Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii
© Valentín-Berríos et al; licensee BioMed Central Ltd. 2009
Received: 16 December 2008
Accepted: 19 May 2009
Published: 19 May 2009
Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition) depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways.
In this work we describe a new G protein α subunit gene in S. schenckii, ssg-2. The cDNA sequence of ssg-2 revealed a predicted open reading frame of 1,065 nucleotides encoding a 355 amino acids protein with a molecular weight of 40.9 kDa. When used as bait in a yeast two-hybrid assay, a cytoplasmic phospholipase A2 catalytic subunit was identified as interacting with SSG-2. The sspla 2 gene, revealed an open reading frame of 2538 bp and encoded an 846 amino acid protein with a calculated molecular weight of 92.62 kDa. The principal features that characterize cPLA2 were identified in this enzyme such as a phospholipase catalytic domain and the characteristic invariable arginine and serine residues. A role for SSPLA2 in the control of dimorphism in S. schenckii is suggested by observing the effects of inhibitors of the enzyme on the yeast cell cycle and the yeast to mycelium transition in this fungus. Phospholipase A2 inhibitors such as AACOCF3 (an analogue of archidonic acid) and isotetrandrine (an inhibitor of G protein PLA2 interactions) were found to inhibit budding by yeasts induced to re-enter the yeast cell cycle and to stimulate the yeast to mycelium transition showing that this enzyme is necessary for the yeast cell cycle.
A new G protein α subunit gene was characterized in S. schenckii and protein-protein interactions studies revealed this G protein alpha subunit interacts with a cPLA2 homologue. The PLA2 homologue reported here is the first phospholipase identified in S. schenckii and the first time a PLA2 homologue is identified as interacting with a G protein α subunit in a pathogenic dimorphic fungus, establishing a relationship between these G proteins and the pathogenic potential of fungi. This cPLA2 homologue is known to play a role in signal transduction and fungal pathogenesis. Using cPLA2 inhibitors, this enzyme was found to affect dimorphism in S. schenckii and was found to be necessary for the development of the yeast or pathogenic form of the fungus.
Sporothrix schenckii is a dimorphic fungus that produces lymphocutaneous lesions in humans and animals. It is the etiologic agent of sporotrichosis, a subcutaneous lymphatic mycosis with a worldwide distribution . In its saprophytic form it develops hyaline, regularly septated hyphae and pyriform conidia which can be found single or in groups in a characteristic daisy-like arrangement. The yeast or parasitic form shows ovoid cells with single or multiple budding.
In S. schenckii, dimorphism is both a proliferative and morphogenetic process. We have reported that in response to different environmental stimuli, S. schenckii unbudded synchronized yeast cells, either proliferate (yeast cell cycle) or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition). Dimorphism in S. schenckii, depends on transmembrane signalling pathways that respond to cell density [2, 3], external pH [2, 3], cyclic nucleotides  and extracellular calcium concentration .
Dimorphism is an adaptation response to changing environmental conditions. The morphology displayed by dimorphic fungi is probably the result of the stimulation of membrane receptors by extracellular ligands. Heterotrimeric (αβγ) guanine nucleotide binding proteins have been associated with membrane receptors and with morphogenetic transition signalling in many eukaryotes, and play a crucial role in fungal morphogenesis as well . They constitute a family of GTP hydrolases involved in signal transduction pathways. These proteins are coupled to membrane receptors (GPCR) that recognize different extracellular signals. The α subunits of the heterotrimeric G proteins bind GTP. The interaction of a ligand with the GPRC initiates the exchange of bound GDP for GTP in the Gα subunit resulting in the dissociation of the heterotrimer into α-GTP and βγ subunits. The dissociated α-GTP subunit and the βγ dimer, relay signals to different targets resulting in changes in cytoplasmic ionic composition or in second messenger levels (e.g., cAMP) that ultimately lead to a cellular response [7–10].
Genes encoding proteins that are similar to the Gα class of the heterotrimeric G proteins have been described in filamentous fungi such as Aspergillus nidulans  and Neurospora crassa [12–14], as well as in fungal plant pathogens like Cryphonectria parasitica [15, 16], Ustilago maydis  and Magnaporthe grisea , among others. In S. schenckii, a 41 kDa Gα subunit homologous to the Gαi subunit and sensitive to inhibition by pertussis toxin was described previously by us . This was the first Gαi subunit described in a pathogenic dimorphic fungus.
In higher eukaryotes, members of the Gα class are known to regulate adenylate cyclase , cGMP phosphodiesterase , phosphoinositide-3-kinase , calcium and potassium channels [22–24], and the activity of phospholipases [9, 25–28]. In fungi, Gα subunits have been shown to regulate adenylate cyclase, morphogenesis and pathogenicity [6, 14, 29, 30]. Most of the studies related to determining the role of the heterotrimeric G protein subunits in fungi involved the observation of the morphological effects produced in the fungus when these genes are deleted [6, 12, 14, 18]. Nevertheless, the full scope of the processes that Gα subunits regulate in fungi is still not known and interactions between these subunits and cellular proteins have seldom been reported in pathogenic fungi.
