Epilepsy monitoring unit practices and safety among NAEC epilepsy centers: A census survey.

OBJECTIVE: An epilepsy monitoring unit (EMU) is a specialized unit designed for capturing and characterizing seizures and other paroxysmal events with continuous video electroencephalography (vEEG). Nearly 260 epilepsy centers in the United States are accredited by the National Association of Epilepsy Centers (NAEC) based on adherence to specific clinical standards to improve epilepsy care, safety, and quality. This study examines EMU staffing, safety practices, and reported outcomes. METHOD: We analyzed NAEC annual report data and results from a supplemental survey specific to EMU practices reported in 2019 from 341 pediatric or adult center directors. Data on staffing, resources, safety practices and complications were collated with epilepsy center characteristics. We summarized using frequency (percentage) for categorical variables and median (inter-quartile range) for continuous variables. We used chi-square or Fisher's exact tests to compare staff responsibilities. RESULTS: The supplemental survey response rate was 100%. Spell classification (39%) and phase 1 testing (28%) were the most common goals of the 91,069 reported admissions. The goal ratio of EEG technologist to beds of 1:4 was the most common during the day (68%) and off-hours (43%). Compared to residents and fellows, advanced practice providers served more roles in the EMU at level 3 or pediatric-only centers. Status epilepticus (SE) was the most common reported complication (1.6% of admissions), while cardiac arrest occurred in 0.1% of admissions. SIGNIFICANCE: EMU staffing and safety practices vary across US epilepsy centers. Reported complications in EMUs are rare but could be further reduced, such as with more effective treatment or prevention of SE. These findings have potential implications for improving EMU safety and quality care.

Associations between testing and treatment pathways in lesional temporal or extratemporal epilepsy: A census survey of NAEC center directors.

OBJECTIVE: The evaluation to determine candidacy and treatment for epilepsy surgery in persons with drug-resistant epilepsy (DRE) is not uniform. Many non-invasive and invasive tests are available to ascertain an appropriate treatment strategy. This study examines expert response to clinical vignettes of magnetic resonance imaging (MRI)-positive lesional focal cortical dysplasia in both temporal and extratemporal epilepsy to identify associations in evaluations and treatment choice. METHODS: We analyzed annual report data and a supplemental epilepsy practice survey reported in 2020 from 206 adult and 136 pediatric epilepsy center directors in the United States. Non-invasive and invasive testing and surgical treatment strategies were compiled for the two scenarios. We used chi-square tests to compare testing utilization between the two scenarios. Multivariable logistic regression modeling was performed to assess associations between variables. RESULTS: The supplemental survey response rate was 100% with 342 responses included in the analyses. Differing testing and treatment approaches were noted between the temporal and extratemporal scenarios such as chronic invasive monitoring selected in 60% of the temporal scenario versus 93% of the extratemporal scenario. Open resection was the most common treatment choice; however, overall treatment choices varied significantly (p < .001). Associations between non-invasive testing, invasive testing, and treatment choices were present in both scenarios. For example, in the temporal scenario stereo-electroencephalography (SEEG) was more commonly associated with fluorodeoxyglucose-positron emission tomography (FDG-PET) (odds ratio [OR] 1.85; 95% confidence interval [CI] 1.06-3.29; p = .033), magnetoencephalography (MEG) (OR 2.90; 95% CI 1.60-5.28; p = <.001), high density (HD) EEG (OR 2.80; 95% CI 1.27-6.24; p = .011), functional MRI (fMRI) (OR 2.17; 95% CI 1.19-4.10; p = .014), and Wada (OR 2.16; 95% CI 1.28-3.66; p = .004). In the extratemporal scenario, choosing SEEG was associated with increased odds of neuromodulation over open resection (OR 3.13; 95% CI 1.24-7.89; p = .016). SIGNIFICANCE: In clinical vignettes of temporal and extratemporal lesional DRE, epilepsy center directors displayed varying patterns of non-invasive testing, invasive testing, and treatment choices. Differences in practice underscore the need for comparative trials for the surgical management of DRE.

Epilepsy center characteristics and geographic region influence presurgical testing in the United States.

