Papers by Juan Carlos Aragon Tomas
Infection and Immunity, 1994
The resistance of Aeromonas salmonicida to complement-mediated killing was investigated by using ... more The resistance of Aeromonas salmonicida to complement-mediated killing was investigated by using different strains and their isogenic mutants that had been previously characterized for their surface components. We found that the classical complement pathway is involved in serum killing of susceptible A. salmonicida strains, while the alternative complement pathway seems not to be involved. All of the A. salmonicida strains are able to activate complement, but the smooth strains (with or without the A-layer) are resistant to complement-mediated killing. The reasons for this resistance are that C3b may be bound far from the cell membrane and that it is rapidly degraded; therefore, the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants are serum sensitive because they bind more C3b than the smooth strains, and if C3b is not completely degraded, then the lytic complex (C5b-9) is formed.
Infection and Immunity, 1999
Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found ... more Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5α. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 ( pla ) and C ( plc ) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggestin...
Journal of Microbiological Methods, 1989
Infection
The capsular polysaccharide (CPS) ofKlebsiella pneumoniae is an important virulence factor. Salic... more The capsular polysaccharide (CPS) ofKlebsiella pneumoniae is an important virulence factor. Salicylate, which inhibits CPS production, was used to expose subcapsular antigens and components that may play an important role in host defense. Salicylate treatment greatly increased phagocytosis of five O1 serotypes by human polymorphonuclear leukocytes with normal rabbit serum and rabbit antisera against purified O1 lipopolysaccharide (O1LPS) as opsonins (p<0.01 or <0.05). Similar results were obtained with rabbit antiserum against a non-encapsulated isogenic strain. To further determine how salicylate increases susceptibility to phagocytosis, the binding of monoclonal antibodies against O1LPS or the LPS core and the binding of complement component C3b were measured by ELISA. The data indicate that salicylate reduced the barrier of CPS in serotypes O1:K1, O1:K10, and O1:K16 and unmasked subcapsular antigenic components in serotypes O1:K2 and O1:K66 so that bound opsonins could react with receptors on phagocytes. Serum bactericidal assays supported this conclusion. Therefore, decapsulating agents such as salicylate accentuate phagocytosis ofK. pneumoniae by making subcapsular antigens and components accessible to immune and nonimmune host defences and vaccination with subcapsular antigens may exhibit optimal protection against lethal infection when combined with salicylate therapy. Kapselpolysaccharid (CPS) vonKlebsiella pneumoniae ist ein bedeutender Virulenzfaktor. Salicylat hemmt dic CPS-Bildung. Es wurde verwendet, um eine stärkere Exposition subkapsulärer Antigene und Komponenten zu bewirken, die eine große Rolle in der Wirtsabwehr spielen dürften. Salicylat-Behandlung führte zu einer ausgeprägten Steigerung der Phagozytose von fünf O1-Serotypen durch menschliche polymorphkernige Leukozyten, wenn normales Kaninchenserum und Kaninchenserum gegen gereinigte O1 Lipopolysaccharide (O1LPS) als Opsonine verwendet wurden (p<0,01 oder <0,05). Ähnliche Beobachtungen wurden mit Kaninchenantiserum gegen einen nicht-kapseltragenden isogenen Stamm gemacht. Um weiter zu erforschen, wie Salicylat die Empfindlichkeit gegenüber Phagozytose steigert, wurde die Bildung monokonaler Antikörper gegen O1LPS oder das Core LPS und die Bindung der Komplement-Komponente C3b mittels ELISA ermittelt. Die Daten weisen darauf hin, daß Salicylat bei den Serotypen O1:K1, O1:K10 und O1:K16 die Schrankenfunktion des CPS reduziert und bei Serotypen O1:K2 und O1:K66 die subkapsulären Antigenkomponenten demaskiert, so daß gebundene Opsonine mit Rezeptoren auf den Phagozyten reagieren können. Die Behandlung mit Substanzen, die zu einer Dekapsulierung führen, wie Salicylat, verstärkt somit die Phagozytose vonK. pneumoniae, indem sie die subkapsulären Antigene und Komponenten den immunologischen und nicht immunologischen Abwehrmechanismen des Wirts zugänglicher macht. Eine Impfung mit subkapsulären Antigenen könnte einen optimalen Schutz gegen eine tödliche Infektion bewirken, wenn sie mit einer Salicylat-Therapie kombiniert wird.
