Mannan Levels Research Articles

Alteration of β-glucan in the emerging fungal pathogen Candida auris leads to immune evasion and increased virulence

Candida auris is an emerging pathogenic yeast that has been categorized as a global public health threat and a critical priority among fungal pathogens. Despite this, the immune response against C. auris infection is still not well understood. Hosts fight Candida infections through the immune system that recognizes pathogen-associated molecular patterns such as β-glucan, mannan, and chitin on the fungal cell wall. In this study, levels of β-glucan and mannan exposures in C. auris grown under different physiologically relevant stimuli were quantified by flow cytometry-based analysis. Lactate, hypoxia, and sublethal concentration of fluconazole trigger a decrease in surface β-glucan while low pH triggers an increase in β-glucan. There is no inverse pattern between exposure levels of β-glucan and mannan in the cell wall architecture among the three clades. To determine the effect of cell wall remodeling on the immune response, a phagocytosis assay was performed, followed by quantification of released cytokines by ELISA. Lactate-induced decrease in β-glucan leads to reduced uptake of C. auris by PMA-differentiated THP-1 and RAW 264.7 macrophages. Furthermore, reduced production of CCL3/MIP-1⍺ but not TNF-⍺ and IL-10 were observed. An in vivo infection analysis using silkworms reveals that a reduction in β-glucan triggers an increase in the virulence of C. auris. This study demonstrates that β-glucan alteration occurs in C. auris and serves as an escape mechanism from immune cells leading to increased virulence.

A chemically induced attenuated strain of Candida albicans generates robust protective immune responses and prevents systemic candidiasis development.

Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a priority. Here, we could for the first time successfully attenuate the virulence of Candida albicans by treating it with a fungistatic dosage of EDTA and demonstrate it to be a potential live whole cell vaccine by using murine models of systemic candidiasis. EDTA inhibited the growth and biofilm formation of C. albicans. RNA-seq analyses of EDTA-treated cells (CAET) revealed that genes mostly involved in metal homeostasis and ribosome biogenesis were up- and down-regulated, respectively. Consequently, a bulky cell wall with elevated levels of mannan and β-glucan, and reduced levels of total monosomes and polysomes were observed. CAET was eliminated faster than the untreated strain (Ca) as found by differential fungal burden in the vital organs of the mice. Higher monocytes, granulocytes, and platelet counts were detected in Ca- vs CAET-challenged mice. While hyper-inflammation and immunosuppression caused the killing of Ca-challenged mice, a critical balance of pro- and anti-inflammatory cytokines-mediated immune responses are the likely reasons for the protective immunity in CAET-infected mice.

A chemically induced attenuated strain of Candida albicans generates robust protective immune responses and prevents systemic candidiasis development

Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a priority. Here, we could for the first time successfully attenuate the virulence of Candida albicans by treating it with a fungistatic dosage of EDTA and demonstrate it to be a potential live whole cell vaccine by using murine models of systemic candidiasis. EDTA inhibited the growth and biofilm formation of C. albicans. RNA-seq analyses of EDTA-treated cells (CAET) revealed that genes mostly involved in metal homeostasis and ribosome biogenesis were up- and down-regulated, respectively. Consequently, a bulky cell wall with elevated levels of mannan and β-glucan, and reduced levels of total monosomes and polysomes were observed. CAET was eliminated faster than the untreated strain (Ca) as found by differential fungal burden in the vital organs of the mice. Higher monocytes, granulocytes, and platelet counts were detected in Ca- vs CAET-challenged mice. While hyper-inflammation and immunosuppression caused the killing of Ca-challenged mice, a critical balance of pro- and anti-inflammatory cytokines-mediated immune responses are the likely reasons for the protective immunity in CAET-infected mice.

