Glucuronoxylomannan from Cryptococcus neoformans Down-regulates the Enzyme 6-Phosphofructo-1-kinase of Macrophages*

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose-and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.
The pathogen Cryptococcus neoformans (Cn) 7 is an encapsulated yeast with worldwide distribution (1)(2)(3). Cn causes cryp-tococcosis, a life-threatening invasive disease with a higher incidence in immunocompromised patients (4,5). The infection usually begins in the lung after inhalation of environmental spores that can disseminate to different cells and tissues. Meningoencephalitis is the most severe condition of cryptococcosis with considerable indices of mortality (5).
Both GXM and galactoxylomannan are constitutively released to the extracellular environment in vitro and in vivo through secretory vesicles (17)(18)(19). In fact, patients with cryptococcosis accumulate GXM in the cerebrospinal fluid and serum, where it is associated with a number of immunomodulatory properties that include down-regulation of proinflammatory cytokine secretion from host cells and reduction of leukocyte migration into inflammatory sites (1, 20 -23).
Macrophages (MO) are considered key cells in cryptococcosis (24 -27). The outcome of phagocytized Cn is critical to pathogenesis. Although MO can kill Cn (27,28), different reports demonstrate that intracellular replication can occur resulting in yeast extrusion followed or not by host cell lysis (29 -31). The mechanism by which Cn survives and replicates within macrophages includes secretion of phospholipases and large amounts of GXM (31). Secreted GXM is also internalized by macrophages (32), and the PS suppresses the host cell proliferative response in a mechanism that includes apoptosis (33). Exposure of macrophages to GXM results in up-regulation of FasL expression, which is at least in part responsible for apoptosis induction in macrophages and T cells (33,34). Although GXM modulates cellular response during infection, the effects of the polysaccharide on the cellular metabolism are completely unclear.
Polysaccharide Isolation by Filtration of Culture Supernatants-C. neoformans cells and debris were removed from culture supernatants by centrifugation, and the resulting supernatant was concentrated ϳ20-fold using an Amicon (Millipore, Danvers, MA) ultrafiltration cell (cutoff ϭ 100 kDa, total capacity of 200 ml) with stirring and Biomax polyethersulfone ultrafiltration discs (76 mm, Millipore) (13). After formation of a viscous film over the filtering disc, the fluid phase was discarded, and the remaining gelatinous material was recovered with a cell scrapper. The final PS was quantified by antibodybased assays using a protocol established previously (38) and modified by Fonseca et al. (39).
Structural Modifications of GXM and Chondroitin Sulfate-Reduction of hexuronic acid carboxyl groups in the GXM was performed as described by Taylor and Conrad (40). About 25 mg of the PS was dissolved in 4 ml of water, and the pH of this solution was adjusted to 4.75 with 0.1 N HCl. Solid 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (75 mg) was added over a period of 10 min, whereas the pH was maintained at 4.75 with 0.1 N HCl. Solid NaBH 4 (300 mg) was added slowly with stirring, and the solution was maintained at 50°C for 2 h. After the addition of several drops of acetic acid to destroy the excess of borohydride, the solution was dialyzed against distilled water and lyophilized to obtain the carboxyl-reduced PS (13,40). The extent of reduction of the carboxyl groups was estimated by the decrease in the carbazole reaction and subsequent formation of glucose (41,42).
For de-O-acetylation, GXM (1.25 mg) was dissolved in 2.5 ml of H 2 0. The pH of this solution was then adjusted to 11.25-11.50 with concentrated NH 4 OH. The solution was then stirred at room temperature for 24 h for further dialysis against water. The remaining sample was lyophilized and further dissolved in the appropriate buffer for use in enzymatic assays.
