SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Candida albicans escapes from the bloodstream by invading the endothelial cell lining of the vasculature. In vitro, C. albicans invades endothelial cells by inducing its own endocytosis. We examined whether this process is regulated by the tyrosine phosphorylation of endothelial cell proteins. We found that endocytosis of wild-type C. albicans was accompanied by the tyrosine phosphorylation of two endothelial cell proteins with molecular masses of 80 and 82 kDa. The phosphorylation of these proteins was closely associated with the endocytosis of C. albicans because these proteins were phosphorylated in response to the endocytosis of both live and killed organisms, but they were not phosphorylated in endothelial cells infected with a poorly endocytosed strain of C. albicans. The tyrosine kinase inhibitors genistein and tyrphostin 47 blocked the phosphorylation of the two endothelial cell proteins and significantly reduced endocytosis of C. albicans. Therefore, C. albicans probably induces its own endocytosis by stimulating the tyrosine phosphorylation of two endothelial cell proteins.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Candida albicans disseminates haematogenously and causes widespread microabscesses in immunocompromised hosts. Based on previous work performed by ourselves and others, we have developed the following model of the events that occur as blood-borne C. albicans cells escape from the intravascular compartment and invades the deep tissues. These organisms enter the bloodstream, either by translocation across the gut or via an indwelling vascular catheter. The blood-borne organisms adhere to and then invade the endothelial cell lining of the vasculature (Rotrosen et al., 1985; Klotz, 1992; Filler et al., 1995; Fu et al., 1998). Invasion into the endothelial cells enables C. albicans to injure and eventually kill these cells (Filler et al., 1995; 1996). Endothelial cell injury disrupts the integrity of the vascular lining and enables the organisms to invade into the deep tissues. Moreover, loss of endothelial cells results in the exposure of the subendothelial cell basement membrane, which can be bound by additional organisms (Klotz and Maca, 1988).

Based on this model, the invasion of endothelial cells by C. albicans is a critical step in the escape of the organism from the intravascular compartment. Therefore, we have been investigating the mechanisms by which C. albicans invades endothelial cell in vitro. We and others have found that this organism invades vascular endothelial cells by inducing its own endocytosis. Both live and killed organisms can be endocytosed and this process can be blocked by inhibitors that block the function of the endothelial cell microfilaments and microtubules (Rotrosen et al., 1985; Filler et al., 1995).

It has been discovered previously that microorganisms such as Shigella flexneri, Listeria monocytogenes, Yersinia pseudotuberculosis and Escherichia coli invade normally non-phagocytic host cells such as epithelial cells by inducing their own endocytosis. The endocytosis of all of these microbial pathogens is regulated by the phosphorylation of tyrosine-containing host cell proteins (reviewed in Finlay and Falkow, 1997). The microorganism binds to a receptor on the cell surface which then stimulates a tyrosine kinase. This tyrosine kinase then phosphorylates one or more proteins on specific tyrosine residues. These phosphorylated proteins then cause the host cell microfilaments to rearrange and begin the process of endocytosis.

The mechanisms that regulate the endothelial cell uptake of any fungal pathogen remain unknown. Therefore, we investigated whether the tyrosine phosphorylation of endothelial cell proteins regulates the endocytosis of C. albicans.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Endothelial cell phosphotyrosine surrounded C. albicans hyphae

Indirect immunofluorescence was used to determine if contact with C. albicans stimulated the accumulation of phosphotyrosine-containing endothelial cell proteins. We observed that phosphotyrosine containing endothelial cell proteins surrounded C. albicans germ tubes that were in the process of being endocytosed (Fig. 1). This co-localization suggested that the tyrosine phosphorylation of these endothelial cell proteins was associated with the endocytosis of C. albicans.

image

Figure 1. Tyrosine phosphorylated endothelial cell proteins co-localize with C. albicans. Images of endothelial cells infected with live C. albicans 36082 for 75 min in the absence (A–C) or presence of 200 µM tyrphostin 47 (D–F). Cells were viewed with confocal microscopy and images were constructed from 0.5 µm optical sections taken along the Z-axis and stacked upon one another. The tyrosine phosphorylated proteins were visualized by indirect immunofluorescence with the anti-phosphotyrosine antibody 4G10 (A and D). The organisms were visualized by staining with Alexa 594-labelled anti-C. albicans antibodies (B and E). Merged images show both phosphotyrosine containing endothelial cell proteins and the organisms (C and F). Arrows in (A and C) indicate the phosphotyrosine that has accumulated around the C. albicans germ tube. Results are representative of two experiments.

