Candida albicans adhesins: Biochemical aspects and virulence

The recognition of host cells by the pathogenic yeast, Candida albicans, is probably an essential step in the pathogenesis of disease development. The interaction of yeast and hyphal mannoproteins with host cell receptors has been studied by a number of laboratories. C. albicans recognizes a variety of host cells as well as host cell extracellular matrix proteins. This observation is not unexpected given the number of sites within and on the body which can be colonized and infected by the organism. Indeed, it would appear that C. albicans has evolved a number of ways in which it recognizes the host. This statement is made with the qualification that the organism uses other processes to infect, such as morphogenesis, phenotypic switching and the production of invasive enzymes, including secreted aspartyl proteases and phosholipases. Recognition of epithelial cells is accomplished through cell surface mannoproteins (adhesins) which bind to carbohydrate-containing receptors. The number of such mannoproteins is not known; pro adhesins exist. The organism also binds to keratinocytes, endothelial cells and matrix proteins, such as fibronectin, laminin, collagen and entactin, and, as such, appears to have a integrin-like cell surface adhesin. In most cases, the adhesin for each of these host proteins is a mannoprotein. The biochemistry of the candidal adhesins has been extensively studied. However, molecular analyses of the encoding genes is only now being studied. Thus, until clean, genetic analyses are complete and strains lacking an adhesin function are constructed, a direct role for the adhesins in pathogenesis can only be inferred. At present, spontaneous, non-adhering strains of the organism have been described which are avirulent in animal models of candidiasis. However, these data only suggest a role for adherence; future studies should be directed towards resolving questions about the role of these proteins in pathogenesis.     

Entamoeba histolytica Contains a β1 Integrin-like Molecule Similar to Fibronectin Receptors from Eukaryotic Cells

Entamoeba histolytica trophozoites do interact with extracellular matrix components in order to invade and finally destroy tissue. An important step in this interaction involves the binding of a 140-kDa membrane protein that binds to fibronectin. The similarity of this amoebic receptor to fibronectin receptors from higher eukaryotic cells was defined by indirect immunofluorescence, western blot and immunohistochemistry. using polyclonal monospecific antibodies raised against the amoebic protein. These results suggest that lower eukaryotic cells have and use a β1 integrin-like molecule as well as mechanisms similar to those present in higher eukaryotic cells during interaction with extracellular matrix components.     

Entamoeba histolytica: monoclonal antibody against the beta1 integrin-like molecule (140 kDa) inhibits cell adhesion to extracellular matrix components

We describe a monoclonal antibody (3C10) against the beta1 integrin-like molecule which immunoprecipitates two polypeptides of 140 and 155 kDa from detergent-soluble extract of Entamoeba histolytica. The 140-kDa polypeptide has been described as a beta subunit of the amoebic fibronectin receptor as it is recognized by an anti-integrin beta1 (human) monoclonal antibody in immunoblot assay. The receptor molecules were localized with the 3C10 monoclonal antibody in intracellular and surface membranes of E. histolytica trophozoites by immunofluorescence and immunogold labeling methods. Significant inhibitions of cell adhesion on extracellular matrix proteins such as fibronectin (56%) (P < 0.001) and collagen (50%) (P < 0.001) and partial inhibition on laminin (23%) (P > 0.1) were achieved by the monoclonal antibody.     

Pacific oyster hemocytes undergo apoptosis following cell-adhesion mediated by integrin-like molecules

Previously, we demonstrated that in the Pacific oyster (Crassostrea gigas, a bivalve mollusc) apoptosis could be induced in hemocytes by treatment with Arg-Gly-Asp (RGD) peptides which are known to function as an integrin ligand. However, it is unclear where the RGD peptides are binding to the C. gigas hemocytes, or what mechanism or molecules are involved, e.g., integrin-ligand interactions. Therefore, this study was undertaken to investigate the binding interactions in C. gigas hemocytes. Initially, to confirm the presence of RGD-recognizing integrin-like molecule(s) on the hemocytes, we assessed the enhancement of spreading ability, and found that spreading ability was enhanced by immobilized human fibronectin, a fibronectin fragment containing the RGD motif, and C. gigas plasma in the presence of divalent cations. Interestingly, viability of the spreading hemocytes dramatically decreased 24 h later and DNA fragmentation with oligonucleosomal laddering of 180-200 bp in length was detected in the dead hemocytes by electrophoresis and TUNEL assay. These results indicated that hemocyte adhesion mediated by integrin-like molecules triggered apoptosis and suggested that integrin-activation contributes to the induction of apoptosis. This is the first report showing the possibility of an integrin functioning in the induction of apoptosis in invertebrate hemocytes.     

