- late the fungus. A recent report has suggested that the ability to switch between different growth forms might be relevant to virulence (Lo et al., 1997). Several factors are assumed to be protective for Candida, one of the most important being the thick glycoprotein-rich yeast wall. It provides rigid- ity, protects against osmotic stress, and its glucan and covalently linked chitin-glucan-rich components resist iysosomal enzymes. In addition, the mannoprotein component of the yeast wall also provides an adhesion func- tion, binding various glycoproteins on the host cell surface (Mar6di et al., 1991 a; Marquis et al., 1991 ; Calderone, 1993). C. albicans is more resistant to myeloperoxidase (MPO)-mediated killing than several less-pathogenic Candida species, a finding which cannot be attributed to a greater inactiva- tion by C. albicans of the MPO-toxic agents, hypochloride and monochloramine (Mar6di et al., 1991b). The role of secreted enzymes in vir- ulence of C. albicans is less well-understood. It can produce different induc- ible aspartyl proteases with pH optimum of 2.2-4.5, whose characteristics depend on the culture conditions. They have a broad substrate specificity, can digest immunoglobulins, collagen, albumin, and also activate the kal- iikrein-kinin system resulting in increased vascular permeability (Lerner et al., 1993). Some Candida strains produce hemolytic factors and phospholi- pases, but their pathogenic role is unexplored (Manns et al., 1994; Ibrahim et al., 1995).

320 R. KAPOSZTA, R. P. Da SILVA, L. MARODI, and S. GORDON

!11. ANTICANDIDAL FUNCTION OF MACROPHAGES

Different forms of candidiasis are often associated with various phagocytic disor- ders, such as NADPH-oxidase or MPO deficiency. These enzymes mediate and amplify the respiratory burst and oxidative killing mechanisms in phagocytes (Lehrer et al., 1969; Cohen et al., 1981; Mar6di et al., 1991 b, 1998). Internalized C. albicans within the phagocytes can be killed by oxygen-dependent toxic metab- olites (Mar6di et al., 1991 b). Hydrogen peroxide can also serve as a substrate for MPO to generate candidacidal hypochlorous acid and chloramines. Internalization of serum-opsonized Candida results in superoxide release by both monocytes and MO, whereas the unopsonised pathogen is less efficient and induces activation of a respiratory burst only in M~ (Mar6di et al., 1991 a). The role of nitric oxide (NO) and reactive nitrogen compounds in killing of Candida is more controversial (Van- quez-Torres et al., 1995; Fang, 1997). NO can be synthesized by NO synthases from arginine, molecular oxygen, and NADPH, and can interact with superoxide anion and other products of the oxygen dependent killing system, forming more toxic peroxynitrites and nitrogen-containing radicals. The possible candidacidal activity of reactive nitrogen compounds requires further investigation. The contri- bution of oxygen independent mechanisms to candidacidal activity of phagocytes is not well-explored. Lysosomal acid hydrolases such as t~-mannosidase, iysozyme, and apolactoferrin have been reported to be fungicidal for C. albicans (Arnold et al., 1980; Marquis et al., 1982, 1991 ). In vitro studies demonstrated that of all the cytokines that are known to activate M~, IFN-y is the most effective to increase the anticandidal activity. Granulocyte macrophage colony-stimulating factor (GM-CSF), M-CSF, interleukin (IL)-I, and IL-3 were also reported to upregulate antifungal capacity of MO (Wang et al., 1989). Recent studies sug- gested that the basis of the effect of IFN-y on the anticandidal activity is the upreg- ulation of the oxygen-dependent killing mechanisms in MO (Mar6di et al., 1991 b, 1994). However, nonoxidative killing mechanisms, and the role of maturation of Candida-phagosomes have not yet been analyzed in IFN-y activated MO. We have demonstrated the in vivo importance of IFN-y in resistance to invasive candidiasis using transgenic mice (K~iposzta et al., 1998).

IV. CELL BIOLOGY OF INTERACTION BETWEEN MACROPHAGES AND C. ALBICANS

A. Binding and Entry Mechanisms of C. albicans Yeasts

To study the cell biology of Ml?J-Candida interaction, we plated biogel-elic- ited routine peritoneal M~ onto glass coverslips in serum-free medium. We added serum-opsonised or unopsonised C. albicans to Mi~ at 4~ to allow cells to bind yeasts and to synchronize the uptake, then warmed the samples at

Cellular Mechanisms of Phagocytosis of Candida by Mufine Macrophages 3 21

Figure 2. C. albicans blastoconidia engulfed by peritoneal MID are associated with F-actin and talin. Cells were fixed and permeabilized at 2 minutes of phagocytosis and stained with FITC-phalloidin to visualize F-actin (A), or with antibody to talin (C). Left panels are a single representative section from the confocal microscopy, right panels are the corresponding phase-contrast image. The arrows point to the entering Candida in the confocal images. Scale: 0.07 lure/1 pixel; the width of each picture = 425 pixels.

