Browsing by MeSH term "Bacterial Outer Membrane Proteins"
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Publication A New Aspergillus fumigatus Typing Method Based on Hypervariable Tandem Repeats Located within Exons of Surface Protein Coding Genes (TRESP)(Public Library of Science (PLOS), 2016-10-04) Garcia-Rubio, Rocio; Gil, Horacio; Monteiro, Maria Candida; Pelaez, Teresa; Mellado, Emilia; Instituto de Salud Carlos IIIAspergillus fumigatus is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For A. fumigatus genotyping, the short tandem repeat method (STRAf) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. A. fumigatus isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated A. fumigatus strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson's index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for A. fumigatus has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks.Publication A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection(American Society for Microbiology (ASM), 2024-05-08) Roberts, Adam J; Ong, Han Boon; Clare, Simon; Brandt, Cordelia; Harcourt, Katherine; Takele, Yegnasew; Ghosh, Prakash; Toepp, Angela; Waugh, Max; Matano, Daniel; Färnert, Anna; Adams, Emily; Moreno, Javier; Mbuchi, Margaret; Petersen, Christine; Mondal, Dinesh; Kropf, Pascale; Wright, Gavin J; Wellcome Trust; Unión Europea. Comisión Europea. H2020; Foundation for Innovative New Diagnostics (Suiza); European and Developing Countries Clinical Trials Partnership (EDCTP); Unión Europea. Comisión Europea. Horizonte Europa. ERA-LEARNVisceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.Publication A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule(American Society for Biochemistry and Molecular Biology (ASBMB), 2012-10-12) Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; Garcia, Ruth; Lasala, Fatima; Jimenez, Mercedes; Mir-Gerrero, Carmen; Morreale, Antonio; Admon, Arie; Lopez, Daniel; Ministerio de Ciencia e Innovación (España); Fundación para la Investigación y la Prevención del Sida en España; Israel Science FoundationThe transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.Publication Abnormal nonshivering thermogenesis in mice with inherited defects of fatty acid oxidation.(American Society for Clinical Investigation (ASCI), 1998-11-01) Guerra, C; Koza, R A; Walsh, K; Kurtz, D M; Wood, P A; Kozak, L P; United States Department of Health and Human ServicesWhen placed in the cold (4 degreesC), BALB/cByJ mice of both genders rapidly lose body temperature as compared with the control strain, C57BL/6J. This sensitivity to cold resembles that previously described for mice with a defect in nonshivering thermogenesis due to the targeted inactivation of the brown adipocyte-specific mitochondrial uncoupling protein gene, Ucp1. Genetic mapping of the trait placed the gene on chromosome 5 near Acads, a gene encoding the short chain acyl CoA dehydrogenase, which is mutated in BALB/cByJ mice. The analysis of candidate genes in the region indicated a defect only in the expression of Acads. Confirmation of the importance of fatty acid oxidation to thermogenesis came from our finding that mice carrying the targeted inactivation of the long chain acyl CoA dehydrogenase gene (Acadl) are also sensitive to the cold. Both of these mutations attenuate the induction of genes normally responsive to adrenergic signaling in brown adipocytes. These results suggest that the action of fatty acids as regulators of gene expression has been perturbed in the mutant mice. From a clinical perspective, it is important to determine whether defects in thermogenesis may be a phenotype in human neonates with inherited deficiencies in fatty acid beta-oxidation.Publication Activity of cefiderocol and innovative β-lactam/β-lactamase inhibitor combinations against isogenic strains of Escherichia coli expressing single and double β-lactamases under high and low permeability conditions(Elsevier, 2024-05) Blanco-Martín, Tania; Alonso-García, Isaac; González-Pinto, Lucía; Outeda-García, Michelle; Guijarro-Sánchez, Paula; López-Hernández, Inmaculada; Perez-Vazquez, Maria; Aracil, Belen; López-Cerero, Lorena; Fraile-Ribot, Pablo; Oliver, Antonio; Vázquez-Ucha, Juan Carlos; Beceiro, Alejandro; Bou, Germán; Arca-Suárez, Jorge; GEMARA/SEIMC-CIBERINFEC