In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.     

Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture.

A substantial proportion of bacteria from five Alexandrium cultures originally isolated from various countries produced sodium channel blocking (SCB) toxins, as ascertained by mouse neuroblastoma assay. The quantities of SCB toxins produced by bacteria and dinoflagellates were noted, and the limitations in comparing the toxicities of these two organisms are discussed. The chemical nature of the SCB toxins in selected bacterial isolates was determined as paralytic shellfish toxins by pre- and postcolumn high-performance liquid chromatography, capillary electrophoresis-mass spectrometry, and enzyme immunoassay.     

Mito‐nuclear discordance at a mimicry color transition zone in bumble bee Bombus melanopygus

As hybrid zones exhibit selective patterns of gene flow between otherwise distinct lineages, they can be especially valuable for informing processes of microevolution and speciation. The bumble bee, Bombus melanopygus, displays two distinct color forms generated by Müllerian mimicry: a northern “Rocky Mountain''’ color form with ferruginous mid‐abdominal segments (B. m. melanopygus) and a southern “Pacific''’ form with black mid‐abdominal segments (B. m. edwardsii). These morphs meet in a mimetic transition zone in northern California and southern Oregon that is more narrow and transitions further west than comimetic bumble bee species. To understand the historical formation of this mimicry zone, we assessed color distribution data for B. melanopygus from the last 100 years. We then examined gene flow among the color forms in the transition zone by comparing sequences from mitochondrial COI barcode sequences, color‐controlling loci, and the rest of the nuclear genome. These data support two geographically distinct mitochondrial haplogroups aligned to the ancestrally ferruginous and black forms that meet within the color transition zone. This clustering is also supported by the nuclear genome, which, while showing strong admixture across individuals, distinguishes individuals most by their mitochondrial haplotype, followed by geography. These data suggest the two lineages most likely were historically isolated, acquired fixed color differences, and then came into secondary contact with ongoing gene flow. The transition zone, however, exhibits asymmetries: mitochondrial haplotypes transition further south than color pattern, and both transition over shorter distances in the south. This system thus demonstrates alternative patterns of gene flow that occur in contact zones, presenting another example of mito‐nuclear discordance. Discordant gene flow is inferred to most likely be driven by a combination of mimetic selection, dominance effects, and assortative mating.     

Consumer versus resource control and the importance of habitat heterogeneity for estuarine bivalves

The relative influence of consumers (top down) and resources (bottom up) on the distribution and abundance of organisms remains a key question in ecology. We examined the relationships between consumer and resource variables along a productivity gradient for a dominant predator–prey interaction in a marine soft‐sediment system. We 1) quantified density and size of the clam Macoma balthica (prey species) in six replicate sites at each of four habitat types (shallow mud, deep mud, muddy sand and detrital mud) in the Rhode River, Chesapeake Bay. We selected one habitat type of high food availability and clam density (shallow mud) and another of low food availability and clam density (muddy sand) for manipulative experiments. Then, we 2) measured M. balthica survival and growth through transplants, 3) measured food availability as sedimentary organic carbon content, 4) quantified predator density, and 5) calculated predator foraging efficiency in the two habitat types. Clam density in the four habitat types differed and was related to sedimentary carbon availability and predator density. One of the habitats, detrital mud, appeared to be a population sink because it only held juvenile Macoma that never survived to reproductive age. Macoma size and growth, and predator (mainly blue crab Callinectes sapidus) densities were positively correlated with productivity and were higher in shallow mud than muddy sand. In contrast, Macoma mortality, local ‘interaction strength’, and predator foraging efficiency were lower in the productive habitat (shallow mud). Thus, predation intensity was inversely correlated with productivity (food availability); consumer and resource effects differed by habitat type; and, at a relatively small spatial scale, consumer and resource forces jointly determined population dynamics in this soft‐sediment marine system.     

Nutrient enrichment and fisheries exploitation: interactive effects on estuarine living resources and their management

Both fisheries exploitation and increased nutrient loadings strongly affect fish and shellfish abundance and production in estuaries. These stressors do not act independently; instead, they jointly influence food webs, and each affects the sensitivity of species and ecosystems to the other. Nutrient enrichment and the habitat degradation it sometimes causes can affect sustainable yields of fisheries, and fisheries exploitation can affect the ability of estuarine systems to process nutrients. The total biomass of fisheries landings in estuaries and semi-enclosed seas tends to increase with nitrogen loadings in spite of hypoxia, but hypoxia and other negative effects of nutrient over-enrichment cause declines in individual species and in parts of systems most severely affected. More thoroughly integrated management of nutrients and fisheries will permit more effective management responses to systems affected by both stressors, including the application of fisheries regulations to rebuild stocks negatively affected by eutrophication. Reducing fishing mortality may lead to the recovery of depressed populations even when eutrophication contributes to population declines if actions are taken while the population retains sufficient reproductive potential. New advances in modeling, statistics, and technology promise to provide the information needed to improve the understanding and management of systems subject to both nutrient enrichment and fisheries exploitation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: J. H. Andersen & D. J. Conley Eutrophication in Coastal Ecosystems: Selected papers from the Second International Symposium on Research and Management of Eutrophication in Coastal Ecosystems, 20–23 June 2006, Nyborg, Denmark     

Molecular Mechanism of BST2/Tetherin Downregulation by K5/MIR2 of Kaposi's Sarcoma-Associated Herpesvirus

