Constituent Of Fungal Cell Walls Research Articles

Cell Wall Integrity Mediated by CfCHS1 Is Important for Growth, Stress Responses and Pathogenicity in Colletotrichum fructicola

Camellia oleifera, a woody plant that produces edible oil, is indigenous to China. The devastating disease of anthracnose inflicts significant financial losses on Ca. oleifera. The primary causative agent of anthracnose on Ca. oleifera is Colletotrichum fructicola. Chitin, a pivotal constituent of fungal cell walls, assumes a critical function in their proliferation and maturation. To study the biological functions of chitin synthase 1(Chs1) in C. fructicola, the CfCHS1 gene knockout mutants, ∆Cfchs1-1 and ∆Cfchs1-2, and their complementary strain, ∆Cfchs1/CfCHS1, of C. fructicola were generated. Our results showed that the colony diameters of wild-type and complement-strain ∆Cfchs1/CfCHS1, mutant ∆Cfchs1-1 and ∆Cfchs1-2 cultured on the CM and MM medium were 5.2, 5.0, 2.2 and 2.4 cm and 4.0, 4.0, 2.1 and 2.6 cm, respectively, which were significantly smaller for the mutant than for the wild type and complement strain; the inhibition rates on the CM medium supplemented with H2O2, DTT, SDS and CR were 87.0% and 88.5%, 29.6% and 27.1%, 88.0% and 89.4%, and 41.7% and 28.7%, respectively, for the mutant strains, ∆Cfchs1-1 and ∆Cfchs1-2, which were significantly higher than those for the other two strains; the rate of hyphal tips with CFW fluorescence in ∆Cfchs1-1 and ∆Cfchs1-2 was 13.3% and 15.0%, which was significantly lower than those for the other two strains; the mutant strains, ∆Cfchs1-1 and ∆Cfchs1-2, lost the ability to produce conidia; the mutant strains showed weaker pathogenicity on wounded and unwounded Ca. oleifera leaves than the wild type and complement strain. The findings of this study suggest that CfChs1 plays a crucial role in the growth and development, stress responses, and pathogenicity of C. fructicola. Thus, this gene could be a potential target for developing novel fungicide.

In-Silico Validation of Prosopis ciniraria Therapeutic Peptides Against Fungal Cell Wall: Better Treatment Strategy for Fungal Diseases.

Prosopis cineraria commonly known as Druce are valuable herb that holds antibacterial role, antifungal properties. We identified different peptides from this plant by deploying CADD (Computer-aided-drug-designing) approaches, these peptide sequences are as follows seq1 (RHDEEEEKAKV),seq3(KSNSTVEISQNVQSVDSSKM),seq4(KQVAEMNKPAVGSKTSDANHDLKS),seq5(KTKSAGNDSIQSTKPVPSALTVDKA),seq6(RELEDSNIHHVAASVVLESKSSRT), and seq8(LYSKVELHPFGLHNLGNSCYANAVFSV), these peptides holds therapeutic properties as shows interaction with chitin, a major constituent of fungal cell wall. Molecular docking was conducted by using AutoDock-Vina tool and the results were found to be promising where all binding energies were found in the range of − 9.1 to − 7.5 kcal/mol, it indicates strong binding of peptide sequences with chitin molecule. Even the toxicity analysis supports the considered peptide sequences to hold therapeutic role against fungus with non-toxic effect on humans. These peptides were successfully predicted as important therapeutic agents of P. cinerariaseed that can initiate chitin breakdown, due to their possible strong interaction with fungal cell wall and it also suggests this medicinal plant holds the key for multiple fungal disease treatments. This study will open new research dimensions and integration of computational biology with microbial pathology that will assist scientific and medical community to develop rapid disease prevention strategies against fungal pathogenesis.

Plasma 1,3-β-d-glucan levels predict adverse clinical outcomes in critical illness.