37~ for 1-120 minutes before fixation in paraformaldehyde. After immun- ostaining with monoclonal antibodies specific for actin-associated proteins or lysosome-associated membrane proteins (Lamps) as primary antibodies and fluoresceinated secondary antibodies, we analyzed the samples by confocal immunofluorescent microscopy. We also used fluorescein isothiocyanate-phal- loidin to label actin. In some cases we labeled the lysosomes with Texas Red-dextran (70,000 Da) for confocal study, or with bovine serum albumin (BSA)-gold for transmission electron microscopy. Optimal binding and inges- tion of Candida by MO required opsonization by serum components including classical and alternative complement proteins, and Candida-specific antibodies (Mar6di et al., 1991; Casadevail, 1995). Unopsonised Candida can be internal- ized mostly, if not entirely, via the mannose receptor (Mar6di et al., 1991a, 1994; K~iposzta et al., 1999). Mannose receptor-mediated binding does not

322 R. KAPOSZTA, R. P. Da SILVA, L. MARODI, and S. GORDON

U

100

80

60

o 40

20

Cytochalasin D Nocodazole

I Phagocytosis of Candida

I~1 Phagocytosis of Beads

Figure 3. Effect of various inhibitors on internalization of C.albicans yeasts and mannosylated latex beads by peritoneal MO. MO were treated with 0.2 mM cytochalasin D, 10 mM nocodazole for 60 minutes, and allowed to ingest Candida blastoconidia or mannosylated latex beads for 60 minutes. Cells were fixed, permeabilized, and stained with FITC-phalloidin to determine the localization of particles using confocal microscopy. Cytochalasin D had the strongest effect on the uptake of both particles, also moderately reduced by nocodazole. Each bar represents the mean + SE of experiments performed in triplicate; 600 infected MO were counted for each coverslip.

result in significant increases in superoxide release or nitrite production by murine peritoneal MO. Our experiments showed that the entry mechanism is relatively rapid, 66.4 • 5.6% of bound yeast cells enter the MO within 10 min- utes, which increases up to 98.1 • 1.6% by 60 minutes. We visualized the out-

Cellular Mechanisms of Phagocytosis of Candida by Murine Macrophages 323

Figure 4. Time-course of vacuolar fusion with Candida- and bead-phagosomes. Peritoneal MO were allowed to ingest Candida blastoconidia for 5 (A-D), 15 (E-H), 30 (I-L) or 60 minutes (M-P). Cells were then fixed, permeabilized, and stained for Lamp, using specific monoclonal antibodies and a secondary antibody conjugated with FITC. The top two rows show the maturation kinetics of Candida phagosomes; panels A, E, I and M show phase-contrast images and panels B, F, J, and N show confocal sections of infected MO stained for Lamp. The bottom two rows show maturation of the bead-phagosomes; panels C, G, K, and O are phase-contrast images and panels D, H, L, and P are confocal sections of macrophage stained for Lamp. Fusion of Candida-phagosomes (arrows) with lysosomes and late endosomes had already started at 5 minutes of phagocytosis (B, F, J, and N), and at 60 minutes germ tube formation could be observed within the phagolysosomes (M and N) (arrow). However, fusion of lysosomes with the bead-phagosomes (arrows) could only be observed after 30 minutes of phagocytosis (L and P). Scale: 0.1 pm/1 pixel; the width of each picture = 300 pixels.

324 R. K,~POSZTA, R. P. Da SILVA, L. MARODI, and S. GORDON

Figure 5. Electron micrographs of Candida uptake by murine peritoneal M~D. Six nm gold-BSA had been internalized by MID overnight and chased into late endocytic structures: lysosomes and late endosomes for 2 hours (arrows). MID were allowed to bind C. albicans at 4~ for 15-30 minutes, then to ingest at 37~ for 2 minutes. At the beginning of phagocytosis, membrane ruffling could be observed with extension of pseudopodia projecting from MID to envelope the yeast. Lysosomes remained around the nucleus at that stage.

line of the MO using FITC-phalloidin, and determined phagocytosis of the yeast using confocal immunofluorescence.