OBJECTIVE: Persons with drug-resistant epilepsy may benefit from epilepsy surgery and should undergo presurgical testing to determine potential candidacy and appropriate intervention. Institutional expertise can influence use and availability of evaluations and epilepsy surgery candidacy. This census survey study aims to examine the influence of geographic region and other center characteristics on presurgical testing for medically intractable epilepsy. METHODS: We analyzed annual report and supplemental survey data reported in 2020 from 206 adult epilepsy center directors and 136 pediatric epilepsy center directors in the United States. Test utilization data were compiled with annual center volumes, available resources, and US Census regional data. We used Wilcoxon rank-sum, Kruskal-Wallis, and chi-squared tests for univariate analysis of procedure utilization. Multivariable modeling was also performed to assign odds ratios (ORs) of significant variables. RESULTS: The response rate was 100% with individual element missingness < 11% across 342 observations undergoing univariate analysis. A total of 278 complete observations were included in the multivariable models, and significant regional differences were present. For instance, compared to centers in the South, those in the Midwest used neuropsychological testing (OR = 2.87, 95% confidence interval [CI] = 1.2-6.86; p = .018) and fluorodeoxyglucose-positron emission tomography (OR = 2.74, 95% CI = = 1.14-6.61; p = .025) more commonly. For centers in the Northeast (OR = .46, 95% CI = .23-.93; p = .031) and West (OR = .41, 95% CI = .19-.87; p = .022), odds of performing single-photon emission computerized tomography were lower by nearly 50% compared to those in the South. Center accreditation level, demographics, volume, and resources were also associated with varying individual testing rates. SIGNIFICANCE: Presurgical testing for drug-resistant epilepsy is influenced by US geographic region and other center characteristics. These findings have potential implications for comparing outcomes between US epilepsy centers and may inject disparities in access to surgical treatment.

Association Between Characteristics of National Association of Epilepsy Centers and Reported Utilization of Specific Surgical Techniques.

BACKGROUND AND OBJECTIVE: Nearly one-third of persons with epilepsy will continue having seizures despite trialing multiple antiseizure medications. Epilepsy surgery may be beneficial in these cases, and evaluation at a comprehensive epilepsy center is recommended. Numerous palliative and potentially curative approaches exist, and types of surgery performed may be influenced by center characteristics. This article describes epilepsy center characteristics associated with epilepsy surgery access and volumes in the United States. METHODS: We analyzed National Association of Epilepsy Centers 2019 annual report and supplemental survey data obtained with responses from 206 adult epilepsy center directors and 136 pediatric epilepsy center directors in the United States. Surgical treatment volumes were compiled with center characteristics, including US Census region. We used multivariable modeling with zero-inflated Poisson regression models to present ORs and incidence rate ratios of receiving a given surgery type based on center characteristics. RESULTS: The response rate was 100% with individual element missingness less than 4% across 352 observations undergoing univariate analysis. Multivariable models included 319 complete observations. Significant regional differences were present. The rates of laser interstitial thermal therapy (LITT) were lower at centers in the Midwest (incidence rate ratio [IRR] 0.74, 95% CI 0.59-0.92; p = 0.006) and Northeast (IRR 0.77, 95% CI 0.61-0.96; p = 0.022) compared with those in the South. Conversely, responsive neurostimulation implantation rates were higher in the Midwest (IRR 1.45, 95% CI 1.1-1.91; p = 0.008) and West (IRR 1.91, 95% CI 1.49-2.44; p < 0.001) compared with the South. Center accreditation level, institution type, demographics, and resources were also associated with variations in access and rates of potentially curative and palliative surgical interventions. DISCUSSION: Epilepsy surgery procedure volumes are influenced by US epilepsy center region and other characteristics. These variations may affect access to specific surgical treatments for persons with drug resistant epilepsy across the United States.

Depression readmission risk is elevated in multiple sclerosis compared to other chronic illnesses.

