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Results for “toxin secretion system”

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At least 433 records · Page 24Linked to original sources

PubMed1995

Autocrine regulation of toxin synthesis by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen causing diseases which range from minor skin infection to endocarditis and toxic shock syndrome. The pathogenesis of S. aureus is due primarily to the production of toxic exoproteins, whose synthesis is controlled by a global regulatory system, agr. We show here that agr is autoinduced by a proteinaceous factor produced and secreted by the bacteria and that it is inhibited by a peptide produced by an exoprotein-deficient S. aureus mutant strain. The inhibitor, RIP, competes with the activator, RAP, and may be a mutational derivative. Our results suggest two possible approaches, independent of antibiotics, to the control of S. aureus infections. RIP may prove useful as a direct inhibitor of virulence and RAP as a vaccine against the expression of agr-induced virulence factors; either could interfere with the ability of the bacteria to establish and maintain an infection.

Bacterial Proteins↗

PubMed2003

Intranasal vaccination using interleukin-12 and cholera toxin subunit B as adjuvants to enhance mucosal and systemic immunity to human immunodeficiency virus type 1 glycoproteins.

We have investigated the induction of protective mucosal immunity to human immunodeficiency virus type 1 (HIV-1) isolate 89.6 by intranasal (i.n.) immunization of mice with gp120 and gp140 together with interleukin-12 (IL-12) and cholera toxin subunit B (CTB) as adjuvants. It was found that both IL-12 and CTB were required to elicit mucosal antibody responses and that i.n. immunization resulted in increased total, immunoglobulin G1 (IgG1), and IgG2a anti-HIV-1 antibody levels in serum; increased total, IgG1, IgG2a, and IgA antibody expression in bronchoalveolar lavage fluids; and increased IgA antibody levels in vaginal washes. Levels of anti-HIV-1 antibodies in both sera and secretions were higher in groups immunized with gp140 than in those immunized with gp120. However, only gp120-specific mucosal antibodies demonstrated neutralizing activity against HIV-1 89.6. Taken together, the results show that IL-12 and CTB act synergistically to enhance both systemic and local mucosal antibody responses to HIV-1 glycoproteins and that even though gp140 induces higher antibody titers than gp120, only gp120-specific mucosal antibodies interfere with virus infectivity.

AIDS Vaccines↗

PubMedDate not supplied

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.

Enteropeptidase↗

PubMed2003

Interactions between p-33 and p-55 domains of the Helicobacter pylori vacuolating cytotoxin (VacA).

The VacA toxin secreted by Helicobacter pylori is considered to be an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. VacA monomers self-assemble into water-soluble oligomeric structures and can form anion-selective membrane channels. The goal of this study was to characterize VacA-VacA interactions that may mediate assembly of VacA monomers into higher order structures. We investigated potential interactions between two domains of VacA (termed p-33 and p-55) by using a yeast two-hybrid system. p-33/p-55 interactions were detected in this system, whereas p-33/p-33 and p-55/p-55 interactions were not detected. Several p-33 proteins containing internal deletion mutations were unable to interact with wild-type p-55 in the yeast two-hybrid system. Introduction of these same deletion mutations into the H. pylori vacA gene resulted in secretion of mutant VacA proteins that failed to assemble into large oligomeric structures and that lacked vacuolating toxic activity for HeLa cells. Additional mapping studies in the yeast two-hybrid system indicated that only the N-terminal portion of the p-55 domain is required for p-33/p-55 interactions. To characterize further p-33/p-55 interactions, we engineered an H. pylori strain that produced a VacA toxin containing an enterokinase cleavage site located between the p-33 and p-55 domains. Enterokinase treatment resulted in complete proteolysis of VacA into p-33 and p-55 domains, which remained physically associated within oligomeric structures and retained vacuolating cytotoxin activity. These results provide evidence that interactions between p-33 and p-55 domains play an important role in VacA assembly into oligomeric structures.

Amino Acid Sequence↗

PubMed2026

Occurrence of blaOXA-72 in a clinical isolate of carbapenem-resistant Acinetobacter pittii ST206 in Japan.