Chara — a living sister to the land plants with pivotal enzymic toolkit for mannan and xylan remodelling

AbstractLand‐plant transglycosylases ‘cut‐and‐paste’ cell‐wall polysaccharides by endo‐transglycosylation (transglycanases) and exo‐transglycosylation (transglycosidases). Such enzymes may remodel the wall, adjusting extensibility and adhesion. Charophytes have cell‐wall polysaccharides that broadly resemble, but appreciably differ from land‐plants'. We investigated whether Chara vulgaris has wall‐restructuring enzymes mirroring those of land‐plants.Wall enzymes extracted from Chara were assayed in vitro for transglycosylase activities on various donor substrates — β‐(1→4)‐glucan‐based [xyloglucan and mixed‐linkage glucans (MLGs)], β‐(1→4)‐xylans and β‐(1→4)‐mannans — plus related acceptor substrates (tritium‐labelled oligosaccharides, XXXGol, Xyl6‐ol and Man6‐ol), thus 12 donor:acceptor permutations. Also, fluorescent oligosaccharides were incubated in situ with Chara, revealing endogenous enzyme action on endogenous (potentially novel) polysaccharides.Chara enzymes acted on the glucan‐based polysaccharides with [3H]XXXGol as acceptor substrate, demonstrating ‘glucan:glucan‐type’ transglucanases. Such activities were unexpected because Chara lacks biochemically detectable xyloglucan and MLG. With xylans as donor and [3H]Xyl6‐ol (but not [3H]Man6‐ol) as acceptor, high trans‐β‐xylanase activity was detected. With mannans as donor and either [3H]Man6‐ol or [3H]Xyl6‐ol as acceptor, we detected high levels of both mannan:mannan homo‐trans‐β‐mannanase and mannan:xylan hetero‐trans‐β‐mannanase activity, showing that Chara can not only ‘cut/paste’ these hemicelluloses by homo‐transglycosylation but also hetero‐transglycosylate them, forming mannan→xylan (but not xylan→mannan) hybrid hemicelluloses. In in‐situ assays, Chara walls attached endogenous polysaccharides to exogenous sulphorhodamine‐labelled Man6‐ol, indicating transglycanase (possibly trans‐mannanase) action on endogenous polysaccharides.In conclusion, cell‐wall transglycosylases, comparable to but different from those of land‐plants, pre‐dated the divergence of the Charophyceae from its sister clade (Coleochaetophyceae/Zygnematophyceae/land‐plants). Thus, the ability to ‘cut/paste’ wall polysaccharides is an evolutionarily ancient streptophytic trait.

Convergent and divergent roles of the glucose-responsive kinase SNF4 in Candida tropicalis

ABSTRACT The sucrose non-fermenting 1 (SNF1) complex is a heterotrimeric protein kinase complex that is an ortholog of the mammalian AMPK complex and is evolutionally conserved in most eukaryotes. This complex contains a catalytic subunit (Snf1), a regulatory subunit (Snf4) and a scaffolding subunit (Sip1/Sip2/Gal73) in budding yeast. Although the function of AMPK has been well studied in Saccharomyces cerevisiae and Candida albicans, the role of AMPK in Candida tropicalis has never been investigated. In this study, we focused on SNF4 in C. tropicalis as this fungus cannot produce a snf1Δ mutant. We demonstrated that C. tropicalis SNF4 shares similar roles in glucose derepression and is necessary for cell wall integrity and virulence. The expression of both SNF1 and SNF4 was significantly induced when glucose was limited. Furthermore, snf4Δ strains exhibited high sensitivity to many surface-perturbing agents because the strains contained lower levels of glucan, chitin and mannan. Interestingly, in contrast to C. albicans sak1Δ and snf4Δ, C. tropicalis snf4Δ exhibited phenotypes for cell aggregation and pseudohypha production. These data indicate that SNF4 performs convergent and divergent roles in C. tropicalis and possibly other unknown roles in the C. tropicalis SNF1-SNF4 AMPK pathway.

Protective effects of peptides on the cell wall structure of yeast under osmotic stress.

