Functional near infrared spectroscopy detects cortical activation changes concurrent with memory loss in postmenopausal women with Type II Diabetes.

Older adults with Type II Diabetes Mellitus (DM) experience mild cognitive impairment, specifically in the domain of recall/working memory. No consistent causative structural cortical deficits have been identified in persons with DM (PwDM). Memory deficits may be exacerbated in older adult females, who are at the highest risk of cardiovascular decline due to DM. The focus of the current study was to evaluate functional cortical hemodynamic activity during memory tasks in postmenopausal PwDM. Functional Near Infrared Spectroscopy (fNIRS) was used to monitor oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) during memory-based tasks in a cross-sectional sample of postmenopausal women with DM. Twenty-one community-dwelling DM females (age = 65 ± 6 years) and twenty-one age- and sex-matched healthy controls (age = 66 ± 6 years) were evaluated. Working memory performance (via N-back) was evaluated while study participants donned cortical fNIRS. Health state, metabolic data, and menopausal status data were also collected. Deficits in working memory accuracy were found in the DM group as compared to controls. Differences in HbO responses emerged in the DM group. The DM group exhibited altered PFC activity magnitudes and increased functional cortical activity across ROIs compared to controls. HbO and HbR responses were not associated with worsened health state measures. These data indicate a shift in cortical activity patterns with memory deficits in postmenopausal PwDM. This DM-specific shift of HbO is a novel finding that is unlikely to be detected by fMRI. This underscores the value of using non-MRI-based neuroimaging techniques to evaluate cortical hemodynamic function to detect early mild cognitive impairment.

Expert athletes activate somatosensory and motor planning regions of the brain when passively listening to familiar sports sounds.

The present functional magnetic resonance imaging study examined the neural response to familiar and unfamiliar, sport and non-sport environmental sounds in expert and novice athletes. Results revealed differential neural responses dependent on sports expertise. Experts had greater neural activation than novices in focal sensorimotor areas such as the supplementary motor area, and pre- and postcentral gyri. Novices showed greater activation than experts in widespread areas involved in perception (i.e. supramarginal, middle occipital, and calcarine gyri; precuneus; inferior and superior parietal lobules), and motor planning and processing (i.e. inferior frontal, middle frontal, and middle temporal gyri). These between-group neural differences also appeared as an expertise effect within specific conditions. Experts showed greater activation than novices during the sport familiar condition in regions responsible for auditory and motor planning, including the inferior frontal gyrus and the parietal operculum. Novices only showed greater activation than experts in the supramarginal gyrus and pons during the non-sport unfamiliar condition, and in the middle frontal gyrus during the sport unfamiliar condition. These results are consistent with the view that expert athletes are attuned to only the most familiar, highly relevant sounds and tune out unfamiliar, irrelevant sounds. Furthermore, these findings that athletes show activation in areas known to be involved in action planning when passively listening to sounds suggests that auditory perception of action can lead to the re-instantiation of neural areas involved in producing these actions, especially if someone has expertise performing the actions.

Functional neuroimaging of sensorimotor cortices in postmenopausal women with type II diabetes.

Significance: Deficits in sensorimotor function in persons with type II diabetes mellitus (PwDM) have traditionally been considered a result of peripheral nerve damage. Emerging evidence has suggested that factors outside of nerve damage due to type II diabetes mellitus, such as impaired hemodynamic function, contribute significantly to both sensory and motor deficits in PwDM. Aim: The focus of the current study was to evaluate functional cortical hemodynamic activity during sensory and motor tasks in PwDM. Approach: Functional near-infrared spectroscopy was used to monitor oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) across the cortex during sensory and motor tasks involving the hands. Results: Decline in HbO across sensory and motor regions of interest was found in PwDM with simultaneous deficits in manual motor tasks, providing the first evidence of functional cortical hemodynamic activity deficits relating to motor dysfunction in PwDM. Similar deficits were neither specifically noted in HbR nor during evaluation of sensory function. Health state indices, such as A 1 c , blood pressure, body mass index, and cholesterol, were found to clarify group effects. Conclusions: Further work is needed to clarify potential sex-based differences in PwDM during motor tasks as well as the root of reduced cortical HbO indices but unchanged HbR indices in PwDM.

