A kinase-independent function of PAK is crucial for pathogen-mediated actin remodelling.

The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor ß-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.

Locating the rotator cable during subacromial arthroscopy: bursal- and articular-sided anatomy.

BACKGROUND: The rotator cable (RCa) is an important articular-sided structure of the cuff capsular complex that helps prevent suture pull out during rotator cuff repairs (RCRs) and plays a role in force transmission. Yet, the RCa cannot be located during bursal-sided RCRs. The purpose of this study is to develop a method to locate the RCa in the subacromial space and compare its bursal- and articular-sided dimensions. METHODS: In 20 fresh-frozen cadaveric specimens, the RCa was found from the articular side, outlined with stitches, and then evaluated from the bursal side using an easily identifiable reference point, the intersection of a line bisecting the supraspinatus (SS) tendon and posterior SS myotendinous junction (MTJ). Four bursal-sided lengths were measured on the SS-bisecting line as well as the RCa's outside anteroposterior base. For the articular-sided measurements, the rotator cuff capsular complex was detached from bone and optically scanned creating 3D solid models. Using the 3D models, 4 articular-sided lengths were made, including the RCa's inside and outside anteroposterior base. RESULTS: The RCa's medial arch was located 9.9 ± 5.6 mm from the reference point in 10 intact specimens and 4.1 ± 2.4 mm in 10 torn specimens (P = .007). The RCa's width was 10.9 ± 2.1 mm, and the distance from the lateral edge of the RCa to the lateral SS insertion was 13.9 ± 4.8 mm. The bursal- and articular-sided outside anteroposterior base measured 48.1 ± 6.4 mm and 49.6 ± 6.5 mm, respectively (P = .268). The average inside anteroposterior base measurement was 37.3 ± 5.9 mm. DISCUSSION: The medial arch of the RCa can be reliably located during subacromial arthroscopy using the reference point, analogous to the posterior SS MTJ. The RCa is located 10 mm in intact and 4 mm in torn tendons (P = .007) from the posterior SS MTJ. If the above 6-mm shift in location of the RCa is not taken into consideration during rotator cuff suture placement, it could negatively affect time zero repair strength. The inside anteroposterior base of the RCa measures on average 37 mm; therefore, rotator cuff tears measuring >37 mm are at risk of rupturing part or all of the RCa's 2 humeral attachments, which if not recognized and addressed could impact postoperative function.

MYO6 is targeted by Salmonella virulence effectors to trigger PI3-kinase signaling and pathogen invasion into host cells.

To establish infections, Salmonella injects virulence effectors that hijack the host actin cytoskeleton and phosphoinositide signaling to drive pathogen invasion. How effectors reprogram the cytoskeleton network remains unclear. By reconstituting the activities of the Salmonella effector SopE, we recapitulated Rho GTPase-driven actin polymerization at model phospholipid membrane bilayers in cell-free extracts and identified the network of Rho-recruited cytoskeleton proteins. Knockdown of network components revealed a key role for myosin VI (MYO6) in Salmonella invasion. SopE triggered MYO6 localization to invasion foci, and SopE-mediated activation of PAK recruited MYO6 to actin-rich membranes. We show that the virulence effector SopB requires MYO6 to regulate the localization of PIP3 and PI(3)P phosphoinositides and Akt activation. SopE and SopB target MYO6 to coordinate phosphoinositide production at invasion foci, facilitating the recruitment of cytoskeleton adaptor proteins to mediate pathogen uptake.

Arf6 Can Trigger Wave Regulatory Complex-Dependent Actin Assembly Independent of Arno.

The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.

Arf6 coordinates actin assembly through the WAVE complex, a mechanism usurped by Salmonella to invade host cells.

ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1-WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.

The Drosophila Arf1 homologue Arf79F is essential for lamellipodium formation.

