Cryptococcus gattii evades CD11b-mediated fungal recognition by coating itself with capsular polysaccharides.

Cryptococcus gattii is a capsular pathogenic fungus causing life-threatening cryptococcosis. Although the capsular polysaccharides (CPs) of C. gattii are considered as virulence factors, the physiological significance of CP biosynthesis and of CPs themselves is not fully understood, with many conflicting data reported. First, we demonstrated that CAP gene deletant of C. gattii completely lacked capsule layer and its virulence, and that the strain was susceptible to host-related factors including oxidizing, hypoxic, and hypotrophic conditions in vitro. Extracellular CPs recovered from culture supernatant bound specifically to C. gattii acapsular strains, not to other fungi and immune cells, and rendered them the immune escape effects. In fact, dendritic cells (DCs) did not efficiently uptake the CP-treated acapsular strains, which possessed no visible capsule layer, and a decreased amount of phosphorylated proteins and cytokine levels after the stimulation. DCs recognized C. gattii acapuslar cells via an immune receptor CD11b- and Syk-related pathway; however, CD11b did not bind to CP-treated acapsular cells. These results suggested that CPs support immune evasion by coating antigens on C. gattii and blocking the interaction between CD11b and C. gattii cells. Here, we describe the importance of CPs in pathogenicity and immune evasion mechanisms of C. gattii.

Light Signaling Regulates Aspergillus niger Biofilm Formation by Affecting Melanin and Extracellular Polysaccharide Biosynthesis.

Light is an important signal source in nature, which regulates the physiological cycle, morphogenetic pathways, and secondary metabolites of fungi. As an external pressure on Aspergillus niger, light signaling transmits stress signals into the cell via the mitogen-activated protein kinase (MAPK) signaling pathway. Studying the effect of light on the biofilm of A. niger will provide a theoretical basis for light in the cultivation of filamentous fungi and industrial applications. Here, the characterization of A. niger biofilm under different light intensities confirmed the effects of light signaling. Our results indicated that A. niger intensely accumulated protective mycelial melanin under light illumination. We also discovered that the RlmA transcription factor in the MAPK signaling pathway is activated by light signaling to promote the synthesis of melanin, chitin, and other exopolysaccharides. However, the importance of melanin to A. niger biofilm is rarely reported; therefore, we knocked out key genes of the melanin biosynthetic pathway-Abr1 and Ayg1 Changes in hydrophobicity and electrostatic forces resulted in the decrease of biofilm caused by the decrease of melanin in mutants.IMPORTANCE As an important industrial filamentous fungus, Aspergillus niger can perceive light. The link between light signaling and A. niger biofilm is worthy of further study since reports are lacking in this area. This study found that light signaling promotes biofilm production in A. niger, wherein melanin plays an important role. It was further discovered that the RlmA transcription factor in the mitogen-activated protein kinase (MAPK) signaling pathway was mediated by light signaling to promote the synthesis of melanin and extracellular polysaccharides. These findings set the stage for light signal regulation of biofilm in filamentous fungi and provide a theoretical basis for the development of a new light-controlled biofilm method to improve biofilm-based industrial fermentation.

Non-coding RNAs as cell wall regulators in Saccharomyces cerevisiae.

The cell wall of Saccharomyces cerevisiae is an extracellular organelle crucial for preserving its cellular integrity and detecting environmental cues. The cell wall is composed of mannoproteins attached to a polysaccharide network and is continuously remodelled as cells undergo cell division, mating, gametogenesis or adapt to stressors. This makes yeast an excellent model to study the regulation of genes important for cell wall formation and maintenance. Given that certain yeast strains are pathogenic, a better understanding of their life cycle is of clinical relevance. This is why transcriptional regulatory mechanisms governing genes involved in cell wall biogenesis or maintenance have been the focus of numerous studies. However, little is known about the roles of long non-coding RNAs (lncRNAs), a class of transcripts that are thought to possess little or no protein coding potential, in controlling the expression of cell wall-related genes. This review outlines currently known mechanisms of lncRNA-mediated regulation of gene expression in S. cerevisiae and describes examples of lncRNA-regulated genes encoding cell wall proteins. We suggest that the association of currently annotated lncRNAs with the coding sequences and/or promoters of cell wall-related genes highlights a potential role for lncRNAs as important regulators of the yeast cell wall structure.

Transcriptome analysis of three cultivars of Poria cocos reveals genes related to the biosynthesis of polysaccharides.