Download figure to PowerPoint

C.albicans stimulated tyrosine phosphorylation of two endothelial cell proteins

To determine which endothelial cell proteins were tyrosine phosphorylated in response to C. albicans, we probed immunoblots of endothelial cell proteins with an antiphosphotyrosine monoclonal antibody. We found that exposure of endothelial cells to C. albicans for 60 and 90 min induced the tyrosine phosphorylation of two proteins with molecular masses of approximately 80 and 82 kDa (Fig. 2). These two proteins were identified only in the Triton X-100 insoluble fraction, indicating that they were associated with the endothelial cytoskeleton. Maximal phosphorylation of these proteins occurred at approximately 60 min and declined by 90 min, as endocytosis neared completion.

image

Figure 2. C. albicans stimulates the tyrosine phosphorylation of two endothelial cell proteins. Immunoblot of Triton X-100 insoluble proteins extracted from endothelial cells after exposure to live C. albicans 36082 for the indicated times. Blot was probed with anti-phosphotyrosine antibody 4G10. Results are representative of three separate experiments. Lanes with a + above them represent endothelial cells that were infected with C. albicans. Numbers below the blot indicate the percentage of organisms that were endocytosed at each time point.

Download figure to PowerPoint

We did not detect any consistent induction of tyrosine phosphorylation of the Triton X-100 soluble proteins at any time point tested (data not shown). Also, no tyrosine phosphorylated proteins were detectable in either Triton X-100 soluble or insoluble fractions prepared from C. albicans that were adherent to plastic in the absence of endothelial cells (data not shown).

Tyrosine phosphorylation of the two endothelial cell proteins was closely associated with the endocytosis of C. albicans

To verify the relationship between the tyrosine phosphorylation of the two endothelial cell proteins and the endocytosis of C. albicans, we compared the response of endothelial cells incubated with either live or killed germinated wild-type C. albicans. Killed organisms were used because they are known to be endocytosed by endothelial cells, but they do not injure these cells (Filler et al., 1995). We confirmed that the killed, wild-type organisms were endocytosed by the endothelial cells and discovered that they induced the phosphorylation of the two endothelial cell proteins (Fig. 3A). However, the phosphorylation induced by the killed organisms was slightly weaker than that induced by the live organisms. Therefore, the phosphorylation of these proteins is associated with the endocytosis of C. albicans, and may also be weakly induced by endothelial cell injury.

image

Figure 3. Tyrosine phosphorylation of the 80 and 82 kDa endothelial cell proteins is specifically associated with the endocytosis of wild-type C. albicans.

A. Immunoblot of endothelial cell proteins collected after exposure to live or killed C. albicans 36082, or live C. albicans V6 for 90 min.

B. Immunoblot of endothelial cell proteins extracted after infection with the indicated strains of C. albicans for 75 min.

Blots were probed with the anti-phosphotyrosine antibody 4G10. Results are representative of three independent experiments. Percentage of organisms of each strain that was endocytosed by the endothelial cells is indicated below each of the immunoblots.

Download figure to PowerPoint

We also examined the response of endothelial cells to live C. albicans V6. This non-germinating mutant adheres normally to endothelial cells, but is poorly endocytosed and does not cause endothelial cell injury. Candida albicans V6 did not induce the tyrosine phosphorylation of any endothelial cell proteins and very few cells of this mutant were endocytosed (Fig. 3A). These results demonstrate that the tyrosine phosphorylation of the two endothelial cell proteins is stimulated by the endocytosis, but not just the adherence of the organism.

To investigate whether the tyrosine phosphorylation of the two endothelial cell proteins was induced by other strains of C. albicans, we tested two additional clinical isolates. All of these isolates stimulated the same pattern of tyrosine phosphorylation (Fig. 3B).

Tyrosine kinase inhibitors blocked Candida-induced tyrosine phosphorylation and reduced endothelial cell endocytosis

The preceding results suggested that tyrosine phosphorylation of the two endothelial cell proteins may govern the endocytosis of C. albicans. Based on this model, inhibiting the phosphorylation of these proteins with tyrosine kinase inhibitors should reduced endocytosis. We found that two tyrosine kinase inhibitors, genistein and tyrphostin 47, blocked the phosphorylation of the endothelial cell proteins in response to C. albicans (Figs 1D–F and 4A). In addition, these inhibitors significantly reduced the percentage of organisms endocytosed by endothelial cells (Fig. 4B). However, they had no effect on the percentage of organisms that were bound by the endothelial cells (data not shown). These findings demonstrate that the endocytosis of C. albicans by endothelial cells is at least partially regulated by the tyrosine phosphorylation of the 80 and 82 kDa endothelial cell proteins.

image

Figure 4. Genistein and tyrphostin 47 inhibit endothelial cell tyrosine phosphorylation and endocytosis of C. albicans. Endothelial cells were infected with live C. albicans 36082 in the presence of either 125 µM genistein or 200 µM tyrphostin 47.

