Biomedical subjects

T Chakraborty

Publications and source records attributed to T Chakraborty.

At least 19 recordsLinked to original sources

PubMed2000

FimE-catalyzed off-to-on inversion of the type 1 fimbrial phase switch and insertion sequence recruitment in an Escherichia coli K-12 fimB strain.

We have investigated the capacity of a well-defined Escherichia coli fimB strain, AAEC350 (a derivative of MG1655), to express type 1 fimbriae under various growth conditions. The expression of type 1 fimbriae is phase-variable due to the inversion of a 314-bp DNA segment. Two tyrosine recombinases, FimB and FimE, mediate the inversion of the phase switch. FimB can carry out recombination in both directions, whereas the current evidence suggests that FimE-catalyzed switching is on-to-off only. We show here that AAEC350 is in fact capable of off-to-on phase switching and type 1 fimbrial expression under aerobic static growth conditions. The phase switching is mediated by FimE, and allows emerging fimbriate AAEC350 to outgrow their non-fimbriate counterparts by pellicle formation. Following inversion of the phase switch, this element can remain phase-locked in the on orientation due to integration of insertion sequence elements, viz. IS1 or IS5, at various positions in either the fimE gene or the phase switch.

Agglutination↗

PubMed2000

Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier.

The efficacious delivery of eukaryotic expression plasmids to inductive cells of the immune system constitutes a key prerequisite for the generation of effective DNA vaccines. Here, we have explored the use of bacteria as vehicles to orally deliver expression plasmids. Attenuated Salmonella typhimurium aroA harbouring eukaryotic expression plasmids that encoded virulence factors of Listeria monocytogenes were administered orally to BALB/c mice. Strong cytotoxic and helper T cell responses as well as antibody production were elicited even after a single administration. Mice immunised four times with Salmonella that carried a eukaryotic expression plasmid encoding the secretory listerial protein listeriolysin were protected against a subsequent lethal challenge with this pathogen. A single dose was already partially protective. The efficiency of this vaccination procedure was due to transfer of the expression plasmid from the bacterial carrier to the mammalian host. Evidence for such an event could be obtained in vivo and in vitro. Expression of the desired antigen in various lymphoid tissues was already detectable 1 day after administration of the DNA vaccine and persisted for at least 1 month in spleen and mesenteric lymph nodes. Induction of cytotoxic and helper T cell responses was observed in all mouse strains tested including outbred strains whereas antibodies were mainly detected in BALB/c. Furthermore, we could show that immunogenicity could be improved by increasing the invasiveness of the bacterial carrier.

Administration, Oral↗

PubMed2000

Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex.

The recruitment of actin to the surface of intracellular Listeria monocytogenes and subsequent tail formation is dependent on the expression of the bacterial surface protein ActA. Of the different functional domains of ActA identified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility. Mutational analysis of this domain which abolished actin recruitment by intracellular Listeria monocytogenes identified two arginine residues within the 146-KKRRK-150 motif that are essential for its activity. More specifically, recruitment of the Arp2/3 complex to the bacterial surface, as assessed by immunofluorescence staining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells with latrunculin B, which abrogated actin filament formation, did not affect association of ActA with p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and precedes, actin polymerisation. Our data suggest that binding of the Arp2/3 complex is mediated by specific interactions dependent on arginine residues within the 146-KKRRK-150 motif present in ActA.

Actin-Related Protein 2↗

PubMed1999

Affinity mass spectrometry-based approaches for the analysis of protein-protein interaction and complex mixtures of peptide-ligands.

Combined applications of affinity purification procedures and mass-spectrometric analyses (affinity mass spectrometry or affinity-directed mass spectrometry) have gained broad interest in various fields of biological sciences. We have extended these techniques to the purification and analysis of closely related peptides from complex mixtures and to the characterization of binding motifs and relative affinities in protein-protein interactions. The posttranslational modifications in the carboxy-terminal region of porcine brain tubulin are used as an example for the applicability of affinity mass spectrometry in the characterization of complex patterns of related peptides. We also show that affinity mass spectrometry allows the mapping of sequential binding motifs of two interacting proteins. Using the ActA/Mena protein-protein complex as a model system, we show that we can selectively purify Mena-binding peptides from a tryptic digest of ActA. The results from this assay are compared to data sets obtained earlier by classical methods using synthetic peptides and molecular genetic experiments. As a further expansion of affinity mass spectrometry, we have established an internally standardized system that allows comparison of the affinities of related ligands for a given protein. Here the affinities of two peptide ligands for the monoclonal tubulin-specific antibody YL1/2 are determined in terms of half-maximal competition.

Amino Acid Sequence↗

PubMed1999

The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes.

Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton.

Actin-Related Protein 2↗

PubMed1999

Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands.

