Genotypes and serotype distribution of macrolide resistant invasive and non- invasive Streptococcus pneumoniae isolates from Lebanon

<p>Abstract</p> <p>Background</p> <p>This study determined macrolide resistance genotypes in clinical isolates of <it>Streptococcus pneumoniae </it>from multiple medical centers in Lebanon and assessed the serotype distribution in relation to these mechanism(s) of resistance and the source of isolate recovery.</p> <p>Methods</p> <p>Forty four macrolide resistant and 21 macrolide susceptible <it>S. pneumoniae </it>clinical isolates were tested for antimicrobial susceptibility according to CLSI guidelines (2008) and underwent molecular characterization. Serotyping of these isolates was performed by Multiplex PCR-based serotype deduction using CDC protocols. PCR amplification of macrolide resistant <it>erm </it>(encoding methylase) and <it>mef </it>(encoding macrolide efflux pump protein) genes was carried out.</p> <p>Results</p> <p>Among 44 isolates resistant to erythromycin, 35 were resistant to penicillin and 18 to ceftriaxone. Examination of 44 macrolide resistant isolates by PCR showed that 16 isolates harbored the <it>erm</it>(B) gene, 8 isolates harbored the <it>mef </it>gene, and 14 isolates harbored both the <it>erm</it>(B) and <it>mef </it>genes. There was no amplification by PCR of the <it>erm</it>(B) or <it>mef </it>genes in 6 isolates. Seven different capsular serotypes 2, 9V/9A,12F, 14,19A, 19F, and 23, were detected by multiplex PCR serotype deduction in 35 of 44 macrolide resistant isolates, with 19F being the most prevalent serotype. With the exception of serotype 2, all serotypes were invasive. Isolates belonging to the invasive serotypes 14 and 19F harbored both <it>erm</it>(B) and <it>mef </it>genes. Nine of the 44 macrolide resistant isolates were non-serotypable by our protocols.</p> <p>Conclusion</p> <p>Macrolide resistance in <it>S. pneumoniae </it>in Lebanon is mainly through target site modification but is also mediated through efflux pumps, with serotype 19F having dual resistance and being the most prevalent and invasive.</p

Interaction du domaine nucleocapside de la polyprotéine Gag du VIH-1 avec la protéine cellulaire Unr : implication sur la traduction IRES-dépendante du virus

The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC), as a mature protein (NCp7) or as a domain of the polyprotein Gag, plays several important roles in both the early and late phase of the infection. NC is a nucleic acid chaperone protein with two zinc fingers. We searched for new cellular protein partners of NCp7 and identified the RNA binding protein Unr, Upstream of N-ras, whose interaction with both Gag and NCp7 was confirmed. Unr interaction with Gag is RNA dependent and mediated by its NC domain. Unr is an ITAF (IRES trans-acting factor) regulating the translation driven by several IRESs. The HIV-1 genomic mRNA harbors two IRESs elements: one of them found within the HIV-1 5’-Untranslated region drives HIV-1 mRNA translation when the cap-dependent translation is diminished due to the infection-induced cell cycle arrest. Using a dual luciferase assay, Unr was shown to act as an ITAF, increasing the HIV-1 IRES dependent translation. Point mutations of the HIV-1 IRES in a consensus Unr binding motif were found to alter both the IRES activity and its activation by Unr suggesting a strong dependency of the IRES on Unr. Unr stimulation effect is furthermore counteracted by NCp7, but not by Gag overexpression, which increases the IRES activity in an additive manner to Unr suggesting a differential Unr effect on the early and late phases of the infection. Finally, knockdown of Unr in HeLa cells leads to a decline in infection by a non-replicative lentivector proving its functional implication in the early phase.La protéine de nucléocapside (NC) du virus de l’immunodéficience humaine (VIH-1) joue de nombreux rôles dans les phases précoce et tardive de l’infection. La NC est une protéine à deux doigts de zinc, chaperonne des acides nucléiques. Nous avons cherché de nouveaux partenaires cellulaires de la NCp7 et identifié une protéine de liaison aux ARNs, Upstream of N-ras (Unr), dont l’interaction avec Gag et NCp7 a été confirmée. L’interaction entre Gag et Unr est dépendante de l’ARN et médiée par le domaine NC. Unr est une ITAF (IRES transacting factor) régulant la traduction médiée par plusieurs IRESs cellulaires et viraux. L’ARN génomique du VIH-1 possède deux IRESs dont un localisé dans la région non traduite en 5’ qui permet aux ARNm viraux de conserver un fort niveau de traduction lorsque la traduction coiffe-dépendante de la cellule est affaiblie par l’arrêt du cycle viral induit par l’infection. En utilisant un système de dual luciférase, nous avons montré qu’Unr est une ITAF dont la surexpression stimule l’IRES VIH-1. Des mutations ponctuelles de cet IRES, dans un motif consensus de liaison à Unr, altèrent à la fois l’activité de l’IRES et sa réponse à Unr suggérant que l’activité IRES dépend fortement de Unr. L’effet d’Unr sur l’IRES est inhibé par la surexpression de NCp7 mais pas par celle de Gag dont l’effet stimulateur sur l’IRES est additif de celui d’Unr suggérant un rôle d’Unr différent dans les phases précoce et tardive de l’infection. Pour finir, le knockdown de l’expression d’Unr entraîne une diminution significative de l’infection par un pseudovirus non réplicatif soulignant l’implication fonctionnelle d’Unr dans la phase précoce

