Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: null phenotypes of the tar and tap genes.

The tar and tap genes are located adjacent to one another in an operon of chemotaxis-related functions. They encode methyl-accepting chemotaxis proteins implicated in tactic responses to aspartate and maltose stimuli. The functional roles of these two gene products were investigated by isolating and characterizing nonpolar, single-gene deletion mutants at each locus. Deletions were obtained by selecting for loss or a defective Mu d1 prophage inserted in either the tar or tap gene. The extent of the tar deletions was determined by genetic mapping with Southern hybridization. Representative deletion mutants were surveyed for chemotactic responses on semisolid agar and by temporal stimulation in a tethered cell assay to assess flagellar rotational responses to chemoeffector compounds. The tar deletion strains exhibited complete loss of aspartate and maltose responses, whereas the tap deletion strains displayed a wild-type phenotype under all conditions tested. These findings indicate that the tap function is unable to promote chemotactic responses to aspartate and maltose, and its role in chemotaxis remains unclear.     

Tumoricidal responses in vitro of peritoneal macrophages from conventionally housed and germ-free nude mice

Peritoneal macrophages from untreated nude mice were nonspecifically cytotoxic to tumor cells in vitro and were more responsive to chemotactic stimuli than macrophages from normal mice or from phenotypically normal littermates of nude mice. Tumoricidal and chemotactic responses of activated macrophages from nude mice were quantitatively comparable to responses of macrophages from BCG-infected normal mice. Peritoneal macrophages from germ-free nude mice, however, were not tumoricidal in vitro. These observations suggest that environmental stimuli, rather than thymic deficiency per se, induced activated macrophages in nude mice.     

Role of membrane potential and calcium in chemotactic sensing by bacteria

Changes in Escherichia coli membrane potential in response to a wide variety of chemotactic stimuli were measured using a permeant, lipophilic cation (tetraphenylphosphonium chloride) and a fluorescent dye (3-propyl-2-(5-(3-propyl-2(3H)-benzothiazolylidene)-1,3-pentadienyl)iodide). Some attractants and repellants caused permanent, monotonic depolarizations or hyperpolarizations. Not all chemoeffectors, however, produced potential changes, and the direction of change did not correlate with physiological responses to these compounds. Moreover, changes were observed in a number of chemotactic mutants. From these results, we conclude that perturbations in membrane potential effected by chemical stimuli are not related to chemotactic sensing. These findings, and the close correlation between cytoplasmic ionic conditions and membrane potential, led us to examine the role of calcium in chemotaxis. By growing cells in the presence of a calcium chelator, we were able to lower cellular calcium levels over tenfold, with no change in behavior. These results indicate that sensory transduction in these cells is not mediated by this cation.     

Tumor necrosis factor-alpha regulates expression of receptors for formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and platelet-activating factor. Dissociation from priming in human polymorphonuclear neutrophils.

TNF-alpha enhances polymorphonuclear responses to many stimuli, including chemotactic peptide FMLP. It also promotes expression of FMLP receptors and thus may prime polymorphonuclear neutrophils to this and other agonists by up-regulating signal recognition molecules. However, we find that the cytokine's actions on FMLP receptors lagged priming of FMLP-induced degranulation. Moreover, TNF-alpha enhanced degranulation responses to leukotriene B4 and platelet-activating factor but paradoxically down-regulated leukotriene B4 receptors and only transiently up-regulated platelet-activating factor receptors. Hence, TNF-alpha has pleiotropic effects on receptor expression; these effects diverge from priming; and a large part of the primed state must reflect enhancement of post-receptor events.     

N-terminal half of CheB is involved in methylesterase response to negative chemotactic stimuli in Escherichia coli.