Study Group on the activity and resistance mechanisms to new β-lactams and β-lactamase inhibitors (PROTECT); Instituto de Salud Carlos III; Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Merck, Sharp & Dohme; Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas); RETICS-Investigación en Patología Infecciosa (REIPI-ISCIII) (España); Plan Nacional de I+D+i (España); Xunta de Galicia (España); Ministerio de Ciencia e Innovación (España)Objectives: To analyse the impact of the most clinically relevant β-lactamases and their interplay with low outer membrane permeability on the activity of cefiderocol, ceftazidime/avibactam, aztreonam/avibactam, cefepime/enmetazobactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/xeruborbactam and meropenem/nacubactam against recombinant Escherichia coli strains. Methods: We constructed 82 E. coli laboratory transformants expressing the main β-lactamases circulating in Enterobacterales (70 expressing single β-lactamase and 12 producing double carbapenemase) under high (E. coli TG1) and low (E. coli HB4) permeability conditions. Antimicrobial susceptibility testing was determined by reference broth microdilution. Results: Aztreonam/avibactam, cefepime/zidebactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam were active against all E. coli TG1 transformants. Imipenem/relebactam, meropenem/vaborbactam, cefepime/taniborbactam and cefepime/enmetazobactam were also highly active, but unstable against most of MBL-producing transformants. Combination of β-lactamases with porin deficiency (E. coli HB4) did not significantly affect the activity of aztreonam/avibactam, cefepime/zidebactam, cefiderocol or meropenem/nacubactam, but limited the effectiveness of the rest of carbapenem- and cefepime-based combinations. Double-carbapenemase production resulted in the loss of activity of most of the compounds tested, an effect particularly evident for those E. coli HB4 transformants in which MBLs were present. Conclusions: Our findings highlight the promising activity that cefiderocol and new β-lactam/β-lactamase inhibitors have against recombinant E. coli strains expressing widespread β-lactamases, including when these are combined with low permeability or other enzymes. Aztreonam/avibactam, cefiderocol, cefepime/zidebactam and meropenem/nacubactam will help to mitigate to some extent the urgency of new compounds able to resist MBL action, although NDM enzymes represent a growing challenge against which drug development efforts are still needed.Publication Affinity of cefotiam for the alternative penicillin binding protein PBP3SAL used by Salmonella inside host eukaryotic cells(Oxford University Press, 2023-02-01) Cestero, Juan J; Castanheira, Sónia; González, Henar; Zaragoza, Oscar; García-Del Portillo, Francisco; Ministerio de Ciencia e Innovación (España); Unión EuropeaBackground: Following the invasion of eukaryotic cells, Salmonella enterica serovar Typhimurium replaces PBP2/PBP3, main targets of β-lactam antibiotics, with PBP2SAL/PBP3SAL, two homologue peptidoglycan synthases absent in Escherichia coli. PBP3SAL promotes pathogen cell division in acidic environments independently of PBP3 and shows low affinity for β-lactams that bind to PBP3 such as aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefalotin. Objectives: To find compounds with high affinity for PBP3SAL to control Salmonella intracellular infections. Methods: An S. Typhimurium ΔPBP3 mutant that divides using PBP3SAL and its parental wild-type strain, were exposed to a library of 1520 approved drugs in acidified (pH 4.6) nutrient-rich LB medium. Changes in optical density associated with cell filamentation, a read-out of blockage in cell division, were monitored. Compounds causing filamentation in the ΔPBP3 mutant but not in wild-type strain-the latter strain expressing both PBP3 and PBP3SAL in LB pH 4.6-were selected for further study. The bactericidal effect due to PBP3SAL inhibition was evaluated in vitro using a bacterial infection model of cultured fibroblasts. Results: The cephalosporin cefotiam exhibited higher affinity for PBP3SAL than for PBP3 in bacteria growing in acidified LB pH 4.6 medium. Cefotiam also proved to be effective against intracellular Salmonella in a