The development of carbapenem resistance in Acinetobacter spp., which are recognized as significant opportunistic pathogens, is of critical importance as it poses challenges to therapy and the control of healthcare-associated infections in clinical settings. This study investigated the genetic characteristics of a carbapenem-resistant A. pittii clinical isolate from a university hospital using whole-genome sequencing. The A. pittii strain SU8507, which was detected in the abdominal drainage fluid of a patient, exhibited resistance to imipenem (MIC: 128 μg/mL) and meropenem (MIC: 64 μg/mL) and produced positive results by the CIMTris method. A. pittii SU8507, belonging to ST206, harbored the blaOXA-72 and new variants of intrinsic blaOXA-213-like gene blaOXA-1222, which lacks an upstream insertion sequence element, and blaADC-1-like gene blaADC-343. The blaOXA-72 gene, flanked by XerC/XerD-like recombination sites, was located on a plasmid pSU8507, sized at 10,913 bp, carrying 13 predicted protein-coding genes. Complete pSU8507 containing mobA, repB, and the yoeB-yefM toxin-antitoxin genes, showed 98.9% nucleotide sequence identity and 75% coverage with plasmid pA2702 of the A. baylyi strain A2702, but a low BLAST MAX score. SU8507 harbored virulence genes involved in biofilm formation, types II and VI secretion systems, type IV pilus system, and serum resistance. This study describes the first isolation of an OXA-72-producing, carbapenem-resistant A. pittii clinical isolate in Japan. Given that the isolation rate of carbapenem-resistant Acinetobacter spp. Remains low in Japan, it is crucial to expand the scope of rapid, accurate carbapenemase detection to include not only A. baumannii, but also non-baumannii Acinetobacter spp.

Humans↗

PubMed2003

Influence of the murine oestrous cycle on the induction of mucosal immunity.

PROBLEM: To determine if the stage of oestrous cycle, at the time of immunization, affects the magnitude of mucosal and systemic immunity. METHOD OF STUDY: Female BALB/c mice were immunized with tetanus toxoid and cholera toxin by the oral, intranasal and transcutaneous routes. Groups of mice were immunized at proestrus, oestrus, postestrus and diestrus. Antibodies in serum and mucosal secretions were determined by ELISA and T cell responses by lymphocyte proliferation assay. RESULTS: Oral immunization at the oestradiol dominant stage of cycle (oestrus and proestrus) significantly enhanced TT-specific IgG and IgA levels in female reproductive tract (FRT) secretions and TT-specific IgA levels in faecal extracts. Transcutaneous immunization at diestrus enhanced TT-specific IgG in faecal extracts. TT-specific T cell proliferation is greatest following intranasal immunization at proestrus and transcutaneous immunization at diestrus, particularly in the caudal and lumbar lymph nodes draining the FRT and colon. CONCLUSIONS: Reproductive cycle-associated changes in the endogenous sex hormones oestradiol and progesterone influence the levels of vaccine-induced immunity in the FRT and distal colon following oral and transcutaneous immunization.

Administration, Cutaneous↗

PubMed1991

Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene.

Female mites of the species Pyemotes tritici inject an extremely potent venom into their insect prey that causes muscle-contraction and paralysis. These mites are able to paralyse insects 150,000 times their size and their venom is effective in a broad range of insect species. A toxin (TxP-I) associated with the mite venom apparatus causes immediate muscle-contractive paralysis when injected into insects but not mice. In this report, we describe the cloning, sequencing and expression of a complementary DNA (Tox-34) encoding TxP-I. Insect cells infected with a recombinant baculovirus (vEV-Tox34) expressing Tox-34 secrete three polypeptides related to TxP-I which cause paralysis on injection. Larvae infected with vEV-Tox34 become paralysed during infection, thus reflecting the potential application of this toxin gene in insect biocontrol methods. The toxin gene expression system will also allow further exploration of the neurophysiological basis of its insect-specific effects.

Amino Acid Sequence↗

PubMed2001

The presence of superantigens and complex host responses in severe sepsis may need a broad therapeutic approach.

Patients with sepsis can progress into septic shock, disseminated intravascular coagulation, and multiorgan dysfunction syndrome. Various materials secreted by or released from microorganisms such as bacteria initiate these processes. In some bacteria, certain antigens and toxins may cause a 100-fold greater or supernormal activation of monocytes and T lymphocytes, leading to activation of the cascade systems in the host. This can explain the extremely rapid progress of the sepsis into septic shock seen in some patients. In Group A streptococci, more than 100 different toxins have been identified, about 5 of which (superantigens) cause an extremely fast immunological response. Because the toxins and antigens can activate so many different cascade systems in the host, the clinical picture is extremely complex, and little benefit is derived from therapy, which interferes with only 1 or 2 of the parameters in the patient with sepsis. Instead, reversal of the septic shock requires the removal or inhibition of several toxins or substances activating the cascade systems. A broader therapeutic approach may be the use of apheresis (plasma exchange).

