Pili and the interaction of Aeromonas species with humanperipheral blood polymorphonuclear cells

Abstract The interaction of differentiallu piliated Aeromonas strains expressing pili of two broadly different morphologic types (short, rigid (S/R) and/or long, wavy (L/W)) with human peripheral blood mononuclear leukocytes (PMN) was investigated to determine whether host defense cells might exert a selective pressure on pili expression in vivo accounting for the different pili phenotypes of clinical and environmental strains. A majority of Aeromonas veronii biotype sobria strains from water (6/6) and faeces (8/11) readily associated with PMN (>60% PMN with adherent and/or internalised bacteria), irrespective of their degree, or predominant type, of piliation. Rigid pili of Aeromonas species did not promote interaction with PMN. However, the majority (55%) of strains which interacted well with PMN were adherent to HEp-2 cells. Interactio with PMN is unlikely to be the reason few S/R pili are seen on faecal strains, but it may be a selective pressure on L/W adhesive pili, or other OMP adhesins, resulting in the shedding of strains which have lost critical adhesins.     

Functional Activity of Blood Polymorphonuclear Leukocytes as an Oxidative Stress Biomarker in Human Subjects

In the present work, we studied the role of polymorphonuclear leukocytes (PMN) in aged individuals and coronary heart disease (CHD)-bearing patients, two physiopathological processes associated with overproduction of reactive oxygen species (ROS). The effects of antioxidant supplementation on the functional activity of PMN from CHD patients were also determined. The function of PMNs was evaluated by measuring of phagocytosis, killing activity, and ROS production. Luminol amplified chemiluminescence (CL) was used to estimate ROS production by stimulated PMNs. Total cholesterol and the LDL-cholesterol fraction from CHD patients were found to be higher than those recommended, returning to normal levels after antioxidant therapy. PMN CL of CHD patients was found to be higher than the associated control groups. Antioxidant therapy administrated to CHD patients lead to an increase in the killing activity accompanied by a decrease in PMN CL of these subjects. The study also showed that killing activity of PMN from human subjects over 60 years was significantly lower than the activity measured in younger subjects. PMN CL produced after stimulation was found to be positively correlated with the increasing age of human subjects (r = .946, p < .01).     

The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells

The influence of the two surface structures of Neisseria meningitidis, capsule and pili, in bacterial interactions with human endothelial cells was investigated. Increased association correlated with the presence of pili on bacteria while capsule type had no apparent effect. Strains expressing both Class I and Class II pili associated with endothelial cells in significantly larger numbers compared with the non-piliated variants of the same strains (greater than 10x). Variants of Neisseria gonorrhoeae strain P9 expressing antigenically distinct pili also associated with endothelial cells in larger numbers (greater than 30x) compared with the non-piliated variant. Electron microscopic studies confirmed these data and showed that gonococci were internalized more frequently compared with meningococci. One consequence of increased association was an increase in the cytopathic effect of bacteria on the target cells.     

Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells

The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus-deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep-2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili-expressing strains. In addition to these interclass functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep-2 cells ('non-adherent' phenotype; adherence of less than 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non-adherent Pil- and Pil+ bacteria by selection on Chang cells (adherence of 10-25 bacteria per cell). In contrast to epithelial cells, all variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (greater than 50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus-facilitated interactions of N. meningitidis with endothelial and epithelial cells.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

Evidence that hsp90 is involved in the altered interactions of Acanthamoeba castellanii variants with bacteria

There are many similarities between the interactions of environmental protozoa with pathogenic bacterial species and those observed in mammalian macrophages. Since single-celled protozoa predate mammalian hosts, it is likely that interactions in environmental biofilms have selected for many of the bacterial virulence mechanisms responsible for human disease. In order to better understand bacterial-phagocyte interactions, we developed a selection for Acanthamoeba castellanii variants that are more resistant to killing by bacterial pathogens. We identified four amoebal clones that display decreased phagocytosis of bacteria but no difference in uptake of latex beads compared to wild-type amoebae. These amoebal variants display differences in cellular morphology, partial resistance to killing by bacteria, more bactericidal activity, and higher frequencies of lysosome fusion with the bacterial vacuole. Three proteins are present at lower levels in these variants than in wild-type amoebae, and matrix-assisted laser desorption ionization-time of flight mass spectrometry allowed identification of two of them as actin and hsp90. We found that specific inhibitors of hsp90 produce a similar phenotypic effect in macrophages. These data suggest that hsp90 plays a role in phagocytic and, possibly, bactericidal pathways that affect interactions of phagocytic cells with bacteria.     

