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Title: Xylose donor transport is critical for fungal virulence}
Article:
Abstract:
Cryptococcus neoformans, an AIDS-defining opportunistic pathogen, is the leading cause of fungal meningitis worldwide and is responsible for hundreds of thousands of deaths annually. Cryptococcal glycans are required for fungal survival in the host and for pathogenesis. Most glycans are made in the secretory pathway, although the activated precursors for their synthesis, nucleotide sugars, are made primarily in the cytosol. Nucleotide sugar transporters are membrane proteins that solve this topological problem, by exchanging nucleotide sugars for the corresponding nucleoside phosphates. The major virulence factor of C. neoformans is an anti-phagocytic polysaccharide capsule that is displayed on the cell surface; capsule polysaccharides are also shed from the cell and impede the host immune response. Xylose, a neutral monosaccharide that is absent from model yeast, is a significant capsule component. Here we show that Uxt1 and Uxt2 are both transporters specific for the xylose donor, UDP-xylose, although they exhibit distinct subcellular localization, expression patterns, and kinetic parameters. Both proteins also transport the galactofuranose donor, UDP-galactofuranose. We further show that Uxt1 and Uxt2 are required for xylose incorporation into capsule and protein; they are also necessary for C. neoformans to cause disease in mice, although surprisingly not for fungal viability in the context of infection. These findings provide a starting point for deciphering the substrate specificity of an important class of transporters, elucidate a synthetic pathway that may be productively targeted for therapy, and contribute to our understanding of fundamental glycobiology.
Introduction:
Glycans are critical for the normal development, growth, and viability of organisms across all kingdoms of life. The extensive glycoconjugate repertoire of Cryptococcus neoformans, a ubiquitous environmental fungus, enables this pathogen to cause serious respiratory disease in the setting of immune compromise. This pulmonary infection often progresses to a lethal meningoencephalitis, even with treatment, leading to several hundred thousand deaths each year[1–3]. The major virulence factor of C. neoformans, a polysaccharide capsule, acts as a physical barrier against host defenses when associated with the cell wall and as an immune modulator when shed into the extracellular space[4, 5]. This material consists primarily of two complex polysaccharides, glucuronoxylomannan( GXM) and glucuronoxylomannanogalactan( GXMGal)[4]. The more abundant capsule component, GXM, is a linear mannose( Man) polymer with single glucuronic acid( GlcA) and xylose( Xyl) side chains[6]. The second polysaccharide, GXMGal, consists of a galactose backbone modified with single galactofuranose( Galf) residues and galactomannan side chains bearing a variable number of GlcA and Xyl residues[7–9]. Beyond the capsule, C. neoformans glycoconjugates include proteins with N- and O-linked glycans that resemble the corresponding mannose structures of the model yeast Saccharomyces cerevisiae, although they are further modified with Xyl or Xyl-phosphate residues[10–13]. Cryptococcal glycosphingolipids range from simple mannose modification of lipids to more complex structures that also incorporate galactose( Gal) and Xyl[14], and the cryptococcal cell wall consists of glucans, chitin, chitosan, and mannoproteins, many of which bear GPI anchors[15]. These glycans play integral structural and regulatory roles to facilitate fungal survival and pathogenesis[16]. Consistent with the abundant glycosylation of C. neoformans, a significant portion of its genetic machinery and metabolic energy is dedicated to glycan synthesis. These synthetic reactions typically occur in the secretory pathway, although they rely on nucleotide sugar donors that are synthesized in the cytosol[17]. The charged donors enter the luminal space via nucleotide sugar transporters( NSTs), which exchange activated sugars for the corresponding nucleoside monophosphates[18, 19]. NSTs thus mediate a limiting step in glycan biosynthesis, and are consequently required for cryptococcal viability and pathogenicity[20–22]. Our focus is on defining glycan synthesis in C. neoformans, motivated by its unique biology and critical role in a deadly disease. Cryptococcal NSTs comprise a key subset of this machinery, which has stimulated us to identify these proteins and their functions. This effort is complicated by the observations that NST homology is not always a reliable predictor of substrate specificity and that NSTs may be functionally redundant. Individual NSTs also range from highly specific single-substrate transporters to more promiscuous, multi-substrate proteins[23–28]. NST substrate specificity may also be modulated by localization to a particular cellular compartment and/or association with other glycan biosynthetic enzymes[21, 29]. In prior work, protein structure predictions and homology facilitated identification of the cryptococcal NSTs responsible for GDP-Man[21, 30] and UDP-Gal[20, 22] transport. We have now used product analysis and mass spectrometry based assays to discover Uxt1 and Uxt2, which both transport UDP-Xyl and UDP-Galf although they exhibit distinct subcellular localization, expression patterns, and kinetic parameters. Cells without these two proteins lack Xyl in all analyzed glycoconjugates and exhibit growth defects and metabolic abnormalities that are present to a lesser extent in single mutant strains. We further made the unexpected finding that transporter function is required for virulence in a mouse model of disease, but not for persistence in that context.