Fluorescence Microscopy-Macrophage were plated onto wells of a 24-well plate covered with glass coverslips (5 ϫ 10 5 cells per well). GXM (10 g/ml, final concentration) was added to the macrophage monolayers and incubated for 6 or 24 h. The cells were then washed with PBS, fixed with 4% paraformaldehyde in PBS for 30 min, and then blocked with PBS containing 5% BSA for 1 h at room temperature. After washing, the cells were incubated with a mouse monoclonal antibody raised to GXM (mAb 18B7, 1 g/ml, generously provided by Dr. Arturo Casadevall, Albert Einstein College of Medicine) followed by anti-PFK rat polyclonal antibodies produced as previously described (43). Cells were washed again and then incubated with secondary antibodies for 1 h at room temperature. Texas Red-labeled anti-rat IgG (Millipore, São Paulo, Brazil) was used (at 1:500 dilution) to recognize anti-PFK antibodies, and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Sigma) was used (at 1:500 dilution) to detect mAb 18B7. Preimmune serum of the rats used to produce the anti-PFK antibody was used as control for this antibody. Cells that were not incubated with GXM were used as the control for the mAb 18B7 staining. Controls lacking incubation with primary antibodies were also used to evaluate the nonspecific labeling of the secondary antibodies used. Glass coverslips were placed in mounting medium (50% glycerol and 50 mM N-propyl gallate in PBS) over glass slides. Fluorescence confocal microscopy images were collected as described previously (35) with an Axiovert 200M motorized inverted microscope. Detection setup consisted of a LSM 510 META NLO system (Carl Zeiss). Samples were excited with the 488-nm laser line of the 50-milliwatt air-cooled argon ion laser under the control of an acousto-optical tunable filter set to 13% transmission. Excitation light was directed to the sample by a dichroic mirror (HFT 488/543) and through a Zeiss Plan-Apochromat 63ϫ/1.4 oil differential interference contrast objective lens. The fluorescence light collected by the objective was sent through the dichroic mirror, split with a secondary dichroic mirror NFT 545, and selected by band pass filters BP500 -530 (FITC) and BP565-615 (TRITC) for two-channel acquisition. Conventional fluorescence and visible microscopy were performed using an Axioplan 2 fluorescence microscope (Zeiss). Images were acquired using a Color View SX digital camera and processed with the software system analySIS (Soft Image System).
Cell Viability Assay, Glucose Consumption, and Lactate Production-Cell viability assay, evaluation of glucose consumption, and lactate production were performed as described previously (44). Briefly, RAW 264.7 cells were grown in DMEM medium supplemented with 10% FBS until confluence was achieved. The medium was removed, fresh medium containing the desired concentrations of GXM was added, and cells were returned to the incubator for 24 h. After this incubation, the medium was removed and used to evaluate the glucose consumption and lactate production, whereas the remaining cells were used for cell viability evaluation through mitochondrial reduction of MTT reagent. Glucose consumption was performed assessing the glucose content that remained in the culture media using a coupled enzyme system containing glucose oxidase/catalase (Glucox 500, Doles Ltda, GO, Brazil). Lactate production was evaluated assessing the lactate content in the culture media, incubating the media in the presence of lactate dehydrogenase and NAD ϩ , and measuring the formation of NADH spectrophotometrically at 340 nm. As for the cell viability assay, cells were washed twice with PBS and 20 l of 5 mg/ml MTT reagent (3,4,5-dimethiazol-2,5-diphenyltetrazolium bromide, Sigma) was added. After 3 h at the incubator, the reagent was removed, and the formazan crystals formed were dissolved in 200 l of DMSO. Afterward, the formazan content was evaluated in a microplate reader set at 560 nm with the background at 670 nm subtracted.
Radiometric Assay for PFK Activity-PFK activity was measured by the method described in Sola-Penna et al. (45) with the modifications introduced in Zancan and Sola-Penna (46, 47) using a reaction medium containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 5 mM (NH 4 ) 2 SO 4 , 1 mM [␥-32 P]ATP (4 Ci/nmol), 1 mM F6P, and 1 g/ml purified PFK. Modifications to pH and the concentrations of ATP, F6P, and PFK are specified for each experiment in the figure legends. The reaction was stopped by the addition of a suspension of activated charcoal in 0.1 M HCl and 0.5 M mannitol. After centrifugation, the supernatant, which contained [1-32 P]fructose 1,6-bisphosphate, was analyzed in a liquid scintillation counter. Appropriate controls in the absence of fructose 6-phosphate were performed and subtracted from all measurements to discount ATP hydrolysis. One milliunit was considered as the formation of 1 nmol of fructose-1,6-bisphosphate/min.
