Septins Regulate Bacterial Entry into Host Cells

Background

Septins are conserved GTPases that form filaments and are required in many organisms for several processes including cytokinesis. We previously identified SEPT9 associated with phagosomes containing latex beads coated with the Listeria surface protein InlB.

Methodology/Principal Findings

Here, we investigated septin function during entry of invasive bacteria in non-phagocytic mammalian cells. We found that SEPT9, and its interacting partners SEPT2 and SEPT11, are recruited as collars next to actin at the site of entry of Listeria and Shigella. SEPT2-depletion by siRNA decreased bacterial invasion, suggesting that septins have roles during particle entry. Incubating cells with InlB-coated beads confirmed an essential role for SEPT2. Moreover, SEPT2-depletion impaired InlB-mediated stimulation of Met-dependent signaling as shown by FRET.

Conclusions/Significance

Together these findings highlight novel roles for SEPT2, and distinguish the roles of septin and actin in bacterial entry.     

The Entry of Sodium into Human Red Blood Cells in Vivo

The kinetics of sodium, movement into human red blood cells has been studied in vivo with ²⁴Na. When human serum albumin^-131I is used to measure the percentage of plasma trapped in the packed red blood cells after centrifugation, approximately 30 % of red blood cell sodium is found to equilibrate immediately with plasma. It is concluded that this immediately exchangeable compartment of red blood cell sodium is an experimental artefact, associated with the use of labeled albumin for measuring plasma trapping. This immediately exchangeable fraction disappears when sucrose-¹⁴C is used to measure plasma trapping. The experimental results were examined by compartmental analysis, using an analogue computer. The results obtained, when plasma trapping was measured with sucrose-¹⁴C could be simulated by the use of models containing two compartments, arranged in series or in parallel. The errors of the techniques used and the possible physical basis for the results are discussed.     

Entry of Vesicular Stomatitis Virus into L Cells

Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of (3)H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm.     

The Virological Synapse Facilitates Herpes Simplex Virus Entry into T Cells

The virological synapse (VS) is a specialized molecular structure that facilitates the transfer of certain lymphotropic viruses into uninfected T cells. However, the role of the VS in the transfer of nonlymphotropic viruses into T cells is unknown. Herpes simplex virus (HSV) has been shown in vitro to infect T cells and modulate T-cell receptor function, thereby suppressing T-cell antiviral function. However, whether such infection of T cells occurs in vivo is unknown. Here, we examined whether T-cell infection could be observed in human HSV disease and investigated the mechanism of HSV entry into T cells. We found that HSV-infected T cells were readily detectable during human disease, suggesting that infection and modulation of T-cell function plays a role in human immunopathology. HSV infection of both CD4⁺ and CD8⁺ T cells occurred much more efficiently via direct cell-to-cell spread from infected fibroblasts than by cell-free virus. Activation of T cells increased their permissivity to HSV infection. Cell-to-cell spread to T cells did not require HSV glycoproteins E and I (gE and gI), which are critical for cell-to-cell spread between epithelial cells. Transfer of HSV to T cells required gD, and the four known entry receptors appear to be contributing to viral entry, with a dominant role for the herpesvirus entry mediator and nectin-1. VS-like structures enriched in activated lymphocyte function-associated antigen 1 (LFA-1) were observed at the point of contact between HSV-infected fibroblasts and T cells. Consistent with spread occurring via the VS, transfer of HSV was increased by activation of LFA-1, and cell-to-cell spread could be inhibited by antibodies to LFA-1 or gD. Taken together, these results constitute the first demonstration of VS-dependent cell-to-cell spread for a predominantly nonlymphotropic virus. Furthermore, they support an important role for infection and immunomodulation of T cells in clinical human disease. Targeting of the VS might allow selective immunopotentiation during infections with HSV or other nonlymphotropic viruses.The virological synapse (VS) is a specialized molecular structure that facilitates the transfer of certain lymphotropic viruses, such as human immunodeficiency virus (HIV) and human T-cell leukemia virus type 1 (HTLV-1), into uninfected T cells (22, 28, 38). Entry and infection of T cells by HIV or HTLV-1 via the VS is far more efficient than infection by cell-free virus, and thus this structure plays a critical role in the pathogenesis of these viruses. The organization of the VS is in many respects similar to the immunological synapse (IS), in particular, to the immature IS. The VS is highly enriched in the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) and its ligands intercellular adhesion molecule 1 (ICAM-1) and ICAM-3 (29); however, it does not possess the CD3-enriched central region associated with the mature IS (28, 47). While the VS is critical to the pathogenesis of HIV and HTLV-1, it remains an unanswered question whether the VS is also involved in T-cell infection by other viruses, especially those not typically considered lymphotropic.Herpes simplex virus (HSV) is a remarkably successful human pathogen that establishes lifelong latency in neurons of the dorsal root ganglia. HSV can efficiently reactivate from the latent state and transmit to new hosts despite the presence of preformed immunity. HSV is thought to achieve this feat by employing a number of sophisticated immune evasion mechanisms (33), many of which are directed at the cellular arm of the immune response. In one such potential mechanism, HSV has evolved the ability to enter and infect T cells. Although T cells do not support efficient viral replication (25), infection by HSV profoundly modulates T-cell receptor (TCR) signaling, which prevents T-cell cytotoxic function (55) and alters cytokine production profiles toward an interleukin-10-dominated immunosuppressive phenotype (54). However, it is unknown whether and to what extent HSV infection of T cells occurs during human HSV disease. Furthermore, the dominant mechanisms by which HSV might gain access to lesion-infiltrating T cells have not been elucidated.Here, we evaluated T-cell infection during human HSV infections, the mechanisms by which HSV enters T cells, the relative involvement of cell-cell spread versus cell-free virus in T-cell infection, and the role of the VS in the infection of T cells by HSV. The demonstration of infection of T cells in human HSV disease and of a dominant role for the VS in entry of HSV into T cells suggests that the VS is important in the pathogenesis of nonlymphotropic as well as lymphotropic viruses. Thus, the VS may be a unique pharmacologic target to allow improved immune control of a wide variety of viral infections.     

Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.     

Entry of Methylammonium and Ammonium Ions into Chara Internodal Cells

Low concentrations of ammonia and methylamine greatly affectmembrane transport by, and the electrical properties of, cellsof Chara corallina (=C. australis). In the presence of theseamines, influx of Cl^– and efflux of K⁺ increase and alarge depolarizing current flows through the cell membrane. Measurements with [¹⁴C]methylamine show that methylamine isabsorbed rapidly over a wide pH range, and that the absorptionisotherm is complex. Methylamine influx is not affected by presenceor absence of Cl^–, K⁺, or Na⁺, but is decreased by additionof . The depolarizing current is associated with a small increase in membrane conductance, except at highpH, and both these effects are reversible. The current showssaturation with increasing amine concentration; when methylamineis 10–12 times more concentrated than ammonia, it producesa current of the same magnitude. The results show that the amines enter the cells as cations( or CH₃) except where external pH is high, and that a specific, selective electrogenicporter is involved. There is no need to invoke active transport,as there is no evidence that internal amine concentrations exceedthe equilibrium (Nernst) concentrations.     

Role of DC-SIGN in Lassa Virus Entry into Human Dendritic Cells

The arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with high mortality in humans. Antigen-presenting cells, in particular dendritic cells (DCs), are early and preferred targets of LASV, and their productive infection contributes to the virus-induced immunosuppression observed in fatal disease. Here, we characterized the role of the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) in LASV entry into primary human DCs using a chimera of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that differentiation of human primary monocytes into DCs enhanced virus attachment and entry, concomitant with the upregulation of DC-SIGN. LASV and rLCMV-LASVGP bound to DC-SIGN via mannose sugars located on the N-terminal GP1 subunit of LASVGP. We provide evidence that DC-SIGN serves as an attachment factor for rLCMV-LASVGP in monocyte-derived immature dendritic cells (MDDC) and can accelerate the capture of free virus. However, in contrast to the phlebovirus Uukuniemi virus (UUKV), which uses DC-SIGN as an authentic entry receptor, productive infection with rLCMV-LASVGP was less dependent on DC-SIGN. In contrast to the DC-SIGN-mediated cell entry of UUKV, entry of rLCMV-LASVGP in MDDC was remarkably slow and depended on actin, indicating the use of different endocytotic pathways. In sum, our data reveal that DC-SIGN can facilitate cell entry of LASV in human MDDC but that its role seems distinct from the function as an authentic entry receptor reported for phleboviruses.     