A. Immunoblot of endothelial cells proteins extracted after 90 min of infection and probed with the anti-phosphotyrosine antibody 4G10.

B. Percentage of organisms endocytosed by the endothelial cells, as determined by differential fluorescence assay. Results are mean ± SD of at least three separate experiments, each performed in triplicate. *P < 0.0001 compared with endothelial cells infected with C. albicans in the absence of tyrosine kinase inhibitors.

Download figure to PowerPoint

Inhibiting tyrosine phosphorylation protected endothelial cells from injury byC. albicans

After C. albicans is endocytosed by endothelial cells, it injures and eventually kills these cells (Filler et al., 1995). Therefore, we examined the effects of genistein and tyrphostin 47 on the extent of endothelial cell injury caused by C. albicans. Both tyrosine kinase inhibitors protected the endothelial cells from injury by C. albicans (Fig. 5). The concentrations of genistein and tyrphostin 47 that resulted in a 50% reduction in endothelial cell injury were 66 µM and 97 µM respectively.

image

Figure 5. Inhibiting tyrosine kinase activity blocks endothelial cell injury caused by C. albicans. Endothelial cells were infected with live C. albicans 36082 for 3 h in the presence of the indicated concentrations of genistein and tyrphostin 47. The extent of endothelial cell injury was measured by the specific release of 51Cr. Results are the mean ± SD of at least three separate experiments performed in triplicate. *P < 0.02, **P < 0.0001 compared with endothelial cells infected with C. albicans in the absence of tyrosine kinase inhibitors.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The tyrosine phosphorylation of host cell proteins during the invasion of microbial pathogens is the subject of intense investigation. The majority of studies on the invasion of normally non-phagocytic host cells by these pathogens have focused on epithelial cells. Much less is known about the role of tyrosine phosphorylation in the uptake of microorganisms by endothelial cells. Furthermore, to our knowledge, this is the first report of a fungal pathogen inducing tyrosine phosphorylation in a normally non-phagocytic host cell.

We found that the endocytosis of C. albicans by endothelial cells was associated with the phosphorylation of two proteins with molecular masses of approximately 80 and 82 kDa. Adherence of C. albicans to host cells is known to induce the tyrosine phosphorylation of candidal proteins (Bailey et al., 1995). However, two lines of evidence indicate that 80 and 82 kDa proteins that were phosphorylated in response to C. albicans are likely of endothelial cell origin. First, these phosphoproteins were not detected in preparations of C. albicans that were exposed to bare plastic in the absence of endothelial cells. Second, exposure of endothelial cells to killed organisms stimulated the phosphorylation of these two proteins (Fig. 3A).

The phosphorylation of the two endothelial cell proteins was closely associated with the endocytosis of C. albicans. The poorly endocytosed strain of C. albicans, V6 did not stimulate tyrosine phosphorylation of any endothelial cell proteins (Fig. 3A). Furthermore, the two tyrosine kinase inhibitors, genistein and tyrphostin 47 blocked the phosphorylation of the 80 and 82 kDa proteins and reduced the endothelial cell uptake of C. albicans (Fig. 4). Thus, the phosphorylation of at least one of these proteins is likely required to induce the endocytosis of C. albicans by endothelial cells. Confirmation of this hypothesis awaits further characterization of these proteins.

The two tyrosine kinase inhibitors blocked the phosphorylation of the 80 and 82 kDa proteins almost completely, however, they reduced the endocytosis of C. albicans by only 41% to 63% (Fig. 4B). This incomplete inhibition by genistein and tyrphostin 47 indicates that there may be multiple signal transduction pathways that regulate the endocytosis of C. albicans by endothelial cells.