PBP3SAL-dependent manner. Conversely, cefuroxime, which has higher affinity for PBP3, showed decreased effectiveness in killing intracellular Salmonella. Conclusions: Antibiotics with affinity for PBP3SAL, like the cephalosporin cefotiam, have therapeutic value for treating Salmonella intracellular infections.Publication The antioxidant uncoupling protein 2 stimulates hnRNPA2/B1, GLUT1 and PKM2 expression and sensitizes pancreas cancer cells to glycolysis inhibition(Elsevier, 2016-12) Brandi, Jessica; Cecconi, Daniela; Cordani, Marco; Torrens-Mas, Margalida; Pacchiana, Raffaella; Dalla Pozza, Elisa; Butera, Giovanna; Manfredi, Marcello; Marengo, Emilio; Oliver, Jordi; Roca, Pilar; Dando, Ilaria; Donadelli, MassimoSeveral evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose. Through a bidimensional electrophoresis analysis, we identify 19 protein species differentially expressed after treatment with the UCP2 inhibitor genipin and, by bioinformatic analyses, we show that these proteins are mainly involved in metabolic processes. In particular, we demonstrate that the antioxidant UCP2 induces the expression of hnRNPA2/B1, which is involved in the regulation of both GLUT1 and PKM2 mRNAs, and of lactate dehydrogenase (LDH) increasing the secretion of L-lactic acid. We further demonstrate that the radical scavenger N-acetyl-L-cysteine reverts hnRNPA2/B1 and PKM2 inhibition by genipin indicating a role for reactive oxygen species in the metabolic reprogramming of cancer cells mediated by UCP2. We also observe an UCP2-dependent decrease in mtOXPHOS complex I (NADH dehydrogenase), complex IV (cytochrome c oxidase), complex V (ATPase) and in mitochondrial oxygen consumption, suggesting a role for UCP2 in the counteraction of pancreatic cancer cellular respiration. All these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation with the concomitant metabolic shift from mtOXPHOS to the glycolytic pathway.Publication Association between variation of circulating 25-OH vitamin D and methylation of secreted frizzled-related protein 2 in colorectal cancer.(2020-06-09) Boughanem, Hatim; Cabrera-Mulero, Amanda; Hernández-Alonso, Pablo; Clemente-Postigo, Mercedes; Casanueva, Felipe F; Tinahones, Francisco José; Morcillo, Sonsoles; Crujeiras, Ana B; Macias-Gonzalez, ManuelColorectal cancer (CRC) results from the accumulation of epigenetic and genetic changes in colon cells during neoplasic transformation, which the activation of Wingless (Wnt) signaling pathway is a common mechanism for CRC initiation. The Wnt pathway is mainly regulated by Wnt antagonists, as secreted frizzled-related protein (SFRP) family. Indeed, SFRP2 is proposed as a noninvasive biomarker for CRC diagnosis. Vitamin D also antagonizes Wnt signaling in colon cancers cells. Several studies showed that vitamin D was able to alter DNA methylation, although this mechanism is not yet clear. Therefore, the aim of this study was to find an association between circulating 25-OH vitamin D (30th percentile of vitamin D) and the SFRP2 methylation. A total of 67 CRC patients were included in the study. These patients were subdivided into two groups based on their 30th percentile vitamin D (20 patients were below, and 47 participants were above the 30th percentile of vitamin D). We investigated the SFRP2 methylation in peripheral blood mononuclear cells (PBMCs), visceral adipose tissue (VAT), CRC tumor tissue, and adjacent tumor-free area. We also determined the relationship between SFRP2 methylation and methylation of carcinogenic and adipogenic genes. Finally, we tested the effect of vitamin D on the SFRP2 methylation in human colorectal carcinoma cell lines 116 (HCT116) and studied the association of neoadjuvant therapy under the 30th percentile vitamin D with SFRP2 promoter methylation. SFRP2 methylation in tumor area was decreased in patients who had higher levels of vitamin D. SFRP2 promoter methylation was positively correlated in tumor area with insulin and homeostasis model assessment of insulin resistance (HOMA-IR) but negatively correlated with HDL-c. SFRP2 methylation was also correlated with T cell lymphoma invasion and metastasis 1 (TIAM1) methylation in tumor area and CCAAT/enhancer-binding protein alpha (C/EBPα) in VAT. Treatment with vitamin D did not affect