P. aeruginosa Biofilms in CF Infection.

Pseudomonas aeruginosa is an opportunistic pathogen of immunocompromised hosts. In cystic fibrosis (CF), P. aeruginosa causes acute and chronic lung infections that result in significant morbidity and mortality. P. aeruginosa possesses several traits that contribute to its ability to colonize and persist in acute and chronic infections. These include high resistance to antimicrobials, ability to form biofilms, plethora of virulence products, and metabolic versatility. In P. aeruginosa, a cell-to-cell communication process termed quorum sensing (QS) regulates many of these factors that contribute to its pathogenesis. Recent evidence suggests that the CF lung environment presents a specialized niche for P. aeruginosa. The relationship of P. aeruginosa QS, biofilm formation, and the CF lung environment is discussed.

Review of Catheter-Associated Urinary Tract Infections and In Vitro Urinary Tract Models.

Catheter-associated urinary tract infections (CAUTIs) are one of the most common nosocomial infections and can lead to numerous medical complications from the mild catheter encrustation and bladder stones to the severe septicaemia, endotoxic shock, and pyelonephritis. Catheters are one of the most commonly used medical devices in the world and can be characterised as either indwelling (ID) or intermittent catheters (IC). The primary challenges in the use of IDs are biofilm formation and encrustation. ICs are increasingly seen as a solution to the complications caused by IDs as ICs pose no risk of biofilm formation due to their short time in the body and a lower risk of bladder stone formation. Research on IDs has focused on the use of antimicrobial and antibiofilm compounds, while research on ICs has focused on preventing bacteria entering the urinary tract or coming into contact with the catheter. There is an urgent need for in vitro urinary tract models to facilitate faster research and development for CAUTI prevention. There are currently three urinary tract models that test IDs; however, there is only a single very limited model for testing ICs. There is currently no standardised urinary tract model to test the efficacies of ICs.

Same day discharge after thyroidectomy is safe and effective.

Historically, thyroidectomies have been performed as inpatient operations due to concerns of postoperative bleeding and symptomatic hypocalcemia. We aim to demonstrate that outpatient thyroidectomy can be performed safely. METHODS: This report outlines a 7-year retrospective analysis (2009-2016) of outpatient vs inpatient thyroidectomies, with outcomes including hematoma, blood loss, recurrent laryngeal nerve injury, symptomatic hypocalcemia, and postoperative emergency room (ER) visits. RESULTS: A total of 1460 thyroidectomies were performed: 1272 (87%) outpatient and 188 (13%) inpatient. Five outpatients: 4 total thyroidectomies (TT), 1 TT with a central lymph node dissection (CLND), and 1 partial thyroidectomy (PT) developed postoperative hematomas (0.34%) at post-discharge hour 3, 9, 10, 13, and 42. Average time to discharge was 2 hours and 37 minutes. Hematomas were evacuated successfully in the operating room under local anesthesia with a 2-day average hospital stay. There were no differences between TT, thyroid lobectomy (TL), and PT procedures for postoperative hematoma (p=0.17). Outpatient compared to inpatient thyroidectomy was more likely to have been performed in patients with lower American Society of Anesthesia scores (2.3 vs 2.9, p<0.0001), less mean blood loss (74 vs 227 ml, p<0.0001), lesser age (52 vs 56 years, p=0.0012), less extensive dissection (p<0.0001), and fewer RLN injuries (2.4% vs 8.5%, p<0.0001). There was no difference between outpatient and inpatient symptomatic hypocalcemia (6.3% vs 9.6%, p=0.09), 30-day postoperative ER visits (8.8% vs 9.6%, p=0.73), and postoperative hematoma (0.39% vs 0%, p=0.39). There was one inpatient mortality from stroke. CONCLUSION: Postoperative hematomas can be managed safely without life-threatening complications suggesting outpatient thyroidectomy can be performed safely by an experienced surgeon, and adverse sequelae dealt with in a safe and effective manner.

Quorum sensing: dynamic response of Pseudomonas aeruginosa to external signals.

