Linkage of adhesion, morphogenesis, and virulence in Candida albicans

Linkage of adhesion, morphogenesis, and virulence in Canclida albicans MARGARET K. HOSTEI"rER MINNEAPOLIS, MINNESOTA

Abbreviations: b p = base pair; IgG = i m m u n o g l o b u l i n G; IgM = i m m u n o g l o b u l i n M; kbp = kilobase pair; RGD : arginine-gtycine-aspartic a c i d

W

~hether one confronts Candida in a case of congenital candidiasis, as the cause of fatal esophagitis in an HIV-infected patient, or as a bloodstream pathogen in a neutropenic host, Candida albicans is the organism most frequently responsible for fungal infection in the i m m u n o c o m p r o m i s e d patient. 1 Its predilection to penetrate mucosal surfaces or to invade the bloodstream depends on its ability to "read" the host's microenvironment and to execute 3 strategies of pathogenesis: (1) adhesion to the epithelium of the gastrointestinal or genitourinary tract, (2) replication at the mucosal barrier to colonize the host, and then, as host defenses fail, (3) invasion. Throughout this pathogenic cascade, morphology anticipates pathology. Adhesion involves the attachment of blastospores to an epithelial surface; in Fig 1 (left panel) we see them budding as they divide and colonize. 2 As host defenses fail and invasion supervenes, we see (Fig 1, right panel) the malignant transformation of yeast to germ tube with the extension of the cell b o d y that drives through the epithelial barrier. 3 This cunning capacity to change shape enables a supremely well-equipped microorganism to read and respond to

From the Department of Pediatrics, Universityof Minnesota. Supported by National Institutes of Health grantsAI25827,HD00850, and HD33692 and by an endowment from the Minnesota American Legion and Women'sAuxiliaryHeart Research Foundation. Presented at the Seventieth Annual Meeting of the Central Society for Clinical Research, September 26, 1997, Chicago, IL. Portions of this article also appear in a review article in Pediatric Research 1996;39:569-73. Submitted for publicationApril 1, 1998; accepted June 3, 1998. Reprint requests: Margaret K. Hostetter, MD, Professor of Pediatrics, Yale University School of Medicine, Director Yale Child Health Research Center, 464 CongressAvenue,New Haven, CT 06520-8081. J Lab Clin Med 1998;132:258-63. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5/1/92174 258

its environment and to press its advantage against the enfeebled host. Is it possible that these processes are linked in C. albicans--that is, that an adhesin is also implicated in morphogenesis and virulence? Current candidal adhesins-and the list is enlarging day by d a y - - i n c l u d e a [31,2 tetramannose epitope defined by Li and Cutler 4 and a fimbrial adhesin characterized by Irvin, 5 A gene encoding a putative surface protein of 30 kd involved in adhesion to plastic has been cloned by Barki et al. 6 Putative receptors for laminin, fibronectin, or other proteins of the extracellular matrix would be superb candidates to link these two processes, save that fibronectin- and laminin-binding proteins do not appear to be implicated in morphogenesis (Table 1).7,8 Among genes implicated in morphogenesis, we have the candidal homologs of the M A P K pathway in S. cerevisiae: STE20, STE7, and STE12. 9-12 None of these, however, encodes a surface protein. PHR1, cloned by William Fonzi, encodes a cell-wall protein that is required for hyphal transformation at pH 7.5; aphrl null mutant is reduced in virulence in a murine model. 13-14 This gene product would be a good candidate for an adhesin, but its role in this process has not been tested. Because no single gene appears on both lists, we therefore focused on another surface protein, known to be expressed in Candida blastospores and hyphae, that displays some intriguing similarities with the vertebrate integrins a M and ~X, the leukocyte adhesion glycoproteins. 15 Our working hypothesis was that there was an integrin-like protein in C. albicans, and we used the wellcharacterized structure of leukocyte integrin heterodimers--c~ and l] subunits--as our paradigm. The leukocyte adhesion glycoproteins, also known as the ~2 integrins, are expressed as heterodimeric transmembrane proteins on a wide variety of cells including neutrophils, monocytes, and macrophages. In vertebrates, integrins allow cells to adhere and to change shape; for

