Phosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasite Plasmodium falciparum establishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile of P. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. The Plasmodium genome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted in Plasmodium berghei, and in vitro growth of P. falciparum was sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential in Plasmodium blood stages. For intraparasitic PI3P localization, transgenic P. falciparum that expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function in Plasmodium appear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.Phosphatidylinositol is a crucial phospholipid in eukaryotic cells. It is a structural membrane lipid, and phosphorylation of the hydroxyl groups of its inositol head group by specific lipid kinases leads to the production of seven different phosphoinositide species, which have been found to be enriched in distinct cellular compartments. They play key roles in a multitude of cellular processes, such as membrane traffic, cell motility, cytoskeletal reorganization, DNA synthesis, the cell cycle, adhesion, and signal transduction (9). Approximately 1% of total lipids in mammalian cells are phosphoinositides, mainly phosphatidylinositol 4-monophosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] (45). Derivatives phosphorylated at the 3 position are considerably less abundant in mammalian cells. Phosphatidylinositol 3-monophosphate (PI3P) is a ubiquitous lipid in eukaryotic cells and is present in small amounts in mammalian cells (classically Ͻ15% of PI4P), while PI3P is as abundant as PI4P in the yeast Saccharomyces cerevisiae (2). It has been suggested that one of the functions of these lipids is to establish membrane identity (46); PI4P predominates at the Gol...

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The availability of specific and rapid detection methods is essential for monitoring the health status of farmed species, particularly in viral diseases as in this case early diagnosis is a critical factor in containing disease outbreaks. Three complementary genome-based methods were developed for the detection of Macrobrachium rosenbergii nodavirus (MrNV), i.e. dot-blot hybridization, in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). Detection limits were established for dot-blot hybridization and RT-PCR and are c. 7 fg and 8 pg of viral RNA, respectively. In situ hybridization indicated that infection was confined to the striated muscle tissue. As a result of its sensitivity, RT-PCR can be used for in-depth investigations to examine the extent of the viral infection and establish the onset of infection in hatcheries. The application of RT-PCR on samples collected from prawn farms in China showed the possible use of this method in routine health monitoring.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins interact with CD4 and chemokine receptors on T cells to deliver signals that trigger either activation, anergy, or apoptosis. However, the molecular mechanisms driving these responses remain poorly understood. In this study we demonstrate that apoptosis is induced upon HIV-1 envelope binding to the chemokine receptor CXCR4. Cells expressing a mutant form of CXCR4 with a C-terminal deletion were also sensitive to HIV-1 envelope-mediated apoptosis, indicating that the cytoplasmic tail of CXCR4 is not required to induce the apoptotic pathway. The specificity of this process was analyzed using several inhibitors of gp120-CD4-CXCR4 interaction. Monoclonal antibodies directed against the gp120-binding site on CD4 (ST4) and against CXCR4 (MAB173) prevented the apoptotic signal in a dose-dependent manner. The cell death program was also inhibited by SDF-1alpha, the natural ligand of CXCR4, and by suramin, a G protein inhibitor that binds with a high affinity to the V3 loop of HIV-1 gp120 envelope protein. These results highlight the role played by gp120-binding on CXCR4 to trigger programmed cell death. Next, we investigated the intracellular signal involved in gp120-induced apoptosis. This cell death program was insensitive to pertussis toxin and did not involve activation of the stress- and apoptosis-related MAP kinases p38(MAPK) and SAPK/JNK but was inhibited by a broad spectrum caspase inhibitor (z-VAD.fmk) and a relatively selective inhibitor of caspase 3 (z-DEVD.fmk). Altogether, our results demonstrate that HIV induces a caspase-dependent apoptotic signaling pathway through CXCR4.

Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

To elucidate the structural features of the mussel defensin MGD1 required for antimicrobial activity, we synthesized a series of peptides corresponding to the main known secondary structures of the molecule and evaluated their activity towards Gram-positive and Gram-negative bacteria, and filamentous fungi. We found that the nonapeptide corresponding to residues 25-33 of MGD1 (CGGWHRLRC) exhibited bacteriostatic activity once it was cyclized by a non-naturally occurring disulfide bridge. Longer peptides corresponding to the amino acid sequences of the a-helical part or to the b-strands of MGD1 had no detectable activity. The bacteriostatic activity of the sequence 25-33 was strictly dependent on the bridging of Cys25 and Cys33 and was proportional to the theoretical isoelectric point of the peptide, as deduced from the variation of activity in a set of peptide analogues of the 25-33 sequence with different numbers of cationic charges. By using confocal fluorescence microscopy, we found that the cyclic peptides bound to Gram-positive bacteria without apparent lysis. However, by using a fluorescent dye, we observed that dead bacteria had been permeated by the cyclic peptide 25-33. Sequence comparisons in the family of arthopod defensins indicate that MGD1 belongs to a subfamily of the insect defensins, characterized by the constant occurrence of both positively charged and hydrophobic amino acids in the loop. Modelling studies showed that in the MGD1 structure, positively charged and hydrophobic residues are organized in two layered clusters, which might have a functional significance in the docking of MGD1 to the bacterial membrane.

Mounting evidence suggests that the gut-to-lung axis is critical during respiratory viral infections. We herein hypothesized that disruption of gut homeostasis during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may associate with early disease outcomes. To address this question, we took advantage of the Syrian hamster model. Our data confirmed that this model recapitulates some hallmark features of the human disease in the lungs. We further showed that SARS-CoV-2 infection associated with mild intestinal inflammation, relative alteration in intestinal barrier property and liver inflammation and altered lipid metabolism. These changes occurred concomitantly with an alteration of the gut microbiota composition over the course of infection, notably characterized by a higher relative abundance of deleterious bacterial taxa such as Enterobacteriaceae and Desulfovibrionaceae. Conversely, several members of the Ruminococcaceae and Lachnospiraceae families, including bacteria known to produce the fermentative products short-chain fatty acids (SCFAs), had a reduced relative proportion compared to non-infected controls. Accordingly, infection led to a transient decrease in systemic SCFA amounts. SCFA supplementation during infection had no effect on clinical and inflammatory parameters. Lastly, a strong correlation between some gut microbiota taxa and clinical and inflammation indices of SARS-CoV-2 infection severity was evidenced. Collectively, alteration of the gut microbiota correlates with disease severity in hamsters making this experimental model valuable for the design of interventional, gut microbiota-targeted, approaches for the control of COVID-19. Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019; SCFAs, short-chain fatty acids; dpi, day post-infection; RT-PCR, reverse transcription polymerase chain reaction; IL, interleukin. ACE2, angiotensin converting enzyme 2; TMPRSS2, transmembrane serine protease 2.

Cellular stress activates multiple mitogen-activated protein kinase (MAPK) cascades and immediate-early gene (IEG) transcription. To address how these events are linked, we investigated the endogenous signaling/transcription factor network driving IEG activation by arsenite and anisomycin in the human osteosarcoma cell line HOS/TE-85. Induction of IEG transcription by both stresses corresponded temporally with the phosphorylation of the regulatory factors Elk-1 and cAMP response element-binding protein (CREB), along with activation of the extracellular signal-regulated kinase (ERK), stressactivated protein kinase (SAPK) and p38 MAPK cascades. To assess the role of the different cascades, they were selectively inhibited with PD98059, SP600125 and SB203580, respectively. This implicated all three cascades in Elk-1 phosphorylation after arsenite treatment, whereas ERK and SAPK inhibition diminished this, and IEG mRNA levels, downstream of anisomycin. SB blocked phosphorylation of both serum response factor (SRF) and CREB, and strongly reduced IEG activation by both stresses. Combining PD with SB further reduced arsenite induction of IEG transcription. Thus, all three MAPK cascades mediate anisomycin-and arseniteinduced signaling to IEG promoters in HOS cells through the differential targeting of Elk-1, SRF and CREB.