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Results for “toxin secretion system”

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PubMed1978

Effect of heat stable and heat labile Escherichia coli enterotoxins and cholera toxin in combination with theophylline on unidirectional sodium and chloride flux in the small intestine of weanling swine.

The effect of heat stable and heat labile Escherichia coli enterotoxins or cholera toxin in combination with theophylline on net water, sodium and chloride and unidirectional sodium and chloride fluxes was examined in acute isolated loops of jejunum of weanling swine. The effect of heat stable enterotoxin in combination with theophylline was determined in loops located in the proximal jejunum, while combinations of theophylline and either heat labile enterotoxin or cholera toxin were studied in the distal jejunum. In each situation the addition of theophylline resulted in an additive rather than a synergistic increment of intestinal secretory activity. This study implies that the intestinal adenyl cyclase system and enterotoxin induced intestinal secretion may not be directly related in the swine small intestine.

Animals↗

PubMed1988

GTP-dependent inhibition of insulin secretion by epinephrine in permeabilized RINm5F cells. Lack of correlation between insulin secretion and cyclic AMP levels.

The mechanism by which alpha 2-adrenergic agonists inhibit exocytosis was investigated in electrically permeabilized insulin secreting RINm5F cells. In this preparation alpha 2-adrenoceptors remain coupled to adenylate cyclase, since basal- and forskolin-stimulated cyclic AMP production was lowered by epinephrine and clonidine by 30-50%. Cyclic AMP levels did not correlate with the rate of insulin secretion. Thus, at low Ca2+, forskolin enhanced cyclic AMP levels 5-fold without eliciting secretion, and Ca2+-stimulated secretion was associated with decreased cyclic AMP accumulation. Epinephrine (plus propranolol) inhibited Ca2+-induced insulin secretion in a GTP-dependent manner. The maximal inhibition (43%) occurred at 500 microM GTP. Clonidine also inhibited Ca2+-stimulated secretion. Replacement of GTP by GDP or by the nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate as well as treatment of the cells with pertussis toxin prior to permeabilization abolished epinephrine inhibition of insulin secretion. Pertussis toxin did not affect Ca2+-stimulated secretion. Insulin release stimulated by 1,2-didecanoyl glycerol was also lowered by epinephrine suggesting an effect distal to the activation of protein kinase C (Ca2+/phospholipid-dependent enzyme). These results taken together with the ability of epinephrine to inhibit ionomycin-induced insulin secretion in intact cells suggest that alpha 2-adrenergic inhibition is distal to the generation of second messengers. A model is proposed for alpha 2-adrenoceptor coupling to two effector systems, namely the adenylate cyclase and the exocytotic site in insulin-secreting cells.

Adenylate Cyclase Toxin↗

PubMed2003

The ADP-ribosylating toxin, AexT, from Aeromonas salmonicida subsp. salmonicida is translocated via a type III secretion pathway.

AexT is an extracellular ADP ribosyltransferase produced by the fish pathogen Aeromonas salmonicida subsp. salmonicida. The protein is secreted by the bacterium via a recently identified type III secretion system. In this study, we have identified a further 12 open reading frames that possess high homology to genes encoding both structural and regulatory components of the Yersinia type III secretion apparatus. Using marker replacement mutagenesis of aopB, the A. salmonicida subsp. salmonicida homologue of yopB in Yersinia, we demonstrate that the bacterium translocates the AexT toxin directly into the cytosol of cultured fish cells via this type III secretion pathway. An acrV mutant of A. salmonicida subsp. salmonicida displays a calcium-blind phenotype, expressing and secreting significant amounts of AexT even in the presence of CaCl2 concentrations as high as 10 mM. This acrV mutant is also unable to translocate AexT into the cytosol of fish cells, indicating AcrV is involved in the translocation process. Inactivation of either the aopB or acrV gene in A. salmonicida subsp. salmonicida (resulting in an inability to translocate AexT) is accompanied by a loss of cytotoxicity that can be restored by trans complementation. Finally, we present data indicating that preincubation of the wild-type bacteria with antibodies directed against recombinant AcrV-His protein provides fish cells protection against the toxic effects of the bacterium.

ADP Ribose Transferases↗

PubMed1986

Oxidative inactivation of pneumolysin by the myeloperoxidase system and stimulated human neutrophils.

