Identification and characterization of Tc1/mariner-like DNA transposons in genomes of the pathogenic fungi of the Paracoccidioides species complex

<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>(Eukaryota, Fungi, Ascomycota) is a thermodimorphic fungus, the etiological agent of paracoccidioidomycosis, the most important systemic mycoses in Latin America. Three isolates corresponding to distinct phylogenetic lineages of the <it>Paracoccidioides </it>species complex had their genomes sequenced. In this study the identification and characterization of class II transposable elements in the genomes of these fungi was carried out.</p> <p>Results</p> <p>A genomic survey for DNA transposons in the sequence assemblies of <it>Paracoccidioides</it>, a genus recently proposed to encompass species <it>P. brasiliensis </it>(harboring phylogenetic lineages S1, PS2, PS3) and <it>P. lutzii </it>(<it>Pb01-like </it>isolates), has been completed. Eight new <it>Tc1/mariner </it>families, referred to as Trem (<b>Tr</b>ansposable <b>e</b>lement <b>m</b>ariner), labeled A through H were identified. Elements from each family have 65-80% sequence similarity with other <it>Tc1/mariner </it>elements. They are flanked by 2-bp TA target site duplications and different termini. Encoded DDD-transposases, some of which have complete ORFs, indicated that they could be functionally active. The distribution of Trem elements varied between the genomic sequences characterized as belonging to <it>P. brasiliensis </it>(S1 and PS2) and <it>P. lutzii</it>. TremC and H elements would have been present in a hypothetical ancestor common to <it>P. brasiliensis </it>and <it>P. lutzii</it>, while TremA, B and F elements were either acquired by <it>P. brasiliensis </it>or lost by <it>P. lutzii </it>after speciation. Although TremD and TremE share about 70% similarity, they are specific to <it>P. brasiliensis </it>and <it>P. lutzii</it>, respectively. This suggests that these elements could either have been present in a hypothetical common ancestor and have evolved divergently after the split between <it>P. brasiliensis </it>and <it>P. Lutzii</it>, or have been independently acquired by horizontal transfer.</p> <p>Conclusions</p> <p>New families of <it>Tc1/mariner </it>DNA transposons in the genomic assemblies of the <it>Paracoccidioides </it>species complex are described. Families were distinguished based on significant BLAST identities between transposases and/or TIRs. The expansion of Trem in a putative ancestor common to the species <it>P. brasiliensis </it>and <it>P. lutzii </it>would have given origin to TremC and TremH, while other elements could have been acquired or lost after speciation had occurred. The results may contribute to our understanding of the organization and architecture of genomes in the genus <it>Paracoccidioides</it>.</p

Cerâmicas medievais do Museu de Francisco Tavares Proença Jr.

BACKGROUND: To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. METHODS AND FINDINGS: Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. CONCLUSIONS: This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization

Cell adhesion and lysosome exocytosis-inducing properties of SAP-CE associated with <i>T. cruzi</i> metacyclic trypomastigote internalization.

<p>(A) Increasing amounts of the purified recombinant protein SAP-CE or GST were added to 96-well plates covered with HeLa cells. After fixation and washes in PBS, the cells were incubated with MAb-SAP (diluted 1∶100) and with anti-mouse IgG peroxidase conjugate. The bound protein was revealed by <i>o</i>-phenylenediamine. Values are the means ± standard deviations of triplicates. (B) HeLa cells were incubated for 30 min with or without the recombinant protein SAP-CE or GST (40 μg/mL) and then incubated with metacyclic forms. After incubation for 1 h, cells were washed in PBS, fixed, and stained with Giemsa. The number of internalized parasites was counted in 500 cells. The values represent the means ± standard deviations of three independent experiments performed in duplicate. SAP-CE significantly inhibited parasite invasion (*p<0.05). (C) Semi-confluent HeLa cell monolayers were incubated in absence or in the presence of GST or the purified recombinant protein SAP-CE (20 μg/mL) for 60 min. The supernatant was collected and the release of β-hexosaminidase measured. Exocytosis was expressed as a percentage of the total β-hexosaminidase activity (supernatant + cell extract). Values are the means ± standard deviations of four independent experiments performed in duplicate. β-hexosaminidase activity was significantly higher in the presence of SAP-CE (*p<0.05). (D) HeLa cells were incubated with or without the purified recombinant protein SAP-CE (20 μg/mL) and processed for indirect immunofluorescence using anti-Lamp-2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA. Scale bar, 10 µm.</p

Release of SAP proteins into the extracellular medium.

