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ABSTRACT: Invasive infection with Entamoeba histolytica causes intestinal and hepatic amoebiasis. In liver, parasites cross the endothelial barrier before abscess formation in the parenchyma. We focussed on amoebae interactions with human hepatic endothelial cells, the latter potentially playing a dual role in the infection process: as a barrier and as modulators of host defence responses. We characterized early responses of a human liver sinusoidal endothelial cell line to virulent and virulence-attenuated E. histolytica. Within the first minutes human cells start to retract, enter into apoptosis and die. In the presence of virulent amoebae, expression of genes related to cell cycle, cell death and integrin-mediated adhesion signalling was modulated, and actin fibre, focal adhesion kinase and paxillin localizations changed. Effects of inhibitors and amoeba strains not expressing pathogenic factors amoebapore A and cysteine protease A5 indicated that cell death and cytoskeleton disorganization depend upon parasite adhesion and amoebic cysteine proteinase activities. The data establish a relation between cytotoxic effects of E. histolytica and altered human target cell adhesion and suggest that interference with adhesion signalling triggers endothelial cell retraction and death. Understanding the roles of integrin signalling in endothelial cells will provide clues to unravel host-pathogen interactions during amoebic liver infection.
Cellular Microbiology 07/2011; 13(7):1091-106. · 5.46 Impact Factor
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ABSTRACT: Interleukin-7 (IL-7) responses are impaired in CD4(+) T cells from HIV-infected patients, which may play a significant role in the loss of CD4(+) T-cell homeostasis. We set to investigate the nature of IL-7-dependent signaling defects in patients with progressive HIV-1 infection.
IL-7 signaling was compared in CD4(+) T cells from viremic patients with a viral load more than 10,000 copies of HIV RNA/ml (n = 23) and from healthy blood donors (n = 23). Phosphorylation of the transcription factor STAT5 on the regulatory serine S726 and the key tyrosine Y694 was monitored by intracellular flow cytometry. Phospho-STAT5 relocalization to the nucleus was analyzed by quantitative immunofluorescence imaging.
In control CD4(+) T cells, S726 phosphorylation was mostly constitutive and inducible by IL-7 to a limited extent (1.3x, P < 0.05). In contrast, phosphorylation at Y694 was highly inducible by IL-7 (12.6x, P < 0.0001). Progressive HIV infection led to hyperphosphorylation of both S726 and Y694 in naive CD4(+) T cells, with these changes correlating together (R = 0.66, P = 0.01). Quantitative image analysis revealed an impairment in the nuclear relocalization of both forms of phospho-STAT5 in patient cells (P < 0.005 for S726; P < 0.05 for Y694). The nuclear relocalization defect correlated with increased HLA-DR expression (R = 0.75, P < 0.01), suggesting a role for chronic immune activation in perturbed IL-7 signal transduction.
HIV infection perturbs IL-7 signaling by impairing the access of STAT5 to the nuclear compartment. This defect may contribute to the loss of CD4(+) T-cell populations in patients with chronically high immune activation.
AIDS (London, England) 06/2011; 25(15):1843-53. · 4.91 Impact Factor
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ABSTRACT: The WHIM syndrome, which features high susceptibility to human papillomavirus (HPV) infection, is a rare immunodeficiency associated with autosomal dominant heterozygous mutations of the CXCR4 chemokine receptor. CXCL12 and its receptors, CXCR4 and CXCR7, are linked to tumorigenesis, and we reported that abnormal expression of CXCL12 in epidermal keratinocytes correlates with HPV infection. However, the HPV-related pathologies observed in WHIM patients remain mechanistically unexplained. We show that keratinocytes immortalized by oncogenic HPV16 or HPV18 upregulate CXCL12 and its receptors in a manner dependent upon expression of the viral proteins E6 and E7. Autocrine signaling activated by CXCL12-engagement of its receptors controls motility and survival of the infected cells. Strikingly, expression of a WHIM syndrome-related gain-of-function CXCR4 mutant confers transforming capacity to HPV18-immortalized keratinocytes. These results establish a pivotal role for CXCL12 signaling in HPV-mediated transformation and provide a mechanistic basis for understanding HPV pathogenesis in WHIM syndrome.
Cell host & microbe 12/2010; 8(6):523-33. · 13.02 Impact Factor
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ABSTRACT: The specialised DNA polymerase μ (pol μ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol μ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol μ⁻(/)⁻ mouse embryonic fibroblasts (MEFs) we show that lack of pol μ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol μ. Here we show pronounced senescence in pol μ⁻(/)⁻ MEFs, associated with high levels of the tumor-suppressor p16(INK4A) and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol μ⁻(/)⁻ MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol μ. Pol μ⁻(/)⁻ MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.
DNA repair 10/2010; 9(11):1187-99. · 4.20 Impact Factor
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ABSTRACT: Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin-3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high-content and high-throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.
Cellular Microbiology 04/2010; 12(4):545-56. · 5.46 Impact Factor
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ABSTRACT: Septins are filament-forming GTPases implicated in several cellular functions, including cytokinesis. We previously showed that SEPT2, SEPT9, and SEPT11 colocalize with several bacteria entering into mammalian non-phagocytic cells, and SEPT2 was identified as essential for this process. Here, we investigated the function of SEPT11, an interacting partner of SEPT9 whose function is still poorly understood. In uninfected HeLa cells, SEPT11 depletion by siRNA increased cell size but surprisingly did not affect actin filament formation or the colocalization of SEPT9 with actin filaments. SEPT11 depletion increased Listeria invasion, and incubating SEPT11-depleted cells with beads coated with the Listeria surface protein InlB also led to increased entry as compared with control cells. Strikingly, as shown by fluorescence resonance energy transfer, the InlB-mediated stimulation of Met signaling remained intact in SEPT11-depleted cells. Taken together, our results show that SEPT11 is not required for the bacterial entry process and rather restricts its efficacy. Because SEPT2 is essential for the InlB-mediated entry of Listeria, but SEPT11 is not, our findings distinguish the roles of different mammalian septins.
Journal of Biological Chemistry 03/2009; 284(17):11613-21. · 4.77 Impact Factor
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ABSTRACT: Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3(-/-) mice -- in which brain tissue was less severely infected -- had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV-induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.
PLoS Pathogens 03/2009; 5(2):e1000315. · 9.13 Impact Factor
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ABSTRACT: 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.
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.
Together these findings highlight novel roles for SEPT2, and distinguish the roles of septin and actin in bacterial entry.
PLoS ONE 02/2009; 4(1):e4196. · 4.09 Impact Factor