Spectrophotometric Assay for PFK Activity-PFK activity was assayed as described previously (48) in a medium containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl 2 , 5 mM (NH 4 ) 2 SO 4 , the indicated concentrations of fructose 6-phosphate and ATP, 0.5 mM NADH, 2 milliunits/ml aldolase, 2 milliunits/ml triosephosphate isomerase, 2 milliunits/ml ␣-glycerophosphate dehydrogenase, and 0.5 g/ml protein for purified PFK or 50 g/ml protein for cell lysate in a final volume of 200 l. Other reagents used are indicated for each experiment. The reaction was started by the addition of protein, and NADH oxidation was followed by measuring the decrease in absorbance at 340 nm in a microplate reader. Blanks in the absence of the coupled enzymes were performed to control nonspecific NADH oxidation.
Intrinsic Fluorescence Measurements-Intrinsic fluorescence measurements of PFK were performed as described previously (49) using the same conditions described for the radioassay. Excitation wavelength was fixed at 280 nm, and fluorescence emission was scanned from 300 to 400 nm. The center of mass of the intrinsic fluorescence spectra (CM) was calculated using where is the wavelength, and I is the fluorescence intensity at a given . Center of mass is used to evaluate the oligomeric state of PFK because the dissociated enzyme exposes its tryptophans to the aqueous milieu to a greater extent than the oligomer; thus, the fluorescence emitted by these tryptophans is of lower energy. Consequently, the center of mass of a population of tetramers is smaller than that of a population of dimers, as confirmed in many recent publications (44, 50 -53).
Statistics and Calculations-Statistical analyses were performed using the software SigmaPlot 10.0 integrated with SigmaStat 3.51 (Systat, CA). Student's t test or one-tailed analysis of variance were used to evaluate the significance of different numerical values. p Ͻ 0.05 was considered to be statistically significant.
Kinetic parameters for the effects of ATP on PFK were calculated considering the two components for PFK modulation by this metabolite. The first component is the stimulatory component for the substrate saturation curve, in which PFK exhibits an allosteric pattern that is described by the equation where v is the PFK activity at a given concentration of ATP ([ATP]), V max app is the apparent maximal velocity calculated, K 0.5 is the affinity constant for this component, and n s is the cooperativity index for this component. The second component is the inhibitory component that can be adjusted by the equation where v is the PFK activity at a given concentration of ATP which was fitted to the experimental data through non-linear regression for the effects of ATP on PFK activity. Kinetic parameters for the effects of F6P on PFK were calculated through non-linear regression using the experimental data to fit the parameters of the equation where v is the PFK activity calculated for a given concentration of F6P ([F6P]), V max is the maximal velocity calculated at saturating concentrations of F6P, K 0.5 is the affinity constant for F6P, which is equal to the concentration of F6P responsible for half-activation of the PFK by F6P, and n is the cooperativity index for this phenomenon.

RESULTS
GXM Decreases Glycolytic Flux-Incubation of RAW 264.7 cells for 24 h in the presence of increasing concentrations of GXM (0, 1, 10, and 100 g/ml) promoted a dose-dependent decrease in cell viability, as assessed through the number of cells that were not permeable to trypan blue (Fig. 1A). In parallel to this effect, mitochondrial reductive function, determined by the ability of the cells to reduce the MTT reagent (Fig. 1B), glucose consumption (Fig. 1C), and lactate production (Fig. 1D) were decreased. These data indicate that GXM alters cell metabolism, decreasing the glycolytic flux. Aiming at investigating the mechanism by which GXM inhibited the glycolytic flux, we evaluated the activity of the major regulatory enzyme within glycolysis, 6-phosphofructo-1-kinase (PFK) (52,53) under the same conditions used for the experiments described in Fig. 1. Incubation of RAW 264.7 for 24 h with increasing concentrations of GXM also promoted the inhibition of PFK activity in a dose-dependent fashion ( Fig. 2A). This effect is also dependent on the preincubation time, as can be seen in Fig. 2B. After the 1-h incubation, a significant PFK inhibition was observed after exposure to macrophages to 100 g/ml GXM. After the 3-h exposure, however, all the concentrations of GXM tested caused PFK inhibition (Fig. 2B). These data are indicative that inhibition of the enzyme is not dependent on translational events and can occur due to a direct effect of GXM over PFK.