The chemotactic receptor-transducer proteins of Escherichia coli are responsible for directing the swimming behavior of cells by signaling for either straight swimming or tumbling in response to chemostimuli. The signaling states of these proteins are affected not only by the concentrations of various stimuli but also by the extent to which they have been methylated at specific glutamyl residues. The activities of a chemotaxis-specific methyltransferase (CheR) and a chemotaxis-specific methylesterase (CheB) are regulated in response to chemotactic stimuli to enable sensory adaptation to unchanging levels of stimuli by appropriately shifting the signaling states of the transducer proteins. For CheB this regulation involves a feedback loop that requires some of the components making up the chemotactic signal transduction machinery of the cell. This feedback loop causes the methylesterase activity of CheB to decrease transiently in response to attractant stimuli and to increase transiently in response to negative stimuli (repellent addition or attractant removal). In this report we demonstrate that the methylesterase response to negative stimuli involves the N-terminal half of the CheB protein, whereas the response to positive stimuli does not require this segment of the protein. Both aspects of the methylesterase response to positive stimuli does not require this segment of the protein. Both aspects of the methylesterase response require CheA. In addition, we demonstrate that mutant forms of CheB lacking methylesterase activity can adversely affect the swimming behavior and chemotactic ability of cells and can markedly diminish modulation of the wild-type methylesterase activity in response to negative stimuli. The significance of these results is discussed in relation to the recent demonstration of phosphoryl transfer from CheA to CheB (J. F. Hess, K. Oosawa, N. Kaplan, and M. I. Simon, Cell 53:79-87, 1988) and the discovery of sequence homology between the N-terminal half of CheB and CheY (A. Stock, D. E. Koshland, Jr., and J. Stock, Proc. Natl. Acad. Sci. USA 82:7989-7993, 1985).     

HIV-1 and its envelope glycoprotein down-regulate chemotactic ligand receptors and chemotactic function of peripheral blood monocytes

Peripheral blood monocytes from AIDS patients exhibit defective migratory responses to chemotactic stimuli in vitro and to inflammatory sites in vivo. In studies presented here, normal monocytes were infected with the HIV-1/HTLV-IIIBa-L isolate in vitro and evaluated for chemotactic responsiveness. Within 2 days after viral exposure, but before evidence of virus production in the monocytes, chemotactic activity was significantly impaired. Decreased chemotactic activity was associated with modulation of receptors for the chemotactic ligands, C5a and FMLP, on the monocyte cell surface. In addition to HIV-1, monocytes treated with purified HIV-1 envelope glycoprotein gp120 demonstrated a comparable modulation of chemotactic ligand receptors and migratory function. In addition, the HIV-1 or HIV-1 gp120-treated monocytes were induced to undergo differentiation as monitored by HLA-DR expression. Immunoprecipitation of the gp120 with a specific antibody reversed its effects on monocyte chemotaxis and HLA-DR expression. Taken together, these data indicate that the initial interaction of HIV-1 with the monocyte is not passive, but that the binding of HIV-1 and/or HIV-1 gp120 to the CD4R on monocytes transduces a signal leading to transient monocyte activation.     

Chemotaxis of rat lymphocytes.

Rat lymphocytes obtained from spleens, lymph nodes, and thymus glands showed migratory responses to a variety of factors including fluids from mixed lymphocyte culture fluids from concanavalin A-stimulated cells, fluids from phagocytizing macrophages, and to anti-rat IgG. Migratory responses to the last factor were bimodal over a dose range of anti-Ig; at high concentrations of anti-Ig, the response appeared to be nonspecific, whereas, at low concentrations, the responses seemed to be chemotactic in character. When lymphocytes from spleens, lymph nodes, and thymic glands were compared, qualitative and quantitative differences on the responses were evident with use of the three attractants. When spleen lymphocytes were separated into T cell- and B cell-enriched fractions, T cells responded to the culture fluids from mixed lymphocyte cultures, whereas B cells seemed to respond poorly, if at all. Only B cells responded to anti-Ig. These findings may explain, at least in part, the accumulation of lymphoid cells at sites of inflammatory stimuli.     

Chemosensory-induced motor behaviors in fish

Chemical sensory signals play a crucial role in eliciting motor behaviors. We now review the different motor behaviors induced by chemosensory stimuli in fish as well as their neural substrate. A great deal of research has focused on migratory, reproductive, foraging, and escape behaviors but it is only recently that the molecules mediating these chemotactic responses have become well-characterized. Chemotactic responses are mediated by three sensory systems: olfactory, gustatory, and diffuse chemosensory. The olfactory sensory neuron responses to chemicals are now better understood. In addition, the olfactory projections to the central nervous system were recently shown to display an odotopic organization in the forebrain. Moreover, a specific downward projection underlying motor responses to olfactory inputs was recently described.     