Three peptides (LL, LML, and LLL) were used to examine their influences on the osmotic stress tolerance and cell wall properties of brewer's yeast. Results suggested that peptide supplementation improved the osmotic stress tolerance of yeast through enhancing the integrity and stability of the cell wall. Transmission electron micrographs showed that the thickness of yeast cell wall was increased by peptide addition under osmotic stress. Additionally, quantitative analysis of cell wall polysaccharide components in the LL and LLL groups revealed that they had 27.34% and 24.41% higher chitin levels, 25.73% and 22.59% higher mannan levels, and 17.86% and 21.35% higher β-1,3-glucan levels, respectively, than the control. Furthermore, peptide supplementation could positively modulate the cell wall integrity pathway and up-regulate the expressions of cell wall remodeling-related genes, including FKS1, FKS2, KRE6, MNN9, and CRH1. Thus, these results demonstrated that peptides improved the osmotic stress tolerance of yeast via remodeling the yeast cell wall and reinforcing the structure of the cell wall. KEY POINTS: • Peptide supplementation improved yeast osmotic stress tolerance via cell wall remodeling. • Peptide supplementation enhanced cell wall thickness and stability under osmotic stress. • Peptide supplementation positively modulated the CWI pathway under osmotic stress.

Magnesium impairs Candida albicans immune evasion by reduced hyphal damage, enhanced β-glucan exposure and altered vacuole homeostasis.

With a limited arsenal of available antifungal drugs and drug-resistance emergence, strategies that seek to reduce Candida immune evasion and virulence could be a promising alternative option. Harnessing metal homeostasis against C. albicans has gained wide prominence nowadays as a feasible antifungal strategy. Herein, the effect of magnesium (Mg) deprivation on the immune evasion mechanisms of C. albicans is demonstrated. We studied host pathogen interaction by using the THP-1 cell line model and explored the avenue that macrophage-mediated killing was enhanced under Mg deprivation, leading to altered cytokine (TNFα, IL-6 and IL10) production and reduced pyroptosis. Insights into the mechanisms revealed that hyphal damage inside the macrophage was diminished under Mg deprivation. Additionally, Mg deprivation led to cell wall remodelling; leading to enhanced β-1,3-glucan exposure, crucial for immune recognition, along with concomitant alterations in chitin and mannan levels. Furthermore, vacuole homeostasis was disrupted under Mg deprivation, as revealed by abrogated morphology and defective acidification of the vacuole lumen. Together, we demonstrated that Mg deprivation affected immune evasion mechanisms by: reduced hyphal damage, enhanced β-1,3-glucan exposure and altered vacuole functioning. The study establishes that Mg availability is indispensable for successful C. albicans immune evasion and specific Mg dependent pathways could be targeted for therapy.

The Mnn10/Anp1-dependent N-linked outer chain glycan is dispensable for Candida albicans cell wall integrity.

Candida albicans cell wall glycoproteins, and in particular their mannose-rich glycans, are important for maintaining cellular integrity as well as host recognition, adhesion, and immunomodulation. The asparagine (N)-linked mannose outer chain of these glycoproteins is produced by Golgi mannosyltransferases (MTases). The outer chain is composed of a linear backbone of ∼50 α1,6-linked mannoses, which acts as a scaffold for addition of ∼150 or more mannoses in other linkages. Here, we describe the characterization of C. albicans OCH1, MNN9, VAN1, ANP1, MNN10, and MNN11, which encode the conserved Golgi MTases that sequentially catalyze the α1,6 mannose outer chain backbone. Candida albicans och1Δ/Δ, mnn9Δ/Δ, and van1Δ/Δ mutants block the earliest steps of backbone synthesis and like their Saccharomyces cerevisiae counterparts, have severe cell wall and growth phenotypes. Unexpectedly, and in stark contrast to S. cerevisiae, loss of Anp1, Mnn10, or Mnn11, which together synthesize most of the backbone, have no obvious deleterious phenotypes. These mutants were unaffected in cell morphology, growth, drug sensitivities, hyphal formation, and macrophage recognition. Analyses of secreted glycosylation reporters demonstrated that anp1Δ/Δ, mnn10Δ/Δ, and mnn11Δ/Δ strains accumulate glycoproteins with severely truncated N-glycan chains. This hypo-mannosylation did not elicit increased chitin deposition in the cell wall, which in other yeast and fungi is a key compensatory response to cell wall integrity breaches. Thus, C. albicans has evolved an alternate mechanism to adapt to cell wall weakness when N-linked mannan levels are reduced.