BACKGROUNDThe fungal cell wall constituent 1,3-β-d-glucan (BDG) is a pathogen-associated molecular pattern that can stimulate innate immunity. We hypothesized that BDG from colonizing fungi in critically ill patients may translocate into the systemic circulation and be associated with host inflammation and outcomes.METHODSWe enrolled 453 mechanically ventilated patients with acute respiratory failure (ARF) without invasive fungal infection and measured BDG, innate immunity, and epithelial permeability biomarkers in serially collected plasma samples.RESULTSCompared with healthy controls, patients with ARF had significantly higher BDG levels (median [IQR], 26 pg/mL [15–49 pg/mL], P < 0.001), whereas patients with ARF with high BDG levels (≥40 pg/mL, 31%) had higher odds for assignment to the prognostically adverse hyperinflammatory subphenotype (OR [CI], 2.88 [1.83–4.54], P < 0.001). Baseline BDG levels were predictive of fewer ventilator-free days and worse 30-day survival (adjusted P < 0.05). Integrative analyses of fungal colonization and epithelial barrier disruption suggested that BDG may translocate from either the lung or gut compartment. We validated the associations between plasma BDG and host inflammatory responses in 97 hospitalized patients with COVID-19.CONCLUSIONBDG measurements offered prognostic information in critically ill patients without fungal infections. Further research in the mechanisms of translocation and innate immunity recognition and stimulation may offer new therapeutic opportunities in critical illness.FUNDINGUniversity of Pittsburgh Clinical and Translational Science Institute, COVID-19 Pilot Award and NIH grants (K23 HL139987, U01 HL098962, P01 HL114453, R01 HL097376, K24 HL123342, U01 HL137159, R01 LM012087, K08HK144820, F32 HL142172, K23 GM122069).

The Lymphocytic Scavenger Receptor CD5 Shows Therapeutic Potential in Mouse Models of Fungal Infection.

Invasive fungal diseases represent an unmet clinical need that could benefit from novel immunotherapeutic approaches. Host pattern recognition receptors (e.g., Toll-like receptors, C-type lectins, or scavenger receptors) that sense conserved fungal cell wall constituents may provide suitable immunotherapeutic antifungal agents. Thus, we explored the therapeutic potential of the lymphocyte class I scavenger receptor CD5, a nonredundant component of the antifungal host immune response that binds to fungal β-glucans. Antifungal properties of the soluble ectodomain of human CD5 (shCD5) were assessed in vivo in experimental models of systemic fungal infection induced by pathogenic species (Candida albicans and Cryptococcus neoformans). In vitro mechanistic studies were performed by means of fungus-spleen cell cocultures. shCD5-induced survival of lethally infected mice was dose and time dependent and concomitant with reduced fungal load and increased leukocyte infiltration in the primary target organ. Additive effects were observed in vivo after shCD5 was combined with suboptimal doses of fluconazole. Ex vivo addition of shCD5 to fungus-spleen cell cocultures increased the release of proinflammatory cytokines involved in antifungal defense (tumor necrosis factor alpha and gamma interferon) and reduced the number of viable C. albicans organisms. The results prompt further exploration of the adjunctive therapeutic potential of shCD5 in severe invasive fungal diseases.

Cordycepin, a metabolite of Cordyceps militaris, reduces immune-related gene expression in insects

