Candida albicans, the causal agent of candidiasis, poses health threats to immunocompromised hosts and candidiasis-affected neonates. Systemic infection of C. albicans involved penetration of vasculature in mammalian cells through self-induced tyrosine-assisted-endocytosis. N-cadherin, a 130 kDa transmembrane protein, was isolated from Candida albicans SC5314 using affinity purification method. N-cadherin’s endocytotic role was elucidated by quantifying endocytosed cells of the human umbilical cord vein and CHO-K1 following method on flow cytometry. Immunfluorecence microscopy and transfection using N-cadherin siRNA duplex oligonucleotides determined localization and endocytotic role of N-cadherin respectively. N-cadherin facilitates endocytosis and the process involves actin rearrangement, Ca2+ assisted ligand-binding, and signaling of T2pk2 moiety. N-cadherin is expressed throughout the endothelial cell surface area. N-cadherin to N-cadherin interaction is specific. The hyphal cells, unlike the blastospores were active participants in endocytosis. Architectural changes of the microfilaments, particularly actin, accompanied the endocytotic activity. Furthermore, it was found that adhesion and endocytosis were strictly separate events. N-cadherin may be regulated by intracellular factors during ‘out-in’ signaling. T2pk2 is involved in the expression of ligand where the receptor, N-cadherin, attaches.
Further future research on N-cadherin and associated receptor roles is still necessary to clarify the transmembrane’s mechanism.
N-Cadherin and Endocytosis in Candida albicans
Candida albicans, diploid yeast-like fungi, are harmless commensals of the human body; however, it can also be the causal agent of opportunistic and genital infections known as candidiasis or moniliasis or trush. General-purpose genotype strains (GPGs) of C. albicans has been demonstrated to cause the pathogenic disease more significantly compared to other strains and the predominating effect may be attributed to the ability of the strains to replace the existing commensals (Schmidt and others 1999) and to the differences in the gene region located on 60 alleles of ALS7 ORF or the so-called hypermutable contingency genes (Zhang and others 2003).
The hymatogenous distribution of C. albicans causes allergies in healthy individuals and microabscesses in immunocompromized individuals. Normally, the organism inhabits different sites of the host which includes many organs, the bloodstream and the mucosa. In immunocompromised individuals, the organism thrives on the mucosal lining of the oral cavity, esophagus (oropharyngeal c.), vagina (vulvovaginal c.), and gastrointestinal tract (cutaneous c.) and this opportunistic pathogen contributes to a significant amount of patient morbidity, and at worse, lethality upon systemic infection.
Dissemination of the organism arise by population of the mucosal surfaces through epithelial cell adhesion, with subsequent penetration of the endothelial and epithelial cell barriers by inducing endocytosis. Overcoming the defenses of the vascular endothelium is of huge importance to the systemic infection and thus, putting to light the interaction between the organism and the endothelial cells and associated studies/methods on preventing the passage of the pathogens. To overcome humoral host defense, the extracellular proteinase excreted by the C. albicans degrades (e.g. proteolysis) humoral defenses such as Fc portion of immunoglobulin G, and complement C3 which debilitate host defense and facilitate ease of transfection (Kaminishi and others 1995).
Pathogen virulence is relative to the dimorphic state of the C. albicans and associated host defense factors. C. albicans can also attack the central nervous system (CNS) aside from the well-studied virulence of the pathogenic organism on neonates and immunocompromised individuals. The hyphal form of the organism is the infective state. Lossinsky and others (2006) studied he transmigration of the yeast and the hyphal morphologies in an in vivo and cell culture model of human brain microvascular endothelial cells. Intraperitoneal inoculation patterned the hyphal infection of kidneys, liver, spleen, abdominal tissues and other nasally-connected tissues. Meningitis was notably absent with yeast inoculation and in the case of brain endothelium, neutrophilic meningitis was possible for the non-yeast state. Additionally, intracellular budding and pseudohyphal formation inside the human brain microvascular endothelial cells does not affect its’ monolayer integrity.