Pneumolysin, a hemolytic toxin from Streptococcus pneumoniae, is a member of the group of thiol-activated, oxygen-labile cytolysins produced by various Gram-positive bacteria. The toxin activity of pneumolysin, as determined by lysis of 51Cr-labeled human erythrocytes, was destroyed on exposure to the neutrophil enzyme myeloperoxidase, hydrogen peroxide, and a halide (chloride or iodide). Detoxification required each component of the myeloperoxidase system and was prevented by the addition of agents that inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The entire myeloperoxidase system could be replaced by sodium hypochlorite at micromolar concentrations. Toxin inactivation was a function of time of exposure to the myeloperoxidase system (less than 1 min), the rate of formation of H2O2 (0.05 nmol/min), and the concentration of toxin employed. Toxin that had been inactivated by the myeloperoxidase system was reactivated on incubation with the reducing agent dithiothreitol. Pneumolysin was also inactivated when incubated with human neutrophils (10(5)) in the presence of a halide and phorbol myristate acetate, an activator of neutrophil secretion and oxygen metabolism. Toxin inactivation by stimulated neutrophils was blocked by azide, cyanide, or catalase, but not by superoxide dismutase. Neutrophils from patients with impaired oxygen metabolism (chronic granulomatous disease) or absent myeloperoxidase (hereditary deficiency) failed to inactivate the toxin unless they were supplied with an exogenous source of H2O2 or purified myeloperoxidase, respectively. Thus, inactivation of pneumolysin involved the secretion of myeloperoxidase and H2O2, which combined with extracellular halides to form agents (e.g., hypochlorite) capable of oxidizing the toxin. This example of oxidative inactivation of a cytolytic agent may serve as a model for phagocyte-mediated detoxification of microbial products.

Bacterial Proteins↗

PubMed1990

Interrelationship between signals transduced by phytohemagglutinin and interleukin 1.

In the murine cell line LBRM-331A5, phytohemagglutinin (PHA) induces secretion of the T cell growth factor interleukin 2 (IL2). IL1 augments PHA-induced IL2 production. In this cell line, PHA stimulates a number of biochemical changes including phospholipid hydrolysis, increases in cytosolic free calcium [( Ca2+]i), membrane hyperpolarization, cytosolic alkalinization, and tyrosine phosphorylation of specific substrates. Using LBRM cells, we have studied the interrelationship between these events and the secretion of IL2. Increases in [Ca2+]i triggered by PHA or following addition of ionomycin result in membrane hyperpolarization but are not required for PHA-induced cytosolic alkalinization or tyrosine phosphorylation. Addition of IL1 to PHA-stimulated cells did not affect any of the biochemical parameters, although it significantly augmented PHA-induced IL2 secretion. Increasing [Ca2+]i with ionomycin did not trigger IL2 secretion, increases in cytosolic pH, or tyrosine phosphorylation in the presence or absence of IL1. Preventing increases in cytosolic pH did not alter PHA-induced changes in [Ca2+]i or membrane potential. These data are compatible with PHA including activation of phospholipase C and production of inositol phosphates resulting in both release of Ca2+ from internal stores and transmembrane uptake of Ca2+ as well as activation of protein kinase C. However, unlike other growth factor or mitogen-stimulated systems, the changes stimulated by PHA and IL1 in LBRM cells including IL2 secretion are not regulated by a pertussis toxin-sensitive G protein.

Adenosine Diphosphate Ribose↗

PubMed2002

Lectin binding patterns of alveolar epithelium and subepithelial seromucous glands of the bronchi in sepsis and controls--an approach to characterize the non-specific immunological response of the human lung to sepsis.