Hypocrealean entomopathogenic fungi (EPF) (Sordariomycetes, Ascomycota) are natural regulators of insect populations in terrestrial environments. Their obligately-killing life-cycle means that there is likely to be strong selection pressure for traits that allow them to evade the effects of the host immune system. In this study, we quantified the effects of cordycepin (3′-deoxyadenosine), a secondary metabolite produced by Cordyceps militaris (Hypocreales, Cordycipitaceae), on insect susceptibility to EPF infection and on insect immune gene expression. Application of the immune stimulant curdlan (20 µg ml−1, linear beta-1,3-glucan, a constituent of fungal cell walls) to Drosophila melanogaster S2r+ cells resulted in a significant increase in the expression of the immune effector gene metchnikowin compared to a DMSO-only control, but there was no significant increase when curdlan was co-applied with 25 µg ml−1 cordycepin dissolved in DMSO. Injection of cordycepin into larvae of Galleria mellonella (Lepidoptera: Pyralidae) resulted in dose-dependent mortality (LC50 of cordycepin = 2.1 mg per insect 6 days after treatment). Incubating conidia of C. militaris and Beauveria bassiana (Hypocreales, Cordycipitaceae; an EPF that does not synthesize cordycepin) with 3.0 mg ml−1 cordycepin had no effect on the numbers of conidia germinating in vitro. Co-injection of G. mellonella with a low concentration of cordycepin (3.0 mg ml−1) plus 10 or 100 conidia per insect of C. militaris or B. bassiana caused a significant decrease in insect median survival time compared to injection with the EPF on their own. Analysis of predicted vs. observed mortalities indicated a synergistic interaction between cordycepin and the EPF. The injection of C. militaris and B. bassiana into G. mellonella resulted in increased expression of the insect immune effector genes lysozyme, IMPI and gallerimycin at 72 h post injection, but this did not occur when the EPF were co-injected with 3.0 mg ml−1 cordycepin. In addition, we observed increased expression of IMPI and lysozyme at 48 h after injection with C. militaris, B. bassiana and sham injection (indicating a wounding response), but this was also prevented by application of cordycepin. These results suggest that cordycepin has potential to act as a suppressor of the immune response during fungal infection of insect hosts.

Patulin, ergosterol and Howard mold count (HMC): Which one is the best quality indicator for tomatoes?

Ergosterol, an important constituent of fungal cell wall, was started to be used as a quality parameter for the tomatoes and derivatives instead of Howard mold count (HMC). In addition, patulin is another important microbiological quality indicator of molds such as Penicillium, Aspergillus, and Byssochlamys. In this study, it is aimed to identify the relationship between HMC, patulin, and ergosterol in juices produced from the tomatoes which has the surface rotten of 0% (sound), 25%, 50%, 75%, and 100%. As the rotten proportion increased, HMC, ergosterol, and patulin values increased. Linear correlations (r) between HMC, ergosterol, patulin, and rotten proportion were calculated as 0.99, 0.99, and 0.98, respectively. In addition, linear correlations between HMC and ergosterol (r=0.95), HMC and patulin (r=0.95) and ergosterol and patulin (r=0.98) were found. In contrast, non-linear correlation between pH, brix and titratable acidity, and the rotten proportion was determined. PRACTICAL APPLICATIONS: The manuscript has provided the relationship between HMC, patulin, and ergosterol in juices produced from the tomatoes which have the surface rotten of 0% (sound), 25%, 50%, 75%, and 100%. Linear correlations between rotten proportions of tomatoes and HMC, ergosterol, patulin and between HMC and ergosterol, HMC and patulin, and ergosterol and patulin were determined in juices produced from rotten tomatoes. Patulin indicated correlation with HMC and ergosterol in juices produced from rotten tomatoes. In addition to HMC and ergosterol, patulin has great potential for the assessment of tomato products as associated with the rotten proportion.

A Mouse Model of Trichophyton Inflammation Based on Trichophytin-induced Contact Hypersensitivity.

