| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1Division of Hematology–Oncology, Mailstop 57, Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA 2Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA 3,4Division of Infectious Diseases3 and Department of Surgery4, Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA
Correspondence Ambrose Y. Jong ajong{at}chla.usc.edu
Received August 28, 2002
Accepted December 6, 2002
 |
Abstract |
|---|
 TOP Abstract INTRODUCTIONMETHODSRESULTSDISCUSSIONAcknowledgementsREFERENCES |
|---|
-aminocaproic acid and aprotinin, can effectively block plasmin-binding activity. Thirdly, as with many plasminogen receptors, binding of Candida enolase to plasmin(ogen) is lysine-dependent, whereas little inhibition occurred with arginine, aspartate and glutamate. Fourthly, immobilized enolase enhanced plasminogen's affinity for streptokinase at least tenfold, as demonstrated by its activation of plasmin activity. To elucidate the biological significance of this result, it was demonstrated that the plasmin(ogen)-bound Candida cells were able to induce fibrinolysis activity in a matrix-gel assay. Furthermore, plasmin-bound Candida cells had an increased ability to cross an in vitro blood–brain barrier system. The results given here indicate that Candida enolase is a plasminogen- and plasmin-binding protein and that the interaction of C. albicans enolase with the plasminogen system may contribute to invasion of the tissue barrier.
These authors contributed equally to this work.
Abbreviations: EACA, -aminocaproic acid; ECM, extracellular matrix; HBMEC, human brain microvascular endothelial cells; Ni-NTA, nickel nitrilotriacetic acid; SAP, secreted aspartyl proteinase; TEER, transendothelial electric resistance; tPA, tissue-type plasminogen activator.
|
INTRODUCTION |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONAcknowledgementsREFERENCES |
|---|
Many pathogens can penetrate normal tissue barriers, such as vascular basement membranes and other organized extracellular matrices. Several lines of evidence suggest that pathogens can use microbial or host-derived enzymes, particularly proteinases, in their invasion processes. Candida also uses a proteolytic activity to facilitate its penetration. A secreted aspartyl proteinase (SAP) gene family that contains nine members (SAP1–9) has been identified (De Bernardis et al., 1995; Sanglard et al., 1997; Ibrahim et al., 1998; Naglik et al., 1999; Staib et al., 1999, 2000). At least one phospholipase (PLB1) is involved in invasion (Leidich et al., 1998). Gene disruption experiments revealed that these gene products did not account for all Candida infections (Hube et al., 1997). Additional degradation mechanisms, such as host-derived enzymes, may participate in the complicated invasive process.
Plasminogen is abundant in human plasma and extracellular fluids (
0.15 mg ml-1). It is converted to the proteolytic form, plasmin, by eukaryotic activators such as tissue-type plasminogen activator (tPA) and urokinase, and by prokaryotic activators such as streptokinase and staphylokinase (Malke et al., 1994). Plasmin has important functions in mammals, such as the degradation of extracellular matrix (ECM) proteins, blood clot dissociation (fibrinolysis) and cellular migration. It is also involved in cancer metastasis (Chapman, 1997). Some pathogens are able to recruit host plasminogen via their surface components or so-called plasminogen receptors (Korhonen et al., 1997), resulting in an increased invasive capacity for the micro-organisms. The identified pathogenic plasminogen receptors generally fall into two major categories: (a) filamentous protein structures that are morphologically similar to fibrin–fimbriae proteins; and (b) non-filamentous surface proteins with enzymic activity and multiple-binding properties. The non-filamentous plasminogen receptors are usually abundant proteins with a relatively low affinity for plasminogen, which recognizes the lysine-binding sites of a receptor molecule (Lähteenmäki et al., 1995). For example, enolase has been characterized as a plasmin(ogen)-binding protein on the surface of pathogenic streptococci (Pancholi & Fischetti, 1998).
