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Biomedical subjects

R Rappuoli

Publications and source records attributed to R Rappuoli.

At least 199 records · Page 11Linked to original sources
PubMed1994

Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins.

Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity. This cavity consists of 18 contiguous amino acids that form an alpha-helix bent over a beta-strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophilic residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins.

ADP Ribose Transferases↗
PubMed1994

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis.

Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41-->Phe, Ala-45-->Tyr or Glu, Val-53-->Tyr, Val-60-->Gly, Ser-68-->Pro, His-70-->Pro, Val-97-->Tyr and Ser-114-->Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54-->Lys or Ala, Tyr-59-->Met, Ser-68-->Lys, Ala-72-->Arg, His or Asp and Arg-192-->Asn. The results provide a further understanding of the structure-function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.

Amino Acid Sequence↗
PubMed1994

Formaldehyde treatment of proteins can constrain presentation to T cells by limiting antigen processing.

Proteins to be used as vaccines are frequently treated with formaldehyde, although little is known about the effects of this treatment on protein antigenicity. To investigate the effect of formaldehyde treatment on antigen recognition by T cells, we compared the in vitro T-cell response to proteins that have been formaldehyde treated with the response to untreated proteins. We found that peripheral blood mononuclear cells from individuals vaccinated with three formaldehyde-treated proteins (pertussis toxin, filamentous hemagglutinin, pertactin) of Bordetella pertussis showed little or no response to the formaldehyde-treated proteins but proliferated very well in response to the corresponding untreated protein. These findings were further confirmed with CD4+ T-cell clones specific for defined epitopes of the bacterial proteins. We found that some epitopes are presented poorly or not at all when formaldehyde-treated proteins are used, whereas other epitopes are equally presented to T-cell clones when either formaldehyde-treated or untreated antigens are used. However, T-cell recognition could be restored by either antigen degradation before formaldehyde treatment or heat denaturation after such treatment. Parallel digestion with trypsin of both formaldehyde-treated and untreated proteins showed that fragments generated from the two forms of the same antigen were different in size. These results demonstrate that formaldehyde treatment can constrain antigen presentation to T cells and that this may be due to an altered proteolytic processing of formaldehyde-treated proteins.

Adult↗
PubMed1994

Pertussis toxin activates platelets through an interaction with platelet glycoprotein Ib.

Platelets present a unique model to study the B-oligomer effects of pertussis toxin because they become activated in response to the B oligomer but are not susceptible to ADP-ribosylation by the holotoxin. In these studies, the B oligomer of pertussis toxin caused concentration-dependent platelet activation, as determined by increases in intracellular calcium concentration, dense granule secretion, and platelet aggregation. Stirring was required for pertussis toxin to increase intracellular calcium. A monoclonal antibody against platelet glycoprotein Ib abolished increases in intracellular calcium concentration and increased the latency and reduced the slope of the aggregation response elicited by the B oligomer. Pertussis toxin also evoked [14C]serotonin release from platelets, and this effect was inhibited, though not eliminated, by an antibody against platelet glycoprotein Ib. Binding of pertussis toxin to glycoprotein Ib was observed after nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that the B oligomer of pertussis toxin induces platelet activation mediated, at least in part, by an interaction with platelet glycoprotein Ib.

Blood Platelets↗
PubMed1994

Pathogenesis of Helicobacter pylori and perspectives of vaccine development against an emerging pathogen.

Infection of the stomach and the duodenum by Helicobacter pylori is the major cause of acute and chronic gastroduodenal pathologies in humans and increases the risk of gastric cancer. The recognition of the infectious nature of the illness is having a major impact in the management of the disease that is shifting from the treatment of symptoms by anti-H2 blockers to the eradication of the bacterial infection by antibiotic regimen. Experience with other bacterial diseases, suggests that antibiotic treatment will select resistant strains that in the long term will make the antibiotics infective. Vaccination that classically is the most effective way to prevent and control infectious diseases in large population, could be used to prevent infection and possibly also to treat the disease. Here we summarize the studies on the identification and characterization of the virulence factors that are important for the pathogenesis of the bacterium and that may be candidate components for a vaccine. Animal models of the infection are also described.

Adhesins, Bacterial↗
PubMed1993

Cell vacuolization induced by Helicobacter pylori: inhibition by bafilomycins A1, B1, C1 and D.

All available bafilomycins (A1, B1, C1 and D) inhibit and revert macroscopic vacuolization induced by Helicobacter pylori cell-free extracts. Bafilomycin A1 displays the highest activity, followed by bafilomycin B1, C1 and D. The different potency of bafilomycins correlates with their ability to inhibit the vacuolar-type ATPase (V-ATPase) and to dissipate the membrane pH gradient of intracellular acidic organelles. These results suggest that bafilomycins should be considered as possible therapeutic agents in the treatment of gastritis.