Of importance, we observed that although genistein and tyrphostin 47 partially inhibited endocytosis, they completely blocked Candida-induced endothelial cell injury. These results strongly suggest that the tyrosine phosphorylation of endothelial cell proteins induced by C. albicans is essential for endothelial cell injury to occur. Also, although both live and killed organisms were endocytosed to the same extent, the live organisms stimulated stronger tyrosine phosphorylation than did killed organisms. Because live organisms injure endothelial cells, whereas killed organisms do not (Filler et al., 1995), it is possible that endothelial cell injury is a weaker stimulus for the tyrosine phosphorylation of the two endothelial cell proteins.

There is a remote possibility that the tyrosine kinase inhibitors blocked the synthesis of one or more candidal factors required for induction of endothelial cell injury. Genistein is known to inhibit mitogen-activated protein (MAP) kinases in mammalian cells (Tang et al., 1998) and tyrphostin 47 may actually stimulate MAP kinase activity (Agbotounou et al., 1994). In C. albicans, MAP kinases are important in regulating hyphal formation (Kohler and Fink, 1996; Leberer et al., 1996; Lo et al., 1997). We have shown previously that the formation of true hyphae is required for C. albicans to be endocytosed by and injure endothelial cells (Phan et al., 2000). In the current experiments, we observed that neither inhibitor had any visible effect on candidal growth or hyphal formation under the conditions tested (data not shown). Nevertheless, we cannot completely rule out the possibility that these inhibitors altered the ability of C. albicans to cause endothelial cell injury.

Many microbial pathogens are known to stimulate their own uptake by endothelial cells in vitro. These pathogens include Streptococcus pneumoniae, Toxoplasma gondii, Citrobacter freundii, Listeria monocytogenes and Staphylococcus aureus (Hamill et al., 1986; Woodman et al., 1991; Cundell et al., 1995; Greiffenberg et al., 1997; Badger et al., 1999). The role of tyrosine phosphorylation in the endocytosis of these microorganisms has not been investigated in detail. Prasadarao et al., 1997 have found that the endocytosis of E. coli by brain microvascular endothelial cells is regulated by the tyrosine phosphorylation of host cell proteins. There are significant differences between the endocytosis induced by E. coli and C. albicans. First, E. coli is endocytosed only by brain microvascular endothelial cells and not by human umbilical vein endothelial cells, as is C. albicans (Prasadarao et al., 1996). Second, metabolically inactive E. coli cells are not taken up by endothelial cells (Prasadarao et al., 1993), whereas killed C. albicans are endocytosed. Third, E. coli stimulates the tyrosine phosphorylation of endothelial cell proteins and is endocytosed after 10–20 min of infection (Prasadarao et al., 1997; 1999), compared with C. albicans which induces tyrosine phosphorylation and its own endocytosis only after 60 min of contact with the endothelial cells. Finally, the uptake of E. coli by brain microvascular endothelial cells is associated with the tyrosine phosphorylation of a MAP kinase (Prasadarao et al., 1997). Candida albicans did not induce any detectable phosphorylation of MAP kinases, which would have been discernible in the Triton X-100 soluble fraction. These differences between the two organisms strongly suggest that the endocytosis of C. albicans by endothelial cells is mediated by a unique mechanism.

The uptake of E. coli by brain microvascular endothelial cells, but not umbilical vein endothelial cells demonstrates that endothelial cells obtained from the vasculature of different organs may respond differently to a microbial pathogen. Thus, it is possible that endothelial cells from different organs or different types of blood vessels may have distinct responses to C. albicans. These differences may contribute to the tropism of blood-borne C. albicans for specific organs such as the kidneys and brain.

The identity of the 80 and 82 kDa proteins that are phosphorylated in response to C. albicans remains to be determined. Known tyrosine kinase substrates with similar molecular masses include, cortactin, the 85 kDa regulatory subunit of class IA phosphatidylinositide 3-kinase, and the ezrin, radixin and moesin family of proteins (Takeuchi et al., 1994; Dehio et al., 1995; Fawaz et al., 1997; Lowry et al., 1998; Wymann and Pirola, 1998; Skoudy et al., 1999). Experiments are in progress to determine whether any of these candidate proteins are phosphorylated in endothelial cells that are infected with C. albicans. Elucidating the signalling pathways that regulate the uptake of C. albicans by endothelial cells may lead to the development of therapeutic strategies to prevent vascular invasion during the initiation of haematogenously disseminated candidal infections.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Reagents

All of the reagents used, unless otherwise stated, were purchased from Sigma-Aldrich Chemical Company, St Louis, MO.