SFRP2 methylation in HCT116 cell line. Finally, neoadjuvant treatment was correlated with higher circulating 25-OH vitamin D and SFRP2 methylation under linear regression model. Our results showed that higher circulating vitamin D is associated with low SFRP2 promoter methylation. Therefore, our results could suggest that vitamin D may have an epigenetic effect on DNA methylation. Finally, higher vitamin D could contribute to an improvement response to neoadjuvant treatment.Publication Biofilm formation avoids complement immunity and phagocytosis of Streptococcus pneumoniae(American Society for Microbiology (ASM), 2013-07) Domenech, Mirian; Ramos-Sevillano, Elisa; García, Ernesto; Moscoso, Miriam; Yuste, Jose Enrique; Ministerio de Economía y Competitividad (España); Centro de Investigación Biomedica en Red - CIBERStreptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.Publication Biofilm formation displays intrinsic offensive and defensive features of Bacillus cereus(Springer, 2020-01-15) Caro-Astorga, Joaquín; Frenzel, Elrike; Perkins, James R.; Álvarez-Mena, Ana; de Vicente, Antonio; Ranea, Juan A. G.; Kuipers, Oscar P.; Romero, Diego; [Caro-Astorga,J; Álvarez-Mena,A; de Vicente,A; Romero,D] Departamento de Microbiología, Universidad de Málaga, Málaga, Spain. [Frenzel,E; Kuipers,OP] Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands. [Perkins,JR] Research Laboratory, IBIMA, Regional University Hospital of Malaga-UMA, Málaga, Spain. [Perkins,JR; Ranea,JAG] CIBER de Enfermedades Raras (CIBERER), ISCIII Madrid, Spain. [Ranea,JAG] Department of Molecular Biology and Biochemistry, Universidad de Málaga, Málaga, Spain.Biofilm formation is a strategy of many bacterial species to adapt to a variety of stresses and has become a part of infections, contaminations, or beneficial interactions. In this study, we demonstrate that profound physiological changes permit Bacillus cereus to switch from a floating to a sessile lifestyle, to undergo further maturation of the biofilm and to differentiate into the offensive or defensive features. We report that floating and biofilm cells are populations that differentiate metabolically, with members of each subpopulation developing different branches of certain metabolic pathways. Secondly, biofilm populations rearrange nucleotides, sugars, amino acids, and energy metabolism. Thirdly, this metabolic rearrangement coexists with: the synthesis of the extracellular matrix, sporulation, reinforcement of the cell wall, activation of the ROS detoxification machinery and production of secondary metabolites. This strategy contributes to defend biofilm cells from competitors. However, floating cells maintain a fermentative metabolic status that ensures a higher aggressiveness against hosts, evidenced by the production of toxins. The maintenance of the two distinct subpopulations is an effective strategy to face different environmental conditions found in the life styles of B. cereus.Publication Carbapenemase-producing enterobacteriaceae in Spain in 2012(American Society for Microbiology (ASM), 2013-12) Oteo-Iglesias, Jesus; Saez, David; Bautista, Veronica; Fernandez-Romero, Sara; Hernández-Molina, Juan Manuel; Perez-Vazquez, Maria; Aracil, Belen; Campos, Jose; Instituto de Salud Carlos III; Ministerio de Economía y Competitividad (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); RETICS-Investigación en Patología Infecciosa (REIPI-ISCIII) (España)We report the epidemiological impact of carbapenemase-producing Enterobacteriaceae (CPE) in Spain in 2012. Of the 237 carbapenemases detected, 163 were from the OXA-48 group, 60 were from VIM-1, 8 were from KPC-2, 5 were from IMP, and 1 was from NDM-1. Interhospital spread of carbapenemase-producing Klebsiella pneumoniae was due to a limited number of multilocus sequence types (MLST) and carbapenemase types, including ST15-VIM-1, ST11-OXA-48, ST405-OXA-48, ST101-KPC-2, and ST11-VIM-1. The number of CPE cases in Spain has increased sharply in recent years, due mainly to the emergence of OXA-48.Publication Cardiac electrical defects in progeroid mice and Hutchinson-Gilford progeria syndrome patients with nuclear lamina alterations(National Academy of Sciences, 2016) Rivera-Torres, Jose; Calvo, Conrado