Short contiguous peptides harboring proline-rich motifs are frequently involved in protein-protein interactions, such as associations with Src homology 3 (SH3) and WW domains. Although patches of aromatic residues present in either domain interact with polyprolines, their overall structures are distinct, suggesting that additional protein families exist that use stacked aromatic amino acids (AA domains) to bind polyproline motifs [1] [2] [3]. A polyproline motif (E/DFPPPPTD/E in the single-letter amino-acid code), present in the ActA protein of the intracellular bacterial pathogen Listeria monocytogenes, serves as a ligand for the Ena/VASP protein family --the vasodilator-stimulated phosphoprotein (VASP), the murine protein Mena, Drosophila Enabled (Ena) and the Ena/VASP-like protein Evl [4] [5] [6] [7]. These share a similar overall structure characterized by the two highly conserved Ena/VASP homology domains (EVH1 and EVH2) [5]. Here, using three independent assays, we have delineated the minimal EVH1 domain. Mutations of aromatic and basic residues within two conserved hydrophilic regions of the EVH1 domain abolished binding to ActA. Binding of an EVH1 mutant with reversed charges could partially be rescued by introducing complementary mutations within the ligand. Like SH3 domains, aromatic residues within the EVH1 domain interacted with polyprolines, whereas the ligand specificity of either domain was determined by reciprocally charged residues. The EVH1 domain is therefore a new addition to the AA domain superfamily, which includes SH3 and WW domains.

Amino Acid Sequence↗

PubMed1999

The actin-based motility of intracellular Listeria monocytogenes is not controlled by small GTP-binding proteins of the Rho- and Ras-subfamilies.

In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies. These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures. We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins. Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L. monocytogenes and had no effect on the actin-based motility of this bacterial parasite. Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.

Actins↗

PubMed1999

Physical and genetic map of the Listeria monocytogenes EGD serotype 1/2a chromosome.

Listeria monocytogenes is a facultative intracellular pathogen responsible for both invasive and non-invasive food-borne illness in animals and humans. In this study, macrorestriction analysis following pulsed-field gel electrophoresis was used to show that Listeria monocytogenes serovar 1/2a strain EGD has a single chromosome containing eight NotI fragments of 1100, 850, 365, 320, 275, 40, 30 and 20 kb in size and 11 AscI fragments of 860, 470, 410, 360, 320, 250, 110, 80, 50, 30 and 20 kb. The total genome therefore comprises 3000 +/- 50 kb. The creation of a physical and genetic map of the Listeria genome was achieved by generating NotI linking clones and their use in subsequent hybridisation analysis. Using isogenic mutants harbouring additional artificial NotI restriction sites, we were able to precisely map the positions of all currently known virulence genes on the chromosome.

Chromosome Mapping↗

PubMed1999

Molecular and cell biological aspects of infection by Listeria monocytogenes.

Bacterial pathogens have developed many subtle mechanisms to overcome and exploit cellular processes within the infected eukaryotic host cell. Listeria monocytogenes, a facultative intracellular non-spore forming gram-positive pathogen, uses a number of strategies to ursurp and harness host cell processes to invade, proliferate, move intracellularly and effect cell-to-cell spread during the course of infection. In this review progress in elucidating mechanisms by which the bacteria recruit and use components of the host actin-based cytoskeleton to generate intracellular motility is presented. Analysis of this fascinating property is giving us unexpected glimpses into the molecular mechanisms of complex cellular functions, here in particular, of actin-based cellular motility. Apart from an understanding of the fundamental biology of living processes these studies provide us with novel strategies to combat and halt infections by intracellular bacteria.

Actins↗

PubMed1999

Role of Listeria monocytogenes exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C in activation of human neutrophils.

Polymorphonuclear leukocytes (PMN) are essential for resolution of infections with Listeria monocytogenes. The present study investigated the role of the listerial exotoxins listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) in human neutrophil activation. Different Listeria strains, mutated in individual virulence genes, as well as purified LLO were used. Coincubation of human neutrophils with wild-type L. monocytogenes provoked PMN activation, occurring independently of phagocytosis events, with concomitant elastase secretion, leukotriene generation, platelet-activating factor (PAF) synthesis, respiratory burst, and enhanced phosphoinositide hydrolysis. Degranulation and leukotriene formation were noted to be solely dependent on LLO expression, as these features were absent when the LLO-defective mutant EGD- and the avirulent strain L. innocua were used. These effects were fully reproduced by a recombinant L. innocua strain expressing LLO (INN+) and by the purified LLO molecule. LLO secretion was also required for PAF synthesis. However, wild-type L. monocytogenes was more potent in eliciting PAF formation than mutants expressing LLO, suggesting the involvement of additional virulence factors. This was even more obvious for phosphoinositide hydrolysis and respiratory burst: these events were provoked not only by INN+ but also by the LLO-defective mutant EGD- and by a recombinant L. innocua strain producing listerial PlcA. We conclude that human neutrophils react to extracellularly provided listerial exotoxins by rapid cell activation. Listeriolysin is centrally involved in triggering degranulation and lipid mediator generation, and further virulence factors such as PlcA apparently contribute to trigger neutrophil phosphoinositide hydrolysis and respiratory burst. In this way, listerial exotoxins may influence the host defense against infections with L. monocytogenes.