Dissection of the anti-Candida albicans mannan immune response using synthetic oligomannosides reveals unique properties of \u03b2-1,2 mannotriose protective epitopes

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and β anomery complemented with a synthetic β-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or β linkages, excepted for MAb B6.1 (protective epitope) reacting with β-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients’ sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing β- and α-Man epitopes and detection of antibodies against β-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and β-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/β ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

Iron alters the cell wall composition and intracellular lactate to affect Candida albicans susceptibility to antifungals and host immune response

Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of β-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. Although iron has recently been shown to affect β-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin; and increased β-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate mitochondrial functioning; leading to a reduction in accumulation of lactate that signals through the transcription factor Crz1 to induce β-1,3-glucan masking in C. albicans We show here that iron-induced changes in β-1,3-glucan exposure are lactate-dependent; and high iron causes β-1,3-glucan exposure by preventing lactate-induced, Crz1-mediated inhibition of activation of the fungal MAPK Cek1. Furthermore, despite exhibiting enhanced antifungal resistance, high iron C. albicans cells had reduced survival upon phagocytosis by macrophages. Our results underscore the role of iron as an environmental signal in multiple signaling pathways that alter cell wall architecture in C. albicans, thereby affecting its survival upon exposure to antifungals and host immune response.

Loss of Kex2 Affects the Candida albicans Cell Wall and Interaction with Innate Immune Cells.

The secretory pathway in Candida albicans involves the protein translocation into the lumen of the endoplasmic reticulum and transport to the Golgi complex, where proteins undergo posttranslational modifications, including glycosylation and proteolysis. The Golgi-resident Kex2 protease is involved in such processing and disruption of its encoding gene affected virulence and dimorphism. These previous studies were performed using cells without URA3 or with URA3 ectopically placed into the KEX2 locus. Since these conditions are known to affect the cellular fitness and the host–fungus interaction, here we generated a kex2Δ null mutant strain with URA3 placed into the neutral locus RPS1. The characterization of this strain showed defects in the cell wall composition, with a reduction in the N-linked mannan content, and the increment in the levels of O-linked mannans, chitin, and β-glucans. The defects in the mannan content are likely linked to changes in Golgi-resident enzymes, as the α-1,2-mannosyltransferase and α-1,6-mannosyltransferase activities were incremented and reduced, respectively. The mutant cells also showed reduced ability to stimulate cytokine production and phagocytosis by human mononuclear cells and macrophages, respectively. Collectively, these data showed that loss of Kex2 affected the cell wall composition, the protein glycosylation pathways, and interaction with innate immune cells.

Evaluation of the effect of β-mannanase supplementation and mannans on nursery pig growth performance and serum acute-phase protein concentrations

The objective was to evaluate the effects of dietary mannan and β-mannanase supplementation on growth performance and serum acute-phase proteins in nursery pigs. Pigs (n = 480) were blocked by initial body weight (6.6 ± 0.4 kg), and 12 pens per treatment were randomly assigned to one of four treatments in a 2 × 2 factorial arrangement for 28 d. Two levels of dietary mannan (0.4% vs. 2.8%) were offered with and without 0.05% endo-1,4-β-mannanase. Serum was collected (one pig per pen) for haptoglobin and C-reactive protein (CRP) analysis on days 0 and 28. There were no significant interactions between mannan level and β-mannanase supplementation. High-mannan diet decreased average daily gain (P = 0.027) and average daily feed intake (P = 0.024) compared with low-mannan diets with no effect of β-mannanase (P > 0.10). Haptoglobin and CRP were not affected by mannan level or β-mannanase supplementation (P ≥ 0.160). Therefore, dietary β-mannans as high as 2.8% did not induce a systemic innate immune response. Thus, the levels of β-mannan found in practical diets (<2%) are unlikely to induce an immune response; therefore, supplementation of β-mannanase to avoid this response is not warranted.

