Candida albicans cell wall mannosidases, Dfg5 and Dcw1, regulate chitin synthesis

In Candida albicans chitin synthesis is important for cell wall integrity and may also have a role in emergence of drug-resistance. Our past studies showed that cell wall mannosidases, Dfg5 and Dcw1, regulate HOG MAPK signaling. In this study, we investigated how Dfg5 and Dcw1 regulate chitin synthesis by affecting HOG, PKC and Calcium-Calcineurin signaling pathways. DFG5 and DCW1 heterologous mutants (ES1 & ES195) and a conditional mutant (ES195+methionine/cysteine) were utilized. WT SC5314 served as negative control and Hog1 knock-out mutant as positive control. Fluorescence microscopy of calcofluor white (CFW) stained mutant and control strains was performed to observe chitin accumulation. Quantitative PCR analysis was performed to measure the relative expression of chitin synthases CHS1, CHS2, CHS3 and CHS8. Incubation with chitinase was done to determine cell separation using light microscopy and scanning electron microscopy (SEM) analysis. Fluorescence microscopy showed significantly increased chitin accumulation in the mutants as compared to wild type. Chitin accumulation was observed mainly at the budding sites indicating a cause for defective cell separation phenotype. Incubation with chitinase led to cell separation in the mutants. CHS2, CHS3 and CHS8 expression was observed to be significantly upregulated in the conditional mutant and HOG1 mutant as compared to the wild type. This upregulation was also observed when the cell wall integrity PKC pathway was activated. However, activation of the Calcium-calcineurin pathway downregulated chitin synthase expression in the mutants. Our data indicates that Dfg5 and Dcw1 regulate expression of chitin synthases via HOG MAPK, PKC and Calcium-calcineurin signaling pathways.

A Novel Role for Histatin 5 in Combination with Zinc to Promote Commensalism in C. albicans Survivor Cells

Candida albicans is maintained as a commensal by immune mechanisms at the oral epithelia. Oral antifungal peptide Histatin 5 (Hst 5) may function in innate immunity, but the specific role Hst 5 plays in C. albicans commensalism is unclear. Since Zn-binding potentiates the candidacidal activity of Hst 5, we hypothesized that Hst 5+Zn would elicit a unique fungal stress response to shape interactions between C. albicans and oral epithelial cells (OECs). We found that Hst 5+Zn but not Hst 5 alone resulted in the activation of cell wall integrity (CWI) signaling, and deletion mutants were then used to determine that CWI-mediated chitin synthesis was protective against killing. Using flow cytometry, we confirmed that Hst 5+Zn-treated cells had significantly elevated levels of cell-wall chitin, mannan and β-1,3 glucan compared to Hst 5-treated cells. We then tested the activation of host signaling components involved in C. albicans cell-wall recognition. The immunoblot assay of C. albicans-exposed oral epithelial cells showed increased activation of EphA2 and NF-κB but not EGFR. Interestingly, C. albicans treated with Hst 5+Zn induced the global suppression of pro-inflammatory cytokine release from OECs, but an increase in negative regulator IL-10. Hst 5+Zn-treated cells were more adherent but ultimately less invasive to OECs than control cells, thus indicating lowered virulence. Therefore, Hst 5+Zn-treated C. albicans cells are discerned by epithelial monolayers, but are less virulent and promote anti-inflammatory signaling, suggesting that Hst 5+Zn in combination could play a role in regulating commensalism of oral C. albicans through cell wall reorganization.

Is the suckling period and application pattern relevant for fluazuron against tick infestation in cows and their suckling calves?