Bacterial Toxins↗

PubMed1999

Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation.

Antigen 43 (Ag43), the product of the flu gene, is a surface-displayed autotransporter protein of Escherichia coli. Ag43 is responsible for the autoaggregation and flocculation of static liquid cultures of many E. coli strains. The expression of Ag43 has been reported to be phase variable and controlled by the product of the oxyR gene. Type 1 fimbriae are thin adhesive thread-like surface organelles responsible for bacterial receptor recognition and tissue colonization. Like that of Ag43, the expression of type 1 fimbriae is phase variable. Interestingly, previous results have suggested that the expression of type 1 fimbriae and the expression of Ag43 are mutually exclusive. In the present report, we show, by use of well-defined mutants, that fimbriation abolishes Ag43-mediated autoaggregation but does not affect Ag43 expression. Autoaggregation is shown to require an intercellular Ag43-Ag43 interaction, and the physical presence of fimbriae on the cells seems to abrogate this interaction. The Ag43 or OxyR status does not appear to influence fimbria expression, and our results suggest that the expression of Ag43 and the expression of fimbriae are independent processes.

Adhesins, Bacterial↗

PubMed1999

Type 1 fimbriation and phase switching in a natural Escherichia coli fimB null strain, Nissle 1917.

Escherichia coli Nissle 1917 has been used as a probiotic against intestinal disorders for many decades. It is a good colonizer of the human gut and has been reported to be able to express type 1 fimbriae. Type 1 fimbriae are surface organelles which mediate alpha-D-mannose-sensitive binding to various host cell surfaces. The expression is phase variable, and two tyrosine recombinases, FimB and FimE, mediate the inversion of the fimbrial phase switch. Current evidence suggests that FimB can carry out recombination in both directions, whereas FimE-catalyzed switching is on to off only. We show here that under liquid shaking growth conditions, Nissle 1917 did not express type 1 fimbriae, due to a truncation of the fimB gene by an 1,885-bp insertion element. Despite its fimB null status, Nissle 1917 was still capable of off-to-on switching of the phase switch and expressing type 1 fimbriae when grown under static conditions. This phase switching was not catalyzed by FimE, by truncated FimB, or by information residing within the insertion element. No further copies of fimB seemed to be present on the chromosome of Nissle 1917, suggesting that another tyrosine recombinase in Nissle 1917 is responsible for the low-frequency off-to-on inversion of the phase switch that is strongly favored under static growth conditions. This is the first report documenting the non-FimB- or non-FimE-catalyzed inversion of the fim switch.

Amino Acid Sequence↗

PubMed1999

Bradycardia, reversible panconduction defect and syncope following self-medication with a homeopathic medicine.

Alkaloid extracts from the plant Aconitum species have been used in various forms of herbal remedies predominantly as anti-inflammatory and analgesic agents. Many of these alkaloids are extremely potent cardiotoxins and documented cases of various arrhythmias with fatal outcomes have been reported. We report a case of self-medication with 'tincture of aconite' resulting in severe bradycardia, reversible panconduction defect evidenced by sinus inactivity, atrioventricular dissociation with idiojunctional rhythm and left bundle branch block pattern resulting in hypotension and syncope. Complete reversal of ECG findings with marked improvement in symptoms was noted within a few hours. Herbal medicines containing aconite alkaloids may result in severe cardiotoxicity, and strict regulatory measures are warranted to curb unsupervised use for therapeutic purposes.

Aconitum↗

PubMed1998

Inhibition of gastric mucosal prostaglandin synthetase activity by mercaptomethylimidazole, an inducer of gastric acid secretion--plausible involvement of endogenous H2O2.

