Effect of Intermittent High-Mechanical Index Impulses on Left Ventricular Strain.

BACKGROUND: Intermittent high-mechanical index (MI) impulses from a transthoracic ultrasound transducer are recommended for regional wall motion analysis and assessment of myocardial perfusion following intravenous administration of ultrasound enhancing agents (UEAs). High-MI impulses (>1.0) applied in this setting have also been shown to increase microvascular blood flow through a purinergic signaling pathway, but their effects on left ventricular (LV) myocardial function are unknown. Therefore, the aim of this study was to investigate the effect of transthoracic intermittent high-MI impulses during intravenous UEA infusion in patients with normal and abnormal resting systolic function. METHODS: Fifty patients referred for echocardiography to evaluate LV systolic function during continuous infusion of UEAs (Definity 3% infusion) were prospectively assigned to low-MI (<0.2) imaging alone (group 1) or low-MI (<0.2) imaging with intermittent high-MI impulses (five frames, 1.8 MHz, MI = 1.0-1.2) applied at least two times in each apical window to clear myocardial contrast (group 2). Global longitudinal strain (GLS) measurements were obtained at baseline before UEA administration and at 5-min intervals up to 10-min after infusion completion. RESULTS: There were no differences between groups with respect to age, gender, resting GLS, biplane LV ejection fraction, or cardiac risk factors. Resting GLS in group 1 was -15.5 ± 5.2% before UEA infusion and -15.5 ± 5.4% at 10 min after UEA infusion. In comparison, GLS increased in group 2 (-15.3 ± 5.0 before infusion and -16.8 ± 4.8% at 10 min, P < .00001). Improvements in GLS were seen in patients with normal and abnormal systolic function. Regional analysis demonstrated that the increase in strain in patients with abnormal LV ejection fractions was primarily in the apical segments (-12.0 ± 2.7% before infusion and -13.4 ± 3.4% at 10 min, P = .001). CONCLUSIONS: High-MI impulses during infusion of a commercially available contrast agent can improve LV systolic function and may have therapeutic effect in patients with LV dysfunction.

Safety and Efficacy of Cardiac Ultrasound Contrast in Children and Adolescents for Resting and Stress Echocardiography.

BACKGROUND: Small pilot studies of ultrasound contrast (UC) echocardiography in children have suggested that it is safe; therefore, larger scale evaluation of safety and efficacy in this population is of particular interest. METHODS: This was a retrospective study (January 2005 to June 2014). Known intracardiac shunt was the only exclusion criterion. UC echocardiography was performed on commercially available ultrasound systems using Definity (3% infusion). When indicated, real-time myocardial contrast echocardiography was performed at rest and stress, with examination of myocardial contrast replenishment, plateau intensity, and wall motion. The primary outcome was short-term safety and efficacy (<24 hours). RESULTS: In all, 113 patients (55% male; mean age, 17.8 ± 3 years; age range, 5-21 years) underwent UC echocardiography for left ventricular opacification or stress wall motion and perfusion analysis. Diagnosis categories were congenital heart disease (30%), acquired heart disease (21%), and other (suspected cardiac complications of disease of other organ systems; 49%). Twelve patients (11%) with right ventricular systolic pressures > 40 mm Hg received UC without complications; four of these (33%) had the highest right ventricular-right atrial gradient recorded with ultrasound contrast-enhanced Doppler. Myocardial perfusion and/or UC echocardiography-detected wall motion abnormalities were seen in 13 patients (12%); four had confirmed correlations by angiography or magnetic resonance imaging. There were 13 instances of adverse events or reported symptoms during UC echocardiography. All symptoms and events were transient, all patients completed protocols, and there were no immediate sequelae. CONCLUSIONS: These data demonstrate the usefulness and safety of UC echocardiography in children and adolescents for a wide variety of indications. UC echocardiography provided myocardial perfusion and wall motion information important in clinical decision making.

Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa.

