Transcriptional and Proteomic Responses to Carbon Starvation in Paracoccidioides

Abstract

BackgroundThe genus Paracoccidioides comprises human thermal dimorphic fungi, which cause paracoccidioidomycosis (PCM), an important mycosis in Latin America. Adaptation to environmental conditions is key to fungal survival during human host infection. The adaptability of carbon metabolism is a vital fitness attribute during pathogenesis.Methodology/Principal FindingsThe fungal pathogen Paracoccidioides spp. is exposed to numerous adverse conditions, such as nutrient deprivation, in the human host. In this study, a comprehensive response of Paracoccidioides, Pb01, under carbon starvation was investigated using high-resolution transcriptomic (RNAseq) and proteomic (NanoUPLC-MSE) approaches. A total of 1,063 transcripts and 421 proteins were differentially regulated, providing a global view of metabolic reprogramming during carbon starvation. The main changes were those related to cells shifting to gluconeogenesis and ethanol production, supported by the degradation of amino acids and fatty acids and by the modulation of the glyoxylate and tricarboxylic cycles. This proposed carbon flow hypothesis was supported by gene and protein expression profiles assessed using qRT-PCR and western blot analysis, respectively, as well as using enzymatic, cell dry weight and fungus-macrophage interaction assays. The carbon source provides a survival advantage to Paracoccidioides inside macrophages.Conclusions/SignificanceFor a complete understanding of the physiological processes in an organism, the integration of approaches addressing different levels of regulation is important. To the best of our knowledge, this report presents the first description of the responses of Paracoccidioides spp. to host-like conditions using large-scale expression approaches. The alternative metabolic pathways that could be adopted by the organism during carbon starvation can be important for a better understanding of the fungal adaptation to the host, because systems for detecting and responding to carbon sources play a major role in adaptation and persistence in the host niche.

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose

“…The response is conserved over all substrates analysed here, indicating a universal process consistent with the carbon starvation experience. In line with this, enrichment of almost all of these GO-terms was also observed in the 5 days glucose culture where glucose is depleted and increased transcription of genes linked to fatty acid metabolism in response to carbon starvation has been reported for Aspergilli [16,55] as well as in other fungi [56,57]. A role for carbon starvation in induction of plant polysaccharide degradative enzymes was previously established in A. niger [34,38], A. nidulans [36,37,58] and N. crassa [28,59], which involved low-level upregulation of a subset of genes encoding CAZymes active on plant polysaccharides, the 'scouting enzymes' .…”

Analysis ofParacoccidioideslutziimitochondria: a proteomic approach

“…The proteomic approach was performed using NanoUPLC‐MS E as previously described (Murad and Rech, ; Murad et al, ) and adapted for samples of Paracoccidioides species in our laboratory (Bailão et al, ; Lima et al, , ; Oliveira et al, ). The peptide and protein data were generated by the PLGS process and data from analysis are shown in Figure S6.…”

“…The samples, in biological duplicates (70 µg), were analysed using nanoscale liquid chromatography coupled with tandem mass spectrometry (NanoUPLC‐MS E ) as previously described (Lima et al, ; Murad and Rech, ) in three experimental replicates, totalling six runs. Briefly, 50 mM ammonium bicarbonate was added to the sample, followed by addition of 25 μL of RapiGEST TM (0.2% v/v; Waters Corporation, Milford, MA, USA).…”

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time

“…These compounds have been shown to provide some antioxidant benefit when supplemented to cultured neuronal cells (Sawa et al 2017), though a more likely explanation is the use of these compounds in the synthesis of amino acids and/or their derivatives involved in oxidative stress remediation. Increases in glucose and fructose under stress in both isolates may also be reflective of higher levels of metabolic demand for simple sugars, and the beginnings of carbon starvation leading to gluconeogenesis (Dijkema et al 1985; Lima et al 2014), particularly at 7 DAI (Figure 3).…”