OBJECTIVE: Assess readmissions for depression or suicide attempt (SA) after MS admission versus other chronic inflammatory illnesses. METHODS: This retrospective cohort study identified MS, asthma, rheumatoid arthritis (RA), depression, and SA in the 2013 National Readmissions Database by International Classification of Diseases codes. Index admissions (MS, n = 7698; asthma, n = 93,590; RA, n = 3685) and depression or SA readmission rates were analyzed. Hazard ratios (HRs) estimated 1-year depression/SA readmission hazard, comparing MS to asthma or RA, adjusting for age, sex, psychiatric comorbidity, substance abuse, tobacco use, income, and index hospitalization characteristics. RESULTS: MS had more baseline depression (24.7%) versus asthma (15.6%) and RA (14.6%). Ninety-day depression readmission rate was higher in MS (0.5%) than asthma (0.3%) and RA (0.03%). Depression readmission HR was higher after MS admission versus asthma (HR = 1.37, 95% confidence interval (CI) = 1.00-1.86, p = 0.0485) and RA (HR = 4.68, 95% CI = 1.60-13.62, p = 0.0047). HR was not different for SA readmission across groups. Depression readmission HR was more than double in MS patients with psychiatric disease or substance abuse versus RA or asthma patients with either comorbidity. CONCLUSION: Depression readmission risk after MS hospitalization was elevated versus asthma/RA. Substance use and baseline psychiatric comorbidity were more strongly associated with depression readmission in MS patients.

Seizures and status epilepticus may be risk factor for cardiac arrhythmia or cardiac arrest across multiple time frames.

OBJECTIVE: To determine if Emergency Department (ED) or inpatient encounters for epilepsy or status epilepticus are associated with increased odds of cardiac arrhythmia or cardiac arrest over successively longer time frames. METHODS: The State Inpatient and ED Databases (from New York, Florida, and California) are statewide datasets containing data on 97% of hospitalizations and ED encounters from these states. In this retrospective, case-crossover study, we used International Classification of Diseases, Ninth Revision, Clinical Modification codes to identify index cardiac arrhythmia encounters. Exposures were inpatient or ED encounters for epilepsy or status epilepticus. The case-crossover analysis tested whether an epilepsy or status epilepticus encounter within various case periods (1, 3, 7, 30, 60, 90, and 180 days prior to index encounter) was associated with subsequent ED or inpatient encounter for cardiac arrhythmia, as compared to control periods of equal length one year prior. RESULTS: The odds ratio (OR) for cardiac arrhythmia after an epilepsy encounter was significant at all time intervals (OR range 2.37-3.36), and highest at 1 day after epilepsy encounter (OR 3.63, 95% confidence interval [CI] 1.66-7.93, p = 0.0013). The OR after status epilepticus was significant at 7- to 180-day intervals (OR range 2.25-2.74), and highest at 60 days (OR 2.74, CI 2.09-3.61, p < 0.0001). SIGNIFICANCE: Epilepsy and status epilepticus events are associated with increased odds of subsequent cardiac arrhythmia or cardiac arrest over multiple chronic timeframes. Increased cardiac surveillance may be warranted to minimize morbidity and mortality in patients with epilepsy.

Recognizing and refuting the myth of tongue swallowing during a seizure.

OBJECTIVE: There is a harmful myth that persists in modern culture that one should place objects into a seizing person's mouth to prevent "swallowing the tongue." Despite expert guidelines against this, the idea remains alive in popular media and public belief. We aimed to investigate the myth's origins and discredit it. METHODS: A medical and popular literature review was conducted for the allusions to "swallowing one's tongue" and practice recommendations for and against placing objects into a seizing person's mouth. Current prevalence of these beliefs and relevant anatomy and physiology were summarised. RESULTS: The first English language allusions to placing objects in a patient's mouth occurred in the mid-19th century, and the first allusions to swallowing one's tongue during a seizure occurred in the late 19th century. By the mid-20th century, it was clear that some were recommending against the practice of placing objects in a patient's mouth to prevent harm. Relatively recent popular literature and film continue to portray incorrect seizure first aid through at least 2013. There is ample modern literature confirming the anatomical impossibility of swallowing one's tongue and confirming the potential harm of putting objects in a patient's mouth. CONCLUSION: One cannot swallow their tongue during a seizure. Foreign objects should not be placed into a seizing person's mouth. We must continue to disseminate these ideas to our patients and colleagues. As neurologists, we have an obligation to champion safe practices for our patients, especially when popular media and culture continue to propagate dangerous ones.

Exacerbation of hepatic cirrhosis may trigger admission for epilepsy and status epilepticus.