Cancer patients are known to be highly susceptible to Pseudomonas aeruginosa (Pa) infection, but it remains unknown whether alterations at the tumor cell level can contribute to infection. This study explored how cellular changes associated with tumor metastasis influence Pa infection using highly metastatic MTLn3 cells and non-metastatic MTC cells as cell culture models. MTLn3 cells were found to be more sensitive to Pa infection than MTC cells based on increased translocation of the type III secretion effector, ExoS, into MTLn3 cells. Subsequent studies found that higher levels of ExoS translocation into MTLn3 cells related to Pa entry and secretion of ExoS within MTLn3 cells, rather than conventional ExoS translocation by external Pa. ExoS includes both Rho GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT) enzyme activities, and differences in MTLn3 and MTC cell responsiveness to ExoS were found to relate to the targeting of ExoS-GAP activity to Rho GTPases. MTLn3 cell migration is mediated by RhoA activation at the leading edge, and inhibition of RhoA activity decreased ExoS translocation into MTLn3 cells to levels similar to those of MTC cells. The ability of Pa to be internalized and transfer ExoS more efficiently in association with Rho activation during tumor metastasis confirms that alterations in cell migration that occur in conjunction with tumor metastasis contribute to Pa infection in cancer patients. This study also raises the possibility that Pa might serve as a biological tool for dissecting or detecting cellular alterations associated with tumor metastasis.

Patient outcome following 2 different stress imaging approaches: a prospective randomized comparison.

OBJECTIVES: The study sought to prospectively compare patient outcome after stress real-time myocardial contrast echocardiography (RTMCE) versus conventional stress echo (CSE), where contrast is used to optimize wall motion (WM) analysis. BACKGROUND: Myocardial perfusion imaging with RTMCE may improve the detection of coronary artery disease (CAD), and predict patient outcome. METHODS: Patients with intermediate to high pre-test probability referred for dobutamine or exercise stress echocardiography were prospectively randomized to either RTMCE or CSE. Definity contrast was used for CSE only when endocardial border delineation was inadequate (63% of studies). Studies were interpreted by either an experienced contrast reviewer (R1; n = 1257), or 4 Level 3 echocardiographers (R2) with basic contrast training (n = 806). Death, nonfatal myocardial infarction (MI), and revascularizations were recorded at follow-up. RESULTS: Follow-up was available in 2,014 patients (median 2.6 years). Mean age was 59 ± 13 years (53% women). An abnormal RTMCE was more frequently observed than an abnormal CSE (p < 0.001), and more frequently resulted in revascularization (p = 0.004). Resting WM abnormalities were also more frequently seen with RTMCE (p < 0.01), and were an independent predictor of death/nonfatal MI (p = 0.005) for RTMCE, but not CSE. The predictive value of a positive study, whether with CSE or RTMCE, was significant for both R1 and R2 reviewers in predicting the combined endpoint, but R1 was better than R2 at predicting patients at risk for death or nonfatal MI. CONCLUSIONS: Perfusion imaging with RTMCE improves the detection of CAD during stress echocardiography, and identifies those more likely to undergo revascularization following an abnormal study.

Regulation of Rab5 function during phagocytosis of live Pseudomonas aeruginosa in macrophages.

Pseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment of P. aeruginosa infection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages of P. aeruginosa invasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated, P. aeruginosa inhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of live P. aeruginosa. These studies provide evidence that live P. aeruginosa cells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5.

Prospective randomized comparison of conventional stress echocardiography and real-time perfusion stress echocardiography in detecting significant coronary artery disease.

BACKGROUND: Although retrospective studies have suggested that myocardial perfusion and wall motion analysis with real-time myocardial contrast echocardiography (RTMCE) improves the detection of coronary artery disease (CAD) during dobutamine or exercise stress echocardiography, a prospective randomized comparison with conventional stress echocardiography that did not use RTMCE has not been performed. METHODS: A total of 1,776 patients with preserved resting left ventricular wall motion undergoing dobutamine or exercise stress echocardiography for suspicion of CAD were randomized to either non-RTMCE, for which contrast was used only for the approved indication of enhancing left ventricular opacification, or RTMCE, for which contrast infusion was used in all cases to examine both wall motion and myocardial perfusion. Comparisons in test positivity, and positive predictive value in those subsequently referred for quantitative coronary angiography, were performed. RESULTS: Patients randomized to RTMCE had significantly higher test positivity (22% for RTMCE vs 15% with non-RTMCE, P = .0002). The increased test positivity occurred without a difference in positive predictive value in predicting >50% diameter stenoses by quantitative coronary angiography (67% for non-RTMCE, 73% for RTMCE). The mechanism for increased detection of CAD with RTMCE was mostly due to the detection of subendocardial wall thickening abnormalities that would have gone undetected when examining transmural wall thickening. CONCLUSIONS: RTMCE improves the detection of CAD during dobutamine and exercise stress echocardiography, mainly by the detection of subendocardial ischemia.