Fibronectin and heparin binding domains of latent TGF-beta binding protein (LTBP)-4 mediate matrix targeting and cell adhesion

Latent transforming growth factor (TGF)-β binding proteins are extracellular matrix (ECM) proteins involved in the regulation of TGF-β sequestration and activation. In this study, we have identified binding domains in LTBP-4, which mediate matrix targeting and cell adhesion. LTBP-4 was found to possess heparin binding activity, especially in its N-terminal region. The C-terminal domain of LTBP-4 supported fibroblast adhesion, a property reduced by soluble heparin. In addition, we found that LTBP-4 binds directly to fibronectin (FN), which was indispensable for the matrix assembly of LTBP-4. The FN binding sites were also located in the N-terminal region. Interestingly, heparin was able to reduce the binding of LTBP-4 to FN. In fibroblast cultures, LTBP-4 colocalized first with FN and subsequently with fibrillin-1, pointing to a role for FN in the early assembly of LTBP-4. In FN −/− fibroblasts, LTBP-mediated ECM targeting was disturbed, resulting in increased TGF-β activity. These results revealed new molecular interactions which are evidently important for the ECM targeting, but which also are evidence of novel functions for LTBP-4 as an adhesion molecule.     

Involvement of extracellular matrix and integrin-like proteins on conidial adhesion and appressorium differentiation in Magnaporthe oryzae

Conidial adhesion and appressorium formation of Magnaporthe oryzae on the rice surface are important early events in the infection process. As an initiative step to understand the mechanisms underlying these cellular processes at a biochemical level, the effect of a human fibronectin antibody (HFA) and RGD peptides on conidial adhesion and appressorium formation was evaluated. HFA inhibited conidial adhesion and appressorium formation in a dosage-dependent manner. RGD peptides also inhibited these cellular events. Conidial adhesion and appressorium formation inhibited by RGD peptides were restored by chemicals involved in the cyclic AMP-dependent signaling pathway. These results suggest that extracellular matrix proteins might be involved in conidial adhesion and appressorium formation through integrin-like receptor mediation and modulation of cAMP-dependent signaling in the cells.     

Adherence of Candida albicans to components of the subendothelial extracellular matrix

Candida albicans yeasts adhered avidly to extracellular matrix (ECM) proteins, type IV collagen, laminin, and fibronectin immobilized on plastic. Type IV collagen showed an increase of adherence of 400% above control values; laminin, 300%; and fibronectin, 150%. In addition, all three (in quantities of 0.02-200 micrograms/well of a culture tray) bound yeasts in a dose-response fashion. Adherence was inhibited when the proteins were preincubated with specific antibody, except with type IV collagen. Soluble laminin or fibronectin inhibited yeast adherence to the same proteins by 36 and 94%, respectively. Soluble fibronectin bound to the yeast surface and in so doing inhibited subsequent yeast adherence to fibronectin by 66%. By comparison, Candida albicans yeasts adhered in smaller numbers to glycosaminoglycans (GAGs). Keratan sulfate, hyaluronic acid, chondroitin sulfate, Type B, and heparin actually decreased yeast adherence compared to control from 10% to 25%.     

Integrin-like substrate adhesion in RTG-2 cells, a fibroblastic cell line derived from rainbow trout

Fluorometric cell attachment assays together with competitive inhibitors of adhesion were used to probe for the presence of integrins, a diverse family of heterodimeric cell-surface glycoproteins involved in cell-cell and cell-extracellular matrix adhesion, in the fibroblastic rainbow trout cell line, RTG-2. The adhesive properties of this cell line were evaluated. RTG-2 cells adhered poorly to TC plastic in the absence of serum but as little as 2.5% fetal bovine serum allowed over 75% of the cells to attach after 5 h. Surfaces coated with the extracellular matrix proteins collagen I, collagen IV, fibrin, fibrinogen, or fibronectin were able to support attachment of RTG-2 cells. Adhesion of RTG-2 cells to fibronectin varied linearly with fibronectin coating densities in the range 0 to 65 ng/mm(2). Oligopeptides containing the sequence Arg-Gly-Asp (RGD) caused dose-dependent inhibition of adhesion to microtiter plates coated with fibrin, fibrinogen, and fibronectin, whereas attachment to collagen I and collagen IV was less severely affected. In all cases, peptides containing Arg-Gly-Glu (RGE) or Asp-Gly-Arg (DGR) sequences caused no reduction of cell attachment. Since many integrins mediate adhesion by binding to RGD sequences in their target ligands, these results suggest the presence of integrin-like adhesion molecules on the surface of RTG-2 cells.     