A delayed or deficient immunological protection as well as an overstimulation of the mucosal immune system may act as possible additional promoters of sepsis-induced lung injury in patients suffering from a severe septic condition. Lectin-binding patterns in pulmonary tissue samples obtained at autopsy from septic patients and control individuals were studied using 11 carbohydrate-specific lectins (Con A, UEA, GSA I, GSA II, MPA, PNA, Jac, WGA, MAA, LPA, and SNA). There were no differences in the secretory product of serous parts of bronchial glands detectable in the two study groups, whereas lectin binding patterns of alveolar epithelium and mucous parts of subepithelial seromucous glands were different in sepsis cases when compared to controls. Apart from differences in binding sites for alpha-mannose, N-acetyl-neuraminic acid and alpha-(2-6)-galactose (as detected by different expression for Con A, MAA and SNA) in the two study groups, the main finding was that no binding sites for alpha-N-acetyl-galactosamine (as investigated by MPA immunoreactivity) could be detected on alveolar epithelial cells and mucous parts of subepithelial seromucous glands in sepsis cases in contrast to the presence of such binding sites in the control cases. We hypothesize that the finding of an altered secretory product of alveolar epithelial cells and bronchial glands in sepsis may be a result of specific carbohydrate deprivation or consumption, respectively, possibly due to direct bacterial effects or pathogenetic events in response to bacterial toxins during the complex cascade of the host's systemic inflammatory response in sepsis. The altered type of mucus glycoprotein physiologically secreted by alveolar epithelium and mucous parts of subepithelial seromucous glands of the bronchi with subsequent loss of a considerable proportion of protection of the mucosal barrier in sepsis may play an important additional role in the development of sepsis-induced lung injury.

Adult↗

PubMed2005

Expression and purification of Pseudomonas aeruginosa SecA N-terminal domain: stimulation of ATPase activity of the SecAL43P mutant protein.

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium which secretes a wide range of hydrolytic enzymes, toxins, and virulence factors into the extracellular medium. Although P. aeruginosa possesses numerous specific systems for the export of proteins across its double-membrane envelopes, the Sec system is still the major and essential mechanism. However, very little is known about its molecular basis. We constructed, cloned, and expressed the N-terminal 236 amino acids of PaSecA domain (PaSecAN236), and SecAL43P mutants of P. aeruginosa in Escherichia coli BL21.19 (secA(ts)). Here, we describe the purification of PaSecAN236 by using osmotic shock as the first step to efficiently release targeted protein from cells, followed by cation-exchange and size exclusion columns to obtain homogeneous PaSecAN236. The purified PaSecA N-terminal domain was functional in stimulating the ATPase activity of mutant SecAL43P protein of P. aeruginosa.

Adenosine Triphosphatases↗

PubMed2006

Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids.

EpsE is a cytoplasmic component of the type II secretion system in Vibrio cholerae. Through ATP hydrolysis and an interaction with the cytoplasmic membrane protein EpsL, EpsE supports secretion of cholera toxin across the outer membrane. In this study, we have determined the effect of the cytoplasmic domain of EpsL (cyto-EpsL) and purified phospholipids on the ATPase activity of EpsE. Acidic phospholipids, specifically cardiolipin, bound the copurified EpsE/cyto-EpsL complex and stimulated its ATPase activity 30-130-fold, whereas the activity of EpsE alone was unaffected. Removal of the last 11 residues (residues 243-253) from cyto-EpsL prevented cardiolipin binding as well as stimulation of the ATPase activity of EpsE. Further mutagenesis of the C-terminal region of the EpsL cytoplasmic domain adjacent to the predicted transmembrane helix suggested that this region participates in fine tuning the interaction of EpsE with the cytoplasmic membrane and influences the oligomerization state of EpsE thereby stimulating its ATPase activity and promoting extracellular secretion in V. cholerae.

Adenosine Triphosphatases↗

PubMed2002

Detoxification of cholera toxin without removal of its immunoadjuvanticity by the addition of (STa-related) peptides to the catalytic subunit. A potential new strategy to generate immunostimulants for vaccination.

Peptides related to the heat-stable enterotoxin STa were fused to the N terminus of the A-subunit of cholera toxin (CTA) to explore whether peptide additions could help generate detoxified cholera toxin (CT) derivatives. Proteins carrying APRPGP (6-CTA), ASRCAELCCNPACPAP (16-CTA), or ANSSNYCCELCCNPACTGCYPGP (23-CTA) were genetically constructed. Using a two-plasmid system these derivatives were co-expressed in Vibrio cholerae with cholera toxin B-subunit (CTB) to allow formation and secretion of holotoxin-like molecules (engineered CT, eCTs). Purified eCTs maintained all normal CT properties yet they were more than 10-fold (eCT-6), 100-fold (eCT-16), or 1000-fold (eCT-23) less enterotoxic than wild-type CT. The inverse correlation between enterotoxicity and peptide length indicated sterical interference with the ADP-ribosylating active site in CTA. This interpretation agreed with greater than 1000-fold reductions in cAMP induction, with reductions, albeit not proportional, in in vitro agmatine ADP-ribosylation, and was supported by molecular simulations. Intranasal immunization of mice demonstrated that eCTs retained their inherent immunogenicity and ability to potentiate immune responses to a co-administered heterologous protein antigen, although in variable degrees. Therefore, the addition of STa-related peptides to CTA reduced the toxicity of CT while partly preserving its natural immunoadjuvanticity. These results suggest peptide extensions to CTA are a useful alternative to site-directed mutagenesis to detoxify CT. The simplicity of the procedure, combined with efficient expression and assembly of derivatives, suggests this approach could allow for large scale production of detoxified, yet immunologically active CT molecules.

