Emmanuelle Perret

Human Immunodeficiency Virus Type 1 Entry into Macrophages Mediated by Macropinocytosis

ABSTRACT Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.

The HIV-1 DNA flap stimulates HIV vector- mediated cell transduction in the brain

During HIV-1 reverse transcription, central initiation of the plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) lead to the formation of a three-stranded DNA structure: the HIV-1 central DNA flap. We recently reported that the DNA flap acts as a cis-active determinant of HIV-1 genome nuclear import. Commonly employed HIV-1–derived vectors (HR vectors) lack the central DNA flap. Here we report that the insertion of this DNA flap sequence into HR vectors (TRIP vectors) improves gene transduction in neural cells, ex vivo and in vivo, in rat brain. When neural cells are exposed to increasing concentrations of TRIP vector particles, transgene expression correlates with the dose of vector. This effect contrasts with the plateau observed when using an HR vector. We further demonstrate that the increase of in vivo transduction efficiency obtained with TRIP vectors is due to the stimulation of their genome nuclear import.

An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus

Pulmonary infections due to Aspergillus fumigatus result from the development of a colony of tightly associated hyphae in contact with the air, either in the alveoli (invasive aspergillosis) or in an existing cavity (aspergilloma). The fungal ball observed in vivo resembles an aerial colony obtained in agar medium in vitro more than a mycelial mass obtained in liquid shaken conditions that have been classically used to date to study A. fumigatus physiology. For this reason, we embarked on an analysis of the characteristics of A. fumigatus colonies grown in aerial static conditions. (i) Under static aerial conditions, mycelial growth is greater than in shaken, submerged conditions. (ii) The colony surface of A. fumigatus revealed the presence of an extracellular hydrophobic matrix that acts as a cohesive linkage bonding hyphae into a contiguous sheath. (iii) The extracellular matrix is composed of galactomannan, α1,3 glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. (iv) A. fumigatus colonies were more resistant to polyenes than shake, submerged mycelium. This is the first analysis of the three dimensional structure of a mycelial colony. Knowledge of this multicellular organization will impact our future understanding of the pathobiology of aerial mold pathogens.

Constrained Intracellular Survival of Mycobacterium tuberculosis in Human Dendritic Cells

Dendritic cells (DCs) are likely to play a key role in immunity against Mycobacterium tuberculosis, but the fate of the bacterium in these cells is still unknown. Here we report that, unlike macrophages (Mφs), human monocyte-derived DCs are not permissive for the growth of virulent M. tuberculosis H37Rv. Mycobacterial vacuoles are neither acidic nor fused with host cell lysosomes in DCs, in a mode similar to that seen in mycobacterial infection of Mφs. However, uptake of the fluid phase marker dextran, and of transferrin, as well as accumulation of the recycling endosome-specific small GTPase Rab11 onto the mycobacterial phagosome, are almost abolished in infected DCs, but not in Mφs. Moreover, communication between mycobacterial phagosomes and the host-cell biosynthetic pathway is impaired, given that <10% of M. tuberculosis vacuoles in DCs stained for the endoplasmic reticulum-specific proteins Grp78/BiP and calnexin. This correlates with the absence of the fusion factor N-ethylmaleimide-sensitive factor onto the vacuolar membrane in this cell type. Trafficking between the vacuoles and the host cell recycling and biosynthetic pathways is strikingly reduced in DCs, which is likely to impair access of intracellular mycobacteria to essential nutrients and may thus explain the absence of mycobacterial growth in this cell type. This unique location of M. tuberculosis in DCs is compatible with their T lymphocyte-stimulating functions, because M. tuberculosis-infected DCs have the ability to specifically induce cytokine production by autologous T lymphocytes from presensitized individuals. DCs have evolved unique subcellular trafficking mechanisms to achieve their Ag-presenting functions when infected by intracellular mycobacteria.

Reversal of pathology in the entire brain of mucopolysaccharidosis type VII mice after lentivirus-mediated gene transfer

Gene transfer vectors derived from human immunodeficiency virus (HIV-1) efficiently transduce nondividing cells and remain stably integrated in their genome. Long-term expression of reporter genes has been documented after intracerebral injection of these vectors. Using a HIV-based vector, we looked for a reversal of brain damage in the beta-glucuronidase-deficient mucopolysaccharidosis type VII mouse, an animal model of human lysosomal storage diseases. The vector suspension was injected stereotactically in the brain of 10-week-old animals, an age at which storage lesions are patent in glia, perivascular cells, and neurons. Either a single intrastriatal injection or multiple injections in both cerebral hemispheres and in the cerebellum were performed. Local tolerance, enzyme delivery, and correction of storage lesions were investigated by comprehensive analysis of serial sections of the entire brain of mice killed 6 or 16 weeks postinjection. Histochemical staining detected enzyme activity in widely distributed areas, the size of which increased with time. Clearance of lysosomal storage extended far beyond enzyme-positive areas. In mice receiving multiple injections of the vector, complete correction or significant reduction of the pathology was observed in every section, suggesting disease regression in the entire brain. These results may have implications for the treatment of neurological symptoms in lysosomal storage diseases.