<p>(A) Metacyclic trypomastigotes (CL strain) were incubated overnight in PBS at 28°C (1.0×10⁸ parasites/mL). After centrifugation, the conditioned medium (CM) was filtered and analyzed by western blot using MAb-SAP (diluted 1∶100). (B) Epimastigotes and metacyclic trypomastigotes (Dm28c) were incubated for 6 h at 28°C in DMEM or TAU3AAG (1.0×10⁸ parasites/mL), respectively. After centrifugation the conditioned medium was filtered and submitted to ultracentrifugation according to the protocol described by Bayer-Santos et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083864#pone.0083864-BayerSantos1" target="_blank">[14]</a>. Vesicles and soluble-protein fractions (2 μg of protein from each fraction) were analyzed by western blot using MAb-SAP (diluted 1∶100) or a monoclonal antibody against the flagellar calcium-binding protein (FCaBP). The relative molecular masses (kDa) of the immunoreactive proteins are shown on the right. V2, fraction enriched in plasma membrane-derived vesicles/ectosomes; V16, fraction enriched in exosomes; and VF, vesicle-free fraction enriched in soluble proteins.</p

Expression of SAP proteins in the developmental forms of the <i>T. cruzi</i> CL strain.

<p>(A) The levels of SAP transcripts in epimastigotes (Epi) and metacyclic trypomastigotes (Meta) were estimated by qRT-PCR using primers that amplified a conserved 135 bp fragment shared by all SAP genes. The values, which were calculated after normalization with GAPDH transcripts and using epimastigotes as the reference sample (SAP/GAPDH ratio  = 1), are the means ± standard deviations of four independent experiments performed in triplicate. The difference between epimastigotes and metacyclic trypomastigotes was significant (*p<0.0001). (B) SAP expression was determined by quantitative western blot using total protein extracts from epimastigotes and metacyclic trypomastigotes (15 µg protein/lane) reacted with MAb-SAP (diluted 1∶100). As loading control, α-tubulin was used. (C) Difference in size of SAP variants expressed in the different <i>T. cruzi</i> developmental forms. Total protein extracts from epimastigotes (3.0××10⁷ cells), metacyclic trypomastigotes (1.0×10⁸ cells), extracellular amastigotes (3.0×10⁷ cells) and tissue culture-derived trypomastigotes (1.0×10⁸ cells) were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with MAb-SAP (diluted 1∶100). The relative molecular masses (kDa) of the immunoreactive proteins are shown on the right.</p

SAP transcripts isolated from epimastigotes, metacyclic trypomastigotes and extracellular amastigotes of the <i>T. cruzi</i> CL strain by RT-PCR amplification.

<p>1) Accession number according to the TriTrypDB database.</p><p>(2) Localization of SAP genes based on the 41 chromosome-sized scaffolds <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083864#pone.0083864-Weatherly1" target="_blank">[25]</a>.</p><p>(3) SAP transcripts shared by epimastigotes, metacyclic trypomastigotes and extracellular amastigotes.</p><p>(4) The accession numbers represent the three copies of the same gene.</p

SAP proteins secreted into the extracellular medium by epimastigotes and metacyclic trypomastigotes (clone Dm28c) identified by mass spectrometry.

<p>(1) Accession number according to the TriTrypDB database.</p><p>(2 and 3) To validate the quality of protein identification, the following parameters were used: (2) Xcorr (CrossCorr/avg [AutoCorr offset = -75 to 75]) ≥1.5, 2.0, and 2.5, for singly, doubly and triply charged peptides, respectively. (3) DC<i>n</i> (Xcorr₁ – Xcorr₂/Xcorr₁) ≥0.1.</p><p>(4) Sample enriched in plasma membrane-derived vesicles/ectosomes.</p><p>(5) Sample enriched in exosomes.</p><p>(6) Sample enriched in soluble proteins (vesicle-free, VF).</p