Comparative protein analysis of the chitin metabolic pathway in extant organisms: A complex network approach

BackgroundFusarium circinatum is a pressing threat to the cultivation of many economically important pine tree species. Efforts to develop effective disease management strategies can be aided by investigating the molecular mechanisms involved in the host-pathogen interaction between F. circinatum and pine species. Pinus tecunumanii and Pinus patula are two closely related tropical pine species that differ widely in their resistance to F. circinatum challenge, being resistant and susceptible respectively, providing the potential for a useful pathosystem to investigate the molecular responses underlying resistance to F. circinatum. However, no genomic resources are available for P. tecunumanii. Pathogenesis-related proteins are classes of proteins that play important roles in plant-microbe interactions, e.g. chitinases; proteins that break down the major structural component of fungal cell walls. Generating a reference sequence for P. tecunumanii and characterizing pathogenesis related gene families in these two pine species is an important step towards unravelling the pine-F. circinatum interaction.ResultsEight reference based and 12 de novo assembled transcriptomes were produced, for juvenile shoot tissue from both species. EvidentialGene pipeline redundancy reduction, expression filtering, protein clustering and taxonomic filtering produced a 50 Mb shoot transcriptome consisting of 28,621 contigs for P. tecunumanii and a 72 Mb shoot transcriptome consisting of 52,735 contigs for P. patula. Predicted protein sequences encoded by the assembled transcriptomes were clustered with reference proteomes from 92 other species to identify pathogenesis related gene families in P. patula, P. tecunumanii and other pine species.ConclusionsThe P. tecunumanii transcriptome is the first gene catalogue for the species, representing an important resource for studying resistance to the pitch canker pathogen, F. circinatum. This study also constitutes, to our knowledge, the largest index of gymnosperm PR-genes to date.

beta-Glucans are polymers of D-glucose which represent major structural components of fungal cell walls. It was shown previously that fungi interact with macrophages through beta-glucan receptors, thereby inducing release of tumor necrosis factor alpha (TNF-alpha). Additional studies demonstrated that vitronectin, a host adhesive glycoprotein, binds to fungi and enhances macrophage recognition of these organisms. Since vitronectin contains a carbohydrate-binding region, we postulated that vitronectin binds fungal beta-glucans and subsequently augments macrophage TNF-alpha release in response to this fungal component. To study this, we first determined the release of TNF-alpha from alveolar macrophages stimulated with fungal beta-glucan. Maximal TNF-alpha release occurred with moderate concentrations of beta-glucan (100 to 200 micrograms/ml), whereas higher concentrations of beta-glucan (> or = 500 micrograms/ml) caused apparent suppression of the TNF-alpha activity released. This suppression of TNF-alpha activity by high concentrations of beta-glucan was mediated by the particulate beta-glucan binding soluble TNF-alpha, through the lectin-binding domain of the cytokine, rendering the TNF-alpha less available for measurement. Next, we assessed the interaction of vitronectin with beta-glucan. Binding of 125I-vitronectin to particulate fungal beta-glucan was dose dependent and specifically inhibitable by unlabeled vitronectin. Furthermore, treatment of beta-glucan with vitronectin substantially augmented macrophage TNF-alpha release in response to this fungal component. These findings demonstrate that fungal beta-glucan can directly modulate TNF-alpha release from macrophages. Further, these studies indicate that the host adhesive glycoprotein vitronectin specifically binds beta-glucan and augments macrophage cytokine release in response to this fungal element.

Chitin is a major structural component of fungal cell walls (Bartnicki-Garcia 1968; Cabib and Shematek 1981). In filamentous fungi, it contributes to the structure of both lateral walls and septa. In the yeast, Saccharomyces cerevisiae, it is mainly found in the primary septum, although a small amount appears to be dispersed throughout the cell wall (Molano et al. 1980; Horisberger and Vonlanthen 1977; Mol and Wessels 1987). This localization suggested that the formation of chitin and of the chitinous septum would make a good model for the study of mechanisms of morphogenesis. We approached this problem by investigating the synthesis of chitin at the molecular level in the hope that the mechanisms by which chitin synthetase activity is controlled would also be operative in regulating in vivo the formation of the structure supported by the polysaccharide.