Organisms

Candida albicans ATCC 36082, was obtained from the American Type Culture Collection (Rockville, MD). Candida albicans 15153 was a blood isolate obtained from the clinical microbiology laboratory at Harbor-UCLA Medical Center. Candida albicans SC5314 was generously supplied by W. A. Fonzi (Georgetown University, Washington, DC). All of these strains are clinical isolates and all of them are known to be endocytosed by and cause injury to endothelial cells in vitro (Filler et al., 1995; Ibrahim et al., 1995; Phan et al., 2000). The non-germinating mutant, C. albicans V6 was generously provided by Dr Helen Buckley, Temple Medical School, Philadelphia, PA (Buckley et al., 1982). All organisms were grown overnight on a rotating drum at room temperature in yeast nitrogen base broth (Difco Laboratories, Detroit, MI) supplemented with 0.5% (wt/vol) dextrose as previously described (Filler et al., 1991; 1995). After incubation, the blastospores were harvested by centrifugation and washed twice in Dulbecco's phosphate-buffered saline (PBS) (Irvine Scientific, Santa Anna, CA). The organisms were counted with a haemacytometer and adjusted to the desired concentrations in RPMI 1640 medium (Irvine Scientific).

Germinated organisms were obtained by incubating blastospores of C. albicans ATCC 36082 in RPMI 1640 on a rotary shaker at 37°C for 90 min. The germinated organisms were killed by exposure to 20µM sodium periodate for 30 min at room temperature and then washed extensively in PBS (Filler et al., 1995). An aliquot of the organisms was inoculated onto Sabouraud dextrose agar (Difco) to confirm that all of the organisms had been killed.

Endothelial cells

Endothelial cells were harvested from human umbilical veins by the method of Jaffe et al. (1973). The cells were grown in M-199 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, Calabasas, CA), 10% defined bovine calf serum (Hyclone, Logan, UT), and 2 mM l-glutamine with penicillin and streptomycin (Irvine Scientific). For use in the experiments, third passage cells were grown either in multiwell tissue culture plates (Falcon, Lincoln Park, NJ), or on glass coverslips coated with fibronectin (Collaborative Biomedical Products, Bedford, MA). All incubations were at 37°C in 5% CO2.

Endothelial cell adherence

The effects of the tyrosine kinase inhibitors, genistein and tyrphostin 47 on the adherence of C. albicans to endothelial cells was determined by our previously described method (Ghannoum et al., 1992). Briefly, endothelial cells in six-well tissue culture plates were preincubated in either 125 µM genistein for 20 min or 200 µM tyrphostin 47 for 2 h. Control wells were incubated in parallel with the diluent, DMSO at a final concentration of 0.2%. Next, 102C. albicans blastospores were added to each well and incubated for 30 min. The tyrosine kinase inhibitors were present during the entire incubation and the inoculum was confirmed by colony counting. At the end of the incubation period, the medium was aspirated from each well and the non-adherent organisms were removed by rinsing with Hanks' balanced salt solution in a standardized manner. The wells were then overlaid with Sabouraud dextrose agar and the number of adherent organisms was determined by colony counting. Adherence was expressed as a percentage of the original inoculum, and each experiment was performed in triplicate on three separate occasions.

Endocytosis assay

The endocytosis of C. albicans by the endothelial cells was quantified as described (Fratti et al., 1996; Tsuchimori et al., 2000). Confluent endothelial cells on 12 mm diameter glass coverslips were incubated with tyrphostin 47, genistein or diluent alone as in the adherence assays. As a negative control, 0.3 µM cytochalasin D was added to selected coverslips just before the addition of C. albicans to inhibit endocytosis (Filler et al., 1995). Each coverslip was then incubated with 105 organisms. After a 2 h incubation, the medium was aspirated and the cells were fixed with 3% paraformaldehyde. The non-endocytosed organisms were labelled by incubating the coverslips for 1 h with Alexa 594-conjugated rabbit antibodies directed against C. albicans (Biodesign International, Kennebunk, ME). Next, the endothelial cell were made permeable by incubating them with 0.1% Triton X-100 in PBS (v/v). Finally, both the endocytosed and non-endocytosed organisms were stained with 1% Uvitex in PBS (v/v) (Levitz et al., 1987). The coverslips were examined with a Zeiss Axiovert 10 microscope equipped for epifluorescence (Carl Zeiss, Thornwood, NY), and the number of endocytosed organisms was determined by subtracting the number of Alexa 594-labelled organisms (non-endocytosed) from the number of organisms labelled with Uvitex (total organisms). All coverslips were examined by a blinded observer and at least 100 organisms per coverslip were analysed. Each experiment was performed in triplicate.