Activation of plasminogen by tPA proceeds poorly in solution but is dramatically enhanced by immobilization of plasminogen on fibrin, eukaryotic tissue surfaces or plasminogen receptors. Immobilization of plasminogen onto lysine-containing surfaces is associated with considerable molecular conformational changes that result in higher susceptibility to tPA-mediated activation and higher resistance to the physiological inhibitors present in human plasma (Irigoyen et al., 1999). Thus, plasminogen receptors are involved in the activation of a mechanism that generates a targeted, localized and transient proteolytic activity. Although plasminogen activation has been studied intensively, degradation of mammalian tissue compounds by fungus-bound plasmin and its role in tissue invasion are poorly understood. It is important to identify plasminogen receptors that contribute to Candida invasion of the host tissue barriers.
Enolase is an abundant enzyme in the glycolytic pathway, yet it may have alternative functions on the cell surface in addition to its enzymic activity. For example, it is also a major structural component of turtle lens, 
-crystallin (Wistow et al., 1988). S. cerevisiae enolase has the ability to bind polynucleotides (al-Giery & Brewer, 1992). Moreover, human (Andronicos et al., 1997), rat (Nakajima et al., 1994) and streptococcal (Pancholi & Fischetti, 1998) 
-enolases are cell-surface plasminogen-binding proteins. Their binding to plasminogen can enhance plasminogen activation by urokinase and prevent 2-antiplasmin from binding. C. albicans enolase associates with glucan, an abundant immunodominant antigen in the cell wall (Angiolella et al., 1996), and the enzyme can be secreted in the growth medium. In vivo, increased levels of fungal-specific enolase have also been found in patients with invasive candidiasis. Although it has been suggested that Candida enolase is a major participant in systemic infections of candidiasis (van Deventer et al., 1994), it is unclear how Candida enolase contributes to the pathogenesis of candidiasis.
In this paper, we report that Candida bound to both plasminogen and plasmin in a saturable and specific manner. The binding, at least in part, was mediated by enolase. In addition, the binding of plasminogen to the immobilized enolase enhanced its affinity for streptokinase up to tenfold, indicating that binding/activation was mediated in part by the enolase. Moreover, plasminogen/plasmin-bound Candida enhanced fibrinolysis on the matrix gel and also had increased ability to cross an in vitro blood–brain barrier system.
|
METHODS |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONAcknowledgementsREFERENCES |
|---|
URA3 : : IMM434/URA3 : : IMM434) was kindly provided by Drs J. Pla and C. Nombela, University of Complutense, Madrid, Spain; strains CAF2 (isogenic to CAI4, except that it also contains one functional URA3 gene) and CHK21 (CaHK1, both CaHK1 genes were deleted from CAI4 strain) were kindly provided by Dr Mikio Arisawa, Nippon Roche K. K. Research Center, Kanagawa, Japan. Strain CAJ4 is a spontaneous mutant of CAI4 with a slower growth rate. Candida cells were grown aerobically at 30 °C in rich YPD broth (1 % yeast extracts, 2 % peptone and 2 % glucose) or Sabouraud medium (Difco). Yeast cells were harvested at exponential phase, washed and resuspended in PBS for the plasminogen/plasmin binding assay.
125I iodination of plasminogen.
Plasminogen was purified from human serum by using a Lysine Sepharose 4B column (Amersham Biosciences; no. 17-0690-01). A pellet of IODO-Beads Iodination reagent (Pierce Biotechnology; no. 28665) was rinsed with 0.5 ml PBS, dried with a towel and added to 1 mCi (3.7 x 107 Bq) 125I (50 µl) at room temperature for 5 min. Subsequently, 0.5 ml plasminogen (2 mg ml-1) was added to the activated solution and further incubated at room temperature for 10 min. Unincorporated 125I was removed by passing the sample through a PD-10 desalting column (Amersham Biosciences; no. 17-0851-01). The labelled plasminogen that was collected exhibited a specific activity of 1.2–1.6 x 106 c.p.m. mg-1.