GXM Co-localizes with PFK within Macrophages-As documented previously, GXM binds to and is taken up by macrophages (32,54), accumulating in the cytosol. Considering that GXM inhibits PFK-1 activity by a mechanism that precedes translational events, we investigated whether the polysaccharide and PFK-1 co-localize in GXM-treated host cells. As described previously (32,54), GXM accumulated in the cytosol (Fig. 3A). As expected, PFK was also abundantly detected in the cytoplasm (Fig. 3B). Image merging revealed a clear superposition of the cellular distribution of a subpopulation of GXM and PFK (Fig. 3C). Analysis of distinct z-sections confirmed that a fraction of the protein and a fraction of the PS co-localize within the cytosol (Fig. 3D). The large cellular compartment lacking staining corresponded to the nucleus, as revealed by control experiments using DAPI (data not shown). The specificity of the antibodies used was tested in cells that were not treated with GXM. No fluorescence signal was detected in the cytosol of these cells upon the same treatment that generated the image presented in Fig. 3A (supplemental Fig. S1). The faint fluorescence signal observed for both secondary antibodies in the nuclear area (Fig. S1, C and D) is due to some unspecific labeling of the secondary antibodies used. This is clear when the cells were incubated only with the secondary antibodies, lacking the initial incubation with anti-PFK or anti-GXM antibodies (supplemental Fig. S2). Moreover, this weak fluorescence labeling was observed solely when the acquisition gain is at the maximal level. This fact explains why it was not observed in Fig.  3, where the strong fluorescence signal of specific labeling of PFK and GXM in the cytosol was observed, and the detector gain was adjusted for this signal. The specificity of the anti-PFK antibody was evaluated using RAW 264.7 extracts submitted to SDS-PAGE and Western blotting, where a single staining with ϳ85 kDa was observed. Moreover, the cells were also treated with serum from rats used to grow the anti-PFK antibody before the immunization of the animals. After incubation of these cells with both secondary antibodies, the faint signal observed was comparable with that shown in supplemental Fig.  S2 where only secondary antibodies were employed (supplemental Fig. S3). These data confirm the high specificity of the antibodies used and prove that the fluorescence signals shown in Fig. 3, A and B, are relative to the presence of GXM and PFK in the cytosol of RAW 264.7.
PFK Activity Is Directly Influenced by GXM-To investigate whether GXM could exert a direct action over PFK, we evaluated the effects of the PS on the activity of the purified enzyme. Incubation of PFK in the presence of increasing concentrations of GXM resulted in a dose-dependent inhibition of the enzyme (Fig. 4A, filled circles). Removal of the O-acetyl groups of the PS did not affect its inhibitory properties over PFK (Fig. 4A, empty   circles). In contrast, glucuronyl-associated negative charge of the PS was apparently required for inhibitory activity. This conclusion was based on the fact that partial reduction of the carboxyl groups of glucuronic acids (35% reduction) resulted in attenuation of PS inhibitory properties (Fig. 4A, filled triangles). Carboxyl reduction, in fact, was linearly correlated with attenuation of the GXM-induced PFK inhibition (Fig. 4B). To evaluate whether the inhibitory property of GXM on PFK activity was specific or not, other anionic PS were tested (Table 1). We observed that hyaluronic acid and chondroitin sulfate also inhibited PFK at distinct levels. Hyaluronic acid promoted a discrete inhibition, whereas chondroitin sulfate was as effective as GXM. Carboxyl reduction of chondroitin sulfate also attenuated its inhibitory effects, corroborating that the negative charges are important for PFK inhibition by the PS. This conclusion was supported by the fact that non-charged polysaccharides, such as glycogen and chitosan, did not alter PFK activity.
The inhibition promoted by GXM was not dependent on substrate (F6P and ATP) concentration (Fig. 4, C and D, respectively) and resulted in the decrease of the affinity of the enzyme for both substrates as indicated by the increased affinity constants (K 0.5 ) for F6P and for ATP at its catalytic site ( Table 2). On the other hand, upon incubation with 100 g/ml GXM, PFK exhibited an increased affinity for ATP at the inhibitory allosteric site, as supported by the decreased inhibition constant (I 0.5 ) for ATP (Table 2). Upon incubation with GXM, PFK also presented a lower maximal velocity (V max ). These modifications on the kinetic parameters for the substrates interaction with PFK are compatible with those described when PFK oligomers dissociates from full active tetramers into weakly active dimers (52,53).