The WAVE regulatory complex (WRC) drives the polymerisation of actin filaments located beneath the plasma membrane to generate lamellipodia that are pivotal to cell architecture and movement. By reconstituting WRC-dependent actin assembly at the membrane, we recently discovered that several classes of Arf family GTPases directly recruit and activate WRC in cell extracts, and that Arf cooperates with Rac1 to trigger actin polymerisation. Here, we demonstrate that the Class 1 Arf1 homologue Arf79F colocalises with the WRC at dynamic lamellipodia. We report that Arf79F is required for lamellipodium formation in Drosophila S2R+ cells, which only express one Arf isoform for each class. Impeding Arf function either by dominant-negative Arf expression or by Arf double-stranded RNA interference (dsRNAi)-mediated knockdown uncovered that Arf-dependent lamellipodium formation was specific to Arf79F, establishing that Class 1 Arfs, but not Class 2 or Class 3 Arfs, are crucial for lamellipodia. Lamellipodium formation in Arf79F-silenced cells was restored by expressing mammalian Arf1, but not by constitutively active Rac1, showing that Arf79F does not act via Rac1. Abolition of lamellipodium formation in Arf79F-silenced cells was not due to Golgi disruption. Blocking Arf79F activation with guanine nucleotide exchange factor inhibitors impaired WRC localisation to the plasma membrane and concomitant generation of lamellipodia. Our data indicate that the Class I Arf GTPase is a central component in WRC-driven lamellipodium formation.

Analysis of CYP2D6 genotype and response to tetrabenazine.

Tetrabenazine is effective in the treatment of the chorea associated with Huntington disease and other hyperkinetic movement disorders. Following oral administration, tetrabenazine is hepatically transformed into 2 active metabolites that are CYP2D6 substrates. There are 4 CYP2D6 genotypes: poor metabolizers, intermediate metabolizers, extensive metabolizers, and ultrarapid metabolizers. CYP2D6 genotyping was performed on sequential subjects treated with tetrabenazine, but results were not known at the time of titration. Duration of titration to a stable dose, total daily dose, response rating scores, and adverse events were retrospectively collected and subsequently analyzed. Of 127 patients, the majority (n = 100) were categorized as extensive metabolizers, 14 as intermediate metabolizers, 11 as poor metabolizers, and 2 as ultrarapid metabolizers. Ultrarapid metabolizer patients needed a longer titration (8 vs 3.3, 4.4, and 3 weeks, respectively; P < .01) to achieve optimal benefit and required a higher average daily dose than the other patients, but this difference did not reach statistical significance. The treatment response was less robust in the intermediate metabolizer group when compared with the extensive metabolizer patients (P = .013), but there were no statistically significant differences between the various groups with regard to adverse effects. Our findings demonstrate that, aside from the need for a longer titration in the ultrarapid metabolizers, there are no distinguishing features of patients with various CYP2D6 genotypes, and therefore the current recommendation to systematically genotype all patients prescribed more than 50 mg/day of tetrabenazine should be reconsidered.

Salmonella invasion of a cell is self-limiting due to effector-driven activation of N-WASP.

Salmonella Typhimurium drives uptake into non-phagocytic host cells by injecting effector proteins that reorganize the actin cytoskeleton. The host actin regulator N-WASP has been implicated in bacterial entry, but its precise role is not clear. We demonstrate that Cdc42-dependent N-WASP activation, instigated by the Cdc42-activating effector SopE2, strongly impedes Salmonella uptake into host cells. This inhibitory pathway is predominant later in invasion, with the ubiquitin ligase activity of the effector SopA specifically interfering with negative Cdc42-N-WASP signaling at early stages. The cell therefore transitions from being susceptible to invasion, into a state almost completely recalcitrant to bacterial uptake, providing a mechanism to limit the number of internalized Salmonella. Our work raises the possibility that Cdc42-N-WASP, known to be activated by numerous bacterial and viral species during infection and commonly assumed to promote pathogen uptake, is used to limit the entry of multiple pathogens.

Is Delayed Anterior Cruciate Ligament Reconstruction Associated With a Risk of New Meniscal Tears? Reevaluating a Longstanding Paradigm.

