Ruth Bryan

Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8.

Respiratory epithelial cells play a crucial role in the inflammatory response during Pseudomonas aeruginosa infection in the lungs of patients with cystic fibrosis. In this study, we determined whether the binding of specific Pseudomonas gene products (pilin, flagellin) to their receptors on respiratory epithelial cells would result in production of the neutrophil chemoattractant IL-8. Piliated wild-type organisms, purified pili, or antibody to the pilin receptor (asialoGM1) evoked significant production of IL-8 by immortalized airway epithelial cells, whereas nonpiliated organisms were less able to bind to respiratory epithelial cells and stimulated much less IL-8 secretion (P < 0.01). A piliated, nonflagellated strain was also associated with decreased binding and a diminished level of IL-8 production when compared to wild-type organisms. Isogenic, nonadherent rpoN mutants, lacking pilin and flagellin, did not bind or elicit an IL-8 response. In addition, the IL-8 response was four-fold higher in a cystic fibrosis cell line compared with its corrected cell line. The Pseudomonas autoinducer, an exoproduct secreted during chronic infection, was found to stimulate IL-8 in a dose-dependent manner. P. aeruginosa adhesins, which are necessary for initial infection, directly stimulate IL-8 production by respiratory epithelial cells and therefore play a major role in the pathogenesis of Pseudomonas infection in patients with cystic fibrosis. The inflammatory response is subsequently perpetuated by Pseudomonas autoinducer which is secreted during chronic infection.

Activation of NF-kappaB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells.

PMN-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease. Epithelial cells respond to organisms such as Pseudomonas aeruginosa, the major pathogen in CF, by expressing the leukocyte chemokine IL-8. Experiments were performed using several different types of respiratory epithelial cells that demonstrate that ligation of ceramide-associated receptors on epithelial surfaces by P. aeruginosa pili is a major stimulus for the translocation of transcription factor nuclear factor (NF)-kappaB and initiation of IL-8 expression by epithelial cells. Using electrophoretic mobility shift assays and Western hybridizations, nuclear NF-kappaB was found shortly after epithelial cells were stimulated by either whole organisms, isolated pili, or antibody to the pilin receptor asialoGM1. IB3 cells, which express mutations in cystic fibrosis transmembrane conductance regulator (CFTR) (DeltaF508/W1282X), were noted to have significantly greater amounts of endogenous nuclear NF-kappaB, but not the transcription factor C/EBP, than CF cells corrected by episomal copies of normal CFTR (C-38) or IB3 cells grown at a permissive temperature (25 degreesC). Activation of NF-kappaB and subsequent IL-8 expression in epithelial cells can result from activation of at least two pathways: an exogenous signaling cascade that is activated by ligation of ceramide-associated adhesins such as P. aeruginosa pilin, or endogenous stimulation, suggested to be a consequence of cell stress caused by the accumulation of mutant CFTR in the endoplasmic reticulum.

The organization of the Caulobacter crescentus flagellar filament.

The structural organization of the flagellar filament of Caulobacter crescentus, as revealed by immunoelectron microscopy, shows five antigenically distinct regions within the hook-filament complex. The first region is the hook. The second region is adjacent to the hook and is approximately 10 nm in length. On the basis of its location in the hook-filament complex, this region may contain hook-associated proteins. Next to this is the third region, which is approximately 60 nm in length. Antibody decoration experiments using mutant strains with deletions of the structural gene for the 29 x 10(3) Mr flagellin (flgJ) showed that the presence of this region is correlated with the expression of the 29 x 10(3) Mr flagellin gene. The next region (region IV), of length approximately 1 to 2 microns, appears to contain the 27.5 x 10(3) Mr flagellin, but at its distal end includes, in gradually increasing amounts, the 25 x 10(3) Mr flagellin. The rest of the filament (region V) is made up predominantly, if not completely, of the 25 x 10(3) Mr flagellin. Except for the hook, there are no morphological features that would otherwise distinguish these regions. A functional flagellum, having the wild-type length and morphology, is assembled by mutant strains deficient in the 29 x 10(3) Mr flagellin and 27.5 x 10(3) Mr flagellin.