Chemotaxis in Escherichia coli: construction and properties of lambda tsr transducing phage.

The tsr gene of Escherichia coli, located at approximately 99 min on the chromosomal map, encodes a methyl-accepting protein that serves as the chemoreceptor and signal transducer for chemotactic responses to serine and several repellents. To determine whether any other chemotaxis or motility genes were located in the tsr region, we constructed and characterized two lambda tsr transducing phages that each contain about 12 kilobases of chromosomal material adjacent to tsr. lambda tsr70 carries sequences from the promoter-proximal side of tsr; lambda tsr72 carries sequences from the promoter-distal side of tsr. Restriction maps of the bacterial inserts in these phages and Southern hybridization analyses of the bacterial chromosome indicated that the tsr gene is transcribed in the counterclockwise direction on the genetic map. Insert deletions were isolated in lambda tsr70 and transferred into the host chromosome to examine the null phenotype of tsr. All such strains exhibited wild-type swimming patterns and chemotactic responses to a variety of stimuli, but were specifically defective in serine taxis and other Tsr-mediated responses. In addition, UV programming experiments demonstrated that Tsr and several of its presumptive degradation products were the only bacterial proteins encoded by lambda tsr70 and lambda tsr72 that required host FlbB/FlaI function for expression. These findings indicate that there are probably no other chemotaxis-related genes in the tsr region. A series of tsr point mutations were isolated by propagating lambda tsr70 on a mutD host and used to construct a fine-structure map of the tsr locus. These mutations should prove valuable in exploring structure-function relationships in the Tsr transducer.     

Linear gramicidin activates neutrophil functions and the activation is blocked by chemotactic peptide receptor antagonist

The activation of functional responses in rabbit peritoneal neutrophils by gramicidin and the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine methyl ester, was studied. Gramicidin activated superoxide generation, lysosomal enzyme release and a decrease in fluorescence of chlortetracycline-loaded cells, as for the chemotactic peptide. The maximum intensities of the responses by gramicidin were lower than that by chemotactic peptide. Responses by both these peptides could be inhibited by t-butyloxycarbonyl-methionyl-leucyl-phenylalanine, a chemotactic peptide receptor antagonist. Gramicidin gave responses at low doses comparable to that of the chemotactic peptide.     

Stimulus-specificity of the chemotactic deactivation of human neutrophils by lipoxygenase products of arachidonic acid

Preincubation of human neutrophils with chemotactic concentrations of 5(S)-hydroxy-eicosatetraenoic acid (5-HETE) or 5(S), 12(R)-dihydroxy-6,14 cis-8,10 trans-eicosatetraenoic acid (leukotriene B4) induces a state of preferential chemotactic unresponsiveness to the homologous factor, termed deactivation, and less suppression of the responses to other chemotactic stimuli. The ratio of the concentrations required for maximal chemotactic deactivation of neutrophils to that which stimulates chemotaxis optimally is greater for 5-HETE and leukotriene B4 than for peptide and protein factors. In contrast to other chemotactic factors, 5-hydroperoxy-eicosatetraenoic acid (5-OOHETE) induces neutrophil chemotactic deactivation that is independent of the nature of the subsequent stimulus and is more slowly reversible after elimination of the fluid-phase deactivating factor. The unique characteristics of the chemotactic deactivation of human neutrophils by 5-OOHETE may be attributable in part to its endogenous metabolism to potent deactivating factors or to covalent derivatization of subcellular structures of the neutrophils by the highly reactive 5-OOHETE.     

Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis

We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signalling and cytoskeletal proteins using Western blotting.Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils.An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor.In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.     

Intracellular calcium redistribution and its relationship to fMet-Leu-Phe, leukotriene B4, and phorbol ester induced rabbit neutrophil degranulation

The addition of low concentrations (less than 10(-7) M) of the calcium ionophore A23187 to rabbit neutrophils releases the intracellular pool of calcium previously shown in radioactive steady-state and chlortetracycline fluorescence studies to be mobilized by chemotactic factors. A23187 at these concentrations elicits no functional responses from these cells. However, A23187, added before chemotactic factors such as fMet-Leu-Phe and leukotriene B4, inhibits the ability of the latter stimuli to induce, in the presence of cytochalasin B, an exocytotic release of the neutrophil's cytoplasmic granules. These results imply that the chemotactic-factor-induced release of intracellular calcium is a necessary event for the optimal activation of the neutrophils. Phorbol ester-induced neutrophil degranulation on the other hand is unaffected by exposure to A23187, thereby completely dissociating its mechanism of action from rises in cytoplasmic free calcium.     