Modulation of the host immune response by a transient intracellular stage of Mycobacterium ulcerans: the contribution of endogenous mycolactone toxin

Mycobacterium ulcerans (Mu), the aetiological agent of Buruli ulcer, is an extracellular pathogen producing the macrolide toxin mycolactone. Using a mouse model of intradermal infection, we found that Mu was initially captured by phagocytes and transported to draining lymph nodes (DLN) within host cells. Similar to Buruli ulcers in humans, the infection site eventually became ulcerated with tissue necrosis and extracellular bacteria, at later stages. In contrast to Mycobacterium bovis BCG (BCG), Mu did not disseminate to the spleen. However, mice infected with Mu or BCG developed comparable primary cellular responses to mycobacterial antigens in DLN and spleen. The role of mycolactone in this sequence of events was examined with a mycolactone‐deficient (mup045) mutant of Mu. Mup045 bacilli were better internalized than wild‐type (wt) bacteria by mouse phagocytes in vitro. Moreover, infection with wt but not mup045 Mu led to inhibition of TNF‐α expression, upregulation of MIP‐2 chemokine, and host cell death within 1 day. Our results suggest that mycolactone expression during the intracellular life of Mu may contribute to immune evasion by inhibiting phagocytosis, provoking apoptosis of antigen presenting cells and altering the establishment of an appropriate inflammatory reaction.

Lipoprotein Access to MHC Class I Presentation During Infection of Murine Macrophages with Live Mycobacteria

Following uptake by macrophages, live mycobacteria initially reside within an immature phagosome that resists acidification and retains access to recycling endosomes. Glycolipids are exported from the mycobacterial phagosome and become available for immune recognition by CD1-restricted T cells. The aim of this study was to explore the possibility that lipoproteins might similarly escape from the phagosome and act as immune targets in cells infected with live mycobacteria. We have focused on a 19-kDa lipoprotein from Mycobacterium tuberculosis that was previously shown to be recognized by CD8+ T cells. The 19-kDa Ag was found to traffic separately from live mycobacteria within infected macrophages by a pathway that was dependent on acylation of the protein. When expressed as a recombinant protein in rapid-growing mycobacteria, the 19-kDa Ag was able to deliver peptides for recognition by MHC class I-restricted T cells by a TAP-independent mechanism. Entry into the class I pathway was rapid, dependent on acylation, and could be blocked by killing the mycobacteria by heating before infection. Although the pattern of 19-kDa trafficking was similar with different mycobacterial species, preliminary experiments suggest that class I presentation is more efficient during infection with rapid-growing mycobacteria than with the slow-growing bacillus Calmette-Guérin vaccine strain.

Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

A quantitative method for measuring phototoxicity of a live cell imaging microscope.

Fluorescence-based imaging regimes require exposure of living samples under study to high intensities of focused incident illumination. An often underestimated, overlooked, or simply ignored fact in the design of any experimental imaging protocol is that exposure of the specimen to these excitation light sources must itself always be considered a potential source of phototoxicity. This can be problematic, not just in terms of cell viability, but much more worrisome in its more subtle manifestation where phototoxicity causes anomalous behaviors that risk to be interpreted as significant, whereas they are mere artifacts. This is especially true in the case of microbial pathogenesis, where host-pathogen interactions can prove especially fragile to light exposure in a manner that can obscure the very processes we are trying to observe. For these reasons, it is important to be able to bring the parameter of phototoxicity into the equation that brings us to choose one fluorescent imaging modality, or setup, over another. Further, we need to be able to assess the risk that phototoxicity may occur during any specific imaging experiment. To achieve this, we describe here a methodological approach that allows meaningful measurement, and therefore relative comparison of phototoxicity, in most any variety of different imaging microscopes. In short, we propose a quantitative approach that uses microorganisms themselves to reveal the range over which any given fluorescent imaging microscope will yield valid results, providing a metrology of phototoxic damage, distinct from photobleaching, where a clear threshold for phototoxicity is identified. Our method is widely applicable and we show that it can be adapted to other paradigms, including mammalian cell models.

Glucocorticoid treatment induces expression of small heat shock proteins in human satellite cell populations: consequences for a desmin-related myopathy involving the R120G alpha B-crystallin mutation.

A missense mutation (R120G) of the molecular chaperone alpha B-crystallin has recently been linked to a familial form of desmin-related myopathy characterized by intrasarcoplasmic aggregates of desmin. It was previously demonstrated that the mutant R120G had a defective chaperone-like function. However, the cellular and physiopathological consequences of R120G mutant expression in human muscle cells are as yet unclear. Thus, we developed a cellular model for the study of this R120G alpha B-crystallin-related desmin-related myopathy. We demonstrate that dexamethasone enhances alpha B-crystallin and HSP27 expression in normal and desmin-related myopathy-derived muscle cells. In the undifferentiated desmin-related myopathy satellite cell population no intracytoplasmic aggregates were observed. However, in differentiated satellite cells derived from a related myopathy patient, we observed an enhanced plasma membrane localization of alpha B-crystallin following glucocorticoid. We also observed that the protective effect against stress of alpha B-crystallin is altered upon glucocorticoid-induced small heat shock protein expression for the desmin-related myopathy-derived cells.