The prevalence of Trichophyton-induced superficial skin mycosis is very high among human patients. Dermatophytes generally infect the epidermis, especially the stratum corneum, forming scales, hyperkeratosis, and vesicles. The important roles played by the immune system in Trichophyton infection are detection of fungal invasion and elimination of fungi.These immune mechanisms are presumed to involve not only innate immunity but also acquired immunity. Therefore, there is a substantial need for studies on treatment methods based on new basic knowledge, and the elucidation of immunological mechanisms of Trichophyton-induced inflammatory reactions is especially important.However, since Trichophyton cannot colonize on the mouse skin, we tried to develop a model for Trichophyton inflammation induced by trichophytin extracted from Trichophyton mentagrophytes using a method based on contact hypersensitivity.Trichophytin is a crude extract that mainly contains fungal cell wall constituents including β-glucan and zymosan. In this model, TLR2, TLR4, and dectin-1 were highly expressed, and production of IL-17A and IL23 was observed. This indicates that we succeeded in inducing fungal-specific inflammation in the mice.In this review, we introduce a mouse Trichophyton inflammation model developed to investigate the immunological mechanisms of Trichophyton-induced inflammatory reactions. In addition, we report results of evaluation of anti-inflammatory and anti-itching effects of anti-fungal agents using the inflammation model.

Genome Analysis of Fimbriiglobus ruber SP5T, a Planctomycete with Confirmed Chitinolytic Capability.

Members of the bacterial order Planctomycetales have often been observed in associations with Crustacea. The ability to degrade chitin, however, has never been reported for any of the cultured planctomycetes although utilization of N-acetylglucosamine (GlcNAc) as a sole carbon and nitrogen source is well recognized for these bacteria. Here, we demonstrate the chitinolytic capability of a member of the family Gemmataceae, Fimbriiglobus ruber SP5T, which was isolated from a peat bog. As revealed by metatranscriptomic analysis of chitin-amended peat, the pool of 16S rRNA reads from F. ruber increased in response to chitin availability. Strain SP5T displayed only weak growth on amorphous chitin as a sole source of carbon but grew well with chitin as a source of nitrogen. The genome of F. ruber SP5T is 12.364 Mb in size and is the largest among all currently determined planctomycete genomes. It encodes several enzymes putatively involved in chitin degradation, including two chitinases affiliated with the glycoside hydrolase (GH) family GH18, GH20 family β-N-acetylglucosaminidase, and the complete set of enzymes required for utilization of GlcNAc. The gene encoding one of the predicted chitinases was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The genome also contains genes required for the assembly of type IV pili, which may be used to adhere to chitin and possibly other biopolymers. The ability to use chitin as a source of nitrogen is of special importance for planctomycetes that inhabit N-depleted ombrotrophic wetlands.IMPORTANCE Planctomycetes represent an important part of the microbial community in Sphagnum-dominated peatlands, but their potential functions in these ecosystems remain poorly understood. This study reports the presence of chitinolytic potential in one of the recently described peat-inhabiting members of the family Gemmataceae, Fimbriiglobus ruber SP5T This planctomycete uses chitin, a major constituent of fungal cell walls and exoskeletons of peat-inhabiting arthropods, as a source of nitrogen in N-depleted ombrotrophic Sphagnum-dominated peatlands. This study reports the chitin-degrading capability of representatives of the order Planctomycetales.

ECHINOCANDINS VERSUS DATED ANTIFUNGALS IN COMBINATION AGAINST OPPORTUNISTIC MYCOTIC INFECTIONS

Scientific and clinical reports globally demonstrated that the opportunistic mycotic infections are at major risk to the human fitness. In past few decades, development of resistance in microbes to existing antifungals, has emphasized on the search of new antimycotic drugs. As a matter of fact "echinocandins" are new categories of broad-spectrum antifungal enlighten a hope in this direction. Echinocandins are bulky lipopeptides that inhibits the production of β-[1,3]-glucan "a major constituent of fungal cell wall" which ultimately leads to the death of fungal pathogens. In vitro as well as in vivo published reports have demonstrated that the echinocandins exhibit fungicidal activity against most Candida spp while fungistatic against Aspergillus spp and exclusively found to be more effective when tested in combination with polyenes/azoles. Present article is an expert views on the recent and historical literature available on the antifungal therapies with accessing their impact on the human health. Emphasis is given on the utility of the echinocandins as potential antifungal agent by discussing recent examples of clinical and laboratory studies including the use of improved proteomics approaches to know a bit more about the interaction of human host and fungal pathogens.

