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Aspergillus oxylipin signaling and quorum sensing pathways depend on g protein-coupled receptors.

Affeldt KJ, Brodhagen M, Keller NP - Toxins (Basel) (2012)

Bottom Line: Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation.A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production.The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Department of Medical Microbiology and Immunology, 1550 Linden Drive, Madison, WI 53706, USA.

ABSTRACT
Oxylipins regulate Aspergillus development and mycotoxin production and are also involved in Aspergillus quorum sensing mechanisms. Despite extensive knowledge of how these oxylipins are synthesized and what processes they regulate, nothing is known about how these signals are detected and transmitted by the fungus. G protein-coupled receptors (GPCR) have been speculated to be involved as they are known oxylipin receptors in mammals, and many putative GPCRs have been identified in the Aspergilli. Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation. A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production. A. flavus encodes two putative GprD homologs, GprC and GprD, and we demonstrate here that they are required to transition to a high-density development state, as well as to respond to spent medium of a high-density culture. The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

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(a) Conidia produced by low density and high density cultures were counted; (b) Sclerotia were collected from low density and high density cultures, and their dry weight was measured; (c) Aflatoxin (AF) was extracted from low density and high density cultures, separated by thin layer chromatography (TLC), and visualized under 366-nm light. The absolute intensities of the AF spots were calculated as described in the Experimental section. For all three graphs in (a), (b), and (c), the data were analyzed using one-way ANOVA and a Tukey post-test. Different letters represent statistically significant differences (p < 0.05), with lowercase letters used for low-density data and uppercase letters used for high-density data; (d) A sample of the plates is shown, containing a full set of high-density plates (top four panels) both before (“Unwashed”) and after (“Washed”) washing off conidia. The wild type at low density (lowest panel) is also included for a point of reference; (e) The TLC plates for low and high density cultures are shown here. An AF standard was run on either side of the plates.
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toxins-04-00695-f003: (a) Conidia produced by low density and high density cultures were counted; (b) Sclerotia were collected from low density and high density cultures, and their dry weight was measured; (c) Aflatoxin (AF) was extracted from low density and high density cultures, separated by thin layer chromatography (TLC), and visualized under 366-nm light. The absolute intensities of the AF spots were calculated as described in the Experimental section. For all three graphs in (a), (b), and (c), the data were analyzed using one-way ANOVA and a Tukey post-test. Different letters represent statistically significant differences (p < 0.05), with lowercase letters used for low-density data and uppercase letters used for high-density data; (d) A sample of the plates is shown, containing a full set of high-density plates (top four panels) both before (“Unwashed”) and after (“Washed”) washing off conidia. The wild type at low density (lowest panel) is also included for a point of reference; (e) The TLC plates for low and high density cultures are shown here. An AF standard was run on either side of the plates.

Mentions: Considering the critical role of oxylipins in A. flavus quorum sensing development and the possibility that GprC/D could be involved in oxylipin perception, the A. flavus GprC and GprD mutants were examined for density dependent development. The fungi were grown at low (103 spores per plate) and high (107 spores per plate) densities, and production of conidia, sclerotia, and AF were measured after seven days (Figure 3). At low density, there were subtle differences between the mutants and wild type (NRRL3357), but the most striking differences were seen at high density. Here, production of conidia was high in the wild type, but it was reduced 2.3 fold in the ∆gprC strain and was almost completely absent in the KD::gprCD mutant (both p < 0.001, Figure 3a,d). Conversely, sclerotia production was minimal in the wild type at high density. As seen in Figure 3b,d, all three mutants exhibited profound increases in sclerotia production at high density. The ∆gprC and ∆gprD strains produced 35.8 and 30.9 fold more sclerotia, respectively, than the wild type, while the KD::gprCD strain produced 60.7 fold more sclerotia than the wild type, approximately doubling what the two single mutants produced (all p < 0.001). AF biosynthesis was also regulated in response to population density changes, being up-regulated at low density and down-regulated at high density as previously reported [16]. The ∆gprD and KD::gprCD mutants showed modest increases in AF compared to the wild type at low density. Most remarkably, the KD::gprCD mutant produced more AF at high density than wild type AF production at the favorable low density conditions (Figure 3e).