Type IV-like pili formed by the type II secreton: specificity,composition, bundling,polar localization,and surface presentation of peptides

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).     

Phagocytic killing of Candida albicans by different murine effector cells

Three major phagocytic populations in the mouse were tested in vitro for killing of Candida albicans by means of 51Cr release assay: early inflammatory peritoneal polymorphonuclear cells (PMN), unfractionated or adherent spleen cells and resident peritoneal macrophages (PEC). Considerable candidacidal activity was found in the early inflammatory neutrophil and adherent spleen cell populations. On the contrary, only limited activity was found to be associated with resident peritoneal macrophages. The phagocytic killing apparently involved multiple mechanisms.     

Validation of different chemilumigenic substrates for detecting extracellular generation of reactive oxygen species by phagocytes and endothelial cells

Chemiluminescence is a widely used tool to detect extracellular generation of reactive oxygen species (ROS). In the present study we tested four different chemilumigenic substrates (CLS)—luminol, isoluminol, lucigenin and pholasin—to detect extracellular CL in different cell types: polymorphonuclear leukocytes (PMN); DMSO‐differentiated HL‐60 cells; murine macrophages (RAW 264.7); and TNFα‐stimulated human endothelial cells (HUVEC). Extracellular ROS production was calculated by subtracting intracellular CL response in the presence of superoxide dismutase and catalase from the overall CL response in the absence of enzymes. CL varied considerably in dependence on the CLS and the stimulus used to evoke ROS generation. Luminol (oxidized LDL and zymosan stimulation) and isoluminol (FMLP and PMA stimulation) were the most effective CLS for PMN. Using 5 µmol/L lucigenin as CLS, small but consistent CL responses could be obtained in macrophages stimulated with PMA, zymosan or oxidized LDL. FMLP‐stimulated extracellular CL in H‐60 cells, HUVEC and macrophages was detected with the greatest sensitivity by pholasin. Our results demonstrate that none of the investigated CLS consistently yielded the highest CL quantum, either in different cell types with one stimulating agent or by different stimulating agents in one cell type. To get the highest CL quantum in experimental studies, we recommend optimizing the CLS depending on the cell type and the ROS‐generating stimulus used. Copyright © 2003 John Wiley & Sons, Ltd.     

Biochemical comparison of pili from variants of Neisseria gonorrhoeae P9

Four different types of pili produced by variants of Neisseria gonorrhoeae P9 were isolated and characterized. The pili differed in subunit molecular weight with SDS-PAGE and in subunit isoelectric point on agarose gels. Isoelectric points of the major molecular species in Triton-agarose gels of octylglucoside solubilized pili were: delta, pI 6.5; alpha, pI 6.0; beta, pI 5.3 and gamma, pI 5.5. Amino acid analyses of pili showed close homology between different types but a reduction in the content of aspartate and serine was notable in the low molecular weight delta pili; also beta and gamma maps of tryptic/chymotryptic digests of pili with several major peptides apparently common to all four pilus types.     

Effects of beta-carotene, selenium and vitamin A on in vitro polymorphonuclear leukocytic activity in peripartal buffalo (Bubalus bubalus)