As mentioned above, adhesion of C. albicans on the endothelial cells play an important role in the transfection process. The adhesins include Als1p, Als3p, Als5p ( Glee and others 2003), hydrophobic cell wall proteins (Calderon and Braun 1991), and a 53 integrin-like receptor which possibly attaches to the vitronectin and other Arg-Gly-Asp-containing proteins on the endothelial cell surface of the mammalian cells (Santoni and others 2001). Cell-surface hydrophobicity (CSH) phenotype of the SHH1 gene is regulated via temperature gradient and differential surface location (Singleton and Hazen 2004). Enolase, a protein present in the cell surface of the C. albicans, binds with plasminogen/plasmin; the binding interaction between the two is lysine-dependent. Interaction between the two facilitates in vitro fibrinolytic activity of the C. albicans (Jong and others 2003). Gene factors and regulation of C. albicans assists in the mammalian cell adhesion. EAP1 gene in C. albicans enhanced filamentous growth of hyphae in kidney cells which is regulated by transcription factor Efg1p and the cyclic AMP-dependent protein kinase pathway (Li and Palecek 2003).
Invasion and injury of endothelial cells by Candida albicans blastospores were meager (Phan and others 2000) compared to hyphal invasion during organism-induced endocytosis prior to systemic infection. Fungal viability is not a requirement to induce endocytosis although the process needs the presence of endothelial cytoskeleton, microfilaments and microtubules (Filler and others 1995). Endocytosis of the wild type pathogen strain is associated with tyrosine phosphorylation of two endothelial cell proteins with molecular masses of 80 and 82 kDa (Belanger and others 2002).
Studies on endothelial cell receptors during endocytosis are considerably few and as such, the paper aims to elucidate the ‘receptor’ role of N-cadherin based on the study of Phan and his colleagues. Phan and others (2005) attempted to isolate the endothelial cell surface proteins of Candida albicans strain SC5314. Endothelial cells from human umbilical cord vein and CHO-K1 were utilized for the study. Quantifying endocytosed organism of the endothelial and the CHO cells proceeded by differential fluorescence assay. The isolation of cell surface proteins from the viable blastospores or hyphae of Candida albicans proceed by affinity purification method. Eluted surface proteins were identified using SDS-PAGE separation followed by 130 kDa excision and microsequencing using matrix-assisted laser desorption ionization time-of-flight. Indirect immunofluorescence using anti-N-cadherin monoclonal antibody revealed cadherin localization. Cell expression of N-cadherin (neural c.) and VE-cadherin (vascular epithelium c.) were quantified by flow cytometry. Endothelial cells were transfected with N-cadherin siRNA duplex oligonucleotides to determine effect of down regulation on endocytosis. Identifying the presence of an endothelial receptor on the cell surface of the C. albicans is the central point of the Phan’s study. The general hypothesis of the study is the presence of an endothelial receptor on the cell surface that mediates the endocytosis of the pathogen.
This paper aims to construct a review on Phan and others’ cadherin study and compare it with existing related studies on endothelial cell receptors. General feasibility/reliability of the study and its application is also assessed.
Phan and others (2005) hypothesized that there is direct contact between an endothelial cell receptor and a ligand expressed on the exterior of hyphae and that this endothelial contact is Ca2+- dependent.
To test extracellular Ca2+ dependency involved in the self-induced endocytosis of C. albicans, a calcium chelator was added to the media. The addition of EGTA, a calcium chelator, to the incubating medium [RPMI 1640] contributed to the cell adherence and hyphal endocytosis and increased the number of the cell-associated organisms in the endothelial cells. There was 60% overall reduction of the endocytosed cells when compared with control endothelial cells which were not exposed to the EGTA chelator. Concomitant addition of magnesium did not contribute to the chelating effect of EGTA indicating that endocytotic effect is calcium-dependent. Thus, the interaction between the endothelium and the cell receptor is Ca2+ dependent.
Recent finding on S. aureus indicate that the serum components influenced the endocytosis of the pathogen particularly that of endothelial cell contact/adhesion. Non-viable hyphae of C. albicans SC5314, was incubated in serum- and non-serum conditions following exposure to the CHO-K1 and endothelial cells with subsequent measure of endocytosis. A 40% decrease in the number of endocytosed organisms were observed for the serum-incubated C. albicans hyphae. This indicates that the serum proteins have an inhibitory effect on receptor-mediated endocytosis and thus, are unlikely candidate of endothelial receptors.
To identify endothelial cell proteins that bound Candida albicans hyphae in a Ca2+-dependent manner, affinity purification method was performed for the blastospores, viable, and non-viable hyphae. The affinity purification procedure revealed that pathogenic blastospores bound only to the endothelial cell protein with a molecular mass equivalent to 105kDa whereas the viable and non-viable hyphae bound to a 135-kDa protein as well as the 105-kDa protein. Non-viable hypahae incubated in the serum condition has no profound effect on the binding of endothelial cell proteins. Serum proteins, which were known to have an interaction with the surface components for endocytosis of S. aureus, have no contributory effect to the endocytosis of C. albicans.