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Fig 1. Adhesion of C. albicans blastosporesto corneal epithelium (leflpanel). (FromRay TL, Payne CD, Infect Immun 1988;56:1942-9. By permission.)Penetration of C. albicans germ tubes through epithelial barrier (right panel). (FromWilbornWH, Montes LF, JAMA 1980;244:2294-7. By permission.)

Table I. C a n d i d a t e m o l e c u l e s Adhesion

Morphogenesis

[31,2 tetramannose Fimbrial adhesion 30 kd antigen Receptors for laminin and fibronectin

STE20=CST20 STE7=HST7 STE12=CPH1 CPH1/EFG1 PHR1

example, in the neutrophil the expression of a M is critical for activation and adherence to the endothelial surface and for the morphologic changes associated with diapedesis.16 Two of these proteins--aM, also known as Mac-l, C D l l b , or CR3; and c~X, also known as p150,95, C D l l c , or CR4--share homology with antigens on the C. albicans surface. 15 In the extracellular portion of these proteins, just distal to the amino-terminus, czM and c~X display a ligand-binding domain for the recognition of extracellular matrix proteins. Many but not all of these ligands contain the sequence RGD. Also in the extracellular domain are 3 divalent cation binding sites required for integrin-mediated adhesion in vertebrates, and these conform to the EF-hand motif. At the carboxy-terminus there is a hydrophobic transmembrane domain and a signature sequence, KVGFFK, that demarcates the transition from the membrane to the cytoplasmic tail, which contains a single tyrosine residue. 17 The first evidence for integrin-like proteins in C. albicans came from Heidenreich and Dierich 18 and subse-

quently from Edwards. 19 Our laboratory used monoclonal antibodies against the integrins czM and ~X in flow cytometry to quantitate surface expression of this integrin-like protein with several dozen yeast isolates. C. albicans exhibited the greatest fluorescence; fluorescence was reduced in C. tropicalis, C. parapsiIosis, C. krusei, and C. glabrata; and fluorescence was negligible in S. cerevisiae. 15 This hierarchy of surface expression mirrors the frequency of isolation of these species in immunocompromised hosts. Thus a surface determinant on C. albicans is recognized by monoclonal antibodies against vertebrate integrin ~-subunits. Within the constraints of available monoclonal antibodies, we have found no evidence for a [32-subunit in C. albicans. One might also predict that expression of this integrin-like protein on C. albicans should permit this yeast to masquerade as a leukocyte and thereby elude phagocytosis. Indeed, that is just what occurs. After preincubation with glucose to increase surface expression of the integrin-like protein on C. albicans, phagocytosis of yeast cells by polymorphonuclear leukocytes was significantly decreased. 20 We next used these same monoclonal antibodies to identify the candidal protein by affinity purification; a single band of 185 kd eluted under nonreducing conditions thereby approximating the molecular weight of a M (165 kd) and otX (150 kd). If indeed this 185 kd protein were functionally akin to czM and c~X, it must meet 3 criteria: (1) it must play a role in adhesion; (2) it must recognize the complement fragment iC3b as lig-