The limulus factor G reacts with (1-->3)-beta-D-glucan, a major structural component of fungal cell walls. The Fungitec G test is a colorimetric assay that measures the concentration of (1-->3)-beta-D-glucan and is used as a serodiagnostic test for deep mycosis. Wako-WB003 is another assay for (1-->3)-beta-D-glucan that determines the change in turbidity of the gelatin reaction of limulus factor G with (1-->3)-beta-D-glucan. In five rabbits inoculated intravenously with 1 x 10(7) CFU of Candida albicans, the concentration of (1-->3)-beta-D-glucan measured by the fungitec G test increased gradually reaching a peak of 660.9 +/- 427.9 pg/ml (mean +/- SD) 4 days after inoculation, but to 42.225 +/- 41.275 ng/ml on day 6 in the Wako-WB003 test. In one rabbit challenged intravenously with 5 x 10(6) CFU of C. albicans, (1-->3)-beta-D-glucan increased to 101.5 pg/ml on day 4 on the fungitec G test, whereas the level remained below the detection limit of the Wako-WB003 test throughout the course of the disease. We also detected high concentrations of (1-->3)-beta-D-glucan in 11 patients with candidemia, 4 with suspected candidemia, 1 with invasive pulmonary aspergillosis, and 12 patients with aspergilloma. The concentration of (1-->3)-beta-D-glucan measured by the Fungitec G test was > 150, > 1006.8; 312.1, and 55.6 +/- 37.4 pg/ml (range, 20.1-138.0 pg/ml), and by the Wako-WB003 test > 153.000, > 17.70, 153.000 and 2.645 +/- 7.248 ng/ml (range, < 25.20 ng/ml) in these patients, respectively. In contrast, the concentration of (1-->3)-beta-D-glucan in 9 patients with pulmonary cryptococcosis and 6 with superficial candida colonization ranged from < 13.2 and < 15.3 pg/ml in the Fungitec G test and < 0.53 and < 0.12 ng/ml in Wako-WB003 test. There was a weak relationship between the concentration of (1-->3)-beta-D-glucan measured by the Fungitec G test and Wako-WB003 test (r = 0.521). Our results indicate that the sensitivity of the Wako-WB003 test is lower than that of the Fungitec G test.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Phosphorylation of PBL19 mediated by CERK1 in response to chitin is crucial for enhancing plant defense against fungal pathogens, underscoring the importance of chitin signaling in plant immune responses. Fungal pathogens pose a serious threat to global agriculture, causing substantial yield losses. Plants have evolved complex defense mechanisms, including the recognition of chitin, a major component of fungal cell walls, to activate immune responses that are crucial for defense against pathogens. Protein phosphorylation plays a pivotal role in regulating these responses, fine-tuning immune signaling pathways. Here, we investigate PBL19, a member of the RLCK VII subfamily in Arabidopsis thaliana, known for its pivotal role in chitin signaling pathways. Our study reveals that upon chitin perception, PBL19 undergoes phosphorylation-mediated by the receptor CERK1. Through mass spectrometry analysis, we identified eight specific phosphorylation sites on PBL19. Functional characterization revealed their crucial role in enhancing fungal resistance mechanisms in plants. This study advances our understanding of how plants employ phosphorylation-mediated signaling to combat fungal pathogens, providing insights into fundamental mechanisms of plant immunity.

BackgroundChitinase, a key enzyme family within the pathogenesis-related (PR) protein, plays a crucial role in plant defense by degrading chitin, a major component of fungal cell walls. The HvCHT genes in barley are involved in responses to biotic and abiotic stresses, although their full range of functions is not yet fully understood.ResultsIn this study, we identified 24 potential HvCHT genes through a genome-wide analysis. The comparative synteny analysis showed conserved relationships between HvCHT genes and their homologs in Sorghum bicolor, Oryza sativa, and Arabidopsis thaliana. Chromosomal mapping, gene structure, characterization, protein motif analysis, and miRNA regulation were performed to gain insight into the genetic structures of these genes. Segmental duplication events observed in the HvCHT family suggest an important role in the evolutionary development of these genes. Additionally, cis-regulatory element analysis revealed the presence of light-responsive elements, and regulators for Abscisic acid, methyl jasmonate (MeJA), salicylic acid, and gibberellins, indicating potential involvement in stress responses. Transcriptomic data showed differential expression of HvCHT genes in response to salt stress, with distinct patterns observed in leaf and root tissues. Furthermore, the genes defensive responses to drought stress and Fusarium infection were characterized across multiple time points. Notably, qRT-PCR analysis confirmed the upregulation of HvCHT1, HvCHT4, and HvCHT17, highlighting their potential involvement in stress-related pathways.ConclusionThese findings provide a comprehensive overview of the HvCHT genes role in barley defense mechanisms, underlining their regulatory functions in biotic and abiotic stressors. The results lay the groundwork for future functional studies on HvCHT genes, with the potential to enhance stress tolerance in crops.Clinical trial numberNot applicable.

SummaryChitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.

Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially complements a yeast beta-1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high beta-1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5 expression in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant.

Analysis of Turkey’s airport network structure and centrality in the opening-out period after the first wave of COVID-19: A complex network approach

Pulmonary surfactant inactivation by β-D-glucan and protective role of surfactant protein A

Purpose: The present study investigates the survival and success of new organizations in the light of complex network theory. Methodology: The empirical data was collected using the survey method from the technology park companies are analyzed with social network analysis. Two main methods were used in this study: descriptive statistics and social network analysis. Findings: The findings indicate that new nodes appearing because of splitting up of bigger nodes from present or other related networks have a higher degree of centrality. In practice, this means that companies founded by former members of large-scale companies from these networks are more successful due to the ease in providing the flow of resources and information through previous links. This suggests that the imprint effect can be observed in the appearance, lifecycle, and performance of new nodes in complex networks. Originality: The literature lacks studies on new organizations’ lifecycle in complex networks despite the existence of studies about new organizations in organizational networks. This study examines the appearance, success, and survival of new organizations in networks by complex network approaches such as dynamism, dissipative structures, and uncertainties.

A new method for similarity analysis of protein sequences is presented in this paper. On the basis of positions, proportion difference and various physicochemical properties of 20 kinds of amino acid in different protein sequences, representative information was extracted from protein sequence and converted into a numeric vector, thus further similarities of protein sequences were analyzed by studying the similarities between vectors. To facilitate the comparison between protein sequences of different length, every protein sequence is first mapped to a fixed-length vector, of which the vector information is relative position of amino acids. Then percentage of 20 kinds of amino acids in the sequence and 3 physicochemical properties are combined to constitute physicochemical information vector. Finally, a one-dimensiona l feature vector with 80 elements(feat ure vector) representing a protein sequence is synthesized. The shortest distance method was applied for cluster analysis on feature vectors so as to analyze similarities in protein sequences. In the numerical experiment part of the article, similarity analysis was conducted for 9 different species of the mitochondrial NADH dehydrogenase. The result of numerical experiment is consistent with the biological fact, which validates the effectiveness of model to a certain extent.

Voltage Control in Distributed Generation Systems Based on Complex Network Approach

SUMMARY Chitin and a β-linked glucan were the major chemical components of Aspergillus nidulans cell walls. Other monomeric residues identified in enzymic and acid hydrolyses of whole cell walls and cell-wall fractions included galactose, mannose, glucuronic acid and galactosamine. The β-glucan contained (1 → 3) and (1 → 6) linkages and was two-thirds digested by an exo-β-D-1,3 glucanase prepared from a cell-wall lysing Streptomyces species. An α-glucan was identified as a cell-wall component and it also contained (1 → 3) linkages. This latter polysaccharide was distinguishable from nigeran (an α-1,3; α-1,4 glucan present in other Aspergillus species) by infrared spectroscopy and by its low susceptibility to hydrolysis by an endo-α-1,3; α-1,4 glucan glucanohydrolase. Both glucans were alkali-soluble, but the β-glucan was completely solubilized only after acid extraction of the wall. The N-acetylglucosamine to galactosamine ratio in the A. nidulans cell wall was 1.32 and the two hexosamines were shown to be constituents of distinct polymers. The remaining cell wall was accounted for by protein, lipids, readily extractable and bound, and, in the wild-type, melanin. The melanin was distributed throughout the cell wall but was associated particularly with the chitin fraction. The pigment has been partially characterized chemically and contains indolic residues; this result does not substantiate earlier views that indolic melanins are peculiar to the animal kingdom. Melanin appears to be a finite heteropolymer both in terms of its molecular size and its chemistry.