Adenosine Triphosphatases↗
PubMed1993

Adhesion of Bordetella pertussis to eukaryotic cells requires a time-dependent export and maturation of filamentous hemagglutinin.

Bordetella pertussis, the human pathogen of whooping cough, when grown at 22 degrees C is nonvirulent and unable to bind eukaryotic cells. In response to a temperature shift to 37 degrees C, the bacterium acquires the ability to bind eukaryotic cells in a time-dependent fashion. By studying in vitro the temperature-induced transition, from the nonvirulent to the virulent state, we found that binding to CHO cells is mediated by the Arg-Gly-Asp-containing domain of filamentous hemagglutinin (FHA), a protein with multiple binding specificities. This protein is synthesized as a 367-kDa polypeptide within 10 min after temperature shift, but requires 2 hr before it is detected on the bacterial cell surface and starts to bind CHO cells. Mutations affecting the cell surface export of FHA abolish bacterial adhesion to CHO cells, while mutations in the outer membrane protein pertactin strongly reduce binding. This suggests that multiple chaperon proteins are required for a correct function of FHA. Finally, several hours after maximum binding efficiency is achieved, the N-terminal 220-kDa portion of FHA that contains the binding regions is cleaved off, possibly to release the bacteria from the bound cells and facilitate spreading. The different forms of FHA may play different roles during bacterial infection.

Adhesins, Bacterial↗
PubMed1993

Priming to heat shock proteins in infants vaccinated against pertussis.

To investigate whether and in which proportion normal individuals experience a priming to microbial heat shock proteins (hsp), the presence of antibodies to two mycobacterial hsp was tested in serum sample from 2- to 4-mo-old children before and at different times after vaccination with the trivalent vaccine against tetanus, diphtheria, and pertussis (DTP). We show that 88.9% of infants vaccinated with DTP developed antibody responses to mycobacterial hsp. Such a response was due to the whole-cell pertussis component of the vaccine, because it was not observed in infants receiving an acellular pertussis vaccine. Antibodies and cells reactive to the mycobacterial 65-kDa hsp were also found in mice immunized with DTP. Interestingly, whole-cell pertussis vaccine-induced anti-hsp antibodies cross-reacted with the Escherichia coli GroEL hsp, and at a some extent with the human 60-kDa hsp, belonging to the same hsp family. These data suggest that priming of the immune system to hsp is a common phenomenon occurring very early in life.

Animals↗
PubMed1993

Cell penetration of diphtheria toxin. Reduction of the interchain disulfide bridge is the rate-limiting step of translocation in the cytosol.

The pathway of cell penetration of diphtheria toxin (DT) was studied in Vero cells by following the kinetics of uptake, reduction, degradation, and sub-cellular distribution of 125I-DT in the absence or presence of bafilomycin A1 (baf-A1), a powerful inhibitor of the endosomal H(+)-ATPase. After a lag phase of 4 min, DT, bound to Vero cells, reached an acidic intracellular compartment, where about one-third of it underwent a transition to a state competent for subsequent reduction and membrane translocation. After further 4 min, this DT fraction was reduced in a baf-A1-insensitive reaction and DT-A, the intracellularly active protomer of DT, was immediately released in the cytosol. The present data indicate that cell-mediated reduction of the toxin is the rate-determining step of the DT cell intoxication process. Subcellular fractionation showed that DT underwent the low pH-driven conformational change in an early endosome, distinct from the subsequent endosomal compartment where reduction took place. DT-B remained endosome-bound and was proteolyzed at low pH as well as the portion of DT which was not reduced after the exposure to low pH in early endosomes.

Animals↗
PubMed1993

Environmental regulation of virulence factors in Bordetella species.

Many bacteria respond in a coordinate manner to environmental changes. External stimuli, sensed by receptors, are transduced to regulatory proteins which participate in well defined pathways of gene expression by varying their structure and mode of action. The network of environmental signal transduction is responsible for a fine and continuous communication between the host and the pathogenic bacteria. As a result, the gene expression machinery of the pathogen is modified continuously, in order to establish the optimal conditions for bacterial survival and multiplication.

Amino Acid Sequence↗
PubMed1993

Absence of protective immunity against diphtheria in a large proportion of young adults.

