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PubMed2005

Functional analysis of early secreted antigenic target-6, the dominant T-cell antigen of Mycobacterium tuberculosis, reveals key residues involved in secretion, complex formation, virulence, and immunogenicity.

Proteins of the 6-kDa early secreted antigenic target (ESAT-6) secretion system-1 of Mycobacterium tuberculosis are not only strongly involved in the anti-mycobacterial Th1-host immune response but are also key players for virulence. In this study, protein engineering together with bioinformatic, immunological, and virulence analyses allowed us to pinpoint regions of the ESAT-6 molecule that are critical for its biological activity in M. tuberculosis. Mutation of the Trp-Xaa-Gly motif, conserved in a wide variety of ESAT-6-like proteins, abolished complex formation with the partner protein CFP-10, induction of specific T-cell responses, and virulence. Replacement of conserved Leu residues interfered with secretion, coiled-coil formation, and virulence, whereas certain mutations at the extreme C terminus did not affect secretion but caused attenuation, possibly because of altered ESAT-6 targeting or trafficking. In contrast, the mutation of several residues on the outer surface of the four-helical bundle structure of the ESAT-6.CFP-10 complex showed much less effect. Construction of recombinant BCG expressing ESAT-6 with a C-terminal hexahistidine tag allowed us to co-purify ESAT-6 and CFP-10, experimentally confirming their strong interaction both in and outside of the mycobacterial cell. The strain induced potent, antigen-specific T-cell responses and intermediate in vivo growth in mice, suggesting that it remained immunogenic and biologically active despite the tag. Together with previous NMR data, the results of this study have allowed a biologically relevant model of the ESAT-6.CFP-10 complex to be constructed that is critical for understanding the structure-function relationship in tuberculosis pathogenesis.

Antigens, Bacterial↗
PubMed1974

Colchicine-induced inhibition of lipoprotein and protein secretion into the serum and lack of interference with secretion of biliary phospholipids and cholesterol by rat liver in vivo.

Rats were injected with colchicine and the secretion of triglycerides into the serum was studied for 90 min after injection of [(14)C]palmitic acid and Triton WR 1339. The release of labeled and chemically determined triglyceride was reduced to about 20-30% of control values. The effect of colchicine on serum triglyceride levels was not dependent on the presence of Triton and was similar in males and females and in fed and fasted rats. The effect was dose dependent and was reversible 6-7 h after injection of 0.05 mg/100 g body weight. Colchicine inhibited also the release of labeled proteins into the serum but did not affect the amount of [(3)H]leucine incorporated into liver proteins. Within 4 h of colchicine treatment there was an 80% fall in serum very low density lipoproteins (VLDL), a 30% fall in serum high density lipoproteins (HDL), and no change in the d > 1.21 protein level, but reduction in the appearance of labeled proteins was encountered in all serum fractions. Colchicine had no effect on the rate of bile flow and on the secretion of phospholipids and cholesterol into the bile. In the hepatocyte there was accumulation of Golgi-derived secretory vesicles, containing nascent VLDL particles; these vesicles were seen also in the vicinity of the sinusoidal cell surface, but the space of Disse contained few or no VLDL particles. There was an apparent reduction in microtubules and some increase in microfilaments. It is suggested that microtubules affect the secretion of lipoproteins and proteins into the serum by maintaining the organization of the plasma membrane required for its fusion with secretory vesicles. The lack of effect of colchicine on biliary lipid secretion indicates that the latter is not dependent on vesicular transport.

Animals↗
PubMed1976

The modulation of lymphocyte functions by molecules secreted by macrophages. II. Conditions leading to increased secretion.

Cultures of peritoneal exudate cells rich in macrophages were studied for the secretion of lymphostimulatory molecules. Two conditions produced increased secretion: (a) addition to the cultures of various agents that readily interacted with macrophages, such as latex particles, antibody-coated red cells, endotoxin, Listeria organisms, or Be salt; or (b) addition of activated lymphocytes. In the first case the increased activity was found during the first 24 or 48 h after uptake of the stimuli. Increased activity was found in normal or peptone-stimulated macrophages but not in macrophages after injection of endotoxin or thioglycollate. The addition of T lymphocytes from Listeria-infected mice to macrophage cultures increased greatly the activities. This increase was also produced by addition to antigen-primed T cells together with antigen. The lymphocytes by themselves did not secrete active factors. The lymphostimulatory activities were tested on thymocyte DNA synthesis and on antibody formation in vitro. The latter assay was done on spleen cells from immunized mice where one striking effect was the stimulation of differentiation to antibody-secreting cells. Some dissociation of both activities (thymocyte DNA synthesis and B-cell differentiation) was observed with selected culture fluids.