Spatial control of actin polymerization during neutrophil chemotaxis

Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients.     

Isolation and characterisation of chemotactic mutants of Chlamydomonas reinhardtii obtained by insertional mutagenesis

The swimming behaviour of the green flagellated protist Chlamydomonas reinhardtii is influenced by several different external stimuli including light and chemical attractants. Common components are involved in both the photo- and chemo-sensory transduction pathways, although the nature and organisation of these pathways are poorly understood. To learn more about the mechanism of chemotaxis in Chlamydomonas, we have generated nonchemotactic strains by insertional mutagenesis. The arginine-requiring strain arg7-8 was transformed with DNA carrying the wild-type ARG7 gene. Of the 8630 arginine-independenttransformants obtained, five are defective in their chemotaxis towards various sugars. Two of the mutants (CTX2 and CTX3) are blocked only in their response to xylose. Mutant CTX1 is blocked in its response to xylose, maltose and mannitol, but displays normal taxis to sucrose. Mutants CTX4 and CTX5 lack chemotactic responses to all sugars tested. CTX1, CTX4 and CTX5 represent novel chemotactic phenotypes not previously obtained using ultra-violet or chemical mutagenesis. Genetic analysis confirms that each mutation maps to a single nuclear locus that is unlinked to the mating-type locus. Further analysis of CTX4 indicates that the mutant allele is tagged by the transforming ARG7 DNA. CTX4 appears to be defective in a component specific for chemotactic signal transduction since it exhibits wild-type photobehavioural responses (phototaxis and photoshock) as well as the wild-type responses of EGTA-induced trans-flagellum inactivation and acid-induced deflagellation. Insertional mutagenesis has thus permitted the generation of novel chemotactic mutants that will be of value in the molecular dissection of the signalling machinery.     

Human neutrophil chemotactic and degranulating activities of leukotriene B5 (LTB5) derived from eicosapentaenoic acid

Leukotriene B4 exhibited 10- to 30-fold greater chemotactic potency for human neutrophils than eicosapentaenoic acid-derived leukotriene B5, as assessed in modified Boyden micropore filter chambers. In contrast, leukotrienes B4 and B5 were equipotent stimuli of human neutrophil lysosomal degranulation in vitro, as quantified by the release of beta-glucosaminidase. Analyses of competitive inhibition of the binding of [3H] leukotriene B4 to neutrophils indicated that leukotriene B4 binds with a 500-fold greater association constant than leukotriene B5 to a subclass of high-affinity receptors, which appears to transduce chemotactic responses efficiently, while leukotrienes B4 and B5 bind equally well to low-affinity receptors.     

Effect of temperature on Pseudomonas fluorescens chemotaxis.

The effects of temperature and attractants on chemotaxis in psychrotrophic Pseudomonas fluorescens were examined using the Adler capillary assay technique. Several organic acids, amino acids, and uronic acids were shown to be attractants, whereas glucose and its oxidation products, gluconate and 2-ketogluconate, elicited no detectable response. Chemotaxis toward many attractants was dependent on prior growth of the microorganism with these compounds. However, the organic acids, malate and succinate, caused strong chemotactic responses regardless of the carbon source used for growth of the bacteria. The temperature at which the cells were grown (30 or 5 degrees C) had no significant detectable effect on chemotaxis to the above attractants. The temperature at which the cells were assayed appeared to affect the rate but the extent of the chemotactic response, nor the concentration response curves. The ratios of the rate of accumulation of cells to the attractant malate were approximately 2, 4, and 1 at 30, 17, and 5 degrees C, respectively. Strong chemotactic responses were observed with cells assayed at temperatures approaching 0 degree C and appeared to be functional over a broad temperature range of 3 to 35 degrees C.     

Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-alpha, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.