OBJECTIVE: To determine whether acute exacerbations of cirrhotic liver disease are associated with higher odds of readmission for epilepsy or status epilepticus. METHODS: The New York State Inpatient Database is a statewide dataset containing data on 97% of hospitalizations for New York State. In this retrospective, case-crossover design study, we used International Classification of Diseases, Ninth Revision, Clinical Modification codes to identify index status epilepticus and epilepsy admissions. The primary exposure was defined as admission due to an acute exacerbation of cirrhotic liver disease. The case-crossover analysis tested whether exposure to a hepatic exacerbation within progressively longer case periods (14, 30, 60, 90, 120, 150, and 180 days before index admission), compared to control periods 1 year before the case period, was associated with readmission for epilepsy or status epilepticus. RESULTS: The odds ratio for subsequent admission for epilepsy after exposure to an acute exacerbation of cirrhotic liver disease was significant in the 30-day window at 2.072 (95% confidence interval [CI] = 1.095-3.92, P = .0252) and peaked in the 150-day window at 2.742 (95% CI = 1.817-4.137, P < .0001). In the status epilepticus group, all case periods demonstrated significantly elevated odds of subsequent admission following hepatic exacerbation. SIGNIFICANCE: Hepatic exacerbations are associated with increased odds for hospital admissions for epilepsy and status epilepticus across several timeframes.

Characteristics and Outcomes of Retinal Artery Occlusion: Nationally Representative Data.

Background and Purpose- There are few large studies examining comorbidities, outcomes, and acute interventions for patients with retinal artery occlusion (RAO). RAO shares pathophysiology with acute ischemic stroke (AIS); direct comparison may inform emergent treatment, evaluation, and secondary prevention. Methods- The National Readmissions Database contains data on ≈50% of US hospitalizations from 2013 to 2015. We used International Classification of Diseases, Ninth Revision, codes to identify and compare index RAO and AIS admissions, comorbidities, and interventions and Clinical Comorbidity Software codes to identify readmissions causes, using survey-weighted methods when possible. Cumulative risk of all-cause readmission after RAO ≤1 year was estimated by Kaplan-Meier analysis. Results- Among 4871 RAO and 1 239 963 AIS admissions, patients with RAO were less likely (P<0.0001) than patients with AIS to have diabetes mellitus (RAO, 24.3% versus AIS, 36.8%), congestive heart failure (9.1% versus 14.8%), atrial fibrillation (15.5% versus 25.2%), or hypertension (62.2% versus 67.6%) but more likely to have valvular disease (13.3% versus 10.5%) and tobacco usage (38.6% versus 32.9%). In RAO admissions, thrombolysis was administered in 2.9% (5.8% in central RAO subgroup, versus 8.0% of AIS), therapeutic anterior chamber paracentesis in 1.0%, thrombectomy in none; 1.4% received carotid endarterectomy during index admission, 1.6% within 30 days. Nearly 1 in 10 patients with RAO were readmitted within 30 days and were more than twice as likely as patients with AIS to be readmitted for dysrhythmia or endocarditis. Readmission for stroke after RAO was the highest within the first 150 days after index admission, and risk was higher in central RAO than in branch RAO. Conclusions- Patients with RAO had high prevalence of many stroke risk factors, particularly valvular disease and smoking, which can be addressed to minimize subsequent risk. Despite less baseline atrial fibrillation, RAO patients were more likely to be readmitted for atrial fibrillation/dysrhythmias. A variety of interventions was administered. AIS risk is the highest shortly after RAO, emphasizing the importance of urgent, thorough neurovascular evaluation.

Increased risk of hospital admission for mood disorders following admission for epilepsy.

OBJECTIVE: To determine if epilepsy admissions, compared to admissions for other medical causes, are associated with a higher readmission risk for mood disorders. METHODS: The Nationwide Readmissions Database is a nationally representative dataset comprising 49% of US hospitalizations in 2013. In this retrospective cohort study, we used ICD-9-CM codes to identify medical conditions. Index admissions for epilepsy (n = 58,278) were compared against index admissions for stroke (n = 215,821) and common medical causes (n = 973,078). Readmission rates (per 100,000 index admissions) for depression or bipolar disorders within 90 days from discharge for index hospitalization were calculated. Cox regression was used to test for associations between admission type (defined in 3 categories as above) and readmission for depression or bipolar disorder up to 1 year after index admission, in univariate models and adjusted for age, sex, psychiatric history, drug abuse, income quartile of patient's zip code, and index hospitalization characteristics. RESULTS: The adjusted hazard ratio (HR) for readmission for depression in the epilepsy group was elevated at 2.80 compared to the stroke group (95% confidence interval [CI] 2.39-3.27, p < 2 × 10-16), and 2.09 compared to the medical group (95% CI 1.88-2.32, p < 2 × 10-16). The adjusted HR for readmission for bipolar disorder in the epilepsy group was elevated at 5.84 compared to the stroke group (95% CI 4.56-7.48, p < 2 × 10-16), and 2.46 compared to the medical group (95% CI 2.16-2.81, p < 2 × 10-16). CONCLUSION: Admission for epilepsy was independently associated with subsequent hospital readmission for mood disorders. The magnitude of elevated risk in this population suggests that patients admitted with epilepsy may warrant targeted psychiatric screening during their hospital admission.

