Pseudomonas aeruginosa quorum sensing molecule N-3-oxo-dodecanoyl-l-homoserine lactone activates human platelets through intracellular calcium-mediated ROS generation

Pseudomonas aeruginosa, an opportunistic pathogen release N-3-oxo-dodecanoyl-l-homoserine lactone (3-oxo-C₁₂HSL) and N-butyryl-l-homoserine lactone (C₄-HSL) quorum sensing (QS) molecules to regulate various virulence factors responsible for infection in the host. 3-oxo-C₁₂ HSL not only regulates the bacterial gene expression but also modulates the host cell system. Thus, it is pertinent to evaluate the effect of these QS molecules on blood platelets which is responsible for the maintenance of hemostasis and thrombus formation. Here, in the present study, we showed that 3-oxo-C₁₂ HSL activates platelets in a dose-dependent manner and induces intracellular calcium-mediated reactive oxygen species (ROS) release, whereas no such effect was observed with C₄-HSL. 3-oxo-C₁₂ HSL stimulated ROS release was mediated by NADPH oxidase. Results confirmed the involvement of phospholipase C (PLC) and IP₃ receptor behind intracellular calcium-mediated ROS generation. The impact of 3-oxo-C₁₂ HSL on platelet activation suggests that it could interfere and alter the normal function of platelet in individuals infected with P. aeruginosa.     

The Pseudomonas aeruginosa autoinducer N-3-oxododecanoyl homoserine lactone accelerates apoptosis in macrophages and neutrophils

Quorum-sensing systems are critical regulators of the expression of virulence factors of various organisms, including Pseudomonas aeruginosa. Las and Rhl are two major quorum-sensing components, and they are regulated by their corresponding autoinducers, N-3-oxododecanoyl homoserine lactone (3-oxo-C(12)-HSL) and N-butyryl-L-homoserine lactone (C(4)-HSL). Recent progress has demonstrated the potential of quorum-sensing molecules, especially 3-oxo-C(12)-HSL, for modulation of the host immune system. Here we show the specific ability of 3-oxo-C(12)-HSL to induce apoptosis in certain types of cells. When bone marrow-derived macrophages were incubated with synthetic 3-oxo-C(12)-HSL, but when they were incubated not C(4)-HSL, significant loss of viability was observed in a concentration (12 to 50 micro M)- and incubation time (1 to 24 h)-dependent manner. The cytotoxic activity of 3-oxo-C(12)-HSL was also observed in neutrophils and monocytic cell lines U-937 and P388D1 but not in epithelial cell lines CCL-185 and HEp-2. Cells treated with 3-oxo-C(12)-HSL revealed morphological alterations indicative of apoptosis. Acceleration of apoptosis in 3-oxo-C(12)-HSL-treated cells was confirmed by multiple criteria (caspases 3 and 8, histone-associated DNA fragments, phosphatidylserine expression). Structure-activity correlation experiments demonstrated that the fine structure of 3-oxo-C(12)-HSL, the HSL backbone, and side chain length are required for maximal activity. These data suggest that Pseudomonas 3-oxo-C(12)-HSL specifically promotes induction of apoptosis, which may be associated with 3-oxo-C(12)-HSL-induced cytotoxicity in macrophages and neutrophils. Our data suggest that the quorum-sensing molecule 3-oxo-C(12)-HSL has critical roles in the pathogenesis of P. aeruginosa infection, not only in the induction of bacterial virulence factors but also in the modulation of host responses.     

Phospholipase C gamma negatively regulates Rac/Cdc42 activation in antigen-stimulated mast cells

The Rho GTPases Rac and Cdc42 play a central role in the regulation of secretory and cytoskeletal responses in antigen-stimulated mast cells. In this study, we examine the kinetics and mechanism of Rac and Cdc42 activation in the rat basophilic leukemia RBL-2H3 cells. The activation kinetics of both Rac and Cdc42 show a biphasic profile, consisting of an early transient peak at 1 min and a late sustained activation phase at 20-40 min. The inhibition of phospholipase C (PLC)gamma causes a twofold increase in Rac and Cdc42 activation that coincides with a dramatic production of atypical filopodia-like structures. Inhibition of protein kinase C using bisindolylmaleimide mimics the effect of PLCgamma inhibition on Rac activation, but not on Cdc42 activation. In contrast, depletion of intracellular calcium leads to a complete inhibition of the early activation peak of both Rac and Cdc42, without significant effects on the late sustained activation. These data suggest that PLCgamma is involved in a negative feedback loop that leads to the inhibition of Rac and Cdc42. They also suggest that the presence of intracellular calcium is a prerequisite for both Rac and Cdc42 activation.     

