Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores

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

Yeast two-hybrid analyses—<i>P</i>. <i>brasiliensis</i>/<i>S</i>. <i>cerevisiae</i> gene interactions.

<p>Colonies that grew on SD-Ade/-His/-Leu/-Trp drop-out plates and had α-galactosidase activity, as determined by a blue colouration of the colonies growing on α-X-gal supplemented media, are defined as positive (+) and those that did not as (-). All the gene sequences were swapped between the pGADT7 and pGBKT7 vectors and only those giving a positive-reaction in both vectors were scored as positive for an interaction. The ScCyr1^(1–365), ScCyr1^(350–480), ScCyr1^(600–800) are the N-terminal, Gα and Ras association domains, respectively; whilst ScGpr1(679–961) the ScGpa2 association domain. A set of control reactions was undertaken at the same time to validate the test reactions: positive control, pGADT7- Ag with pGBKT7-P53; negative controls, pGBKT7-Lam with pGADT7, pGADT7-TPK2^1-270, pGADT7-TPK2^1-583, pGADT7-TPK2^265-583 and pGADT7-GPG1, and pGBKT7-TPK1 with pGADT7, pGBKT7, pGADT7-Lam, pGBKT7-Lam and pGBKT7-PbActin. NT- Not tested.</p><p>Yeast two-hybrid analyses—<i>P</i>. <i>brasiliensis</i>/<i>S</i>. <i>cerevisiae</i> gene interactions.</p

PbTupA induces hyperfilamentous growth that can be repressed by PbGpb1.

<p>The <i>S</i>. <i>cerevisiae</i> diploid strain MLY61a/α (WT) and its <i>TPK2Δ</i> mutant XPY5a/α (XPY) were transformed with the <i>PbTUPA</i>, <i>ScTUP1</i>, <i>PBTPK2</i> and <i>PbGPB1</i> as indicated. To allow selection, generally, constructs for the expression of the PbTupA-mRFP, ScTup1-mRFP, PbTpk2-GFP and PbGpb1-GFP fusion proteins were used and the transformants, selected on the basis of their green and/or red fluorescence. <b>(A)</b> The cells were analysed for pseudohyphal growth in SLAD agar containing 50 μM (upper panel) or 200 μM (lower panel) ammonium sulfate. Single colonies from the agar plate were observed at 20x magnification in an Eclipse E-400 microscope. The scale bar is 50μm. WT cells expressing PbTupA were hyperfilamentous; whilst those expressing ScTup1 did not produce pseudohyphae. XPY cells expressing PbTupA produced few pseudohyphae; whilst those expressing PbTupA with PbTpk2, but not a kinase defective K301R derivative, were hyperfilamentous, indicating the requirement for a functional PKA. The co-expression of PbGpb1 with PbTupA repressed the filamentous growth of the XPY/<i>PBTPK2</i> but not the WT cells, indicating that PbGpb1 specifically inhibits PbTpk2. <b>(B)</b> A bar chart showing <i>FLO11</i> transcript levels for the indicated transformants. The measured quantity of the <i>FLO11</i> mRNA in each of the treated samples is the relative abundance to the value for <i>actin</i>. The data represent the average of 3 measurements. <b>(C)</b> The <i>S</i>. <i>cerevisiae</i> diploid strain XPY95a/α, a <i>FLO8Δ</i> mutant, was transformed with the <i>PbTUPA-mRFP and the transformants</i> selected on the basis of their red fluorescence (lower panel), and analysed for pseudohyphal growth in SLAD agar containing 50 μM (upper panel) ammonium sulfate. PbTupA was in capable of inducing either the development of pseudohyphae (upper panel) or invasive growth (lower panel), which are the hallmarks of the parent strain MLY61a/α (WT) transformed with <i>PbTUPA-mRFP</i>. This data suggests that PbTupA works up-stream of Flo8 to induce hyperfilamentous growth.</p

A model for the regulation of the fungal cAMP-signaling pathway by the Gβ and TupA proteins.