1,3-β-D-Glucan contamination of common antimicrobials.

1,3-β-D-Glucan (BDG) is a fungal cell wall constituent used in the diagnosis of invasive fungal infections. BDG testing, although endorsed by the European Organization for Research and Treatment of Cancer, suffers from limited specificity. False-positive results have been linked to haemodialysis membranes, blood products, antineoplastic agents and antimicrobial use. The aim of this study was to determine whether false-positive BDG results in the context of antimicrobial use are caused by BDG present in infusion solutions. We obtained 35 antimicrobial drugs (30 antibiotics and 5 antifungals) and analysed their BDG content using two different assays. Twenty-five antimicrobials (20 antibiotics and all the tested antifungals) contained enough BDG to trigger a positive test. Depending on the substance, BDG varied between 9 and 2818 pg/mL. A majority of the available antimicrobial substances contained BDG, potentially limiting the utility of BDG testing in the context of prior exposure to these drugs. As the cumulative effects of repeated BDG exposure are unknown, efforts to reduce contamination should be considered.

The N-acetylglucosamine catabolic gene cluster in Trichoderma reesei is controlled by the Ndt80-like transcription factor RON1.

SummaryChitin is an important structural constituent of fungal cell walls composed of N‐acetylglucosamine (GlcNAc) monosaccharides, but catabolism of GlcNAc has not been studied in filamentous fungi so far. In the yeast C andida albicans, the genes encoding the three enzymes responsible for stepwise conversion of GlcNAc to fructose‐6‐phosphate are clustered. In this work, we analysed GlcNAc catabolism in ascomycete filamentous fungi and found that the respective genes are also clustered in these fungi. In contrast to C . albicans, the cluster often contains a gene for an Ndt80‐like transcription factor, which we named RON1 (regulator of N‐acetylglucosamine catabolism 1). Further, a gene for a glycoside hydrolase 3 protein related to bacterial N‐acetylglucosaminidases can be found in the GlcNAc gene cluster in filamentous fungi. Functional analysis in T richoderma reesei showed that the transcription factor RON1 is a key activator of the GlcNAc gene cluster and essential for GlcNAc catabolism. Furthermore, we present an evolutionary analysis of Ndt80‐like proteins in Ascomycota. All GlcNAc cluster genes, as well as the GlcNAc transporter gene ngt1, and an additional transcriptional regulator gene, csp2, encoding the homolog of N eurospora crassa CSP2/GRHL, were functionally characterised by gene expression analysis and phenotypic characterisation of knockout strains in T . reesei.

Fungal chitinases: function, regulation, and potential roles in plant/pathogen interactions

In the past decades our knowledge about fungal cell wall architecture increased tremendously and led to the identification of many enzymes involved in polysaccharide synthesis and remodeling, which are also of biotechnological interest. Fungal cell walls play an important role in conferring mechanic stability during cell division and polar growth. Additionally, in phytopathogenic fungi the cell wall is the first structure that gets into intimate contact with the host plant. A major constituent of fungal cell walls is chitin, a homopolymer of N-acetylglucosamine units. To ensure plasticity, polymeric chitin needs continuous remodeling which is maintained by chitinolytic enzymes, including lytic polysaccharide monooxygenases N-acetylglucosaminidases, and chitinases. Depending on the species and lifestyle of fungi, there is great variation in the number of encoded chitinases and their function. Chitinases can have housekeeping function in plasticizing the cell wall or can act more specifically during cell separation, nutritional chitin acquisition, or competitive interaction with other fungi. Although chitinase research made huge progress in the last decades, our knowledge about their role in phytopathogenic fungi is still scarce. Recent findings in the dimorphic basidiomycete Ustilago maydis show that chitinases play different physiological functions throughout the life cycle and raise questions about their role during plant-fungus interactions. In this work we summarize these functions, mechanisms of chitinase regulation and their putative role during pathogen/host interactions.

