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At least 793 records · Page 44Linked to original sources
PubMed1999

Differential regulation of Salmonella typhimurium type III secreted proteins by pathogenicity island 1 (SPI-1)-encoded transcriptional activators InvF and hilA.

Salmonella enterica encodes a type III protein secretion system within a pathogenicity island (SPI-1) that is located at centisome 63 of its chromosome. This system is required for the ability of these bacteria to stimulate cellular responses that are essential for their pathogenicity. Expression of components and substrates of this system is subject to complex regulatory mechanisms. These mechanisms involve the function of HilA and InvF, two transcriptional regulatory proteins encoded within SPI-1. In this study, we examined the functional relationship between these two regulatory proteins. We found that strains carrying loss-of-function mutations in either hilA or invF differ in their ability to stimulate cellular responses. An S. typhimurium hilA mutant strain retained considerable signaling capacity that resulted in significant levels of internalization into host cells. In contrast, introduction of a nonpolar loss-of-function mutation in invF rendered S. typhimurium significantly impaired in its ability to enter host cells. Consistent with these different phenotypes, we found that HilA and InvF control the expression of different genes. HilA regulates the expression of components of the type III secretion machinery, whereas InvF controls the expression of type III secreted proteins encoded outside of SPI-1. We also found that the expression of secreted proteins encoded within SPI-1 are under the control of both HilA and InvF. Our results therefore indicate that InvF and HilA differentially control the expression of components and substrates of the invasion-associated type III secretion system.

Bacterial Proteins↗
PubMed1998

[Renin-aldosterone system in secretion of cortisol in patients with arterial hypertension in European North].

Renin activity (RA), concentration of aldosterone and hydrocortisone in plasma were measured by radioimmunoassay in 78 males with arterial hypertension living in the Far North. RA and aldosterone concentrations were high in patients with borderline arterial hypertension irrespectively of hemodynamic type of the disease. Hydrocortisone levels in them were normal. In hypertension stage I and II RA and hydrocortisone concentrations were normal, while aldosterone levels have risen. Renin-aldosterone index showed high RA in all hemodynamic types of hypertension.

Adult↗
PubMed2000

Virulence factors of Helicobacter pylori responsible for gastric diseases in Mongolian gerbil.

Helicobacter pylori infection induces various gastroduodenal diseases. We examined the role of two genes, vacA and cagE, in the gastric pathogenesis induced by H. pylori using a long-term (62 wk) animal model. Reportedly, both genes are associated with the virulence of H. pylori: vacA encodes vacuolating cytotoxin, and cagE, with other genes in the cag pathogenicity islands, encodes a type IV secretion system. Mongolian gerbils were challenged in this study by a wild-type TN2 strain and its isogenic mutants of cagE or vacA. The wild-type and vacA mutants induced severe gastritis, whereas cagE mutants induced far milder changes. Gastric ulcer was induced at the highest rate (22/23) by the wild-type TN2, followed by the vacA mutant (19/28). No ulcer was found in the gerbils infected with the cagE mutant (0/27) or in controls (0/27). Intestinal metaplasia was also found in the gerbils infected with the wild-type (14/23) or vacA mutant (15/28). Gastric cancer developed in one gerbil with wild-type infection and in one with vacA mutant infection. In conclusion, the knocking out of the cagE gene deprived wild-type H. pylori of the pathogenicity for gastritis and gastric ulcer, suggesting that the secretion system encoded by cag pathogenicity island genes plays an essential role.

Adenocarcinoma↗
PubMed2001

cDNA-AFLP analysis unravels a genome-wide hrpG-regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria.

