The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a human pathogenic fungus that switches from a saprobic mycelium to a pathogenic yeast. Consistent with the morphological transition being regulated by the cAMP-signalling pathway, there is an increase in cellular cAMP levels both transiently at the onset (< 24 h) and progressively in the later stages (> 120 h) of the transition to the yeast form, and this transition can be modulated by exogenous cAMP. We have cloned the cyr1 gene encoding adenylate cyclase (AC) and established that its transcript levels correlate with cAMP levels. In addition, we have cloned the genes encoding three Gα (Gpa1–3), Gβ (Gpb1) and Gγ (Gpg1) G proteins. Gpa1 and Gpb1 interact with one another and the N-terminus of AC, but neither Gpa2 nor Gpa3 interacted with Gpb1 or AC. The interaction of Gpa1 with Gpb1 was blocked by GTP, but its interaction with AC was independent of bound nucleotide. The transcript levels for gpa1, gpb1 and gpg1 were similar in mycelium, but there was a transient excess of gpb1 during the transition, and an excess of gpa1 in yeast. We have interpreted our findings in terms of a novel signalling mechanism in which the activity of AC is differentially modulated by Gpa1 and Gpb1 to maintain the signal over the 10 days needed for the morphological switch

MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -3

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -5

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -2

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p>nd prey vectors were simultaneously transformed into yeast strain AH109 and plated out on SD/–Leu/–Trp for 3 days. Yeast colonies that grew on SD/–Leu/Trp were restreaked on SD/–Ade/–His/–Leu/–Trp and incubated for a further 3 days. The results growths indicate that pGAD- and pGAD- could, but pGAD- and pGAD- could not, directly interact with pGBK-. In a series of negative controls, pGBK- could not interact with pGAD-, pGADT7-T, pGAD- and pGAD-. B. Pull-down assays to demonstrate that Gpa1 and Gpb1 both interact with Gpb1. GST and GST-Cyr were purified from bacteria, loaded onto glutathione sepharose beads before incubation with translated S-Gpa1 and 10 mM nucleotide. After washing the beads, the proteins were eluted by the addition of 4× NuPAGE LDS sample buffer, followed by boiling at 90°C for 5 min, and separated on a 4–12% NuPAGE gel under denaturing conditions. Bound Gpa1 was detected as a gel band by autoradiography. Lanes 2, 4 and 6 establish that Gpa1 binds to immobilized Cyr1, but there was little difference in apparent affinity after incubation with GTP (lane 2), no nucleotide (lane 4) or GDP (lane 6). Negative controls, using immobilized GST, are shown in lanes 3, 5, 7, 10 and 13. A control using immobilized GST-Gpb1 to pull-down S-Gpa1, in the presence of 10 mM GDP, shows a more intense band, suggesting that Gpa1-GDP is bound with higher affinity to Gpb1 than to Cyr. Using translated S-Cyr (lane 11), an interaction with immobilized GST-Gpb1 (lane 12) was detected

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -1

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of -0

<p><b>Copyright information:</b></p><p>Taken from "The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of "</p><p></p><p>Molecular Microbiology 2007;65(3):761-779.</p><p>Published online 01 Aug 2007</p><p>PMCID:PMC2064555.</p><p>© 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd</p