Background: Delayed anterior cruciate ligament (ACL) reconstruction has been associated with an increased risk of meniscal tears. However, studies comparing early versus delayed ACL reconstruction have not clearly demonstrated that meniscal tears diagnosed arthroscopically are new injuries as opposed to concomitant injuries sustained during ACL rupture. Purpose: To determine whether and how delay of ACL reconstruction is associated with risk of "new" meniscal tears (defined as those visualized arthroscopically that had not been detected on magnetic resonance imaging [MRI]) in adult and pediatric patients. Study Design: Cohort study; Level of evidence, 3. Methods: We retrospectively identified patients who underwent primary ACL reconstruction between 2013 and 2022 at our institution. To ensure that MRI reflected initial intra-articular pathology, we included only patients who had an MRI scan within 3 weeks after injury (173 pediatric and 369 adult patients). Multivariate Poisson regression was performed to calculate the adjusted relative risk (ARR) of new meniscal tears after delayed (≥8 weeks from injury) operative treatment. Results: The mean (± SD) time from injury to MRI was 1.0 ± 0.8 weeks for pediatric patients and 1.1 ± 0.7 weeks for adults. Less than half of the meniscal tears observed arthroscopically had been absent on initial MRI. New medial meniscal tears occurred in 15% of pediatric patients and 16% of adults. New lateral meniscal tears occurred in 48% of pediatric patients and 34% of adults. Among pediatric patients, delayed ACL reconstruction was associated with higher risk of new medial tears (ARR, 3.9; 95% CI, 1.5-10) but not lateral tears (ARR, 0.8; 95% CI, 0.4-1.5). In contrast, adults had no significant increase in risk of meniscal tears associated with operative delay. Conclusion: Delayed ACL reconstruction may be acceptable in adults, who may be less active and less injury-prone than children and adolescents.

Convergence spasm in conversion disorders: prevalence in psychogenic and other movement disorders compared with controls.

BACKGROUND: Convergence spasm refers to transient ocular convergence, miosis and accommodation associated with disconjugate gaze mimicking abducens palsy. While it may be a manifestation of brainstem pathology, this sign is often associated with conversion (somatisation) disorders and, if unrecognised as a sign of a psychogenic disorder, it may lead to unnecessary and occasionally invasive evaluation. METHODS: To better characterise this neuro-ophthalmologic sign, 36 subjects were studied, 13 with psychogenic movement disorders, 11 with organic movement disorders and 12 normal controls. Patients were recorded during a manoeuvre to elicit convergence spasm and the videotapes were rated by two blinded raters on a scale of 0=normal, 1=mild convergence spasm and 2=marked convergence spasm. RESULTS: Convergence spasm was present in 9/13 (69%) psychogenic movement disorders cases, 4/11 (36%) non-psychogenic movement disorders cases and 4/12 (33%) controls (p=0.049 when psychogenic vs non-psychogenic disorders or controls were compared). Inter-rater reliability analysis of the presence (rating 1 or 2) versus absence (rating 0) showed good agreement (27/36 or 75%; kappa 0.491, SE 0.141, p=0.002). Analysis for the presence of marked convergence spasm (rating 2) yielded agreement in 32/36 (88.9%) examinations (kappa 0.652, SE 0.154, p<0.001) with a specificity of 87% (sensitivity 15%). CONCLUSION: Convergence spasm may provide benefit in the clinical examination of psychogenic movement disorders patients.

Deep brain stimulation hardware complications in patients with movement disorders: risk factors and clinical correlations.

BACKGROUND: Deep brain stimulation (DBS) has proven to be an effective treatment for Parkinson's disease (PD) and other movement disorders, but its usefulness is limited by complications related to the hardware. METHODS: We reviewed the records of all our patients treated with DBS from January 1996 to August 2010 and analyzed those with hardware complications and reasons for surgical revision. RESULTS: A total of 512 patients underwent 856 electrode implantations during the study period. A total of 297 (58%) patients had PD, 127 (24.8%) had essential tremor (ET), 40 (7.8%) had dystonia, and 48 (9.37%) had another movement disorder. The mean age at the first electrode implantation was 57.6 ± 14 years and patients were followed for a mean of 3.9 ± 2.8 years. A total of 44 patients (8.6%) had a hardware complication or system revision. Lead fracture was the most common complication and occurred in 13 (2.5%) patients, followed by infections (n = 10, 1.9%), electrode misplacement (n = 10, 1.9%), electrode migration (n = 9, 1.75%), and other complications (n = 2 , 0.39%). Patients with ET had a higher risk of hardware complications compared to those with PD, 13 vs. 7% (OR 2.03; p = 0.042). CONCLUSIONS: DBS is a safe intervention with a relatively low rate of hardware complications.