Discussion:
C. neoformans encodes an unusual pair of highly homologous UDP-Xyl/UDP-Galf transporters, which together are critical for virulence. Uxt1 and Uxt2 are unique for their high affinity for UDP-Xyl( Fig 2), with KM values almost two orders of magnitude lower than those of the Arabidopsis UDP-Xyl transporters[36]. Despite transporting the same nucleotide sugars, the two proteins are not completely functionally redundant, likely due to differences in expression, enzyme kinetics, and localization. Beyond nucleotide sugars known to occur in C. neoformans, in vitro Uxt1 and Uxt2 also transport UDP-Arap and UDP-Araf( Fig 2), potentially enabled by the similar structures of Xyl and Ara( D-Xyl and L-Ara are epimers). While the NSTs most closely related to Uxt1 and Uxt2( At UAfT1-4) are highly specific for UDP-Araf[37], the substrate range of the cryptococcal proteins most closely resembles that of plant UDP-Xyl transporters( UXT1-3;[36]) despite their sequence divergence( S1B Fig). These observations highlight the importance of using rigorous biochemical analysis to test functional assumptions based on homology. Since UDP-Ara is not found in cryptococcal cells and abrogating UDP-Galf synthesis does not alter cryptococcal growth or virulence[8], the phenotypes associated with loss of Uxt1 and Uxt2 likely result from disruption of UDP-Xyl transport into the secretory compartment. Notably, capsule material was still produced( Fig 5) even when no Xyl was detected in GXM because both transporters were absent( Fig 1A). This suggests that Xyl incorporation is not required for GXM backbone synthesis or elongation, or for incorporation of GlcA. However, lack of the Xyl donor did reduce the amount of shed capsule material by over 75%( S5C Fig). Since Xyl constitutes only 20–30% of the capsule mass, loss of this moiety alone does not explain this reduction. Instead, it may be a direct effect of the reduced Xyl incorporation, if these side chains are needed for capsule recognition by synthetic or trafficking machinery, or an indirect effect, for example if synthetic enzymes must be xylosylated to function efficiently. Lack of UDP-Xyl transport also yielded thinner capsules( Fig 5B, S5A Fig) with abnormal fiber morphology( Fig 5C, S5D Fig); this presumably results from the lack of Xyl substitution, which may be required for proper conformation or organization of capsule polysaccharides. Why does C. neoformans express two UDP-Xyl transporters? Judging by the severity of mutant phenotypes( Fig 5) and the gene expression levels( Fig 3), Uxt1 is the major transporter of the pair, but loss of both is required to eliminate Xyl incorporation( Fig 1). These data exclude the possibility of a third UDP-Xyl transporter of any significance, while highlighting the unequal contribution of these two proteins. One factor in this inequity is likely the higher affinity and catalytic efficiency for UDP-Xyl transport of Uxt1 compared to Uxt2( Fig 2). Another is probably their distinct regulatory patterns, with UXT1 expressed constitutively, while UXT2 expression levels is upregulated in response to greater glycan biosynthetic demands( Fig 3). Curiously, expression of the two genes was not optimally regulated to enable compensation in the single mutants: expression of UXT1 did not change in response to the loss of UXT2 even in capsule-inducing conditions, and the normal UXT2 induction was muted in the absence of UXT1( S8 Fig). Future studies will address this regulatory relationship. The distinct roles of Uxt1 and Uxt2 also potentially reflect their association with other glycan synthetic proteins, such as glycosyltransferases. We found no evidence of association with specific xylosyltransferase( s), as for example preferential loss of β-1, 2 or β-1, 4 linked Xyl in the GXM of either mutant( S1 Table). However, the full cryptococcal glycan repertoire is not known; future studies may enable us to identify specific protein or lipid modifications enabled by each enzyme. Another factor in the dominant role of Uxt1 is likely its localization to the Golgi( Fig 4B), the probable site of capsule and protein xylosylation[10, 11, 14], in contrast