Distribution of PDLIM1 at actin-rich structures generated by invasive and adherent bacterial pathogens.

The enteric bacterial pathogens Listeria monocytogenes (Listeria) and enteropathogenic Escherichia coli (EPEC) remodel the eukaryotic actin cytoskeleton during their disease processes. Listeria generate slender actin-rich comet/rocket tails to move intracellularly, and later, finger-like membrane protrusions to spread amongst host cells. EPEC remain extracellular, but generate similar actin-rich membranous protrusions (termed pedestals) to move atop the host epithelia. These structures are crucial for disease as diarrheal (and systemic) infections are significantly abrogated during infections with mutant strains that are unable to generate the structures. The current repertoire of host components enriched within these structures is vast and diverse. In this protein catalog, we and others have found that host actin crosslinkers, such as palladin and α-actinin-1, are routinely exploited. To expand on this list, we set out to investigate the distribution of PDLIM1, a scaffolding protein and binding partner of palladin and α-actinin-1, during bacterial infections. We show that PDLIM1 localizes to the site of initial Listeria entry into cells. Following this, PDLIM1 localizes to actin filament clouds surrounding immotile bacteria, and then colocalizes with actin once the comet/rocket tails are generated. Unlike palladin or α-actinin-1, PDLIM1 is maintained within the actin-rich core of membrane protrusions. Conversely, α-actinin-1, but not PDLIM1 (or palladin), is enriched at the membrane invagination that internalizes the Listeria-containing membrane protrusion. We also show that PDLIM1 is a component of the EPEC pedestal core and that its recruitment is dependent on the bacterial effector Tir. Our findings highlight PDLIM1 as another protein present within pathogen-induced actin-rich structures.

Localization of alpha-actinin-4 during infections by actin remodeling bacteria.

Bacterial pathogens cause disease by subverting the structure and function of their target host cells. Several foodborne agents such as Listeria monocytogenes (L. monocytogenes), Shigella flexneri (S. flexneri), Salmonella enterica serovar Typhimurium (S. Typhimurium) and enteropathogenic Escherichia coli (EPEC) manipulate the host actin cytoskeleton to cause diarrheal (and systemic) infections. During infections, these invasive and adherent pathogens hijack the actin filaments of their host cells and rearrange them into discrete actin-rich structures that promote bacterial adhesion (via pedestals), invasion (via membrane ruffles and endocytic cups), intracellular motility (via comet/rocket tails) and/or intercellular dissemination (via membrane protrusions and invaginations). We have previously shown that actin-rich structures generated by L. monocytogenes contain the host actin cross-linker α-actinin-4. Here we set out to examine α-actinin-4 during other key steps of the L. monocytogenes infectious cycle as well as characterize the subcellular distribution of α-actinin-4 during infections with other model actin-hijacking bacterial pathogens (S. flexneri, S. Typhimurium and EPEC). Although α-actinin-4 is absent at sites of initial L. monocytogenes invasion, we show that it is a new component of the membrane invaginations formed during secondary infections of neighboring host cells. Importantly, we reveal that α-actinin-4 also localizes to the major actin-rich structures generated during cell culture infections with S. flexneri (comet/rocket tails and membrane protrusions), S. Typhimurium (membrane ruffles) and EPEC (pedestals). Taken together, these findings suggest that α-actinin-4 is a host factor that is exploited by an assortment of actin-hijacking bacterial pathogens.

Development and Assessment of Novel Lung Ultrasound Interpretation Criteria for the Detection of Interstitial Lung Disease in Systemic Sclerosis.

OBJECTIVE: Interstitial lung disease (ILD) is a frequent complication of systemic sclerosis (SSc), and ILD screening, characterization, and monitoring are important for therapeutic decision-making and prognostication. Lung ultrasonography (US) is a potential alternative imaging modality for ILD detection. In this study, our objective was to develop and test a novel lung US examination technique and interpretation criteria for detecting SSc-ILD. METHODS: Lung US acquisition was performed by collecting short US movies at 14 lung positions. Lung US interpretation criteria for SSc-ILD detection focused on visualized pleural changes. To assess the performance of our methodology for SSc-ILD detection, we prospectively enrolled SSc patients with high-resolution computed tomography (HRCT) imaging within 3 months of lung US. Lung US examinations were scored independently by 2 blinded readers (1 ultrasonographer and 1 nonultrasonographer). The sensitivity and specificity for SSc-ILD detection were assessed, and agreement was measured with Cohen's kappa statistic. RESULTS: To test the performance of our lung US acquisition technique and interpretation criteria, 20 SSc patients were evaluated by lung US (278 lung zones) and HRCT. HRCT confirmed ILD in 9 patients (45%). Lung US was positive for SSc-ILD in 11 patients (55%) with a sensitivity of 100% and specificity of 82% versus HRCT, with perfect agreement between the 2 readers (κ = 1). Analysis by individual lung zones found excellent agreement between readers, with 93.8% concordance and κ = 0.82. CONCLUSION: We developed a novel lung US examination technique and interpretation criteria that are highly sensitive and specific for SSc-ILD detection in an SSc cohort, affording perfect agreement between ultrasonographer and nonultrasonographer readers.

Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading.

Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens.IMPORTANCEListeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1-based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1-based internalization event can exceed the theoretical size limit for this endocytic pathway.

NR2B phosphorylation at tyrosine 1472 contributes to brain injury in a rodent model of neonatal hypoxia-ischemia.

BACKGROUND AND PURPOSE: The NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor is phosphorylated by the Src family kinase Fyn in brain, with tyrosine (Y) 1472 as the major phosphorylation site. Although Y1472 phosphorylation is important for synaptic plasticity, it is unknown whether it is involved in NMDA receptor-mediated excitotoxicity in neonatal brain hypoxia-ischemia (HI). This study was designed to elucidate the specific role of Y1472 phosphorylation of NR2B in neonatal HI in vivo and in NMDA-mediated neuronal death in vitro. METHODS: Neonatal mice with a knockin mutation of Y1472 to phenylalanine (YF-KI) and their wild-type littermates were subjected to HI using the Vannucci model. Brains were scored 5 days later for damage using cresyl violet and iron staining. Western blotting and immunoprecipitation were performed to determine NR2B tyrosine phosphorylation. Expression of NADPH oxidase subunits and superoxide production were measured in vivo. NMDA-induced calcium response, superoxide formation, and cell death were evaluated in primary cortical neurons. RESULTS: After neonatal HI, YF-KI mice have reduced expression of NADPH oxidase subunit gp91phox and p47phox and superoxide production, lower activity of proteases implicated in necrotic and apoptotic cell death, and less brain damage when compared with the wild-type mice. In vitro, YF-KI mutation diminishes superoxide generation in response to NMDA without effect on calcium accumulation and inhibits NMDA and glutamate-induced cell death. CONCLUSIONS: Upregulation of NR2B phosphorylation at Y1472 after neonatal HI is involved in superoxide-mediated oxidative stress and contributes to brain injury.

Lactams as EP4 prostanoid receptor subtype selective agonists. Part 1: 2-Pyrrolidinones-stereochemical and lower side-chain optimization.

A series of 7-[(5R)-substituted 2-oxo-1-pyrrolidinyl]-heptanoic acids were prepared, their isomeric purity determined, and pharmacologically evaluated. Lactams with affinity for the EP(4) receptor displayed agonist behavior. The lower side-chain of the lactam template could be substituted to afford ligands (e.g., 17, 24, 30, 31, and 33) of high potency and greater than 1000-fold affinity for EP(4) versus the other EP prostanoid receptors.