Plasminogen/plasmin binding assay.
Candida cells (1 x 107 cells ml-1) were pre-incubated with 1 ml 125I-plasminogen (105 c.p.m.) diluted in PBS with aprotinin (5 µg ml-1) and PMSF (5 µg ml-1) at 37 °C for 1 h. The cell suspension was washed three times with PBS/0.2 % Triton and the Candida cells were subjected to 
-counting. In the case of the plasmin assay, plasmin activity was measured spectrophotometrically. Briefly, 10 µl plasmin (5 mg ml-1; Sigma) was incubated with Candida cells. Washed cell pellets were resuspended in 0.5 ml PBS that contained 6 µl chromogenic substrate [N-(p-tosyl)-Gly–Pro–Lys p-nitroanilide acetate salt (5 mg powder in 0.5 ml PBS) (Sigma; T-6140)]. Formation of the product, p-nitroanilide, was measured at 405 nm with an extinction coefficient of 9.65. The reaction was incubated at 37 °C and OD405 was measured at different time-intervals.
Expression of Candida enolase in Escherichia coli.
The Candida enolase gene was isolated by PCR from genomic DNA by using primers J-203 (5'-ACAGGATCCTACGCCACTAAAATCCAC-3') and J-204 (5'-AACCTCGAGTTGAGAAGCCTTTTGGAA-3'). The resulting 1.32 kbp DNA fragment contained the complete ORF of the enolase gene, flanked with BamHI and XhoI restriction sites. The DNA fragment was subcloned into the BamHI and XhoI sites of the E. coli expression vector pET-28b (Novagen). Candida enolase was expressed in E. coli and purified by nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography (QIAexpress system; Qiagen). The enolase retained its enzymic activity when expressed at 25–30 mg protein (l culture)-1. The assay for the enzymic activity of enolase was performed at 37 °C in HEPES buffer (100 mmol l-1, pH 7), containing (l-1): 3.3 mmol MgSO4, 0.2 mmol NADH.H+, 0.3 mmol 2-phosphoglycerate, 1.2 mmol ADP, 10 IU lactate dehydrogenase and 3 IU pyruvate kinase in a final reaction volume of 1 ml. The reaction was initiated by adding 100 µl test solution that contained enolase. Enolase activity was measured as the reduction of NADH.H+ to NAD+. A decrease in absorbance at 340 nm was recorded as change in A340 min-1 by using a spectrophotometer (PerkinElmer).
Studies of immobilized enolase/plasminogen association by activation of streptokinase activity.
Candida enolase with a 6 x His tag was expressed in E. coli and purified by using QIAexpress Ni-NTA resin (Qiagen) to >95 % homogeneity as described above. For binding studies, enolase or plasminogen was diluted to 25 µg ml-1 with sodium carbonate (pH 9.6, 50 mmol l-1) and 100 µl was added to each well of a 96-well polystyrene ELISA plate that was then incubated at 37 °C for 4 h. The enolase- or plasminogen-coated plates were transferred to 4 °C overnight. These coated plates were then washed four times with buffer NET (0.25 % gelatin, 0.15 M NaCl, 5 mmol EDTA l-1, 0.05 % Tween and 50 mmol Tris l-1, pH 8.0). After washing, 200 µl buffer NET was added to each well. To measure the change in affinity of plasminogen for streptokinase (ICN Biochemicals), three sets of experiments were performed in parallel. For enolase-bound plasminogen (set I), 100 µl plasminogen (50 µg ml-1 in PBS) was added to an enolase-coated plate at 37 °C for 4 h. Unbound plasminogen was removed by washing four times with PBS. Streptokinase (10 µl; final concn 10 nM to 10 µM) and 190 µl plasmin chromogenic substrate (0.5 mg N-(p-tosyl)-Gly–Pro–Lys p-nitroanilide ml-1 in PBS, pH 7.2) were added to the enolase-bound plasminogen set (set I). In parallel, the plasminogen-coated plates without enolase (set II) and plain plates that contained aqueous plasminogen (set III) were used. The plates were incubated at 37 °C for 30 min and increases in A405 were recorded. The maximum absorbance of each set was taken to be the maximum activation of plasminogen by streptokinase.