Detailed characterization of a long-term rodent model of critical illness and recovery.

To characterize a long-term model of recovery from critical illness, with particular emphasis on cardiorespiratory, metabolic, and muscle function. Randomized controlled animal study. University research laboratory. Male Wistar rats. Intraperitoneal injection of the fungal cell wall constituent, zymosan or n-saline. Following intervention, rats were followed for up to 2 weeks. Animals with zymosan peritonitis reached a clinical and biochemical nadir on day 2. Initial reductions were seen in body weight, total body protein and fat, and muscle mass. Leg muscle fiber diameter remained subnormal at 14 days with evidence of persisting myonecrosis, even though gene expression of regulators of muscle mass (e.g., MAFbx, MURF1, and myostatin) had peaked on days 2-4 but normalized by day 7. Treadmill exercise capacity, forelimb grip strength, and in vivo maximum tetanic force were also reduced. Food intake was minimal until day 4 but increased thereafter. This did not relate to appetite hormone levels with early (6 hr) rises in plasma insulin and leptin followed by persisting subnormal levels; ghrelin levels did not change. Serum interleukin-6 level peaked at 6 hours but had normalized by day 2, whereas interleukin-10 remained persistently elevated and high-density lipoprotein cholesterol persistently depressed. There was an early myocardial depression and rise in core temperature, yet reduced oxygen consumption and respiratory exchange ratio with a loss of diurnal rhythmicity that showed a gradual but incomplete recovery by day 7. This detailed physiological, metabolic, hormonal, functional, and histological muscle characterization of a model of critical illness and recovery reproduces many of the findings reported in human critical illness. It can be used to assess putative therapies that may attenuate loss, or enhance recovery, of muscle mass and function.

Genomic analyses and transcriptional profiles of the glycoside hydrolase family 18 genes of the entomopathogenic fungus Metarhizium anisopliae.

Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen Metarhizium anisopliae, chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the M. anisopliae genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named chit1, chi2 and chi3. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-N-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different M. anisopliae developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of M. anisopliae chitinases and ENGases and a more detailed study concerning the enzymes’ roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.

Exposure to Beta-(1,3)-D-glucan in house dust at age 7-10 is associated with airway hyperresponsiveness and atopic asthma by age 11-14.

BackgroundMould exposure has been linked to childhood asthma and bronchial hyper-responsiveness. Few studies have assessed beta-(1,3)-d-glucan (beta-glucan), a significant fungal cell wall constituent, in relation to asthma in adolescence.ObjectiveTo determine whether house dust-derived beta-glucan exposure at age 7–10 is associated with the development and persistence of atopic and non-atopic asthma, and bronchial hyper-responsiveness (BHR) by age 11–14.MethodsDust samples were collected from the 1995 Study of Asthma, Genes, and Environment (SAGE) birth cohort. This cohort was derived from Manitoba provincial healthcare administrative records of children high and low risk for asthma. Samples were collected from the homes of 422 children at age 7–10 and analyzed using beta-glucan and endotoxin-specific Limulus Amoebocyte Lysate assays. Asthma, atopy, and BHR status of each child were also assessed at ages 7–10 and 11–14.ResultsAt age 7–10, beta-glucan dust levels in the home were associated with persistent atopic asthma at age 11–14 (OR 1.79 for each unit increase in levels, 95% CI 1.14–2.81), independent of endotoxin exposure, and Alternaria or Cladosporium sensitization. The likelihood of BHR almost doubled with unit increases in dust beta-glucan in asthmatic children. In children without asthma, exposure to high beta-glucan levels at age 7–10 also elevated risk for BHR in adolescence (OR 1.74, 95% CI 1.05–2.89). New-onset atopic asthma was twice more likely following high beta-glucan exposure in children without asthma but the association did not reach statistical significance. No associations were evident with concurrent asthma phenotype at age 7–10 or non-atopic asthma at age 11–14.ConclusionThese findings implicate home beta-glucan exposure at school-age as a risk factor for persistent atopic asthma and new-onset BHR. The higher prevalence of BHR in urban adolescents may be propagated by this home exposure.