Adhesion and surface-aggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber

Adhesive interactions between Candida albicans and oral bacteria are generally thought to play a crucial role in the microbial colonization of denture acrylic, which may lead to denture stomatitis. This study investigated the influence of saliva on the adhesive interactions between C. albicans and Streptococcus sanguis or Actinomyces naeslundii on denture acrylic. First, bacteria were allowed to adhere to the acrylic surface from a flowing suspension, and subsequently yeasts were flowed over the acrylic surface. The organisms were assayed in the presence or absence of human whole saliva. All experiments were carried out in a parallel plate flow chamber and enumeration was done in situ with an image analysis system. In the absence of adhering bacteria, adhesion of C. albicans from buffer was more extensive than from saliva. However, in the presence of adhering bacteria, yeast adhesion from saliva was increased with respect to adhesion of yeasts from buffer, indicating that specific salivary components constitute a bridge between bacteria and yeasts. In all cases, yeast aggregates consisting of 3 to 5 yeast cells were observed adhering to the surface. A surface physico-chemical analysis of the microbial cell surfaces prior to and after bathing the microorganisms in saliva, suggests that this bridging is mediated by acid-base interactions since all strains show a major increase in electron-donating surface free energy parameters upon bathing in saliva, with no change in their zeta potentials. The surface physico-chemical analysis furthermore suggests that S. sanguis and A. naeslundii may use a different mechanism for adhesive interactions with C. albicans in saliva.     

Perlecan inhibits smooth muscle cell adhesion to fibronectin: role of heparan sulfate

Smooth muscle cell migration, proliferation, and deposition of extracellular matrix are key events in atherogenesis and restenosis development. To explore the mechanisms that regulate smooth muscle cell function, we have investigated whether perlecan, a basement membrane heparan sulfate proteoglycan, modulates interaction between smooth muscle cells and other matrix components. A combined substrate of fibronectin and perlecan showed a reduced adhesion of rat aortic smooth muscle cells by 70-90% in comparison to fibronectin alone. In contrast, perlecan did not interfere with cell adhesion to laminin. Heparinase treated perlecan lost 60% of its anti-adhesive effect. Furthermore, heparan sulfate as well as heparin reduced smooth muscle cell adhesion when combined with fibronectin whereas neither hyaluronan nor chondroitin sulfate had any anti-adhesive effects. Addition of heparin as a second coating to a preformed fibronectin matrix did not affect cell adhesion. Cell adhesion to the 105- and 120 kDa cell-binding fragments of fibronectin, lacking the main heparin-binding domains, was also inhibited by heparin. In addition, co-coating of fibronectin and (3)H-heparin showed that heparin was not even incorporated in the substrate. Morphologically, smooth muscle cells adhering to a substrate prepared by co-coating of fibronectin and perlecan or heparin were small, rounded, lacked focal contacts, and showed poorly developed stress fibers of actin. The results show that the heparan sulfate chains of perlecan lead to altered interactions between smooth muscle cells and fibronectin, possibly due to conformational changes in the fibronectin molecule. Such interactions may influence smooth muscle cell function in atherogenesis and vascular repair processes.     