Developing PHarmacie-R: A bedside risk prediction tool with a medicines management focus to identify risk of hospital readmission.

BACKGROUND: The imperative to identify patients at risk of medication-related harm has never been greater. Hospital clinicians cannot easily predict risk of readmission or harm. Candidate variables associated with medication-related harm derived from the literature or significantly represented in a complex patient cohort have been previously described by PHarmacie-4. With a focus on polypharmacy and high-risk medicines in vulnerable patient cohorts, PHarmacie-4 was easy to use and highlighted risks. However it over-estimated risk, reducing its usefulness in stratifying risk of readmission. OBJECTIVE: Develop a risk prediction tool built into a smart phone app, enabling clinicians to identify and refer high-risk patients for an early post-discharge medicines review. Demonstrate usability, real world application and validity in an independent dataset. METHODS: A retrospective, observational study was conducted with 1201 randomly selected patients admitted to Sir Charles Gairdner Hospital between June 1, 2016 to December 31, 2016. Patient characteristics and outcomes of interest were reported, including unplanned hospital utilisation at 30, 60 and 90 days post-discharge. Using multivariable logistic regression modelling, an algorithm was developed, built into a smart phone app and used and validated in an independent dataset. RESULTS: 738 patients (61%) were included in the derivation sample. The best predictive performance was achieved by PHarmacie-R (C-statistic 0.72, 95% CI 0.68-0.75) which included PHarmacie-4 risk variables, a non-linear effect of age, unplanned hospital utilisation in the preceding six months and gender. The independent validation dataset had a C-statistic of 0.64 (95% CI 0.56-0.72). CONCLUSION: PHarmacie-R is the first readmission risk prediction tool, built into a smart phone app, focussing on polypharmacy and high-risk medicines in vulnerable patients. It can assist clinical pharmacists to identify medical inpatients who may benefit from early post-discharge medication management services. External validation is needed to enable application in other clinical settings.

Partial Distal Biceps Avulsion Results in a Significant Loss of Supination Force.

BACKGROUND: Partial avulsions of the short and/or long head of the distal biceps tendon cause pain and loss of strength. The goal of the present study was to quantify the loss of supination and flexion strength following a series of surgical releases designed to simulate partial and complete short and long head traumatic avulsions. METHODS: Mechanical testing was performed to measure supination moment arms and flexion force efficiency on 18 adult fresh-frozen specimens in pronation, neutral, and supination. The distal biceps footprint length was divided into 4 equal segments. In 9 specimens (the distal-first group), the tendon was partially cut starting distally by releasing 25%, 50%, and 75% of the insertion site. In the other 9 specimens (the proximal-first group), the releases started proximally. Mechanical testing was performed before and after each release. RESULTS: Significant decreases in the supination moment arm occurred after a 75% release in the distal-first release group; the decrease was 24% in pronation (p = 0.003) and 10% in neutral (p = 0.043). No significant differences in the supination moment arm (p ≥ 0.079) or in flexion force efficiency (p ≥ 0.058) occurred in the proximal-first group. CONCLUSIONS: A simulated complete short head avulsion significantly decreased the supination moment arm and therefore supination strength. CLINICAL RELEVANCE: A mechanical case can be made for repair of partial distal biceps tendon avulsions when the rupture involves ≥75% of the distal insertion site.

Impact of STN-DBS on life and health satisfaction in patients with Parkinson's disease.

OBJECTIVE: Advanced Parkinson's disease (PD) is associated with various motor and non-motor symptoms which adversely impact health-related quality of life (HRQoL). Subthalamic nucleus (STN) deep brain stimulation (DBS) has been reported to improve some dimensions of HRQoL in appropriately selected candidates. Prior studies of HRQoL following DBS have used instruments comprising a predetermined list of questions which assess issues that are generally relevant in PD, but that may not be of equal or consistent importance to all individuals. In this study, we evaluate the effect of STN DBS on quality of life using the QLS(M), a modular questionnaire in which satisfaction scores for each item are weighted in light of patient-rated importance. METHODS: We prospectively analysed QLS(M) scores in 21 patients with PD (11 men, mean age 61.5+/-8.6 years) before STN DBS surgery and at a mean 7.4+/-1.5, and again at a mean 16.6+/-6.8 months postoperatively. RESULTS: Following STN DBS, patients experienced an improvement in HRQoL as measured by various items of the movement disorder and health modules of the QLS(M). Specifically, QLS(M) items pertaining to energy level/enjoyment of life, independence from help, controllability/fluidity of movement and steadiness when standing and walking showed significant improvements, although items concerning general life issues (eg, occupational function, interpersonal relationships, leisure activities) did not improve. CONCLUSION: Following STN DBS, symptomatic and functional improvements translate into higher HRQoL, with high satisfaction in domains related to movement disorders and general health.

