Polar clustering of the chemoreceptor complex in Escherichia coli occurs in the absence of complete CheA function.

Bacterial chemotaxis requires a phosphorelay system initiated by the interaction of a ligand with its chemoreceptor and culminating in a change in the directional bias of flagellar rotation. Chemoreceptor-CheA-CheW ternary complexes mediate transduction of the chemotactic signal. In vivo, these complexes cluster predominantly in large groups at the cell poles. The function of chemoreceptor clustering is currently unknown. To gain insight into the relationship between signaling and chemoreceptor clustering, we examined these properties in several Escherichia coli mutant strains that produce CheA variants altered in their ability to mediate chemotaxis, autophosphorylate, or bind ATP. We show here that polar clustering of chemoreceptor complexes does not require functional CheA protein, although maximal clustering occurred only in chemotactically competent cells. Surprisingly, in cells containing a minimum of 13 gold particles at the cell pole, a significant level of clustering was observed in the absence of CheA, demonstrating that CheA is not absolutely essential for chemoreceptor clustering. Nonchemotactic cells expressing only CheA(S), a C-terminal CheA deletion, or CheA bearing a mutation in the ATP-binding site mediated slightly less than maximal chemoreceptor clustering. Cells expressing only full-length CheA (CheA(L)) from either a chromosomal or a plasmid-encoded allele displayed a methyl-accepting chemotaxis protein localization pattern indistinguishable from that of strains carrying both CheA(L) and CheA(S), demonstrating that CheA(L) alone can mediate polar clustering.

TNP-ATP and TNP-ADP as probes of the nucleotide binding site of CheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli.

The interaction of CheA with ATP has important consequences in the chemotaxis signal transduction pathway of Escherichia coli. This interaction results in autophosphorylation of CheA, a histidine protein kinase. Autophosphorylation of CheA sets in motion a chain of biochemical events that enables the chemotaxis receptor proteins to communicate with the flagellar motors. As a result of this communication, CheA allows the receptors to control the cell swimming pattern in response to gradients of attractant and repellent chemicals. To probe CheA interactions with ATP, we investigated the interaction of CheA with the fluorescent nucleotide analogues TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] and TNP-ADP. Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar Kd values) and caused a marked enhancement of the fluorescence of the TNP moiety of these modified nucleotides. Analysis of titration experiments indicated a binding stoichiometry of two molecules of TNP-ATP (TNP-ADP) per CheA dimer and suggested that the two binding sites on the CheA dimer operate independently. Binding of TNP-ATP to CheA was inhibited by ATP, and analysis of this inhibition indicated that the CheA dimer binds 2 molecules of ATP. Competition experiments also indicated that CheA binds TNP-ATP considerably more tightly than it binds unmodified ATP. Binding of TNP-ADP to CheA was inhibited by ADP in a similar manner. TNP-ATP was not a substrate for CheA and served as a potent inhibitor of CheA autophosphorylation (Ki < 1 microM). The glycine-rich regions (G1 and G2) of CheA and other histidine protein kinases have been presumed to play important roles in ATP binding and/or catalysis of CheA autophosphorylation, although few experimental tests of these functional assignments have been made. Here, we demonstrate that a CheA mutant protein with Gly-->Ala substitutions in G1 and G2 has a markedly reduced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatography. This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity. Surprisingly, a distinct single-site substitution in G2 (Gly470-->Lys) had no observable effect on the affinity of CheA for ATP and ADP, despite the fact that it rendered CheA completely inactive as an autokinase. This mutant protein also bound TNP-ATP and TNP-ADP with affinities and stoichiometries that were indistinguishable from those observed with wild-type CheA. These results provide some preliminary insight into the possible functional roles of G1 and G2, and they suggest that TNP-nucleotides are useful tools for exploring the effects of additional mutations on the active site of CheA.

Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA.

Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis. The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB. CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators. The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation. The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems. On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP. We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo. We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2. In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all. These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer.

pH dependence of CheA autophosphorylation in Escherichia coli.

Chemotaxis by cells of Escherichia coli and Salmonella typhimurium depends upon the ability of chemoreceptors called transducers to communicate with switch components of flagellar motors to modulate swimming behavior. This communication requires an excitatory pathway composed of the cytoplasmic signal transduction proteins, CheAL, CheAS, CheW, CheY, and CheZ. Of these, the autokinase CheAL is most central. Modifications or mutations that affect the rate at which CheAL autophosphorylates result in profound chemotactic defects. Here we demonstrate that pH can affect CheAL autokinase activity in vitro. This activity exhibits a bell-shaped dependence upon pH within the range 6.5 to 10.0, consistent with the notion that two proton dissociation events affect CheAL autophosphorylation kinetics: one characterized by a pKa of about 8.1 and another exhibiting a pKa of about 8.9. These in vitro results predict a decrease in the rate of CheAL autophosphorylation in response to a reduction in intracellular pH, a decrease that should cause increased counterclockwise flagellar rotation. We observed such a response in vivo for cells containing a partially reconstituted chemotaxis system. Benzoate (10 mM, pH 7.0), a weak acid that when undissociated readily traverses the cytoplasmic membrane, causes a reduction of cytoplasmic pH from 7.6 to 7.3. In response to this reduction, cells expressing CheAL, CheAS, and CheY, but not transducers, exhibited a small but reproducible increase in the fraction of time that they spun their flagellar motors counterclockwise. The added presence of CheW and the transducers Tar and Trg resulted in a more dramatic response. The significance of our in vitro results, their relationships to regulation of swimming behavior, and the mechanisms by which transducers might affect the pH dependence of CheA autokinase activity are discussed.