The ultrastructure of Candida albicans infections

Scrapings of Candida albicans plaques from the tongue and buccal mucosa of patients with oral candidiasis were examined by electron microscopy. In addition, urine sediment from patients with infection of their catheterized urinary tracts was similarly examined. Three types of C. albicans-oral epithelial cell interactions were noted: a loose adherence apparently mediated by a ruthenium red positive matrix, a "tight" adherence where no space could be seen between the host and yeast cell. and invasion of host cells by yeast hyphal elements. Adhesion of Candida blastospores to hyphal elements and adhesion of bacteria to Candida cells was also frequently observed. Urine sediments from patients with mixed bacteria-yeast infections demonstrated adhesion of the bacteria to the yeast cells. This phenomenon was also demonstrated in in vitro experiments and fibrous ruthenium red material invariably occupied the zone of adhesion. Phagocytosis of yeast by polymorphonuclear leukocytes was found in urinary, but not in oral. candidiasis. Our in vivo and in vitro observations indicate that a ruthenium red positive matrix covers the surfaces involved in the yeast to yeast, yeast to host, and yeast to bacteria adhesion.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii , we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule. The SfbI repeat region exclusively bound to the 30 kDa N-terminal fragment of fibronectin, whereas the SfbI spacer region exclusively bound to the 45 kDa collagen-binding fragment of fibronectin. In the case of native surface-expressed SfbI protein, an induced fit mode of bacteria–fibronectin interaction was identified. We demonstrate that binding of the 30 kDa fibronectin fragment to the repeat region of SfbI protein co-operatively activates the adjacent SfbI spacer domain to bind the 45 kDa fibronectin fragment. The biological consequence arising from this novel mode of fibronectin targeting was analysed in eukaryotic cell invasion assays. The repeat region of SfbI protein is mediating adherence and constitutes a prerequisite for subsequent invasion, whereas the SfbI spacer domain efficiently triggers the invasion process of streptococci into the eukaryotic cell. Thus, we were able to dissect bacterial adhesion from invasion by manipulating one protein. SfbI protein therefore represents a highly evolved prokaryotic molecule that exploits the host factor fibronectin not only for extracellular targeting but also for its subsequent activation that leads to efficient cellular invasion.     

Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion,migration and capillary tube formation

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell–matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell — fibronectin interaction resulting in impaired endothelial cell migration and tubular formation.     

High molecular weight,cell surface-associated glycoprotein (fibronectin) lost in maglinant transformation

Fibronectin is a polymorphic glycoprotein found in blood and tissues of vertebrates and in cultures of adherent vertebrate cells. There are several forms of fibronectin is composed of two high molecular weight subunits held together by forms found in tissues and on and around the surfaces of cultured cells. Soluble fibronectin is composed of two high molecular weight subunits held together by disulfide bonds. Insoluble fibronectin may be covalently cross-linked in larger complexes. Fibronectin has affinities for collagen, fibrin, heparin, and cell surfaces. in culture, fibronectin in growth medium may mediate attachment of cells to substratum, and fibronectin synthesized by cells may mediate adhesion to substratum. The widespread occurrence of fibronectin in basal lamina indicates that many different cell types in vivo abut against a fibronectin-containing matrix. Cultured transformed cells usually lack cell-surface fibronectin, also called large, external transformation-sensitive (LETS) protein. The failure of transformed cells to synthesize or bind fibronectin is paralleled (at least in some systems) by failures to synthesize or bind collagen and proteoglycans. Abnormal synthesis of fibronectin and other matrix components and abnormal interactions with the tissue matrix may account for several phenotypic characteristics of transformed cultutred cells and for some of the malignant behavior of neoplastic cells in vivo.     

The effects of chemokine, adhesion and extracellular matrix molecules on binding of mesenchymal stromal cells to poly(l-lactic acid)

Abstract Background aims. Mesenchymal stromal cells (MSC) are pluripotent adult stem cells capable of osteogenesis and chondrogenesis to form bone and cartilage. This characteristic gives them the potential for bone and cartilage regeneration. Synthetic polymers have been studied to examine whether they could be used as a scaffold for tissue engineering. In the current study a two-dimensional (2-D) poly(l-lactic acid) (PLLA) scaffold was treated with chemokine, adhesion and extracellular matrix molecules with the aim of using biologic molecules to improve the attachment of human MSC. Methods. MSC were isolated from human bone marrow and applied to a 2-D PLLA scaffold. Chemokines ligand (CXCL12 and CXCL13), adhesion molecules [P-selectin, vascular cell adhesion molecule (VCAM)-1 and heparin] and extracellular matrix molecules (fibronectin and type IV collagen) were coated on the scaffold and their effects on the number of MSC that adhered were recorded. Results. When used alone CXCL12 and CXCL13 enhanced MSC adhesion, as did VCAM-1, P-selectin, fibronectin and collagen, but not heparin. The effects of VCAM-1, P-selectin and heparin were enhanced by the addition of CXCL12. Incubation of MSC with antibodies to integrins α4 and α5β1 inhibited their adhesion to VCAM-1 and fibronectin-treated PLLA respectively, suggesting that these integrins were involved in the MSC interactions. Conclusions. The use of certain chemokines and adhesion and extracellular matrix molecules, alone or in combination, is beneficial for the attachment of MSC to PLLA, and may be helpful as natural molecules in scaffolds for regenerative medicine.     