Examining the role of actin-plasma membrane association in Pseudomonas aeruginosa infection and type III secretion translocation in migratory T24 epithelial cells.

The opportunistic pathogen Pseudomonas aeruginosa targets wounded epithelial barriers, but the cellular alteration that increases susceptibility to P. aeruginosa infection remains unclear. This study examined how cell migration contributes to the establishment of P. aeruginosa infections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulate P. aeruginosa infection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, and P. aeruginosa cells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowed P. aeruginosa to be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation by P. aeruginosa cells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection with P. aeruginosa expressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment of P. aeruginosa and ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalized P. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities on P. aeruginosa internalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability of P. aeruginosa and ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment of P. aeruginosa infection. Our studies also highlight the contribution of ExoS to the opportunistic nature of P. aeruginosa infection through its ability to exert cytotoxic effects that interrupt T3S translocation and P. aeruginosa internalization, which in turn limit the P. aeruginosa infectious process.

Bacterial biofilm diversity in contact lens-related disease: emerging role of Achromobacter, Stenotrophomonas, and Delftia.

PURPOSE: Multi-species biofilms associated with contact lens cases and lenses can predispose individuals to contact lens-related inflammatory complications. Our study used culture-independent methods to assess the relationship between the severity of contact lens-related disease and bacteria residing in biofilms of contact lens cases and lenses. METHODS: Contact lens cases and lenses from 28 patients referred to the West Virginia University Eye Institute and diagnosed as having mild keratitis, keratitis with focal infiltrates, or corneal ulcers were processed and evaluated for bacterial composition based on 16S ribosomal RNA gene sequencing. Cases and lenses from nine asymptomatic contact lens wearers were processed in a manner similar to controls. Relationships between disease severity, bacterial types, and bacterial diversity were evaluated statistically. RESULTS: Disease severity and presenting visual acuity correlated with an increase in the diversity of bacterial types isolated from contact lens cases. A significant difference also was observed in the number of bacterial types associated with the three clinical groups. Achromobacter, Stenotrophomonas, and Delftia were prevalent in all disease groups, and Achromobacter and Stenotrophomonas were present in one asymptomatic control. Scanning electron microscopy revealed that Achromobacter and Stenotrophomonas formed a biofilm on the surface of contact lenses. CONCLUSIONS: Culture-independent methods identified an association between disease severity and bacterial diversity in biofilms isolated from cases and lenses of patients with contact lens-related corneal disease. Achromobacter, Stenotrophomonas, and Delftia were predominant bacteria identified in our study, drawing attention to their emerging role in contact lens-related disease.

Ultrasound contrast and real-time perfusion in conjunction with supine bicycle stress echocardiography for comprehensive evaluation of surgically corrected congenital heart disease.

AIMS: We sought to evaluate the efficacy of ultrasound contrast (UC) and low mechanical index real-time perfusion (RTP) in the haemodynamic and anatomic assessment of repaired congenital heart disease (CHD) at rest and during supine bicycle stress echocardiography (BSE). METHODS AND RESULTS: Patients with CHD (n = 51, median age 21.5 years) were prospectively studied. All had compromised image quality, 20 (39%) had arrhythmias, and 10 (20%) had pacemakers. RTP was performed at rest and during BSE using Definity and Contrast Pulse Sequencing, with assessment of Doppler pressure gradients. Diagnoses included tetralogy of Fallot (n = 27), transposition of the great arteries (TGA) atrial switch (n = 10), TGA arterial switch (n = 2), aortic valve disease (n = 4), Fontan (n = 4), and Kawasaki disease (n = 4). UC with RTP improved endocardial border definition, with increased number of left ventricular (LV) and right ventricular (RV) segments visualized at rest (P < 0.0001) and during stress. LV ejection fraction (EF) and RV fractional area change (FAC) were measurable at rest and peak stress, RV FAC correlating closely with same-day magnetic resonance EFs (r = 0.72; P < 0.001). UC enhanced Doppler signals, enabling subpulmonary ventricular systolic pressure measurements at rest and stress. In six patients, marked elevations of subpulmonary ventricular systolic pressure were detected with UC during BSE, and quantifiable ventricular dysfunction. No adverse events occurred, other than transient low back pain in one patient. CONCLUSION: UC at rest and with supine BSE enables safe and comprehensive assessment of anatomy, haemodynamics, and biventricular functional and perfusion reserve in adolescents and young adults with surgically modified CHD.

