BclA-mediated CFH recruitment inhibited downstream complement activation <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Complement hemolytic assay. Spores were incubated with 20% NHS and centrifuged. The supernatants (1:10 diluted) were used to perform complement hemolytic assays using opsonized sheep erythrocytes (EA-SRBC). Data shown was from at least three independent experiments. (<b>B</b>) Determination of C5a levels in human serum incubated with the different spores. GVB⁰ buffer containing 20% NHS was pre-treated with buffer only (no antibody), OX24, or control IgG1, followed by incubation with 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA spores. C5a levels in the supernatants were measured using the Human Complement Component C5a DuoSet. Data shown was combined from two independent experiments, each with duplicate wells. (<b>C</b>) Determination of C5a levels in mouse BAL fluid. C57BL/6 were i.n. inoculated with 7702 (n = 8), Δ<i>bclA</i> (n = 8), Δ<i>bclA</i>/BclA (n = 8) spores or PBS (n = 6). BAL fluids were collected 6 hours later and C5a level in the supernatant determined using the Mouse Complement Component C5a DuoSet. Data shown were combined from two independent experiments, each with duplicate wells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01. ****, <i>p</i> < 0.0001, <i>t</i> test.</p

A Novel C₂H₂ Transcription Factor that Regulates <i>gliA</i> Expression Interdependently with GliZ in <i>Aspergillus fumigatus</i>

<div><p>Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of <i>Aspergillus</i> species, <i>Trichoderma</i> species, and <i>Penicillium</i> species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn₂Cys₆ binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal species. Using a high-copy inducer screen in <i>A. fumigatus</i>, our lab has identified a novel C₂H₂ transcription factor, which plays an important role in regulating the gliotoxin biosynthetic cluster. This transcription factor, named GipA, induces gliotoxin production when present in extra copies. Furthermore, loss of <i>gipA</i> reduces gliotoxin production significantly. Through protein binding microarray and mutagenesis, we have identified a DNA binding site recognized by GipA that is in extremely close proximity to a potential GliZ DNA binding site in the 5′ untranslated region of <i>gliA</i>, which encodes an efflux pump within the gliotoxin cluster. Not surprisingly, GliZ and GipA appear to work in an interdependent fashion to positively control <i>gliA</i> expression.</p></div

High-copy expression of <i>gipA</i> induces gliotoxin production.

<p>All cultures were grown in repressing conditions. Total RNA was isolated and quantified by dot blot analysis in triplicate. Gliotoxin levels were quantified by RP-HPLC using a standard curve and calculated as mg gliotoxin/g dry mycelial mass. All data sets are normalized to AMA.GL. (a) mRNA transcript levels of several gliotoxin cluster genes after 48 hrs of growth relative to AMA.GL. The results of one representative experiment of three independent experiments are shown as mean ± SD. (b) Gliotoxin levels in growth medium relative to AMA.GL. The data presented is an average of three biological replicates, shown as mean ± SD. The asterisk (*) indicates a statistically significant difference (p-value<0.05), compared to AMA.GL, calculated by one-way ANOVA and Tukey comparison test.</p

<i>B</i>. <i>anthracis</i> spore surface protein BclA mediated CFH binding to spores.

<p>Spores were incubated with purified human CFH in PBS buffer containing D-alanine. Spore-bound CFH was determined by flow cytometry (<b>A</b>), solid phase binding assay (<b>B</b>) and Western blot (<b>C</b>). Flow cytometry results were combined from at least three independent experiments. Solid phase binding assay results were combined from two independent experiments, each with duplicate wells. Western blots shown were representative of at least three independently performed experiments. (<b>D</b>) Recombinant BclA protein (rBclA) bound to immobilized human CFH in a concentration-dependent manner. Results were combined from three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; <i>t</i> test.</p

Schematic of the three rounds of the high-copy inducer screen.

<p>(a) Round one: transformation of the genomic library into Af293.1-GL. (b) Round two: transformation of single plasmids from the genomic library into Af293.1-GL. (c) Round three: transformation of single genes into Af293.1-GL.</p

BclA impaired protective immunity against lethal <i>B</i>. <i>anthracis</i> challenges.

<p>C57BL/6 mice were i.n. inoculated with a sub-lethal dose of ~1×10⁸ spores of 7702, Δ<i>bclA</i> or vehicle control once (<b>A</b>) or twice (<b>B</b>). Mice were then challenged with ~1×10¹⁰ 7702 spores by i.p. injection 15 days after the last i.n. inoculation and monitored for survival. Data shown in (<b>A</b>) were combined from two independent experiments, with n = 7, 8, and 10 for ctrl-7702, 7702–7702, and Δ<i>bclA</i>-7702, respectively. Data shown in (<b>B</b>) were combined from two independent experiments, with n = 6, 8 and 12 for ctrl-7702, 7702–7702, and Δ<i>bclA</i>-7702, respectively. Log-rank test was used to statistical comparison of the survival curves.</p

BclA significantly promoted spore persistence in the mouse lungs.

