Phenotypic characterization of specific CD8⁺ T cells of infected and/or AdASP-2 immunized A/Sn mice.

<p>A/Sn mice were infected or immunized as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002699#ppat-1002699-g003" target="_blank">Figure 3A</a>. Splenic cells were collected nineteen days after infection/immunization. The histograms show FACS analysis on CD8⁺ cells (Gr. 1) and H-2K^k-TEWETGQI⁺ CD8⁺ cells (Gr. 2, 3, and 4) and the indicated marker (blue). Control cells were from naive mice (red lines). Results of CD44, KLRG1 and CD183 staining are presented as MFI and frequencies of the CD44^High, KLRG1^High or CD183^High cells, respectively. On the other hand, results of CD27 and CD62L staining are presented as MFI and frequencies of the CD27^Low or CD62L^Low cells. Representative analyses are shown from pools of cells from 3 mice. Stainings were performed 2 or more times with identical results.</p

Schematic representation of the ASP-2 antigen and its expression in intra-cellular amastigotes of <i>T. cruzi</i>.

<p><b>A</b>) The protein is a prototypical member of the <i>trans</i>-sialidase family of <i>T. cruzi</i> surface antigens containing a putative signal peptide at the N-terminal region, 2 ASP-box sequences, and a VTV-box at the C-terminal domain. The protein is attached to the membrane through a glycophosphatidylinositol anchor. <b>B</b>) ASP-2 expression in intra-cellular amastigotes was determined by immunofluorescence with the specific MAb K22. HeLa cells were infected for 48 h with trypomastigotes of the Y strain. After fixation, indirect immunofluorescence and DAPI staining were performed and imaged under fluorescence microscopy. Bar, 14 µM. Photomicrography kindly provided by Dr. Clara Claser (Singapore Immunology Network -SIgN, Singapore).</p

<i>Ureaplasma diversum</i> Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host

<div><p>Whole genome sequencing and analyses of <i>Ureaplasma diversum</i> ATCC 49782 was undertaken as a step towards understanding <i>U</i>. <i>diversum</i> biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)—Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of <i>U</i>. <i>diversum</i> was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable <i>U</i>. <i>diversum</i>. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, <i>U</i>. <i>diversum</i> has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis.</p></div

Gene synteny and phylogeny.

<p>(A) Syntenic maps of ureaplasma genomes. Sybil map used <i>U</i>. <i>diversum</i> ATCC 49782 as a reference genome. (B) Phylogenetic trees based on 32 concatenated proteins of <i>Mollicutes</i>.</p

Coding DNA sequences (CDSs) of <i>Ureaplasma diversum</i> ATCC 49782 genome classified by TIGR role category.

<p>Coding DNA sequences (CDSs) of <i>Ureaplasma diversum</i> ATCC 49782 genome classified by TIGR role category.</p

Virulence and pathogenicity mechanisms.

<p>(A) Virulence map of <i>U</i>. <i>diversum</i> ATCC 49782. (B) Schematic representation of the urease gene cluster from <i>U</i>. <i>diversum</i> ATCC 49782. Structural subunits: <i>ure</i>A (gudiv_255), <i>ure</i>B (gudiv_254), and <i>ure</i>C (gudiv_253). Accessory proteins <i>ure</i>E (gudiv_252), <i>ure</i>F (gudiv_251), <i>ure</i>G (gudiv_250), and <i>ure</i>D (gudiv_249) (C) Diagram of <i>Ureaplasma diversum</i> Multiple-Banded Antigen-like protein (MBA-like—gudiv_653) and locus and similarity of MBA-like with the human ureaplasmal Multiple-Banded Antigen (MBA) (Accession number: AF055358.2).</p

Metabolic map of <i>U</i>. <i>diversum</i> ATCC 49782.

<p>A view of the transporters and main metabolism pathways. Metabolic products are shown in black and ureaplasma proteins in green. Enzymes that were not found in <i>U</i>. <i>diversum</i> are shown in red.</p

General features of the genome of <i>Ureaplasma diversum</i> ATCC 49782 compared to human ureaplasmas and other members of <i>Mycoplasma</i>, <i>Acholeplasma</i> and <i>Phytoplasma</i> species.

<p>General features of the genome of <i>Ureaplasma diversum</i> ATCC 49782 compared to human ureaplasmas and other members of <i>Mycoplasma</i>, <i>Acholeplasma</i> and <i>Phytoplasma</i> species.</p