Fungal Cell Wall Preparation Research Articles

NADPH Oxidase Controls Neutrophil Infiltration in the Lung in Early Response to Fungal Cell Walls Via LTB4

The leukocyte NADPH oxidase generates superoxide, the precursor to reactive oxygen species important for host defense and immunoregulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by serious bacterial and fungal infections and aberrant inflammation. Aspergillus pneumonia is frequent, and associated with pyogranulomatous inflammation. Of note, even sterile fungal cell walls elicit increased neutrophilic inflammation in CGD mice, demonstrating that that NADPH oxidase can limit inflammation independent of its antimicrobial effects. Leukotriene B4 (LTB4) is a potent inflammatory mediator, acting as a chemoattractant and activating other polymorphonuclear leukocyte (PMN) functions. PMN themselves are prominent source of LTB4, and sense LTB4 through its receptor BLT1, which stimulates additional LTB4 synthesis by autocrine and paracrine routes. This leads to feed forward amplification of LTB4 production locally and formation of PMN clusters. Ca2+ acts as an important second messenger in PMN, including regulation of LTB4 synthesis. Activated human CGD PMN have increased intracellular Ca2+ levels compared to PMN from healthy donors due to electrogenic effects of the NADPH oxidase (Geiszt M. et al JBC 1997). Intracellular Ca2+ overload in PMN is speculated to contribute to increased PMN pro-inflammatory activity, but specific pathways and in vivo relevance are not well defined. Therefore, we therefore investigated the production of LTB4 by murine CGD PMN and the role of LTB4 in PMN recruitment and lung hyperinflammation following pulmonary challenge with zymosan, a sterile fungal cell wall preparation. Zymosan-stimulated PMN from Cybb-null mice, a model for X-linked CGD, produced higher LTB4 in vitro compared to wild type (WT), even in the presence of SOD and catalase to counteract oxidative degradation. Higher LTB4 production correlated with higher levels of intracellular Ca2+ in stimulated X-CGD PMN. The increased LTB4 produced by X-CGD PMN was dependent on cell density and the LTB4 receptor BLT1, consistent with a feed-forward loop that amplified LTB4 production. Zymosan-stimulated X-CGD PMN also formed larger and more numerous clusters in vitro compared to WT PMN. Cluster formation was abrogated by inhibiting LTB4 synthesis with the 5-lipoxygenase (5-LO) inhibitor zileuton or by the BLT1 receptor antagonist U75302, demonstrating that cluster formation was LTB4 and BLT1 dependent. We next examined whether LTB4 regulated the response to zymosan in the lung. Following intranasal administration of zymosan, X-CGD mice had higher PMN numbers in bronchoalveolar lavage (BAL), larger PMN foci by lung histology, and increased lung LTB4 as compared to WT mice. These differences were evident by 8 hr and progressed over the first 24 hr post-challenge, in contrast to WT mice where inflammation plateaued at 8 hr. Treatment with zileuton or U75302 just prior to zymosan significantly reduced lung PMN numbers for both WT and X-CGD mice at 8 hr post challenge, and lung PMN remained substantially reduced in X-CGD mice at 24 hrs. Moreover, delaying administration of zileuton to 8 hr after zymosan challenge also significantly decreased PMN inflammation in X-CGD but not WT mice at 24 hrs, indicating that ongoing synthesis of LTB4 otherwise continued to promote PMN recruitment in CGD mice. These results demonstrate that PMN production of LTB4 in response to fungal cell walls is limited by NADPH oxidase via its effects on intracellular Ca2+. In oxidase-deficient CGD mice, LTB4 plays a major role in driving excessive PMN recruitment to the lung in the early response to zymosan. This study is the first to implicate LTB4 in promoting neutrophilic lung inflammation in response to fungal cell walls in CGD, likely by an amplified feed-forward loop involving increased production of LTB4 by CGD PMN. LTB4 could be a potential therapeutic target to ameliorate CGD hyperinflammation. Disclosures No relevant conflicts of interest to declare.

Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.

Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Patients with cystic fibrosis (CF) experience chronic or recurrent bacterial and fungal lung infections. Many patients with CF cannot effectively clear Aspergillus from their lungs. This may result in IgE sensitization and the development of allergic bronchopulmonary aspergillosis, or invasive infections, such as Aspergillus bronchitis. Lung disease in patients with CF is associated with neutrophil-dominated inflammation and elevated levels of the serine protease, neutrophil elastase (NE). Various C-type lectin-like receptors (CLRs), including Dectin-1 and Dectin-2, are involved in the immune response to Aspergillus. Here, we show that purified NE cleaves Dectin-1 in an isoform-specific manner. Bronchoalveolar lavage fluid from patients with CF, which contains high NE activity, induces Dectin-1 cleavage. Similarly, filtrate from a protease-producing strain of Aspergillus fumigatus induces isoform-specific cleavage of Dectin-1. Dectin-1 knockout (KO) cells and NE-treated cells demonstrated reduced phagocytosis of zymosan, a fungal cell wall preparation. In addition, NE cleaves 2 other CLRs, Dectin-2 and Mincle, and fungal-induced cytokine production was reduced in Dectin-1 KO cells, Dectin-2 KO cells, and NE-treated cells. Thus, Dectin-1 and Dectin-2 cleavage by NE and/or A. fumigatus–derived proteases results in an aberrant antifungal immune response that likely contributes to disease pathology in patients with CF.—Griffiths, J. S., Thompson, A., Stott, M., Benny, A., Lewis, N. A., Taylor, P. R., Forton, J., Herrick, S., Orr, S. J., McGreal, E. P. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

Spore germination of Trichoderma atroviride is inhibited by its LysM protein TAL6

LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They have general N-acetylglucosamine binding properties and therefore bind to chitin and related carbohydrates. In plants, plasma-membrane-bound proteins containing LysM motifs are involved in plant defence responses, but also in symbiotic interactions between plants and microorganisms. Filamentous fungi secrete LysM proteins that contain several LysM motifs but no enzymatic modules. In plant pathogenic fungi, for LysM proteins roles in dampening of plant defence responses and protection from plant chitinases were shown. In this study, the carbohydrate-binding specificities and biological function of the LysM protein TAL6 from the plant-beneficial fungus Trichodermaatroviride were investigated. TAL6 contains seven LysM motifs and the sequences of its LysM motifs are very different from other fungal LysM proteins investigated so far. The results showed that TAL6 bound to some forms of polymeric chitin, but not to chito-oligosaccharides. Further, no binding to fungal cell wall preparations was detected. Despite these rather weak carbohydrate-binding properties, a strong inhibitory effect of TAL6 on spore germination was found. TAL6 was shown to specifically inhibit germination of Trichoderma spp., but interestingly not of other fungi. Thus, this protein is involved in self-signalling processes during fungal growth rather than fungal-plant interactions. These data expand the functional repertoire of fungal LysM proteins beyond effectors in plant defence responses and show that fungal LysM proteins are also involved in the self-regulation of fungal growth and development.

Effect of polysaccharides in slow filters integrated into closed hydroponic greenhouse systems

SummarySlow filtration is a widespread and environmentally sound method for the disinfestation of nutrient solutions in closed hydroponic systems. The efficacy of slow filters can be explained in part by straining and adsorption of pathogens onto filter material surfaces. Both mechanisms are influenced by choice of filter material, flow rate of the nutrient solution, biomass and extent of extracellular polymers on the filter skin. In a 2-year study, the total polysaccharide content and enzyme activity of the filter skin, and filter efficacy were monitored. Closed hydroponic systems using the nutrient film technique connected to slow filters were run with or without weekly inoculation with a fungal cell wall preparation (10.5 g m–2 of filter surface). This organic matter amendment increased the amount of total polysaccharide and enzyme activity within the filter skin. The fungal cell wall preparation did not accumulate, but was degraded rapidly. The total polysaccharide content (tPS; in mg g–1) of the filter skin increased linearly with time at 34.50 mg g–1 + 0.26 mg g–1 d–1 × days (d) (R2 = 0.556; P = 0.02) for untreated filters, and at 43.40 mg g–1 + 0.70 mg g–1 d–1 × days (d) (R2 = 0.826; P = 0.000) for treated filters. The extracellular polysaccharide content in the filter skin, determined after 16 and 20 weeks of slow filter operating time, ranged from 87.9 – 109.7 mg g–1 mineral wool (rockwool) sampled. The amount of extracellular polysaccharide was not influenced by the total polysaccharide content of the filter skin, by enzyme activity, or by addition of the fungal cell wall preparation. There was a trend towards higher filter efficacies when fungal cell wall preparations were added to the filter skin (treated: 98.3% ± 0.96; untreated: 97.9% ± 1.8); however, on the basis of the present data, no correlation between the assessed parameters and filter efficacies were observed.

