Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis

ABSTARCT: Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available.

Overall results of the I.D. and immunoblot reactivity of recombinant <i>P. lutzii</i> (rPlp43), native or recombinant gp43 (gp43, gp43r3) and their Endo H-deglycosylated forms (EH-gp43, EH-gp43r3) with PCM patients' sera from Brazilian Southeastern (S) and Midwestern (M) regions (Table S1).

<p>*Sera groups: 1, 1S–50S and 82M–95M; 2, 51M–81M; 3, 96M–98M.</p>#<p>ID titers with purified gp43 or Pb339 culture supernantant are specified in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003111#pntd.0003111.s002" target="_blank">Table S2</a>.</p><p>Overall results of the I.D. and immunoblot reactivity of recombinant <i>P. lutzii</i> (rPlp43), native or recombinant gp43 (gp43, gp43r3) and their Endo H-deglycosylated forms (EH-gp43, EH-gp43r3) with PCM patients' sera from Brazilian Southeastern (S) and Midwestern (M) regions (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003111#pntd.0003111.s001" target="_blank">Table S1</a>).</p

(A) Representative immunoblot reactions of PCM patients' sera (1∶1,000) with rPlp43 and Endo H-deglycosylated (EH-) gp43.

<p>The overall results are shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003111#pntd-0003111-t001" target="_blank">Table 1</a> for sera groups 1, 2, 3, and individual sera 99M, 100M, and101M. On the right, CBB-stained SDS-PAGE gel showing the amount of antigen used in the reactions. Results with Endo H-deglycosylated gp43r3 were similar to (EH-)gp43 and are not shown. (<b>B</b>) <b>PCR amplification of </b><i>HSP70</i><b> using primers for the </b><i>P. lutzii</i><b> gene as shown in an agarose gel.</b> Partial <i>HSP70</i> PCR (529 bp) amplification shows that clinical isolates Pb51M and Pb52M are <i>P. lutzii</i>. DNA extracted from Pb01 (<i>P. lutzii</i>) and Pb18 (<i>P. brasiliensis</i>) were used as species control. Crt-, in the absence of DNA.</p

Amino acid sequence alignment and molecular models show differences between Plp43 (from Pb01) and gp43 (from Pb18).

<p>Differences in amino acid residues can be seen. Known sequence features are indicated: SP (signal peptide), MAb3e epitope (D/purple), T-cell P10 epitope (grey shade with boxed antigenic core; A/navy), N-glycosylation site (N-gly, C/green), and NEP glucanase active site (Glu, B). The sequences corresponding to primers gp01S and gp01AS used to amplify Pl<i>P43</i> ORF are shown. In the molecular models below, arrows (1–4) point to remarkable differences between Plp43 and gp43; caps letters indicate the motifs above specified. Yellow, beta-sheets; magenta, alpha-helix.</p

Recombinant rPlp43 is not glycosylated and seems to be enzymatically active.

<p>(<b>A</b>) Heterologous expression of rPlp43 in <i>P. pastoris</i> culture supernatants from methanol-induced recombinant yeasts containing the Pl<i>P43</i> insert or not (EV, empty vector). (<b>B</b>) SDS-PAGE profile of rPlp43 and control gp43 before (−) and after (+) treatment with Endo H. (<b>C</b>) Glucanase activity against PNPG in culture supernatants from <i>P. pastoris</i> expressing rPlp43 when compared with that, at equivalent total protein amount, of yeasts containing empty vector (EV), wild type <i>P. pastoris</i> and <i>S. cerevisiae</i>. Purified gp43 was used as protein negative control at equivalent protein amount to rPlp43, as estimated in SDS-PAGE gels. BSA was also included as negative control at higher amounts. PBS+rPlp43 (without substrate) and PNPG+enzyme buffer (without substrate) were used as negative controls. Migration of standard molecular masses is indicated.</p

Immunoblot reactivity of anti-gp43 monoclonal antibodies with gp43 and rPlp43.