N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa

In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures of Y. pseudotuberculosis and P. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL), N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as the N-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, and N-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37 degrees C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require an N-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.     

Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils

Phagocytosis is a complex process engaging a concerted action of signal-transduction cascades that leads to ingestion, subsequent phagolysosome fusion, and oxidative activation. We have previously shown that in human neutrophils, C3bi-mediated phagocytosis elicits a significant oxidative response, suggesting that activation of the small GTPase Rac is involved in this process. This is contradictory to macrophages, where only Fc receptor for immunoglobulin G (FcgammaR)-mediated activation is Rac-dependent. The present study shows that engagement of the complement receptor 3 (CR3) and FcgammaR and CR3- and FcgammaR-mediated phagocytosis activates Rac, as well as Cdc42. Furthermore, following receptor-engagement of the CR3 or FcgammaRs, a downstream target of these small GTPases, p21-activated kinase, becomes phosphorylated, and Rac2 is translocated to the membrane fraction. Using the methyltransferase inhibitors N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine, we found that the phagocytic uptake of bacteria was not Rac2- or Cdc42-dependent, whereas the oxidative activation was decreased. In conclusion, our results indicate that in neutrophils, Rac2 and Cdc42 are involved in FcR- and CR3-induced activation and for properly functioning signal transduction involved in the generation of oxygen radicals.     

Dominant role of paraoxonases in inactivation of the Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl)-L-homoserine lactone

The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosa's quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 > PON1(192R) > PON1(192Q) > PON3. PON2 exhibited a high specific activity of 7.6 +/- 0.4 micromols/min/mg at 10 microM 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium- and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.     

Isolation and characterization of quorum‐sensing signalling molecules in Pseudomonas aeruginosa isolates recovered from nosocomial infections

Pseudomonas aeruginosa is one of the most common pathogens in nosocomial infections. Many studies have documented the role of quorum‐sensing (QS) systems in antibiotic tolerance of P. aeruginosa. N‐acyl homoserine lactones (AHLs) serve as QS signalling molecules and can be a target for modulating bacterial pathogenicity. In this study, nosocomial isolates of P. aeruginosa were characterized for the presence of different types of QS signalling molecules. AHLs were solvent extracted and quantified by determination of β‐galactosidase activity using the Escherichia coli MG4 reporter strain. Further characterization was performed by analytical thin layer chromatography coupled with detection using the Agrobacterium tumefaciens A136 biosensor strain. All P. aeruginosa isolates produced AHLs, but there were differences in the quantity and nature of AHLs. We identified AHLs belonging to C4‐homoserine lactone (HSL), C6‐HSL, C8‐HSL, C10‐HSL and C12‐HSL. AHL profiling of P. aeruginosa isolates showed differences in the amounts and types of AHLs, suggesting differences in the virulence factors and the potential for infection. Our results may be investigated further using animal model systems.     

Pseudomonas aeruginosa Quorum Sensing Molecule N-(3-Oxododecanoyl)-l-Homoserine-Lactone Induces HLA-G Expression in Human Immune Cells

HLA-G is a nonclassical class I human leukocyte antigen (HLA) involved in mechanisms of immune tolerance. The objective of this study was to determine whether N-(3-oxododecanoyl)-l-homoserine lactone (3O-C₁₂-HSL), a quorum sensing molecule produced by Pseudomonas aeruginosa, could modify HLA-G expression to control the host immune response. We evaluated the ability of 3O-C₁₂-HSL to induce HLA-G expression in primary immune cells, monocytes (U937 and THP1), and T-cell lines (Jurkat) in vitro and analyzed the cellular pathway responsible for HLA-G expression. We studied the HLA-G promoter with a luciferase assay and interleukin-10 (IL-10) and p38/CREB signaling with enzyme-linked immunosorbent assay and immunofluorescence, respectively. We observed that 3O-C₁₂-HSL is able to induce HLA-G expression in human monocytes and T cells. We showed that the induction of HLA-G by 3O-C₁₂-HSL is p38/CREB and IL-10 dependent. 3O-C₁₂-HSL treatment is able to arrest only the U937 cell cycle, possibly due to the peculiar expression of the ILT2 receptor in the U937 cell line. Our observations suggest HLA-G as a mechanism to create a protected niche for the bacterial reservoir, similar to the role of HLA-G molecules during viral infections.     