Increased risk of hospital admission for ICD-9-CM psychotic episodes following admission for epilepsy.

OBJECTIVE: To determine whether epilepsy admissions are associated with a higher readmission risk for psychotic episodes compared to admissions for other medical causes. METHODS: The Nationwide Readmissions Database is a nationally representative dataset from 2013. We used International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes to identify medical conditions. There were 58 278 index admissions for epilepsy, and this group was compared against admissions for stroke (n = 215 821) and common medical causes (pneumonia, urinary tract infection [UTI], congestive heart failure [CHF], and chronic obstructive pulmonary disease [COPD], n = 973 078). Readmission rates for psychotic episodes within 90 days from discharge for index hospitalizations were calculated. Cox regression was used to test for associations between admission type and readmission for psychotic episodes up to 1 year after index admission, in univariate models and adjusted for multiple medical, social, and psychiatric variables. RESULTS: Up to 90 days from index admission, there were 683/100 000 readmissions for psychotic episodes in the epilepsy group, 92/100 000 in the stroke group, and 58-206/100 000 in the medical group. The relative rate of readmission in the epilepsy group was highest in the first 30 days following index admission (311/100 000). Unadjusted hazard ratio (HR) for readmission for psychotic episodes within 1 year in the epilepsy group compared to the stroke group was 6.58 (95% confidence interval [CI] 5.69-7.61, P < 2 × 10-16 ), and 4.41 compared to the medical group (95% CI 4.00-4.85, P < 2 × 10-16 ). The fully adjusted HR for readmission in the epilepsy group remained elevated at 3.63 compared to the stroke group (95% CI 3.08-4.28, P < 2 × 10-16 ), and 1.95 compared to the medical group (95% CI 1.76-2.15, P < 2 × 10-16 ). Confounding factors most strongly associated with psychosis readmission were documented psychosis history at the time of index admission, younger age, and lower income quartile. SIGNIFICANCE: An epilepsy admission was independently associated with subsequent hospital readmission for psychotic episodes, even after adjustment for confounding variables.

Risk of readmission for suicide attempt after epilepsy hospitalization.

OBJECTIVE: The objective of this study was to examine if epilepsy admissions are associated with a higher readmission risk for suicide attempt, independent of psychiatric comorbidity, compared with index admissions for other medical causes. METHODS: The Nationwide Readmissions Database is a nationally representative dataset containing data from roughly 15 million hospital discharges. Analysis of International Classification of Disease Clinical Modification 9 (ICD-9-CM) codes in the year 2013 revealed 58,278 index admissions for epilepsy; this group was compared with admissions for stroke (N=215,821) and common medical causes (N=973,078). Ninety-day readmission rates for suicide attempts were calculated. Cox regression tested for associations between admission type and suicide attempt readmissions up to 1year following index admission. RESULTS: There were 402/100,000 readmissions for suicide attempt within 90days from index admission in the group with epilepsy; 43/100,000 in the stroke group; and between 37 and 89/100,000 in the medical group. Unadjusted hazard ratios (HR) for suicide readmissions within 1year in the group with epilepsy compared with the stroke group were 9.61 (95% confidence interval (CI): 7.69-11.90, p<2.0×10-16) and 5.02 compared with the medical group (95% CI: 4.40-5.73, p<2.0×10-16). The HR for readmission in the group with epilepsy, after adjustment for sociodemographic and psychiatric variables, were elevated at 4.91 compared with the stroke group (95% CI: 3.83-6.27, p<2.0×10-16), and 2.66 compared with the medical group (95% CI: 2.32-3.05, p<2.0×10-16). CONCLUSION: Independent of psychiatric comorbidities, epilepsy admissions may be independently associated with more than a threefold increased risk of hospital readmission for suicide in the year following index admission in comparison with patients recently hospitalized because of stroke or other common medical disorders.