A recent study suggests that the opportunistic pathogen Pseudomonas aeruginosa can actively monitor the host immune system. The P. aeruginosa outer membrane protein OprF was found to bind specifically to the cytokine interferon-gamma (IFN-gamma), and this interaction upregulated production of virulence factors through a cell-cell communication system known as quorum sensing (QS). Taken together with previous findings that P. aeruginosa QS can alter the host immune response (e.g. by activation of IFN-gamma), these data illustrate an exciting new element of bacteria-host interactions in which the P. aeruginosa quorum-sensing system both senses and modulates the host immune state.

Protein and bacterial fouling characteristics of peptide and antibody decorated surfaces of PEG-poly(acrylic acid) co-polymers.

The potential for base poly(ethylene glycol) graft poly(acrylic acid) PEG-g-PA copolymers and surface-modified PEG-g-PA materials to inhibit random protein fouling and bacterial adhesion are investigated. PEG-g-PA co-polymers were synthesized that inhibited non-specific protein and cellular adhesion. PEG-g-PA co-polymers were then covalently modified with either cell adhesion peptides (YRGDS, YEILDV) or fragments of antibodies to monocyte/macrophage integrin receptors (Anti-VLA4, Anti-beta1, Anti-beta2, and Anti-CD64) known to enhance macrophage adhesion and, perhaps, modulate their activation. Materials produced in this work were characterized using: hydrophobicity by contact angle; angle-resolved X-ray Photoelectron Spectroscopy to confirm the presence of PEG in the bulk material and the surface; degree of hydration; differential scanning calorimetry; and thermal gravimetric analysis. To evaluate the non-fouling efficacy of the various modified surfaces, three proteins, human serum albumin, human fibronectin (Fraction I) and human immunoglobulin were 125I labeled. Samples of base PEG-g-PA and PEG-g-PA, modified with various peptides, were exposed to solutions containing either 2 or 200 microg/ml of one of the labeled proteins at 37 degrees C for 24 h. PEG-g-PA substrata modified with directly bound peptides exhibited protein adsorption that varied depending upon the surface bounded peptide. PEG-g-PA modified with peptides linked by linear PEG tethers reduced protein adsorption at 24 h by approximately 45% in comparison to PEG-g-PA. Peptides linked by way of StarPEO and StarlikePEO tethers further decreased protein adsorption in comparison to PEG-g-PA. The ability of peptide:PEOtethers to inhibit protein adsorption appeared to be a function of type and surface coverage of the PEO tether and not influenced by the amount or molecular structure the tethered peptide. Peptides directly coupled to the PEG-g-PA increased the amount of protein fouling relative to controls and there appeared to be some dependency of the amount of protein adsorption on which peptide was tethered. Two 14C-labeled pathogens, Staphylococcus epidermidis and Pseudomonas aeruginosa, were used to quantify the degree of bacterial adhesion using two types of laminar flow cell chambers; one that provided invasive sampling of the target substrata and one that provided non-invasive microscopic surveillance of adhering bacterial cells. Attachment of both species to PEG-g-PA and peptide-modified PEG-g-PA was reduced compared to the basic poly(acrylic acid). Presence of peptides on the surface, whether directly bound or bound by the PEO tether did not influence adhesion of P. aeruginosa relative to controls. S. epidermidis adhesion rates increased slightly for those materials where peptides were directly bound to the surface but were reduced relative to base PEG-g-PA when peptides were bound by PEO tethers. All PEG-g-PA surfaces modified with fragments of monoclonal antibodies dramatically enhanced bacterial initial adhesion rates and maximum extent of attachment.

Monocyte/macrophage interactions with base and linear- and star-like PEG-modified PEG-poly(acrylic acid) co-polymers.