Endothelial cell damage

The extent of endothelial cell injury caused by C. albicans was determined by our standard chromium release assay using endothelial cells grown in 24-well tissue culture plates (Filler et al., 1995). The inoculum was 105C. albicans blastospores per well and the incubation time was 3 h. The effects of genistein and tyrphostin 47 on endothelial cell injury was determined as described for the adherence assay, except that the genistein was used at concentrations ranging from 15.5 to 125 µM and the tyrphostin 47 was tested at 50–200 µM. Experiments were performed in triplicate on at least 3 different days.

Indirect immunofluorescence

Indirect immunofluorescence was utilized to determine the spatial relationship among the C. albicans cells and the phosphotyrosine containing endothelial cell proteins. Confluent endothelial cells on 12 mm-diameter glass coverslips were incubated with 2 × 105 organisms for 75 min. Next, the cells were washed with 0.4 mM Na3VO4 in PBS and then fixed with 3% paraformaldehyde. The cells were permeablized with 0.1% Triton X-100 and then washed three times in PBS containing 1% FBS. The organisms were stained by inverting the coverslips onto Alexa 594-conjugated anti-C. albicans antibodies for 45 min. Next, the coverslips were rinsed extensively in PBS. Tyrosine-phosphorylated proteins were detected by incubating the cells with 10 µg ml−1 of a monoclonal antibody directed against phosphotyrosine (Clone 4G10; Upstate Biotechnology, Lake Placid, NY) followed by a fluorescein isothiocyanate-labelled goat anti-mouse antibody. The samples were viewed by confocal microscopy and the final images were produced by combining 0.5 µm optical sections that encompassed the entire thickness of the endothelial cell.

Immunoblotting

Immunoblotting was used to detect endothelial cell proteins that were tyrosine phosphorylated in response to C. albicans. Endothelial cell extracts were prepared by the method of Palmer et al., 1997. Briefly, endothelial cells in six-well tissue culture plates were incubated for selected time intervals with 4 × 106 live C. albicans 36082. The inoculum was increased to 8 × 106 organisms per well to provide maximal stimulation when either non-germinating or fixed organisms were used. After incubation, the plates were cooled on ice, and all subsequent steps where performed at 4°C. The endothelial cells were rinsed three times with PBS containing 0.4 mM Na3VO4, 1 mM NaF, and 0.1 mg of phenylethylsulphonyl fluoride per ml. Next, they were removed from the plate by scraping in 1.5 ml of this same buffer. The cells were collected by centrifugation at 12 000 g for 1 min, and then lysed by a 30-min incubation in 100µl of lysis buffer (50 mM Tris-HCL (pH 7.6) containing 0.4 mM Na3VO4, 1 mM NaF, 1% Triton X-100, and 0.1 mg of phenylethylsulphonyl fluoride, 0.01 mg of leupeptin and 0.01 mg of pepstatin per ml. To separate the Triton X-100 soluble and insoluble fractions, the lysate was centrifuged at 14 000 g for 30 min. The supernatant was collected and the pellet was resuspended in 40µl of lysis buffer. The two fractions were mixed with Laemmli buffer (Laemmeli, 1970), boiled for 7 min, cleared by centrifugation and the proteins were separated by SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Boehringer Mannheim, Indianapolis, IN) by electroblotting. Phosphotyrosine-containing proteins were detected by enhanced chemiluminescence (Boehringer Mannheim) using the anti-phosphotyrosine monoclonal antibody. All experiments were performed at least three times using endothelial cells from different donors.