Animals↗
PubMed1988

C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha.

We have demonstrated that purified C5a is a potent stimulus to human PBMC secretion of TNF-alpha, IL-1 beta, and IL-1 alpha, which proceeds in a dose-dependent fashion. At a given concentration of C5a, TNF-alpha and IL-1 beta secretion did not differ significantly; both were secreted in significantly greater quantity than IL-1 alpha. Clinical conditions such as Gram-positive and Gram-negative bacterial infections, trauma, and immune complex diseases activate complement. Through the mediation of TNF and IL-1 secreted in response to C5a, these diverse disorders can share common features of fever, coagulopathy, acute phase protein production, and disordered metabolism.

Complement C5↗
PubMed2000

Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion.

Escherichia coli K-12, the most widely used laboratory bacterium, does not secrete proteins into the extracellular medium under standard growth conditions, despite possessing chromosomal genes encoding a putative type II secretion machinery (secreton). We show that in wild-type E.coli K-12, divergent transcription of the two operons in the main chromosomal gsp locus, encoding the majority of the secreton components, is silenced by the nucleoid-structuring protein H-NS. In mutants lacking H-NS, the secreton genes cloned on a moderate-copy-number plasmid are expressed and promote efficient secretion of the endogenous, co-regulated endochitinase ChiA. This is the first time that secretion of an endogenous extracellular protein has been demonstrated in E.coli K-12.

Bacterial Proteins↗
PubMed1991

Protein secretion by the mouse blastocyst: differences in the polypeptide composition secreted into the blastocoel and medium.

To initiate studies on the protein microenvironment of the mouse blastocoel, we examined the electrophoretic profile of newly synthesized proteins secreted into the blastocoel, as well as those secreted into the medium. Although most polypeptides reveal no relative enrichment, some proteins (e.g., proteins of Mr = 155,000 and 33,000) are enriched in the blastocoel relative to those secreted apically into the medium. In addition, some proteins (e.g., proteins of Mr = 102,000 and 40,000) are enriched in the medium relative to the blastocoel. The relative amount of newly synthesized protein secreted into the blastocoel is about 2.5% of total protein synthesis. In addition, the trophectoderm and inner cell mass contribute to these proteins. Results of these studies suggest a potential function for these blastocoel-enriched proteins in inner cell mass development.

Animals↗
PubMed2006

Digestive calcitonin-secreting tumors of the foregut: comparison with non-calcitonin-secreting tumors.

OBJECTIVES: Digestive calcitonin-secreting endocrine tumors are very rare lesions of the foregut. This study was undertaken to compare the characteristics and the prognosis of these tumors and those of non-calcitonin-secreting endocrine tumors. METHODS: All patients with a digestive endocrine tumor of the foregut followed up in Reims University Hospital and whose serum calcitonin levels were determined between 1988 and 2004 were included. Clinical and tumor characteristics of calcitonin-positive and calcitonin-negative patients were compared. RESULTS: Thirty-two patients were included. Among the five (15.6%) with high calcitonin levels (median: 340 pg/ml, range: 42-7460 pg/ml), only one tumor was functioning (diarrhea). Significant differences between patients with positive and negative calcitonin levels were, respectively: liver metastases [5 (100%) versus 11 (40.7%); P=0.04], type according to the World Health Organization 2000 histological classification [notably 4 (80%) versus 3 (12.5%) poorly differentiated endocrine carcinomas; P=0.02] and Ki67 proliferation index [median: 25% (range: 20-30%) versus 7% (0-80%); P=0.03]. The only calcitonin-positive well-differentiated endocrine carcinoma had a high proliferation index (30%). Survival also differed significantly (P=0.001), as all calcitonin-positive patients died, with a median survival of 22.6 months (range: 1.2-27.2 months), versus five (18.5%) calcitonin-negative patients. Median follow-up period for the latter was 42.3 months (range: 3.4-208 months). CONCLUSIONS: The secretion of calcitonin appears predictive of a poor prognosis. Digestive endocrine calcitonin-secreting tumors correspond histopathologically to poorly differentiated or well-differentiated carcinomas with high proliferation indexes.