The Hrp type III protein secretion system is essential for pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria. Expression of the hrp gene cluster is controlled by HrpG, a two-component response regulator, and HrpX, an AraC-type transcriptional activator. Using the cDNA-AFLP technique, 30 hrpG-induced (hgi) and five hrpG-repressed (hgr) cDNA fragments were identified, defining a large hrpG-regulon in X. campestris pv. vesicatoria. Expression of most genes in the hrpG-regulon was dependent on hrpX. Seven cDNA fragments map to the known hrp gene cluster and flanking regions. All other genes appear to be scattered over the chromosome and endogenous plasmids. Sequence analysis identified genes encoding putative extracellular proteases, a putative transcriptional regulator and XopJ and XopB (Xanthomonas outer proteins), homologues of YopJ from Yersinia spp. and the avirulence protein AvrPphD of Pseudomonas syringae respectively. XopB is secreted by the Hrp type III secretion system. Analysis of deletion mutants in several hgi genes revealed a new virulence locus. This study demonstrates that cDNA-AFLP is a powerful tool to study prokaryotic transcriptomes and to identify genes contributing to Xanthomonas virulence and putative effector proteins.

Bacterial Proteins↗
PubMed2003

Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas.

Pseudomonas aeruginosa is the agent of opportunistic infections in immunocompromised individuals and chronic respiratory illnesses in cystic fibrosis patients. Pseudomonas aeruginosa utilizes a type III secretion system for injection of toxins into the host cell cytoplasm through a channel on the target membrane (the 'translocon'). Here, we have functionally and structurally characterized PopB and PopD, membrane proteins implicated in the formation of the P.aeruginosa translocon. PopB and PopD form soluble complexes with their common chaperone, PcrH, either as stable heterodimers or as metastable heterooligomers. Only oligomeric forms are able to bind to and disrupt cholesterol-rich membranes, which occurs within a pH range of 5-7 in the case of PopB/PcrH, and only at acidic pH for PcrH-free PopD. Electron microscopy reveals that upon membrane association PopB and PopD form 80 A wide rings which encircle 40 A wide cavities. Thus, formation of metastable oligomers precedes membrane association and ring generation in the formation of the Pseudomonas translocon, a mechanism which may be similar for other pathogens that employ type III secretion systems.

Amino Acid Sequence↗
PubMed1997

Synchronization by low-amplitude light-dark cycles of 24-hour pineal and plasma melatonin rhythms of hatchling European starlings (Sturnus vulgaris).

In young European starlings, as in other avian species, high-amplitude 24-hr rhythms in plasma and pineal melatonin are already present around the time of hatching. In chickens this rhythmicity results at least partly from the light sensitivity of the melatonin-producing and -secreting system. In contrast to the chicken, the starling is a hole-nesting bird, and it seemed questionable whether the low light intensities in the nest are sufficient to synchronize perinatal melatonin rhythms. We therefore exposed starling eggs to light cycles roughly simulating those measured in nest-boxes, i.e., an 11-hr phase of complete darkness and a 13-hr phase consisting of 15 min of dim light (10 lux) alternating with 30 min of darkness. For one group the photophase lasted from 0600 to 1900 hr; for the other group the photophase lasted from 1800 to 0700 hr. In approximately 10-hr-old hatchlings of both groups, plasma and pineal melatonin concentrations were high during the dark phase and low during the light phase. We conclude that perinatal low-amplitude light intensity changes of the kind experienced by hatching starlings in the field are sufficient for synchronizing the melatonin-producing and -secreting system in the pineal and possibly other organs.

Animals↗
PubMed2006

Differential expression of VirB9 and VirB6 during the life cycle of Anaplasma phagocytophilum in human leucocytes is associated with differential binding and avoidance of lysosome pathway.

Anaplasma phagocytophilum, an obligate intracellular bacterium, is the aetiologic agent of human granulocytic anaplasmosis (HGA). A. phagocytophilum virB/D operons encoding type IV secretion system are expressed in cell culture and in the blood of HGA patients. In the present study, their expression across the A. phagocytophilum intracellular developmental cycle was investigated. We found that mRNA levels of both virB9 and virB6 were upregulated during infection of human neutrophils in vitro. The antibody against the recombinant VirB9 protein was prepared and immunogold and immunofluorescence labelling were used to determine the VirB9 protein expression by individual organisms. Majority of A. phagocytophilum spontaneously released from the infected host cells poorly expressed VirB9. At 1 h post infection, VirB9 was not detectable on most bacteria associated with neutrophils. However, VirB9 was strongly expressed by A. phagocytophilum during proliferation in neutrophils. In contrast, with HL-60 cells, approximately 80% of A. phagocytophilum organisms associated at 1 h post infection expressed VirB9 protein and were colocalized with lysosome-associated membrane protein-1 (LAMP-1), whereas, VirB9-undetectable bacteria were not colocalized with LAMP-1. These results indicate developmental regulation of expression of components of type IV secretion system during A. phagocytophilum intracellular life cycle and suggest that bacterial developmental stages influence the nature of binding to the hosts and early avoidance of late endosome-lysosome pathway.