Interaction of the nucleocapsid domain of the Human lmmunodeficiency Virus type-1 with the cellular protein Unr : implication in viral IRES dependent translation

La protéine de nucléocapside (NC) du virus de l’immunodéficience humaine (VIH-1) joue de nombreux rôles dans les phases précoce et tardive de l’infection. La NC est une protéine à deux doigts de zinc, chaperonne des acides nucléiques. Nous avons cherché de nouveaux partenaires cellulaires de la NCp7 et identifié une protéine de liaison aux ARNs, Upstream of N-ras (Unr), dont l’interaction avec Gag et NCp7 a été confirmée. L’interaction entre Gag et Unr est dépendante de l’ARN et médiée par le domaine NC. Unr est une ITAF (IRES transacting factor) régulant la traduction médiée par plusieurs IRESs cellulaires et viraux. L’ARN génomique du VIH-1 possède deux IRESs dont un localisé dans la région non traduite en 5’ qui permet aux ARNm viraux de conserver un fort niveau de traduction lorsque la traduction coiffe-dépendante de la cellule est affaiblie par l’arrêt du cycle viral induit par l’infection. En utilisant un système de dual luciférase, nous avons montré qu’Unr est une ITAF dont la surexpression stimule l’IRES VIH-1. Des mutations ponctuelles de cet IRES, dans un motif consensus de liaison à Unr, altèrent à la fois l’activité de l’IRES et sa réponse à Unr suggérant que l’activité IRES dépend fortement de Unr. L’effet d’Unr sur l’IRES est inhibé par la surexpression de NCp7 mais pas par celle de Gag dont l’effet stimulateur sur l’IRES est additif de celui d’Unr suggérant un rôle d’Unr différent dans les phases précoce et tardive de l’infection. Pour finir, le knockdown de l’expression d’Unr entraîne une diminution significative de l’infection par un pseudovirus non réplicatif soulignant l’implication fonctionnelle d’Unr dans la phase précoce.The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC), as a mature protein (NCp7) or as a domain of the polyprotein Gag, plays several important roles in both the early and late phase of the infection. NC is a nucleic acid chaperone protein with two zinc fingers. We searched for new cellular protein partners of NCp7 and identified the RNA binding protein Unr, Upstream of N-ras, whose interaction with both Gag and NCp7 was confirmed. Unr interaction with Gag is RNA dependent and mediated by its NC domain. Unr is an ITAF (IRES trans-acting factor) regulating the translation driven by several IRESs. The HIV-1 genomic mRNA harbors two IRESs elements: one of them found within the HIV-1 5’-Untranslated region drives HIV-1 mRNA translation when the cap-dependent translation is diminished due to the infection-induced cell cycle arrest. Using a dual luciferase assay, Unr was shown to act as an ITAF, increasing the HIV-1 IRES dependent translation. Point mutations of the HIV-1 IRES in a consensus Unr binding motif were found to alter both the IRES activity and its activation by Unr suggesting a strong dependency of the IRES on Unr. Unr stimulation effect is furthermore counteracted by NCp7, but not by Gag overexpression, which increases the IRES activity in an additive manner to Unr suggesting a differential Unr effect on the early and late phases of the infection. Finally, knockdown of Unr in HeLa cells leads to a decline in infection by a non-replicative lentivector proving its functional implication in the early phase