Temporal regulation and overlap organization of two Caulobacter flagellar genes

The biogenesis of the bacterial flagellum and chemotaxis apparatus in both Escherichia coli and Caulobacter crescentus requires the ordered expression of over 40 genes whose expression is controlled by a trans-acting regulatory hierarchy. In C. crescentus, additional control mechanisms ensure that the transcription of these genes is initiated at the correct time in the cell cycle. We demonstrate here that two flagellar genes, flaE and flaY, whose products function in trans to modulate the level of transcription of other flagellar genes, are themselves temporally controlled. DNA sequence analysis of the 3413 base-pairs encompassing the flaE and flaY coding sequences and the 5 regulatory region showed that flaE encodes a protein of 16,000 Mr and flaY a protein of 17,000 Mr. Evidence that flaE and flaY are transcribed as a polycistronic message includes (1) the polar effect of Tn5 insertions; (2) deletion analysis showing that the flaE promoter is essential for complementation of both flaE and flaY alleles; and (3) nuclease S1 assays showing protection of a transcript spanning both genes. The transcript start site in front of flaE was determined and the -10 region conforms to the E. coli sigma 28 promoter consensus sequence. Nuclease S1 analysis also revealed a protected fragment whose size was consistent with a transcript initiating in vivo at a consensus nif promoter sequence in front of the flaY gene. The entire promoter region and an upstream consensus sequence that might be a regulatory element for the flaY gene lies within the carboxyl-terminal coding sequence of the flaE gene.

Genetic regulatory hierarchy in Caulobacter development

Publisher Summary This chapter describes the Caulobacter regulatory cascade that controls flagellar morphogenesis and then compares the observed mechanisms to those that appear in other prokaryotic developmental systems. It also focuses on the freshwater Caulobacter crescentus, which is the best known species of the group. Caulobacters are dimorphic, Gram-negative bacteria which are found in fresh water, salt water, and soils. Caulobacters are well adapted to survival under the conditions of limited nutrients. The stalked cell attaches to the surfaces using the holdfast at the end of the stalk, whereas the motile cell (swarmer) can undergo chemotaxis, swimming toward a more favorable location. The chapter concludes that, ecologically, Caulobacter exploits two survival strategies each generation: one cell remains and one swims away.

Temporal and Spatial Control of Flagellar and Chemotaxis Gene Expression during Caulobacter Cell Differentiation

Each Caulobacter cell division yields daughter cells that differ from one another both structurally and functionally. By focusing on the biogenesis of the polar flagellum and the proteins of the chemosensory system, several laboratories have now defined an extensive network of genes whose temporal expression is controlled in the predivisional cell. The differential turn-on of these genes contributes to the generation of asymmetry in the predivisional cell in that the products of these genes are targeted to specific cellular locations. To define the mechanisms that mediate this temporal and spatial control, fla genes whose products are not known were accessed by the insertion of transposon-carried drug resistance markers. The transposons were altered so that upon insertion into the chromosome, transcription fusions are formed in which the promoter regions of fla genes drive the expression of the downstream promoter-less drug resistance genes. Assays of the differential placement of the promoter-less drug resistance proteins (encoded within the interrupted fla genes) allow us to determine whether the positioning of the fla gene products is controlled by signal sequences in their proteins, by specific mRNA-targeting sequences in the 5-regulatory regions of these genes, or by specific transcription from only one of the two newly replicated chromosomes in the predivisional cell.

Staphylococcus aureus virulence in the respiratory tract:agr and sar dependent activation of epithelial Ca2+ fluxes and IL-8 expression |[diams]| 849

S. aureus is a common respiratory pathogen in cystic fibrosis particularly in young patients. In this study we used well characterized mutants of S. aureus RN6390 to identify which virulence factors are important in the pathogenesis of pulmonary disease and the induction of inflammation in the lung. A neonatal mouse model of acute pulmonary infection was used to compare the virulence of the parental strain, and genetically constructed mutants lacking either of the global regulatory genes sar (Staphylococcus accessory regulator) agr (accessory gene regulator) or both regulatory loci. Whereas the parental strain caused pneumonia, as defined by recovery of >103cfu/100 μl of lung tissue, in 100% of the 10-14 day old BALBc/ByJ mice (n=30) when introduced intranasally, 100% bacteremia and 30% mortality; the agr sar mutant infected 75% of the inoculated mice (n=32), caused bacteremia in 38% and 0% mortality (P<0.01). Infection associated with a single mutation in sar gave an intermediate phenotype, with 100% infection, but 0% mortality. To examine the mechanisms through which S. aureus cause pneumonia, we compared the ability of the different strains to activate signal transduction in fura-2 loaded 16HBEo- human respiratory epithelial cells, by monitoring the changes in intracellular Ca2+ after exposure to the bacteria. Adherence of the wild type strain promptly stimulated oscillating Ca2+ waves, while there was minimal response to the sar mutant, and no response to the sar, agr mutant. However, the sar, agr, and double mutant strains were able to stimulate IL-8 expression from human epithelial cells lines in a dose dependent fashion in the presence of 0.1% fetal bovine serum, to levels comparable to that elicited by an IL-1β positive control. Neither S. aureus culture supernatant, nor dead organisms stimulated an IL-8 response. These results suggest that S. aureus has multiple surface components which trigger epithelial responses, and while the agr and sar regulatory loci are clearly implicated in virulence in vivo, even avirulent mutants can elicit a significant proinflammatory response from respiratory epithelial cells. S. aureus colonization in children with CF, can contribute to ongoing airway inflammation, even in the absence of chronic infection.