Adenosine Diphosphate Ribose↗

PubMed2000

Mutations in the extracellular protein secretion pathway genes (eps) interfere with rugose polysaccharide production in and motility of Vibrio cholerae.

Vibrio cholerae is the causal organism of the diarrheal disease cholera. The rugose variant of V. cholerae is associated with the secretion of an exopolysaccharide. The rugose polysaccharide has been shown to confer increased resistance to a variety of agents, such as chlorine, bioacids, and oxidative and osmotic stresses. It also promotes biofilm formation, thereby increasing the survival of the bacteria in the aquatic environments. Here we show that the extracellular protein secretion system (gene designated eps) is involved directly or indirectly in the production of rugose polysaccharide. A TnphoA insertion in epsD gene of the eps operon abolished the production of rugose polysaccharide, reduced the secretion of cholera toxin and hemolysin, and resulted in a nonmotile phenotype. We have constructed defined mutations of the epsD and epsE genes that affected these phenotypes and complemented these defects by plasmid clones of the respective wild-type genes. These results suggest a major role for the eps system in pathogenesis and environmental survival of V. cholerae.

Alkaline Phosphatase↗

PubMed2006

Escherichia coli cyclomodulin Cif induces G2 arrest of the host cell cycle without activation of the DNA-damage checkpoint-signalling pathway.

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins and effector proteins, the cyclomodulins, that deregulate the host cell cycle. Upon injection into HeLa cells by the enteropathogenic Escherichia coli (EPEC) type III secretion system, Cif induces a cytopathic effect characterized by the recruitment of focal adhesion plates and the formation of stress fibres, an irreversible cell cycle arrest at the G(2)/M transition, and sustained inhibitory phosphorylation of mitosis inducer, CDK1. Here, we report that the reference typical EPEC strain B171 produces a functional Cif and that lipid-mediated delivery of purified Cif into HeLa cells induces cell cycle arrest and actin stress fibres, implying that Cif is necessary and sufficient for these effects. EPEC infection of intestinal epithelial cells (Caco-2, IEC-6) also induces cell cycle arrest and CDK1 inhibition. The effect of Cif is strikingly similar to that of cytolethal distending toxin (CDT), which inhibits the G(2)/M transition by activating the DNA-damage checkpoint pathway. However, in contrast to CDT, Cif does not cause phosphorylation of histone H2AX, which is associated with DNA double-stranded breaks. Following EPEC infection, the checkpoint effectors ATM/ATR, Chk1 and Chk2 are not activated, the levels of the CDK-activating phosphatases Cdc25B and Cdc25C are not affected, and Cdc25C is not sequestered in host cell cytoplasm. Hence, Cif activates a DNA damage-independent signalling pathway that leads to inhibition of the G(2)/M transition.

Animals↗

PubMed1997

Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the vagina.

Forty-five volunteers were vaccinated twice intranasally with 10, 100, or 1,000 microg of cholera toxin B subunit (CTB). Blood and nasal and vaginal secretions were collected before and 1 week after the second vaccination from all volunteers, and the specific and total immunoglobulin A (IgA) and IgG titers were determined by enzyme-linked immunosorbent assay. Samples were also taken 6 months (n = 16) and 1 year (n = 14) after the vaccination. The 10- and 100-microg doses were well tolerated by the volunteers, but the 1,000-microg dose induced increased secretions from the nose and repetitive sneezings for several hours. The CTB-specific serum IgA and IgG increased 21- and 7-fold, respectively, 1 week after vaccination with the medium dose and increased 61- and 37-fold, respectively, after the high dose. In nasal secretions the specific IgA and IgG increased 2- and 6-fold after the medium dose and 2- and 20-fold after the high dose, respectively. In vaginal secretions the specific IgA and IgG increased 3- and 5-fold after the medium dose and 56- and 74-fold after the high dose, respectively. The lowest dose did not induce any significant antibody titer increases in serum or in secretions. The specific IgA and IgG levels in secretions were still elevated after 6 months but were decreasing 1 year after the vaccination. These results show that intranasal vaccination of humans with CTB induces strong systemic and mucosal antibody responses and suggest that CTB may be used as a carrier for antigens that induce protective immunity against systemic as well as respiratory and genital infections.