A large number of G protein coupled receptors have been observed to induce activation of phospholipase A2 in higher eukaryotic systems . The PLA2 superfamily can be classified according to cellular location or biological properties . The phospholipase A superfamily includes the calcium dependent-secretory PLA2 (sPLA2), the calcium independent-intracellular PLA2 (iPLA2) and the cytosolic PLA2 (cPLA2). They differ in terms of calcium requirements, substrate specificity, molecular weight and lipid modification. The sPLA2 is usually a 13 to 15 kDa protein while the cPLA2 is a 85 kDa protein in human macrophages. The cPLA2 possesses characteristics that suggest that it is associated to receptor-activated signal transduction cascades . This PLA2 is known to translocate to the membrane in response to an increase in intracellular calcium concentration . Cytosolic PLA2 hydrolyses the sn-2 position of phospholipids, resulting in the release of lysophospholipids and free fatty acids. The most commonly released fatty acid is arachidonic acid, which in turn is converted to eicosanoids that regulate multiple processes including calcium channels, mitogenic signals and most important, the inflammatory response of macrophages [31, 32, 35, 36].
The present study was undertaken to identify the presence of and characterize additional Gα subunits in S. schenckii, to identify any important interacting partners of the new Gα subunit, and finally to determine the involvement if any of the interacting protein, in this case cPLA2, in the control of dimorphism in this fungus. Here we give details of the identification and sequencing of the ssg-2 gene, including gene organization, the presence and position of introns, derived amino acid sequence and conserved polypeptide-encoded domains. Using SSG-2 as bait, we identified a cPLA2 homologue interacting with this G protein α subunit. We give the genomic sequence of this gene and the complete derived amino acid sequence. We also report the effects on the yeast to mycelium transition and the yeast cell cycle of phopholipase effectors in S. schenckii.
This work constitutes the first report of the presence of multiple G protein α subunits in S. schenckii, the presence of a cPLA2 homologue interacting with this G protein α subunit, and the involvement of cPLA2 in the control of dimorphism in S. schenckii. In addition to being a very important determinant of pathogenicity in fungi and other organisms, cPLA2 is shown to have a direct effect in the control of dimorphism in this fungus. This information will ultimately help us construct the signal transduction pathway leading from the G proteins onward and the role of G proteins and its interacting partners in fungal pathogenesis.
Identification of the ssg-2 gene
Bioinformatic characterization of SSG-2
The derived amino acid sequence (GenBank accession number AAL57853) revealed a Gα subunit of 355 amino acids as shown in Figure 1B. The calculated molecular weight of the ssg-2 gene product was 40.90 kDa. Blocks analysis of the amino acid sequence of SSG-2 revealed a G-protein alpha subunit signature from amino acids 37 to 276 with an E value of 5.2e-67 and a fungal G-protein alpha subunit signature from amino acids 61 to 341 with an E value of 3.3e-28 . SSG-2 has the motifs encoding the GTPase domain  with the corresponding consensus sequences involved in GTP binding shaded in gray in Figure 1B. The phosphate binding loop which includes the sequence GXGXXGKS is found in SSG-2 as GSGESGKS. The magnesium binding residues with the consensus sequence DXXG is present as DVGG in SSG-2, while the guanine ring binding sites are those with the consensus sequence NKXD is present as NKVD. The TXAT consensus sequence is present as TQAT in SSG-2. Another region involved in phosphate binding includes the consensus sequence RXXT that in SSG-2 is present as RTKT. In addition to these conserved domains, the protein derived from the ssg-2 cDNA sequence has the N-terminal glycine that is myristoylated in Gα subtypes and is needed for membrane association. The 5 residues that identify the adenylate cyclase interaction site according to BLAST analysis  are in red in Figure 1, these include I187, K212, I215, H216, and E 219. The putative receptor binding site includes amino acids L318 to R334 and is shown in blue letters in Figure 1.
Comparison of G protein alpha subunit homologues to SSG-2 of S. schenckii
Yeast two-hybrid screening
Two independent yeast two-hybrid screenings, using different S. schenckii yeast cells cDNA libraries were done with the complete coding sequence of SSG-2 as bait. In both screenings, 3 blue colonies growing in quadruple drop out (QDO) medium (SD/-Ade/-His/-Leu/-Trp/X-α-gal) were identified as containing the same PLA2 homologue insert. The expression of the Ade+, His+ phenotypes and α galactosidase activity are considered by the manufacturer as corroborative of true interactions. The inserts from all three colonies were found to contain the carboxy-terminal residues of a protein homologous to PLA2's from A. nidulans. Our results indicated that the last 162 amino acids of the S. schenckii cPLA2 homologue interacted with SSG-2.
