JOAQUIN IBAR SANCHEZ - Academia.edu (original) (raw)

Papers by JOAQUIN IBAR SANCHEZ

Current opinion in investigational drugs (London, England : 2000), 2003

A large proportion of pathogens either invade through, or cause disease at mucosal surfaces. Many... more A large proportion of pathogens either invade through, or cause disease at mucosal surfaces. Many new generation mucosal vaccine candidates lack important immunostimulatory features of the original pathogens and thus often do not elicit sufficiently strong immune responses. Despite numerous efforts, there is a profound lack of available agents with mucosal immunomodulatory and adjuvant activity. Immunomodulatory adjuvants are often derived from pathogens and thus efficiently activate the innate immune system leading to subsequent development of strong, specific acquired immunity. In this review, recent advances in mucosal immunomodulators/adjuvants are described with special emphasis on recently developed detoxified cholera toxin and Escherichia coli heat labile enterotoxin derivatives, and the newly described Toll-like receptor ligands CpG DNA and imidazoquinoline compounds. These agents hold much promise as useful mucosal immunomodulators/adjuvants for induction of strong innate i...

The Indian journal of medical research, 2011

After De΄s pivotal demonstration in 1959 of a diarrhoeogenic exo-enterotoxin in cell-free culture... more After De΄s pivotal demonstration in 1959 of a diarrhoeogenic exo-enterotoxin in cell-free culture filtrates from Vibrio cholerae (of classical biotype), much insight has been gained about cholera toxin (CT), which is arguably now the best known of all microbial toxins. The subunit structure and function of CT, its receptor (the GM1 ganglioside), and its effects on the cyclic AMP system and on intestinal secretion were defined in the 1970s, and the essential aspects of the genetic organization in the 1980s. Recent findings have generated additional perspectives. The 3D-crystal structure of CT has been established, the CT-encoding operon has been shown to be carried by a non-lytic bacteriophage, and in depth knowledge has been gained on how the bacterium controls CT gene expression in response to cell density and various environmental signals. The mode of entry into target cells and the intracellular transport of CT are becoming clearer. CT has become the prototype enterotoxin and a w...

Progress in Vaccinology, 1989

Enteric infection with enterotoxinogenic Escherichia coli (ETEC) is one of the most important cau... more Enteric infection with enterotoxinogenic Escherichia coli (ETEC) is one of the most important causes of diarrhea in developing countries and is also the most frequent cause of diarrhea among travelers. Illness resulting from ETEC infection is characterized by watery diarrhea, often accompanied by low grade fever, abdominal cramps, malaise, and vomiting. In its most severe form ETEC infection may result in cholera-like disease leading to severe dehydration and sometimes death.

The Scientific World Journal, 2014

We applied the so-called chemical kinetics approach to complex bacterial growth patterns that wer... more We applied the so-called chemical kinetics approach to complex bacterial growth patterns that were dependent on the liquid-surface-area-to-volume ratio (SA/V) of the bacterial cultures. The kinetic modeling was based on current experimental knowledge in terms of autocatalytic bacterial growth, its inhibition by the metabolite CO2, and the relief of inhibition through the physical escape of the inhibitor. The model quantitatively reproduces kinetic data of SA/V-dependent bacterial growth and can discriminate between differences in the growth dynamics of enteropathogenicE. coli,E. coli JM83, andSalmonella typhimuriumon one hand andVibrio choleraeon the other hand. Furthermore, the data fitting procedures allowed predictions about the velocities of the involved key processes and the potential behavior in an open-flow bacterial chemostat, revealing an oscillatory approach to the stationary states.

Proceedings of the National Academy of Sciences, 1984

A genetically engineered Vibrio cholerae strain from which the cholera toxin genes had previously... more A genetically engineered Vibrio cholerae strain from which the cholera toxin genes had previously been deleted was used as a host in which to study the expression and secretion of related toxins and their subunits. Recombinant plasmids encoding heat-labile enterotoxins (LTs) from Escherichia coli of human and porcine origin were expressed in the V. cholerae host, and this resulted in the secretion of the LTs into the extracellular milieu. The secreted LTs were isolated and it was found that the A subunits of human and porcine LT were "unnicked" polypeptides, which indicates that nicking is not obligatory for toxin secretion. V. cholerae strains were also constructed that harbored plasmids encoding either the A or the B subunits of human LT (A+B-, or A-B+). Approximately 90% of the B subunits were secreted from the A-B+ strain, while all of the A subunits expressed by the A+B- strain remained cell associated. This implies that strains synthesizing both subunits assemble the...

