Biomedical subjects

Philippe Gros

Publications and source records attributed to Philippe Gros.

At least 37 records · Page 2Linked to original sources

PubMed2005

The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages.

Bacteriophages are the most abundant life forms in the biosphere. They play important roles in bacterial ecology, evolution, adaptation to new environments, and pathogenesis of human bacterial infections. Here, we report the complete genomic sequences, and predicted proteins of 27 bacteriophages of the Gram-positive bacterium Staphylococcus aureus. Comparative nucleotide and protein sequence analysis indicates that these phages are a remarkable source of untapped genetic diversity, encoding 2,170 predicted protein-encoding ORFs, of which 1,402 cannot be annotated for structure or function, and 522 are proteins with no similarity to other phage or bacterial sequences. Based on their genome size, organization of their gene map and comparative nucleotide and protein sequence analysis, the S. aureus phages can be organized into three groups. Comparison of their gene maps reveals extensive genome mosaicism, hinting to a large reservoir of unidentified S. aureus phage genes. Among the phages in the largest size class (178-214 kbp) that we characterized is phage Twort, the first discovered bacteriophage (responsible for the Twort-D'Herelle effect). These phage genomes offer an exciting opportunity to discern molecular mechanisms of phage evolution and diversity.

Chromosome Mapping↗

PubMed2005

A mutation in the Icsbp1 gene causes susceptibility to infection and a chronic myeloid leukemia-like syndrome in BXH-2 mice.

BXH-2 mice develop a fatal myeloid leukemia by a two-step mutagenic process. First, a BXH-2-specific recessive mutation causes a myeloproliferative syndrome. Second, retroviral insertions alter oncogenes or tumor suppressors, resulting in clonal expansion of leukemic cells. We have identified a recessive locus on chromosome 8 (Myls) that is responsible for myeloproliferation in BXH-2. This Myls interval has been narrowed down to 2 Mb and found to contain several positional candidates, including the interferon consensus sequence-binding protein 1 gene (Icsbp, also known as interferon regulatory factor 8 [IRF8]). We show that BXH-2 mice carry a mutation (915 C to T) resulting in an arginine-to-cysteine substitution at position 294 within the predicted IRF association domain of the protein. Although expression of Icsbp1 mRNA transcripts is normal in BXH-2 splenocytes, these cells are unable to produce interleukin 12 and interferon-gamma in response to activating stimuli, confirming that R294C behaves as a loss-of-function mutation. Myeloproliferation in BXH-2 mice is concomitant to increased susceptibility to Mycobacterium bovis (BCG) despite the presence of resistance alleles at the Nramp1 locus. These results suggest a two-step model for chronic myeloid leukemia in BXH-2, in which inactivation of Icsbp1 predisposes to myeloproliferation and immunodeficiency. This event is required for retroviral replication, and subsequent insertional mutagenesis that causes leukemia in BXH-2 mice.

Amino Acid Substitution↗

PubMed2005

Hiyama cross-coupling of chloro-, fluoro-, and methoxypyridyltrimethylsilanes: room-temperature novel access to functional bi(het)aryl.

The incorporation of chloro, fluoro, or methoxy substituents on the pyridine ring of pyridyltrimethylsilanes allowed us to perform efficient Hiyama cross-coupling with various (het)aryl halides. The reactions proceeded smoothly at room temperature leading to the corresponding functional bis(het)aryl in fair to excellent yields. The presence of pyridine nitrogen alpha to the trimethylsilyl group was requisite to achieve the cross-coupling. [Reaction: see text]

Journal Article↗

PubMed2005

Single gene effects in mouse models of host: pathogen interactions.

Inbred mouse strains have been known for many years to vary in their degree of susceptibility to different types of infectious diseases. The genetic basis of these interstrain differences is sometimes simple but often complex. In a few cases, positional cloning has been used successfully to identify single gene effects. The natural resistance-associated macrophage protein 1 (Nramp1) gene (Slc11a1) codes for a metal transporter active at the phagosomal membrane of macrophages, and Nramp1 mutations cause susceptibility to Mycobacterium, Salmonella, and Leishmania. Furthermore, recent advances in gene transfer technologies in transgenic mice have enabled the functional dissection of gene effects mapping to complex, repeated parts of the genome, such as the Lgn1 locus, causing susceptibility to Legionella pneumophila in macrophages. Finally, complex traits such as the genetically determined susceptibility to malaria can sometimes be broken down into multiple single gene effects. One such example is the case of pyruvate kinase, where a loss-of-function mutation was recently shown by our group to be protective against blood-stage infection with Plasmodium chabaudi. In all three cases reviewed, the characterization of the noted gene effect(s) has shed considerable light on the pathophysiology of the infection, including host response mechanisms.