The center of mass of the intrinsic fluorescence emission spectrum of PFK is a powerful tool to evaluate the distribution of enzyme tetramers and dimers (35, 49 -53, 55, 56). Due to exposure to a polar environment, intrinsic fluorescence emission by dimers presents lower energy than by tetramers, and thus, the center of mass of the intrinsic fluorescence spectrum shifts toward the red region of the spectrum when tetramers dissociate into dimers. Therefore, we evaluated the center of mass of PFK intrinsic fluorescence emission spectra in the presence of increasing concentrations of GXM. The polysaccharide promoted a dose-dependent shift in the center of mass (Fig. 5A,  main panel), promoting no significant alterations on the emission spectra (Fig. 5A, inset), which is compatible with the dissociation of PFK tetramers into dimers. To reinforce the hypothesis that the inhibitory effects of GXM on PFK are due to the dissociation of the enzyme tetrameric conformation into dimers, we plotted a correlation between the center of mass of intrinsic fluorescence spectra (an indicative of dimerization) and the enzyme activity upon its incubation with GXM and its carboxyl-reduced derivative (Fig. 5B). This plot showed a strong negative correlation (r 2 ϭ 0.9997) supporting that the two phenomena are closely related.
GXM Inhibition Is Influenced by Regulatory Physiological Ligands of PFK-Alterations on the oligomeric equilibrium between PFK tetramers and dimers represent a common mechanism of regulation of the enzyme, which is affected by many physiologic ligands, such as ATP bound at the inhibitory allosteric site and its counteracting ligand, fructose 2,6-bisphosphate (F2,6BP) (52,53), calmodulin (49,51), and lactate (50) among others. Therefore, we evaluated the effects of GXM on PFK activity in the presence of several modulators (activators   and inhibitors) that promote their effects through altering the equilibrium between PFK tetramers and dimers. These experiments were performed under two experimental conditions; that is, when the catalytic site of the enzyme for ATP was saturated (1 mM ATP; Fig. 6A) and when the inhibitory allosteric site for ATP was saturated (5 mM ATP; Fig. 6B). GXM alone inhibited the enzyme in both conditions as already shown in Fig. 4C. This inhibition was attenuated by PFK activators F2,6BP and ADP and completely abrogated upon phosphorylation of the enzyme by PKA or binding of CaM (Fig. 6). Moreover, GXM potentiated the inhibitory effects of citrate and lactate under both conditions tested (Fig. 6).

DISCUSSION
During infection Cn secretes large amounts of GXM, which efficiently interferes with host immune response (for review, see Refs. 1, 21, 57, and 58). In this context, MOs are believed to be one of the many recipient cells for secreted polysaccharide (59). The receptors and kinetics of GXM internalization by macrophages have been studied as well as its effects on macrophage response, such as modulation of cytokines and nitric oxide production (22,54,60). GXM uptake culminates in apoptosis (33,54). However, metabolic changes that occur right after GXM internalization have never been investigated. In this study we demonstrate the ability of GXM to regulate the glycolytic enzyme PFK. We believe this phenomenon could represent a new pathogenic mechanism by which C. neoformans could interfere with the physiology of host cells. Lipopolysaccharide was demonstrated to regulate PFK activity (61), but to our knowledge this is the first report showing that a microbial polysaccharide inhibits PFK activity in host cells.
Our data suggested that the mechanism by which GXM interferes with PFK at initial periods of incubation involves the dissociation of the enzyme active tetramers into inactive dimers. Currently we have no tools to evaluate whether this dissociation occurs inside the cells, but the literature clearly shows that PFK dimer-tetramer equilibrium is affected by many intracellular components, such as filamentous actin, microtubules, and several metabolites (62). Therefore, it would be reasonable to hypothesize that GXM may inhibit PFK in cellular systems through the dissociation of the tetramers of the enzyme.
