Limits...
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.

Show MeSH

Related in: MedlinePlus

(a) Samples were treated with EtOH (control) or an equivalent volume of 13(S)-HpODE dissolved in EtOH to achieve the final sample concentrations listed; (b) Samples were treated with EtOH (control) or an equivalent volume of pure oxylipin dissolved in EtOH to achieve a final sample concentration of 10 μM. For both (a) and (b), tissues were harvested as described, and cAMP concentrations were measured. Differing letters above bars in (a) denote treatments significantly different from one another (p≤ 0.05; one-tailed paired Student’s T-test). Differences from the EtOH control in (b) are denoted as follows: *p < 0.05; **p < 0.01, determined by one-tailed paired Student’s T-tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475224&req=5

toxins-04-00695-f001: (a) Samples were treated with EtOH (control) or an equivalent volume of 13(S)-HpODE dissolved in EtOH to achieve the final sample concentrations listed; (b) Samples were treated with EtOH (control) or an equivalent volume of pure oxylipin dissolved in EtOH to achieve a final sample concentration of 10 μM. For both (a) and (b), tissues were harvested as described, and cAMP concentrations were measured. Differing letters above bars in (a) denote treatments significantly different from one another (p≤ 0.05; one-tailed paired Student’s T-test). Differences from the EtOH control in (b) are denoted as follows: *p < 0.05; **p < 0.01, determined by one-tailed paired Student’s T-tests.

Mentions: A hallmark of G protein signaling is alteration of cAMP levels; indeed, binding of 9(S)-HODE to the mammalian GPCR, G2A, inhibits cAMP accumulation [35]. Because the role of cAMP in development had already been described for the genetic model A. nidulans [42,43,44,45,46], the first Aspergillus species studied for oxylipin developmental effects [19], we measured cAMP levels in tissues of this fungus exposed to pure plant oxylipins. We first examined the wild type response to increasing concentrations of 13(S)-HpODE and found the cAMP burst to be released in a dose-dependent manner. Fungal tissues were treated with 12.5 μL EtOH containing increasing concentrations of 13(S)-HpODE with a resulting increasing production of cAMP (all p < 0.05; Figure 1a). Previously, individual oxylipin species were measured in homogenized A. nidulans tissues at approximately 30 to 110 nmol/g dry weight [12]. In the current study, assuming a water content of 70% in fungal tissues, 33 nanomoles oxylipin were added per gram dry weight when oxylipins were added at 100 nM and correspondingly more at higher concentrations (Figure 1a). At 10 μM, the cAMP response appeared to be at saturation (as also seen for 9(S)-HODE perceived by G2A [35]), and increasing from 10 μM to 100 μM 13(S)-HpODE did not cause a significant difference in cAMP response (p = 0.6), so oxylipins were applied at 10 μM in subsequent experiments.


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

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

(a) Samples were treated with EtOH (control) or an equivalent volume of 13(S)-HpODE dissolved in EtOH to achieve the final sample concentrations listed; (b) Samples were treated with EtOH (control) or an equivalent volume of pure oxylipin dissolved in EtOH to achieve a final sample concentration of 10 μM. For both (a) and (b), tissues were harvested as described, and cAMP concentrations were measured. Differing letters above bars in (a) denote treatments significantly different from one another (p≤ 0.05; one-tailed paired Student’s T-test). Differences from the EtOH control in (b) are denoted as follows: *p < 0.05; **p < 0.01, determined by one-tailed paired Student’s T-tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

toxins-04-00695-f001: (a) Samples were treated with EtOH (control) or an equivalent volume of 13(S)-HpODE dissolved in EtOH to achieve the final sample concentrations listed; (b) Samples were treated with EtOH (control) or an equivalent volume of pure oxylipin dissolved in EtOH to achieve a final sample concentration of 10 μM. For both (a) and (b), tissues were harvested as described, and cAMP concentrations were measured. Differing letters above bars in (a) denote treatments significantly different from one another (p≤ 0.05; one-tailed paired Student’s T-test). Differences from the EtOH control in (b) are denoted as follows: *p < 0.05; **p < 0.01, determined by one-tailed paired Student’s T-tests.
Mentions: A hallmark of G protein signaling is alteration of cAMP levels; indeed, binding of 9(S)-HODE to the mammalian GPCR, G2A, inhibits cAMP accumulation [35]. Because the role of cAMP in development had already been described for the genetic model A. nidulans [42,43,44,45,46], the first Aspergillus species studied for oxylipin developmental effects [19], we measured cAMP levels in tissues of this fungus exposed to pure plant oxylipins. We first examined the wild type response to increasing concentrations of 13(S)-HpODE and found the cAMP burst to be released in a dose-dependent manner. Fungal tissues were treated with 12.5 μL EtOH containing increasing concentrations of 13(S)-HpODE with a resulting increasing production of cAMP (all p < 0.05; Figure 1a). Previously, individual oxylipin species were measured in homogenized A. nidulans tissues at approximately 30 to 110 nmol/g dry weight [12]. In the current study, assuming a water content of 70% in fungal tissues, 33 nanomoles oxylipin were added per gram dry weight when oxylipins were added at 100 nM and correspondingly more at higher concentrations (Figure 1a). At 10 μM, the cAMP response appeared to be at saturation (as also seen for 9(S)-HODE perceived by G2A [35]), and increasing from 10 μM to 100 μM 13(S)-HpODE did not cause a significant difference in cAMP response (p = 0.6), so oxylipins were applied at 10 μM in subsequent experiments.

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