Examinando por Autor "Ruano Gallego, D."

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A multiplex antigen microarray for simultaneous IgG and IgM detection against SARS-CoV-2 reveals higher seroprevalence than reported
(Society for Applied Microbiology, 2021-04-30) Ruano Gallego, D.; García Villadangos, M.; Moreno Paz, M.; Gómez Elvira, J.; Postigo, M.; Simón Sacristan, M.; Reyburn, H. T.; Carolis, C.; Rodrigo, N.; Codeseira, Y. B.; Rueda, P.; Zúñiga, Sonia; Enjuanes, L.; Parro García, V.; Agencia Estatal de Investigación (AEI); Consejo Superior de Investigaciones Científicas (CSIC); Comunidad de Madrid (CM); 0000-0002-2163-2088; 0000-0002-3928-3592; 0000-0003-1245-3253; 0000-0002-9068-9846; 0000-0002-4041-3788; 0000-0003-2855-1595; 000-0003-4240-1139; 0000-0002-6050-2446; 0000-0001-8120-7262; 0000-0002-7735-3766; 0000-0003-2549-6826; 0000-0002-0854-0226; 0000-0003-3738-0724; Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
The surge of SARS-CoV-2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID-19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 μg ml-1) and IgG (~0.017 μg ml-1) and is scalable and cost-effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma-based treatments, high-throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency.
Restringido
Type III secretion system effectors form robust and flexible intracellular virulence networks
(American Association for the Advancement of Science, 2021-03-12) Ruano Gallego, D.; Sánchez Garrido, J.; Kozik, Z.; Núñez Berrueco, E.; Cepeda Molero, M.; Mullineaux Sanders, C.; Clark, J. N.; Slater, S. L.; Wagner, N.; Glegola Madejska, I.; Roumeliotis, T. I.; Pupko, T.; Fernández, L. Á.; Rodríguez Patón, A.; Choudhary, J. S.; Frankel, G.; Agencia Estatal de Investigación (AEI); Medical Research Council (MRC); Ruano Gallego, D. [0000-0002-2163-2088]; Sánchez Garrido, J. [0000-0002-5847-4167]; Kozik, Z. [0000-0001-6713-5776]; Núñez Barrueco, E. [0000-0003-3995-6176]; Cepeda Molero, M. [0000-0002-0635-928X]; Mullineaux Sanders, C. [0000-0001-8995-7615]; Clark, J. N. [0000-0002-6207-8466]; Slater, S. L. [0000-0001-9990-5624]; Wagner, N. [0000-0001-7759-3009]; Glegola Madejska, I. [0000-0003-1270-8498]; Pupko, T. [0000-0001-9463-2575]; Fernández, L. Á. [0000-0001-5920-0638]; Rodríguez Patón, A. [0000-0001-7289-2114]; Frankel, G. [0000-0002-0046-1363]
INTRODUCTION Infections with many Gram-negative pathogens, including Escherichia coli, Salmonella, Shigella, and Yersinia, rely on the injection of effectors via type III secretion systems (T3SSs). The effectors hijack cellular processes through multiple mechanisms, including molecular mimicry and diverse enzymatic activities. Although in vitro analyses have shown that individual effectors can exhibit complementary, interdependent, or antagonistic relationships, most in vivo studies have focused on the contribution of single effectors to pathogenesis. Citrobacter rodentium is a natural mouse pathogen that shares infection strategies and virulence factors with the human pathogens enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). The ability of these pathogens to colonize the gastrointestinal tract is mediated by the injection of effectors via a T3SS. Although C. rodentium infects 31 effectors, the prototype EPEC strain E2348/69 translocates 21 effectors. RATIONALE The aim of this study was to test the hypotheses that, rather than operating individually, the T3SS effectors form robust intracellular networks that can sustain large contractions and that expanded effector repertoires play a role in distinct disease phenotypes and host adaption. RESULTS We tested the effector-network paradigm by infecting mice with >100 C. rodentium effector mutant combinations. First, using machine learning prediction algorithms, we discovered additional effectors, NleN and NleO. We then sequentially deleted effector genes from two distinct starting points to reach sustainable endpoints, which resulted in strains missing 19 unrelated effectors (CR14) or 10 effectors involved in the modulation of innate immune responses in intestinal epithelial cells (IECs) (CRi9). Moreover, we deleted Map and EspF, which target the mitochondria and disrupt tight junctions. Unexpectedly, all strains colonized the colon and activated conserved metabolic and antimicrobial processes in the IECs while eliciting distinct cytokine and immune cell infiltration responses. In particular, although infection with C. rodentium Δmap/ΔespF failed to induce secretion of interleukin-22 (IL-22), CR14 and CRi9 triggered heightened secretion of IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) and of IL-22, interferon-γ (IFN-γ), and IL-17 from colonic explants, respectively. Nonetheless, infection with CR14 or CRi9 induced protective immunity against secondary infections. Although Tir, EspZ, and NleA are essential, other effectors exhibit context-dependent essentiality in vivo. Moreover, C. rodentium expressing the effector repertoire of EPEC E2348/69 failed to efficiently colonize mice. We used curated functional information and our in vivo data to train a machine learning model that predicted values for colonization efficiency of previously uncharacterized mutant combinations. Notably, a mutant with a low predicted value, lacking only nleF, nleG8, nleG1, nleB, and espL, failed to colonize. CONCLUSION Our analysis revealed that T3SS effectors form robust networks, which can sustain substantial contractions while maintaining virulence, and that the composition of the effector network contributes to host adaptation. Alternative effector networks within a single pathogen triggered markedly different immune responses yet induced protective immunity. CR14 did not tolerate any further contraction, which suggests that this network reached its robustness limit with only 12 effectors. As the robustness limits of other effector networks depend on the contraction starting point and the order of the deletions, machine learning models could transform our ability to predict alternative network functions. Together, this study demonstrates the robustness of T3SS effector networks and the ability of IECs to withstand drastic perturbations while maintaining antibacterial functions.