Animals↗

PubMed2005

The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain.

The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C(4) (LTC(4)) is one of the highest affinity substrates of MRP1. In this study, we have synthesized and characterized a novel photoreactive azido analogue of LTC(4) (AALTC(4)). The specificity of AALTC(4) binding to MRP1 was confirmed using an LTC(4)-specific monoclonal antibody. Moreover, binding with radioiodinated [(125)I]AALTC(4) (or IAALTC(4)) to MRP1 was dramatically competed with unmodified LTC(4) and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC(4) binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC(4) binding to MRP1. Using AALTC(4) together with a panel of epitope-specific and LTC(4)-specific monoclonal antibodies, we identified LTC(4) binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPr1, QCRL1, and MRPm6) showed LTC(4) binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC(4), indicating that MSD0 contains an LTC(4) binding site. Higher resolution mapping of additional LTC(4) binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC(4) and digested with trypsin to isolate specific regions of MRP1 that interact with LTC(4). These results confirmed that sequences in MSD0 interact with IAALTC(4). Other regions that were photoaffinity labeled by IAALTC(4) include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC(4) binding domains in MRP1 and provide the first direct evidence for LTC(4) binding within MSD0.

Amino Acid Sequence↗

PubMed2005

Naip5/Birc1e and susceptibility to Legionella pneumophila.

Genetic analysis in mice is a powerful approach for the identification of genes and proteins that have a key role at the interface of the host-pathogen interaction. The Lgn1 locus has been found to control the intracellular replication of Legionella pneumophila in murine macrophages. Using functional complementation in transgenic mice, the Naip5/Birc1e gene has been identified as responsible for the Lgn1 effect. The classification of Naip5/Birc1e as a member of the NLR protein family suggests that Naip5/Birc1e acts as an intracellular sensor of L. pneumophila. The nature of the signal transduced by Naip5/Birc1e in response to Legionella products is of great interest but is currently unknown. Here, several possible scenarios are presented.

Animals↗

PubMed2005

Erythrocyte variants and the nature of their malaria protective effect.

The malaria threat to global health is exacerbated by widespread drug resistance in the Plasmodium parasite and its insect vector, and the lack of an efficacious vaccine. Infection with Plasmodium parasites can cause a wide spectrum of pathologies, from a transient mild form of anaemia to a severe and rapidly fatal cerebral disease. Epidemiological studies in humans and experiments in animal models have shown that genetic factors play a key role in the onset, progression, type of disease developed and ultimate outcome of malaria. The protective effect of polymorphic variants in erythrocyte-specific structural proteins or metabolic enzymes against the blood-stage of the disease is one of the clearest illustrations of this genetic modulation, and has suggested co-evolution of the Plasmodium parasite with its human host in areas of endemic disease. Here, we present a brief overview of erythrocyte polymorphisms with biological relevance to malaria pathogenesis, and current work on the mechanism(s) by which these mediate their protective effect. The recent addition of erythrocyte pyruvate kinase to this group of protective genes will also be discussed.

Animals↗

PubMed2005

Interleukin-15 enhances innate and adaptive immune responses to blood-stage malaria infection in mice.

Compared to C57BL/6 wild-type mice, interleukin-15(-/-) (IL-15(-/-)) mice showed delayed clearance of Plasmodium chabaudi AS infection, lower type 1 cytokine production, impaired dendritic cell and NK cell functions, and lower titers of malaria-specific antibodies. Thus, IL-15 supports early control and timely resolution of blood-stage malaria through promotion of Th1-dependent innate and adaptive immune responses.

Animals↗

PubMed2005

Primitive neuroectodermal tumor of the stomach.

Ewing sarcoma/primitive neuroectodermal tumor is classically a tumor of the soft tissue or bone in children and young adults, but several cases have been described in patients of all ages. Within the last decade, the clinicopathologic spectrum of Ewing sarcoma/primitive neuroectodermal tumor has been markedly expanded by recognition that the tumor may also have a visceral origin. We describe a case of primitive neuroectodermal tumor arising in the stomach of a 66-year-old woman. The neoplasm was excised using a radical surgical procedure. Microscopically, the tumor was made up of solid nests and sheets of round cells. Immunohistochemically, the tumor cells showed immunoreactivity for CD99, S100, neuron-specific enolase, and vimentin. A multiplex real-time polymerase chain reaction assay detected an EWS-ERG fusion. To our knowledge, this is the first description of a primitive neuroectodermal tumor arising in the stomach.