Adolescent↗
PubMed1990

The secreted aspartate proteinase of Candida albicans: physiology of secretion and virulence of a proteinase-deficient mutant.

It was established that Candida albicans grew rapidly in a simple medium containing yeast extract (0.2%, w/v) plus glucose (2%, w/v). These cultures were in or near to a state of nitrogen limitation and the concentration of secreted aspartate proteinase increased rapidly (within 3-4 h) on addition of BSA. Synthesis and secretion were apparently controlled both positively (induction by albumin or, more probably, the peptides produced from it) and negatively (repression by NH4Cl). A small intracellular pool of the enzyme was detected during production of the enzyme and this pool decreased with the cessation of synthesis and secretion. A stable mutant, IR24, was isolated which secreted less than 0.3% of the amount of the proteinase exported by the parent strain ATCC 10261. The LD50 values for mutant IR24 and the parent strain administered intravenously to mice were greater than 1.0 x 10(9) and 1.6 x 10(6) c.f.u. kg-1 respectively.

Animals↗
PubMed2004

SsaM and SpiC interact and regulate secretion of Salmonella pathogenicity island 2 type III secretion system effectors and translocators.

The type III secretion system (TTSS) encoded by Salmonella Pathogenicity Island 2 (SPI-2) is required for systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. The SPI-2 TTSS is activated after internalization of bacteria by host cells, and translocates effector proteins into and across the vacuolar membrane, where they interfere with several host cell functions. Here, we investigated the function of SsaM, a small protein encoded within SPI-2. An ssaM deletion mutant had virulence and intracellular replication defects comparable to those of a SPI-2 TTSS null mutant. Although the ssaM mutant was able to secrete the effector protein SseJ in vitro, it failed to translocate SseJ into host cells, and to secrete the translocon proteins SseB, SseC and SseD in vitro. This phenotype is similar to that of a strain carrying a mutation in the SPI-2 gene spiC, whose product is reported to be an effector involved in trafficking of the Salmonella vacuole in macrophages. Both ssaM and spiC mutants were found to oversecrete the SPI-2 effector proteins SseJ and PipB in vitro. Fractionation assays and immunofluorescence microscopy were used to investigate the localization of SsaM and SpiC in macrophages. No evidence for translocation of these proteins was obtained. The similar phenotypes of the ssaM and spiC mutants suggested that they might be involved in the same function. Pull-down and co-immune precipitation experiments showed that SpiC and SsaM interact within the bacterial cell. We propose that a complex involving SsaM and SpiC distinguishes between translocators and effector proteins, and controls their ordered secretion through the SPI-2 TTSS.

Animals↗
PubMed2005

A secreted anti-activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri.

Bacteria of Shigella spp. are responsible for shigellosis in humans and use a type III secretion (TTS) system to enter epithelial cells and trigger apoptosis in macrophages. Transit of translocator and effector proteins through the TTS apparatus is activated upon contact of bacteria with host cells. Transcription of approximately 15 genes encoding effectors is regulated by the TTS apparatus activity and controlled by MxiE, an AraC family activator, and its coactivator IpgC, the chaperone of IpaB and IpaC translocators. Using a genetic screen, we identified ospD1 as a gene whose product negatively controls expression of genes regulated by secretion activity. OspD1 associates with the chaperone Spa15 and the activator MxiE and acts as an anti-activator until it is secreted. The mechanism regulating transcription in response to secretion activity involves an activator (MxiE), an anti-activator (OspD1), a co-anti-activator (Spa15), a coactivator (IpgC) and two anti-coactivators (IpaB and IpaC) whose alternative and mutually exclusive interactions are controlled by the duration of the TTS apparatus activity.

Bacterial Proteins↗
PubMed2005

The Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion.

Activation of bacterial virulence-associated type III secretion systems (T3SSs) requires direct contact between a bacterium and a eukaryotic cell. In Yersinia pestis, the cytosolic LcrG protein and a cytosolic YopN-TyeA complex function to block T3S in the presence of extracellular calcium and prior to contact with a eukaryotic cell. The mechanism by which the bacterium senses extracellular calcium and/or cell contact and transmits these signals to the cytosolic compartment is unknown. We report here that YscF, a small protein that polymerizes to form the external needle of the T3SS, is essential for the calcium-dependent regulation of T3S. Alanine-scanning mutagenesis was used to identify YscF mutants that secrete virulence proteins in the presence and absence of calcium and prior to contact with a eukaryotic cell. Interestingly, one of the YscF mutants that exhibited constitutive T3S was unable to translocate secreted proteins across the eukaryotic plasma membrane. These data indicate that the YscF needle is a multifunctional structure that participates in virulence protein secretion, in translocation of virulence proteins across eukaryotic membranes and in the cell contact- and calcium-dependent regulation of T3S.