False-positive Candida and Aspergillus antigen testing in recipients of allogeneic haematopoietic cell transplantation due to administration of parenteral nutrition and fixed combinations of piperacillin-tazobactam.

False positivity of antigen immunoassays used as an early diagnostic tool to detect invasive fungal infections is known. Interpretation of the assay needs the identification of sources which could affect the specificity of the test. We focused on the influence of parenteral nutrition (PN) and piperacillin-tazobactam (TZP) on fungal immunoassays. Measurable amounts of Candida antigen mannan were detected in several compounds of PN and TZP in a previous in vitro study. In the current study, 84 patients undergoing allogeneic haematopoietic cell transplantation receiving either TZP, PN or both were monitored with Aspergillus and Candida antigen assay. Six patients were analysed closer in a kinetic analysis with more frequent blood sampling to detect mannan. PN in diverse compositions as well as TZP did not increase significantly the amount of mannan and the Aspergillus antigen in serum. We could not confirm the positive results of the in vitro study. Physicians should be aware that mannan antigenemia due to drug infusion could be a transient issue and should be considered in the interpretation of fungal immunoassays, although we could not find clinically relevant effects on mannan levels.

Effect of Nicotinamide Against Candida albicans.

Nicotinamide (NAM) has a long history in clinical applications and can be safely used for treating various diseases. In recent years, NAM was found to exhibit antimicrobial activities, inhibiting the growth of Plasmodium falciparum, Mycobacterium tuberculosis, and human immunodeficiency virus (HIV). Here we investigated the activity of NAM against Candida albicans, one of the most prevalent human fungal pathogens. Our results showed that NAM exhibited significant antifungal activity against C. albicans, including fluconazole-resistant isolates. NAM could also effectively suppress biofilm formation. In addition, NAM exhibited antifungal activity against non-Candida albicans species and Cryptococcus neoformans. Combination of NAM and fluconazole showed an even strong antifungal activity. The antifungal activity of NAM was further confirmed in a mouse model of disseminated candidiasis. Confocal laser scanning microscopy revealed that NAM increased cell wall β-glucans exposure and chitin content while decreased mannan level. Furthermore, by screening the C. albicans homozygous deletion mutant library, the C. albicans mutant lacking GIN4, which encodes a septin regulatory protein kinase and is essential for the maintenance of cell wall integrity, was identified to be high sensitive to NAM. These findings suggested that NAM might exhibit antifungal activities through affecting cell wall organization.

Experimental Candida albicans osteomyelitis: Microbiologic, antigenic, histologic, and 18FDG-PET-CT imaging characteristics in a newly established rabbit model.

Candida osteomyelitis is a debilitating disease that is difficult to diagnose and treat. As there are no animal models or prospective studies for this uncommon infection, little is known about the pathogenesis, diagnosis, or treatment. We therefore sought to establish an animal model for the study of the pathophysiology, diagnostic modalities, and therapeutic interventions of Candida osteomyelitis. We developed a modified version of the Norden rabbit model of tibial osteomyelitis, in which the right tibia was inoculated intraoperatively with different inocula of C. albicans or normal saline as control. On days 7, 14, and 21 after inoculation, the animals underwent bone radiography, 18-fluoro-2-deoxy-D-glucose positron emission tomography combined with computed tomography (PET/CT) scan, and blood sampling for blood cultures, blood counts, erythrocyte sedimentation rate, and Candida mannan antigen serum levels. On day 21, animals were euthanized, and infected tibias harvested for culture and histology. Among eight evaluable animals inoculated with 1 × 106 to 1 × 107 cfu, histology and bone cultures established the presence of Candida osteomyelitis in seven, with a host response of neutrophils, mononuclear cells, multinucleate giant cells, fibrosis, and necrosis. Infected animals demonstrated radiological signs of osteomyelitis with significantly increased tracer uptake in 18FDG-PET/CT scans (P < .01) and elevated serum mannan levels (P < .01). All blood cultures were negative. Indices of inflammation were only slightly increased. In conclusion, we report successful establishment of a new animal model of Candida albicans osteomyelitis that may be applicable to advancing our understanding of the pathophysiology, diagnostic modalities, and treatment of this debilitating infection.

