Cloning, functional characterization and screening of potential inhibitors for Chilo partellus chitin synthase A using in silico, in vitro and in vivo approaches.

Chitin synthase (CHS) is one of the crucial enzymes that play an essential role in chitin synthesis during the molting process, and it is considered to be the specific target to control insect pests. Currently, there are no potent inhibitors available in the market, which specifically target this enzyme. Pyrimidine nucleoside peptide, nikkomycin Z, binds to nucleotide-binding sites of fungal and insect CHS. But, their mode of action is still fragmentary due to the lack of a 3Dstructure of CHS. Chilo partellus is a severe pest insect of major food crops such as maize and sorghum, in an attempt to target integument expressed cuticular CpCHS. The CpChsA cDNA was cloned, and subsequently, their developmental and tissue-specific expression was studied. The 3D structure of the CHS catalytic domain was modeled, after which natural compounds were screened using a virtual screening workflow and resulted in the identification of five hit molecules. Molecular dynamics simulations were performed to investigate the dynamics and interactions of hits with CpCHS. The obtained results revealed that the compounds kasugamycin, rutin and robinin could act as potent inhibitors of CpCHS. All three molecules were observed to significantly reduce the chitin production as validated using in vitro and in vivo studies. Thus, this study aims to provide a set of novel inhibitor molecules against CpCHS for controlling the pest population. Communicated by Ramaswamy H. Sarma.

Structural characterization of a putative chitin synthase gene in Phytophthora spp. and analysis of its transcriptional activity during pathogenesis on potato and soybean plants.

Although chitin is a major component of the fungal cell wall, in oomycetes (fungal-like organisms), this compound has only been found in very little amounts, mostly in the cell wall of members of the genera Achlya and Saprolegnia. In the oomycetes Phytophthora infestans and P. sojae the presence of chitin has not been demonstrated; however, the gene putatively encoding chitin synthase (CHS), the enzyme that synthesizes chitin, is present in their genomes. The evolutionary significance of the CHS gene in P. infestans and P. sojae genomes is not fully understood and, therefore, further studies are warranted. We have cloned and characterized the putative CHS genes from two Phytophthora spp. and multiple isolates of P. infestans and P. sojae and analyzed their phylogenetic relationships. We also conducted CHS inhibition assays and measured CHS transcriptional activity in Phytophthora spp. during infection of susceptible plants. Results of our investigations suggest that CHS contains all the motifs that are typical in CHS genes of fungal origin and is expressed, at least at the mRNA level, during in vitro and in planta growth. In infected tissues, the highest levels of expression occurred in the first 12 h post inoculation. In addition, results from our inhibition experiments appear to suggest that CHS activity is important for P. infestans normal vegetative growth. Because of the considerable variation in expression during infection when compared to basal expression observed in in vitro cultures of non-sporulating mycelium, we hypothesize that CHS may have a meaningful role in Phytophthora pathogenicity.

Antifungal curcumin promotes chitin accumulation associated with decreased virulence of Sporothrix schenckii.

Curcumin, a yellow polyphenol compound, is known to possess antifungal activity for a range of pathogenic fungi. However, the fungicidal mechanism of curcumin (CUR) has not been identified. We have occasionally found that chitin redistributes to the cell wall outer layer of Sporothrix schenckii (S. schenckii) upon sublethal CUR treatment. Whether CUR can affect chitin synthesis via the protein kinase C (PKC) signaling pathway has not been investigated. This study describes a direct fungicidal activity of CUR against S. schenckii demonstrated by the results of a checkerboard microdilution assay and, for the first time, a synergistic effect of CUR with terbinafine (TRB). Furthermore, the results of real-time PCR showed that sublethal CUR upregulated the transcription of PKC, chitin synthase1 (CHS1), and chitin synthase3 (CHS3) in S. schenckii. The fluorescence staining results using wheat germ agglutinin-fluorescein isothiocyanate (WGA-FITC) and calcofluor white (CFW) consistently showed that chitin exposure and total chitin content were increased on the conidial cell wall of S. schenckii by sublethal CUR treatment. A histopathological analysis of mice infected with CUR-treated conidia showed dampened inflammation in the local lesion and a reduced fungal burden. The ELISA results showed proinflammatory cytokine secretion at an early stage from macrophages stimulated by the CUR-treated conidia. The present data led to the conclusion that CUR is a potential antifungal agent and that its fungicidal mechanism may involve chitin accumulation on the cell wall of S. schenckii, which is associated with decreased virulence in infected mice.

Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies.

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall ß-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca(2+)-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

Chitin synthesis in Saccharomyces cerevisiae in response to supplementation of growth medium with glucosamine and cell wall stress.

In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.

Homology of Rhizobium meliloti NodC to polysaccharide polymerizing enzymes.

Rhizobium bacteria form nitrogen-fixing nodules on legume roots. As part of the nodulation process, they secrete Nod factors that are beta-1,4-linked oligomers of N-acetylglucosamine. These factors depend on nodulation (nod) genes, but most aspects of factor synthesis are not yet known. We show here that one gene, nodC, shows striking similarity to genes encoding proteins known to be involved in polysaccharide synthesis in yeast and bacteria, specifically chitin and cellulose synthases, as well as a protein with unknown function in Xenopus embryos, DG42. This similarity is consistent with a role for the NodC protein in the formation of the beta-1,4-linkage in Nod factors.