<p>Reactivity of rPlp43 was tested with five different anti-gp43 MAbs (indicated) in immunoblot, using gp43 as positive control. On the right, CBB-stained SDS-PAGE gel showing the amount of antigen used in the assay. The results were the same for HisPlp43 expressed as inclusion bodies in bacteria.</p

Environmental Isolates of Burkholderia pseudomallei in Ceará State, Northeastern Brazil▿

Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons

Cyclopalladated Compound 7a Induces Apoptosis- and Autophagy-Like Mechanisms in Paracoccidioides and Is a Candidate for Paracoccidioidomycosis Treatment

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species, is the main cause of death due to systemic mycoses in Brazil and other Latin American countries. Therapeutic options for PCM and other systemic mycoses are limited and time-consuming, and there are high rates of noncompliance, relapses, toxic side effects, and sequelae. Previous work has shown that the cyclopalladated 7a compound is effective in treating several kinds of cancer and parasitic Chagas disease without significant toxicity in animals. Here we show that cyclopalladated 7a inhibited the in vitro growth of Paracoccidioides lutzii Pb01 and P. brasiliensis isolates Pb18 (highly virulent), Pb2, Pb3, and Pb4 (less virulent) in a dose-response manner. Pb18 was the most resistant. Opportunistic Candida albicans and Cryptococcus neoformans were also sensitive. BALB/c mice showed significantly lighter lung fungal burdens when treated twice a day for 20 days with a low cyclopalladated 7a dose of 30 μg/ml/day for 30 days after intratracheal infection with Pb18. Electron microscopy images suggested that apoptosis- and autophagy-like mechanisms are involved in the fungal killing mechanism of cyclopalladated 7a. Pb18 yeast cells incubated with the 7a compound showed remarkable chromatin condensation, DNA degradation, superoxide anion production, and increased metacaspase activity suggestive of apoptosis. Autophagy-related killing mechanisms were suggested by increased autophagic vacuole numbers and acidification, as indicated by an increase in LysoTracker and monodansylcadaverine (MDC) staining in cyclopalladated 7a-treated Pb18 yeast cells. Considering that cyclopalladated 7a is highly tolerated in vivo and affects yeast fungal growth through general apoptosis- and autophagy-like mechanisms, it is a novel promising drug for the treatment of PCM and other mycoses

Down regulation of Pb<i>GP43</i> correlated with reduced fungal burden in lungs of infected BALB/c mice.

<p>Graphs represent CFU (expressed as log₁₀ values) recovered from lung homogenates 50 days after intratracheal infection with 1×10⁶ viable yeasts of PbWt, PbEV or Pb<i>GP43</i> aRNA1. Eight mice per fungal strain were used and error bars indicate standard deviations. <i>P</i> values (**<i>p</i><0.0071, ***<i>p</i><0.0002) represent significant differences in comparison with the PbWt or PbEV.</p

Inhibition of Pb<i>GP43</i> expression using an aRNA plasmid and <i>A. tumefaciens</i>-mediated transformation.

<p>(<b>A</b>) Pb<i>GP43</i> anti-sense cassette. Three anti-sense oligonucleotides were produced (AS1, AS2, AS3) based on Pb339 (PbWt) genomic sequence, as detailed in Materials and Methods, and cloned under the control of the <i>H. capsulatum CBP-1</i> promoter (P). The constructs were sub-cloned into the T-DNA region of the binary vector pUR5750 harboring the <i>E. coli HPH</i> resistance gene driven by the GPDA promoter from <i>A. nidulans</i> and bearing transciptional terminators (T) from <i>cat-B</i> and <i>TRPC</i>; LB, left border; RB right border. (<b>B</b>) PCR fragments amplified with <i>HPH</i> specific primers to yield a 1,000-bp internal fragment. We used as template DNA from Pb<i>GP43</i> aRNA (AS3, clones 1, 2, 3), PbWt and PbEV. Strains were tested before and after exhaustive subculturing in selective and non-selective medium with similar results. MW, DNA molecular marker. (<b>C</b>) Pb<i>GP43</i> expression levels in PbWt, PbEV, and Pb<i>GP43</i> aRNA (1 and 2) after 1 month and over 18 months of subculture. Gene expression levels obtained by RT-qPCR were normalized with the internal control <i>TUB2</i>; *, <i>p</i><0.05 when compared with PbWt and PbEV. (<b>D</b>) Inhibition of Pb<i>GP43</i> expression did not alter the morphology of Pb<i>GP43</i> aRNA1 that showed round multibudding yeasts similar to control strains PbWt and PbEV. Cellular morphology of exponentially growing yeast cells was visualized by fluorescence microscopy using calcofluor white staining. White bars correspond to 5 µm.</p