Sporadic Creutzfeldt-Jakob Disease With Unilateral Symptoms in the Setting of Metastatic Renal Cell Carcinoma.

INTRODUCTION: Although it is not rare for magnetic resonance imaging findings in Creutzfeldt-Jakob disease to be asymmetric, unilateral clinical syndromes are uncommonly reported and may confound diagnosis. In addition, neurological paraneoplastic syndromes are not common in renal cell carcinoma, though there are cases reported, often without an offending antibody isolated. CASE REPORT: A 66-year-old man was admitted with 1 month of left-sided numbness and "loss of control" of the left arm. Examination revealed action-induced irregular jerking movements of the left arm. Mental status testing was normal. Magnetic resonance imaging brain revealed patchy areas of restricted diffusion along the cerebral cortices. Screening computed tomographic scans revealed innumerable lung nodules compatible with metastases, as well as a renal mass consistent with renal cell carcinoma. Lumbar puncture was performed and cerebrospinal fluid was sent for paraneoplastic autoantibody evaluation and protein 14-3-3. Over the next week the patient developed dystonic posturing of the left arm, left leg jerking movements, a right arm action tremor, and cognitive impairment. Paraneoplastic autoantibodies were negative. Protein 14-3-3 was elevated and brain biopsy revealed spongiform encephalopathy with positive immunoblotting. The patient died about 2 months from symptom onset. CONCLUSIONS: Creutzfeldt-Jakob disease can present with entirely unilateral myoclonus and numbness, without specific complaints of cognitive impairment. Not every difficult or unclear neurological syndrome in a patient with metastatic cancer is a paraneoplastic syndrome.

More Time Is Taken to Administer Tissue Plasminogen Activator in Ischemic Stroke Patients with Earlier Presentations.

BACKGROUND: In ischemic stroke, administration of tissue plasminogen activator (tPA) within 4.5 hours from the time last known well (LKW) improves outcomes, with better outcomes seen with earlier administration. However, for patients presenting early, a perception of significant remaining time within this window may lead to delayed tPA administration. We hypothesized that cases with a shorter LKW-to-stroke team activation (code) time will have a longer "code-to-tPA" administration time. METHODS: In the Mount Sinai Hospital Stroke Registry (2009-2015), 122 patients received tPA. The patients were divided by "LKW-to-code" time into 3 groups: 0-59 minutes (n = 38), 60-119 minutes (n = 49), and 120 minutes or more (n = 35). The code-to-tPA time was compared among these groups, adjusting for age, sex, National Institutes of Health Stroke Scale (NIHSS) score, and race-ethnicity. RESULTS: The average code-to-tPA time was 80 minutes in the 0-59 minutes group, 67 minutes in the 60-119 minutes group, and 52 minutes in the 120 minutes or more group (analysis of variance P < .0001). There was an average 28-minute difference (P = .021) between the 0-59 and 120 minutes or more groups. CONCLUSION: There was a significant negative correlation between the LKW-to-code time and the code-to-tPA time that was independent of age, sex, NIHSS score, and race-ethnicity.

Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex.

A type 3 secretion system is used by many bacterial pathogens to inject proteins into eukaryotic cells. Pathogens insert a translocon complex into the target eukaryotic membrane by secreting two proteins known as translocators. How these translocators form a translocon in the lipid bilayer and why both proteins are required remains elusive. Pseudomonas aeruginosa translocators PopB and PopD insert pores into membranes forming homo- or hetero-complexes of undetermined stoichiometry. Single-molecule fluorescence photobleaching experiments revealed that PopD formed mostly hexameric structures in membranes, whereas PopB displayed a bi-modal distribution with 6 and 12 subunits peaks. However, individually the proteins are not functional for effector translocation. We have found that when added together, the translocators formed distinct hetero-complexes containing 8 PopB and 8 PopD molecules. Thus, the interaction between PopB and PopD guide the assembly of a unique hetero-oligomer in membranes.

Efficient isolation of Pseudomonas aeruginosa type III secretion translocators and assembly of heteromeric transmembrane pores in model membranes.

Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins undergo the transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH-dependent manner as observed by two independent assays, time-resolved Förster resonance energy transfer and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins, we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH <5.0, suggesting that acidic residues might be involved in the initial membrane binding and/or insertion. Altogether, the experimental setup described here represents an efficient method for the reconstitution and analysis of membrane-inserted translocators.