Abstract
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.
Highlights
Tococcosis, a life-threatening invasive disease with a higher incidence in immunocompromised patients [4, 5]
The remaining capsular mass is formed by galactoxylomannan, an ␣1– 6-linked galactan containing mannosyl, glucuronyl, and xylosyl substitutions (2, 8 –11)
During infection Cn secretes large amounts of GXM, which efficiently interferes with host immune response
Summary
Materials—ATP, fructose 6-phosphate (F6P), fructose 2,6bisphosphate, ADP, 3Ј-5Ј-adenosine monophosphate cyclic nucleotide (cAMP), cAMP-dependent protein kinase (PKA; EC 2.7.11.11), chitosan, glycogen, hyaluronic acid, chondroitin sulfate, lactate, citrate, and calmodulin (CaM) were purchased from Sigma. 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 MgCl2, 5 mM (NH4)2SO4, 1 mM [␥-32P]ATP (4 Ci/nmol), 1 mM F6P, and 1 g/ml purified PFK. 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 MgCl2, 5 mM (NH4)2SO4, 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. 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 v ϭ
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