Modulation of postsurgical cell infiltration and fibrinolytic activity by tolmetin in two species.

Previous studies showed that tolmetin administered as a single instillate intraperitoneally at the end of surgery can reduce adhesion formation. In this report, studies on the mechanism by which this occurs were conducted. The effects of tolmetin administered intraperitoneally on red (RBC) and white blood cell (WBC) number, macrophage and polymorphonuclear neutrophil infiltration, protease activity in lavage fluid, and the fibrinolytic activity of a biopsy of nonsurgical and traumatized peritoneal sidewall were examined. Tolmetin was shown to increase the number of RBC at one postoperative time point in rabbits, but not in rats. In addition, tolmetin administration elevated the number of WBC harvested from the peritoneum predominantly through an increase in macrophage number. Administration of tolmetin also modulated the level of protease and protease inhibitor activity in the lavage fluid harvested from the peritoneum. The most pronounced change was a decrease in the level of plasminogen activator inhibitor activity. In addition, acute administration of tolmetin to rats elevated the level of fibrinolytic activity at the site of trauma as measured by in vitro cultures. In summary, intraperitoneal administration of tolmetin, a nonsteroidal anti-inflammatory drug which reduces adhesion formation in both rats and rabbits, at the end of surgery modulated the number of WBCs in the peritoneal cavity and the protease and protease inhibitory activities present in the peritoneal lavage fluid and peritoneum after surgery.

Peritoneal lavage fluid protease levels after in vivo administration of tolmetin in hyaluronic acid.

Tolmetin sodium in a hyaluronic acid carrier (tolmetin-HA) was previously shown to reduce adhesion formation and alter the rate and extent to which wound repair cells enter the peritoneal cavity after surgery. In this study, the effect of tolmetin-HA on the levels of protease activities present in lavage fluid from the peritoneal cavity was determined at various postsurgical times. Collagenase activity in peritoneal lavage fluid was suppressed at 6, 12, and 24 hr after administration of tolmetin-HA in comparison to control. Elastase activity levels were biphasic with peak levels at 6 and 72 hr in lavage fluid from controls compared to peak levels at 6 and 48 hr in lavage fluid from treated rabbits. Plasminogen activator activity present in lavage fluid was significantly decreased at 48 hr after surgery in the tolmetin-HA-treated rabbits compared to controls. However, no alteration in the level of plasminogen activator inhibitor activity was observed in either the tolmetin-HA-treated or control rabbits. These data suggest that tolmetin-HA treatment altered the levels of neutral protease activity present in the peritoneal cavity and may therefore effect the proteolytic potential in the peritoneal cavity after surgery.

In vivo administration of tolmetin in hyaluronic acid modulates protease levels in postsurgical macrophage-conditioned media.

Tolmetin sodium in a hyaluronic acid carrier (tolmetin-HA) was previously shown to reduce adhesion formation and alter the kinetics and levels of cellular influx into the peritoneal cavity after surgery. In this study, the effect of tolmetin-HA on the level of protease activity in macrophage-conditioned media was determined. The level of collagenase activity in macrophage-conditioned media was suppressed at 12 and 24 h after administration of tolmetin-HA. Alternatively, the peak level of elastase activity measured in macrophage-conditioned media was unchanged after tolmetin-HA treatment, but the kinetics of expression of maximal protease activity was delayed from 12 h in the control surgical rabbits to 24 h in tolmetin-HA-treated rabbits. Elevated plasminogen activator activity was detected in acid-treated conditioned media from the tolmetin-HA-treated rabbits when compared to control levels. However, no alteration in the level of plasminogen activator inhibitor activity was present in conditioned media of macrophages harvested from tolmetin-HA-treated rabbits compared to controls. These data suggest that tolmetin-HA treatment altered the levels of neutral protease activity secreted by postsurgical macrophages and may therefore elevate the fibrinolytic potential of the peritoneal cavity after surgery.

Follicle regulatory protein: a novel marker for granulosa cell cancer patients.