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and; and (3) it must recognize RGD-containing peptides. Dr. Catherine Bendel quantitated the binding of radio labeled yeast cells to monolayers of human cervical epithelium. 2] As predicted, C, albicans adhered well, while other yeast species, especially S. cerevisiae, were virtually nonadherent. Next, Dr. Bendel made the surprising observation that both IgG and IgM monoclonal antibodies against the vertebrate integrins c~M and o~X significantly inhibited epithelial adhesion. 21 What substrate might this "candidal integrin" recognize on human epithelial cells? Studies from our laboratory identified two possibilities: the C3 fragment iC3b and fibronectin, both of which contain RGD sequences. Dr. Bendel then demonstrated that candidal adhesion to epithelium could be inhibited both by purified iC3b and by RGD peptides derived from this protein but not from fibronectin. For example, RGD peptides derived from the amino acid sequence of iC3b but less than 9 amino acids in length, or a series of RGD peptides from fibronectin (7 to 23 amino acids), failed to block adhesion of C. albicans to epithelial cells. An RGK peptide was similarly ineffective. In contrast, peptides of 9, 10, or 15 amino acids encompassing the RGD sequence in iC3b successfully blocked the adhesion of C. albicans to human epithelium. In these latter experiments, adhesion was not completely obliterated--thus testifying to the fact that a smart pathogen will have a number of ways to hold on--but reasonably small concentrations of 15-mer RGD peptides (1.0 mg/mL) did indeed reduce adhesion by almost 50%. One can model this adhesive interaction with the "tapdancing yeasts" (Fig 2), in which C. albicans sports its integrin-like protein like an antenna. Below is the yeast cell epithelium, busily synthesizing the hemispheric iC3b molecules that stud its surface, each containing an RGD sequence. Like many of the c~-subunits among the vertebrate integrins, the integrin-like protein in C. albicans recognizes the RGD site and specific flanking residues in iC3b, and adhesion occurs. Species such as C. parapsilosis, C. glabrata, C. krusei, or S. cerevisiae fail to express an integrin-like protein and therefore lack the "adhesion advantage" that it confers. To identify the gene, we were initially stymied because the amino terminus of the purified protein was blocked, and internal peptides did n o t yield unambiguous sequence. From a cDNA clone encoding human c~M, Nianjun Tao used a 314 bp fragment spanning the transmembrane domain as a probe to screen an EcoRI digest of C. albicans and found strong hybridization with a 3.5 kbp fragment. She then made a restricted library of C. albicans EcoRI fragments from 3.0 to 3.8 kbp. The 5 clones isolated by sib selection were then further refined by hybridization with a degenerate oligonucleotide encoding the sequence KVGFFK, which marks the divi-

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Fig 2. In this diagram schematizing 1 type of adhesive mechanism, Intlp extends from the C. albicans surface like an antenna and recognizes the RGD tripeptide and flanking residues in iC3b molecules (hemispheres) synthesized and displayed by epithelial cells. (From Pediatr Res 1994;36:692-98.)

sion between transmembrane domain and cytoplasmic tail in virtually all c~-integrin subunits.]7, 22 We reasoned that many C. albicans proteins might have a transmembrahe domain but that only integrin-like proteins should have this C-terminal sequence. The net result was 1 clone, INT1, that spanned an open reading frame of 4992 bp, sufficient to encode a protein of 1664 amino acids with a predicted molecular weight of 188 kd. The hybridizing sequence encoding KKRFFK was found just proximal to the C-terminus, thereby confirming the identification of the correct clone. Other sites of interest included (1) a ligand-binding domain that is related both to the A domain in ~ and c~X23 and to the fibrinogen-binding domain in Staphylococcus aureus clumping factor, 24 (2) 2 divalent cation binding sites that conform to the EF hand motif, (3) a hydrophobic C-terminal sequence of 25 amino acids, and (4) a presumptive cytoplasmic tail with conserved tyrosine residue. And intriguingly, in contrast to vertebrate integrins, the Candida protein exhibited its own RGD site at amino acids 1149 through 1151. 22 We named this gene INT1 because we had isolated it with integrin probes, but of course, the overall amino acid homology of only 18% meant that there was no close structural relationship with vertebrate integrins. However, if we turn instead to functional relationships, then I hope to show how I n t l p integrates adhesion, morphogenesis, and virulence. First we made polyclonal antibodies to 20-mer peptides spanning the second divalent cation binding site and the RGD domain and used these to localize the protein. There was intense surface fluorescence on blas-