Defects in intracellular trafficking of fungal cell wall synthases lead to aberrant host immune recognition.

The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the β-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.

Aspartyl proteases in Candida glabrata are required for suppression of the host innate immune response

A family of 11 cell surface-associated aspartyl proteases (CgYps1–11), also referred as yapsins, is a key virulence factor in the pathogenic yeast Candida glabrata. However, the mechanism by which CgYapsins modulate immune response and facilitate survival in the mammalian host remains to be identified. Here, using RNA-Seq analysis, we report that genes involved in cell wall metabolism are differentially regulated in the Cgyps1–11Δ mutant. Consistently, the mutant contained lower β-glucan and mannan levels and exhibited increased chitin content in the cell wall. As cell wall components are known to regulate the innate immune response, we next determined the macrophage transcriptional response to C. glabrata infection and observed differential expression of genes implicated in inflammation, chemotaxis, ion transport, and the tumor necrosis factor signaling cascade. Importantly, the Cgyps1–11Δ mutant evoked a different immune response, resulting in an enhanced release of the pro-inflammatory cytokine IL-1β in THP-1 macrophages. Further, Cgyps1–11Δ–induced IL-1β production adversely affected intracellular proliferation of co-infected WT cells and depended on activation of spleen tyrosine kinase (Syk) signaling in the host cells. Accordingly, the Syk inhibitor R406 augmented intracellular survival of the Cgyps1–11Δ mutant. Finally, we demonstrate that C. glabrata infection triggers elevated IL-1β production in mouse organs and that the CgYPS genes are required for organ colonization and dissemination in the murine model of systemic infection. Altogether, our results uncover the basis for macrophage-mediated killing of Cgyps1–11Δ cells and provide the first evidence that aspartyl proteases in C. glabrata are required for suppression of IL-1β production in macrophages.

Isolation and Characterization of Mannanase-producing Bacillus cereus Isolated from the Hindgut of Termites

Abstract: Background and Objective: Mannanase is a mannan-degrading enzyme that can be produced by microorganisms such as bacteria. Mannanase is widely used in the animal feed industry with the aim of improving the quality of feed ingredients containing high levels of mannan, such as palm kernel meal. This study aimed to isolate, characterize and produce mannanase from bacteria found in the hindgut of termites. Materials and Methods: Bacteria were isolated by culturing on solid media containing 0.5% locust bean gum for 24 h at 30°C. The mannanolytic index was then measured by the addition of 2% congo red, which was then washed with 1 M NaCl. Mannanase activity was measured by the dinitrosalicylic acid method, while the protein content was measured by the Bradford method. Results: A total of 8 isolates were obtained. The 3 isolates with the highest mannanolytic indices were selected (isolates V9, AZ and AD). Isolate V9 exhibited the highest mannanase activity of 29.50 U mL–1 after 88 h of incubation with a specific activity of 110.32 U mg–1. The activity of the mannanase was optimal at pH 8 and at an incubation temperature of 50°C. Based on 16S-DNA analysis, isolate V9 was identified as Bacillus cereus. Conclusion: Bacillus cereus, which is isolated from the hindgut of termites, has fairly high mannanase activity and has the potential to hydrolyse mannan in feed ingredients such as palm kernel meal.