Anti-Bacterial Agents↗

PubMed1998

Specific-antibody-secreting cells in the rectums and genital tracts of nonhuman primates following vaccination.

To determine optimal strategies to induce specific-antibody-secreting cells (specific ASC) in the rectal and vaginal mucosae, we immunized monkeys with a prototype mucosal immunogen, cholera toxin (CT), given locally or via gastric or parenteral administration. Repeated rectal or vaginal CT immunizations induced strong mucosal and systemic ASC responses. The mucosal responses were, however, confined to the immunization sites and comprised high levels of both specific antitoxin immunoglobulin A (IgA) and IgG. Large numbers of specific IgA and IgG ASC were detected in cell suspensions from dissociated genital and rectal tissues, demonstrating local accumulation of effector B cells at these sites. Intragastric immunization with CT did not per se give rise to cervicovaginal or rectal ASC responses but did prime for a rectal IgA ASC response to local booster immunization. Both rectal and vaginal immunizations also induced circulating blood IgG ASC and IgA ASC. In conclusion, these results show that local administration of antigen to the rectal or vaginal mucosa results in higher ASC responses than systemic or distant mucosal delivery. Furthermore, both the vaginal and the rectal mucosae can serve as inductive sites for systemic ASC responses. These observations should be relevant to the development of vaccines against sexually transmitted diseases such as that caused by human immunodeficiency virus.

Administration, Intranasal↗

PubMed1991

Brefeldin-A enhancement of ricin A-chain immunotoxins and blockade of intact ricin, modeccin, and abrin.

After binding, the protein toxins ricin, abrin, and modeccin are endocytosed and processed through the cell's vesicular system in a poorly understood fashion, prior to translocation to the cytosol. The role of the Golgi apparatus in toxin processing was studied using brefeldin-A (BFA), a fungal metabolite which blocks Golgi function. At concentrations that inhibit secretion of interleukin-2 (IL-2), BFA blocks ricin, modeccin, and abrin intoxication of a lymphocyte derived cell line (Jurkat). Paradoxically, BFA enhances the toxicity of two ricin A-chain immunotoxins targeted against distinct cell surface determinants. BFA concentrations which are optimal for immunotoxin enhancement are below those needed to affect ricin intoxication or IL-2 secretion. BFA blockade of ricin does not involve effects on ricin endocytosis, toxin translocation to the cytosol, or the enzymatic activity of toxin A-chain. In contrast, BFA has no effect on immunotoxin processing but does enhance the immunotoxin translocation step. It is concluded that: 1) intact Golgi function is required for holotoxin processing. 2) Intact Golgi function is not required for holotoxin translocation. 3) Golgi function is tightly linked to immunotoxin translocation. 4) BFA has effects on vesicular routing in addition to the block of Golgi function in secretion which has been reported.

Abrin↗

PubMedDate not supplied

Action of New World scorpion venom and its neurotoxins in secretion.

New World scorpion venom contains protein toxins specific for ion channels in the plasmalemma of excitable cells. The effects were examined of whole venoms from Tityus serrulatus, T. bahiensis and T. stigmurus, and some purified toxins in isolated nerve endings (synaptosomes) and pancreatic acinar cells. Both systems initiated exocytosis in a dose-dependent response to the venom or its bioactive protein toxins. Actions differed, however, such that pancreatic acinar cells required Ca2+ while cerebrocortical synaptosomes responded by a Ca(2+)-dependent mechanism, except in the case of one toxin, IV-5, that elicited a Ca(2+)-independent response. Membrane depolarization caused by scorpion venom toxins was measured via radioisotopic discharge of tetra[3H]phenylphosphonium bromide. The role of protein kinase C in second-messenger coupling in pancreatic acinar cells is favored over ion-exclusive routes characteristic of synaptosomes.

Animals↗

PubMed1998

Nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues.

Female rhesus macaques were nasally immunized with p55gag (p55) of SIV and cholera toxin as a mucosal adjuvant. Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum. Furthermore, high numbers of p55-specific IgA and IgG Ab-forming cells were induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4+ T cells in both systemic and mucosal compartments expressed IFN-gamma and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.