K3/MIR1 and K5/MIR2 of Kaposi''s sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.Bone marrow stromal cell antigen 2 (BST2) was recently identified as a host cell restriction factor that prevents the release of retroviral and filoviral particles from infected host cells (23). Human immunodeficiency virus type 1 (HIV-1) counteracts this antiviral function of BST2 by expressing the viral auxiliary protein VPU (41, 53). In the absence of VPU, virus particles are prevented from budding off the cellular membrane in cells that express BST2, resulting in virions being tethered to the plasma membrane. BST2 was therefore renamed tetherin (41), although questions still remain as to whether BST2 acts as the actual tether and whether BST2-dependent tethering occurs in all BST2-expressing cell types (36). Independently, BST2 was shown to be induced by type I and type II interferons (IFNs) (7), suggesting that BST2 is part of the innate antiviral response triggered in infected cells.Using a quantitative membrane proteomic approach, we observed that BST2 is underrepresented in plasma membranes from cells expressing not only VPU (14) but also the K5 protein of Kaposi''s sarcoma-associated herpesvirus (KSHV) (4). K5 is a viral homologue of a family of cellular transmembrane ubiquitin ligases, termed membrane-associated RING-CH (MARCH) proteins (3), that mediate the ubiquitination of the cytoplasmic portion of transmembrane proteins (reviewed in reference 40). Each member of this family targets a subset of cellular membrane proteins with both unique and shared specificities (4, 56). One of the functions of cellular MARCH proteins is to modulate antigen presentation by mediating the ubiquitin-dependent turnover of major histocompatibility complex (MHC) class II molecules in dendritic cells, B cells, and monocytes/macrophages (43, 52). In contrast, viral homologues of MARCH proteins encoded by KSHV, murine herpesvirus 68, and the leporipoxvirus myxomavirus all share the ability to mediate the destruction of MHC-I (reviewed in reference 16) but not MHC-II molecules. Thus, one of the functions of the viral proteins is to promote viral escape from immune clearance by CD8⁺ T lymphocytes (50). Furthermore, each viral MARCH homologue specifically eliminates additional host cell proteins, so each plays multiple roles in viral pathogenesis. KSHV carries two viral MARCH proteins, K3 and K5, also known as MIR1 and MIR2, which both support viral escape from T-cell, NK-cell, and NKT-cell recognition by eliminating the corresponding ligands from the surfaces of infected cells (reviewed in reference 10). In endothelial cells (ECs), K5 additionally downregulates EC-specific adhesion molecules that play an essential role in the formation of adhesive platforms and adherens junctions (31, 32). Since Kaposi''s sarcoma is a tumor of EC origin, K5 might thus also contribute to tumorigenesis by disrupting normal EC barrier function and by modulating the interaction of ECs with inflammatory leukocytes.The downregulation of BST2 by K5 further suggests that K5 also counteracts innate antiviral responses, which might benefit KSHV. However, most transmembrane proteins targeted by viral or cellular MARCH proteins are type I transmembrane proteins that belong to the immunoglobulin superfamily. In contrast, BST2 is a type II transmembrane protein that is also glycosylphosphatidylinositol (GPI) anchored (25). Thus, BST2 has a short cytoplasmic amino terminus followed by an outside-in transmembrane domain, a large glycosylated extracellular portion, and a GPI anchor. The additional propensity of BST2 to form homodimers (44) was speculated to be crucial for the tethering function of BST2 in that self-association of BST2 molecules in the viral envelope with plasma membrane BST2 could prevent viral exit (19). The unusual topology of BST2 and its multimerization raised the question of whether BST2 is a bona fide target of K5 or whether its downregulation is a downstream effect of K5 eliminating other transmembrane proteins. Additionally, it is not clear whether BST2 would be downregulated in the context of a normal viral infection and, particularly, whether virally expressed K5 would be able to overcome the high expression levels of BST2 observed upon IFN induction. We now demonstrate that KSHV efficiently downregulates IFN-induced BST2 both during primary infection and upon reactivation from latency in ECs. IFN-induced BST2 is ubiquitinated by K5 upon exiting the endoplasmic reticulum (ER) and is rapidly degraded by a pathway that is sensitive to proteasome inhibitors but resistant to inhibitors of lysosomal acidification. These data suggest that despite its unusual topology, BST2 is directly targeted by K5. We further demonstrate that BST2 reduces KSHV release upon inhibition of K5 expression by small interfering RNA (siRNA), suggesting that BST2 is part of the IFN-induced innate immune response to KSHV. Thus, in addition to contributing to viral evasion of cellular immune responses and remodeling EC function, K5 also counteracts the innate immune defense of the host cell.     

Hepatocellular protection by nitric oxide or nitrite in ischemia and reperfusion injury

Ischemia and reperfusion (I/R)-induced liver injury occurs in several pathophysiological disorders including hemorrhagic shock and burn as well as resectional and transplantation surgery. One of the earliest events associated with reperfusion of ischemic liver is endothelial dysfunction characterized by the decreased production of endothelial cell-derived nitric oxide (NO). This rapid post-ischemic decrease in NO bioavailability appears to be due to decreased synthesis of NO, enhanced inactivation of NO by the overproduction of superoxide or both. This review presents the most current evidence supporting the concept that decreased bioavailability of NO concomitant with enhanced production of reactive oxygen species initiates hepatocellular injury and that endogenous NO or exogenous NO produced from nitrite play important roles in limiting post-ischemic tissue injury.     

Neurotrophic peptide aldehydes: solid phase synthesis of fellutamide B and a simplified analog

A combination of solid phase and solution phase synthetic methods have been used to complete the total synthesis of the neurotrophic lipopeptide aldehyde fellutamide B (2). The beta-hydroxy aliphatic tail was prepared by regioselective reductive opening of a cyclic sulfate, and later coupled to a solid phase resin. The synthetic compound was then examined in cytotoxicity and nerve growth factor (NGF) induction assays. A simplified analog of fellutamide B also showed activity.