Flocculation in industrial strains ofSaccharomyces cerevisiae: role of cell wall polysaccharides and lectin-like receptors

Yeast flocculation is the reversible aggregation of yeast cells promoted by the interaction between lectin-like protein receptors with mannose side chains on adjacent cell walls. Flocculation is governed by several physiological factors, including the type of nutrient sugar available to yeast. We grew four industrial strains of Saccharomyces cerevisiae, representing applications in the brewing, winemaking and bioethanol sectors, to late stationary phase and quantified the cellular content of mannans, glucans and lectin-like proteins on yeast cell surfaces. Results indicated that brewing and champagne strains showed moderate to high flocculation ability when grown with glucose, fructose, maltose or galactose, whereas winemaking and fuel alcohol strains only showed moderate flocculation when grown on maltose and galactose. All yeast strains studied were weakly flocculent when grown on mannose. With regard to lectin-like receptors, their number played a more important role in governing yeast flocculation than the mannan and glucan contents in yeast cell walls. We conclude that all the industrial strains of S. cerevisiae belonged to New-Flo type on the basis of their flocculation behaviour observed when cultured on different sugars. Quantification of yeast cell wall polysaccharides and receptor sites indicates that mannan and glucan levels remain almost constant, irrespective of the strain under investigation. The main difference in flocculation characteristics in industrial yeast strains appears to be due to variations in concentrations of lectin-like cell surface receptors. Our findings may benefit brewers, winemakers and other yeast-based technologies in design of media to prevent premature flocculation during fermentation. Copyright © 2017 The Institute of Brewing & Distilling

Effects of mannan level and β-mannanase supplementation on growth performance, apparent total tract digestibility and blood metabolites of growing pigs

The exogenous enzymes are less consistent in their effects as their beneficial effects depend upon the types and level non-starch polysaccharides (NSP) present in the diets. Therefore, exogenous enzymes should be selected on the basis of types and amount of the NSP in the pig diets. The objectives of the present experiments were to investigate the effects of dietary level of mannan and β-mannanase supplementation on growth performance, apparent total tract digestibility (ATTD) of energy and nutrients, and blood metabolites of growing pigs. In Exp. 1, 96 barrows were randomly allotted to four treatments on the basis of BW. There were four replicates in each treatment with six pigs per replicate. The dietary treatments were a corn–soybean meal (SBM)-based control diet and three other diets consisted of the control diet supplemented with 400, 800 or 1600 U of β-mannanase/kg diet. The final BW, average daily gain (ADG) and blood glucose increased (linear, P<0.05) with increasing concentrations of dietary β-mannanase. The ATTD of dry matter (DM), gross energy (GE) and β-mannan was higher (linear, P<0.05) with increase in dietary β-mannanase concentrations. In Exp. 2, 288 barrows were allotted to six treatments in a 2×3 factorial arrangement of mannan level (high v. low) and addition of β-mannanase (0, 400 or 800 U/kg diet). There were four replicates in each treatment with 12 pigs/replicate. Pigs were fed corn–SBM-based low-mannan diet (6.1 g/kg) or high-mannan (25.2 g/kg) diet in which corn and SBM were partially replaced with 50 g/kg diet palm kernel meal. All diets were fed in meal form for 42 days. Pigs fed diets supplemented with β-mannanase had greater (P<0.05) final BW, ADG, feed to gain (F : G), the ATTD of DM, GE, and β-mannan and blood glucose concentration compared with pigs fed diets without β-mannanase. In addition, the final BW, ADG, F : G and the ATTD of GE and β-mannan were reduced (P<0.05) in low mannan level. The dietary level of mannan and the β-mannanase supplementation had no effects (P>0.05) on the concentrations of blood total cholesterol, triacylglycerides and blood urea nitrogen. These results indicate that supplementation of β-mannanase to low- or high-mannan diets have potential to improve the performance of growing pigs. In addition, palm kernel meal may partially replace corn and SBM without reducing pig performance if β-mannanase is added to diet.