Poly(ethylene glycol):poly(acrylate) PEG-g-PA co-polymers were made that inhibited nonspecific protein and cellular adhesion. PEG-g-PA co-polymers were then covalently modified with either cell adhesion peptides or fragments of antibodies to monocyte/macrophage integrin receptors (anti-VLA4, anti-beta(1), anti-beta(2), and anti-CD64) known to enhance macrophage adhesion and, perhaps, modulate their activation. Peptides were either directly conjugated to the base material or linked by way of PEO-star tethers. Fragments of the antibody region containing the antigen-binding site (Fab' fragments) were coupled to other PEG-g-PA samples using the sulhydryl end groups on Fab' fragments to amine-bearing PEO stars. Macrophage adhesion rates, phagocytic response (oxidative burst), and cytokine expression were determined for each PEG-g-PA material. Luminol-enhanced chemiluminescence was used as a semiquantitative indication of monocyte-macrophage phagocytic activation (oxidative burst). Macrophage cytokine expression in response to control, base, and modified materials was determined by ELISAs for TNF-alpha, IL-1 beta, IL-6, and IL-8. Tissue culture poly(styrene) (TCPS)-mediated the greatest number of adherent monocyte/macrophage cells relative to PEG-g-PA materials. Both YRGDS and YEILDV peptides, whether directly or indirectly (via StarPEO) conjugated to PEG-g-PA, increased adhesion versus controls. Fab' fragments of all four antibodies also promoted enhanced adhesion versus controls. Fab'StarPEO materials presented two orders of magnitude fewer ligands per surface unit area than peptide star materials (10(8) vs. 10(10)), but were able to adhere similar numbers of cells. For surfaces presenting Fab'(VLA-4) or YEILDV, both of which may both bind to a cell's VLA-4 receptor, the Star:VLA4 surface showed a greater number of adherent monocyte/macrophages. This result suggests that the Fab' had a higher affinity to the cell receptor than a corresponding minimal peptide binding sequence. All materials exhibited low oxidative burst (luminescence counts per minute, LCPM) per cell DNA without the addition of exogenous stimuli (LCPM/DNA < 100). Directly conjugated peptide materials, poly(propylene) (PP), and TCPS showed the lowest levels of LCPM/DNA without the addition of exogenous stimulus (LCPM/DNA < 20). There was no correlation between LCPM/DNA ratios, with and without added LPS stimulus, versus the individual substrates. Monocyte/macrophages adherent to TCPS substrata showed the overall highest stimulatory potential in cytokine expression response to exogenous LPS, followed by PP > PEG-g-PA > StarPEO. Cells adherent to peptide-modified materials and Fab'-modified materials were overall less stimulated. The method of presenting the peptides (i.e., directly or via Star PEO) influenced the level of cytokine secreted by the adherent macrophage.

Poly(ethylene glycol)-polyacrylate copolymers modified to control adherent monocyte-macrophage physiology: interactions with attaching Staphylococcus epidermidis or Pseudomonas aeruginosa bacteria.

The ability of various surface modifications of poly(ethylene glycol)-graft-polyacrylate (PEG-g-PA) copolymers (tethered adhesion peptides and fragments of monoclonal antibodies) to modulate monocyte-macrophage cell interactions with surface colonizing bacteria is reported. The PEG-g-PA copolymers were made to inhibit nonspecific protein and cellular adhesion. The copolymers were then covalently modified with either cell adhesion peptides (YRGDS, YEILDV, or YRGES) or fragments of antibodies to monocyte-macrophage integrin receptors (anti-VLA4, anti-beta(1), anti-beta(2), and anti-CD64), which are known to enhance macrophage adhesion and perhaps modulate their activation. Cytokine expression and phagocytosis response by surface adherent monocyte-macrophages to Staphylococcus epidermidis and Pseudomonas aeruginosa bacteria were quantified. The cytokine expression (interleukins 6 and 1 beta) of adherent macrophages in response to the modified polymers only and to bacterial challenges were quantified by dynamic ELISA assays. The adherent macrophage phagocytic response (oxidative burst) to various materials is compared to oxidative responses to both opsonized and nonopsonized S. epidermidis and P. aeruginosa bacteria. The efficiency of adherent macrophages to ingest and kill both species was determined using radiolabeled and fluorescent labeled bacterial cell ingestion studies as a function of the PEG-g-PA surface modification. Materials modified with adhesion peptides marginally enhanced (2x) macrophage attachment versus controls but, upon bacterial challenges, these materials predisposed adherent macrophages to overexpress proinflammatory cytokines and to exhibit a significant phagocytic response. Conversely, PEG-g-PA materials modified by fragments of monoclonal antibodies significantly enhanced (7x) macrophage adhesion but, upon bacterial challenge, "per cell" cytokine expression levels were reduced compared to peptide modified materials. Macrophages adhering to antibody fragment modified surfaces also exhibited sustained enhanced phagocytic response and higher bacterial killing efficiencies when compared with peptide modified materials.