Proceedings of the National Academy of Sciences, 1988

In this paper we study the assembly, in vivo and in vitro, of a family of hexameric, heat-labile ... more In this paper we study the assembly, in vivo and in vitro, of a family of hexameric, heat-labile enterotoxins produced by diarrheagenic bacteria. The toxins, which consist of an A subunit and five B subunits, are assembled by a highly coordinated process that ensures secretion of the holotoxin complex. We show that (i) oxidation of cysteine residues in the B subunits is a prerequisite step for in vivo formation of B-subunit pentamers, (ii) reduction of dissociated B subunits in vitro abolishes their ability to reassemble, (iii) the kinetics of B-pentamer assembly in vivo can be mimicked under defined conditions in vitro, (iv) A subunits cannot associate with fully assembled B pentamers in vitro, and (v) A subunits cause an approximately 3-fold acceleration in the rate of B-subunit pentamerization in vivo, implying that A subunits play a coordinating role in the pathway of holotoxin assembly. The last finding is likely to be of general significance, since it provides a mechanism for ...

Microbiology, 1989

The expression of two cell-bound haemagglutinins, one sensitive to L-fucose (FSHA) and the other ... more The expression of two cell-bound haemagglutinins, one sensitive to L-fucose (FSHA) and the other to D-mannOSe (MSHA), on Vibrio cholerae 0 1 strains of both the classical and the El Tor biotypes was studied by (i) agglutination of chicken and human group 0 erythrocytes in the presence of L-fucose or D-mannose, (ii) binding of the bacteria to L-fucose-and D-mannOSecoated agarose beads, and (iii) agglutination of the bacteria by 'biotype-specific' antisera. All of the 12 classical strains studied that were isolated before 1979 gave FSHA of human 0 erythrocytes whereas only 6 of 17 classical strains isolated during recent epidemics expressed FSHA; a few of the classical strains expressed MSHA in addition to FSHA. All the El Tor strains gave MSHA of chicken erythrocytes and one strain also expressed FSHA. Both the cellbound HAS were optimally expressed during the exponential phase of growth; FSHA markedly decreased during the late exponential phase while the MSHA usually persisted into the stationary phase. The expression of FSHA and MSHA correlated very well with the direct binding of vi brios to fucose-and mannose-coated agarose beads, respectively. Antiserum 'specific' for classical vibrios agglutinated classical strains expressing FSHA and also the El Tor strain exhibiting FSHA. Similarly, the anti-El Tor serum agglutinated all El Tor strains and also classical strains expressing MSHA, suggesting that the 'biotype-specific' sera were specific for the biotype-associated cell-bound HAS.

Microbial Pathogenesis, 2014

Vibrio cholerae O1 classical, El Tor and O139 are the primary biotypes that cause epidemic choler... more Vibrio cholerae O1 classical, El Tor and O139 are the primary biotypes that cause epidemic cholera, and they also express cholera toxin (CT). Although classical V. cholerae produces CT in various settings, the El Tor and O139 strains require specific growth conditions for CT induction, such as the so-called AKI conditions, which consist of growth in static conditions followed by growth under aerobic shaking conditions. However, our group has demonstrated that CT production may also take place in shallow static cultures. How these type of cultures induce CT production has been unclear, but we now report that in shallow culture growth conditions, there is virtual depletion of dissolved oxygen after 2.5 h of growth. Concurrently, during the first three to 4 h, endogenous CO 2 accumulates in the media and the pH decreases. These findings may explain CT expression at the molecular level because CT production relies on a regulatory cascade, in which the key regulator AphB may be activated by anaerobiosis and by low pH. AphB activation stimulates TcpP synthesis, which induces ToxT production, and ToxT directly stimulates ctxAB expression, which encodes CT. Importantly, ToxT activity is enhanced by bicarbonate. Therefore, we suggest that in shallow cultures, AphB is activated by initial decreases in oxygen and pH, and subsequently, ToxT is activated by intracellular bicarbonate that has been generated from endogenous CO 2. This working model would explain CT production in shallow cultures and, possibly, also in other growth conditions.

Microbial Pathogenesis, 2004

Up to 28-fold differences in vacA expression in Helicobacter pylori strains grown in vitro were d... more Up to 28-fold differences in vacA expression in Helicobacter pylori strains grown in vitro were demonstrated by real time quantitative RT-PCR. These large differences in expression were unrelated to putative -35 and -10 motifs or to other untranslated sequences upstream of the ATG start site. The lack of correlation between promoter sequences and the vacA expression levels suggest the potential existence of a bacterial strain-specific factor, as earlier proposed by others on the basis of reporter gene fusions.

Journal of Bacteriology, 2004

The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in t... more The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.

Gene, 1993

A highly conserved neutralizing epitope from the surface protein VP4 (amino acids 296-313) of hum... more A highly conserved neutralizing epitope from the surface protein VP4 (amino acids 296-313) of human rotaviruses was genetically fused to the B subunit of cholera toxin (CTB). Synthetic oligodeoxyribonucleotides encoding the VP4 peptide were inserted between the 3&amp;#39; end of the DNA that codes for the leader peptide, and the 5&amp;#39; end of the gene encoding mature CTB. The hybrid protein synthesized in Escherichia coli was found to maintain the ability of CTB to pentamerize, and to adhere to its cell receptor, the GM1 ganglioside. The chimera was efficiently recognized by a monoclonal antibody (mAb) directed at CTB and by a virus-neutralizing mAb against the VP4 peptide. The hybrid polypeptide was shown to induce high titers of serum antibodies (Ab) against CTB and the synthetic VP4 peptide following subcutaneous immunization; paradoxically, however, the Ab obtained did not recognize the virus by an enzyme-linked immunosorbent assay method, nor had detectable neutralizing activity. Potential implications of these results for future design and evaluation of fusion proteins as immunogens are discussed.