Actin-Dependent Receptor Colocalization Required for Human Immunodeficiency Virus Entry into Host Cells

Human immunodeficiency virus (HIV) envelope binds CD4 and a chemokine receptor in sequence, releasing hydrophobic viral gp41 residues into the target membrane. HIV entry required actin-dependent concentration of coreceptors, which could be disrupted by cytochalasin D (CytoD) without an effect on cell viability or mitosis. Pretreatment of peripheral blood mononuclear cells, but not virus, inhibited entry and infection. Immunofluorescent confocal microscopy of activated cells revealed CD4 and CXCR4 in nonoverlapping patterns. Addition of gp120 caused polarized cocapping of both molecules with subsequent pseudopod formation, while CytoD pretreatment blocked these membrane changes completely.     

6-O- and N-Sulfated Syndecan-1 Promotes Baculovirus Binding and Entry into Mammalian Cells

Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.     

Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism.     

Geranylgeraniol Promotes Entry of UT-2 Cells into the Cell Cycle in the Absence of Mevalonate

Although UT-2 cells, a mutant clone of Chinese hamster ovary cells, have been shown to require mevalonate for growth due to a deficiency in 3-hydroxy-3-methylglutaryl-CoA reductase, the precise mevalonate-derived product(s) essential for proliferation has not been identified. These studies show that UT-2 cells proliferate in the presence of free geranylgeraniol (GG-OH), as well as mevalonate. Cell growth was optimal when the culture medium was supplemented with 5–10 μMGG-OH. Under these growth conditions [³H]GG-OH is actively incorporated into UT-2 proteins. Prominent [³H]geranylgeranylated polypeptides in the size range (19–27 kDa) of the small GTP-binding proteins are observed by SDS–PAGE. Analysis of the butanol-soluble products released from the metabolically labeled proteins by digestion with Pronase E reveals that the proteins contain [³H]geranylgeranylated cysteine residues. Even though [³H]farnesol is also incorporated into cysteinyl residues of a different set of UT-2 proteins, farnesol added at 10 μMdid not satisfy the mevalonate requirement for cell growth. These results show that UT-2 cells divide in the presence of exogenously supplied GG-OH, providing evidence that one or more geranylgeranylated proteins are essential for entry of UT-2 cells, and probably other mammalian cells, into the cell cycle.     

The Assimilation of Ammonia by Nitrogen-starved Cells of Chlorella vulgaris: Part II. The Assimilation of Ammonia to other Compounds

Ammonia is rapidly assimilated by nitrogen-starved Chlorellacells and converted into soluble organic nitrogenous compounds.During the first 20 minutes of assimilation most of the ammoniawhich has been used can be accounted for as free or combined-amino-nitrogen and amide-nitrogen. Later a smaller proportionof the assimilated ammonia is found in these fractions whileappreciable quantities of basic amino-acids are formed. Theassimilation of ammonia is accompanied by an increase of therates of oxygen absorption and carbon dioxide production; thevalue of the respiratory quotient decreases. Non-reducing sugarand acid-hydrolysable polysaccharide are metabolized rapidly. Possible interpretations of these facts are discussed.     

Ammonia Induces Starch Degradation in Chlorella Cells

When ammonia was added to cells of Chlorella which had fixed¹⁴CO₂ photo synthetically, ¹⁴C which had been incorporated intostarch was greatly decreased. A similar effect was observedwhen potassium nitrate and sodium nitrite were added. The ammonia-induceddecrease in ¹⁴C-starch was observed in all species of Chlorellatested. With cells of C. vulgaris 11h, most of the radioactivityin starch was recovered in sucrose, indicating that ammoniainduces the conversion of starch into sucrose. The percent of¹⁴C recovered in sucrose differed from species to species andpractically no recovery in sucrose was observed in C. pyrenoidosa.In most species tested, the enhancing effects of blue lightand ammonia on O₂ uptake as well as the ammonia effect on starchdegradation were greater in cells which had been starved inphosphate medium in the dark than in non-starved cells. In contrast,the enhancing effect of ammonia on dark CO₂ fixation was muchgreater in non-starved cells. C. pyrenoidosa was unique in thatblue light did not show any effect on its O₂ uptake. (Received August 15, 1984; Accepted November 16, 1984)