Bacterial Outer Membrane Proteins↗
PubMed2006

Surface anchoring of a bacterial adhesin secreted by the two-partner secretion pathway.

In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. In the present study we sought to determine the mechanism by which HMW1 is anchored to the bacterial surface. In initial experiments we found that HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. Further analysis established that HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Mutagenesis and polyethylene glycol maleimide labelling revealed that anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region. Immunolabelling studies demonstrated that the immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains in the periplasm or is buried in HMW1B. Coexpression of HMW1 lacking the C-terminal 20 amino acids and wild-type HMW1 supported the conclusion that the C-terminus of HMW1 occupies the HMW1B pore. These observations may have broad relevance to proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.

Adhesins, Bacterial↗
PubMed1999

Cytokine secretion of periodontal ligament fibroblasts derived from human deciduous teeth: effect of mechanical stress on the secretion of transforming growth factor-beta 1 and macrophage colony stimulating factor.

The periodontal ligament may play an important role in tooth eruption, root development and resorption. The tissue physiologically receives mechanical force during mastication. We focused on the effects of intermittent mechanical strain on the cytokine synthesis of periodontal ligament (PDL) fibroblasts in vitro. The cells were derived from human periodontal ligament of deciduous teeth (HPLF-Y) and permanent teeth (HPLF). The two kinds of PDL cells and human gingival fibroblasts (HGF) were cultured in flexible bottomed culture plates. The cells were mechanically stretched at 5% elongation, 3-cycles/min for 24 h on d 7 in culture using a Flexercell strain unit. After the stretching, we measured DNA content and alkaline phosphatase activity in the cell layer, transforming growth factor beta 1 (TGF-beta 1) and macrophage colony stimulating factor (M-CSF) contents in the conditioned medium. The TGF-beta 1 level in the conditioned medium of HPLF was significantly higher than that of HPLF-Y and HGF. It was stimulated by mechanical stretching only on HPLF, whereas no significant effect was observed on HPLF-Y and HGF. M-CSF secretion was inhibited by the stretching on all of HPLF, HPLF-Y and HGF. 1 alpha, 25 dihydroxy vitamin D3 (D3) stimulated M-CSF secretion into the culture medium of both HPLF and HPLF-Y, but the stretching inhibited M-CSF secretion and completely blocked the enhancement by D3. These data suggest that periodontal ligament cells synthesize and secrete the molecules as autocrine or paracrine factors that affect bone remodelling and root resorption and the level of those factors change in response to mechanical stress.

Alkaline Phosphatase↗
PubMed1989

Modification of 24-hour growth hormone secretion after continuous subcutaneous infusion of growth hormone-releasing hormone (GHRH (1-29)NH2) in short children with low 24-hour growth hormone secretion.

Six short children with low 24-hour growth hormone (GH) secretion were treated with continuous subcutaneous infusion of GHRH (1-29)NH2 for 3 weeks using a portable infusion pump. Restoration of pulsatile GH secretion was observed in all three children treated with 40 micrograms/kg/day of GHRH, but in only one of the three children treated with 20 micrograms/kg/day. All parameters of 24-hour GH secretion increased, but in five children the magnitude of the GH response was greater on day 1 than on day 21 of GHRH treatment. This decrease was not observed in the single child who responded to a low dose of GHRH (20 micrograms/kg/day); on the contrary, the response in this patient was greater after 21 days of GHRH treatment. Plasma levels of GHRH (1-29)NH2 were significantly higher on day 21 than on day 1 of treatment, suggesting altered pharmacokinetics over time. The effect of GHRH treatment on growth could not be determined because of the short duration of the study, but the data obtained on 24-hour GH secretion and GHRH metabolism suggest that a long-acting analogue of GHRH could be useful for the treatment of GH deficient or insufficient children.

Adolescent↗
PubMed1982

Stimulus-secretion coupling in rat mast cells: inactivation of extracellular calcium dependent secretion.