Properties of the chitinase of the antifungal biocontrol agent Streptomyces lydicus WYEC108

An extracellular chitinase from culture filtrates of Streptomyces lydicus WYEC108, a broad spectrum antifungal biocontrol agent, was characterized and purified. Its role in the antifungal activity of this actinomycete was studied. Low constitutive levels of the enzyme were observed when cultures were grown with both simple and complex carbon substrates. The optimal temperature and substrate concentration for maximal chitinase production were 25–30°C and 0.4–0.8 g ml −1 chitin, respectively. High chitinase production was obtained when 1% colloidal chitin was present in the medium as a growth substrate. Activity was induced by n-acetylglucosamine or n,n′-diacetylchitobiose (GlcNac) 2 and repressed by glucose, xylose, arabinose, raffinose, and carboxymethyl cellulose. Strong catabolite repression of the chitinase was observed. Addition of pectin, laminarin, starch, or β-glucan to the chitin-containing medium, however, increased chitinase production. Probing the S. lydicus genomic DNA with the chiA gene from S. lividans has localized the gene to a 2.5 kb DNA fragment of genomic DNA. The chitinase appears to play a role in the antifungal activities of S. lydicus WYEC108. Production was greatly enhanced when cells were grown in a medium containing colloidal chitin supplemented with certain fungal cell wall preparations, in particular those from Pythium or Aphanomyces species. Crude fungal cell walls were lysed by partially purified chitinase. While S. lydicus also produces one or more antifungal antibiotics, its chitinase probably plays a significant role in the in vivo antifungal biocontrol activity of this rhizosphere-colonizing actinomycete.

Cell suspension cultures of spruce (Picea abies): inactivation of extracellular enzymes by fungal elicitor-induced transient release of hydrogen peroxide (oxidative burst)

Elicitation of suspension culture cells of spruce [Picea abies (L.) Karst] with a fungal cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii Bubak induced inactivation of extracellular enzymes. Extracellular peroxidase, β-glucosidase and acid phosphatase, secreted by the cells during growth, and also α-amylase and pectinase from Aspergillus strains, added to an elicited cell culture, were inactivated. Inactivation is caused by an elicitor-mediated transient release of H2O2 from the cells (oxidative burst). H2O2 released into the medium was determined with ABTS (2,2'-Azino-bis-(3-ethylbenthiazoline-6-sulfonate)) (formation of blue colour) and with phenol red (destruction of pH indicator). The release started only minutes after beginning of elicitation and its inactivating effect existed for more than 1 day. Release of H2O2 is a biphasic process with a first smaller maximum at 1 h, followed by a second larger increase, peaking at 5–6 h and returning to approximately the control levels thereafter. Also H2O2 is transiently released in small quantities from cell incubations in the absence of elicitor as a stress response of the cells to manipulations of the cultures. Extracellular enzymes secreted into the medium could also be inactivated by direct addition of exogenous H2O2. Catalase prevents inactivation of the secreted extracellular enzymes, however, to a limited extent only because, as a result of contact of cells and medium, catalase becomes inactivated. The ionophores A 23187 and cycloheximide induced release of H2O2 and, when present together with elicitor, induction was synergistically increased.