Sequencing of the sspla 2 gene
Bioinformatic characterization of SSPLA2
The PANTHER Classification System identified this protein as a member of the cytosolic phospholipase A2 family (PTHR10728) (residues 132–827) with an extremely significant E value of 6.4 e-97 . BLAST analysis of the derived amino acid sequence of the S. schenckii SSPLA2, showed a phospholipase domain extending from amino acids 177 to 750 . Pfam analysis shows similar results, and in this domain the PLA2 signature GXSG [G, S] (Pfam: Family PLA2_B PF 01735) is present as GVSGS in the active site (highlighted green in Figure 4B) [41, 42]. The amino acids needed for catalytic activity R235, S263 and D553 are given in red in this same figure . S263 is essential for the formation of arachidonyl serine needed for the transfer of the arachidonyl group to glycerol or to water. The amino acids D511 to L523, D583 to G595 and D738 to A750 (highlighted in yellow) comprise putative EF hand domains of the protein (76% identity, probability, 3.33e-06). In Figure 4B a putative calmodulin binding domain was identified from amino acids Q806 to L823 using the Calmodulin Target Database  and highlighted in gray. A serine protease, subtilase family, aspartic acid active site motif was identified using Scan Prosite with an E value of 5.283e-07 from amino acids 549 to 559 and is shaded in blue green in Figure 4B. This motif is characteristic of both yeast and fungal cPLA2 homologues .
Effects of PLA2 effectors on the yeast to mycelium transition and the yeast cell cycle
The heterotrimeric G protein family ranks among the most important protein families identified as intracellular recipients of external signalling. The present study was conducted in order to describe new Gα subunit encoding genes in S. schenckii, identify any important protein interacting with this G alpha subunit and determine the effects on dimorphism in S. schenckii of the protein or proteins identified.
The results presented here, together with our previous report  corroborate the existence of more than one heterotrimeric G protein α subunit gene in S. schenckii. Unpublished results indicate that this protein is one of at least 3 Gα subunits present in S. schenckii. In this sense, S. schenckii is behaving more like the filamentous fungi and plant pathogens such as N. crassa , C. parasitica  and M. grisea , where genes that encode 3 different Gα subunits similar to the Gα class of animals rather than to the GPA group present in yeasts and plants. Computational sequence and phylogenetic analysis of the Gα subunits in filamentous fungi shows the existence of 3 distinct subfamilies of G protein alpha subunits . According to the classification offered by Li and collaborators, SSG-2 belongs to Group III of the fungal G protein alpha subunits . The Group III considered by them to be Gαs analogues because they positively influence cAMP levels although they have more sequence similarity to Gαi .
The nucleotide and amino acid sequence analysis of this new G protein α subunit gene are different from the previously identified ssg-1 gene. The nucleotide conservation of the coding region of ssg-2 is less than 50% when compared to that of the previously reported ssg-1 gene, confirming that ssg-1 and ssg-2 are two different genes (data not shown). The derived amino acid sequence of ssg-2 is 50% identical to that of SSG-1, but they have differences in the motifs that are characteristic of the G protein alpha subunits, the most important difference being that SSG-2 lacks the cysteine residue in domain 5 that characterizes the pertussis binding domain of SSG-1 (TCADT). For this reason, SSG-2 belongs to the Gα class but cannot be strictly considered a Gαi, even though it is 46% identical to mammalian Gαi class members. This shows the high degree of conservation in Gα subunits even among phylogenetically distant organisms.
The work done in order to identify the role of Gα subunits in the filamentous fungi has been mainly concerned with the phenotypes observed when these genes are knocked-out (as reviewed by ). In this paper a different approach was used. We wanted to identify important protein-protein interactions between SSG-2 and the complex signalling system that regulates the flow of information from the environment through the heterotrimeric G proteins into the cell in S. schenckii. Using the yeast two-hybrid technique we identified a cPLA2 homologue as interacting with SSG-2 in two independent experiments, using two different cDNA libraries. This SSG-2-PLA2 interaction was also confirmed by co-immunoprecipitation. Up to date, protein-protein interactions of these Gα subunits have not been reported in the pathogenic fungi, and the exact proteins with which these Gα subunits interact have not been identified. This is the first report of a cytosolic PLA2 homologue interacting with a G protein α subunit in a pathogenic dimorphic fungus, suggesting a functional relationship between these two important proteins. Other proteins interact with SSG-2 (unpublished results), but the SSG-2-PLA2 interaction is very important as it connects this G protein α subunit with both pathogenicity and lipid signal transduction in fungi .