Tourette's syndrome in adults.

Tourette's syndrome (TS) is defined as motor and phonic tics starting before age 18 years, and therefore most studies have focused on childhood TS, whereas the disorder in adults has not been well characterized. We reviewed medical records of all new TS patients referred to our Movement Disorders Clinic over the past 5 years, 19 years or older on initial evaluation and compared them with 100 TS patients 18 years or younger. The mean age at initial visit of 43 adult TS patients was 58.8 ± 6.7 years, whereas the mean age at initial visit of children with TS was 12.9 ± 2.0 years. Of the adult TS patients, 35 (81.4%) had a history of tics with onset before the age of 18 years (mean age at onset: 8.5 ± 3.4 years), with 8 (18.6%) reporting first occurrence of tics after the age of 18 years (mean age at onset: 37.8 ± 13.2 years). Only two (4.7%) patients reported tic onset after the age of 50 years. Adult patients with TS had significantly more facial and truncal tics, and a greater prevalence of substance abuse and mood disorders, but fewer phonic tics, and lower rates of attention-deficit hyperactivity disorder and oppositional behavior than children with TS. Adult TS largely represents reemergence or exacerbation of childhood-onset TS. During the course of TS, phonic and complex motor tics, self-injurious behaviors, and attention-deficit hyperactivity disorder tend to improve, but facial, neck, and trunk tics dominate the adult TS phenotype. In addition, adults with TS are more likely to exhibit substance abuse and mood disorders compared with children with TS.

Correlation between Kinesia system assessments and clinical tremor scores in patients with essential tremor.

The primary aim of this study was to determine whether scores on The Essential Tremor Rating Assessment Scale (TETRAS) correlate with quantitative assessments using the Kinesia™ (CleveMed) system in patients with essential tremor (ET). Patients sequentially evaluated and diagnosed with ET at the Parkinson's Disease Center and Movement Disorders Clinic, Baylor College of Medicine were enrolled in the study. The Kinesia portable device was attached to the wrist and subjects were instructed to hold their arms in an outstretched position and then touch their nose while data were wirelessly transmitted to a computer. Subjects were rated on the arm where the system was placed using specific TETRAS items. A linear regression model was constructed for each task using the logarithmic values of both clinical scores and objective motion data parameters to compute a Kinesia score. Twenty subjects underwent complete clinical TETRAS and Kinesia quantitative assessments. TETRAS clinical scores significantly correlated with predicted Kinesia quantitative variables for postural (r = 0.738; P < 0.001) and kinetic (r = 0.57; P = 0.009) tremor. We conclude that the Kinesia system may, therefore, have a utility in quantitative assessments of ET when combined with standard clinical assessment.

Comparison of epitaxial graphene on Si-face and C-face 4H SiC formed by ultrahigh vacuum and RF furnace production.

We present X-ray photoelectron spectroscopy, van der Pauw Hall mobilities, low-temperature far-infrared magneto transmission (FIR-MT), and atomic force microscopy (AFM) results from graphene films produced by radiative heating in an ultrahigh vacuum (UHV) chamber or produced by radio frequency (RF) furnace annealing in a high vacuum chemical vapor deposition system on Si- and C-face 4H SiC substrates at 1200-1600 degrees C. Although the vacuum level and heating methods are different, graphene films produced by the two methods are chemically similar with the RF furnace annealing typically producing thicker graphene films than UHV. We observe, however, that the formation of graphene on the two faces is different with the thicker graphene films on the C-face RF samples having higher mobility. The FIR-MT showed a 0(-1) --> 1(0) Landau level transition with a square root B dependence and a line width consistent with a Dirac fermion with a mobility >250,000 cm(2) x V(-1) x s(-1) at 4.2 K in a C-face RF sample having a Hall-effect carrier mobility of 425 cm(2) x V(-1) x s(-1) at 300 K. AFM shows that graphene grows continuously over the varying morphology of both Si and C-face substrates.