Poria cocos (P. cocos) polysaccharides (PCPs) are used to improve immunity and possess antitumor activities. We compared three cultivars of P. cocos (5.78, XJ 28 and JHYH) PCP contents. Then we determined that malZ, galA, SORD, gnl and bglX are key enzymes within the PCP biosynthetic pathway by using HiSeq2500 transcriptome and qRT-PCR validation. Our results provide more detailed information about the PCP biosynthesis pathway at the molecular level in P. cocos and establish the functions for the molecular breeding to produce polysaccharides in general for therapeutic use in Chinese medicinal plants.

Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and ß-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3.

Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall ß-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18-/- and Dectin-1-/- murine neutrophils, we found that the ß-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that ß-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4-/- mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4-/- neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18-/- neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4-/- corneas; however, we found no difference between C57BL/6 and PAD4-/- mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.

Fungal melanin stimulates surfactant protein D-mediated opsonization of and host immune response to Aspergillus fumigatus spores.

Surfactant protein D (SP-D), a C-type lectin and pattern-recognition soluble factor, plays an important role in immune surveillance to detect and eliminate human pulmonary pathogens. SP-D has been shown to protect against infections with the most ubiquitous airborne fungal pathogen, Aspergillus fumigatus, but the fungal surface component(s) interacting with SP-D is unknown. Here, we show that SP-D binds to melanin pigment on the surface of A. fumigatus dormant spores (conidia). SP-D also exhibited an affinity to two cell-wall polysaccharides of A. fumigatus, galactomannan (GM) and galactosaminogalactan (GAG). The immunolabeling pattern of SP-D was punctate on the conidial surface and was uniform on germinating conidia, in accordance with the localization of melanin, GM, and GAG. We also found that the collagen-like domain of SP-D is involved in its interaction with melanin, whereas its carbohydrate-recognition domain recognized GM and GAG. Unlike un-opsonized conidia, SP-D-opsonized conidia were phagocytosed more efficiently and stimulated the secretion of proinflammatory cytokines by human monocyte-derived macrophages. Furthermore, SP-D-/- mice challenged intranasally with wildtype conidia or melanin ghosts (i.e. hollow melanin spheres) displayed significantly reduced proinflammatory cytokines in the lung compared with wildtype mice. In summary, SP-D binds to melanin present on the dormant A. fumigatus conidial surface, facilitates conidial phagocytosis, and stimulates the host immune response.

Regulated Release of Cryptococcal Polysaccharide Drives Virulence and Suppresses Immune Cell Infiltration into the Central Nervous System.

Cryptococcus neoformans is a common environmental yeast and opportunistic pathogen responsible for 15% of AIDS-related deaths worldwide. Mortality primarily results from meningoencephalitis, which occurs when fungal cells disseminate to the brain from the initial pulmonary infection site. A key C. neoformans virulence trait is the polysaccharide capsule. Capsule shields C. neoformans from immune-mediated recognition and destruction. The main capsule component, glucuronoxylomannan (GXM), is found both attached to the cell surface and free in the extracellular space (as exo-GXM). Exo-GXM accumulates in patient serum and cerebrospinal fluid at microgram/milliliter concentrations, has well-documented immunosuppressive properties, and correlates with poor patient outcomes. However, it is poorly understood whether exo-GXM release is regulated or the result of shedding during normal capsule turnover. We demonstrate that exo-GXM release is regulated by environmental cues and inversely correlates with surface capsule levels. We identified genes specifically involved in exo-GXM release that do not alter surface capsule thickness. The first mutant, the liv7Δ strain, released less GXM than wild-type cells when capsule was not induced. The second mutant, the cnag_00658Δ strain, released more exo-GXM under capsule-inducing conditions. Exo-GXM release observed in vitro correlated with polystyrene adherence, virulence, and fungal burden during murine infection. Additionally, we found that exo-GXM reduced cell size and capsule thickness under capsule-inducing conditions, potentially influencing dissemination. Finally, we demonstrated that exo-GXM prevents immune cell infiltration into the brain during disseminated infection and highly inflammatory intracranial infection. Our data suggest that exo-GXM performs a distinct role from capsule GXM during infection, altering cell size and suppressing inflammation.

Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase.