<p>The classical cAMP-signaling pathway, prevalent in dimorphic fungi, consists of a G-protein coupled receptor (GPCR), which is associated with a trimeric complex of the Gα, Gβ and Gγ proteins. <b>(A)</b> The GPCR responds to extracellular stimuli activating Gα, which exchanges GDP for GTP, and dissociates from the receptor and GβGγ complex. <b>(B)</b> The Gα-protein interacts with, and activates, adenylate cyclase (AC), which produces the signalling molecule cAMP. The Gβ protein can also interact with AC. Since the expression of Gβ is up-regulated during the morphological transition to the yeast form [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136866#pone.0136866.ref019" target="_blank">19</a>], this excess of free Gβ could act to curtail the signalling process. cAMP binds to Protein Kinase A (PKA), which is composed of two regulatory subunits (Bcy1) and two catalytic subunits (Tpk2). cAMP binds to the Bcy1 subunits inducing their release from the Tpk2 subunits. <b>(C)</b> Our data indicates that the WD/β-propeller proteins Gβ and TupA bind to Tpk2: Gβ binds to the C-terminal, catalytic domain, of Tpk2, whilst TupA, a transcriptional co-regulator, binds to the N-terminal domain of Tpk2. <b>(D)</b> Tpk2 is targeted to the nucleus, where it can interact with and phosphorylate transcription factors (TF) that form a transcriptional complex with RNA polymerase (RNAP) (lefthand figure). This complex is responsible for the expression of genes necessary for the morphological change (e.g. filamentous growth). TupA is also targeted to the nucleus, but may do so in complex with Tpk2, where it interacts with its target transcription factors (righthand figure). By binding to Tpk2, TupA may define those TFs to be phosphorylated and/or facilitate their phosphorylation; and/or facilitate specific interactions with the target DNA or transcription complex. Whatever the mechanism, the effect of TupA is to act as a strong inducer of the morphological change (e.g. hyperfilamentous growth). <b>(E)</b> The interaction of Gβ with Tpk2 facilitates its trafficking to the nucleus and, presumably via binding to the catalytic C-terminal domain, can suppress its kinase activity and the resulting filamentous gowth (lefthand figure). Although our data indicates that Gβ inhibits the kinase activity of Tpk2, the possibility that it alters the specificity of Tpk2 for target transcription factors cannot be excluded. We hypothesis that the TupA/Tpk2/TF complex is regulated by binding of Gβ, which inhibits the kinase activity of Tpk2 and/or alters its specificity (righthand figure). Whilst the binding of TupA and Gβ to Tpk2 could be mutually exclusive, which would provide a mechanism for the inhibitory action of Gβ, it seems likely that the complex is held together via multiple interactions (e.g. with TupA binding to both Tpk2 and a common TF) and that Gβ inhibits or alters the specificity for gene expression of the transcriptional complex. The phosphorylation of the TF is shown to occur before the interaction with the RNAP on the DNA for clarity, but phosphorylation could occur after the interaction.</p

Yeast two-hybrid analyses–<i>P</i>. <i>brasiliensis</i>/<i>P</i>. <i>brasiliensis</i> gene interactions.

<p>Colonies that grew on SD-Ade/-His/-Leu/-Trp drop-out plates and had α-galactosidase activity, as determined by a blue colouration of the colonies growing on α-X-gal supplemented media, are defined as positive (+) and those that did not as (-). All the gene sequences were swapped between the pGADT7 and pGBKT7 vectors and only those giving a positive-reaction in both vectors were scored as positive for an interaction. In addition, Tpk2^(1–583) was tested for interactions with PbCyr1^(600–1316), PbCyr1^{(1302–1871)}, PbCyr1^{(1649–2100)} but none were detected. A set of control reactions was undertaken at the same time to validate the test reactions: positive control, pGADT7- Ag with pGBKT7-P53; negative controls, pGBKT7-Lam with pGADT7, pGADT7-TPK2^1-270, pGADT7-TPK2^1-583, pGADT7-TPK2^265-583 and pGADT7-GPG1, and pGBKT7-TPK1 with pGADT7, pGBKT7, pGADT7-Lam, pGBKT7-Lam and pGBKT7-PbActin. NT- Not tested.</p><p>Yeast two-hybrid analyses–<i>P</i>. <i>brasiliensis</i>/<i>P</i>. <i>brasiliensis</i> gene interactions.</p

The subcellular localization of PbTpk2 and PbGpb1: PbGpb1 is targeted to the nucleus in cells expressing PbTpk2.