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CAF2

CAG3

CAG5

MILK-TWEEN Fig 3. Intlp-dependent effects on filamentous growth in wild-type C. albicans (CAF2), intl/intl homozygous null mutant (CAG3), and INT1 re-integrant (CAG5). (From Gale C, Bendel C, McClellan M, Hauser M, Becker J, BermmaJ, et al. Science 1998;279:1355-8. By permission.)

tospores, germ tubes, and hyphae, with the suggestion of preferential fluorescence as the cell body elongated. This was our first inkling that Intlp might be involved in morphogenesis. Incubation with preimmune IgG did not lead to fluorescence. In addition, these studies confirmed that Intlp was a surface molecule in C. albicans and that both the divalent cation binding site and the RGD domain were extracellular. Southern blotting under high stringency detected INT1 only in C. albicans, not in other Candida species or in S. cerevisiae. However, because there are so many putative adhesins in C. albicans, we thought it better to express the candidal gene in S. cerevisiae and try to make a nonadherent yeast stick. Dr. Cheryl Gale therefore inserted the open reading frame of INT1 behind the GAL1/GALIO promoter of S. cerevisiae. Her plasmid, pCG01, could then be used to transform Saccharomyces, and expression of the Candida gene would be induced with 2% galactose. Northern blots showed the expected message of 5.5 kb at 6 and 24 hours after galactose induction in S. cerevisiae transformants bearing the Candida gene but no message in the transformants bearing vector alone. 22 However, when Dr. G a l e took a look under the microscope at her transformants, we were astounded. Dr. Gale's parent strain of Saccharomyces transformed with plasmid a l o n e - - n o candidal insert--exhibited the customary spherical shape with an occasional budding yeast. Remarkably, however, Saccharomyces transformants expressing the candidal gene after 6 hours' growth in galactose sprouted elongated projections like germ tubes. 22 (We have nicknamed them I~OSeS.)

We performed a number of control experiments to ensure that the expression of INT1 was the cause of this morphologic change. If one cured Saccharomyces of the plasmid, there were no noses. If one grew Saccharomyces in glucose, no noses sprouted. Or if Saccharomyces was grown in glucose in a noninducing concentration of galactose (0.02%), there were no noses.

If one expressed a gene from Chlamydomonas instead of INT1 behind the same promoter, no noses appeared. Thus from these studies we can say conclusively that expression of the Candida INT1 gene in S. cerevisiae led to the induction of germ tubes. 22 Subsequently we have expressed INT1 in haploid and diploid Saccharomyces in several strain backgrounds, even with its candidal promoter, and have always elicited germ tubes. Western blotting confirmed that the candidal protein Intlp was present as early as 4 hours into the experiment, before the development of germ tubes at 6 hours. Moreover, immunoprecipitation of Intlp from S. cerevisiae after biotinylation of surface proteins confirmed its surface location in S. cerevisiae. 25 A nuclear protein, Raplp, was not detected with this method, thereby indicating specificity for surface proteins. Dr. Bendel therefore used these S. cerevisiae transformants in her adhesion assay to confirm that Intlp mediated adhesion to monolayers of human cervical epithelial cells. 25 S. cerevisiae containing vector alone failed to adhere. However, expression of the candidal protein I n t l p in S. cerevisiae conferred a substantial degree of adhesion (30%), even when compared with naturally adherent C. albicans (45%). The failure of adhesion of a S. Cerevisiae cdcl2 mutant, which makes multiple elongated buds, confirmed that INT1 expression, not morphologic change per se, was responsible for these adhesive capabilitiesY Inhibition of adhesion by Intlp-specific antibodies was also demonstrated. Although nonimmune rabbit IgG failed to inhibit this process, adhesion of C. albicans and of Intlp-expressing S. cerevisiae was substantially inhibited by antibodies specific to the second divalent cation binding site or to the RGD domain in Intlp. 25 These studies confirmed that the expression of INT1 in S. cerevisiae endows a nonadherent yeast with the ability to adhere and enables morphologic switching, thereby linking adhesion and morphogenesis in this yeast. But now, back to Candida albicans. Intriguing