Administration, Intranasal↗

PubMed1988

The adenylate cyclase system and calcitonin secretion from perfused dog thyroid lobes.

The aim of the study was to assess the involvement of the adenylate cyclase system in calcitonin (CT) secretion from thyroidal C-cells. The cAMP analogues Br-cAMP (10(-6) and 10(-4) mol/l) and DB-cAMP (10(-4) mol/l) and the activators of adenylate cyclase cholera toxin (0.1 microgram/ml and 5 micrograms/ml) and forskolin (10(-7) mol/l and 10(-5) mol/l) were infused for 6 min periods in perfused dog thyroid lobes. CT was measured in thyroid effluent by radioimmunoassay. Br-cAMP and cholera toxin did not alter basal CT secretion. DB-cAMP had a minimal stimulatory effect and forskolin 10(-5) mol/l a moderate stimulatory effect. This was much less than the effect of increasing perfusate Ca++ from 1.5 to 2.0 mmol/l. 10(-4) mol/l Br-cAMP increased the response to Ca++ with approximately 50 per cent. These results suggest that the activity of the adenylate cyclase system of the C-cells by itself is of little importance for CT secretion, but that it may have a role as modulator of the response to Ca++.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1984

Adrenergic mediation of the intestinal antisecretory action of opiates administered into the central nervous system.

The antisecretory effects of the stable enkephalin analogs, [D-Ala2-Met5] enkephalinamide (DAMA) and [D-Ala2-D-Leu5] enkephalin, and the opiate drug morphine were evaluated on fluid secretion induced by cholera toxin in isolated loops of the jejunum and proximal and distal ileum in anesthetized rats. Intracerebroventricular administration of DAMA (0.03-3 micrograms) or [D-Ala2-D-Leu5] enkephalin (1.0-10 micrograms) dose-dependently reduced secretion in the jejunum without affecting fluid movement in the other small intestinal segments. Morphine in doses up to 30 micrograms i.c.v. had no significant antisecretory effects. Intravenous administration of DAMA, at doses up to 3000 micrograms/kg, had little effect on intestinal fluid accumulation. The antisecretory action of DAMA (3 micrograms i.c.v.) was completely blocked by pretreatment with the alpha adrenergic antagonist phentolamine and after peripheral sympathectomy induced by guanethidine. In contrast, DAMA activity was preserved in adrenal demedullated rats. DAMA had no significant effects upon mean arterial blood pressure or on blood acid-base balance. These results suggest that the antisecretory effects of opiates are, at least partly, mediated at sites within the central nervous system. These actions are probably a consequence of increased activity in sympathetic nerve fibers innervating the upper small intestine.

Animals↗

PubMed2005

CCR1 acts downstream of NFAT2 in osteoclastogenesis and enhances cell migration.

UNLABELLED: We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKL-stimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKL-dependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration. INTRODUCTION: We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells. MATERIALS AND METHODS: RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay. RESULTS AND CONCLUSIONS: We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA- or CCR1 siRNA-treated cells were used. Moreover, treatment with a Galpha inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.

Animals↗

PubMed2000

A genomic sample sequence of the entomopathogenic bacterium Photorhabdus luminescens W14: potential implications for virulence.

Photorhabdus luminescens is a pathogenic bacterium that lives in the guts of insect-pathogenic nematodes. After invasion of an insect host by a nematode, bacteria are released from the nematode gut and help kill the insect, in which both the bacteria and the nematodes subsequently replicate. However, the bacterial virulence factors associated with this "symbiosis of pathogens" remain largely obscure. In order to identify genes encoding potential virulence factors, we performed approximately 2,000 random sequencing reads from a P. luminescens W14 genomic library. We then compared the sequences obtained to sequences in existing gene databases and to the Escherichia coli K-12 genome sequence. Here we describe the different classes of potential virulence factors found. These factors include genes that putatively encode Tc insecticidal toxin complexes, Rtx-like toxins, proteases and lipases, colicin and pyocins, and various antibiotics. They also include a diverse array of secretion (e.g., type III), iron uptake, and lipopolysaccharide production systems. We speculate on the potential functions of each of these gene classes in insect infection and also examine the extent to which the invertebrate pathogen P. luminescens shares potential antivertebrate virulence factors. The implications for understanding both the biology of this insect pathogen and links between the evolution of vertebrate virulence factors and the evolution of invertebrate virulence factors are discussed.