THE USE OF CARBOHYDRATES TO BLOCK THE ATTACHMENT OF PSEUDOMONAS AERUGINOSA TO EPITHELIAL CELLS. • 2290

THE USE OF CARBOHYDRATES TO BLOCK THE ATTACHMENT OF PSEUDOMONAS AERUGINOSA TO EPITHELIAL CELLS. • 2290

Rapid Induction of Signal Transduction in Respiratory Epithelial Cells in Response to Pseudomonas aeruginosa Ligands |[diams]| 898

Studies of bronchoalveolar lavage from infants with cystic fibrosis (CF) have demonstrated increased amounts of proinflammatory cytokines and polymorphonuclear leukocytes even in the absence of detectable infection. Decreased clearance of secretions in the respiratory tree may lead to exposure of epithelial cells to environmental organisms that would be efficiently cleared in a normal host. We hypothesized that even transient exposure to Pseudomonas aeruginosa (PA) ligands, which recognize specific glycolipid receptors on respiratory epithelial cells, can lead to activation of host signal transduction pathways. Fluxes in intracellular calcium concentration ([Ca2+]i) are common components of proinflammatory signalling cascades and can be readily monitored by fluorescence imaging. We studied the kinetics of changes in [Ca2+]i of epithelial cells in response to wild-type PA as well as to several mutant strains, purified PA ligands, and antibody to the pilin receptor. Single cell imaging was performed on primary human nasal polyp, bovine tracheal epithelial cells, and SV-40 immortalized respiratory cell lines (1HAEo- and 16HBE). The cells were loaded with fura-2/AM, a calcium-sensitive, ratiometric fluorescent dye. Stimulation of the cells with wild-type PA (strain PAO1) triggered a 100-fold increase in [Ca2+]i that was maintained for several minutes. UV-killed organisms did not elicit a change in [Ca2+]i. Stimulation with a non-piliated strain (PAO-NP) or a non-piliated non-flagellated strain (PAO-NP fliA) led to a greatly attenuated calcium response by the epithelial cells. Purified PAO1 flagellin induced a more rapid increase in [Ca2+]i that returned to baseline in less than one minute. Treatment with purified pilin protein or antibody to asialoGM1 (the pilin receptor) led to a typical [Ca2+]i oscillatory response in the epithelial cells. All of the above responses were attenuated but not eliminated by chelation of extracellular calcium with EGTA, indicating that intracellular stores are likely to be responsible for these signals. These results demonstrate activation of signal transduction pathways in respiratory cells in response to only brief exposure to P. aeruginosa ligands.

Pseudomonas aeruginosa Interactions with Epithelial Cells: Adherence, Invasion and Apoptosis |[diams]| 896

P. aeruginosa is primarily an extracellular pathogen which adheres to cystic fibrosis epithelial cells due to increased numbers of asialyated receptors on the epithelial surface. Recent studies suggest that P. aeruginosa interacts with normal CFTR leading to ingestion by respiratory epithelial cells (Pier et al Science, 1996) which are then shed, possibly due to apoptosis. Both these processes may be relevant to the pathogenesis of infection in cystic fibrosis. We compared the trafficking of P. aeruginosa in CF and normal respiratory epithelial cells in primary culture and in transformed cell lines using flow cytometry, electron microscopy and confocal microscopy. To determine the relative contribution of superficial attachment versus epithelial phagocytosis, epithelial cell lines were grown in a polarized fashion on transwells, and treated with 1×108 cfu/ml of PAO1. When examined by EM polarized cells with tight junctions show minimal P. aeruginosa adherence and no invasion. Epithelial cells with disrupted tight junctions had increased intracellular bacterial invasion and evidence of intracellular replication. Flow cytometry was used to differentiate adherent from ingested bacteria, facilitating rapid analysis of a large number of individual cells. PAO1 labeled with green fluorescent protein (GFP) were incubated with monolayers of IB-3 (CF) and C-38 cells (“corrected”) cell lines. FACS analysis allowed the enumeration of all cells associated with bacteria and secondary labeling with Texas Red detected adherent but not ingested organisms. 18% of the CF cells had ingested organisms, which was half the amount ingested by the corrected cell line (44%). Ingestion of PAO1 was not abolished by cytochalasin-D. Confocal microscopy clearly differentiated green (ingested) from yellow (adherent) bacteria. These results contrasted markedly with the data obtained with primary cells which retained tight junctions and had virtually no internalized bacteria. A TUNEL assay demonstrated that epithelial cells with ingested bacteria were apoptotic within 6 hours. These experiments suggest P. aeruginosa can invade epithelial cells when the integrity of the epithelium is damaged but are only minimally adherent and non-invasive to an intact respiratory epithelium.