Gene, 1991

A simple and effective electroporation method for the transformation of Vibrio cholerae with nonm... more A simple and effective electroporation method for the transformation of Vibrio cholerae with nonmobilizable plasmids is described. Expression plasmids directing the synthesis of fusion proteins with the subunit B of Escherichiu coli heat-labile enterotoxin B (LT-B) were transformed into nontoxinogenic V. cholerue vaccine strains. A protein consisting of two overlapping immunodominant antibody-binding sites of the hepatitis B virus (HBV) middle surface antigen fused to the C terminus of full-length LT-B was secreted into the supematant of I/. cholerae cultures, whereas two other LT-B/HBV fusion proteins were mostly retained within the cells or rapidly degraded in the culture supernatant. While the secretion of fusion proteins with cholera toxin subunit B (CT-B) from V. cholerae has been described, this is to our knowledge the first report describing extracellular secretion of defined foreign epitopes fused to LT-B in V. cholerue. The fusion of guest epitopes to LT-B or CT-B and secretion in P'. cholerue could be an interesting system to rapidly produce pure fusion proteins for immunisation, functional studies or diagnostic procedures. An LT-B/pre-S2 fusion protein purified from the supernatant of recombinant V. cholerue induced serum IgG antibodies against LT-B and against the HBV middle surface antigen in mice after parenteral and oral immunisation. Receptor-specific large-scale purification of cholera toxin on silica beads derivatized with lyso GM1 ganglioside Eur. J. Biochem. 113

Expert Review of Vaccines, 2003

The mucosal immune system consists of an integrated network of lymphoid cells that work in concer... more The mucosal immune system consists of an integrated network of lymphoid cells that work in concert with innate host factors to promote host defence. Mucosal immunization can be used both to protect the mucosal surfaces against colonization and invasion by microbial pathogens and to provide a means for immunological treatment of selected autoimmune, allergic or infectious-immunopathological disorders through the induction of antigen-specific tolerance. The development of mucosal vaccines, whether for prevention of infectious diseases or for oral tolerance immunotherapy, requires efficient antigen delivery and adjuvant systems. Significant progress has recently been made to generate partly or wholly detoxified derivatives of cholera toxin (including the completely nontoxic cholera toxin B subunit) and the closely related Escherichia coli heat-labile enterotoxin, with retained adjuvant activity. Cholera toxin B subunit is a protective component of a widely registered oral vaccine against cholera, and has proven to be a promising vector for either giving rise to anti-infective immunity or for inducing peripheral anti-inflammatory tolerance to chemically or genetically linked foreign antigens administered mucosally. Promising advances have also recently been made in the design of efficient mucosal adjuvants based on bacterial DNA that contains CpG-motifs and various imidazoquinoline compounds binding to different Toll-like receptors on mucosal antigen-presenting cells.

Current Opinion in Immunology, 2005

Recent work has provided new insights into the pathogenesis of the potentially life-threatening d... more Recent work has provided new insights into the pathogenesis of the potentially life-threatening diarrheas caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC): a new mechanism (post-translational degradation), which is involved in the control of cholera toxin expression, has been discovered. Recent evidence also suggests that vibrios upregulate cholera toxin expression in response to intestinal fluid components, and enterotoxin-carrying bacterial outer membrane vesicles might have a function in ETEC pathogenesis. An important role of the environment is supported by the correlation between cholera incidence and elevated sea surface temperature, which supports the notion that the zooplankton is a V. cholerae reservoir. Additionally, environmental lytic cholera phages could influence cholera seasonality by 'terminating' the seasonal epidemic. Finally, the strong herd immunity elicited by an oral cholera vaccine indicates that cholera vaccination could have a significant public health impact.

Cellular and Molecular Life Sciences, 2008

Many notions regarding the function, structure and regulation of cholera toxin expression have re... more Many notions regarding the function, structure and regulation of cholera toxin expression have remained essentially unaltered in the last 15 years. At the same time, recent findings have generated additional perspectives. For example, the cholera toxin genes are now known to be carried by a non-lytic bacteriophage, a previously unsuspected condition. Understanding of how the expression of cholera toxin genes is controlled by the bacterium at the molecular level has advanced significantly and relationships with cell-density-associated (quorum-sensing) responses have recently been discovered. Regarding the cell intoxication process, the mode of entry and intracellular transport of cholera toxin are becoming clearer. In the immunological field, the strong oral immunogenicity of the non-toxic B subunit of cholera toxin (CTB) has been exploited in the development of a now widely licensed oral cholera vaccine. Additionally, CTB has been shown to induce tolerance against co-administered (linked) foreign antigens in some autoimmune and allergic diseases.