EPEC Recruits a Cdc42-Specific GEF, Frabin, To Facilitate PAK Activation and Host Cell Colonization.

Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that tightly adheres to host cells by forming "actin pedestals" beneath the bacteria, a critical step in pathogenesis. EPEC injects effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. We have recently shown that one such effector, EspG, hijacks p21-activated kinase (PAK) and sustains its activated state to drive the cytoskeletal changes necessary for attachment of the pathogen to target cells. This EspG subversion of PAK required active Rho family small GTPases in the host cell. Here we show that EPEC itself promotes the activation of Rho GTPases by recruiting Frabin, a host guanine nucleotide exchange factor (GEF) for the Rho GTPase Cdc42. Cells devoid of Frabin showed significantly lower EPEC-induced PAK activation, pedestal formation, and bacterial attachment. Frabin recruitment to sites of EPEC attachment was driven by EspG and required localized enrichment of phosphatidylinositol 4,5-bisphosphate (PIP2) and host Arf6. Our findings identify Frabin as a key target for EPEC to ensure the activation status of cellular GTPases required for actin pedestal formation.IMPORTANCE Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea in children, especially in the developing world. EPEC initiates infection by attaching to cells in the host intestine, triggering the formation of actin-rich "pedestal" structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, which work together to hijack host signaling pathways to drive pedestal production. Here we show how EPEC hijacks a host protein, Frabin, which creates the conditions in the cell necessary for the pathogen to manipulate a specific pathway that promotes pedestal formation. This provides new insights into this essential early stage in disease caused by EPEC.

Pathogenic Escherichia coli Hijacks GTPase-Activated p21-Activated Kinase for Actin Pedestal Formation.

Enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) are extracellular pathogens that reorganize the host cell cytoskeleton to form "actin pedestals" beneath the tightly adherent bacteria, a critical step in pathogenesis. EPEC and EHEC inject effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. One such effector, EspG, has been reported to bind and activate p21-activated kinase (PAK), a key cytoskeletal regulator, but the function of this interaction and whether it impacts pedestal assembly are unknown. Here, we demonstrate that deletion of espG significantly impairs pedestal formation and attachment by both EPEC and EHEC. This role of EspG is shown to be dependent on its interaction with PAK. Unexpectedly, EspG was able to subvert PAK only in the presence of Rho family small GTPases, which function to both concentrate PAK at the membrane and stimulate PAK activation. Our findings reveal a novel mechanism by which EspG hijacks PAK and sustains its active state to drive bacterial attachment to host cells.IMPORTANCE Enteropathogenic E. coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) remain a significant global health problem. Both EPEC and EHEC initiate infection by attaching to cells in the host intestine, triggering the formation of actin-rich "pedestal" structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, but how they contribute to disease is often less clear. Here, we show how one of these injected proteins, EspG, hijacks a host signaling pathway for pedestal production. This provides new insights into this essential early stage in EPEC and EHEC disease.

Arf GTPase interplay with Rho GTPases in regulation of the actin cytoskeleton.

The Arf and Rho subfamilies of small GTPases are nucleotide-dependent molecular switches that act as master regulators of vesicular trafficking and the actin cytoskeleton organization. Small GTPases control cell processes with high fidelity by acting through distinct repertoires of binding partners called effectors. While we understand a great deal about how these GTPases act individually, relatively little is known about how they cooperate, especially in the control of effectors. This review highlights how Arf GTPases collaborate with Rac1 to regulate actin cytoskeleton dynamics at the membrane via recruiting and activating the Wave Regulatory Complex (WRC), a Rho effector that underpins lamellipodia formation and macropinocytosis. This provides insight into Arf regulation of the actin cytoskeleton, while putting the spotlight on small GTPase cooperation with emerging evidence of its importance in fundamental cell biology and interactions with pathogenic bacteria.