Aged↗

PubMed2004

Selective lithiation of 4-(1H-1-pyrrolyl)pyridine. access to new electron-releasing ligands.

The first lithiation of 4-(1H-1-pyrrolyl)pyridine has been realized. The use of BuLi-containing lithium aggregates induced the selective pyridine ring functionalization by taking advantage of the electron-donor effect of the pyrrole nucleus. Opportune substituents were introduced alpha to the pyridine nitrogen leading to new electron-enriched pyridylphosphine, bipyridine, and terpyridine ligands.

Journal Article↗

PubMed2004

Independent mutations in mouse Vangl2 that cause neural tube defects in looptail mice impair interaction with members of the Dishevelled family.

Mammalian Vangl1 and Vangl2 are highly conserved membrane proteins that have evolved from a single ancestral protein Strabismus/Van Gogh found in Drosophila. Mutations in the Vangl2 gene cause a neural tube defect (craniorachischisis) characteristic of the looptail (Lp) mouse. Studies in model organisms indicate that Vangl proteins play a key developmental role in establishing planar cell polarity (PCP) and in regulating convergent extension (CE) movements during embryogenesis. The role of Vangl1 in these processes is virtually unknown, and the molecular function of Vangl1 and Vangl2 in PCP and CE is poorly understood. Using a yeast two-hybrid system, glutathione S-transferase pull-down and co-immunoprecipitation assays, we show that both mouse Vangl1 and Vangl2 physically interact with the three members of the cytoplasmic Dishevelled (Dvl) protein family. This interaction is shown to require both the predicted cytoplasmic C-terminal half of Vangl1/2 and a portion of the Dvl protein containing PDZ and DIX domains. In addition, we show that the two known Vangl2 loss-of-function mutations identified in two independent Lp alleles associated with neural tube defects impair binding to Dvl1, Dvl2, and Dvl3. These findings suggest a molecular mechanism for the neural tube defect seen in Lp mice. Our observations indicate that Vangl1 biochemical properties parallel those of Vangl2 and that Vangl1 might, therefore, participate in PCP and CE either in concert with Vangl2 or independently of Vangl2 in discrete cell types.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Molecular and cellular mechanisms underlying iron transport deficiency in microcytic anemia.

A mutation of the iron transporter Nramp2 (DMT1, Slc11a2) causes microcytic anemia in mk mice and in Belgrade rats by impairing iron absorption in the duodenum and in erythroid cells, causing severe iron deficiency. Both mk and Belgrade animals display a glycine-to-arginine substitution at position 185 (G185R) in the fourth predicted transmembrane domain of Nramp2. To study the molecular basis for the loss of function of Nramp2(G185R), we established cell lines stably expressing extracellularly tagged versions of wild-type (WT) or mutated transporters. Like WT Nramp2, the G185R mutant was able to reach the plasmalemma and endosomal compartments, but with reduced efficiency. Instead, a large fraction of Nramp2(G185R) was detected in the endoplasmic reticulum, where it was unstable and was rapidly degraded by a proteasome-dependent mechanism. Moreover, the stability of the mutant protein that reached the plasma membrane was greatly reduced, further diminishing its surface density at steady state. Last, the specific metal transport activity of plasmalemmal Nramp2(G185R) was found to be significantly depressed, compared with its WT counterpart. Thus, a singlepoint mutation results in multiple biosynthetic and functional defects that combine to produce the impaired iron deficiency that results in microcytic anemia.

Anemia↗

PubMed2004

Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding.

The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3.

Amino Acid Sequence↗

PubMed2004

Nramp1 deletion does not confer susceptibility to Rhodococcus equi infection in mice.

Rhodococcus equi is an intracellular bacterium that causes pneumonia in immunocompromised people and foals. The Nramp1 gene influences susceptibility to a variety of intracellular bacteria (including mycobacterial species), but not to Mycobacterium tuberculosis. In this study, we demonstrate that mice functionally deleted of the Nramp1 gene were not more susceptible to infection with virulent R. equi (ATCC 33701) than wild-type mice. Susceptibility of mice to infection with the intracellular bacterium R. equi is more similar to that of M. tuberculosis than to other intracellular bacteria, including other mycobacteria.