Animals↗

PubMed2000

Bacterial inhibition of phagocytosis.

The concerted study of molecular mechanisms of phagocytosis and the inhibition of phagocytosis by specific products of extracellular bacterial pathogens has borne considerable fruit. The importance of tyrosine phosphorylation and of the Rho family of GTPases has become clear to cell biologists, but pathogenic bacteria recognized the importance of these signalling pathways in phagocytic cells long ago. The discoveries described in this review are only the beginning. The simultaneous pursuit of the mechanisms and molecules involved in the initiation and regulation of phagocytosis and that pathogenic bacteria use to inhibit phagocytosis will surely identify more interesting pathways on each side of the contest. Are there any obvious possibilities? There are several bacterial factors that have the potential to inhibit known mechanisms of phagocytosis. Clostridium species, for example, make a number of exotoxins of interest. Clostridium botulinum and Clostridium tetani neurotoxins inactivate the regulated secretory machinery by proteolytic cleavage of SNARE proteins, and targets of tetanus toxin and botulinum b toxin inhibit the exocytotic delivery of membrane vesicles needed for phagocytosis of large particles (Hackam et al., 1998). Moreover, the C3 exotoxin of C. botulinum catalyses ADP ribosylation and inactivation of rho family GTPases (Wiegers et al., 1991), and toxins A and B of C. difficile UDP-glucosylate and inactivate rho GTPases and thereby disrupt the actin cytoskeleton (Just et al., 1995a,b). However, as Clostridia lack the machinery for type III secretion, these proteins are not rapidly targeted to the phagocyte cytoplasm. More searching may reveal a pathogen that has combined the type III secretory machinery with clostridia toxin-like substrates. A potentially unique strategy for remaining outside phagocytes is exhibited by Helicobacter pylori, which contain a type IV secretion system. Unopsonized virulent strains of H. pylori bind readily to macrophages but are only internalized after a delay of several minutes. Such a delay appears to be sufficient for the bacteria to remain extracellular (Allen et al., 2000). Elucidation of the mechanism used by H. pylori to delay phagocytosis may reveal one or more novel virulence factors as well as one or more novel targets in the phagocyte that will add to the understanding of a fundamental process in host defence. Another field ripe for further mechanistic investigation is complement receptor-mediated phagocytosis. Dedicated study of the molecular events and molecular mediators of phagocytosis downstream of CR3 is likely to reveal interesting differences from FcgammaR phagocytosis and is just as likely to reveal that microbes have discovered unique mechanisms for circumventing them. Study of extracellular pathogens and the mechanisms that they use to remain outside phagocytic cells has revealed a great deal about the initial encounter between pathogen and phagocyte. We can look forward to additional discoveries about the host-pathogen interactions and the mechanisms and factors that each side uses to battle against the other.

Animals↗

PubMed1993

A model system for the investigation of heterologous protein secretion pathways in Lactococcus lactis.

The capacity of recombinant strains of Lactococcus lactis to secrete a heterologous protein was investigated by constructing two expression-secretion vectors (pLET2 and pLET3) for use with a lactococcal gene expression system driven by the highly active T7 RNA polymerase. The vectors incorporated different lactococcal secretion leaders and translation initiation sequences. When tetanus toxin fragment C (TTFC) was used as a test protein, the quantities of TTFC produced by the pLET2-TTFC strain exceeded the rate of secretion of TTFC into the growth medium. However, nearly all of the soluble TTFC associated with the cell (3.4%) was translocated through the cell membrane. The pLET3-TTFC strain did not accumulate TTFC intracellularly and exhibited growth characteristics and viability identical to the growth characteristics and viability of the control strain. This strain secreted approximately 2.9 mg of TTFC per liter into the growth medium after 6 h of growth under test tube conditions. Our results indicate that L. lactis is capable of secreting substantial amounts of heterologous protein and also confirm the findings of other workers that the cell wall may serve as a functional barrier to the diffusion of some secreted proteins into the growth medium.

Amino Acid Sequence↗