TLR2 mediates the cytokine production induced by ArtinM.

<p>IL-12p40 and IL-10 levels in the cell culture supernatants of adherent spleen cells (A and B) or peritoneal macrophages (C and D) from WT (white bars) and TLR2-KO (black bars) mice were measured by ELISA. Adherent spleen cells were stimulated with ArtinM (156 nM) or P3C4 (1 µg/mL) for 24 h, while the peritoneal macrophages were incubated with ArtinM (39 nM) or P3C4 (1 µg/mL) for 48 h. Statistical comparisons between unstimulated and stimulated cells were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05.</p

ArtinM triggers TLR2-mediated cell activation.

<p>HEK293A cells were transfected with the ectodomain of human TLR2 (A) or human TLR4 (B), and the necessary co-receptors (CD14, CD36, and MD-2), as well as an NF-κB reporter construct and a <i>Renilla</i> luciferase control reporter plasmid. The cells were then stimulated with different concentrations of ArtinM (15.6, 156, and 780 nM) at 37°C for 18 h. In (A), MALP-2 (50 ng/mL) was used as the positive control. In (B), LPS (1 µg/mL) was used as the positive control; the addition of polymyxin B (100 µg/mL) to LPS served as another control. In both A and B, medium and an empty vector were used as the negative controls. The luciferase activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. Statistical comparisons between unstimulated and stimulated cells were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. * p<0.05.</p

ArtinM induces the activation of TLR2/1- and TLR2/6-expressing cells.

<p>HEK293A cells were transfected with TLR2/1 (A and C) or TLR2/6 (B and D), co-receptors, an NF-κB reporter construct, and a <i>Renilla</i> luciferase reporter plasmid as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#pone-0098512-g002" target="_blank">Figure 2</a>. The transfected cells were stimulated with ArtinM (15.6, 156, and 780 nM) at 37°C for 4 h. Medium and cells transfected with an empty vector were used as the negative controls. The positive controls were P3C4 (1 nM) for TLR2/1 activation (A and C) and FSL1 (0.1 nM) for TLR2/6 activation (B and D). The luciferase activity (A and B) was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. IL-8 levels in the culture supernatants (C and D) were measured by ELISA. Statistical comparisons between the cells incubated with medium and the cells stimulated with ArtinM were performed with a one-way analysis of variance followed by Bonferroni's multiple comparison test. * p<0.05.</p

Enhanced TLR2 relative expression by ArtinM-stimulated macrophages.

<p>Peritoneal macrophages from C57BL/6 mice were incubated with ArtinM (39 nM) for 5 h. Medium was used as a negative control and P3C4 (1 µg/mL) was used as a positive control. RNA from macrophages were isolated and used for qRT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">Materials and Methods</a>. The results are expressed as the relative expression of TLR2 after quantification using the ΔΔCt method and normalized to β-actin expression. Statistical comparisons between stimulated cells and unstimulated were performed with one-way analysis of variance followed by Bonferroni's multiple comparison test. ** p<0.01.</p

ArtinM binding to TLR2 depends on sugar recognition.

<p>(A and B) Peritoneal macrophages from C57BL/6 mice were incubated with biotinylated ArtinM after pre-incubation with anti-TLR2 antibody or non-specific IgG. ArtinM binding was detected with streptavidin-FITC and analyzed by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. Results are expressed as the percentage of cells positive for ArtinM binding (A) and MFI (median fluorescence intensity) (B). (C) The dependence of ArtinM-TLR2 binding on carbohydrate recognition used anti-TLR2 antibody coated onto 96-well microplates (5 µg/mL) to capture TLR2 from a cellular extract of peritoneal macrophages. Biotinylated ArtinM (40 µg/mL), previously incubated with the indicated concentrations of Manα1-3 [Manα1-6] Man or Galα(1,6)Galα(1,6)Gluα(1,2)Fru, was added to the wells. After washing, ArtinM binding was detected by neutravidin-AP, and signal was developed with <i>p</i>-nitrophenyl phosphate. Results are expressed in O.D as the mean ± SEM. Statistical comparisons between cells incubated or not with carbohydrates were performed with one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05.</p

Cell signaling molecules that possibly mediate the macrophage activation induced by ArtinM.

<p>Peritoneal macrophages were pretreated with the inhibitors PD98059 (p42/44 MAPK or ERK 1/2), SB202190 (p38 MAPK), SP600125 (c-Jun N-terminal kinase), and LY-294002 (PI3K), or with medium alone (absence of inhibitor) and then stimulated with ArtinM (39 nM). After 48 h, IL-12p40 and IL-10 levels in the culture supernatants were assessed by ELISA. (A) Cytokine production reported as pg/mL (mean ± SEM) and statistical comparison were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05. (B) Inhibition of ArtinM induced cytokine production after pre-incubation with the indicated inhibitors. The results are presented as the percent inhibition, which represents the ratio between uninhibited cells and those pretreated with inhibitors.</p

Molecular Characterization of a Novel Family of <i>Trypanosoma cruzi</i> Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion

<div><p>Background</p><p>The surface coat of <i>Trypanosoma cruzi</i> is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.</p><p>Methodology/ Principal Findings</p><p>Here, we described a novel family of <i>T</i>. <i>cruzi</i> surface membrane proteins (TcSMP), which are conserved among different <i>T</i>. <i>cruzi</i> lineages and have orthologs in other <i>Trypanosoma</i> species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all <i>T</i>. <i>cruzi</i> developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the <i>T</i>. <i>cruzi</i> secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca²⁺ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.</p><p>Conclusion/Significance</p><p>We hypothesized that the productive interaction of <i>T</i>. <i>cruzi</i> with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.</p></div

Synteny of TcSMP genes among <i>T</i>. <i>brucei</i>, T. cruzi and <i>T</i>. <i>grayi</i>.