<p>Mice were i.n. inoculated with sub-lethal doses of various spores. Lungs were collected, homogenized and either dilution plated to determine the total viable bacterial counts, or heated at 68°C and dilution plated to determine the spore counts. (<b>A</b>). C57BL/6 mice were i.n. inoculated with ~1×10⁸ spores of 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA per mouse. Lungs were collected 2 weeks post inoculation. Data shown were combined from at least two independent experiments (7702, n = 12; Δ<i>bclA</i>, n = 7; Δ<i>bclA</i>/BclA, n = 4). (<b>B</b>) Balb/c mice were i.n. inoculated with ~ 1.5 ×10⁷ spores per mouse of <i>B</i>. <i>subitilis</i> containing pDG1662 vector (n = 20) or pDG1662-BclA (n = 20). Lungs were harvested at one week post inoculation. Data shown were combined from two independent experiments. (<b>C</b>). C3^-/- mice were more susceptible to <i>B</i>. <i>anthracis</i> than C57BL/6. Therefore, a sub-lethal dose of ~ 5×10⁵ spores/mouse was used for i.n. inoculation of C3^-/- mice. Lungs were collected at 2 weeks post inoculation. Data shown were combined from at least two independent experiments (7702, n = 14; Δ<i>bclA</i>, n = 12). (<b>D—G</b>) Mice were i.p. inoculated with lethal doses of 7702 or Δ<i>bclA</i> spores. (<b>D</b>) C57BL/6 mice were inoculated with ~1×10⁸ spores/mouse of 7702 (n = 10) or Δ<i>bclA</i> (n = 10) and survival monitored. Data shown were combined from two independent experiments. (<b>E</b>) C3^-/- mice were inoculated with ~5×10⁶ spores/mouse of 7702 (n = 11) or Δ<i>bclA</i> (n = 11). Data shown were combined from two independent experiments. (<b>F</b>) Bacterial burden in the lungs and spleen of C57BL/6 mice inoculated with ~1×10⁸ spores of 7702 (n = 10) or Δ<i>bclA</i> (n = 10) at 48 hours post inoculation. Data shown were combined from two independent experiments. (<b>G</b>) Bacterial burden in the lungs and spleen of C3^-/- mice inoculated with ~5×10⁶ spores of 7702 (n = 6) or Δ<i>bclA</i> (n = 4) at 48 hours post inoculation. Data shown were combined from two independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ****, <i>p</i> < 0.0001; <i>t</i> test. Analysis of survival curves was done using Log-rank test.</p

Layout of two potential GipA binding sites that are embedded in putative GliZ binding sites in the <i>gliZ</i> promoter.

<p>GliZ putative trinucleotide repeats are purple and bolded, the putative GipA binding sites are underlined in orange, and binding cluster 1 additionally contains an AreA recognition element (underlined in yellow).</p

31 secondary metabolism clusters present in <i>A. fumigatus</i>.

<p>The first 18 secondary metabolism clusters are the ones we predict to be positively regulated by GipA, while the last 13 did not have any genes up-regulated >2-fold from the microarray data.</p><p>*Cluster ranges for unknown products are defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004336#pgen.1004336-Perrin1" target="_blank">[52]</a> or were predicted using SMURF at <a href="http://jcvi.org/smurf/index.php" target="_blank">http://jcvi.org/smurf/index.php</a>.</p>1<p>Although several genes in this cluster have been shown to be involved in production of fumiquinazolines, PesL (Afu6g12050) was recently revealed to be necessary for fumigaclavine C production.</p

BclA-mediated CFH recruitment inhibited downstream complement activation <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Complement hemolytic assay. Spores were incubated with 20% NHS and centrifuged. The supernatants (1:10 diluted) were used to perform complement hemolytic assays using opsonized sheep erythrocytes (EA-SRBC). Data shown was from at least three independent experiments. (<b>B</b>) Determination of C5a levels in human serum incubated with the different spores. GVB⁰ buffer containing 20% NHS was pre-treated with buffer only (no antibody), OX24, or control IgG1, followed by incubation with 7702, Δ<i>bclA</i> or Δ<i>bclA</i>/BclA spores. C5a levels in the supernatants were measured using the Human Complement Component C5a DuoSet. Data shown was combined from two independent experiments, each with duplicate wells. (<b>C</b>) Determination of C5a levels in mouse BAL fluid. C57BL/6 were i.n. inoculated with 7702 (n = 8), Δ<i>bclA</i> (n = 8), Δ<i>bclA</i>/BclA (n = 8) spores or PBS (n = 6). BAL fluids were collected 6 hours later and C5a level in the supernatant determined using the Mouse Complement Component C5a DuoSet. Data shown were combined from two independent experiments, each with duplicate wells. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01. ****, <i>p</i> < 0.0001, <i>t</i> test.</p