Medicina cutánea ibero-latino-americana, 1976
After a review of the bibliography on the subject of eccrine sweat gland carcinomas, the authors ... more After a review of the bibliography on the subject of eccrine sweat gland carcinomas, the authors emphasize the confusing terminology used for the designation of these cases and the difficulties for a correct clinical and histological diagnosis of these tumors. According to the data obtained from the study of 7 personal cases, the most characteristic features of the eccrine carcinomas could be the following: 1) From the clinical standpoint--Appearance of a single tumour, lasting unmodified for a long period of time.--Tendency to reccurrence of the neighbouring areas after tumour excision, and to a slow progression through the superficial lymphatic channels.--Appearance of distant metastasis a long time after the original lesion. These metastases are observed, a) on the regional lymph nodes, b) on the superficial lymphatic channels and c) in some cases in the skin by intraepidermal growth. 2) From the histological point of view--Localisation in the deep dermis of the tumoral masses in...
MicrobiologyOpen, 2012
Motility in Aeromonas caviae, in a liquid environment (in broth culture), is mediated by a single... more Motility in Aeromonas caviae, in a liquid environment (in broth culture), is mediated by a single polar flagellum encoded by the fla genes. The polar flagellum filament of A. caviae is composed of two flagellin subunits, FlaA and FlaB, which undergo O-linked glycosylation with six to eight pseudaminic acid glycans linked to serine and threonine residues in their central region. The flm genetic locus in A. caviae is required for flagellin glycosylation and the addition of pseudaminic acid (Pse) onto the lipopolysaccharide (LPS) O-antigen. However, none of the flm genes appear to encode a candidate glycotransferase that might add the Pse moiety to FlaA/B. The motility-associated factors (Maf proteins) are considered as candidate transferase enzymes, largely due to their conserved proximity to flagellar biosynthesis loci in a number of pathogens. Bioinformatic analysis performed in this study indicated that the genome of A. caviae encodes a single maf gene homologue (maf1). A maf mutant was generated and phenotypic analysis showed it is both nonmotile and lacks polar flagella. In contrast to flm mutants, it had no effect on the LPS O-antigen pattern and has the ability to swarm. Analysis of flaA transcription by reverse transcriptase PCR (RT-PCR) showed that its transcription was unaltered in the maf mutant while a His-tagged version of the FlaA flagellin protein produced from a plasmid was detected in an unglycosylated intracellular form in the maf strain. Complementation of the maf strain in trans partially restored motility, but increased levels of glycosylated flagellin to above wild-type levels. Overexpression of maf inhibited motility, indicating a dominant negative effect, possibly caused by high amounts of glycosylated flagellin inhibiting assembly of the flagellum. These data provide evidence that maf1, a pseudaminyl transferase, is responsible for glycosylation of flagellin and suggest that this event occurs prior to secretion through the flagellar Type III secretion system.
eLS, 2010
The bacterial surface contains various structures that are capable of activating the host defence... more The bacterial surface contains various structures that are capable of activating the host defences and consequently inducing host immune responses. Bacterial capsules are one of the most external structures on the bacterial surface, which may completely surround all the antigenic molecules or may be coexpressed with other bacterial antigens. They are involved in a wide range of biological processes, such as prevention of desiccation, adherence and resistance to nonspecific and specific host immunity. Gram-negative and Gram-positive capsular polysaccharides contribute to the bacterial resistance of host immune responses by different mechanisms. Usually, capsular polysaccharides that mask the underlying cell surface structures activate weakly or not at all the immune system, whereas bacterial capsules coexpressed with other bacterial antigens activate the immune system but mask opsonines and prevent complement attack complex formation, as well as phagocytosis. Key Concepts Bacterial capsules completely surround bacterial cell and enhance the ability of bacteria to cause disease. Polysaccharides are polymeric carbohydrate structures, formed of one or more sugar residues repeating units joined together by glycosidic bonds. Antigenic variation modifies microorganism's surface structures and allows them to overcome the immune response. Phagocytosis is the process by which certain cells called phagocytes engulf and digest a solid particle as bacteria. The complement system is a part of the innate immune system and consists of a number of plasma small proteins which circulating as inactive precursors and after their activation cascade are able to serve as opsonins. Keywords: bacterial capsules; polysaccharides; antigenic variation; avoidance of phagocytosis; complement resistance
eLS, 2010