Background Fluazuron is a chitin synthesis inhibitor administered as a pour-on formulation in cattle for tick control. This study analyzes under endemic tick infestation, the incidence of the pour-on application pattern on the plasma levels of fluazuron in calves and cows in the lactation period of the beef cow. Two hundred and ninety-two beef cows around parturition were treated with a commercial pour-on formulation of fluazuron at a rate of 2.5 mg/kg of body weight. A total of 4 treatments were carried out on days 0, 32, 77, and 117. At each administration time, the cows were grouped according to the pour-on administration pattern: long (~ 60 cm pour-on application surface) and short (~ 30 cm pour-on application surface). Fluazuron levels in cows and calves plasma were determined before the third and fourth application for each subgroup (n = 10) by HPLC-MS/MS. During the entire study, cow-calf pairs were maintained under field conditions and qualitatively examined for tick infestation on the day of each treatment. Both treatments (long and short) schemes were designed to prevent the annual persistence of ticks. Results No animals with presence of ticks were identified during the first 117 days of the study, except for three cows and one calf at the time of the third application (day 77). There were no differences after 40 days (day 77) post-treatment of the second application (30 ± 5 ppb vs. 28.5 ± 12 ppb, p > 0.05) and 45 days (day 117) after the third application (147 ± 55 ppb vs 140 ± 46 ppb, p > 0.05) between groups of cows treated with the long or short pour-on application, respectively. Plasma concentration of fluazuron at second and third application was increased (3.3 and 2.9 times, respectively) in calves under free suckling compared to cows. Nevertheless, both groups of cows and calves showed a significant increase in plasma concentration of fluazuron between times (4.9 times, p < 0.0001 and 2.8 times, p < 0.0001, respectively). In both groups, tick prevalence was 0% throughout the trial, except for day 77, which reached 1%. Conclusions The main conclusions of this study were the following: 1) Different administration patterns (long vs. short) did not differ in plasma levels of fluazuron.; 2) Given that only the cows were treated and lactating calves presented higher plasma levels of fluazuron than cows, passage through milk appears to be relevant and possibly due to a cumulative effect and continuous drug intake.

The Antifungal Effects of Citral on Magnaporthe oryzae Occur via Modulation of Chitin Content as Revealed by RNA-Seq Analysis

The natural product citral has previously been demonstrated to possess antifungal activity against Magnaporthe oryzae. The purpose of this study was to screen and annotate genes that were differentially expressed (DEGs) in M. oryzae after treatment with citral using RNA sequencing (RNA-seq). Thereafter, samples were reprepared for quantitative real-time PCR (RT-qPCR) analysis verification of RNA-seq data. The results showed that 649 DEGs in M. oryzae were significantly affected after treatment with citral (100 μg/mL) for 24 h. Kyoto Encyclopedia of Genes and Genomes (KEGG) and a gene ontology (GO) analysis showed that DEGs were mainly enriched in amino sugar and nucleotide sugar metabolic pathways, including the chitin synthesis pathway and UDP sugar synthesis pathway. The results of the RT-qPCR analysis also showed that the chitin present in M. oryzae might be degraded to chitosan, chitobiose, N-acetyl-D-glucosamine, and β-D-fructose-6-phosphate following treatment with citral. Chitin degradation was indicated by damaged cell-wall integrity. Moreover, the UDP glucose synthesis pathway was involved in glycolysis and gluconeogenesis, providing precursors for the synthesis of polysaccharides. Galactose-1-phosphate uridylyltransferase, which is involved in the regulation of UDP-α-D-galactose and α-D-galactose-1-phosphate, was downregulated. This would result in the inhibition of UDP glucose (UDP-Glc) synthesis, a reduction in cell-wall glucan content, and the destruction of cell-wall integrity.

Crosstalk between calcineurin and the cell wall integrity pathways prevents chitin overexpression in Candida albicans

Echinocandins such as caspofungin are front line antifungal drugs that compromise β-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by up-regulation of chitin synthesis, thereby sustaining cell wall integrity. When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a sub-population of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable super high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway.