Fibrin matrix-gel degradation analysis.
The matrix gel was prepared using the plasmin assay method to detect fibrinolysis activity (Tuan et al., 1996). Briefly, 105 Candida cells were pre-incubated with either plasminogen or plasmin for 30 min in the presence or absence of specific inhibitors: 2-antiplasmin (0.1 µg), aprotinin (1 µg) and -aminocaproic acid (EACA; 50 mmol l-1). A serine protease inhibitor, PMSF (50 mmol l-1), was also used. Thereafter, the mixtures were washed three times with PBS to remove free plasminogen/plasmin. The resulting cell pellets were placed in wells of a fibrin substrate matrix gel that contained 1.25 % low-melting-temperature agarose, plasminogen (50 µg ml-1; Sigma), thrombin (0.05 U ml-1; Sigma) and fibrinogen (2 mg ml-1). The gel was incubated in a humidified chamber at 37 °C for 8–12 h until the appearance of clear spots indicated the presence of fibrinolytic activity. Plasmin activity was detected by clear circles in the opaque fibrin indicator gel and the digested pattern was photographed.
Transcytosis of C. albicans across human brain microvascular endothelial cells (HBMEC) in the presence and absence of plasmin.
HBMEC were cultured on 6.5 mm diameter, collagen-coated Transwell polycarbonate tissue culture inserts with a pore size of 8 µm (Corning Life Sciences). HBMEC were seeded at high density (104 cells ml-1) (Badger et al., 1999; Jong et al., 2001). Experiments were carried out 5 days later, when cells formed a confluent monolayer (according to light microscopic examination). This in vitro model of the blood–brain barrier allows separate access to the upper chamber (blood side) and lower chamber (brain side) and permits mimicking of C. albicans penetration into the brain. HBMEC are polarized and exhibit a transendothelial electric resistance (TEER) of at least 300 
cm-2, as measured with an Endohm V/ meter in conjunction with an Endohm chamber (WPI, Florida) as described previously (Badger et al., 1999; Jong et al., 2001). For the transcytosis studies, 106 yeast cells were added to the upper chamber (total volume, 200 µl) and the monolayers were incubated at 37 °C. After 6, 12, 18 and 24 h, 100 µl samples were taken from the lower chamber (an equivalent volume of medium was immediately replaced, maintaining a total bottom volume of 1 ml) and plated. The integrity of the HBMEC monolayer was assessed by TEER throughout the experiments. Results are expressed as cell number, measured as c.f.u., of yeast transcytosed ml-1.
|
RESULTS |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONAcknowledgementsREFERENCES |
|---|
-radioactive counting. Fig. 1(a) showed that all Candida strains tested could bind plasminogen effectively, although slight variations were seen. To further strengthen this observation, we tested whether C. albicans could bind to plasmin, the activated form of plasminogen. Candida was pre-incubated with plasmin and activity of bound plasmin was monitored by chromogenic enzyme assay. Fig. 1(b) shows that the plasmin activity was linear up to 120 min. In the presence of 2-antiplasmin, activity decreased to 80 and 60 % in proportion to the added concentrations (5 and 10 µg per 0.5 ml reaction volume, respectively). As a control, Candida cells alone did not show chromogenic activity in the absence of plasmin. The data show that C. albicans can bind to both plasminogen and plasmin, and that activity of bound plasmin can be blocked by the specific inhibitor 2-antiplasmin. The results also suggest that there is a plasminogen/plasmin receptor(s) present on the surface of Candida cells.