1. Stimulation by compound 48/80 of mast cells deprived of Ca failed to release histamine. Secretion of histamine was elicited from such cells by the subsequent introduction of Ca. 2. Histamine secretion declined as the interval between stimulation by compound 48/80 and the introduction of Ca increased. This decline is called inactivation. 3. The addition of the ionophore, A23187, with Ca restored maximum histamine secretion overcoming inactivation. 4. Increasing the concentration of Ca introduced after stimulation, from 2 to 8 mM, or to 20 mM reduced the amount of histamine released. This reduction was proportional to the interval between stimulation and the introduction of Ca. The addition of A23187 with the higher concentrations of Ca fully restored histamine secretion. 5. Stimulation of mast cells in Ca-free media by the secretagogues polymyxin B or bradykinin, and the subsequent introduction of Ca, resulted in a similar inactivation or decline in histamine release. 6. Mast cells inactivated by compound 48/80 stimulation in Ca-free media showed no increase in histamine release when the secretagogues polymyxin B plus Ca or bradykinin plus Ca were added. However, when A23187 plus Ca was added, a full secretory response was obtained. 7. It is suggested that the process of inactivation involves time-dependent change in the Ca permeability of the mast cell membrane. The concentration of introduced Ca is suggested to influence the regulation of this permeability.

Animals↗
PubMed1994

Production and Secretion of the Polysaccharide Biodispersan of Acinetobacter calcoaceticus A2 in Protein Secretion Mutants.

Biodispersan is an extracellular anionic polysaccharide produced by Acinetobacter calcoaceticus A2 that changes the surface properties of limestone and acts both as a dispersant and as a grinding aid (E. Rosenberg, C. Rubinovitz, A. Gottlieb, S. Rosenhak, and E. Z. Ron, Appl. Environ. Microbiol. 54:317-322, 1988; E. Rosenberg, C. Rubinovitz, R. Legmann, and E. Z. Ron, Appl. Environ. Microbiol. 54:323-326, 1988; E. Rosenberg, Z. Schwartz, A. Tenenbaum, C. Rubinovitz, R. Legmann, and E. Z. Ron, J. Dispersion Sci. Technol. 10:241-250, 1989). Extracellular fluid also contains a high concentration of secreted proteins that create problems in the purification and application of biodispersan. In order to obtain preparations of biodispersan that contained smaller amounts of protein, we selected mutants of strain A2 that were defective in protein secretion. These mutants produced equal, or even higher, levels of total biodispersan compared with those of the parental strain. Moreover, although there was a significant drop in the concentration of extracellular proteins in the medium, the secretion of biodispersan was unaffected. These results suggest that secretion mutants are potentially useful for the production of extracellular polysaccharides.

Journal Article↗
PubMed1994

Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2.

We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively. Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these transconjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively.

Alcaligenes↗
PubMed2003

Functional and morphological development of lymphoid tissues and immune regulatory and effector function in rhesus monkeys: cytokine-secreting cells, immunoglobulin-secreting cells, and CD5(+) B-1 cells appear early in fetal development.

Little is known regarding the timing of immune ontogeny and effector function in fetal humans and nonhuman primates. We studied the organization of lymphocyte and antigen-presenting cell populations in developing lymphoid tissues of rhesus monkey fetuses during the second and third trimesters (65 to 145 days of gestation; term = 165 days). Immunoglobulin-secreting and cytokine-secreting cells were detected at day 80. The thymus, spleen, lymph nodes, and intestinal mucosa were examined for cells expressing CD3, CD5, CD20, CD68, p55, and HLA-DR. In the spleens of 65-day-old fetuses (early second trimester), the overwhelming majority of total lymphocytes were CD5(+) CD20(+) B-1 cells. The remaining lymphocytes were CD3(+) T cells. By day 80, splenic B and T cells were equal in number. Intraepithelial CD3(+) CD5(-) T cells and lamina propria CD20(+) CD5(+) B cells were present in the intestines of 65-day-old fetuses. By day 80, numerous CD20(+) CD5(+) B cells were present in the jejunums and colons and early lymphocyte aggregate formation was evident. The spleens of 80- to 145-day-old fetuses contained immunoglobulin M (IgM)-secreting cells, while IgA-, IgG-, interleukin-6-, and gamma interferon-secreting cells were numerous in the spleens and colons. Thus, by the second trimester, the lymphoid tissues of the rhesus monkey fetus have a complete repertoire of properly organized antigen-presenting cells, T cells, and B cells.

Animals↗