The immune system controls the host–microbe interactions, the outcome of which can range from sym... more The immune system controls the host–microbe interactions, the outcome of which can range from symbiotic coexistence with commensally microbiota, to mild asymptomatic infections, to highly virulent infectious diseases. This control is achieved by two defence mechanisms: the innate immune defence, which consists in a nonspecific mechanism, and the adaptive immunity defence, acquired over time following infections or vaccination. Despite the sophisticated immune system, extracellular and intracellular pathogens have developed numerous, and often ingenious strategies, to evade, interfere or eradicate the effectiveness of host immune defences. Although the strategies used by viral and bacterial pathogens are numerous, there are several general mechanisms shared between these microbial pathogens. The success of each pathogen depends on the coordinated activities of its virulence factors to overcome host barriers to colonisation and its ability to mount an effective anti-immune response within the infected host, which can ultimately result in acute disease or chronic infection. Key Concepts: Immunoglobulin proteases cleave antibodies rendering them nonfunctional and leading to deficiencies in the immune system. Complement regulators prevent complement activation and cell destruction. Interference with major histocompatibility complexes overcomes T-cell recognition. Microbial interference with cytokines modulates intracellular signalling critical to the regulation, proliferation and differentiation of lymphocytes, which drive inflammation. Intracellular resistance allows microorganisms to circumvent humoral defence mechanisms and spread within the host. Immunosuppresion reduces T or B lymphocytes’ defence. Keywords: destruction of immune molecules; cytokine antagonists; intracellular resistance; destruction of immune cells
Reviews in Medical Microbiology, 1998
Research in Microbiology, 1997
The role of the capsular polysaccharide of Aeromonas hydrophila serogroup 0:34 in the adherence t... more The role of the capsular polysaccharide of Aeromonas hydrophila serogroup 0:34 in the adherence to and invasion of fish cell lines S. Merino (l), A. Aguilar cl), X. Rubires (I), N. Abitiu c2), M. Regu6 c2) and J.M. Tom& (I) (*)
PLoS ONE, 2014
Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-... more Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous glycan. Mutants unable to produce WecP or Gne enzymes showed altered motility, and the study of their polar flagellin glycosylation showed that the patterns of glycosylation differed from that observed with wild type polar flagellin. This suggested the involvement of a lipid carrier in glycosylation. A gene coding for an enzyme linking sugar to a lipid carrier was identified in strain AH-3 (WecX) and subsequent mutation abolished completely motility, flagella production by EM, and flagellin glycosylation. This is the first report of a lipid carrier involved in flagella O-glycosylation. A molecular model has been proposed. The results obtained suggested that the N-acetylhexosamines are Nacetylgalactosamines and that the heptasaccharide is completely independent of the O34-antigen lipopolysaccharide. Furthermore, by comparing the mutants with differing degrees of polar flagellin glycosylation, we established their importance in A. hydrophila flagella formation and motility.
PLoS ONE, 2012
We previously reported that A. hydrophila GalU mutants were still able to produce UDP-glucose int... more We previously reported that A. hydrophila GalU mutants were still able to produce UDP-glucose introduced as a glucose residue in their lipopolysaccharide core. In this study, we found the unique origin of this UDP-glucose from a branched aglucan surface polysaccharide. This glucan, surface attached through the O-antigen ligase (WaaL), is common to the mesophilic Aeromonas strains tested. The Aeromonas glucan is produced by the action of the glycogen synthase (GlgA) and the UDP-Glc pyrophosphorylase (GlgC), the latter wrongly indicated as an ADP-Glc pyrophosphorylase in the Aeromonas genomes available. The Aeromonas glycogen synthase is able to react with UDP or ADP-glucose, which is not the case of E. coli glycogen synthase only reacting with ADP-glucose. The Aeromonas surface glucan has a role enhancing biofilm formation. Finally, for the first time to our knowledge, a clear preference on behalf of bacterial survival and pathogenesis is observed when choosing to produce one or other surface saccharide molecules to produce (lipopolysaccharide core or glucan).
Microbial Pathogenesis, 1997
... Susana Merino, Xavier Rubires, Alicia Aguilar and Juan M. Tomás f1. ... 527556. View Record ... more ... Susana Merino, Xavier Rubires, Alicia Aguilar and Juan M. Tomás f1. ... 527556. View Record in Scopus | Cited By in Scopus (88). 9. JM, Tomás, S, Camprubí, S, Merino, MR, Davey and P. Williams, Surface exposure of O1 serotype lipopolysaccharide inKlebsiella pneumoniae. ...
Microbial Pathogenesis, 2005
A 40 kDa C1q-binding outer membrane protein from Aeromonas salmonicida was identified by direct-b... more A 40 kDa C1q-binding outer membrane protein from Aeromonas salmonicida was identified by direct-binding assay with biotinylated C1q, a subcomponent of the complement classical pathway component C1. The 40 kDa porin structural gene from the A450 A. salmonicida typical strain (A+:O+) was cloned in Escherichia coli and sequenced. The amino acid sequence of the 40 kDa A. salmonicida porin, its ability to bind C1q in an antibody independent process, and its immunological cross-reaction with the A. hydrophila AH-3 porin II, allow us to determine the role of this protein in serum susceptibility. Furthermore, we obtained defined A. salmonicida 40 kDa porin insertion mutants in serum sensitive or resistant strains, and we complemented these mutants with a plasmid harboring only the 40 kDa porin gene from A. salmonicida A450 in order to define its role as an important surface molecule involved in serum susceptibility and C1q binding. Similar complementation was obtained using the A. hydrophila AH-3 porin II gene. The 40 kDa porin gene and/or protein was present in all the A. salmonicida typical or atypical strains tested. Furthermore, the A. hydrophila AH-3 porin II seems to be an important molecule for fish immunoprotection against either A. salmonicida or A. hydrophila strains.