Asparagine ( N)-linked glycosylation requires the ordered, stepwise synthesis of lipid-linked oligosaccharide (LLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (Glc3Man9Gn2-PDol) on the endoplasmic reticulum. The fourth and fifth steps of LLO synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities; Alg2 adds both an α1,3- and α1,6-mannose onto ManGlcNAc2-PDol to form the trimannosyl core Man3GlcNAc2-PDol. The biochemical properties of Alg2 are controversial and remain undefined. In this study, a liquid chromatography/mass spectrometry-based quantitative assay was established and used to analyze the MTase activities of purified yeast Alg2. Alg2-dependent Man3GlcNAc2-PDol production relied on net-neutral lipids with a propensity to form bilayers. We further showed addition of the α1,3- and α1,6-mannose can occur independently in either order but at differing rates. The conserved C-terminal EX7E motif, N-terminal cytosolic tail, and 3 G-rich loop motifs in Alg2 play crucial roles for these activities, both in vitro and in vivo. These findings provide insight into the unique bifunctionality of Alg2 during LLO synthesis and lead to a new model in which alternative, independent routes exist for Alg2 catalysis of the trimannosyl core oligosaccharide.-Li, S.-T., Wang, N., Xu, X.-X., Fujita, M., Nakanishi, H., Kitajima, T., Dean, N., Gao, X.-D. Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase.

The water channel protein aquaporin 1 regulates cellular metabolism and competitive fitness in a global fungal pathogen Cryptococcus neoformans.

In this study, an aquaporin protein, Aqp1, in Cryptococcus neoformans, which can lead either saprobic or parasitic lifestyles and causes life-threatening fungal meningitis was identified and characterized. AQP1 expression was rapidly induced (via the HOG pathway) by osmotic or oxidative stress. In spite of such transcriptional regulation, Aqp1 was found to be largely unnecessary for adaptation to diverse environmental stressors, regardless of the presence of the polysaccharide capsule. The latter is shown here to be a key environmental-stress protectant for C. neoformans. Furthermore, Aqp1 was not required for the development and virulence of C. neoformans. Deletion of AQP1 increased hydrophobicity of the cell surface. The comparative metabolic profiling analysis of the aqp1Δ mutant and AQP1-overexpressing strains revealed that deletion of AQP1 significantly increased cellular accumulation of primary and secondary metabolites, whereas overexpression of AQP1 depleted such metabolites, suggesting that this water channel protein performs a critical function in metabolic homeostasis. In line with this result, it was found that the aqp1Δ mutant (which is enriched with diverse metabolites) survived better than the wild type and a complemented strain, indicating that Aqp1 is likely to be involved in competitive fitness of this fungal pathogen.

Catalytic Mechanism of a Novel Glycoside Hydrolase Family 16 "Elongating" ß-Transglycosylase.

Carbohydrates are complex macromolecules in biological metabolism. Enzymatic synthesis of carbohydrates is recognized as a powerful tool to overcome the problems associated with large scale synthesis of carbohydrates. Novel enzymes with significant transglycosylation ability are still in great demand in glycobiology studies. Here we report a novel glycoside hydrolase family 16 "elongating" ß-transglycosylase from Paecilomyces thermophila (PtBgt16A), which efficiently catalyzes the synthesis of higher polymeric oligosaccharides using ß-1,3/1,4-oligosaccharides as donor/acceptor substrates. Further structural information reveals that PtBgt16A has a binding pocket around the -1 subsite. The catalytic mechanism of PtBgt16A is partly similar to an exo-glycoside hydrolase, which cleaves the substrate from the non-reducing end one by one. However, PtBgt16A releases the reducing end product and uses the remainder glucosyl as a transglycosylation donor. This catalytic mechanism has similarity with the catalytic mode of amylosucrase, which catalyzes the transglycosylation products gradually extend by one glucose unit. PtBgt16A thus has the potential to be a tool enzyme for the enzymatic synthesis of new ß-oligosaccharides and glycoconjugates.

High-Level Production of Exopolysaccharides by a Cosmic Radiation-Induced Mutant M270 of the Maitake Medicinal Mushroom, Grifola frondosa (Agaricomycetes).

A new Grifola frondosa mutant, M270, was successfully isolated for high production of exopolysaccharides (EPSs) using cosmic radiation-induced mutagenesis. We found that the mutant M270 had a clearer and thicker EPS layer (~10 µm) adhering to mycelia than those of its parent strain 265 after Congo red staining. In the 20-L batch fermentation for M270, 10.3 g/L of EPS and 17.9 g/L of dry mycelia biomass were obtained after 204 hours of fermentation. Furthermore, a main water-soluble fraction (EP1) in the EPS was purified from M270 and then confirmed to be heteroglycan-protein complex with 91% (w/w) total carbohydrates and 9% (w/w) total proteins. Four kinds of monosaccharide-D-mannose, D-glucosamine, D-glucose, and D-xylose-were detected in EP1 with a molar ratio of 17.6:1.8:100:2.5. The molecular mass of the main component in EP1 was 8.9 kDa. The EPS from M270 significantly inhibited the growth of sarcoma 180 solid tumors in mice. This G. frondosa M270 mutant could serve as a better candidate strain for polysaccharide production.