This PLA2 homologue belongs to the Group IV PLA2 family that has been highly conserved throughout evolution. BLAST searches of the amino acid sequence of SSPLA2 against the Homo sapiens database shows that it is phylogenetically related to the human Group IVA PLA2 family. This same analysis using the fungal databases revealed that SSPLA2 is more closely related to the phospholipases of the filamentous fungi than to PLAB of yeasts. The similarity to both human and fungal phospholipases is found primarily in the catalytic domain with a great deal of variation contained in the first and last 200 amino acids. In the catalytic domain we find an important difference between SSPLA2 and the human homologues. The former has one continuous catalytic domain, rather than the more typical cPLA2 structure where two homologous catalytic domains are present, interspaced with unique sequences .
SSPLA2 lacks the C2 motif found in cPLA2 of higher eukaryotes. This domain is involved in the translocation of the enzyme to the membrane in response to an increase in intracellular calcium concentration . Nevertheless, SSPLA2 has 3 putative EF hand motifs suggesting that it could also be calcium modulated. EF hand motifs are also present in the PLA2 homologues of M. grisea, G. zeae, N. crassa and A. nidulans in different areas of these proteins. It is interesting to note that A. nidulans PLA2 has been reported to be responsive to calcium even though it also lacks a C2 domain .
Also contributing to the possible modulation by calcium of this protein is the presence of a putative calmodulin binding domain . As in the case of the EF hand-motifs, analysis of the PLA2 homologues of M. grisea, N. crassa, G. zeae and in A. nidulans show the presence of possible calmodulin binding domains in different areas of the proteins . In S. schenckii the putative calmodulin binding domain is at the C terminal end of the protein, while in M. grisea, N. crassa and G. zeae it is within the first 150 to 250 amino acids.
In addition to the identification of PLA2 as interacting with SSG-2, we inquired as to the effects of PLA2 in S. schenckii dimorphism. As mentioned previously, PLA2 hydrolyses the sn-2 position of phospholipids, resulting in the release of lysophospholipids and free fatty acids. The most commonly released fatty acid is arachidonic acid. We tested the effects of exogenously added arachidonic acid on the kinetics of germ tube formation or the yeast cell cycle in S. schenckii. Our results show that exogenously added arachidonic acid had no significant effect on the kinetics of the yeast to mycelium transition, but a significant stimulation (50%) in the percentage of budding in cells induced to re-enter the yeast cell cycle was observed at 6 h of incubation in the presence of this compound. The observed stimulation of the yeast cell cycle by arachidonic acid is consistent with the inhibitory effects on this same cycle observed in the presence of AACOCF3 and isotetrandrine in S. schenckii, inhibitors of PLA2. These inhibitors have different mechanisms of action as stated previously. AACOCF3 is a competitive inhibitor of PLA2  and an analogue of arachidonic acid, while isotetrandrine interferes with G protein activation of PLA2 . Both AACOCF3 and isotetrandrine increased significantly the percentage of cells with germ tubes at 6 and 9 h after inoculation and decreased budding in cells induced to re-enter the yeast cycle. The AACOCF3 results are consistent with our hypothesis that PLA2 activity is needed for the yeast cell cycle in S. schenckii, specifically at the start of DNA synthesis . Furthermore, the isotetrandine results support the hypothesis that the interaction of SSG-2 with PLA2 is required for these processes to occur.
It is of interest to note that we recently reported similar results in the presence of calmodulin inhibitor W7 and inhibitors of calcium-calmodulin kinase in S. schenckii . Inhibiting calmodulin or calmodulin-dependent kinase also inhibited the re-entry of yeast cells into the cell cycle. We can speculate that by inhibiting the calmodulin dependent kinase we are also inhibiting the migration of cPLA2 to the membrane and/or its activation.
We cannot fully ascertain the functional consequences of the observed interaction between PLA2 and SSG-2 at this time. Future work will help us clarify this relationship. Nevertheless, two important processes that have a bearing in cell cycle progression have been identified as subjected to cPLA2 activity in other systems: 1) the production of biologically active molecules and 2) membrane remodeling .
There is very little information regarding the effects of the primary metabolites released from the action of PLA2 (arachidonic acid and lysophospholipids) in fungi, Arachidonic acid was reported to stimulate adenylate cyclase  in S. cerevisiae. If this is also true for S. schenckii, addition of arachidonic acid to the medium would be expected to stimulate the yeast cell cycle and this was what we observed. We had previously reported that dibutyryl derivatives of cAMP inhibit the yeast to mycelium transition in S. schenckii .