to the ER localization of Uxt2( Fig 4A). The latter is intriguing, as this compartment is upstream of most glycan synthesis. It is possible that Uxt2 has transport-independent functions, or that it supplies novel synthetic processes that have yet to be described. These will be exciting areas for future investigation. The increased sensitivity to stress( Fig 5A) and greater uptake by host phagocytes( Fig 6A) of uxt1Δ were insufficient to alter its behavior in animal infection( Fig 6D). We expected the highly impaired double mutant uxt1Δ uxt2Δ, which cannot transport UDP-Xyl into the secretory pathway, to behave like strains that cannot synthesize UDP-Xyl( uxs1Δ), which are avirulent and completely cleared by 7 days post-infection[38]. Surprisingly, this mutant persisted in the lungs( Fig 6E), suggesting either a cytosolic role for UDP-Xyl or a UDP-Xyl-independent role for Uxs1; these possibilities remain to be investigated. The double mutant population increased very slowly in both A/JCr and C57BL/6 mice, likely due to its slower growth rate under stress( Fig 6C, S4 Fig) and reduced ability to resist host defenses( Fig 6A and 6B). Xyl modifications have been identified as immunodominant epitopes in antibody responses to allergens and pathogens[39, 40], and the absence of Xyl modifications in uxt1Δ uxt2Δ did increase immune detection and clearance of the pathogen in vitro( Fig 6A and 6B). The mutant also remained confined to the lungs of A/JCr mice( S7 Fig) and was slow to cause lethal meningoencephalitis in C57BL/6 mice( S6 Fig). This may reflect an inability to disseminate or to efficiently establish infection at distal sites, or may be the result of active restriction by the immune system. Notably, phagocytes have a multifaceted role in cryptococcal infection, potentially aiding and/or inhibiting fungal survival and dissemination depending on the circumstance[35]. Elucidating the complex interplay between Uxt mutants and the infected host will be the focus of future work. Further studies may also uncover facets of this infection that could be exploited for therapeutic intervention and potentially inform vaccine design. C. neoformans is unusual among yeast for its extensive utilization of Xyl, in capsule polysaccharides, N- and O-linked glycans( including a unique Xyl-phosphate modification), and glycoplipids. By elucidating UDP-Xyl transport, we have expanded our understanding of this aspect of cryptococcal glycan biosynthesis, including the sequence and localization of capsule synthetic events, and of NSTs as a protein family. We have identified the first fungal UDP-Xyl/UDP-Galf transporters and also set the stage for studies of an unusual mutant that may help elucidate mechanisms of cryptococcal pathogenesis and host response.
Cryptococcus neoformans , the leading cause of fungal meningitis , kills almost a quarter of a million people each year . Carbohydrate structures , including a complex polysaccharide capsule , enable this microbe to resist host defenses and cause disease . The machinery to build these structures is located inside of specific subcellular compartments . However , most of the reactions require as precursors activated sugar molecules that are made in the cell cytosol . To bring these compounds , called nucleotide sugars , to the site of synthesis , the cell expresses specific transporters . One precursor of particular interest is UDP-xylose , which is the donor of the sugar xylose for the synthesis of cryptococcal capsule , proteins , and lipids . We have identified two UDP-xylose transporters , the first discovered in a fungal organism , and characterized their biochemical properties and role in biology and infection . We found that although this pair of proteins shares biochemical functions , including additional transport of UDP-galactofuranose , the two transporters differ in terms of where they are in the cell , when they are expressed , and their transport properties . Cryptococcal cells lacking both proteins do not cause disease in mice , demonstrating the importance of this synthetic pathway . These findings elucidate fundamental biology as well as fungal pathogenesis .