Rapid detection of coronary artery stenoses with real-time perfusion echocardiography during regadenoson stress.

BACKGROUND: Real-time myocardial contrast echocardiography permits the detection of myocardial perfusion abnormalities during stress echocardiography, which may improve the accuracy of the test in detecting coronary artery stenoses. We hypothesized that this technique could be used after a bolus injection of the selective A2A receptor agonist regadenoson to rapidly and safely detect coronary artery stenoses. METHODS AND RESULTS: In 100 patients referred for quantitative coronary angiography, real-time myocardial contrast echocardiography was performed during a continuous intravenous infusion of 3% Definity at baseline and at 2-minute intervals for up to 6 minutes after a regadenoson bolus injection (400 µg). Myocardial perfusion was assessed by examination of myocardial contrast replenishment after brief high mechanical index impulses. A perfusion defect was defined as a delay (>2 seconds) in myocardial contrast replenishment in 2 contiguous segments. Wall motion was also analyzed. The overall sensitivity/specificity/accuracy for myocardial perfusion analysis in detecting a >50% diameter stenosis was 80%/74%/78%, whereas for wall motion analysis it was 60%/72%/66% (P<0.001 for differences in sensitivity). Sensitivity for myocardial perfusion analysis was highest on images obtained during the first 2 minutes after regadenoson bolus (P<0.001 compared with wall motion), whereas wall motion sensitivity was highest at the 4-to-6-minute period after the bolus. No significant side effects occurred after regadenoson bolus injection. CONCLUSIONS: Regadenoson real-time myocardial contrast echocardiography appears to be a feasible, safe, and rapid noninvasive method for the detection of significant coronary artery stenoses.

Use of 16S ribosomal RNA gene analyses to characterize the bacterial signature associated with poor oral health in West Virginia.

BACKGROUND: West Virginia has the worst oral health in the United States, but the reasons for this are unclear. This pilot study explored the etiology of this disparity using culture-independent analyses to identify bacterial species associated with oral disease. METHODS: Bacteria in subgingival plaque samples from twelve participants in two independent West Virginia dental-related studies were characterized using 16S rRNA gene sequencing and Human Oral Microbe Identification Microarray (HOMIM) analysis. Unifrac analysis was used to characterize phylogenetic differences between bacterial communities obtained from plaque of participants with low or high oral disease, which was further evaluated using clustering and Principal Coordinate Analysis. RESULTS: Statistically different bacterial signatures (P<0.001) were identified in subgingival plaque of individuals with low or high oral disease in West Virginia based on 16S rRNA gene sequencing. Low disease contained a high frequency of Veillonella and Streptococcus, with a moderate number of Capnocytophaga. High disease exhibited substantially increased bacterial diversity and included a large proportion of Clostridiales cluster bacteria (Selenomonas, Eubacterium, Dialister). Phylogenetic trees constructed using 16S rRNA gene sequencing revealed that Clostridiales were repeated colonizers in plaque associated with high oral disease, providing evidence that the oral environment is somehow influencing the bacterial signature linked to disease. CONCLUSIONS: Culture-independent analyses identified an atypical bacterial signature associated with high oral disease in West Virginians and provided evidence that the oral environment influenced this signature. Both findings provide insight into the etiology of the oral disparity in West Virginia.

Role of host cell polarity and leading edge properties in Pseudomonas type III secretion.

Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen Pseudomonas aeruginosa and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchical order of protein involvement was identified that paralleled the architecture of leading edge (LE) focal complexes. Conversely, in HL-60 cells, induction of T3S sensitivity coincided with the onset of LE properties and the development of actin-rich projections associated with polarized cell migration. When LE architecture was examined by immunofluorescent staining for actin, Rac1, IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and phosphatidylinositol 3 kinase (PI3 kinase), intact LE structure was found to closely correlate with host cell sensitivity to P. aeruginosa T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the LE alters membrane properties to favour T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration.

Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane.

Pseudomonas aeruginosa, an important human pathogen, preferentially binds and enters injured cells from the basolateral (BL) surface. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt are necessary and sufficient for P. aeruginosa entry from the apical (AP) surface and that AP addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is sufficient to convert AP into BL membrane (Kierbel, A., A. Gassama-Diagne, K. Mostov, and J.N. Engel. 2005. Mol. Biol. Cell. 16:2577-2585; Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Nat. Cell Biol. 8:963-970). We now show that P. aeruginosa subverts this pathway to gain entry from the AP surface. In polarized monolayers, P. aeruginosa binds near cell-cell junctions without compromising them where it activates and recruits PI3K to the AP surface. Membrane protrusions enriched for PIP3 and actin accumulate at the AP surface at the site of bacterial binding. These protrusions lack AP membrane markers and are comprised of BL membrane constituents, which are trafficked there by transcytosis. The end result is that this bacterium transforms AP into BL membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier.

ADP-ribosylation of cyclophilin A by Pseudomonas aeruginosa exoenzyme S.

The virulence of the opportunistic pathogen Pseudomonas aeruginosa (Pa) is in part mediated by the type III secretion (TTS) of bacterial proteins into eukaryotic hosts. Exoenzyme S (ExoS) is a bifunctional Pa TTS effector protein, with GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities. Known cellular substrates of TTS-translocated ExoS (TTS-ExoS) ADPRT activity include proteins in the Ras superfamily and ERM family proteins. This study describes the ADP-ribosylation of a non-G-protein substrate of TTS-ExoS, cyclophilin A (CpA), a peptidyl-prolyl isomerase (PPIase). Four novel 17 kDa proteins (pI 6.5-6.8) were recognized in a proteomic screen of lysates of human epithelial cells that had been exposed to ExoS-producing Pa, but not an isogenic non-ExoS producing strain. The proteins were identified as isoforms of CpA using MALDI-TOF mass spectrometry and confirmed by Western blotting. Mutagenesis analysis identified arginine 55 and 69 of CpA as sites of ExoS ADP-ribosylation. Examination of the effect of ExoS ADP-ribosylation on CpA function found a moderate (19%) decrease in prolyl isomerization of a Xaa-Pro containing peptides. In comparison, GST-CpA co-immunoprecipitation studies found ExoS ADP-ribosylation of CpA to efficiently inhibit CpA binding to calcineurin/PP2B phosphatase. Our results support that ExoS ADP-ribosylates and affects the function of the cytosolic protein, CpA, with the predominant functional effect relating to interference of CpA-cellular protein interactions.

Examination of the coordinate effects of Pseudomonas aeruginosa ExoS on Rac1.

Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by the Pseudomonas aeruginosa type III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.

Characterization of an ExoS Type III translocation-resistant cell line.

Pseudomonas aeruginosa ExoS is a type III-secreted type III-secreted, bifunctional protein that causes diverse effects on eukaryotic cell function. The coculture of P. aeruginosa strains expressing ExoS with HL-60 myeloid cells revealed the cell line to be resistant to the toxic effects of ExoS. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (TPA) rendered the cell line sensitive to ExoS. To understand the cellular basis for the alteration in sensitivity, undifferentiated and TPA-differentiated HL-60 cells were compared for differences in bacterial adherence, type III secretion induction, and ExoS translocation. These comparisons found that ExoS was translocated more efficiently in TPA-differentiated HL-60 cells than in undifferentiated cells. The studies support the ability of eukaryotic cells to influence P. aeruginosa TTS at the level of membrane translocation.