The effect of different concentrations of three antioxidans on phagocytic and kill activities of blood polymorphonuclear leukocytes (PMN) isolated from buffaloes during the peripartum period (4 weeks before to 7 weeks after parturition) was investigated in this study. Two concentrations of beta-carotene and vitamin A (10(-6) and 10(-5) M) and one concentration of Se (10(-9) M) were used. Phagocytic activity of PMN treated with beta-carotene (10(-6)M) significantly enhanced (P < 0.05) after parturition (Week 0 until Week 3), whereas the kill activity of the same cells significantly (P < 0.05) increased before and after parturition (at Weeks -4, -3, -2, 0, 1, 2 and 3). The concentration of beta-carotene (10(-5) M) enhanced phagocytosis of PMN only at Weeks 0 and 1 and kill activity at Weeks -4, -3, -2, 0, and 1. Selenium (10(-9)M) significantly (P < 0.05) enhanced phagocytic activity of PMN starting from parturition (Week 0) until Week 3 postpartum. Kill activity increased significantly both before (Weeks -4, -3 and -2) and after (Weeks 0, 1, 2, 3 and 4) parturition. Vitamin A (10(-6) M) significantly enhanced phagocytic activity of PMN at Weeks 0, 1, and 2, whereas, the concentration of beta-carotene (10(-5) M) increased phagocytic activity only at Week 0. Kill activity of PMN increased significantly (P < 0.05) at Weeks -1 and 0 (10(-6)M). These results demonstrate that beta-carotene and selenium significantly enhanced phagocytic and kill activities of PMN isolated from buffaloes around parturition in vitro. Vitamin A enhanced phagocytosis and kill activities but not to the same extent as beta-carotene and selenium. Apparently, the in vitro killing activity of PMN is a distinctive function from phagocytosis and both activities may be enhanced by the use of essential nutrients, especially during the peripartum period. Moreover, beta-carotene is more effective as an antioxidant than vitamin A in enhancing the activities of phagocytic cells.     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Antigenic variation of outer membrane protein II in colonial variants of Neisseria gonorrhoeae P9

Antibodies were detected by an enzyme-linked immunosorbent assay (ELISA) in sera from rabbits immunized wtih outer membranes from colonial opacity variants in Neisseria gonorrhoeae P9. ELISA-inhibition experiments with purified antigens revealed approximately equal proportions of antibodies directed against each of the three major surface antigens, lipopolysaccharide, the major outer membrane protein (protein I) and protein II, the variable protein associated with colonial opacity. Inhibition experiments with intact gonococci showed considerable antigenic diversity which could be correlated with differences between the protein II species present. Despite their considerable structural homology, different protein II species from colonial variants of the same strain showed little cross-reactivity with specific anti-protein II sera, thus demonstrating the considerable variation in that part of the antigen which is exposed on the surface of the gonococcus and is closely involved in pathogenic mechanisms.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Evaluation of the innate immune response in pups during canine parvovirus type 1 infection

In two pups (A and B) naturally infected with canine parvovirus type 1 (CPV1) phagocytic responses were evaluated over a period of two weeks (day 0 = T0; day 3 = T1; day 7 = T2; day 14 = T3). CPV1 infection led to a marked reduction of monocyte (MO) phagocytosis in both pups. Also MO killing was impaired and in pup B this function was totally absent. Polymorphonuclear (PMN) phagocytosis values of both pups fluctuated within normal ranges, as well as PMN killing of pup A. In pup B, killing exerted by PMN was absent at T0, then increased but again dropped below normal ranges at T3. The described alterations of phagocytic functions may be regarded as possible viral mechanisms of immune evasion.     

The early effect of sublethal X-irradiation of phagocytic cells in mouse blood and the influence of cystamine as measured by chemiluminescence

The metabolic burst accompanying phagocytosis of granulocytes (PMN) leads to the generation of activated oxygen species such as O-2, H2O2, 1O2 and OH; which give rise to chemiluminescence (CL) in the presence of luminol. Reliable CL-measurements of stimulated PMN can be carried out in freshly drawn mouse blood, when photon counts are related to the number of PMN. Effects of low dose total body X-irradiation were studied using C57B1/6 mice. It was found that 24 and 48 hours after irradiation (0.24-0.95 Gy) CL of whole blood was slightly decreased. If however CL-counts were related to the number of PMN, an enhanced CL per single granulocyte was recorded. The administration of cystamine leads to an immune stimulating effect of unirradiated animals. In animals, who received 0.95 Gy a distinct radioprotective effect of cystamine can be observed.     

A novel post-translational incorporation of tyrosine into multiple proteins in activated human neutrophils. Correlation with phagocytosis and activation of the NADPH oxidase-mediated respiratory burst.