On the other hand, membrane remodeling is also an important function of enzymes such as phospholipases. This process is needed for cell cycle progression and fungal morphogenesis . It has been reported in other systems that in order for the cell cycle to occur there must be a careful balance between membrane phospholipid synthesis and degradation. PLA2 has an important role in the maintenance of this balance [35, 55]. The lipid composition of the membrane is also essential for the correct receptor-protein interactions and plays an important role in signal transduction. G proteins are usually in molar excess when compared to the GPRC's and a large number of inactive GDP-bound heterotrimeric G protein molecules must be available in receptor-rich domains associated to membrane lipids .
G proteins can also affect PLA2 activity by a number of different mechanisms such as: increasing the intracellular calcium concentration, transcriptional regulation and stimulation of phosphorylation through different protein kinases such as protein kinase C and MAP kinases (for a review see ).
The studies presented here constitute the first evidence of the interaction of G protein subunits of fungi with a phospholipase. These results establish for the first time a relationship between G proteins and the pathogenic determinants of fungi. The identification of such an important protein as partners of a G protein alpha subunit in fungi suggests a mechanism by which these G proteins can control pathogenicity in fungi. The existence of the interaction reported here may offer an explanation as to why fungi with decreased G protein alpha subunits such as C. parasitica, hypovirus infection  and M. grisea with disrupted Gα subunit gene, magB, exhibit reduced levels of virulence . This information is essential if we are to understand the disease producing process of fungi. It will also help elucidate the signal transduction pathway leading from the G protein onward and will give us a better insight into signal transduction in pathogenesis and dimorphism in S. schenckii.
We have shown the presence of a new G protein α subunit in S. schenckii, SSG-2. The cDNA sequence of the ssg-2 gene encoded a 355 amino acid Gα subunit of 40.90 kDa containing the 5 consensus domains present in all Gα subunits. The genomic sequence has four introns, whose positions are conserved in the other fungal homologues of this gene.
Yeast two-hybrid analysis using the complete amino acid sequence of SSG-2 identified a PLA2 homologue as an interacting partner of this G protein subunit. This 846 amino acid protein was encoded by an intronless gene. The 92.62 kDa protein encoded by this gene contained all the domains and amino acid residues that characterize cytosolic phospholipase A2.
PLA2 and other phospholipases in fungi have very diverse roles not only as virulence factors but also in membrane homeostasis and signal transduction. Inhibitor studies showed that this PLA2 homologue and its interaction with SSG-2 were necessary for the re-entry of S. schenckii yeast cells into the budding cycle suggesting a role for this important virulence factor in the control of dimorphism in this fungus and for the expression of the yeast form. The effects of PLA2 on the yeast cell cycle could be viewed as resulting from the generation of lipid messenger molecules or from membrane remodelling that affects the G1->S transition and G protein activity.
The relationship reported here between these two proteins, SSG-2 and SSPLA2, constitutes the first report of the interaction of a fungal phospholipase and a G protein subunit and the possible involvement of G protein in fungal virulence and morphogenesis.
Strains and culture conditions
S. schenckii (ATCC 58251) was used for all experiments. The yeast form of this fungus was obtained as described . S. cerevisiae strains AH109 and Y187 were supplied with the MATCHMAKER Two-Hybrid System 3 (Clontech Laboratories Inc., Palo Alto, CA).
Nucleic acids isolation
DNA and RNA were obtained from S. schenckii yeast cells as described previously using the methods of Sherman , and Chomczynski & Sacchi , respectively. Poly A+ RNA was obtained from total RNA using the mRNA Purification Kit from Amersham Biosciences (Piscataway, NJ, USA).
Sequencing the ssg-2 gene
Polymerase chain Reaction and Rapid amplification of cDNA ends (RACE)
S. schenckii DNA (100 ng) was used as template for polymerase chain reaction (PCR) with primers (100–200 ng) targeted to conserved motifs in Gα subunits. The primers used were: GESGKST (fw) 5' ggtgc(c/t)ggtga(a/g)tc(a/c)gg(a/t)aa(a/g)tc 3'; KWIHCF (rev) 5' aagcag tgaatccacttc 3'; TQATDT (rev) 5'gtatcggtagcttgggtc 3'; MGACMS (fw) 5' atggg ggcttgcatgagt 3' and KDSGIL (rev) 5' taggataccggaatctttg 3'. The Ready-to-Go™ PCR Beads (Amersham Biosciences) were used for PCR using the amplification parameters described previously .
PCR products were isolated and cloned using the TOPO TA Cloning System (Invitrogen Corp., Carlsbad, CA, USA) . Plasmid preparations were obtained using the Fast Plasmid TM Mini technology from Eppendorf (Brinkmann Instruments, Inc. Westbury, NY, USA).
