Proteomic and functional analysis identifies galectin-1 as a novel regulatory component of the cytotoxic granule machinery

Secretory granules released by cytotoxic T lymphocytes (CTLs) are powerful weapons against intracellular microbes and tumor cells. Despite significant progress, there is still limited information on the molecular mechanisms implicated in target-driven degranulation, effector cell survival and composition and structure of the lytic granules. Here, using a proteomic approach we identified a panel of putative cytotoxic granule proteins, including some already known granule constituents and novel proteins that contribute to regulate the CTL lytic machinery. Particularly, we identified galectin-1 (Gal1), an endogenous immune regulatory lectin, as an integral component of the secretory granule machinery and unveil the unexpected function of this lectin in regulating CTL killing activity. Mechanistic studies revealed the ability of Gal1 to control the non-secretory lytic pathway by influencing Fas–Fas ligand interactions. This study offers new insights on the composition of the cytotoxic granule machinery, highlighting the dynamic cross talk between secretory and non-secretory pathways in controlling CTL lytic function

Proteomic and functional analysis identifies galectin-1 as a novel regulatory component of the cytotoxic granule machinery

Secretory granules released by cytotoxic T lymphocytes (CTLs) are powerful weapons against intracellular microbes and tumor cells. Despite significant progress, there is still limited information on the molecular mechanisms implicated in target-driven degranulation, effector cell survival and composition and structure of the lytic granules. Here, using a proteomic approach we identified a panel of putative cytotoxic granule proteins, including some already known granule constituents and novel proteins that contribute to regulate the CTL lytic machinery. Particularly, we identified galectin-1 (Gal1), an endogenous immune regulatory lectin, as an integral component of the secretory granule machinery and unveil the unexpected function of this lectin in regulating CTL killing activity. Mechanistic studies revealed the ability of Gal1 to control the non-secretory lytic pathway by influencing Fas?Fas ligand interactions. This study offers new insights on the composition of the cytotoxic granule machinery, highlighting the dynamic cross talk between secretory and non-secretory pathways in controlling CTL lytic function.Fil: Clemente, Tiago. Universidade de Sao Paulo; Brasil. Instituto Nacional de Ciência e Tecnologia ; BrasilFil: Vieira, Narcisio J.. Universidade de Sao Paulo; Brasil. Instituto Nacional de Ciência e Tecnologia ; BrasilFil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Adrain, Colin. Instituto Gulbenkian de Ciência; PortugalFil: Luthi, Alexander. Trinity College; IrlandaFil: Dominguez, Mariana. Universidade de Sao Paulo; BrasilFil: Yon, Monica. Universidade de Sao Paulo; BrasilFil: Barrence, Fernanda C.. Universidade de Sao Paulo; BrasilFil: Riul, Thalita B.. Universidade de Sao Paulo; BrasilFil: Cummings, Richard D.. Harvard Medical School; Estados UnidosFil: Zorn, Thelma. Universidade de Sao Paulo; BrasilFil: Amigorena, Sebastian. Inserm; FranciaFil: Dias Baruffi, Marcelo. Universidade de Sao Paulo; BrasilFil: Rodriguez, Mauricio M.. Universidade de Sao Paulo; BrasilFil: Seamus, J. Martin. Trinity College; IrlandaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Amarante Mendez, Gustavo P.. Trinity College; Irlanda. Universidade de Sao Paulo; Brasil. Instituto Nacional de Ciência e Tecnologia ; Brasi

<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

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

Capsule of <i>U</i>. <i>diversum</i>.

<p>(A) Electron microscopy of cells <i>U</i>. <i>diversum</i> ATCC 49782 obtained in the cultured isolates from mucovulvovaginal bovine semen and treated with red ruthenium dye, showing polysaccharide materials (electrodense external region indicated with arrowheads). Bar 100 nm. (B) Percentage of monosaccharides in capsular components of <i>U</i>. <i>diversum</i> ATCC 49782.</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

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

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