Pseudomonas aeruginosa PA1006, which plays a role in molybdenum homeostasis, is required for nitrate utilization, biofilm formation, and virulence.

Pseudomonas aeruginosa (Pae) is a clinically important opportunistic pathogen. Herein, we demonstrate that the PA1006 protein is critical for all nitrate reductase activities, growth as a biofilm in a continuous flow system, as well as virulence in mouse burn and rat lung model systems. Microarray analysis revealed that ΔPA1006 cells displayed extensive alterations in gene expression including nitrate-responsive, quorum sensing (including PQS production), and iron-regulated genes, as well as molybdenum cofactor and Fe-S cluster biosynthesis factors, members of the TCA cycle, and Type VI Secretion System components. Phenotype Microarray™ profiles of ΔPA1006 aerobic cultures using Biolog plates also revealed a reduced ability to utilize a number of TCA cycle intermediates as well as a failure to utilize xanthine as a sole source of nitrogen. As a whole, these data indicate that the loss of PA1006 confers extensive changes in Pae metabolism. Based upon homology of PA1006 to the E. coli YhhP protein and data from the accompanying study, loss of PA1006 persulfuration and/or molybdenum homeostasis are likely the cause of extensive metabolic alterations that impact biofilm development and virulence in the ΔPA1006 mutant.

Analysis of the hierarchy of quorum-sensing regulation in Pseudomonas aeruginosa.

Quorum-sensing in Pseudomonas aeruginosa is known to regulate several aspects of pathogenesis, including virulence factor production, biofilm development, and antimicrobial resistance. Recent high-throughput analysis has revealed the existence of several layers of regulation within the QS-circuit. To address this complexity, mutations in genes encoding known or putative transcriptional regulators that were also identified as being regulated by the las and/or rhl QS systems were screened for their contribution in mediating several phenotypes, for example motility, secreted virulence products, and pathogenic capacity in a lettuce leaf model. These studies have further elucidated the potential contribution to virulence of these genes within the QS regulon.

Pseudomonas aeruginosa AlgR represses the Rhl quorum-sensing system in a biofilm-specific manner.

AlgR controls numerous virulence factors in Pseudomonas aeruginosa, including alginate, hydrogen cyanide production, and type IV pilus-mediated twitching motility. In this study, the role of AlgR in biofilms was examined in continuous-flow and static biofilm assays. Strain PSL317 (DeltaalgR) produced one-third the biofilm biomass of wild-type strain PAO1. Complementation with algR, but not fimTU-pilVWXY1Y2E, restored PSL317 to the wild-type biofilm phenotype. Comparisons of the transcriptional profiles of biofilm-grown PAO1 and PSL317 revealed that a number of quorum-sensing genes were upregulated in the algR deletion strain. Measurement of rhlA::lacZ and rhlI::lacZ promoter fusions confirmed the transcriptional profiling data when PSL317 was grown as a biofilm, but not planktonically. Increased amounts of rhamnolipids and N-butyryl homoserine lactone were detected in the biofilm effluent but not the planktonic supernatants of the algR mutant. Additionally, AlgR specifically bound to the rhlA and rhlI promoters in mobility shift assays. Moreover, PAO1 containing a chromosomal mutated AlgR binding site in its rhlI promoter formed biofilms and produced increased amounts of rhamnolipids similarly to the algR deletion strain. These observations indicate that AlgR specifically represses the Rhl quorum-sensing system during biofilm growth and that such repression is necessary for normal biofilm development. These data also suggest that AlgR may control transcription in a contact-dependent or biofilm-specific manner.

A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms.

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an approximately 5 x 10(3) strain PAO1 P. aeruginosa. Although bacteria grew to 10(10) cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical-chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.