A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae.

N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae.

Preparation and characterization of a novel extracellular polysaccharide with antioxidant activity, from the mangrove-associated fungus Fusarium oxysporum.

Marine fungi are recognized as an abundant source of extracellular polysaccharides with novel structures. Mangrove fungi constitute the second largest ecological group of the marine fungi, and many of them are new or inadequately described species and may produce extracellular polysaccharides with novel functions and structures that could be explored as a source of useful polymers. The mangrove-associated fungus Fusarium oxysporum produces an extracellular polysaccharide, Fw-1, when grown in potato dextrose-agar medium. The homogeneous Fw-1 was isolated from the fermented broth by a combination of ethanol precipitation, ion-exchange, and gel filtration chromatography. Chemical and spectroscopic analyses, including one- and two-dimensional nuclear magnetic resonance spectroscopies showed that Fw-1 consisted of galactose, glucose, and mannose in a molar ratio of 1.33:1.33:1.00, and its molecular weight was about 61.2 kDa. The structure of Fw-1 contains a backbone of (1 → 6)-linked ß-D-galactofuranose residues with multiple side chains. The branches consist of terminal α-D-glucopyranose residues, or short chains containing (1 → 2)-linked α-D-glucopyranose, (1 → 2)-linked ß-D-mannopyranose, and terminal ß-D-mannopyranose residues. The side chains are connected to C-2 of galactofuranose residues of backbone. The antioxidant activity of Fw-1 was evaluated with the scavenging abilities on hydroxyl, superoxide, and 1,1-diphenyl-2-picrylhydrazyl radicals in vitro, and the results indicated that Fw-1 possessed good antioxidant activity, especially the scavenging ability on hydroxyl radicals. The investigation demonstrated that Fw-1 is a novel galactofuranose-containing polysaccharide with different structural characteristics from extracellular polysaccharides from other marine microorganisms and could be a potential source of antioxidant.

Caspase-8 modulates dectin-1 and complement receptor 3-driven IL-1ß production in response to ß-glucans and the fungal pathogen, Candida albicans.

Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1ß maturation and resistance to fungal dissemination in Candida albicans infection. ß-Glucans are major components of fungal cell walls that trigger IL-1ß secretion in both murine and human immune cells. In this study, we sought to determine the contribution of ß-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1ß production in response to ß-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating ß-glucan-induced IL-1ß processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting ß-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1ß maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1ß production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating ß-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between ß-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.

Overlapping and distinct roles of Aspergillus fumigatus UDP-glucose 4-epimerases in galactose metabolism and the synthesis of galactose-containing cell wall polysaccharides.

The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.

Deletion of the α-(1,3)-glucan synthase genes induces a restructuring of the conidial cell wall responsible for the avirulence of Aspergillus fumigatus.

α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.

Aspergillus galactosaminogalactan mediates adherence to host constituents and conceals hyphal ß-glucan from the immune system.

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall ß-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.

Unusual galactofuranose modification of a capsule polysaccharide in the pathogenic yeast Cryptococcus neoformans.

Galactofuranose (Galf) is the five-membered ring form of galactose. Although it is absent from mammalian glycans, it occurs as a structural and antigenic component of important cell surface molecules in a variety of microbes, ranging from bacteria to parasites and fungi. One such organism is Cryptococcus neoformans, a pathogenic yeast that causes lethal meningoencephalitis in immunocompromised individuals, particularly AIDS patients. C. neoformans is unique among fungal pathogens in bearing a complex polysaccharide capsule, a critical virulence factor reported to include Galf. Notably, how Galf modification contributes to the structure and function of the cryptococcal capsule is not known. We have determined that Galf is ß1,2-linked to an unusual tetrasubstituted galactopyranose of the glucuronoxylomannogalactan (GXMGal) capsule polysaccharide. This discovery fills a longstanding gap in our understanding of a major polymer of the cryptococcal capsule. We also engineered a C. neoformans strain that lacks UDP-galactopyranose mutase; this enzyme forms UDP-Galf, the nucleotide sugar donor required for Galf addition. Mutase activity was required for the incorporation of Galf into glucuronoxylomannogalactan but was dispensable for vegetative growth, cell integrity, and virulence in a mouse model.