|
Binding of purified Candida enolase to plasminogen and plasmin
In order to test whether Candida enolase behaves as a plasminogen/plasmin receptor, we expressed Candida 6 x His-tagged enolase in E. coli for binding studies. The expressed 6 x His-tagged enolase was retained in a nickel-chelating column matrix and used as an affinity column to bind plasminogen or plasmin. As shown in Fig. 2, a saturation curve was observed by titration of the amount of plasmin. In parallel, the empty vector (without the enolase gene) in the same expression system was used as the control; no bound-plasmin activity was detected. Similar association was observed when purified enolase was immobilized on microtitre plates (data not shown). To substantiate the specificity of the observation, we used two specific inhibitors of plasmin, EACA and aprotinin, to determine their effect on plasmin activity. The results (Fig. 3) showed that both EACA and aprotinin blocked plasmin activity effectively.
|
|
In a manner similar to that of human -enolase and other plasminogen receptors (Miles et al., 1991; Andronicos et al., 1997), binding of Candida enolase to plasminogen is lysine-dependent. As shown in Fig. 4, binding was inhibited by more than 85 % when lysine was present in the solution, while < 10 % inhibition occurred with arginine; little effect was observed with aspartate or glutamate in this study (Fig. 4). These results suggest that the binding of plasminogen/plasmin to C. albicans is due, at least in part, to its ability to bind to enolase, and that the binding is lysine-sensitive.
|
Kinetic studies of plasminogen toward streptokinase after binding to different surfaces
One feature of plasmin(ogen) is that binding to a receptor can induce conformational change, affecting its affinity for activators (Irigoyen et al., 1999). We tested whether the binding of plasmin(ogen) to Candida enolase would have such a characteristic. As shown in Fig. 5, the Km value of streptokinase required for plasminogen activation on the solid phase (90 nM) was approximately one-tenth of that of plasmin in the aqueous phase (900 nM). Plasminogen bound to polystrene also had a higher affinity for streptokinase (500 nM), but lower than that of the immobilized enolase. These results indicate that binding of plasminogen to enolase results in the increase in its affinity for streptokinase, a well-known plasminogen activator. These observations, along with binding studies (Figs 2–5), support the hypothesis that Candida enolase plays a role as a receptor for plasminogen.
|
Matrix-gel studies
Fibrinogen is one of the major substrates of plasminogen/plasmin in vivo, and fibrinogen matrix gel has been widely used to examine plasmin activity. We performed matrix-gel studies to test the possible biological significance of Candida enolase–human plasminogen/plasmin association, i.e. whether plasminogen/plasmin-bound Candida cells showed proteolytic activities that mimicked their actions in vivo. In the absence of plasminogen/plasmin, Candida alone showed little fibrinolysis activity (Fig. 6, lane 1); however, association of Candida with plasminogen resulted in a significant increase in fibrinolytic activity (Fig. 6, lane 2). This result suggests that Candida-bound plasminogen can be activated by thrombin, present in the matrix gel, to digest the surrounding fibrinogen. Similar results can be observed by using Candida-bound plasmin (Fig. 6, lane 3). Fibrinogen activities can be inhibited by the general serine protease inhibitor PMSF (Fig. 6, lane 4) and plasmin-specific inhibitors, such as 2-antiplasmin (Fig. 6, lane 5), aprotinin (lane 6) and EACA (lane 7), indicating the specificity of this interaction. Our results suggest that binding of plasminogen/plasmin to the yeast surface may facilitate Candida invasion, which is consistent with the role of plasminogen/plasmin surface-binding functions.