The 5' and 3' ends of the S. schenckii Gα subunit gene were obtained using SMART RACE (BD Biosciences, Clontech, Palo Alto CA, USA). All RACE reactions were carried out as described previously . Primers for RACE were designed based on the sequence obtained previously. Nested primers were designed to improve the original amplification reactions. Bands from the 5' and 3' nested PCR, respectively, were excised from the gel, cloned and sequenced . The following primers were used for 3' RACE: GSP2A (fw) 5' cttgaggaaagcagtcagaaccgaatgatg 3' and GSP2C (fw) 5' gtgaatcgggcacacctcaacttatatcct 3'. The following primers were used for 5' RACE: GSP1E (rev) 5' catcattcggttctgactgctttcctcaag 3'; GSP1D (rev) 5' aaagtcgcagtacgcacggatctcatcgct 3' and SSG-2 5 'UTR primer-1 (rev) 5' tagcagtagaatcttgcattctcgccgt 3' and SSG-2 5' UTR primer-2 (rev) 5' tcctcttcttctgctccacctcctcact 3'.
The complete coding sequence of the ssg-2 gene from cDNA and genomic DNA were obtained using reverse transcriptase polymerase chain reaction (RTPCR) and end to end PCR, respectively. The cDNA obtained using the RETROscript™ First Strand Synthesis kit (Ambion, Applied Biosystems, Foster City, CA, USA) was used as template. The following primers were used: MGACMS (fw)/KDSGIL (rev) primer pair. The sequence of these primers were the following: 5' atgggggcttgcatgagt 3' and 5' aggataccggaatctttg 3', respectively. For the genomic sequence PCR, DNA was used as template and the primers used were the same as those used for RTPCR. The PCR products containing the entire coding sequence, from both the cDNA and genomic templates were cloned and sequenced.
Sequencing the sspla 2 gene
Polymerase chain Reaction and Genome Walker
The 5' sequence of the PLA2 homologue was obtained using a combination of PCR and Genome Walker (Clontech Laboratories Inc., Palo Alto, CA, USA). Genomic DNA was used as template for PCR. For genome walking a Pvu II library of S. schenckii genomic DNA done as described by the manufacturer was used as template for the primary specific PCR reactions using the gene specific primers (GSP) and AP1 primer. The primary PCR reactions were used as template for nested PCR using nested gene specific primers (NGSP) and AP2 primer. The primers used were: YARRFA (NGSP, rev) 5' ccgagagacgatgcaaagcgacgggcgta 3'; SLLVFS (GSP and NGSP, rev) 5' agagaagacgaggagact 3'; GSLSDEIWRE (rev) 5' ctcgcgccaaatctcgtcggacagggatcc 3'; VHPEVQ (GSP, rev) 5' gaactggacttcggggtg 3'; LAKYLDLPA (NGSP, rev) 5' ggcagggaggtcgaggtacttggcgag 3'; (fw) 5' ctcgccaagtacctcgacctccctgccg 3'; DDVPVIA (rev) 5' aacgcaatcacgggcacgtcgtcgg 3'; GVSGSGC (fw) 5' ggagtgagcggttcatgctgg 3'; LYFSSFT (rev) 5' taaacgacgaaaagtacag 3'; PVGVGWPPA (GSP, rev) 5' cggccggcggccagcccacacccactgg 3'; PVGVG (fw) 5' ccagtgggtgtgggctg 3'; DDKIEQ (fw) 5' gacgacattgaacaggccaaagccgac 3'; DKIEQ (rev) 5' ttcaatcttgtcgtccgg 3'; ERHKRERL (rev) 5' cagccgctccctcttgtgccgctcctc 3'; NOVGGR (NGSP, rev) 5' ctccgacttaattaaaat 3'; 3' UTR primer (GSP, rev) 5' atgctgtgtcgccctccgac 3'. The AP1 and AP2 primers supplied by the manufacturer. The touchdown and nested PCR parameters used were those described previously .
DNA sequencing and analysis
All sequencing reactions for the ssg-2 gene were conducted using the ABI PRISM™ 377 automated DNA sequencer (Applied Biosystems) and the Thermo Sequenase II Dye terminator Cycle Sequencing Premix Kit (Amersham Biosciences) as described previously . Sequencing of the sspla 2 gene products was done commercially using the SeqWright sequencing service (Fisher Scientific, Houston, TX, USA)
Bioinformatics Sequence Analysis
The theoretical molecular weights were calculated using the on-line ExPASy tool http://www.expasy.ch/tools/. On-line Prosite Scan (Proscan Search) search was used to identify potential motifs present in SSG-2 and SSPLA2 http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_prosite.html. The protein classification was performed using the PANTHER Gene and Protein Classification System http://www.pantherdb.org and on-line Blocks Analysis Server http://blocks.fhcrc.org/blocks/blocks_search.html. The calmodulin-binding domain was identified using the on line Calmodulin Target Database http://calcium.uhnres.utoronto.ca/ctdb/ctdb/sequence.html. On-line database searches and comparisons for SSG-2 were performed using the Integrated Protein Classification (iProClass) database  and its BLAST algorithm implementation with a cutoff of 10-7, a low complexity filter and the Blosum 62 matrix. The iProClass/UniProt accession numbers of the sequences used for the multiple sequence alignment of G protein subunits were: S. schenckii (SSG-2), Q8TF91; M. grisea (MAGA), O13314; C. parasitica (CPG2), Q00581; N. crassa (GNA3) Q9HFW7; R. necatrix (WGA1/RGA1), Q9HFA3; E. nidulans (GANB), Q9UVK8, and S. schenckii (SSG-1), O74259.