Induction of neutrophil chemotaxis by the quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone

Acyl homoserine lactones are synthesized by Pseudomonas aeruginosa as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.     

Detection of Pseudomonas aeruginosa cell-to-cell signals in lung tissue of cystic fibrosis patients

Chronic Pseudomonas aeruginosa infections lead to progressive lung tissue destruction in cystic fibrosis (CF) patients. Two bacterial cell-to-cell signals, 3-oxo-C(12)-HSL and C(4)-HSL are required for the production of several extracellular virulence factors. 3-oxo-C(12)-HSL is also required for the development of a differentiated biofilm, induces IL-8 production by epithelial cells and possesses immunomodulatory activities. These two signalling molecules are therefore believed to play a role in the pathogenesis of P. aeruginosa infections, but have never been isolated from infected human tissues. We extracted and quantified the two P. aeruginosa cell-to-cell signals from lung tissues of two CF patients infected by P. aeruginosa. 3-oxo-C(12)-HSL and C(4)-HSL were detected in the lung tissues in fmol/gram, respectively pmol/gram concentrations; the ratio C(4)-HSL/3-oxo-C(12)-HSL exceeded 100 in all tissue samples. Random Amplified Polymorphism DNA genotyping revealed that one genotype was present per lung. In vitro the P. aeruginosa isolates from the two lungs produced 3-oxo-C(12)-HSL, whereas some isolates did not produce detectable C(4)-HSL. Our results suggest that both P. aeruginosa cell-to-cell signals were produced in the lung tissue of these two cystic fibrosis patients.     

Quorum sensing mediated response of Achromobacter denitrificans SP1 towards prodigiosin production under phthalate stress

Quorum sensing is a density-dependent chemical process between bacteria, which may be intergenus or intragenus. N-acyl homoserine lactones (HSLs) are a type of small signaling molecules associated with Gram-negative bacteria for monitoring their own population density. The present study unveils the mechanism of HSLs in Achromobacter denitrificans SP1 while transforming di(2-ethylhexyl) phthalate (DEHP) into prodigiosin in a simple basal salt medium. The primary detection of HSLs was done by the colorimetric method. Fourier-transform infrared spectroscopy and liquid chromatography–mass spectrometry-quadrupole time-of-flight confirmed and identified the HSLs. The maximum production of HSLs was observed between 24 and 72 h of incubation, which is noted to be a peak time of DEHP degradation. A total of 57.2% of DEHP was degraded within 30 h and complete degradation was observed within 72 h of incubation. Regulation in the synthesis of various acyl-HSL molecules, viz. 3OC6-HSL in the initial stage of DEHP stress, 3OC8-HSL, and C10-HSL during the time of degradation and 3OC12-HSL on completion of degradation was noticed. The role of HSLs on the production of prodigiosin was confirmed using vanillin as an HSL inhibitor. Through the selective activation of HSL molecules, A. denitrificans SP1 sustain the changing stressful conditions. Supplementation of acyl-HSL signal molecules may boost up the efficacy of A. denitrificans SP1 in both DEHP degradation and prodigiosin production which offers great potential towards the management of DEHP containing plastic wastes.     

Cdc42 is crucial for the establishment of epithelial polarity during early mammalian development.

To study the role of Cdc42 in the establishment of epithelial polarity during mammalian development, we generated murine Cdc42-null embryonic stem cells and analyzed peri-implantation development using embryoid bodies (EBs). Mutant EBs developed endoderm and underlying basement membrane, but exhibited defects of cell polarity, cell-cell junctions, survival, and cavitation. These defects corresponded to a decreased phosphorylation and membrane localization of aPKC, a reduced phosphorylation of GSK3beta, and a diminished activity of Rac1. However, neither Rac1 nor the kinase function of GSK3beta seem to contribute to cell polarization and cell-cell contacts. In contrast, EBs expressing dominant-negative (dn) PKCzeta mimicked well the phenotype of Cdc42-null EBs, suggesting a major role of aPKC in mediating cell polarization downstream of Cdc42. Finally, aggregation experiments with endodermal cell lines suggested that Cdc42 might affect formation of adherens and tight junctions by PKCzeta-dependent regulation of the protein levels of p120 catenin and E-cadherin.     

