Dataset Information

JEC21 CN phosphoproteomic study, Yadav and Heitman

ABSTRACT: Label free quantititative phosphoproteomics analysis following TiO2 enrichment, nanoscale capillary chromatography and high resolution tandem mass spectrometry.

INSTRUMENT(S):

ORGANISM(S): Cryptococcus Neoformans Var. Neoformans Jec21

SUBMITTER: Erik Soderblom

LAB HEAD: Joseph Heitman

PROVIDER: PXD059891 | Pride | 2025-08-21

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20240301_081440_10347_JEC21_Vikas_030124.sne	Other
	ID111890_01_Phos_OA10034_10347_022725.raw	Raw
	ID111891_01_Phos_OA10034_10347_022725.raw	Raw
	ID111892_01_Phos_OA10034_10347_022725.raw	Raw
	ID111893_01_Phos_OA10034_10347_022725.raw	Raw

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Project description:Pochonia chlamydosporia (Goddard) Zare & Gams (Ascomycota, Sordariomycetes, Hypocreales, Pochoniaceae, Pochonia) is a nematophagous fungus with significant potential as a biocontrol agent against animal-parasitic nematode. However, the molecular and cellular mechanisms underlying its infection process remain poorly understood. This study aims to provide a comprehensive investigation of P. chlamydosporia infection dynamics in Parascaris equorum eggs using both microscopic and proteomic approaches. The infection was monitored at three distinct stages (early, middle, and late), with corresponding ultrastructural and molecular changes observed. Microscopic analysis using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and light microscopy (LM) revealed the progressive invasion of P. chlamydosporia into nematode eggs. These observations provided detailed insights into the morphological changes in both fungal structures and nematode eggs, highlighting key infection stages such as fungal attachment, germination, and egg degradation. Furthermore, the observations confirmed the stages of fungal colonization, emphasizing the dynamic host-pathogen interaction at the macroscopic level. To complement these observations, a 4D-DIA-based quantitative proteomics approach was employed to analyze the exoproteomic changes in P. chlamydosporia during infection. A total of 410 differentially expressed proteins (DEPs) were identified across the three infection stages, with 313 proteins upregulated and 403 proteins downregulated. Gene Ontology (GO) enrichment analysis revealed that these DEPs are involved in critical biological processes, including cellular stress response, proteolysis, metabalic process, and hydrolase activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further identified key infection-associated pathways, such as signal transduction, cell wall biosynthesis, energy metabolism, and host-pathogen interactions. These findings suggest that P. chlamydosporia employs a highly coordinated molecular strategy to adapt to and exploit its host. Quantitative PCR (qPCR) validation of key genes involved in signal transduction and immune evasion mechanisms further supported the molecular basis of P. chlamydosporia's parasitic behavior. These findings contribute to our understanding of fungal-nematode interactions and lay a solid foundation for the development of P. chlamydosporia as a sustainable tool for integrated pest management.

Project description:Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of L. donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme is a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

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JEC21 CN phosphoproteomic study, Yadav and Heitman

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