Role of salicylic acid and intracellular Ca 2+ in the induction of chitinase activity in carrot suspension culture

Treatment of carrot suspension culture with a fungal cell wall preparation induced the production of chitinase. This treatment also caused an accumulation of salicylic acid both in the cells and in the extracellular medium. Culture medium of induced cells, which contained elevated levels of salicylic acid, was able to induce chitinase activity in a non-induced suspension culture. Exogenous supply of authentic salicylic acid induced chitinase activity in carrot cells more rapidly than the fungal cell wall. Removal of extracellular Ca2+ by treatment with the calcium chelator, EGTA [ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid], abolished the elicitormediated production of both salicylic acid and chitinase. Verapamil caused similar effects, whereas the addition of the Ca2+ ionophore A23187, enhanced these cellular responses in the absence of elicitor. These observations suggest that Ca2+ plays an important role in the induced production of chitinase and salicylic acid.

A simple procedure for dissolving fungal cell wall preparations for the analysis of neutral sugars

Quantitatively precise solutions of fungal cell walls were obtained by dissolving with 60% (v/v) H2SO4 under normal conditions. Complete dissolution was achieved within 1h when the samples were first ground to a fine powder and suspended in ethanol.

Coordinate- and elicitor-dependent expression of stilbene synthase and phenylalanine ammonia-lyase genes in Vitis cv. Optima

The mechanisms controlling the induction of Stilbene synthase and phenylalanine ammonia-lyase (PAL), two putative key regulatory enzymes of the biosynthetic pathway to stilbene phytoalexins, have been investigated. The induction was studied in cell suspension cultures of grape ( Vitis cv. Optima) by treatment with fungal cell wall. Several independent cDNA clones for PAL and stilbene synthase were isolated from a cDNA library of fungal cell wall-induced grape cells and identified by sequence analysis. The Stilbene synthase cDNA sequence of pSV21 predicted a protein of 392 amino acids and M r 42,791, similar in size to that observed experimentally for immunodetected stilbene synthase. The cDNA sequences of pSV21 and pSV25 differed in 76 bp in the coding region. The sequences of grape stilbene synthase cDNAs exhibited significant homology to the sequence reported for the peanut stilbene synthase cDNA. Both PAL and stilbene synthase mRNA, measured by RNA blot hybridizations, were induced within 1 h of addition of fungal cell wall preparations to the cell cultures, rose to a maximum by the sixth hour, then declined slowly over the next 20 h. The activities of PAL and stilbene synthase were also induced in parallel, but reached their maximum at different times after fungal cell wall addition to the cell cultures. The induction patterns of stilbene synthase and PAL in grape and peanut are discussed.

Specific interaction of aniline blue with (1 → 3)-β- d-glucan

Interaction between aniline blue and curdlan, a (1 → 3)-β- d-glucan, has been studied using absorption and fluorescence spectroscopy. The evidence suggests that a minor, weakly fluorescent component of commercial dyes forms a strongly fluorescent complex with curdlan, with an excitation maximum of 395 nm and an emission maximum of 495 nm. This component was partially purified by TLC on silica gel. Of many polysaccharides surveyed, a number showed weak interactions with the major component of aniline blue but only (1 → 3)-β- d-glucans such as pachyman, curdlan and laminaran induced fluorescence in the minor component. Fluorescence was less with laminaran than with curdlan and decreased with increasing alkali concentration suggesting conformational control of the dye-binding mechanism. As little as 5 μg/ml of curdlan induced easily detectable fluorescence increases in aniline blue, and this was used to demonstrate the presence of (1 → 3)-β- d-glucan in a fungal cell wall preparation. (1 → 3)-β- d-glucan in cereal grain sections was located as bright yellow-green fluorescent particles. In barley these stained particles, located in association with the sub-aleurone endosperm cell wall, showed a fluorescence excitation maximum at 395 nm and emission maximum of 495 nm.

Chitin and related compounds as elicitors of the lignification response in wounded wheat leaves

Chitin, an important component of most fungal cell walls, elicited lignification in wheat leaves when applied as a suspension to wounds. The soluble derivatives ethylene glycol, chitin and chitosan were also active, but glucosamine, N-acetylglucosamine and small chitin oligomers did not elicit the response. The fungal glucan pullulan lacked activity, but laminarin, an algal glucan, was a weak elicitor. The response could also be elicited by fungal cell wall preparations. The possible role of chitin and related wall polymers in inducing host-defence mechanisms in wheat is discussed.