Adjuvants, Immunologic↗

PubMed2003

Getting control of intestinal secretion: thoughts for 2003.

There has been a search for more than 20 years for agents that will directly inhibit intestinal secretory mechanisms and thereby reduce stool volume in patients with high volume watery diarrhoea. Recent work has highlighted the importance of neurohumoral mechanisms in the pathogenesis of diarrhoea, notably the role of 5-hydroxytryptamine, substance P, vasoactive intestinal polypeptide and neural reflexes within the enteric nervous system. Cholera toxin and Escherichia coli enterotoxins are known to invoke these mechanisms in some diarrhoeal states. This new dimension of intestinal pathophysiology has suggested possible novel targets for anti-secretory therapy including, 5-hydroxytryptamine receptor antagonists, substance P antagonists, vasoactive intestinal polypeptide antagonists and the possibility for potentiating the pro-absorptive effects of endogenous enkephalins by use of enkephalinase inhibitors. There now seems to be a real possibility that anti-secretory therapy will become more widely available in the future.

Diarrhea↗

PubMed1996

Atrial natriuretic peptide (ANP)-induced inhibition of glucagon secretion: mechanism of action in isolated rat pancreatic islets.

ANP increases insulin levels in vivo. Because in vitro an ANP-induced increase in cGMP levels of islets of Langerhans was observed but no simultaneous increase in insulin release, secreted glucagon may be a candidate for this second messenger affected by ANP. The inhibitory effect of glucose on glucagon secretion was pronounced by 1.0 nM ANP at 3.0 mM glucose as well as at 5.6 and 8.3 mM glucose. Because in other tissues cGMP (the specific second messenger of ANP1 inhibits Ca2+ channels, the uptake of 45Ca2+ was investigated. ANP (1.0 nM) inhibited 45Ca2+ uptake, which was nearly completely abolished by a pertussis toxin (PT) pretreatment. The inhibition of 45Ca2+ uptake fits to inhibitory ANP effects on glucagon secretion but does not fit to insulin secretion. The glucagon secretion coupling cascade affected by ANP probably involves an increase in cGMP because 8-Br-cGMP (a membrane-permeable cGMP analogue) also decreased glucagon secretion. ANP(4-23), a truncated form of ANP, which is selective for the ANP clearance receptor, also inhibited glucagon secretion. HS-42-1, a guanylate cyclase receptor antagonist, tended to reverse the effect of ANP on glucagon release. The data indicate that in the presence of ANP, the in vivo homeostasis of glucose, though plasma insulin levels are increased, is not due to an ANP-mediated increase in glucagon secretion; ANP has a complex inhibitory effect on glucagon release. The data further indicate that the ANP-induced inhibition of glucagon secretion probably involves the cGMP system, an inhibition of Ca2+ uptake and the involvement of PT-sensitive G-proteins. Moreover, an involvement of the clearance receptor seems to be likely. ANP is a valuable tool for investigating glucagon secretion from pancreatic islets because paracrine effects of insulin can be excluded.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins.

Recombinant immunotoxins (rITs) are highly specific anti-tumor agents composed of monoclonal antibody fragments or other specific carriers coupled to plant or bacterial toxins. A major problem in the purification of rITs is the low periplasmic yield in currently available expression systems. Thus, the aim of this study was the development of a new bacterial expression system for high-level production of rITs. We constructed a series of pET-based vectors for pelB-directed periplasmic secretion or cytoplasmic production under the control of the T7lac promoter. Expression in Escherichia coli BL21(DE3)pLysS allowed a tightly regulated isopropyl beta-d-thiogalactopyranoside (IPTG) induction of protein synthesis. An enterokinase-cleavable poly-histidine cluster was introduced into this setup for purification by affinity chromatography. A major modification resulted from the insertion of a specifically designed multiple cloning site. It contains only rare restriction enzyme recognition sites used for cloning of immunoglobulin variable region genes, as well as unique SfiI and NotI restriction sites for directed insertion of single-chain variable fragments (scFv) available from established bacteriophage systems. For this purpose, we deleted two naturally occurring internal SfiI consensus sites in a deletion mutant of Pseudomonas aeruginosa exotoxin A (ETA'). Each single structural element of the new vector (promoter, leader sequence, purification tag, scFv sequence, selectable marker, and toxin gene) was flanked by unique restriction sites allowing simple directional substitution. The fidelity of IPTG induction and high-level expression were demonstrated using an anti-CD30 scFv (Ki-4) fused to ETA'. These data confirm a bacterial vector system especially designed for efficient periplasmic expression of ETA'-based fusion toxins.