Condensation of the actin filaments and talin, an actin-associated protein, around the entering yeasts could be detected by confocal microscopy (Figure 2) and also by electron microscopy, which strongly suggested that phagocytosis was the main mechanism involved in entry of Candida into MO. Furthermore, inhibition of actin assembly by treatment with 0.2 laM cytochalasin D and

Cellular Mechanisms of Phagocytosis of Candida by Murine Macrophages 325

Figure 6. Dimorphic transformation of C. albicans in MID phagolysosomes. MID were allowed to ingest unopsonised (A, B) and opsonised (C, D) Candida blastoconidia for 90 minutes, then fixed, permeabilized, and processed for Lamp immunostaining using FITC-conjugated secondary antibody. Candida blastoconidia generated germ tubes (arrows) in the phagolysosomes from 60 minutes of ingestion, distending the Lamp + phagolysosome membrane (C), then escaped from the MO and remained weakly Lamp + (A). The left panels show a single representative image of confocal microscopy, the right the corresponding phase-contrast image. Scale: 0.07 Iam/1 pixel; the width of each picture = 425 pixels.

depolymerization of microtubules in MO with 10 laM nocodazole resulted in similar inhibition of uptake of both Candida and latex beads of similar size (Figure 3). We could not find a significant difference in the kinetics of uptake of live and heat-inactivated Candida. These findings indicate that the mecha-

326 R. KAPOSZTA, R. P. Da SILVA, L. MARODI, and S. GORDON

nism involved in uptake of C. albicans by Mt3 is phagocytosis, which requires an intact actin cytoskeleton.

B. Maturation of Candida Phagosomes

We have demonstrated that the newly formed Candida-phagosomes recruit and fuse rapidly with late endosomes and lysosomes and soon acquire Lamp (K,Sposzta et al., 1999). We used mannosylated latex beads of similar size to Can- dida as control particles in kinetic studies. At 10 minutes of phagocytosis two thirds of Candida-phagosomes had already fused with lysosomes. However, man- nosylated bead-phagosomes became progressively Lamp + only after 30 minutes of phagocytosis. Both particles entered MO via the mannose receptor, and the dif- ference in kinetics of phagolysosome formation suggested that rapid recruitment of the lysosomes to Candida-phagosomes is due to the pathogen rather than the cell surface receptor involved in phagocytosis (Figure 4). Fusion of early endo- somes with Candida-phagosomes is also an early event after phagocytosis, as indi- cated by positive staining for transferrin receptor. Electron microscopic studies revealed that binding of Candida induced membrane ruffling in the MO, with extended pseudopodia enveloping the relatively large particles (Figure 5). A thick actin-rich phagocytic coat forms around the yeast, surrounded by early and late endocytic structures, and fusion of lysosomes with Candida-phagosomes started as soon as the actin coat disappeared.

C. Germ Tube Formation and Candida Escape From Macrophages

Under special culture conditions C. albicans yeast cells can grow germ tubes and form hyphae or pseudohyphae (Shepherd et al., 1985; Magee, 1997). We used 5% CO 2, and L-glutamine supplemented media at 37~ in adherent MO culture to induce germ tube formation. After 60 minutes of incubation both extracellular and ingested C albicans had started to form germ tubes, which elongated further and became more easily observed by 90 minutes (Figure 6). The rate of germ tube formation within MO was much lower than extracellularly, and neutralization of lysosomal pH or blocking of vacuolar acidification and fusion could reduce it fur- ther. Germ tubes developed intracellularly within the phagolysosomes, distended the Lamp + vacuolar membrane (Figure 6C), which ruptured and resulted in fungal escape from the MI3 (Figure 6A). This in vitro model of infection resulted in >95% survival of ingested fungi and destruction of Ml3.

D. Entry Mechanisms of Filamentous Form of C. albicans

The invasiveness of filamentous form of Candida can be studied using a variety of drugs that inhibit phagocytosis but do not alter the germ tube formation. In the presence of cytochalasin D, nocodazole, or staurosporine, ingestion of the yeasts

Cellular Mechanisms of Phagocytosis of Candida by Murine Macrophages 3 2 7

Figure 7. Germ tubes of C. albicans penetrate MO and attract Lamp + compartments. Peritoneal MO were treated with 0.2 pM cytochalasin D (A, B), an inhibitor of actin. polymerization, and 0. I IJM thapsigargin (C, D), an inhibitor of endoplasmic reticulum Ca 2+-ATP-ase, and were incubated with Candida blastoconidia for 60 minutes (A, B) or with preformed germ tubes for 15 minutes (C, D) (thapsigargin inhibited germ tube formation). Cells were fixed, permeabilized and processed for Lamp immunostaining, using FITC-labeled secondary antibody. When phagocytosis was blocked the yeast remained outside, but bound to the MO and by 60 minutes of incubation the Candida formed germ tubes that penetrated the cell and fused with Lamp + organelles. Cytochalasin D (A, B) did not inhibit vacuolar movement and fusion, but when cells were treated with thapsigargin the germ tube grew into the cytosol and failed to recruit lysosomes (C, D). The left panels are a single representative image of confocal microscopy, the right, the corresponding phase-contrast image. Arrows point to the penetrating germ tubes in the immunofluorescence images. Scale: 0.09 IJmll pixel; the width of each picture = 384 pixels.