We have reported earlier that mercaptomethylimidazole (MMI), an antithyroid drug of thionamide group, induces gastric acid secretion at least partially through the liberation of histamine, sensitive to cimetidine. Now, we show that the drug has a significant inhibitory effect on the cyclooxygenase and peroxidase activity of the prostaglandin (PG) synthetase of the gastric mucosal microsomal preparation. The effect can also be mimicked by low concentrations of H2O2. While studying the possible intracellular effect of MMI on acid secretion, a cell fraction (F3) enriched in parietal cell was isolated by controlled digestion of the mucosa with protease. This cell fraction is activated by MMI as measured by increased O2 consumption. The activation is sensitive to omeprazole, a proton-pump inhibitor, indicating that the activation is due to increased acid secretion by MMI. MMI was also found to directly inhibit the peroxidase activity of the F3 cell fraction and may thus increase the intracellular level of H2O2. The cyclooxygenase activity of the PG synthetase of the F3 cell fraction is also inhibited by MMI and the effect can be reproduced by low concentrations of H2O2. Both MMI and H2O2 can also inhibit the peroxidase activity of the PG synthetase. We suggest that in addition to the activation of the parietal cell by MMI possibly through endogenous H2O2, MMI induces acid secretion in vivo by inactivating the PG synthetase thereby inhibiting the biosynthesis of PG and removing its inhibitory influence on acid secretion so that the histamine released by MMI can stimulate acid secretion with maximum efficiency.

Animals↗

PubMed1998

Two distinct phospholipases C of Listeria monocytogenes induce ceramide generation, nuclear factor-kappa B activation, and E-selectin expression in human endothelial cells.

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGD delta plcA and EGD delta plcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGD delta plcA or EGD delta plcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-kappa B activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-kappa B activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.

Cells, Cultured↗

PubMed1998

The role of the bacterial membrane protein ActA in immunity and protection against Listeria monocytogenes.

ActA, an essential virulence factor of Listeria monocytogenes, is an integral membrane protein that is required for intracellular motility, cell-to-cell spread, and rapid dissemination of the bacteria in the infected host. To reveal cytotoxic T cell responses against ActA we introduced a recombinant soluble form of ActA into the MHC class I-processing compartment of APC using a variant of listeriolysin mutated within its immunodominant MHC class I epitope. With this experimental system we demonstrate that T cells are induced against ActA during a sublethal infection with L. monocytogenes. However, adoptively transferred cytotoxic CD8+ T cells specific for ActA did not protect mice against a subsequent challenge with this pathogen. This was due to an inability of APC to present ActA by either MHC class I or class II molecules as long as ActA remained tethered to the surface of intracellular viable bacteria. ActA was only presented when L. monocytogenes were engineered to secrete ActA or when the bacteria were killed by antibiotics during the assay. These findings raise questions on the general use of membrane proteins of pathogens as candidates for subunit vaccines.

Adoptive Transfer↗

PubMed1998

Small GTP-binding proteins of the Rho- and Ras-subfamilies are not involved in the actin rearrangements induced by attaching and effacing Escherichia coli.

Attaching and effacing Escherichia coli (AEEC) are extracellular pathogens that induce the formation of actin-rich structures at their sites of attachment to eukaryotic host cells. We analysed whether small GTP-binding proteins of the Rho- and Ras-subfamilies, which control the cellular actin system, are essential for these bacterial-induced microfilament reorganizations. For this purpose we specifically inactivated them using the Clostridium difficile toxins TcdB-10463 and TcdB-1470. Such treatment led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the bacterial-induced actin rearrangements. Our data therefore indicate that the microfilament reorganizations induced by AEEC are independent of those small GTP-binding proteins that under normal conditions control the dynamics and maintenance of the actin cytoskeleton.

Actin Cytoskeleton↗

PubMed1998

Attenuated Listeria monocytogenes carrier strains can deliver an HIV-1 gp120 T helper epitope to MHC class II-restricted human CD4+ T cells.

Listeria monocytogenes is a facultative intracellular pathogen which, following uptake by macrophages, escapes from the phagosome and replicates in the cytoplasm. This property has been exploited using recombinant L. monocytogenes as a carrier for the intracytoplasmic expression of antigens when MHC class I-restricted cytotoxic T lymphocyte responses are required. Much less is known of the ability of these bacteria to trigger MHC class II-restricted responses. Here, we demonstrate that after ingestion of L. monocytogenes expressing a T helper epitope from the gp120 envelope glycoprotein of HIV, human adherent macrophages and dendritic cells can process and present the epitope to a specific CD4+ T cell line in the context of MHC class II molecules. No significant differences were observed when the attenuated strains were trapped in the phagolysosome or impaired in the capacity to spread intracellularly or from cell to cell. Similar results were obtained using carrier proteins that were either secreted, associated with the bacterial surface, or restricted to the bacterial cytoplasm. A dominant expression of the TCR Vbeta 22 gene subfamily was observed in specific T cell lines generated after stimulation with the recombinant strains or with soluble gp120. Our data show that in this in vitro system L. monocytogenes can efficiently deliver antigens to the MHC class II pathway, in addition to the well-established MHC class I pathway. The eukaryotic cell compartment in which the antigen is synthesized, and the mode of display seem to play a minor role in the overall efficiency of epitope processing and presentation.

Antigen Presentation↗