Molecular basis of azithromycin-resistant Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms are extremely recalcitrant to antibiotic treatment. Treatment of cystic fibrosis patients with azithromycin (AZM) has shown promise. We used DNA microarrays to identify differentially expressed transcripts in developing P. aeruginosa biofilms exposed to 2 mug/ml AZM. We report that transcripts for multiple restriction-nodulation-cell division (RND) efflux pumps, known to be involved in planktonic antibiotic resistance, and transcripts involved in type III secretion were upregulated in the resistant biofilms that developed in the presence of AZM. Interestingly, the MexAB-OprM and MexCD-OprJ efflux pumps, but not type III secretion, appear to be integral to biofilm formation in the presence of AZM, as evidenced by the fact that a mutant deleted in both mexAB-oprM and mexCD-oprJ was unable to form a biofilm in the presence of AZM. A mutant deleted in type III secretion was still able to form biofilms in the presence of drug. Furthermore, single mexAB-oprM- and mexCD-oprJ-null mutants were able to form a biofilm in the presence of drug, indicating that either of the pumps can confer resistance to AZM during biofilm development. In contrast to planktonically grown cells, where no mexC expression was detectable regardless of the presence of AZM, biofilms exhibited induction of mexC expression from the outset of their formation, but only in the presence of AZM. mexA, which is constitutively expressed in planktonic cells, was uniformly expressed in biofilms regardless of the presence of AZM. These data indicate that the MexCD-OprJ pump acts as a biofilm-specific mechanism for AZM resistance.

Transcriptome analysis of quorum-sensing regulation and virulence factor expression in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa possesses two well-studied quorum-sensing (QS) systems (las and rhl) that are important in the production of virulence factors, antibiotic sensitivity, and biofilm development. High-density oligonucleotide microarrays were used to further characterize the las QS system and to investigate the effect of environment (planktonic or biofilm mode of growth, absence or presence of oxygen) and nutritional conditions on detection of transcripts encoding QS-regulated virulence factors. Transcriptome results indicate that the QS system is far more complex than previously proposed. Interestingly, we found that many QS-regulated genes encoding virulence products were expressed in all conditions investigated.

Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment.

Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. Two QS systems, known as the las and rhl systems, have been identified previously in the opportunistic pathogen Pseudomonas aeruginosa. High-density oligonucleotide microarrays for the P. aeruginosa PAO1 genome were used to investigate global gene expression patterns modulated by QS regulons. In the initial experiments we focused on identifying las and/or rhl QS-regulated genes using a QS signal generation-deficient mutant (PAO-JP2) that was cultured with and without added exogenous autoinducers [N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone]. Conservatively, 616 genes showed statistically significant differential expression (P

Functional domains of the RhlR transcriptional regulator of Pseudomonas aeruginosa.

The RhlR transcriptional regulator of Pseudomonas aeruginosa, along with its cognate autoinducer, N-butyryl homoserine lactone (C(4)-HSL), regulates gene expression in response to cell density. With an Escherichia coli LexA-based protein interaction system, we demonstrated that RhlR multimerized and that the degree of multimerization was dependent on the C(4)-HSL concentration. Studies with an E. coli lasB::lacZ lysogen demonstrated that RhlR multimerization was necessary for it to function as a transcriptional activator. Deletion analysis of RhlR indicated that the N-terminal domain of the protein is necessary for C(4)-HSL binding. Single amino acid substitutions in the C-terminal domain of RhlR generated mutant RhlR proteins that had the ability to bind C(4)-HSL and multimerize but were unable to activate lasB expression, demonstrating that the C-terminal domain is important for target gene activation. Single amino acid substitutions in both the N-terminal and C-terminal domains of RhlR demonstrated that both domains possess residues involved in multimerization. RhlR with a C-terminal deletion and an RhlR site-specific mutant form that possessed multimerization but not transcriptional activation capabilities were able to inhibit the ability of wild-type RhlR to activate rhlA expression in P. aeruginosa. We conclude that C(4)-HSL binding is necessary for RhlR multimerization and that RhlR functions as a multimer in P. aeruginosa.