328 R. KAPOSZTA, R. P. Da SILVA, L. MARODI, and S. GORDON

+ Inhlbltors

2 min

$-10 mln

60 mln

INVASION

F - Actin Talin

PHAGOEYTOSIS

Transferrln Receptor Lamps HRP. 30'-- Late Endosomes HRP/Dextran. 2h ~ Lysosomes

Lamps

90 rain

(

\

Lamps

Figure 8. Proposed model for internalization of C. albicans by MO cultured with or without phagocytosis inhibitors, and for maturation of the Candida-phagosome. In the presence of inhibitors, the yeast remains extracellular, bound to the MO surface and forms germ tubes penetrating the cell within a Lamp + compartment after 60 minutes of incubation. When phagocytosis is not inhibited, the MO-bound yeast induces membrane ruffling and an F-actin rich, talin + phagocytic coat appears around the particle in the submembranous area. Within 10 minutes the proportion of transferrin receptor + and Lamp + phagosomes increases, and they fuse with horseradish peroxidase (HRP +) vacuoles (chased for 30 minutes), corresponding to late endosomes, as well as HRP + and/or dextran + structures (chased for 2 hours), corresponding to lysosomes. In the phagolysosome, Candida forms germ tubes distending the Lamp + membrane, then escapes from the cell in a thin Lamp + coat.

Cellular Mechanisms of Phagocytosis of Candida by Murine Macrophages 329

was partially blocked, and nonphagocytosed MO-bound Candida cells form germ tubes that penetrate the phagocyte (Figure 7). The invading germ tubes also recruit late endocytic/lysosomal vesicles and soon become Lamp + membrane-bound structures, instead of growing freely in the cytosol. A similar phenotype can be observed when preformed germ tubes are incubated with MO. Disruption of the actin filaments and microtubular network does not prevent the vacuolar movement and fusion. However, in the presence of thapsigargin, a potent inhibitor of endo- plasmic reticulum Ca2+-ATP-ase, invading germ tubes failed to recruit Lamp + organelles (Figure 7).

V. DISCUSSION

The interactions between resident murine peritoneal MO and different forms of Candida--as summarized schematically in Figure 8--provide new insights into the mechanisms of infection and virulence of C. albicans. Uptake of Candida yeasts has the characteristics of phagocytosis, requires intact actin filaments, and a microtubular network. Phagocytic uptake of yeasts by peritoneal MO is rela- tively rapid and involves recruitment of late endocytic/lysosomal organelles. Vac- uolar fusion in MO promotes germ tube formation of Candida, although sprouting is generally suppressed in internalized yeasts. Germ tubes are more invasive, can escape from phagolysosomes, and penetrate intact MO, even when phagocytosis of yeasts is blocked by different inhibitors. Invading germ tubes also recruit Lamp + organelles, which requires active endoplasmic reticulum Ca2+-ATP-ase; however, an intact actin-cytoskeleton is not essential. Inhibition of the lysosomal H+-ATP-ase and of associated lysosomal fusion reduces germ tube formation of Candida within the phagolysosomes. These data suggest that rapid recruitment of late endocytic/lysosomal organelles and vacuolar fusion might be part of the sur- vival strategy and pathogenicity of both forms of C. albicans. This process does not require live organisms and is not specific for the plasma membrane receptors or the entry mechanisms utilized by different morphological stages of the fungus, since both unopsonised yeast bound to the mannose receptor, and serum-opso- nised Candida bound to Fc and complement receptors provoke similarly rapid recruitment of lysosomes. The internalization process, attraction, and rapid fusion with late endocytic/lysosomal organelles, and transmembrane signals induced by C. albicans require more investigation. Better understanding of interactions between C. albicans and MO should clarify pathogenic properties of this fungus and may help in developing more effective anticandidal therapeutic strategies.

ACKNOWLEDGMENTS

This work was supported by grants from the Medical Research Council (06BI), the National Science Foundation of Hungary (OTKA T 025 780), Ministry of Health (ETr 340/96), and

330 R. KAPOSZTA, R. P. Da SILVA, L. MARODI, and S. GORDON

DOTE Mecenatura (Mec-10/96). Facilities for confocai and electron microscopy are funded by an equipment grant from the Wellcome Trust. We thank Mike Hollinshead (Sir William Dunn School of Pathology, Oxford) for technical assistance.

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