N-acylhomoserine lactones antagonize virulence gene expression and quorum sensing in Staphylococcus aureus

Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 muM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.     

An essential function for the calcium-promoted Ras inactivator in Fcgamma receptor-mediated phagocytosis

Fc receptor (FcR)-mediated phagocytosis requires activation of the Rho GTPases Cdc42 and Rac1, but how they are recruited to the FcR is unknown. Here we show that the calcium-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, functions as an adaptor for Cdc42 and Rac1 during FcR-mediated phagocytosis. CAPRI-deficient macrophages had impaired FcgammaR-mediated phagocytosis and oxidative burst, as well as defective activation of Cdc42 and Rac1. CAPRI interacted constitutively with both Cdc42 and Rac1 and translocated to phagocytic cups during FcgammaR-mediated phagocytosis. CAPRI-deficient mice had an impaired innate immune response to bacterial infection. These results suggest that CAPRI provides a link between FcgammaR and Cdc42 and Rac1 and is essential for innate immune responses.     

Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling

Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.     

Disturbed granulocyte macrophage-colony stimulating factor priming of phosphatidylinositol 3,4,5-trisphosphate accumulation and Rac activation in fMLP-stimulated neutrophils from patients with myelodysplasia

The production of reactive oxygen species (ROS) by human neutrophils is imperative for their bactericidal activity. Proinflammatory agents such as granulocyte macrophage-colony stimulating factor (GM-CSF) can prime ROS production in response to chemoattractants such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). In neutrophils from patients suffering from Myelodysplastic syndromes (MDS), a clonal, hematological disorder characterized by recurrent bacterial infections, this GM-CSF priming is severely impaired. In this study, we set out to further delineate the defects in neutrophils from MDS patients. We examined the effect of GM-CSF priming on fMLP-triggered activation of Rac, a small GTPase implicated in neutrophil ROS production. In contrast to healthy neutrophils, activation of Rac in response to fMLP was not enhanced by GM-CSF pretreatment in MDS neutrophils. Furthermore, activation of Rac was attenuated by pretreatment of neutrophils with the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002. Unlike healthy neutrophils, fMLP-induced accumulation of the PI-3K lipid product PI(3,4,5)trisphosphate was not increased by GM-CSF pretreatment in MDS neutrophils. The disturbed Rac and PI-3K activation observed in MDS neutrophils did not appear to reflect a general GM-CSF or fMLP receptor-signaling defect, as fMLP-triggered Ras activation could be primed by GM-CSF in MDS and healthy neutrophils. Moreover, fMLP-induced activation of the GTPase Ral was also normal in neutrophils from MDS patients. Taken together, our data suggest that in neutrophils from MDS patients, a defect in priming of the PI-3K-Rac signaling pathway, located at the level of PI-3K, results in a decreased GM-CSF priming of ROS production.     

Sub-inhibitory concentrations of ceftazidime and tobramycin reduce the quorum sensing signals of Pseudomonas aeruginosa

AIM: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones. METHODS: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli. RESULTS: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P<0.0001). As a percentage of control, C12-HSL was mean 69.4% (SEM 7.3%) at 6% MIC tobramycin, and 31.7% (3.3%) at 25% MIC tobramycin (P=0.0001). C12-HSL was 78.9% (5.3%) at 6% MIC ceftazidime and was 29.7% (1.8%) at 25% MIC ceftazidime (P=0.0001). Both ceftazidime and tobramycin were associated with reduced C4-HSL at 6% MIC and 25% MIC (P<0.03). CONCLUSIONS: SubMIC tobramycin but not ceftazidime reduced elastase production by P. aeruginosa. In contrast, subMIC concentrations of both antimicrobials reduced C12-HSL and C4-HSL. It is unlikely that reduced HSL is the sole explanation for the reduction in elastase.