Actinomycetales Infections↗

PubMed2004

Antimicrobial drug discovery through bacteriophage genomics.

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

Anti-Bacterial Agents↗

PubMed2004

Van Gogh-like2 (Strabismus) and its role in planar cell polarity and convergent extension in vertebrates.

In the past two years, studies of Stbm genes (also known as Vangl2) and the proteins that they encode in mice, flies, frogs and fish have shown that they have a crucial role in regulating planar cell polarity and convergent extension movements. Combined genetic and biochemical analyses have pointed to signaling pathways where Stbm (Vangl2) proteins might act, and have identified several interacting proteins that form a crucial multi-protein signaling complex at the membrane. These studies show that these proteins have a pivotal role in a signaling cascade(s) that has been highly conserved in evolution. This review will summarize recent findings documenting the involvement of Stbm (Vangl2) and associated proteins in planar cell polarity, non-canonical Wnt signaling and convergent extension movements.

Animals↗

PubMed2004

Dysregulated inflammatory response to Candida albicans in a C5-deficient mouse strain.

Experimental infection of inbred mouse strains with Candida albicans provides a good model system to identify host genetic determinants that regulate onset of, response to, and ultimate outcome of disseminated candidiasis. The A/J mouse strain is exquisitely sensitive to infection with C. albicans, while the C57BL/6J strain is relatively resistant, as measured by survival following intravenous injection of Candida blastospores. This differential susceptibility is caused by an A/J-specific loss-of-function mutation in the C5 component of the complement pathway. C5 plays several critical roles in host response to infection, including target lysis and phagocyte recruitment. Therefore, to determine which of its functions were required for host resistance to candidiasis, a detailed comparative analysis of pathophysiology and host response to acute C. albicans infection was conducted in A/J and C57BL/6J mice. C5-sufficient C57BL/6J mice were found to succumb late in infection due to severe kidney pathology, typified by fungal replication and robust neutrophil-based inflammatory response associated with extensive tissue damage. In contrast, A/J mice were moribund within 24 h postinfection but displayed little if any kidney damage despite an inability to mobilize granulocytes and a high fungal load in the kidney. Rather, C5 deficiency in A/J mice was associated with higher levels of circulating cytokines tumor necrosis factor alpha, interleukin-6, monocyte chemotactic protein 1 (MCP-1), MCP-5, and eotaxin in response to C. albicans. Transfer of the C5-defective allele from A/J onto a C57BL/6J genetic background in recombinant congenic strain BcA17 recapitulated the phenotypic aspects of the susceptibility of A/J mice to C. albicans, confirming the causative role of C5 deficiency in the dysregulated cytokine response.

Animals↗

PubMed2003

Genetic control of myeloproliferation in BXH-2 mice.

While studying the unique Nramp1 (Slc11a1)-independent susceptibility to Mycobacterium bovis (BCG) infection of BXH-2 mice, we noted that these mice develop important splenomegaly and enlargement of lymph nodes. Segregation analyses in several F2 crosses showed that splenomegaly segregates as a single recessive trait caused by a novel mutation in BXH-2, independent of the infection. Histologic and fluorescence-activated cell sorter (FACS) analyses indicated that splenomegaly is associated with a large increase in Mac1+/GR1+ (macrophage antigen-1+/granulocyte differentiation antigen 1+) granulocyte precursors in spleen, lymph nodes, and bone marrow, resembling a myeloproliferative syndrome. This is concomitant to extramedullary erythropoiesis in the spleen, as measured by proportion of Ter119+ erythroid cells. The locus controlling this myeloproliferative syndrome and splenomegaly was designated Myls and maps to an 18 centimorgan (cM) region of chromosome 8, which also contains an integrated copy of an N-ecotropic murine leukemia virus (MuLV) provirus (Emv2). The relationship between Myls, expansion of Mac1+/GR1+ cells, and Emv2 was investigated. Homozygosity at Myls is necessary but not sufficient for B-ecotropic virus replication in splenocytes, the extent of which appears to be under separate genetic control. Our results suggest a model in which Myls-dependent myeloproliferation in BXH-2 acts as a predisposing factor for the subsequent development of virally induced myeloid leukemia characteristic of this strain.

Animals↗