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Objectives: The aim of this study was to investigate the structure of a broad and sustained hospital outbreak of OXA-48-producing Klebsiella pneumoniae (KpO48) belonging to sequence type 405 (ST405). Methods: Whole-genome sequencing and comparison of ten ST405 KpO48 isolates obtained from clinical samples in our hospital was performed. Using stringent criteria, 36 single nucleotide polymorphisms (SNPs) were detected (range 0–21 in pairwise comparisons), and allele-specific PCR was used to call the SNPs among a larger set of isolates. Results: Several haplotypes were identified within the population. The haplotypes did not show a spatial structure, but a temporal evolution of sequential haplotype replacements was observed. Conclusions: The dispersed spatial distribution suggests a reservoir formed by a large pool of colonised patients, and the temporal replacement pattern suggests that the sustained outbreak was composed of several small outbreaks that appeared and rapidly dispersed to several units., Ministerio de Economía y Competitividad [grant INNPACTO IPT-2011-0964- 900000 to PG-P and JM]; IdiPAZ [internal grant to JM]; the Instituto de Salud Carlos III, Ministerio de Economía y Compet-itividad [grant PI13/01218 to JM]; and REIPI RD12/0015 from Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, co-financed by the European Development Regional Fund

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-a and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-a and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17, s. This work was supported by grants BFU2007-66443/BMC and BFU2010-19144/BMC from Ministerio de Ciencia e Innovación, a grant from Fundación de Investigación Médica Mutua Madrileña and by RETICS Program RD08/0075-RIER (Red de Inflamacio´n y Enfermedades Reumáticas) from Instituto de Salud Carlos III (to C.C.), a grant from Fundación de Investigación Médica Mutua Madrileña (to M.D.G.L.), and grants PI080794 from Instituto de Salud Carlos III (to M.Y-M) and SAF2007-60578 from Ministerio de Ciencia e Innovación (to E.M.L.). M.D.G.L. was supported by a contract associated to grant SAF2004-01715 from Ministerio de Ciencia e Innovacio´n. S.O. was supported by an I3P predoctoral Fellowship from Consejo Superior de Investigaciones Científicas (CSIC) and by a contract associated to grant BFU2007-66443/BMC from Ministerio de Ciencia e Innovacio´n. A.G. has been supported by a predoctoral Fellowship from Instituto de Salud Carlos III and by grant BFU2007-66443/BMC from Ministerio de Ciencia e Innovación.

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well-established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin-dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti-CD9-specific antibodies resulted in (i) increased beta1 integrin-mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF-alpha, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF-alpha, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.

Resumen Las tetraspaninas son moleculas de la superficie celular de amplia distribucion en los organismos eucarioticos. Poseen como caracteristica estructural peculiar cuatro dominios transmembranales, regiones N- y C-terminales intracitoplasmicas, y dos lazos extracelulares de distinto tamano. Tambien poseen un motivo de secuencia CCG en el lazo extracelular mayor, asi como residuos polares conservados en los dominios transmembranales. Las celulas sanguineas de los mamiferos expresan combinaciones peculiares de distintas tetraspaninas, incluyendo los antigenos de diferenciacion CD9, CD37, CD53, CD81/TAPA-1, CD82, CD151/PETA-3 y CD231/TALLA1. En este trabajo se resumen la estructura y las interacciones de sus regiones citoplasmicas con proteinas del citoesqueleto y senalizadoras, como la proteina cinasa C (PKC) o la Fosfatidil-Inositol 4-cinasa (PI4-K). Sus interacciones especificas con otras tetraspaninas, con integrinas, antigenos de histocompatibilidad, y miembros de la superfamilia de las inmunoglobulinas son tambien revisadas. Las tetraspaninas son proteinas “adaptadoras” o “facilitadoras”. Al formar parte de complejos moleculares, modulan funciones celulares clave que incluyen la fusion celular, la adhesion, la migracion, la diferenciacion y la transduccion de senales. Las tetraspaninas se organizan en una red con distintos niveles de asociacion, determinados por su resistencia a la solubilizacion por detergentes. En concreto, se analizan las tetraspaninas como reguladoras del Sistema Inmunitario gracias a sus interacciones con los receptores de antigeno de los linfocitos T y B, las moleculas de histocompatibilidad de clase I y clase II, y los co-receptores CD2, CD4, CD5, CD8 y CD19. Por ultimo, se revisa detalladamente el papel de la tetraspanina CD9 en la funcion de las celulas linfoides y mieloides, su relevancia en infecciones como el HIV, y la importancia de su asociacion con integrinas en la progresion cancerosa.