Vital Carbohydrate and Lipid Metabolites in Serum Involved in Energy Metabolism during Pubertal Molt of Mud Crab (Scylla paramamosain)

Pubertal molt is a vital stage in the cultivation of mature female crabs in the aquacultural industry of Scylla paramamosain. Since fasting occurs during molting, which requires a large supply of energy, internal energy reserves are critical. However, the dynamics of energy supply during pubertal molt is not clear. This study focuses on the variations of carbohydrates and lipids in serum during the pubertal molt of S. paramamosain via a metabolomics approach. Eleven lipid or carbohydrate metabolic pathways were significantly influenced postmolt. A remarkable decrease in carbohydrates in serum suggested that free sugars were consumed for energy. A significant decrease in glucose and alpha-d-glucosamine 1-phosphate showed that chitin synthesis exhausted glycogen, resulting in insufficient glucose supply. An increase in l-carnitine and acetylcarnitine, and a significant decrease in 15 fatty acyls and 8 glycerophosphocholines in serum indicated that carnitine shuttle was stimulated, and β-oxidation was upregulated postmolt. In addition, astaxanthin, ponasterone A, and riboflavin in serum were significantly decreased postmolt. Eleven potential metabolite biomarkers were identified for pubertal molt. Taken together, carbohydrates and lipids were possibly major energy reserves in pubertal molt. This study suggests that an increase in carbohydrate and lipid levels in crab feed may alleviate the effects of fasting during molt and improve farm productivity in mature female crabs.

The citron homology domain as a scaffold for Rho1 signaling

Aspergillus fumigatus is a human opportunistic pathogen showing emerging resistance against a limited repertoire of antifungal agents available. The GTPase Rho1 has been identified as an important regulator of the cell wall integrity signaling pathway that regulates the composition of the cell wall, a structure that is unique to fungi and serves as a target for antifungal compounds. Rom2, the guanine nucleotide exchange factor to Rho1, contains a C-terminal citron homology (CNH) domain of unknown function that is found in many other eukaryotic genes. Here, we show that the Rom2 CNH domain interacts directly with Rho1 to modulate β-glucan and chitin synthesis. We report the structure of the Rom2 CNH domain, revealing that it adopts a seven-bladed β-propeller fold containing three unusual loops. A model of the Rho1–Rom2 CNH complex suggests that the Rom2 CNH domain interacts with the Rho1 Switch II motif. This work uncovers the role of the Rom2 CNH domain as a scaffold for Rho1 signaling in fungal cell wall biosynthesis.

Atropine Reverts the Neurobehavioral Toxicity Elicited by Acute Exposure to Buprofezin in Sprague Dowley Rats

Buprofezin (BPFN) is a thiadiazine insecticide that inhibits chitin synthesis and the moulting in case of white flies, mealybugs and leaf hoppers. The exposed insects are unable to shed their cuticle and ultimately die as moulting ensue. Neurobehavioral toxic effects elicited by buprofezin remained unclear. Furthermore, the reversal of buprofezin induced neurobehavioral toxicity by atropine was not elaborated. Thus, we explored the neurobehavioral toxic consequences of acute buprofezin exposure in adult male rats and effective reversal of these changes by pretreatment with atropine as an antidote. Acute administration of commercial buprofezin (87.9mg/kg/day through oral gavage with corn oil as vehicle) induce a wide range of neurobehavioral toxicity including damage to pyramidal cells of hippocampal CA1, and CA3,region and behavioral impairments as demonstrated through, loss of motor coordination, locomotor activity, fear loss, hearing, sensorimotor, cognitive and spatial navigation impairment following the exposure .These neurobehavioral toxic effect of acute buprofezin exposure were significantly reversed by the 15 min pre-treatment of atropine antidote before the buprofezin administration. Pre-treated atropine (20mg/kg/day;i.p) attenuates the neurobehavioral toxicity induced by buprofezin in male rats. It was suggested that acute buprofezin exposure elevated the acetylcholine level, by inhibiting the synthesis and release of acetylcholine esterase (AChE) in synapse. But the complete mechanisms are remained to be elucidated