On-line database searches and comparisons for SSPLA2 were performed with the BLAST algorithm http://www.ncbi.nlm.nih.gov/BLAST/ with a cutoff of 10-7, a low complexity filter and the BLOSUM 62 matrix . The Pfam analysis was done on-line using the using the Wellcome Trust Sanger Institute server http://pfam.sanger.ac.uk/. The GenBank accession numbers for the multiple sequence alignment of phospholipases were: A. nidulans (PLA2), XP_663815; S. schenckii (SSPLA2), ACJ04517.1; M. grisea (hypothetical protein), XP_363597; N. crassa (PLA2), XP_962511; C. globosum (hypothetical protein) XP_001223932; P. anserina (hypothetical protein) XP_001909265, and G. zeae (PLA2), XP_382145.
Multiple sequence alignments were built using MCOFFEE http://www.igs.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi. The alignments were visualized using the program GeneDoc http://www.nrbsc.org/downloads/.
MATCHMAKER Two-Hybrid System 3 was used for the yeast two-hybrid assay (Clontech Laboratories Inc., Palo Alto, CA) using all 3 different reporter genes for the confirmation for truly interacting proteins. For the construction of the bait plasmid, ssg-2 cDNA was obtained from poly A+ RNA, transcribed and amplified by RT-PCR using the Ready-to-Go TM Beads (Amersham Biosciences). The RT-PCR product was amplified using primers containing the gene sequence and an additional sequence containing restriction enzyme sites, Xma I and BamH I at the 5' and 3' ends, respectively. The primers used were: Xma I-MGACMS (fw) 5' ccccggggatgggggcttgcatgagt 3' and DSGIL-BamH I (rev) 5' cgcggatccgcgctaggataccggaatctt 3'. The ssg-2 gene PCR product was cloned in frame into the linearized bait plasmid, pGBKT7 (Clontech Laboratories Inc.) using Quick T4 DNA ligase kit (New England Biolabs Inc., Ipswich, MA, USA) and amplified in E. coli by transformation. Sequencing corroborated the sequence, correct orientation, and frame of the inserted gene. The bait containing plasmid was isolated using Fast Plasmid™ Mini technology (Brinkmann Instruments, Inc.) and used to transform competent S. cerevisiae yeast cells (Y187). Competent S. cerevisiae yeast cells were transformed using the YEASTMAKER™ Yeast Transformation System 2 from Clontech (BD Biosciences, Clontech Laboratories Inc.). Tests for autonomous gene activation and cell toxicity were carried out also as described by the manufacturer.
Double stranded cDNA was synthesized from S. schenckii yeast cells Poly A+ RNA using SMART™ Technology Kit (Clontech Laboratories Inc.). The cDNA's were amplified using Long Distance PCR and size selected using the BD CHROMA-SPIN™+TE-400 columns (Clontech Laboratories Inc.).
S. cerevisiae yeast cells AH109 were made competent using the lithium-acetate (LiAc) method mentioned above and transformed with SMART ds cDNA (20 μl) previously amplified by LD-PCR and the linearized pGADT7-Rec (Sma I-linearized plasmid). Transformants were selected in SD/-Leu plates, harvested and used for mating with the bait containing S. cerevisiae strain Y187. Mating of S. cerevisiae yeast cells strains Y187 (Mat-α) and AH109 (Mat-a) was done according to the manufacturer's instructions. The expression of three reporter ADE2, HIS3 and MEL1 genes in the diploids was used as confirmation for true interacting proteins. Diploids expressing interacting proteins were selected in triple drop out medium (TDO), SD/-Ade/-Leu/-Trp. Colonies growing in TDO medium were tested for growth and α-galactosidase production in quadruple drop out medium (QDO), SD/-Ade/-His/-Leu/-Trp/X-α-gal. Re-plating of these positive colonies into QDO medium was done at least 3 times to verify that they maintain the correct phenotype. Colony PCR was also done to corroborate the presence of both plasmids in the diploid cells using the T7/3'BD sequencing primer pair for the pGBKT7/ssg-2 plasmid and the T7/3'AD primer pair for the pGADT7-Rec library plasmid. The PCR products obtained with the T7 Sequencing Primer/3'AD Sequencing Primer pair were cloned and sequenced as described above.