Anaplasma phagocytophilum↗
PubMed2006

Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity.

VirB8-like proteins are essential components of type IV secretion systems, bacterial virulence factors that mediate the translocation of effector molecules from many bacterial pathogens into eukaryotic cells. Based on cell biological, genetic, and x-ray crystallographic data, VirB8 was proposed to undergo multiple protein-protein interactions to mediate assembly of the translocation machinery. Here we report the results of a structure-function analysis of the periplasmic domain of VirB8 from the mammalian pathogen Brucella suis, which identifies amino acid residues required for three protein-protein interactions. VirB8 variants changed at residues proposed to be involved in dimerization, and protein-protein interactions were purified and characterized in vitro and in vivo. Changes at M102, Y105, and E214 affected the self-association as measured by analytical ultracentrifugation and gel filtration. The interaction with B. suis VirB10 was reduced by changes at T201, and change at R230 inhibited the interaction with VirB4 in vitro. The in vivo functionality of VirB8 variants was determined by complementation of growth in macrophages by a B. suis virB8 mutant and by using a heterologous assay of type IV secretion system assembly in Agrobacterium tumefaciens. Changes at Y105, T201, R230, and at several other residues impaired the in vivo function of VirB8, suggesting that we have identified interaction sites of relevance in the natural biological context.

Bacterial Proteins↗
PubMed1993

Functional connection between intracellular and extracellular secretion in species of Euphorbia genus.

In species of the Euphorbia genus the intracellular latex-secretion and the extracellular nectar-secretion are anatomically connected. The functional connection of the secretional systems was proved to be probable by TLC and GC-MSD. The chemical composition of Euphorbia latex and honey was examined with these analytical methods. In our research, the comparative chemical analysis of the latex and the honey of two Euphorbia species (Euphorbia cyparissias L., Euphorbia seguieriana Necker.) was discharged. These species are unusually good melliferous plants in Hungary. Four chemical components that can be found both in the latex and in the honey were detected with ultraviolet light (254 nm), and three general alkaloid reagents (Dragendorff, Meyer and 1% Ce(SO4)2 in 2n H2SO4) were detected by TLC. By means of mass spectrum generated by GC-MSD, the following compounds of Euphorbia honey were identified: butyl-2-methylpropyl phtalat, hexadecane acid, diheptyl phtalat, bis (2-ethyl-hexyl) phtalat, benzenedicarboxylic acid decyl-hexyl ester, benzenedicarboxylic acid isodecyl-octyl ester. The first compounds can also be found in the latex of both examined Euphorbia species. This partial correspondence in the composition of the latex and the honey led to results that suggest a further, functional connection between the two, anatomically connected secretional systems.

Honey↗
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↗
PubMed2003

Bordetella type III secretion induces caspase 1-independent necrosis.

The Bordetella bronchiseptica type III (TIII) secretion system induces cytotoxicity in infected macrophages and epithelial cells. In this report we characterize the cell death phenotype and compare it to the TIII-dependent cytotoxicity induced by Yersinia enterocolitica and Shigella flexneri. Bordetella bronchiseptica strain RB58 was able to induce cell death in J774A.1 macrophages with the same efficiency as Shigella and Yersinia, but only B. bronchiseptica was able to kill epithelial cells in a TIII-dependent manner. Primary macrophages from caspase 1-/- mice were susceptible to RB58-mediated killing, suggesting that unlike Shigella and Salmonella, caspase 1 does not mediate cell death. RB58-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor zVAD, and Western blot analyses of RB58-infected HeLa cells indicated that neither caspase 3 nor 7 was cleaved and PARP remained in its full-length active form. Morphologically the RB58-infected HeLa cells resembled necrotic rather than apoptotic cells, exhibiting cytoplasmic swelling and extensive membrane blebbing in the absence of nuclear changes. The addition of exogenous glycine, which has been shown to prevent necrotic cell death by blocking non-specific ion fluxes across the plasma membrane, blocked RB58-induced cytotoxicity. Addition of cyclosporin A which prevents the opening of the mitochondrial permeability pore, had no effect on RB58-infected cells. We conclude that the B. bronchiseptica TIII secretion system induces a mode of cell death consistent with necrosis that is distinct from that of Yersinia and Shigella.