Statistical analysis

Differences in endothelial cell endocytosis and injury were evaluated by analysis of variance with the Bonferroni correction for multiple comparisons. P-values of ≤ 0.05 were considered to be significant.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank the nurses at Harbor-UCLA Medical Center for collecting umbilical cords and Quynh T. Phan for help with tissue culture. We also appreciate the helpful advice and discussion of Drs John E. Edwards Jr, Nemani V. Prasadarao and Bett J. Eng. Financial support: Public Health Service grants R01 AI19990; P01 AI37194; R29 AI040636; and MO1 RR00425 from the National Institutes of Health and grant 1081-GI3 from the American Heart Association, Greater Los Angeles Affiliate. S. G. Filler was supported by the Burroughs Wellcome Fund New Investigator Award in Molecular Pathogenic Mycology.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Agbotounou, W.K., Mousset, S., Piperno, S., Pierre, M., Jacquemin-Sablon, A., and Pierre, J. (1994) Activation of the mitogen-activated protein kinase cascade by tyrphostin (RG 50864). Biochem Pharmacol 48: 505515.
  • Badger, J.L., Stins, , M.F., and Kim, K.S. (1999) Citrobacter freundii invades and replicates in human brain microvascular endothelial cells. Infect Immun 67: 42084215.
  • Bailey, A., Wadsworth, E., and Calderone, R. (1995) Adherence of Candida albicans to human buccal epithelial cells: host- induced protein synthesis and signaling events. Infect Immun 63: 569572.
  • Buckley, H.R., Price, M.R., and Daneo-Moore, L. (1982) Isolation of a variant of Candida albicans. Infect Immun 37: 12091217.
  • Cundell, D.R., Gerard, N.P., Gerard, C., Idanpaan-Heikkila, I., and Tuomanen, E.I. (1995) Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 377: 435438.
  • Dehio, C., Prevost, M.C., and Sansonetti, P.J. (1995) Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signalling pathway. EMBO J 14: 24712482.
  • Fawaz, F.S., Van Ooij, C., Homola, E., Mutka, S.C., and Engel, J.N. (1997) Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or localization of several host cell proteins including cortactin. Infect Immun 65: 53015308.
  • Filler, S.G., Ibe, B.O., Luckett, P.M., Raj, J.U., and Edwards, J.E. Jr (1991) Candida albicans stimulates endothelial cell eicosanoid production. J Infect Dis 164: 928935.
  • Filler, S.G., Swerdloff, J.N., Hobbs, C., and Luckett, P.M. (1995) Penetration and damage of endothelial cells by Candida albicans. Infect Immun 63: 976983.
  • Filler, S.G., Pfunder, A.S., Spellberg, B.J., Spellberg, J.P., and Edwards, J.E. Jr (1996) Candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells. Infect Immun 64: 26092617.
  • Finlay, B.B., and Falkow, S. (1997) Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61: 136169.
  • Fratti, R.A., Ghannoum, M.A., Edwards, J.E. Jr and Filler, S.G. (1996) Gamma interferon protects endothelial cells from damage by Candida albicans by inhibiting endothelial cell phagocytosis. Infect Immun 64: 47144718.
  • Fu, Y., Rieg, G., Fonzi, W.A., Belanger, P.H., Edwards, J.E. Jr and Filler, S.G. (1998) Expression of the Candida albicans gene ALS1. Saccharomyces cerevisiae induces adherence to endothelial and epithelial cells. Infect Immun 66: 17831786.
  • Ghannoum, M.A., Filler, S.G., Ibrahim, A.S., Fu, Y., and Edwards, J.E. Jr (1992) Modulation of interactions of Candida albicans and endothelial cells by fluconazole and amphotericin B. Antimicrob Agents Chemother 36: 22392244.
  • Greiffenberg, L., Sokolovic, Z., Schnittler, H.J., Spory, A., Bockmann, R., Goebel, W., and Kuhn, M. (1997) Listeria monocytogenes-infected human umbilical vein endothelial cells: internalin-independent invasion, intracellular growth, movement, and host cell responses. FEMS Microbiol Lett 157: 163170.
  • Hamill, R.J., Vann, J.M., and Proctor, R.A. (1986) Phagocytosis of Staphylococcus aureus by cultured bovine aortic endothelial cells: model for postadherence events in endovascular infections. Infect Immun 54: 833836.
  • Ibrahim, A.S., Mirbod, F., Filler, S.G., Banno, Y., Cole, G.T., Kitajima, Y., et al. (1995) Evidence implicating phospholipase as a virulence factor of Candida albicans. Infect Immun 63: 199398.
  • Jaffe, E.A., Nachman, R.L., Becker, C.G., and Minick, C.R. (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52: 27452756.