Activation of human neutrophils by PMA causes a post-translational incorporation of 14C-labeled tyrosine into multiple neutrophil (PMN) proteins, that is distinctly different from the enzymatic tyrosinolation of tubulin in FMLP-stimulated PMN. Post-translational incorporation of other radiolabeled amino acids, including the structurally similar amino acid phenylalanine, does not occur under identical conditions of neutrophil activation, suggesting an involvement of the phenolic hydroxyl group of tyrosine in the PMA-mediated reaction. Similar to the stimulation of PMN tubulin tyrosinolation by FMLP, the PMA-induced incorporation of tyrosine into multiple PMN proteins is closely associated with activation of the NADPH oxidase-mediated respiratory burst in stimulated PMN and can be inhibited by a variety of reducing agents, inhibitors of peroxidase-mediated reactions, and intracellular scavengers of oxygen radicals. Moreover, the PMA-induced post-translational incorporation of tyrosine does not occur in PMN from patients with chronic granulomatous disease and is significantly reduced (50%) in PMN of an individual with myeloperoxidase deficiency. A similar stimulus-induced incorporation of tyrosine into multiple PMN proteins is also observed in PMN exposed to various phagocytic stimuli, and the incorporated radioactivity in cells undergoing phagocytosis is substantially enriched (40- to 50-fold) in isolated PMN phagolysosomes. Consistent with this latter observation, HPLC fractionation of stimulated PMN proteins and analysis of the incorporated radioactivity reveal that the 14C label is primarily associated with PMN membrane proteins. Furthermore, this post-translational incorporation of tyrosine, like that associated with PMA stimulation, is associated with production of oxygen radicals and the generation of protein carbonyl derivatives, which are indicative of oxidative protein modifications via mixed function oxidases. Our findings indicate that tyrosine incorporation into membrane proteins of stimulated PMN is functionally relevant to the physiologic host-defense responses of human neutrophils undergoing phagocytosis.     

Automation and computerization of chemiluminescence: A new methodological approach in the study of human phagocytes

Peripheral blood phagocytic cells (PMNLs) are activated by contact with opsonized particles. Metabolic activation of PMNLs is associated with a remarkable increase in the respiratory burst and generates high energy oxygen compounds which are responsible for the bactericidal activity of PMNLs and for their ability to produce luminol-dependent chemiluminescence (CL). The CL phenomenon is measured by an automated and computerized photoluminometer (Berthold LB950) in whole blood stimulated with opsonized zymosan. This whole blood method of CL measurement has been applied to the study of the phagocytic process and to the investigation of cellular and humoral abnormalities in several pathologies, indicating this assay as a simple, rapid and reliable test.     

Trypanosoma cruzi: parasite-induced release of lysosomal enzymes by human polymorphonuclear leukocytes

The release of beta-glucuronidase and lysozyme from human polymorphonuclear leukocytes (PMN) engaged in phagocytosis and lysis of Trypanosoma cruzi epimastigotes was studied in the presence or absence of chagasic serum. Lysosomal enzyme release was enhanced when parasites were sensitized with serum from a chronic Chagas' patient, increased up to 3 hr of incubation at 28 C, and depended on the PMN:parasite ratio. The release of lysosomal enzymes was determined by the presence of 2 mM cyanide, 2 microM azide, 3 mM amobarbital, and 1 mM phenylbutazone. These drugs inhibited the killing of sensitized T. cruzi by interfering with the oxidative microbicidal mechanisms of PMN without affecting the uptake of the parasites. Lysosomal enzyme release occurred in the presence of cyanide and azide, indicating that in these cases the enzymatic release was unrelated to the killing of the parasites. Amobarbital and phenylbutazone, which stabilize PMN membranes, inhibited the release of beta-glucuronidase and lysozyme by PMN. The addition of 10 micrograms/ml of cytochalasin B inhibited the phagocytosis and killing of sensitized T. cruzi by PMN but increased the enzymatic release by effector cells. Since cytochalasin B did not affect the close contact between PMN and parasites, it appears that the enzymes released to the extracellular milieu were not toxic to noningested parasites. Furthermore, the lysosomal enzymes did not lyse bystander unsensitized parasites. Therefore, the release of lysosomal enzymes during the interaction of T. cruzi epimastigotes and PMN seems to be related to the triggering event of the phagocytic process and does not bear a cause-effect relationship with parasite death.