|
Transcytosis assay of Candida cells in the presence and absence of bound plasmin
To further demonstrate that bound plasmin may facilitate penetration during candidal invasion, we used an in vitro blood–brain barrier system to measure the traversal efficiency of Candida cells in the presence and absence of bound plasmin (Badger et al., 1999; Jong et al., 2001). The TEER was 280–300 cm-2, suggesting that a tight junction was formed. An aliquot of Candida cells, with or without bound plasmin, was loaded on the apical chamber in triplicate. Penetrated cells were measured from the basolateral chamber at different time-intervals. Candida ATCC 10261 is a rapidly growing strain (t1/2: 50 min) and it can adhere to and penetrate HBMEC (Jong et al., 2001). Fig. 7(a) showed that the ability of plasmin-bound Candida ATCC 10261 cells to penetrate the in vitro blood–brain barrier increased by 20–40 % within 24 h. Strain CAJ4 has a slower growing rate (t1/2: 100–110 min); like strain ATCC 10261, this strain can form germ-tubes, which are essential for candidal invasion. Its ability to penetrate is less efficient than that of ATCC 10261 (Fig. 7b). However, the effect of plasmin on penetration is more prominent in this strain, i.e. a greater than fivefold increase was routinely observed. Together, the results suggest that the penetrative ability of Candida cells is increased in the presence of bound plasmin.
|
|
DISCUSSION |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONAcknowledgementsREFERENCES |
|---|
-enolase to plasminogen, as demonstrated by the finding that > 80 % binding is inhibited by the lysine analogue EACA, while only 14 % inhibition occurred with the arginine analogue benzamidine (Miles et al., 1991; Andronicos et al., 1997). Our biochemical studies showed that the binding of plasminogen/plasmin to enolase was conserved among these organisms (Figs 2–4). Our kinetic studies also demonstrated the relationship between Candida enolase and plasminogen (Fig. 5). Previous biochemical studies of human and rat enolase provide good examples of the versatile roles of enolase; however, no information is available to further illustrate its physiological functions in vivo. We performed matrix-gel (Fig. 6) and transcytosis (Fig. 7) studies to provide more evidence that plasmin can execute its protease activity while bound to the yeast surface. Our fibrinolysis and transcytosis studies of Candida enolase provide further insight into its roles in plasminogen/plasmin association. It has been suggested that multiple fungal factors are involved in the pathogenesis of C. albicans infection. These include phenotypic switching of genes (Kennedy et al., 1992), transition between blastospores and hyphal forms (Mitchell, 1998), adherence capacity (Calderone & Braun, 1991; Sundstrom, 1999), hydrolytic enzyme PLB1 (Leidich et al., 1998; Rodier et al., 1999) and secreted aspartyl proteinases SAP1–9 (De Bernardis et al., 1995; Hube et al., 1997; Sanglard et al., 1997; Ibrahim et al., 1998; Naglik et al., 1999; Staib et al., 1999, 2000; Huang & Jong, 2001). Adherent genes may initiate the attachment, whereas the hydrolytic enzymes may assist entry of the pathogen into host cells. Regarding the hydrolytic enzyme activities, an intriguing observation is that a gene family of SAPs has been identified. These enzymes can cleave several proteins that are important in host defences, such as antibodies of both IgG and IgA isotypes (Ruchel, 1986). A possible role for SAPs in the degradation of the ECM has been reported (Morschhäuser et al., 1997). Also, SAPs may promote colonization, penetration and invasion by C. albicans (Kaminishi et al., 1995; Colina et al., 1996; Naglik et al., 1999). Nevertheless, the actual contribution of each of these SAPs to the pathogenesis and severity of the disease remains to be elucidated (Dubois et al., 1998).