Affinity purification methods and sequencing of the eluted surface proteins revealed several matches with the cadherin family and the ‘bounding’ interaction between cadherin and Candida albicans hyphae was detected by probe using a pan-cadherin antibody directed against the conserved C-terminal region of the cadherins. The pan-cadherin antibody recognizes 135-kDa molecules. Figure 1 (Phan and others2005) illustrates interaction of 135-kDa endothelial cell protein and the C. albicans hyphae. N- cadherin and VA cadherin approaches 135-kDa molecular mass and is found to be expressed on the endothelial cells ( Navarro and Dejana 1998).
Monoclonal antibodies were directed against N- cadherin and VA cadherin and Fig 1 illustates the recognition of the 135-kDa endothelial cell protein by N-cadherin. VA-cadherin antibody did not detect the 135-kDa endothelial cell protein.
Fig.1.N-cadherine endothelial cell membrane
extracts binds to C.albicans hyphae (Phan and others 2005).
Verification using indirect immunofluoroscopy revealed localization of C. albicans on endocytosed cell. As illustrated in Figure 2, patterns for localization were found to be similar with the findings from the previous study of Dejana and Navarro wherein the N-cadherin is expressed throughout the endothelial cell surface area and VA-cadherin sublocated only at intercellular junctions.
Fig 2. N-cadherin but not VE-cadherin on intact endothelial
cells co-localizes with C. albicans hyphae that are being endocytosed.
A–N, confocal microscopic images of uninfected endothelial
cells (A–C and H–J) and endothelial cells infected with hyphae of C.
albicans SC5314 (D–G and K–N). The cells were stained for N-cadherin
(A and D), microfilaments (B, E, I, and L), C. albicans (F and M), and
VE-cadherin (H and K) (Phan and others 2005).
Microfilaments, like the actin, co-localized with the N-cadherin at endocytosed hyphae during Candida albicans infection. Often, the N-cadherin aggregates down the whole length of the hyphae, yet it was observed that there was only focal aggregation of actin around the pathogen. VE-cadherin remained localized at its’ intercellular region suggesting minimal roles of the specified cadherin during the endocytosis process.
Aside from the Candida albicans SC5314, in vitro blood isolates, C. ablicans 36082 and 15153, used previously by Belanger and others (2002) tested positive for N-cadherin bounding on the hyphal cells as demonstrated by immunoblotting techniques. Membrane-hyphae binding of the two blood isolates are illustrated in Figure 3.
Fig 3. C. albicans 36082 and 15153 bind to N-cadherin
Normally, signal transduction pathways regulate hyphal formation of the dimorphic pathogen and the lack of such components necessary for ‘signaling’ contributes to the decrease in the endocytotic activity of the endothelial cells (Phan and others 2000). Tpk2?/tpk2? mutant endocytosed approximately half of hyphae compared with the w.t. strains and its reconstituted strain tpk2?/tpk2?::TPK2 although association for both cell strains is still similar. Tpk2?/tpk2? mutant is defective then for endocytotic activity. Unlike the wild type and the reconstituted strain, Tpk2?/tpk2? mutant binds poorly to the N-cadherin although it still binds properly to the 105 kDa protein. Thus, Tpk2, a subunit of protein kinase A and a RAS member, is required for ligand expression on the surface of the endothelial cells and is important for maximal endocytotic activity.
CHO-K1 cells that express little or no cadherins were transfected with N-cadherin siRNA duplex oligonucleotides. CHO cells transfected with human N-cadherin and VE-cadherin , respectively, endocytosed 5x and 2x more compared with vectorless CHO cells. Additionally, the transfectants, endocytosed (by CHO cells) 7x more hyphae than blastospores indicating that N-cadherin binding is specific for the C. albicans hypha. Verification procedures using N-cadherin short-insert- sequence-RNA- transfected endothelial cells reduced the expression of N-cadherin by ninety percent as compared to the random control. The transfected cells endocytosed 34% less hyphae population compared to control siRNA. Down regulation of the N-cadherin expression is thus inhibitory to the endocytosis of Candida albicans
This establishes the role of the N-cadherin as an endothelial cell receptor mediating endocytosis of Candida albicans during infection. It does not support data on adherence of the transmembrane protein on C. albicans hyphae. Endocytosis and adherence are separate processes utilizing different sets of cell surface components.
The capacity of C. albicans to induce its own endocytosis in non-phagocytic host cells and the mechanism involved during the transfection process have positive implications on the developing therapeutic methods to stop/block/inhibit systemic infection caused by the opportunistic pathogen.