S. cerevisiae diploids obtained in the yeast two-hybrid assay were grown in 125 ml flasks containing 25 ml of QDO for 16 h, harvested by centrifugation and resuspended in 4 ml containing phosphate buffer saline (400 μl) with phosphatase inhibitor (400 μl), deacetylase inhibitor (40 μl) (Active Motif North America, Carlsbad, CA, USA) and protease inhibitors cocktail (40 μl) (EDTA-free, Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA). The cells were frozen in a porcelain mortar in liquid nitrogen, glass beads added and the cells broken as described previously . The cell extract was centrifuged and the supernatant used for Co-IP using the Immunoprecipitation Starter Pack (GE Healthcare, Bio-Sciences AB, Bjorkgatan, Sweden) as described by the manufacturer. Briefly, 500 μl of the cell extract (1–2 ug of protein/ml) were combined with 1–5 μl of the anti-cMyc antibody (Clontech, Corp.) and incubated at 4°C for 4 h, followed by the addition of protein G beads and incubated at 4°C overnight in a rotary shaker. The suspension was centrifuged and the supernatant discarded, 500 μl of the wash buffer added followed by re-centrifugation. This was repeated 4 times. The pellet was resuspended in Laemmeli buffer (20 μl) and heated for 5 min at 95°C, centrifuged and the supernatant used for 10% SDS PAGE at 110 V/1 h. Pre-stained molecular weight standards were electrophoresed in outside lanes of the gel (BioRad Corporation, Hercules, CA, USA).
Western blots were done as described by us previously . The electrophoretically separated proteins were transferred to nitrocellulose membranes using the BioRad Trans Blot SystemR for 1 h at 20 volts. After transferring, the nitrocellulose strips were blocked with 3% gelatin in TTBS (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5) at room temperature for 30–60 min. The strips were washed for 5–10 min with TTBS. The TTBS was removed and the strips incubated overnight in the antibody solution containing 20 μg of antibody, anti-cMyc or anti-HA (Clontech, Corp.) was added to each strip. Controls where the primary antibody was not added were included. The antigen-antibody reaction was detected using the Immun-Star™ AP chemiluminescent protein detection system from BioRad Corporation as described by the manufacturer.
Induction of the yeast to mycelium transition
The yeast form of the fungus was obtained from conidia as described previously . Briefly, yeast cell were grown for 5 days from conidia in 125 ml flasks containing 50 ml of medium M with aeration at 35°C. These cells were filtered through sterile Whatman #1 filters (GE Healthcare Life Sciences). This procedure increases the concentration of undbudded singlets to approximately 90%. The cells were collected by filtration using Millipore filters GSWP04700 (0.2 μm) (Millipore Corp. Billerica, MA, USA), washed using basal medium with glucose and used for inoculation to give a final concentration of 105 cells/ml. These cells were induced to form germ tubes in the presence and absence of effectors of PLA2 activity in a basal medium with glucose at pH 4.0 and 25°C. Parallel cultures were inoculated with unbudded yeast cells and at 6 and 9 h after inoculation the content of a flask was filtered for the determination of the percentage of cells with germ tubes for each of the substances tested. These same yeast cells were inoculated to give a final concentration of 107 cells/ml and induced to re-enter the yeast cell cycle as described previously in the presence and absence of effectors of PLA2 in a basal medium with glucose at pH 7.2 and 25°C with aeration. At 6 and 9 h after inoculation samples were taken and the percentage of budding cells was recorded.
The following substances were tested for their effects on the yeast to mycelium transition and the yeast cell cycle: arachidonic acid (40 μM; AACOCF3 (100 μM; Nonadeca-4,7,10,13-tetraenyl-trifluoro-methyl ketone)  and isotetrandrine (50 μM; 6,6',7,12-tetra methoxy-2,2'-dimethyl-berbaman) . These substances were obtained from Calbiochem, EMD Biosciences Inc. (Darmstadt, Germany). The results are expressed as the average percentage of cells with germ tubes or buds at 6 and 9 h of incubation ± one standard deviation of at least three independent determinations. The Student t test was used to determine the statistical significance of the data. A 95% confidence level was used to determine statistical significance.
The authors wish to acknowledge the technical support of Ms. Claribel González in sequencing the sspla 2 gene and the cooperation of graduate student Mr. Jorge Rodríguez with the cloning of PCR products. This investigation was supported by the National Institute of General Medicine, Minority Biomedical Research Support Grant 3S06-GM-008224 and partially by the RISE Program grant R25GM061838. RGM acknowledges funding through NIH NIGMS grant T36GM008789-05 and acknowledges the use of the Pittsburgh Supercomputing Center National Resource for Biomedical Supercomputing resources funded through NIH NCRR grant 2 P41 RR06009-16A1.
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