11 páginas, 8 figuras -- PAGS nros. 746-756, α4β1 integrin is highly expressed in lymphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. α4β1 can be activated by intracellular signals elicited upon T-cell activation by phorbol esters, CD3 crosslinking, or certain chemokine/receptor interactions (inside-out activation). Divalent cations or certain anti- β1 mAbs (i.e., TS2/16) can also bind and activate integrins directly (outside-in activation). In both cases, activation results in increased adhesion and/or affinity for ligands. It is not known if these various stimuli produce the same or different post-adhesion events. To address this, we have studied the cytoskeleton organization and intracellular signaling following activation of α4β1 in Jurkat cells and in human T-lymphoblasts. Treatment with Mn2+, α-CD3 mAb or the chemokine SDF-1α followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM-1 (α4β1 ligands), resulted in cell polarization and migration. In contrast, activation with PMA or TS2/16 induced cell spreading and strong adherence. Video microscopy and Transwell analyses confirmed these results, which correlated with different resistance to detachment under flow. Activation of the small GTPase RhoA or transfection with the constitutively active mutants V14RhoA or V12Rac1, abolished the α4β1-induced cell polarization but did not affect cell spreading. Moreover, Rac1 activity was distinctly modulated by agents that induce a polarized or spread phenotype. The tyrosine kinase Pyk2 was highly phosphorylated upon induction of cell polarity but not during cell spreading. These results reveal novel properties of α4β1 integrin, namely the ability to trigger two types of T-cell cytoskeletal response with different signaling requirements

Tetraspanins associate with several transmembrane proteins forming microdomains involved in intercellular adhesion and migration. Here, we show that endothelial tetraspanins relocalize to the contact site with transmigrating leukocytes and associate laterally with both intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Alteration of endothelial tetraspanin microdomains by CD9–large extracellular loop (LEL)–glutathione S–transferase (GST) peptides or CD9/CD151 siRNA oligonucleotides interfered with ICAM-1 and VCAM-1 function, preventing lymphocyte transendothelial migration and increasing lymphocyte detachment under shear flow. Heterotypic intercellular adhesion mediated by VCAM-1 or ICAM-1 was augmented when expressed exogenously in the appropriate tetraspanin environment. Therefore, tetraspanin microdomains have a crucial role in the proper adhesive function of ICAM-1 and VCAM-1 during leukocyte adhesion and transendothelial migration.

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM-ALCAM) or heterophilic (ALCAM-CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity. © Springer Basel AG 2012., MICINN BFU2010-19144/BMC y SAF2012-34561; Fundación Médica Mutua Madrileña; the RETICS Program RD08/0075-RIER from Instituto de Salud Carlos III; Fundación MAPFRE

Tetraspanins associate on the cell membrane with several transmembrane proteins, including members of the integrin superfamily. The tetraspanin CD9 has been implicated in cell motility, metastasis, and sperm-egg fusion. In this study we characterize the first CD9 conformation-dependent epitope (detected by monoclonal antibody (mAb) PAINS-13) whose expression depends on changes in the activation state of associated beta(1) integrins. MAb PAINS-13 precipitates CD9 under conditions that preserve the association of this tetraspanin with integrins, but not under conditions that disrupt these interactions. Induction of activation of beta(1) integrins by temperature, divalent cation Mn(2+), or mAb TS2/16 correlated with enhanced expression of the PAINS-13 epitope on a variety of cells. Through the use of different K562 myeloid leukemia transfectant cells expressing specific members of the beta(1) integrin subfamily we show that the expression of the PAINS-13 epitope depends on CD9 association with alpha(6)beta(1) integrin. The mAb PAINS-13 reactivity has been mapped to the CD9 region comprising residues 112-154 in the NH(2) half of the large extracellular loop. Also, we show that the CD9 conformation recognized by mAb PAINS-13 is functionally relevant in beta(1) integrin-mediated cellular processes including wound healing migration, tubular morphogenesis, cell adhesion and spreading and in signal transduction involving phosphatidylinositol 3-kinase activation.