J.; Llach, Anna; Guzman-Martinez, Gabriela; Caballero, Ricardo; Gonzalez-Gomez, Cristina; Jimenez-Borreguero, Luis J.; Guadix, Juan A; Osorio, Fernando G; López-Otín, Carlos; Herraiz-Martínez, Adela; Cabello, Nuria; Vallmitjana, Alex; Benítez, Raul; Gordon, Leslie B; Jalife, Jose; Pérez-Pomares, José M; Tamargo, Juan; Delpón, Eva; Hove-Madsen, Leif; Filgueiras-Rama, David; Andres, Vicente; Ministerio de Economía y Competitividad (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Instituto de Salud Carlos III; Unión Europea; Fundación Cajastur; Fundación ProCNICHutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the β-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.Publication Changes in fluoroquinolone-resistant Streptococcus pneumoniae after 7-valent conjugate vaccination, Spain(Centers for Disease Control and Prevention (CDC), 2009-06) de la Campa, Adela G; Ardanuy, Carmen; Balsalobre-Arenas, Maria Luz; Pérez-Trallero, Emilio; Marimón, Jose M; Fenoll, Asuncion; Liñares, Josefina; Ministerio de Ciencia e Innovación (España); Comunidad de Madrid (España)Among 4,215 Streptococcus pneumoniae isolates obtained in Spain during 2006, 98 (2.3%) were ciprofloxacin resistant (3.6% from adults and 0.14% from children). In comparison with findings from a 2002 study, global resistance remained stable. Low-level resistance (30 isolates with MIC 4-8 microg/mL) was caused by a reserpine-sensitive efflux phenotype (n = 4) or single topoisomerase IV (parC [n = 24] or parE [n = 1]) changes. One isolate did not show reserpine-sensitive efflux or mutations. High-level resistance (68 isolates with MIC >or=16 microg/mL) was caused by changes in gyrase (gyrA) and parC or parE. New changes in parC (S80P) and gyrA (S81V, E85G) were shown to be involved in resistance by genetic transformation. Although 49 genotypes were observed, clones Spain9V-ST156 and Sweden15A-ST63 accounted for 34.7% of drug-resistant isolates. In comparison with findings from the 2002 study, clones Spain14-ST17, Spain23F-ST81, and ST8819F decreased and 4 new genotypes (ST9710A, ST57016, ST43322, and ST71733) appeared in 2006.Publication Changes in Group A Streptococcus emm Types Associated with Invasive Infections in Adults, Spain, 2023(Centers for Disease Control and Prevention (CDC), 2023-11) Bellés-Bellés, Alba; Prim, Núria; Mormeneo-Bayo, Saray; Villalon-Panzano, Pilar; Valiente, Mónica; Jover-Sáenz, Alfredo; Aixalà, Núria; Bernet, Albert; López-González, Éric; Prats, Ivan; García-González, MercèAn increase in invasive group A Streptococcus infection was detected in the northeast of Spain in November 2022. A postpandemic decline in the diversity of circulating emm types involved in invasive group A Streptococcus was observed, along with the emergence of emm49 in this geographic area.Publication Characterization of Carbapenemase-Producing Klebsiella oxytoca in Spain, 2016-2017.(American Society for Microbiology (ASM), 2019) Perez-Vazquez, Maria; Oteo-Iglesias, Jesus; Sola-Campoy, Pedro Juan; Carrizo-Manzoni, Hugo; Bautista, Veronica; Lara Fuella, Noelia; Aracil, Belen; Alhambra, Almudena; Martínez-Martínez, Luis; Campos, Jose; Instituto de Salud Carlos III; RETICS-Investigación en Patología Infecciosa (REIPI-ISCIII) (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)There is little information about carbapenemase-producing (CP) Klebsiella oxytoca, an important nosocomial pathogen. We characterized CP K. oxytoca isolates collected from different Spanish hospitals between January 2016 and October 2017. During the study period, 139 nonduplicate CP K. oxytoca isolates were identified; of these, 80 were studied in detail. Carbapenemase and extended-spectrum β-lactamase genes were identified by PCR and sequencing. Genetic relatedness was studied by pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS), carried out on 12 representative isolates, was used to identify the resistome, to elucidate the phylogeny, and to determine the plasmids harboring carbapenemase genes. Forty-eight (60%) isolates produced VIM-1, 30 (37.5%) produced OXA-48, 3 (3.7%) produced KPC-2, 2 (2.5%) produced KPC-3, and 1 (1.2%) produced NDM-1; 4 isolates coproduced two carbapenemases. By PFGE, 69 patterns were obtained from the 80 CP K. oxytoca isolates, and four well-defined clusters were detected: cluster 1 