The schedule of vaccination recommended worldwide for diphtheria, tetanus and other diseases, provides good immunity during childhood. However, little attention has been paid to effective immunity in adults. We have collected sera from 334 Italian Army recruits and tested them for the presence of protective immunity against diphtheria and tetanus. In vivo neutralization assays were performed on rabbits and mice and values below 1/100 IU ml-1 were considered negative. Of the recruits, 22.9% were negative for diphtheria, while only 5.3% had no protective immunity against tetanus. This finding shows that a large proportion of Italian young adults are susceptible to diphtheria, and this could be dangerous if they travel to sites where this disease is still endemic, or if they come into contact with people coming from such areas. A booster vaccination of young adults against diphtheria should become common practice to avoid this risk. To reduce the side effects which are often associated with diphtheria vaccination in adults, we have developed a vaccine which contains a highly purified, non-toxic mutant of diphtheria toxin. This vaccine is combined with tetanus toxoid and can be routinely used as a booster in adults.

Adolescent↗
PubMed1993

Characterization of an IS-like element from Mycobacterium tuberculosis.

A DNA sequence, present in members of the Mycobacterium tuberculosis complex, has been identified and characterized. The distribution of this DNA sequence among mycobacterial species was analysed by DNA hybridization and PCR experiments. As the sequence was detected only in bacteria belonging to the M. tuberculosis complex, it may be useful for the rapid discrimination of mycobacteria. Interestingly, the sequence has some characteristics of an insertion element (IS) and codes for a hypothetical protein with significant homologies to proteins encoded by several IS elements of other organisms, namely IS427 and IS869 from Agrobacterium tumefaciens, IS402 from Pseudomonas cepacia, Tn4811 from Streptomyces lividans and ISRm4 from Rhizobium meliloti. Together, these elements form a previously unrecognized family of transposable elements. This finding suggests the possibility of horizontal gene transfer between pathogenic mycobacteria and other organisms including Gram-negative plant-pathogenic bacteria.

Amino Acid Sequence↗
PubMed1993

Bafilomycin A1 inhibits Helicobacter pylori-induced vacuolization of HeLa cells.

Bafilomycin A1, a specific inhibitor of the vacuolar-type H(+)-ATPase, responsible for acidification of intracellular compartments, prevents the vacuolization of Hela cells induced by H. pylori, with an inhibitory concentration giving 50% of maximal (ID50) of 4 nM. Bafilomycin A1 is also very efficient in restoring vacuolated cells to a normal appearance. The vacuolating activity of Helicobacter pylori is not inhibited by a series of specific inhibitors of vacuolar H(+)-ATPases. These findings indicate that a transmembrane pH gradient is needed for the formation and growth of vacuoles caused by the bacterium and that this pH gradient is due to the activity of a vacuolar ATPase proton pump of HeLa cells.

Anti-Bacterial Agents↗
PubMed1993

Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon.

Pertussis toxin, a major virulence factor of Bordetella pertussis, is an oligomeric protein composed of five different subunits that are exported individually to the periplasmic space by the signal peptide-dependent pathway. After assembly, the protein is exported from the periplasm to the extracellular compartment. We show that pertussis toxin secretion across the outer membrane requires the gene product of at least one gene (ptlC) that is located downstream from the pertussis toxin operon. The amino acid sequence of PtlC shows a high degree of homology to VirB4, a protein encoded by the virB operon, which contains 11 open reading frames that are involved in the transfer of T-DNA from Agrobacterium tumefaciens to the plant cells. This is a novel mechanism of protein export in Gram-negative bacteria.

Agrobacterium tumefaciens↗
PubMed1993

The Hsp60 protein of Helicobacter pylori: structure and immune response in patients with gastroduodenal diseases.

Helicobacter pylori is a human pathogen that has been associated with gastritis, peptic ulcer and gastric carcinoma. The role of the direct action of H. pylori virulence factors and of the induction of autoreactive immunity in the development of chronic gastritis has not been clarified yet. Here we report the cloning and molecular characterization of a gene of H. pylori coding for a protein of 58 kDa, recognized by sera of patients affected by H. pylori-induced gastroduodenal diseases. This antigen is present in all the H. pylori strains tested and it belongs to the Hsp60 family of heat-shock proteins, with high homology with other bacterial and eukaryotic proteins of the same family. This class of homologous proteins has been implicated in the induction of autoimmune disorders in different systems. The presence in infected patients of anti-H. pylori Hsp60 antibodies, potentially cross-reactivity between human Hsp60 and a rabbit antiserum against H. pylori Hsp60 suggest that a role of this protein in gastroduodenal diseases is possible.

Amino Acid Sequence↗
PubMed1993

DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro.

The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes. We show that in Escherichia coli, the PTOX promoter cloned in cis of the bvg locus is activated and environmentally regulated. Cotransformation of E. coli with the bvg locus cloned in a low-copy-number plasmid and with the PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results. If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent. If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished. Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases. In vitro, the transcription of the PTOX promoter is activated on E. coli RNA polymerase supplemented with cell extracts from wild-type B. pertussis. Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized. Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the PTOX promoter in E. coli and that DNA topology may play a role in the induction of transcription.

Bordetella pertussis↗