Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium abscessus.

Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors.

Palmitoylated Cysteines in Chikungunya Virus nsP1 Are Critical for Targeting to Cholesterol-Rich Plasma Membrane Microdomains with Functional Consequences for Viral Genome Replication.

In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication.IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.

Insight into the mechanism of action of EP-39, a bevirimat derivative that inhibits HIV-1 maturation.

Maturation of human immunodeficiency virus type 1 (HIV-1) particles is a key step for viral infectivity. This process can be blocked using maturation inhibitors (MIs) that affect the cleavage of the capsid-spacer peptide 1 (CA-SP1) junction. Here, we investigated the mechanisms underlying the activity of EP-39, a bevirimat (BVM) derivative with better hydrosolubility. To this aim, we selected in vitro EP-39- and BVM-resistant mutants. We found that EP-39-resistant viruses have four mutations within the CA domain (CA-A194T, CA-T200N, CA-V230I, and CA-V230A) and one in the first residue of SP1 (SP1-A1V). We also identified six mutations that confer BVM resistance (CA-A194T, CA-L231F, CA-L231M, SP1-A1V, SP1-S5N and SP1-V7A). To characterize the EP-39 and BVM-resistant mutants, we studied EP-39 effects on mutant virus replication and performed a biochemical analysis with both MIs. We observed common and distinct characteristics, suggesting that, although EP-39 and BVM share the same chemical skeleton, they could interact in a different way with the Gag polyprotein precursor (Pr55Gag). Using an in silico approach, we observed that EP-39 and BVM present different predicted positions on the hexameric crystal structure of the CACTD-SP1 Gag fragment. To clearly understand the relationship between assembly and maturation, we investigated the impact of all identified mutations on virus assembly by expressing Pr55Gag mutants. Finally, using NMR, we have shown that the interaction of EP-39 with a peptide carrying the SP1-A1V mutation (CA-SP1(A1V)-NC) is almost suppressed in comparison with the wild type peptide. These results suggest that EP-39 and BVM could interact differently with the Pr55Gag lattice and that the mutation of the first SP1 residue induces a loss of interaction between Pr55Gag and EP-39.

CFTR Protects against Mycobacterium abscessus Infection by Fine-Tuning Host Oxidative Defenses.

Infection by rapidly growing Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF), a genetic disease caused by a defective CF transmembrane conductance regulator (CFTR). However, the potential link between a dysfunctional CFTR and vulnerability to M. abscessus infection remains unknown. Herein, we exploit a CFTR-depleted zebrafish model, recapitulating CF immuno-pathogenesis, to study the contribution of CFTR in innate immunity against M. abscessus infection. Loss of CFTR increases susceptibility to infection through impaired NADPH oxidase-dependent restriction of intracellular growth and reduced neutrophil chemotaxis, which together compromise granuloma formation and integrity. As a consequence, extracellular multiplication of M. abscessus expands rapidly, inducing abscess formation and causing lethal infections. Because these phenotypes are not observed with other mycobacteria, our findings highlight the crucial and specific role of CFTR in the immune control of M. abscessus by mounting effective oxidative responses.

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development.

The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome- and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.

Biology of Zika Virus Infection in Human Skin Cells.

UNLABELLED: Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. IMPORTANCE: Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor and for cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide the first general insights into the interaction between ZIKV and its mammalian host.

High levels of CD34+CD38low/-CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study.

BACKGROUND: Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34(+)CD38(low/-) compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34(+)CD38(low/-)CD123(+) phenotype. DESIGN AND METHODS: The percentage of CD34(+)CD38(low/-)CD123(+) cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses. RESULTS: A proportion of CD34(+)CD38(low/-)CD123(+) cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34(+)CD38(low/-)CD123(+) higher than 15% retained significance with an odds ratio of 0.33 (0.1-0.97; P=0.044). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells retained prognostic significance for both parameters after multivariate analysis. CONCLUSIONS: The percentage of CD34(+)CD38(low/-)CD123(+) leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.

Methylation of specific CpG sites in the P2 promoter of parathyroid hormone-related protein determines the invasive potential of breast cancer cell lines.