It can be deducted from the previous paragraphs that Phan and his colleagues (2005) considered the following in identifying ‘receptor’ roles in endothelial cells: (1) What is possibly the identity of the cell surface component? (2) What does it require? (3) Is the receptor strain specific? (3) What are the interactions involved in the binding of the receptor to the endothelial cells? (4) What dimorphic state is actively involved during endocytosis? Is it the hyphae or the blastospore? (5) What is the location of the receptor?; and finally (6) What is the role of receptor expression on endocytosis?
It is noted that the methods implicated in the experiment supported that of the query of Phan and others on finding the ID of the receptor. Methods used were advanced and efficient and the in vitro study mimics that of real ‘endocytosis’ in endothelial mammalian cells. Candida albicans take advantage of endocytosis, a non-phagocyte host response, during its complex histopathology.
Opportunistic pathogens have different ways of invading the body and one of them is receptor-mediated endocytosis. Endocytosis normally involves the presence of cell-surface receptors for attachment prior to vesicle formation. In particular opportunistic pathogens Toxoplasma gondii, the causal agent of toxoplasmosis, invades host cell defense using the endocytosis and the adhesion is mediated by microneme proteins (Soldati and others2001). Pseudomonas aeruginosa’s receptor in tracheal cells is sialic acid. S. sanguinis has putative surface proteins (Xu and others 2007) to facilitate endocytosis.
Endocytosis is heavily placated by several studies as a means of C. albicans to penetrate the vasculature of the mammalian cell following systemic infection (Rotrosen and others1985; Belanger, 2002). Cell surface architecture of Candida albicans have specified roles in endocytosis and constituents of the surface consists of polysaccharides like glucan, chitin, and mannan which may either confer structural function and/or antigenic activity. Phan and others (2005) identified N-cadherin, a transmembrane protein to mediate ligand-receptor function of Candida albicans. Past studies by Calderon and Braun (1991) suggested that mannoproteins specifically CR3-like protein confer receptor relations. CR3 roles in endocytosis were elucidated by testing adherence of CR3 mutants on polysterene.
Mannoproteins are dynamically expressed and is stage specific. It is noted that extraction reagents differ between the authors. Calderon and Braun (1991) extracted the mannoproteins containing receptor proteins using zymolyase and dithiothreitol (DDT) whereas Phan and others (2005) used octyl-glucopyranoside and a variety of protease inhibitors in the receptor extraction. This can provide as a dark area for future research on endothelial cell receptor extraction.
Sepulveda and others (1996) reported that ubiquitin-like epitopes display modulating roles in endocytosis of 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. As for the case of regulatory mechanism of N-cadherin, the only thing clear is that it is significantly inhibited by the absence of Ca2+ moiety. This ‘Ca2+ effect’ is consistent with the other members of the family of cadherins. T2pk2 also regulate the adherence to mammalian endothelial cells. AlS3 also binds to the cadherins before endothelial adhesion.
Cell surface interaction is not really clear and multiple host factors are involved. As mentioned above, C3d and other the mannoproteins of 68 and 60 kDa, display multiple biological function and play roles in mediating host protein interaction. Such mannoproteins were classified under F-CAM integrins whereas the cadherins are classified under transmembrane proteins. Endocytosis then, involves several receptors. It is logical to think that endothelial receptors of Candida albicans are multi-faceted, which involves many types of receptors or if not, differentially activates, the endothelial cell receptors, at different stages of growth of the organism—blastospores, hyphae and pseudohyphae.
Lambert and Mege (2000) attempted to explore the mechanism for N-cadherin-mediated surface contact using dimeric N-cadherin-Fc chimera which mimics the structural and functional properties of cadherins. They found that N-cadherin demonstrates N-cadherin-to-N-cadherin specificity as well as accumulations of tyr-phosphorylated proteins, a recruitment and reorganization of actin filaments and local membrane altercation which supports the findings of Phan and others. Their study also revealed the ‘outside-in’ signaling of the chimera from adhesion to adhering accumulation which would then affect cytoskeletal rearrangement and cytoplasmic signal mobilization.
Admittedly, there are very few studies on N-cadherin as an endothelial receptor of C. albicans. Most researches focused on human E-cadherin and its’ function in cell metastasis and
cancer. Phan and others’ study on N-cadherin mediation of endocytosis of C. albicans is relatively weak. The study attempted to explore the endocytotic activity of the N-cadherin at bi-phasic state of C. albicans. Developmental stages of C. albicans—yeast, pseudohyphae, and hyphae may encode for differential activation of the regulatory pathways for endocytosis.