consisted of 11 OXA-48-producing isolates, and the other three clusters included VIM-1-producing isolates (5, 3, and 3 isolates, respectively). In the 12 sequenced isolates, the average number of acquired resistance genes was significantly higher in VIM-1-producing isolates (10.8) than in OXA-48-producing isolates (2.3). All 12 isolates had chromosomally encoded genes of the blaOXY-2 genotype, and by multilocus sequence typing, most belonged to sequence type 2 (ST2). Carbapenemase genes were carried by IncL, IncHI2, IncFII, IncN, IncC, and IncP6 plasmid types. The emergence of CP K. oxytoca was principally due to the spread of VIM-1- and OXA-48-producing isolates in which VIM-1- and OXA-48 were carried by IncL, IncHI2, IncFII, and IncN plasmids. ST2 and the genotype blaOXY-2 predominated among the 12 sequenced isolates.Publication Clinical Isolates of the Spain14-5 Clone of Streptococcus pneumoniae Carry a Recombinant rpoB Gene(American Society for Microbiology (ASM), 2005-11) Ferrandiz-Avellano, Maria-Jose; Perez-Trallero, E.; Marimon, J. M.; de la Campa, Adela GPublication Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor(BioMed Central (BMC), 2014-05-29) Lery, Leticia M. S.; Frangeul, Lionel; Tomas, Anna; Passet, Virginie; Almeida, Ana S.; Bialek-Davenet, Suzanne; Barbe, Valerie; Bengoechea, Jose Antonio; Sansonetti, Philippe; Brisse, Sylvain; Tournebize, RegisBackground: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044. Results: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism. Conclusions: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae.Publication Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.(National Academy of Sciences, 2001-03-13) Walev, I; Bhakdi, S C; Hofmann, F; Djonder, N; Valeva, A; Aktories, K; Bhakdi, SThe pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 10(5)-10(6) molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca(2+)-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.Publication DiiA is a novel dimorphic cell wall protein of Streptococcus pneumoniae involved in invasive disease(Elsevier, 2016-07) Escolano-Martinez, Maria S; Domenech, Arnau; Yuste, Jose Enrique; Cercenado, Maria I; Ardanuy, Carmen; Liñares, Josefina; de la Campa, Adela G; Martin-Galiano, Antonio Javier; Ministerio de Economía y Competitividad (España); Plan Nacional de I+D+i (España); Instituto de Salud Carlos III; Ministerio de Educación (España)Objectives: Many outer multidomain proteins play fundamental virulent roles in an allele-dependent manner. We aimed to investigate the influence of the outer SP1992 protein, here renamed DiiA (Dimorphic invasion-involved A), in pneumococcal disease. Methods: The presence and type of diiA allele was screened by PCR in 560 clinical isolates. Isogenic mutants carrying progressive diiA deletions were constructed and checked in mouse models of infection. DiiA binding to human molecules was carried out by surface plasmon resonance. Results: The diiA gene is exclusive of Streptococcus pneumoniae and included in the core genome. DiiA variants contain one or two imperfect repeats (R1 and R2), an unstructured region and a cell-wall anchor domain. Clonal complexes carrying both repeats were associated with invasive disease, while those carrying R2 preferentially caused non-invasive syndromes in patients with underlying risk factors. Mutants lacking both repeats were less efficient in nasopharyngeal colonization and dissemination from lungs. Moreover, the ΔdiiA defective strain suffered a severe impairment in bacterial proliferation in blood. Purified DiiA bound to collagen and lactoferrin with high affinity. Conclusions: DiiA is a distinctive pneumococcal virulence factor contributing to colonization and long-term invasion in this pathogen.Publication Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2(Multidisciplinary Digital Publishing Institute (MDPI), 2019-10-23) Godoy, César A; Klett, Javier; Di Geronimo, Bruno; Hermoso, Juan A; Guisán, José M; Carrasco-López, César; Consejo Interinstitucional de Ciencia y Tecnologia (Argentina); Ministerio de Economía y Competitividad (España)Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg-1) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg-1), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.