Aspergillus oxylipin signaling and quorum sensing pathways depend on g protein-coupled receptors.

Affeldt KJ, Brodhagen M, Keller NP - Toxins (Basel) (2012)

(a) Conidia produced by low density and high density cultures were counted; (b) Sclerotia were collected from low density and high density cultures, and their dry weight was measured; (c) Aflatoxin (AF) was extracted from low density and high density cultures, separated by thin layer chromatography (TLC), and visualized under 366-nm light. The absolute intensities of the AF spots were calculated as described in the Experimental section. For all three graphs in (a), (b), and (c), the data were analyzed using one-way ANOVA and a Tukey post-test. Different letters represent statistically significant differences (p < 0.05), with lowercase letters used for low-density data and uppercase letters used for high-density data; (d) A sample of the plates is shown, containing a full set of high-density plates (top four panels) both before (“Unwashed”) and after (“Washed”) washing off conidia. The wild type at low density (lowest panel) is also included for a point of reference; (e) The TLC plates for low and high density cultures are shown here. An AF standard was run on either side of the plates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475224&req=5

toxins-04-00695-f003: (a) Conidia produced by low density and high density cultures were counted; (b) Sclerotia were collected from low density and high density cultures, and their dry weight was measured; (c) Aflatoxin (AF) was extracted from low density and high density cultures, separated by thin layer chromatography (TLC), and visualized under 366-nm light. The absolute intensities of the AF spots were calculated as described in the Experimental section. For all three graphs in (a), (b), and (c), the data were analyzed using one-way ANOVA and a Tukey post-test. Different letters represent statistically significant differences (p < 0.05), with lowercase letters used for low-density data and uppercase letters used for high-density data; (d) A sample of the plates is shown, containing a full set of high-density plates (top four panels) both before (“Unwashed”) and after (“Washed”) washing off conidia. The wild type at low density (lowest panel) is also included for a point of reference; (e) The TLC plates for low and high density cultures are shown here. An AF standard was run on either side of the plates.
Mentions: Considering the critical role of oxylipins in A. flavus quorum sensing development and the possibility that GprC/D could be involved in oxylipin perception, the A. flavus GprC and GprD mutants were examined for density dependent development. The fungi were grown at low (103 spores per plate) and high (107 spores per plate) densities, and production of conidia, sclerotia, and AF were measured after seven days (Figure 3). At low density, there were subtle differences between the mutants and wild type (NRRL3357), but the most striking differences were seen at high density. Here, production of conidia was high in the wild type, but it was reduced 2.3 fold in the ∆gprC strain and was almost completely absent in the KD::gprCD mutant (both p < 0.001, Figure 3a,d). Conversely, sclerotia production was minimal in the wild type at high density. As seen in Figure 3b,d, all three mutants exhibited profound increases in sclerotia production at high density. The ∆gprC and ∆gprD strains produced 35.8 and 30.9 fold more sclerotia, respectively, than the wild type, while the KD::gprCD strain produced 60.7 fold more sclerotia than the wild type, approximately doubling what the two single mutants produced (all p < 0.001). AF biosynthesis was also regulated in response to population density changes, being up-regulated at low density and down-regulated at high density as previously reported [16]. The ∆gprD and KD::gprCD mutants showed modest increases in AF compared to the wild type at low density. Most remarkably, the KD::gprCD mutant produced more AF at high density than wild type AF production at the favorable low density conditions (Figure 3e).

Bottom Line: Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation.A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production.The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Department of Medical Microbiology and Immunology, 1550 Linden Drive, Madison, WI 53706, USA.

ABSTRACT
Oxylipins regulate Aspergillus development and mycotoxin production and are also involved in Aspergillus quorum sensing mechanisms. Despite extensive knowledge of how these oxylipins are synthesized and what processes they regulate, nothing is known about how these signals are detected and transmitted by the fungus. G protein-coupled receptors (GPCR) have been speculated to be involved as they are known oxylipin receptors in mammals, and many putative GPCRs have been identified in the Aspergilli. Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation. A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production. A. flavus encodes two putative GprD homologs, GprC and GprD, and we demonstrate here that they are required to transition to a high-density development state, as well as to respond to spent medium of a high-density culture. The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

Show MeSH
Related in: MedlinePlus