<p>Genomic regions around the <i>T</i>. <i>cruzi</i> (CLB) TcSMP paralogs and <i>T</i>. <i>brucei</i> and <i>T</i>. <i>grayi</i> orthologs are shown. Analyses were conducted via TBLASTN using the Artemis Comparison Tool (ACT) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004216#pntd.0004216.ref037" target="_blank">37</a>] with an E value of 500. Comparison among TcChr37 (‘‘P” chromosome assigned to the non-Esmeraldo haplotype and ‘‘S” to the Esmeraldo haplotype), <i>T</i>. <i>brucei</i> chromosome Tb10 and the contig Tgr_12_V1 of <i>T</i>. <i>grayi</i>. Homologous genes are connected by colored lines. The matches and reverse matches are represented in red and in blue, respectively. Grey blocks represent each chromosome. Chromosome markers are drawn in the sense (+) and antisense (-) strands. The numbers indicate the location on chromosomes: <i>T</i>. <i>brucei</i> TREU927 (CHR10—position: 2729733–2761348 nt), CLB <i>T</i>. <i>cruzi</i> (TcChr37-S—positions: 848335–873376 nt and TcChr37-P 848287–882361 nt) and contig of <i>T</i>. <i>grayi</i> (Tgr_12_V1 –position: 1–64810 nt). The locations of the TcSMP paralogs and <i>T</i>. <i>brucei</i> and <i>T</i>. <i>grayi</i> orthologs are depicted in chromosomes by red blocks. Abbreviations: TcSMP (TcSMP—<i>T</i>. <i>cruzi</i>/ PSSA-2—<i>T</i>. <i>brucei</i>); HIRA (HIRA-interacting protein 5, putative); ubiqui (ubiquitin-like modifier-activating enzyme ATG7, putative); RNA-bp (RNA-binding protein, putative); ubiqui E1 (ubiquitin activating E1 enzyme, putative); surf glyco (surface glycoprotein, putative); PRC (paraflagellar rod component, putative); HP (hypothetical protein, conserved); S-methyl (S-methyl-5thioribose kinase); POT (proton-dependent oligopeptide transporter, POT family); tetratrico (tetratricopeptide domain 4); 60S (putative 60S ribosomal protein L6); sterol C (sterol C-24 reductase); IFT 57 (predicted: intraflagellar transporter protein 57 homolog); tRNA-s lig (tRNA-splicing ligase RtcB); PP 2A (protein phosphatase 2A regulatory subunit).</p

Genomic organization of the TcSMP family.

<p>A) Schematic representation of in silico chromosomes TcChr37-P, TcChr37-S and TcChr27-P showing the distribution of TcSMP genes (red boxes). The accession numbers of TcSMP genes in TriTrypDB are indicated above. B) Restriction Southern blot analysis of TcSMP loci. Genomic DNA of CLB was digested with different restriction enzymes, separated on a 0.8% agarose gel and transferred to nylon membrane. Autoradiogram obtained by hybridization with (³²P) TcSMP probe. The enzymes are indicated above each lane. The colored boxes indicate the restriction fragments predicted in chromosomes TcChr37-P, TcChr37-S, and TcChr27-P. C) Karyotype mapping of TcSMP genes. Chromosomal bands of CLB, strain G and <i>T</i>. <i>cruzi marinkellei</i> were separated by PFGE, stained with ethidium bromide, transferred onto nylon membranes and hybridized with TcSMP probe. Numbers on the left correspond to the sizes (Mb) of <i>Hansenula wingei</i> chromosomes used as markers, and on the right the sizes of chromosomal bands hybridizing with (³²P) TcSMP probe.</p

Recombinant protein TcSMP properties in host cell binding, Ca²⁺ signaling, lysosome scattering and inhibition of <i>T</i>. <i>cruzi</i> metacyclic trypomastigote invasion.

<p>A) HeLa cells were incubated with the indicated proteins, at varying concentrations, and binding was evaluated as described in the methods section. Values are the means ± SD of triplicates of one representative assay out of three. B) HeLa cells were grown overnight in DMEM with 10% serum on ibidi multichamber dishes (Hi-Q4, ibidi), and the fluorescence intensity of Fluo-4 was determined as described in the methods section. Images show the basal fluorescence at time zero (left panel) and the maximum fluorescence after stimulation with 40 μg/mL purified TcSMP or GST (right panel). Note the increase in the fluorescence intensity after challenge with TcSMP but not with GST. Bar: 50 μm. C) HeLa cells were grown as in (B) and intracellular calcium was quantified after stimulation (black arrow) with recombinant protein TcSMP or GST. The graph shows the relative concentration of cytoplasmic Ca²⁺ at 400 sec after stimulation, expressed as the maximum peak of total fluorescence minus basal fluorescence at time zero. Thirty cells in three independent experiments were analyzed. Shown on the left side is the difference between the maximum calcium concentration at 400 sec after stimulation with TcSMP and GST (*p = 0.0005). D) HeLa cells were incubated for 30 min with 40 μg/mL of TcSMP or GST, and then processed for confocal fluorescence analysis using anti-LAMP2 antibody and Alexa Fluor 488-conjugated anti-mouse IgG (green), phalloidin-TRITC (red) for actin visualization and DAPI (blue) for DNA, under 63X objective. (Bar: 20 μm). Note the lysosome mobilization to the host cell periphery after treatment with TcSMP. E) HeLa cells were incubated with the indicated recombinant protein at 40 μg/mL. After 30 min, CL strain metacyclic trypomastigotes were added and incubation proceeded for 1 h before fixation and staining with Giemsa. The number of internalized parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. TcSMP, but not GST, significantly inhibited parasite invasion (*p < 0.05).</p