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though it was to have made Saccharomyces adherent and "nosey," we still needed to address whether INT1 links adhesion and morphogenesis in C. albicans. Using techniques pioneered by William Fonzi, 26 Dr. Gale next undertook to disrupt both INT1 alleles in C. albicans, a naturally diploid species, and used Spider medium 9-10 and milk/Tween agar to search for effects on hyphal production. Fig 3 shows representative colonies on milk/Tween agar: (1) the parent strain with lengthy hyphae; (2) the homozygous disruption mutant, with glistening bald cells; and (3) the sinister tendrils of the re-integrant. 25 The same phenotypes were observed on Spider medium: the Medusa-like hyphae of the parent strain and the shining domes of the h o m o z y g o u s disruption mutants. Even the i n t l / i n t i h o m o z y g o u s disruption mutant made hyphae in 20% serum and in RPMI, as do ste12 mutants. 12 Thus our present hypothesis is that I n t l p is not required for hyphal transformation but rather serves as an upstream sensor or antenna that triggers morphologic switching. Using these mutants, Dr. Bendel then demonstrated that successive deletion of INT1 alleles reduced candidal adhesion. 25 As predicted, adhesion of the wild-type strain with both copies of INT1 intact was highest, and adhesion was significantly reduced in the heterozygote, the h o m o z y g o u s disruption mutant, and the re-integrant. Because the homozygous disruption mutant still adhered, we know that there are other adhesins in C. albicans, but Intlp contributes about 30% of the adhesive capabilities. Nonspecific antibodies such as rabbit IgG did not affect adhesion for any candidal strains. However, when I n t l p was expressed on the candidal surface, antibodies to I n t l p inhibited adhesion in the wild type strain, in both heterozygotes, but not in the homozygous disruption mutant, which fails to express Intlp. These studies demonstrated that disruption of INT1 reduces adhesion and suppresses morphogenesis in C. albicans, thereby linking these processes in the pathogenic species as well as in S. cerevisiae. 25 The ultimate question, of course, relates to virulence. Although INT1 links adhesion and morphogenesis in vitro, does the gene product contribute to pathogenicity in vivo? With the collaboration of Jeff Becker of the University of Tennessee, 10 animals each were injected with 105 cells of the parent strain (both INT1 alleles intact), the homozygous disruption mutant (both INT1 alleles disrupted), and a heterozygous re-integrant. All animals injected with the parent strain died by day 8. Ninety percent of the animals injected with the homozygous disruption mutant lived. Animals injected with the re-integrant--just 1 INT1 g e n e - - h a d intermediate mortality at 60%. Thus, INT1 not only links adhesion and morphogenesis, but its absence makes C. albicans virtually avirulent.

In conclusion, how does I n t l p participate in the 3 steps of candidal pathogenesis--adhesion, colonization, and invasion? Mutants with reduced or absent expression of Intlp have reduced adhesion, and antibodies to Intlp inhibit this process in wild-type strains. As for colonization, we are now testing whether Intlp recognizes its own internal RGD site as readily as it does the RGD site on iC3b synthesized by the epithelial cell, thereby allowing yeast cells to attach to each other and to colonize the host. This is eminently testable with RGD peptides and INT1 mutants deleted for the RGD domain now in hand. Last, of course, Dr. Gale's null mutant tells us that the disruption of INT1 alleles suppresses the formation of germ tubes and hyphae. In each case, the protein encoded by INT1 is the antenna, the extracellular hook, that initiates the organism's response to environmental conditions. Fundamental insights into candidal pathogenesis--and the new treatments that they engend e r - a r e waiting for us if we simply follow our noses. REFERENCES

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