Study on the Discovery of Novel Chitinase Inhibitors Based on Natural Products

Chitin, the second most abundant polysaccharide in nature, is a constituent of fungal cell walls, the exoskeletons of crustaceans and insects and the microfilarial sheaths of parasitic nematodes. Chitin has, so far, not been found in mammals. Accumulation of chitin by organisms is modulated by chitin synthase-mediated biosynthesis and by chitinase-mediated hydrolytic degradation. Thus, chitinases are expected to be specific targets for antifungal, insecticidal and antiparasitic agents. Paradoxically, while chitin does not exist in mammals, human chitinase family members, such as acidic mammalian chitinase, have recently been described, and offer significant potential for the treatment of asthma and other related diseases in humans. This review covers the development of two chitinase inhibitors of natural origin, Argifin and Argadin, isolated from the cultured broth of microorganisms in our laboratory. In particular, the practical total synthesis of these natural products and discovery methods that generate only highly-active compounds using a kinetic target (chitinase)-guided synthesis approach (termed in situ click chemistry) are described.

Putative Chitin Synthases from Branchiostoma floridae Show Extracellular Matrix-related Domains and Mosaic Structures

The transition from unicellular to multicellular life forms requires the development of a specialized structural component, the extracellular matrix (ECM). In Metazoans, there are two main supportive systems, which are based on chitin and collagen/hyaluronan, respectively. Chitin is the major constituent of fungal cell walls and arthropod exoskeleton. However, presence of chitin/chitooligosaccharides has been reported in lower chordates and during specific stages of vertebrate development. In this study, the occurrence of chitin synthases (CHSs) was investigated with a bioinformatics approach in the cephalochordate Branchiostoma floridae, in which the presence of chitin was initially reported in the skeletal rods of the pharyngeal gill basket. Twelve genes coding for proteins containing conserved amino acid residues of processive glycosyltransferases from GT2 family were found and 10 of them display mosaic structures with novel domains never reported previously in a chitin synthase. In particular, the presence of a discoidin (DS) and a sterile alpha motif (SAM) domain was found in nine identified proteins. Sequence analyses and homology modelling suggest that these domains might interact with the extracellular matrix and mediate protein–protein interaction. The multi-domain putative chitin synthases from B. floridae constitute an emblematic example of the explosion of domain innovation and shuffling which predate Metazoans.

Salmonella Biofilm Formation on Aspergillus niger Involves Cellulose – Chitin Interactions

Salmonella cycles between host and nonhost environments, where it can become an active member of complex microbial communities. The role of fungi in the environmental adaptation of enteric pathogens remains relatively unexplored. We have discovered that S. enterica Typhimurium rapidly attaches to and forms biofilms on the hyphae of the common fungus, Aspergillus niger. Several Salmonella enterica serovars displayed a similar interaction, whereas other bacterial species were unable to bind to the fungus. Bacterial attachment to chitin, a major constituent of fungal cell walls, mirrored this specificity. Pre-incubation of S. Typhimurium with N-acetylglucosamine, the monomeric component of chitin, reduced binding to chitin beads by as much as 727-fold and inhibited attachment to A. niger hyphae considerably. A cellulose-deficient mutant of S. Typhimurium failed to attach to chitin beads and to the fungus. Complementation of this mutant with the cellulose operon restored binding to chitin beads to 79% of that of the parental strain and allowed for attachment and biofilm formation on A. niger, indicating that cellulose is involved in bacterial attachment to the fungus via the chitin component of its cell wall. In contrast to cellulose, S. Typhimurium curli fimbriae were not required for attachment and biofilm development on the hyphae but were critical for its stability. Our results suggest that cellulose–chitin interactions are required for the production of mixed Salmonella-A. niger biofilms, and support the hypothesis that encounters with chitinaceous alternate hosts may contribute to the ecological success of human pathogens.