Parathyroid hormone-related protein (PTHrP) is upregulated in primary breast cancers and a major candidate for osteoclastic bone resorption present at sites of breast cancer to bone metastases. Using a human model of mammary epithelial cell lines differing in tumorigenicity and PTHrP expression, we investigated the role of epigenetic modifications for PTHrP expression. Quantitative analysis of the DNA methylation patterns at a total of 104 CpGs in the promoter region of PTHrP by pyrosequencing showed the absence of methylation in all analyzed cell lines in the large CpG island upstream of exon 1C. In the second intron of promoter 2 (P2) a region was identified containing 4 CpG nucleotides for which differential methylation correlated with the PTHrP expression level. The functional importance of this control mechanism was confirmed by the ability of the demethylating agent 5'-azacytidine to induce PTHrP mRNA and iPTHrP protein expression in previously non-expressing cell lines and increase their production by metastatic NS2T2A1 cells. In particular, transcription from P2 was activated non-tumoral S1T3 cells upon treatment with 5'-azacytidine. Our findings support the hypothesis that the methylation status of specific CpG dinucleotides is the dominant mechanism involved in silencing of PTHrP expression rather than the overall methylation of the CpG island. Methylation of the PTHrP P2 is a potential marker of breast cancer progression and might be used to evaluate the metastatic potential of breast tumors.

Dual inhibition of PI3K and mTORC1/2 signaling by NVP-BEZ235 as a new therapeutic strategy for acute myeloid leukemia.

PURPOSE: The growth and survival of acute myeloid leukemia (AML) cells are enhanced by the deregulation of signaling pathways such as phosphoinositide 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR). Major efforts have thus been made to develop molecules targeting these activated pathways. The mTOR serine/threonine kinase belongs to two separate complexes: mTORC1 and mTORC2. The mTORC1 pathway is rapamycin sensitive and controls protein translation through the phosphorylation of 4E-BP1 in most models. In AML, however, the translation process is deregulated and rapamycin resistant. Furthermore, the activity of PI3K/Akt and mTOR is closely related, as mTORC2 activates the oncogenic kinase Akt. We therefore tested, in this study, the antileukemic activity of the dual PI3K/mTOR ATP-competitive inhibitor NVP-BEZ235 compound (Novartis). EXPERIMENTAL DESIGN: The activity of NVP-BEZ235 was tested in primary AML samples (n = 21) and human leukemic cell lines. The different signaling pathways were analyzed by Western blotting. The cap-dependent mRNA translation was studied by 7-methyl-GTP pull-down experiments, polysomal analysis, and [(3)H]leucine incorporation assays. The antileukemic activity of NVP-BEZ235 was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation, and survival. RESULTS: The NVP-BEZ235 compound was found to inhibit PI3K and mTORC1 signaling and also mTORC2 activity. Furthermore, NVP-BEZ235 fully inhibits the rapamycin-resistant phosphorylation of 4E-BP1, resulting in a marked inhibition of protein translation in AML cells. Hence, NVP-BEZ235 reduces the proliferation rate and induces an important apoptotic response in AML cells without affecting normal CD34(+) survival. CONCLUSIONS: Our results clearly show the antileukemic efficiency of the NVP-BEZ235 compound, which therefore represents a promising option for future AML therapies.

PTHrP, calcitonin and calcitriol in a case of severe, protracted and refractory hypercalcemia due to a pancreatic neuroendocrine tumor.

A patient with a primary neuroendocrine tumor of the pancreas, presented with severe hypercalcemia. This hypercalcemia of malignancy (HCM) failed to respond to intensive bisphosphonate treatment and needed continuous enhanced diuresis. Only after successful antitumor therapy did the hypercalcemia subside. Hypercalcemia was associated with increased concentrations of plasma PTHrP, calcitonin and 1,25-(OH)(2)D(3). Bone mineral density was markedly increased. We demonstrated the presence of both PTHrP and calcitonin in the tumor at the mRNA and protein level, using RT-PCR, immunohistochemistry and Western blotting. The high levels of plasma PTHrP and the demonstrated predominant renal mechanism in this case of HCM are suspected to be the cause for its refractoriness to bone resorption inhibitors. Our findings furthermore suggest that the tumoral production of calcitonin and PTHrP might have contributed to the increased bone mineral storage of calcium and thus probably attenuated the development of frank hypercalcemia.