<p><b>(A)</b> Cells of <i>S</i>. <i>cerevisiae</i> XPY5a/α (<i>TPK2Δ</i> mutants) were transformed with constructs for the expression of either PbTpk2-GFP or PbTpk2-mRFP fusion proteins and the localization of the proteins detected by confocal microscopy using GFP or mRFP as a marker. The nuclei of the yeast cells were identified by staining with DAPI for confocal microscopy. (a) GFP-expressed from p426MET25, and PbTpk2 N-terminus 1-225-GFP (b) were distributed throughout the cell; PbTpk2 C-terminus 226-583-GFP (c), PbTpk2 FL-mRFP (d) and PbTpk2 (K301R) FL-mRFP (e) were concentrated in the nucleus. <b>(B)</b> Cells of the <i>S</i>. <i>cerevisiae</i> XPY5a/α/ <i>PbTPK2</i> transformant were transformed with a construct for the expression of the Gpb1-GFP fusion protein and the localization of the protein detected by confocal microscopy using GFP as a marker. (a) Gpb1-GFP was distributed throughout the cell in the wild-type (MLY61a/α) strain; (b) Gpb1-GFP was distributed throughout the cell in the XPY5a/α (<i>ΔTPK2</i> mutant) strain; and (c) Gpb1-GFP was concentrated in the nucleus when co-expressed with (full-length) <i>P</i>.<i>brasiliensis</i> Tpk2 (PbTpk2FL) in the <i>TPK</i>2Δ <i>S</i>. <i>cerevisiae</i> cells. In (A) and (B) the upper panel is for the nuclei stained with DAPI, the middle panel for GFP (or mRFP) and the lower panel is the merged DAPI and GFP (or mRFP) images. The scale bar is 5 μm. (C) The localization of PbGpb1, expressed in XPY5a/α (<i>TPK2Δ</i>) <i>S</i>.<i>cerevisiae</i> cells, was determined by immune-gold electron microscopy using antibodies to PbGpb1, revealing that PbGpb1 is localized at the cell membrane, and in the cytoplasm and nucleus.</p

The Gβ protein Gpb1 blocks Tpk2-induced pseudohyphal differentiation.

<p><b>(A)</b> The <i>S</i>. <i>cerevisiae</i> diploid strain XPY5a/α, which had been transformed with <i>PbTPK2</i>^{<i>(1–583)</i>}, was transformed with a construct for the expression of PbGpb1-GFP and the transformants, selected on the basis of their green fluorescence, analysed for pseudohyphal growth in SLAD agar containing 50 μM (upper panel) or 200 μM (lower panel) ammonium sulfate. Single colonies from the agar plate were observed at 20x magnification in an Eclipse E-400 microscope. The scale bar is 50μm. Whilst control cells expressing PbTpk2 were able to form pseudohyphae, those co-transformed with <i>PbGPB1</i> could not. As a control the wild-type strain MLY61a/α was transformed with <i>PbGPB1</i> and shown to still produce pseudohyphae. <b>(B)</b> The <i>S</i>. <i>cerevisiae</i> haploid strain SGY446, which had been transformed with <i>PbTPK2</i>^{<i>(1–583)</i>}, so that it could grow at 37°C, was transformed with a construct for the expression of PbGpb1-GFP and the transformants, selected on the basis of their green fluorescence after growth at 25°C. The SGY446/<i>PbTPK2</i>^{<i>(1–583)</i>}/<i>PbGPB1-GFP</i> transformant was tested for growth at 37°C. In contrast to the control strain SGY446/<i>PbTPK2</i>^{<i>(1–583)</i>}, the SGY446/<i>PbTPK2</i>^{<i>(1–583)</i>}/<i>PbGPB1-GFP</i> transformant could not grow at 37°C. <b>(C)</b> A bar chart showing the relative <i>in vitro</i> kinase activity of <i>P</i>. <i>brasiliensis</i> Tpk2 (PKA) using kempeptide and PbGpb1 as substrates. Note that a slight excess of PbGpb1 causes about a 40% reduction in the kinase activity of PbTpk2 using kempeptide as substrate. <b>(D)</b> A bar chart showing <i>FLO11</i> transcript levels for the indicated transformants. The measured quantity of the <i>FLO11</i> mRNA in each of the treated samples is the relative abundance to the value for <i>actin</i>. The data represent the average of 3 measurements.</p

The expression of <i>PbTUPA</i> causes invasive and aerial growth of <i>S</i>.<i>cerevisiae</i>.

<p><b>(A)</b> The <i>S</i>. <i>cerevisiae</i> diploid strains MLY61a/α (WT) and XPY5a/α, a <i>TPK2Δ</i> mutant, were transformed with the <i>PbTUPA</i>, <i>ScTUP1</i> and <i>PBTPK2</i> as indicated. To allow selection, generally, constructs for the expression of the PbTupA-mRFP, ScTup1-mRFP and PbTpk2-mRFP fusion proteins were used and the transformants, selected on the basis of their fluorescence. The colonies were viewed by Leica M165 FC stereo fluorescence microscopy to identify the invasive growth on the plates. The colony views are shown both before (top panel) and after (bottom panel) washing to remove cells that had not invaded the agar. In response to the expression of PbTupA, WT cells and XPY cells transformed with <i>PbTPK2</i> produced invasive growth. XPY cells expressing PbTupA did not invade the agar, indicating that functional PbTpk2 was required for this phenotype. WT cells expressing ScTup1were non-invasive. <b>(B)</b> XPY cells expressing PbTpk2 and PbTupA developed an aerial stalk (a-c) that topples over to start a new colony (c) and to develop a second stalk (d).</p