Animals↗
PubMed2000

Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes.

Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria. The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA(s)) at the C-terminus, across both membranes of gram-negative bacteria. This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane. This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope. Suitable HlyA(s)-fused antigens are secreted with high efficiency by E. coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica. The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA. This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants. After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules. This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models.

Antigens, Bacterial↗
PubMed1994

An expression system for secretion and purification of a genetically engineered thermostable chimera of protein A and alkaline phosphatase.

A chimera between gene segments of Protein A and a mutated alkaline phosphatase (lysine328 mutated to alanine) of Escherichia coli has been constructed. This chimeric gene was cloned in a T7 promoter-based IPTG-inducible expression vector. The chimeric protein was expressed in E. coli and was efficiently secreted into the periplasm, from which it could be easily purified by a combination of ion-exchange and gel permeation chromatography. The purified chimera was found to be thermostable and exhibited both IgG binding and high alkaline phosphatase activity. It was used as a probe in enzyme-linked immunosorbent assays and results indicate that it is a promising substitute for secondary antibodies in enzyme-linked immunoassays.

Alkaline Phosphatase↗
PubMed2005

Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone.

Human parathyroid hormone (hPTH) is involved in calcium metabolism, and the unique ability of this hormone to stimulate bone growth makes it a promising agent in the treatment of osteoporosis. We have engineered the methylotrophic yeast Pichia pastoris for the production of over 300 mg intact hPTH per liter growth medium. The presence of 10 mM EDTA in the culture medium was essential to obtain this high hormone yield, indicating that metallopeptidases are mainly responsible for the otherwise instability of hPTH. Furthermore, the secretion process of hPTH was considerably improved by coexpression of Saccharomyces cerevisiae protein disulphide isomerase (ScPDI). Since hPTH does not contain any cystein residues, this effect may be indirect or ascribed to the chaperone activity of PDI. Contrary to the situation in S. cerevisiae, use of a protease-deficient host strain provided no additional advantage. The hormone secreted by P. pastoris was not subjected to proteolytic processing by Kex2p in the two internal tribasic sites, nor were any C-terminal truncated hPTH forms detected. However, the P. pastoris hPTH producing transformants cosecreted ubiquitin to the culture medium, possibly as a result of a stress-related response.

Cell Culture Techniques↗
PubMed2003

Differential participation of actin- and tubulin-based vesicle transport systems during secretion in bovine chromaffin cells.

The role of cytoskeletal elements in vesicle transport occurring during exocytosis was examined in adrenal medullary bovine chromaffin cells maintained in culture. Amperometric determination of depolarization-dependent catecholamine release from individual intact cells treated with actin or myosin inhibitors showed alterations in the fast and slow phases of secretion when compared with untreated cells. In contrast, microtubule disassemblers or stabilizers have a moderate effect on secretion, only affecting the release of slow secretory components. In experiments using confocal dynamic microscopy we have observed the drastic effect of actin and myosin inhibitors in abolishing vesicle movement throughout the cytoplasm, and the inhibition of granule mobility in deep perinuclear regions caused by the microtubule stabilizers. Following loss of mobility, vesicles were associated with filaments of F-actin or microtubules. In addition, the mobility of cortical vesicles was affected by actin-myosin inhibitors but not by microtubule inhibitors. The study of cortical cytoskeleton in living cells showed vesicles associated with dense tubular F-actin structures, with microtubules appearing as low density networks. These findings suggest that the distribution and density of both cytoskeletal elements in the cortical region may influence the recruitment of vesicle pools during secretion.

Actins↗