Journal of Biological Chemistry, 2010
The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae... more The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (␣HexN-(1,4)-␣GalA) attached by an ␣1,3 linkage to L-glycero-D-manno-heptopyranose II (L-glycero-␣-D-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is D-glucosamine, whereas in other P. mirabilis strains, it corresponds to D-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of D-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of D-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase. * This work was supported by Plan Nacional de I ϩ D ϩ i and Fondo de Investigaciones Sanitarias grants (Ministerio de Educació n, Ciencia y Deporte, and Ministerio de Sanidad, Spain) and from Generalitat de Catalunya (Centre de Referè ncia en Biotecnologia). □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) HQ259030 and HQ259030. 1 Recipient of a predoctoral fellowship from Generalitat de Catalunya.
Journal of Bacteriology, 2004
The gene cluster ( waa ) involved in Serratia marcescens N28b core lipopolysaccharide (LPS) biosy... more The gene cluster ( waa ) involved in Serratia marcescens N28b core lipopolysaccharide (LPS) biosynthesis was identified, cloned, and sequenced. Complementation analysis of known waa mutants from Escherichia coli K-12, Salmonella enterica , and Klebsiella pneumoniae led to the identification of five genes coding for products involved in the biosynthesis of a shared inner core structure: [ l , d -Hep p IIIα(1→7)- l , d -Hep p IIα(1→3)- l,d -Hep p Iα(1→5)-Kdo p I(4←2)αKdo p II] ( l,d -Hep p , l - glycero - d - manno -heptopyranose; Kdo, 3-deoxy- d - manno -oct-2-ulosonic acid). Complementation and/or chemical analysis of several nonpolar mutants within the S. marcescens waa gene cluster suggested that in addition, three waa genes were shared by S. marcescens and K. pneumoniae , indicating that the core region of the LPS of S. marcescens and K. pneumoniae possesses additional common features. Chemical and structural analysis of the major oligosaccharide from the core region of LPS of an...
Journal of Bacteriology, 2003
To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LP... more To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae , we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment toα - l -glycero- d -manno-heptopyranose II ( l , d -Hep p II) at the O-3 position of anα - d -galactopyranosyluronic acid (α- d -GalA p ) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC , waaF , and wabG mutants were avirulent when tested in differen...
Journal of Bacteriology, 2012
The presence of cell-bound K1 capsule and K1 polysaccharide in culture supernatants was determine... more The presence of cell-bound K1 capsule and K1 polysaccharide in culture supernatants was determined in a series of in-frame nonpolar core biosynthetic mutants from Escherichia coli KT1094 (K1, R1 core lipopolysaccharide [LPS] type) for which the major core oligosaccharide structures were determined. Cell-bound K1 capsule was absent from mutants devoid of phosphoryl modifications on l - glycero - d - manno -heptose residues (HepI and HepII) of the inner-core LPS and reduced in mutants devoid of phosphoryl modification on HepII or devoid of HepIII. In contrast, in all of the mutants, K1 polysaccharide was found in culture supernatants. These results were confirmed by using a mutant with a deletion spanning from the hldD to waaQ genes of the waa gene cluster to which individual genes were reintroduced. A nuclear magnetic resonance (NMR) analysis of core LPS from HepIII-deficient mutants showed an alteration in the pattern of phosphoryl modifications. A cell extract containing both K1 ca...
International Journal of Antimicrobial Agents, 1993
Infection and Immunity, 2001
Aeromonas caviae is increasingly being recognized as a cause of gastroenteritis, especially among... more Aeromonas caviae is increasingly being recognized as a cause of gastroenteritis, especially among the young. The adherence of aeromonads to human epithelial cells in vitro has been correlated with enteropathogenicity, but the mechanism is far from well understood. Initial investigations demonstrated that adherence of A. caviae to HEp-2 cells was significantly reduced by either pretreating bacterial cells with an antipolar flagellin antibody or by pretreating HEp-2 cells with partially purified flagella. To precisely define the role of the polar flagellum in aeromonad adherence, we isolated the A. caviae polar flagellin locus and identified five polar flagellar genes, in the order flaA, flaB, flaG, flaH , and flaJ . Each gene was inactivated using a kanamycin resistance cartridge that ensures the transcription of downstream genes, and the resulting mutants were tested for motility, flagellin expression, and adherence to HEp-2 cells. N-terminal amino acid sequencing, mutant analysis, ...
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Papers by Juan Carlos Aragon Tomas