Sodium channel beta1 and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain

Sequence homology predicts that the extracellular domain of the sodium channel beta1 subunit forms an immunoglobulin (Ig) fold and functions as a cell adhesion molecule. We show here that beta1 subunits associate with neurofascin, a neuronal cell adhesion molecule that plays a key role in the assembly of nodes of Ranvier. The first Ig-like domain and second fibronectin type III-like domain of neurofascin mediate the interaction with the extracellular Ig-like domain of beta1, confirming the proposed function of this domain as a cell adhesion molecule. beta1 subunits localize to nodes of Ranvier with neurofascin in sciatic nerve axons, and beta1 and neurofascin are associated as early as postnatal day 5, during the period that nodes of Ranvier are forming. This association of beta1 subunit extracellular domains with neurofascin in developing axons may facilitate recruitment and concentration of sodium channel complexes at nodes of Ranvier.     

Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions

Remodeling of extracellular matrices occurs during development, wound healing, and in a variety of pathological processes including atherosclerosis, ischemic injury, and angiogenesis. Thus, identifying factors that control the balance between matrix deposition and degradation during tissue remodeling is essential for understanding mechanisms that regulate a variety of normal and pathological processes. Using fibronectin-null cells, we found that fibronectin polymerization into the extracellular matrix is required for the deposition of collagen-I and thrombospondin-1 and that the maintenance of extracellular matrix fibronectin fibrils requires the continual polymerization of a fibronectin matrix. Further, integrin ligation alone is not sufficient to maintain extracellular matrix fibronectin in the absence of fibronectin deposition. Our data also demonstrate that the retention of thrombospondin-1 and collagen I into fibrillar structures within the extracellular matrix depends on an intact fibronectin matrix. An intact fibronectin matrix is also critical for maintaining the composition of cell-matrix adhesion sites; in the absence of fibronectin and fibronectin polymerization, neither alpha5beta1 integrin nor tensin localize to fibrillar cell-matrix adhesion sites. These data indicate that fibronectin polymerization is a critical regulator of extracellular matrix organization and stability. The ability of fibronectin polymerization to act as a switch that controls the organization and composition of the extracellular matrix and cell-matrix adhesion sites provides cells with a means of precisely controlling cell-extracellular matrix signaling events that regulate many aspects of cell behavior including cell proliferation, migration, and differentiation.     

The muscle integrin binding protein (MIBP) interacts with alpha7beta1 integrin and regulates cell adhesion and laminin matrix deposition

Integrins are alphabeta transmembrane receptors that function in key cellular processes, including cell adhesion, differentiation, and extracellular matrix deposition through interactions with extracellular, membrane, and cytoplasmic proteins. We previously identified and cloned a muscle beta1 integrin cytoplasmic binding protein termed MIBP and found that the expression level of MIBP is critical in the decision-making process of terminal myogenic differentiation. We report here that MIBP interacts with the alpha7beta1 integrin but not the alpha5beta1 integrin in C2C12 myoblasts, suggesting an important role of integrin alpha chains in the regulation of the beta1-MIBP interaction. Furthermore, consistent with its selective binding activity toward the alpha7beta1 laminin receptor, we have found that overexpression of MIBP in C2C12 myoblasts resulted in a significant reduction of cell adhesion to laminin and inhibition of laminin matrix deposition. By contrast, neither cell adhesion to fibronectin nor fibronectin matrix deposition was significantly altered in cells overexpressing MIBP. Finally, we show that both the protein level and tyrosine phosphorylation of paxillin, a key signaling molecule involved in the cellular control of myogenic differentiation, are increased by MIBP. These results suggest that MIBP functions in the control of myogenic differentiation by regulating alpha7beta1 integrin-mediated cell interactions with laminin matrix and intracellular signaling through paxillin.