Follicle regulatory protein (FRP) is secreted by the granulosa cell of the ovary and plays a role in modulating follicle development. A dual epitope immunoassay using two murine monoclonal antibodies, isotype IgG1 (raised against porcine FRP), in tandem was developed to measure FRP in serum. The levels of FRP in the serum of women with granulosa cell tumors, normal, menstruating women, and postmenopausal women were determined. The levels of FRP were elevated in the serum of 79% of the women with granulosa cell tumors compared to the normal controls. FRP levels in serial samples from women with granulosa cell tumors generally correlated with the clinical course of the disease. Thus, FRP may provide a useful marker for granulosa cell tumors.

Modulation of macrophage protease activity by acute administration of O,O,S trimethyl phosphorothioate.

Previous studies showed that acute administration of O,O,S trimethyl phosphorothioate (OOS-TMP), a contaminant in malathion, acephate and fenitrothion, led to increases in metabolic activities, such as, secretion of interleukin 1 and nonspecific esterase, of splenic and peritoneal macrophages. In this report, the effect of OOS-TMP administration on the levels of the neutral proteases, elastase, collagenase and plasminogen activator, in cultures supernatants of peritoneal and splenic macrophages is presented. Acute administration of OOS-TMP elevated collagenase levels only at day 3 following treatment with 10 or 20 mg/kg OOS-TMP. Levels of elastase in culture supernatant of peritoneal and splenic macrophages, on the other hand, was elevated at days 1, 3, 5 and 7 following administration of OOS-TMP. The effect on elastase secretion was dose-dependent at days 5 and 7 after treatment. Levels of plasminogen activator activity in the culture supernatants of splenic macrophages was elevated at day 5 following treatment with both doses of OOS-TMP. At days 1 and 3, the level of plasminogen activator inhibitor was suppressed. However, at days 5 and 7 plasminogen activator inhibitory activity was close to control values. These data show that OOS-TMP administration led to an elevation in the levels of neutral proteases in culture supernatants of peritoneal and splenic macrophages. This elevation indicates that acute OOS-TMP administration alters another parameter of macrophage function, which is elevated following exposure to acute inflammatory stimuli.

Modulation of respiratory burst activity and mitogenic response of human peripheral blood mononuclear cells and murine splenocytes and peritoneal cells by malathion.

Previous studies showed that acute administration of noncholinergic doses of malathion in vivo elevated the humoral immune and mitogenic responses but did not alter the generation of the cytotoxic T lymphocyte (CTL) response to alloantigen of splenocytes from treated mice. However, in vitro exposure to malathion suppressed the generation of a CTL response. In this study, the effects of in vivo and in vitro (with and without an NADPH-regenerating liver enzyme system) exposure to malathion on the mitogenic responses of murine splenocytes or respiratory burst activity of peritoneal cells were examined. The effect of in vitro exposure to malathion on the ability of human peripheral blood mononuclear cells (PBMC) to perform these functions was also examined. In vivo exposure to malathion significantly elevated proliferative responses of murine splenocytes to mitogens. Cell separation and reconstitution studies indicated that adherent splenocytes from treated mice could elevate the proliferative responses of nonadherent splenocytes from control mice. Alternatively, in vitro exposure of murine splenocytes or human PBMC to malathion or malathion metabolized by a liver enzyme system suppressed or did not change, respectively, the proliferative responses to mitogens. In addition, cell separation and reconstitution experiments indicated that in vitro exposure to malathion affected nonadherent splenocytes and PBMC. In vivo exposure to malathion also elevated the production of hydrogen peroxide, following stimulation with phorbol myristate acetate, by murine peritoneal cells. In vivo exposure of murine peritoneal cells to malathion suppressed or elevated the respiratory burst activity following exposure to malathion or malathion metabolized by a liver enzyme system, respectively. Exposure of human PBMC to metabolized malathion in vitro enhanced their ability to produce hydrogen peroxide.

Effects of acute administration of O,O,S-trimethyl phosphorothioate on the respiratory burst and phagocytic activity of splenic and peritoneal leukocytes.

An impurity in malathion, O,O,S-trimethyl phosphorothioate (OOS-TMP), was previously shown to be immunosuppressive. The immune cell type which induced immune suppression caused by OOS-TMP at 24 hrs after administration was found to be splenic macrophages. Further characterization of macrophages from OOS-TMP treated mice indicated that OOS-TMP led to macrophage differentiation. In this study, these initial studies were continued and extended to examine the effects of OOS-TMP on splenic and peritoneal macrophages at various times following exposure. Administration of OOS-TMP increased the size heterogeneity of cell volume, phagocytic capability and respiratory burst activity of splenic and peritoneal macrophages. However, by day 7 splenic and peritoneal macrophages from treated animals had size frequency histograms, phagocytic capability and respiratory burst activity similar to control. These data would suggest that macrophages not previously exposed to OOS-TMP migrated to the spleen and peritoneum of treated animals. This migration may allow the restoration of the ability of splenocytes from treated animals to generate an immune response. Alternatively, these data may indicate that 7 days following exposure to OOS-TMP, the differentiative state of the splenic and peritoneal macrophages of treated mice had decayed and hence these cells had regained resident characteristics.