All studies on the endocytosis of Candida albicans are almost exclusively in vivo including Phan and others’ most recent N-cadharin study. Still, it is expected that the lab environment used for the study may mimic that of the natural conditions of Candida albicans in their natural host. The major drawback of in vivo experiments is that there is variation in the methods and reagents used by the different researchers. The heterozygocity of the species C. albicans may vary in the experimental conditions thus affecting the results.
There is apparently no question on the methods adapted by Phan and others. Isolation, adhesion counts, and verification followed by precision techniques of immunoblotting, enzyme immunoassay, transfection, and siRNA down regulation demonstrate efficient methodology. Still, the scope of research is limited. Quantifying endocytosed organisms in the presence of N-cadherin is the central method for Phan and others’ research. Notably, pseudohypha was not counted during endocytosis. Ca2+ dependency was not quantified. It would be a good follow-up of research to used varying amounts of EGTA chelator to determine effect of different quantities of Ca2+ on N-cadherin. Also, since C. albicans is a bloodborne organism, a research on the effect of Fe3+ on N-cadherin mediated endocytosis may be a good venue for research.
Additionally, the Candida albicans strain used for the study is limited. All general-purpose genotype strains should be explored for their endocytotic activity which is a good indicator of pathogen virulence. Another good avenue for research is tracing the N-cadherin mediated endocytosis using human brain endothelial cells. Biogenesis of C. albicans on the brain is not wholly dependent on hyphal formation but there is still the un-answered question on the non-destruction of cell integrity. N-cadherin, being a transmembrane protein, would be affected by factors such as temperature gradient and intracellular factors. This was ignored or excluded in Phan’s research and may be a potential area for future research.
For future studies on ‘N-cadherin’ identity as a mediator in endocytosis and/or ‘endocytosis’ alone, it would be good to dabble on the following areas for research: (1) role of S1r2 gene, the gene that controls phenotypic switching, on endocytosis; (2) N-cadherin roles during the developmental stages of C. albicans; (3) study on the adhesion and endocytotic effect of the many existing identified endothelial receptors of multiple strains of C. albicans along different stages of infection (4) cross-linkage between dimorphism, phenotypic switching and receptor regulation and finally (5) evaluate effect of known receptor-inhibitors on the endocytotic activity of N-cadherin 6) heterozygocity of C.albicans during endocytosis and (7) other cadherins, that may be possibly present like the murine E-caderine.
Most important is the possible application of Phan’s study on the N-cadherin mediating endocytosis. Pharmaceutics may have positive roles correlated to the result of the study. Possibly, stunting and/or inhibiting the systemic infection of the pathogenic Candida albicans on immunocompromised hosts, and on neonates and healthy individuals with moniliasis/trush/candidiasis can be made possible by inhibiting N-cadherin mediation of the endocytosis of the opportunistic pathogen.
IV. Conclusion/Future Research Questions
Opportunistic pathogens pose threats to the human health. Specifically, Candida albicans, the causal agent of candidiasis, poses threats to the health of immunocompromised hosts and candidiasis-affected neonates. Systemic infection of C. albicans involved penetration of the vasculature in mammalian cells through self-induced tyrosine-assisted-endocytosis. Phan and others (2005) identified N-cadherin as the endothelial cell receptor that facilitates endocytosis which involves actin rearrangement, Ca2+ required ligand-binding, and signaling of T2pk2 moiety.
N-cadherin is expressed throughout the endothelial cell surface area. N-cadherin to N-cadherin interaction is specific. Hyphal cells were the active participants in endocytosis whereas blastospores were not that important. Viability of C. albicans is not a requirement to induce endocytosis. Architectural changes of the microfimalent particularly actin accompanied the cytotic activity. Furthermore, adhesion and endocytosis were strictly separate events. N-cadherin may be regulated by intracellular factors during out-in signaling(Lambert and Mege 2000). T2pk2 is involved in the expression of ligand where the receptor, N-cadherin, attaches.
There are several endothelial receptors of C. albicans which play a role in endocytosis. N-cadherin is one of them. The research by Phan and others on N-cadherian as endothelial cell receptor is feasible and is a good source for future researches. The scope of the study is limited though and as previously mentioned, the gaps which are un-answered by the study can provide for future researches on endocytosis and N-cadherin. New studies on heterozygocity and phenotypic switching may also be related to endocytosis. As for the muric N-cadherin, there is still the possibility that other cadherins may still be found on the endothelial mammalian cells.
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