Dynamics of soil amino sugar pools during decomposition processes of corn residues as affected by inorganic N addition

Identifying the impact of inorganic-nitrogen (N) availability on soil amino sugar dynamics during corn (Zea mays L.) residue decomposition may advance our knowledge of microbial carbon (C) and N transformations and the factors controlling these processes in soils. Amino sugars are routinely used as microbial biomarkers to investigate C and N sequestration in microbial residues, and they are also involved in microbial-mediated soil organic matter (SOM) turnover. We conducted a 38-week incubation study using a Mollisol which was amended with corn residues and four levels of inorganic N (i.e., 0, 60.3, 167.2, and 701.9 mg N kg−1 soil). The objective of this study was to examine the effects of inorganic-N availability on fungal and bacterial formation and stabilization of heterogeneous amino sugars during the corn residue decomposition in soil. The surface soil (0–20 cm) used for the experiment was taken from a site in Gongzhuling, Jilin Province of China (43°30′N, 124°48′E). The sampled soils were air-dried, mixed, and passed through a 2-mm sieve. The incubation experiments were carried out at 25 °C, and soil moisture was maintained at 20% gravimetric moisture content by regularly weighing the plastic containers and adding water to the plastic containers during the incubation if necessary. The incubation was destructively sampled at weeks 1, 2, 4, 8, 12, 18, 28, and 38 of the incubation. The whole samples were air-dried, ground (<0.25 mm), and analyzed for amino sugar composition and concentration. Amino sugars (d-glucosamine, d-galactosamine, d-muramic acid, and d-mannosamine) were determined by capillary gas chromatography (GC) after their conversion to aldonitrile acetates (Zhang and Amelung 1996). Soil amino sugar contents and their contribution to SOM accumulation were significantly enhanced by the incorporation of corn residues, and amino sugar concentration maxima were observed during the 38-week-incubation course. Inorganic nitrogen significantly affected the accumulation of amino sugars during the decomposition of corn residues which had a high C/N ratio. It was found that more amino sugars were accumulated with higher inorganic N addition rates. However, when very high N application rates were evaluated, excess inorganic N in soil could not be taken up by microorganisms and some remained as inorganic N in the soil at the end of the incubation. Moreover, the dynamics of the individual amino sugars were different in response to the level of applied inorganic N. Muramic acid, a constituent of bacterial cell walls, was more sensitive to inorganic N supply and showed a faster turnover rate, while glucosamine, a constituent of fungal cell walls, was less sensitive to inorganic N supply. Glucosamine accumulated during the decay process of corn residues, and it was eventually stabilized in the soil. When corn residues were decomposing, individual amino sugar pools were affected by inorganic N addition. Bacterial cell-wall residues played an important role in soil N transformation in the early stages of corn residue decomposition, however, bacterial residues were rapidly remineralized. In the later stages of residue decomposition, amino sugars were assimilated into fungal cell walls which accumulated in soil and contributed to the long-term maintenance of SOM. Therefore, when plant residues with a high C/N ratio are incorporated into the soil, sufficient inorganic N is required for microbial growth. The addition of inorganic N to soils which have received plant residues with C/N ratio ≥30:1 is required to sustain microbial rapid growth; however, oversupply of inorganic N may increase the risk of inorganic N losses from agricultural fields.

Content of (1-3)-β-D-glucan in Common Indoor Air Fungi

RATIONALE: Exposure to (1-3)-β-D-glucan, major fungal cell wall constituent, has been associated with various health effects. It has been speculated whether (1-3)-β-D-glucan exposure can be used as a surrogate for mold exposure. The aim of this study was to analyze the (1-3)-β-D-glucan content of common indoor fungal species.