  • Klotz, S.A. (1992) Fungal adherence to the vascular compartment: a critical step in the pathogenesis of disseminated candidiasis. Clin Infect Dis 14: 340347.
  • Klotz, S.A., and Maca, R.D. (1988) Endothelial cell contraction increases Candida adherence to exposed extracellular matrix. Infect Immun 56: 24952498.
  • Kohler, J.R., and Fink, G.R. (1996) Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development. Proc Natl Acad Sci USA 93: 1322313228.
  • Laemmeli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680685.
  • Leberer, E., Harcus, D., Broadbent, I.D., Clark, K.L., Dignard, D., Ziegelbauer, K., et al. (1996) Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans. Proc Natl Acad Sci USA 93: 1321713222.
  • Levitz, S.M., DiBenedetto, D.J., and Diamond, R.D. (1987) A rapid fluorescent assay to distinguish attached from phagocytized yeast particles. J Immunol Methods 101: 3742.
  • Lo, H.J., Kohler, J.R., DiDomenico, B., Loebenberg, D., Cacciapuoti, A., and Fink, G.R. (1997) Nonfilamentous C. albicans mutants are avirulent. Cell 90: 939949.
  • Lowry, M.B., Duchemin, A.M., Coggeshall, K.M., Robinson, J.M., and Anderson, C.L. (1998) Chimeric receptors composed of phosphoinositide 3-kinase domains and FCgamma receptor ligand-binding domains mediate phagocytosis in COS fibroblasts. J Biol Chem 273: 2451324520.
  • Palmer, L.M., Reilly, T.J., Utsalo, S.J., and Donnenberg, M.S. (1997) Internalization of Escherichia coli by human renal epithelial cells is associated with tyrosine phosphorylation of specific host cell proteins. Infect Immun 65: 25702575.
  • Phan, Q.T., Belanger, P.H., and Filler, S.G. (2000) Role of hyphal formation in interactions of Candida albicans with endothelial cells. Infect Immun 68: 34853490.
  • Prasadarao, N.V., Wass, C.A., Hacker, J., Jann, K., and Kim, K.S. (1993) Adhesion of S-fimbriated Escherichia coli to brain glycolipids mediated by sfaA gene-encoded protein of S-fimbriae. J Biol Chem 268: 1035610363.
  • Prasadarao, N.V., Wass, C.A., and Kim, K.S. (1996) Endothelial cell GlcNAc beta 1–4GlcNAc epitopes for outer membrane protein A enhance traversal of Escherichia coli across the blood–brain barrier. Infect Immun 64: 154160.
  • Prasadarao, N.V., Zhou, J., Wass, C.A., Arditi, M.A., and Kim, K.S. (1997) Tyrosine Protein Kinases are Involved in Escherichia coli Invasion of Brain Microvascular Endothelial Cells. Abstract B-63. 37th Interscience Conference on Antimicrobial Agents and Chemotherapy Toronto. American Society for Microbiology, Washington, D.C, pp. 38.
  • Prasadarao, N.V., Wass, C.A., Stins, M.F., Shimada, H., and Kim, K.S. (1999) Outer membrane protein A-promoted actin condensation of brain microvascular endothelial cells is required for Escherichia coli invasion. Infect Immun 67: 57755783.
  • Rotrosen, D., Edwards, J.E. Jr, Gibson, T.R., Moore, J.C., Cohen, A.H., and Green, I. (1985) Adherence of Candida to cultured vascular endothelial cells: mechanisms of attachment and endothelial cell penetration. J Infect Dis 152: 12641274.
  • Skoudy, A., Nhieu, G.T., Mantis, N., Arpin, M., Mounier, J., Gounon, P., and Sansonetti, P. (1999) A functional role for ezrin during Shigella flexneri entry into epithelial cells. J Cell Sci 112: 20592068.
  • Takeuchi, K., Sato, N., Kasahara, H., Funayama, N., Nagafuchi, A., Yonemura, S., and Tsukita, S. (1994) Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J Cell Biol 125: 13711384.
  • Tang, H., Kerins, D.M., Hao, Q., Inagami, T., and Vaughan, D.E. (1998) The urokinase-type plasminogen activator receptor mediates tyrosine phosphorylation of focal adhesion proteins and activation of mitogen-activated protein kinase in cultured endothelial cells. J Biol Chem 273: 1826818272.
  • Tsuchimori, N., Sharkey, L.L., Fonzi, W.A., French, S.W., Edwards, J.E. Jr and Filler, , S.G. (2000) Reduced virulence of HWP1-deficient mutants of Candida albicans and their interactions with host cells. Infect Immun 68: 19972002.
  • Woodman, J.P., Dimier, I.H., and Bout, D.T. (1991) Human endothelial cells are activated by IFN-gamma to inhibit Toxoplasma gondii replication. Inhibition is due to a different mechanism from that existing in mouse macrophages and human fibroblasts. J Immunol 147: 20192023.
  • Wymann, M.P., and Pirola, L. (1998) Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1436: 127150.