After binding to host receptors, the PS is internalized within few hours (32). Under the conditions used in our experiments a minimum of 4 -5 h was required for visualization of intracellular GXM. We observed an intracellular distribution of GXM after incubation with macrophages, as similarly described by Chang et al. (32). A fraction of the internalized GXM co-localized with part of PFK within macrophages, suggesting that the PS can associate to the enzyme. This observation and the fact that GXM decreases glucose consumption led us to investigate whether GXM interferes with PFK activity.
PFK is the key enzyme regulating glycolysis; therefore, it undergoes a complex regulation by several metabolites and cellular signals (62). Among the molecular mechanisms regulating PFK activity is the stabilization of PFK in distinct oligomeric conformations, where the transition between fully active tetramers and weakly active dimers appears to a key cellular event (62)(63)(64). This transition is involved in the regulation of PFK activity by several modulators, such as its substrates (52,53,62), allosteric ligands (50,62), hormones (35,47,48,55,62,65), other intracellular proteins (49, 51, 62, 66 -70), and drugs (44,56,62,(71)(72)(73)(74). Here, GXM appears as another modulator of this equilibrium, shifting the enzyme toward the dimeric conformation and, thus, inhibiting its catalytic activity. Considering that large amounts of this PS are taken up by macrophages and other host cells, we believe this effect could also be observed in other cell types. This could be considered a new and important pathogenic mechanism, in that the glycolytic pathway is highly dynamic and that major regulators of PFK are known to interfere with each other. In fact, it is clear that the inhibitory effects of GXM can be attenuated, abrogated, or potentiated, depending on which signal rules the enzyme activity. For instance, if the intracellular concentration of F2,6BP rises, e.g. due to insulin signaling (62,75), GXM inhibitory effects would be supposedly less pronounced as F2,6BP favors the formation of tetramers counteracting the formation of dimers favored by GXM. On the other hand, increased intracellular lactate content, which decreases PFK activity and cellular metabolism (50,62,76,77), would potentiate the inhibitory action of GXM over PFK, possibly turning the cell more susceptible to the effects of the polysaccharide. Moreover, cellular stimuli triggering the rise of intracellular calcium concentrations would favor the binding of CaM to PFK, which abrogates the inhibitory effects of GXM on PFK. CaM is described to lock PFK in a fully active dimeric conformation (49,51,62), abolishing the effects of ligands that favor the dissociation of tetramers.
GXM is negatively charged due to multiple residues of glucuronic acid. The network formed by GXM at the Cn surface is at least in part maintained by bridges formed by divalent cations (13). It is reasonable to suppose that free GXM would be able to scavenge mono and divalent cations in the intracellular milieu. Decreasing Ca 2ϩ availability would then be an additional effect caused by GXM uptake by host cells. However, these negative charges are also important in the inhibitory effects of GXM on PFK, as the reduction of the carboxyl groups and the consequent loss of charge attenuate the GXM-induced inhibition of PFK. It is clear that this inhibition strongly correlates with the change in the center of mass of intrinsic fluorescence spectra of the enzyme and that upon reduction of carboxyl groups the remaining inhibitory effects still correlate with the quaternary structure of PFK (see Fig. 5B). Therefore, it is likely that the mechanism through which PFK is inhibited by GXM involves the dissociation of the enzyme tetramers into dimers. Other negatively charged PSs also inhibited PFK to distinct extents. This observation allows us to suggest that negatively charged PSs are general inhibitors of PFK, which may affect the physiopathologic conditions in many other diseases. Some drugs are able to decrease the glycolytic flux through inhibition of PFK activity favoring the dissociation of the tetrameric conformation of the enzyme into dimers. Clotrimazole, an antifungal imidazole derivative, directly interacts with PFK, dissociating the fully active tetramers into dimers and, thus, inhibiting the enzyme (56,71). This is the putative mechanism responsible for the human breast cancer cell line MCF-7 death upon incubation with clotrimazole (43). Furthermore, acetylsalicylic acid decreases the viability of cancer cells by decreasing glucose consumption and lactate production through inhibition of PFK using the same mechanism (44). The present work shows for the first time that a fungal polysaccharide affects MO metabolism in a similar fashion, suggesting that microbes may subvert the metabolism of host cells by a previously unknown pathogenic mechanism.