ADP Ribose Transferases↗

PubMed2001

Therapeutic administration of anti-PcrV F(ab')(2) in sepsis associated with Pseudomonas aeruginosa.

The effects of rabbit-derived polyclonal Ab against PcrV, a protein involved in the translocation of type III secreted toxins of Pseudomonas aeruginosa, was investigated in two animal models of P. aeruginosa sepsis. In a mouse survival study, the i.v. administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P. aeruginosa resulted in the complete survival of the animals. In a rabbit model of septic shock associated with Pseudomonas-induced lung injury, animals treated with anti-PcrV IgG intratracheally or i.v. had significant decreases in lung injury, bacteremia, and plasma TNF-alpha and significant improvement in the hemodynamic parameters associated with shock compared with animals treated in a similar manner with nonspecific control IgG. The administration of anti-PcrV F(ab')(2) showed protective effects comparable to those of whole anti-PcrV IgG. These results document that the therapeutic administration of anti-PcrV IgG blocks the type III secretion system-mediated virulence of P. aeruginosa and prevents septic shock and death, and that these protective effects are largely Fc independent. We conclude that Ab therapy neutralizing the type III secretion system has significant potential against lethal P. aeruginosa infections.

Animals↗

PubMed2004

[Detection of LOS-specific antibody-secreting cells by ELISPOT assay].

AIM: To detect dynamically the response of specific antibody-secreting cells elicited by a detoxified-lipooligosaccharide-cross-reactive mutant (dLOS-CRM) of diphtheria toxin conjugate vaccine for Moraxella catarrhalis (M.cat). METHODS: BALB/c mice were intranasally immunized with dLOS-CRM conjugate vaccine. The specific antibody-secreting cells responding to LOS of M. cat in different inductive and effective sites, including nasally associated lymphoid tissues (NALT), spleen, cervical lymph nodes (CLN), nasal passages (NP), lungs and Peyer's patches (PP) were detected by an enzyme-linked immunospot assay (ELISPOT). The levels of LOS-specific antibodies IgA, IgG and IgM in serum, nasal flush fluid, alveolar douche fluid, saliva and fecal extract were also detected by ELISA. RESULTS: Intranasal immunization with dLOS-CRM plus cholera toxin induced a significantly dose-dependent enhancement of immune response. LOS-specific antibody (IgA,IgG or IgM)-secreting cells were found in NALT, spleens, CLN, NP, lungs and PP with most LOS-specific IgA antibody-secreting cells located in nasal passages, and next, NALT and lungs. It was correlated well with the level of LOS-specific IgA, IgG or IgM antibody titers in nasal flush fluid, alveolar douche fluid, saliva, serum and fecal extract. CONCLUSION: dLOS-CRM can induce specific mucosal and systemic humoral immune response through intranasal immunization. ELISPOT assay is quick, sensitive, specific, and would be a very useful tool to analyze dynamically the mechanism of single antibody-secreting cell response.

Administration, Intranasal↗

PubMed1999

Infiltration of tumors by systemically transferred tumor-reactive T lymphocytes is required for antitumor efficacy.

The systemic transfer of ex vivo-activated tumor-sensitized T lymphocytes can mediate immunologically specific regression of established tumors. However, it has not been conclusively established whether the infiltration of systemically transferred T cells into metastases is required for their effector function. In this study, T cells from lymph nodes draining the murine fibrosarcoma MCA 205 cells were activated ex vivo with anti-CD3 monoclonal antibody and interleukin-2. During the final 24 h of culture, the T cells were treated with pertussis toxin (PTX) to inhibit signaling through G protein-coupled chemokine receptors required for diapedesis. Systemically transferred PTX-treated cells did not have any therapeutic efficacy against 3-day established pulmonary metastases. This lack of efficacy correlated with their failure to infiltrate the tumor parenchyma. However, PTX-treated cells responded to tumor antigen stimulation with IFN-gamma secretion in vitro. More importantly, PTX-treated effector T cells prevented tumor growth when they were admixed with tumor cells and inoculated s.c. These results demonstrate that systemically transferred tumor-reactive T lymphocytes need to infiltrate the tumor parenchyma through the endothelium to initiate tumor regression, but PTX-sensitive proteins are not required for either antigen recognition or effector functions.

Animals↗