The roles of SAPs and bound plasmin may not necessarily be mutually exclusive. It is possible that the plasminogen/plasmin–enolase association may play a role in candidal virulence by causing direct damage to host-cell ECM, possibly by enzymic degradation of ECM proteins or other protein constituents. Such injury would allow fungal hyphal elements to traverse the vascular endothelium more effectively, ultimately increasing the rapidity of dissemination to and colonization of target organs. The fact that Candida surface enolase acts as a plasminogen receptor may provide a functional link between fungal invasion and generation of localized proteolysis in target sites. Our studies do not rule out the existence of other plasminogen receptors on the surface of C. albicans. For example, Streptococcus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can also associate with plasminogen/plasmin (Malke et al., 1994); Candida GAPDH is also a cell-surface protein (Gozalbo et al., 1998). We are currently searching for more plasminogen receptors and investigating their relationship to other proteases.
|
Acknowledgements |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONAcknowledgementsREFERENCES |
|---|
|
REFERENCES |
|---|
|
TOPAbstractINTRODUCTIONMETHODSRESULTSDISCUSSIONAcknowledgementsREFERENCES |
|---|
-enolase) displays characteristics required for a plasminogen binding protein. Biochim Biophys Acta 1337, 27–39.[CrossRef][Medline]-enolase as a candidate plasminogen receptor. Biochemistry 30, 1682–1691.[CrossRef][Medline]-enolase on rat neuronal plasma membrane. J Neurochem 63, 2048–2057.[Medline]-Enolase, a novel strong plasmin(ogen) binding protein on the surface of pathogenic streptococci. J Biol Chem 273, 14503–14515.-Crystallin/-enolase: one gene encodes both an enzyme and a lens structural protein. J Cell Biol 107, 2729–2736.This article has been cited by other articles:
|
|
 |
|
  A. Lama, A. Kucknoor, V. Mundodi, and J. F. Alderete Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated, Fibronectin-Binding Protein of Trichomonas vaginalis Infect. Immun., July 1, 2009; 77(7): 2703 - 2711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. L. Chaffin Candida albicans Cell Wall Proteins Microbiol. Mol. Biol. Rev., September 1, 2008; 72(3): 495 - 544. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Esgleas, Y. Li, M. A. Hancock, J. Harel, J. D. Dubreuil, and M. Gottschalk Isolation and characterization of {alpha}-enolase, a novel fibronectin-binding protein from Streptococcus suis Microbiology, September 1, 2008; 154(9): 2668 - 2679. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Gancedo and C.-L. Flores Moonlighting Proteins in Yeasts Microbiol. Mol. Biol. Rev., March 1, 2008; 72(1): 197 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Mundodi, A. S. Kucknoor, and J. F. Alderete Immunogenic and Plasminogen-Binding Surface-Associated {alpha}-Enolase of Trichomonas vaginalis Infect. Immun., February 1, 2008; 76(2): 523 - 531. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kunert, J. Losse, C. Gruszin, M. Huhn, K. Kaendler, S. Mikkat, D. Volke, R. Hoffmann, T. S. Jokiranta, H. Seeberger, et al. Immune Evasion of the Human Pathogen Pseudomonas aeruginosa: Elongation Factor Tuf Is a Factor H and Plasminogen Binding Protein J. Immunol., September 1, 2007; 179(5): 2979 - 2988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kolberg, A. Aase, S. Bergmann, T. K. Herstad, G. Rodal, R. Frank, M. Rohde, and S. Hammerschmidt Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface. Microbiology, May 1, 2006; 152(Pt 5): 1307 - 1317. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pitarch, A. Jimenez, C. Nombela, and C. Gil Decoding Serological Response to Candida Cell Wall Immunome into Novel Diagnostic, Prognostic, and Therapeutic Candidates for Systemic Candidiasis by Proteomic and Bioinformatic Analyses Mol. Cell. Proteomics, January 1, 2006; 5(1): 79 - 96. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | J MED MICROBIOL | MICROBIOLOGY | J GEN VIROL | ALL SGM JOURNALS |
, 5 µg; 
, 10 µg, respectively). Candida cells only, without Plm or 
). Plasmin substrate, N-(p-tosyl)-Gly–Pro–Lys p-nitroanilide acetate (30 µg), was used for the spectrophotometric measurements.
).
) or directly with the ELISA plate (