Interaction of the nucleocapsid domain of the Human lmmunodeficiency Virus type-1 with the cellular protein Unr : implication in viral IRES dependent translation

La protéine de nucléocapside (NC) du virus de l’immunodéficience humaine (VIH-1) joue de nombreux rôles dans les phases précoce et tardive de l’infection. La NC est une protéine à deux doigts de zinc, chaperonne des acides nucléiques. Nous avons cherché de nouveaux partenaires cellulaires de la NCp7 et identifié une protéine de liaison aux ARNs, Upstream of N-ras (Unr), dont l’interaction avec Gag et NCp7 a été confirmée. L’interaction entre Gag et Unr est dépendante de l’ARN et médiée par le domaine NC. Unr est une ITAF (IRES transacting factor) régulant la traduction médiée par plusieurs IRESs cellulaires et viraux. L’ARN génomique du VIH-1 possède deux IRESs dont un localisé dans la région non traduite en 5’ qui permet aux ARNm viraux de conserver un fort niveau de traduction lorsque la traduction coiffe-dépendante de la cellule est affaiblie par l’arrêt du cycle viral induit par l’infection. En utilisant un système de dual luciférase, nous avons montré qu’Unr est une ITAF dont la surexpression stimule l’IRES VIH-1. Des mutations ponctuelles de cet IRES, dans un motif consensus de liaison à Unr, altèrent à la fois l’activité de l’IRES et sa réponse à Unr suggérant que l’activité IRES dépend fortement de Unr. L’effet d’Unr sur l’IRES est inhibé par la surexpression de NCp7 mais pas par celle de Gag dont l’effet stimulateur sur l’IRES est additif de celui d’Unr suggérant un rôle d’Unr différent dans les phases précoce et tardive de l’infection. Pour finir, le knockdown de l’expression d’Unr entraîne une diminution significative de l’infection par un pseudovirus non réplicatif soulignant l’implication fonctionnelle d’Unr dans la phase précoce.The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC), as a mature protein (NCp7) or as a domain of the polyprotein Gag, plays several important roles in both the early and late phase of the infection. NC is a nucleic acid chaperone protein with two zinc fingers. We searched for new cellular protein partners of NCp7 and identified the RNA binding protein Unr, Upstream of N-ras, whose interaction with both Gag and NCp7 was confirmed. Unr interaction with Gag is RNA dependent and mediated by its NC domain. Unr is an ITAF (IRES trans-acting factor) regulating the translation driven by several IRESs. The HIV-1 genomic mRNA harbors two IRESs elements: one of them found within the HIV-1 5’-Untranslated region drives HIV-1 mRNA translation when the cap-dependent translation is diminished due to the infection-induced cell cycle arrest. Using a dual luciferase assay, Unr was shown to act as an ITAF, increasing the HIV-1 IRES dependent translation. Point mutations of the HIV-1 IRES in a consensus Unr binding motif were found to alter both the IRES activity and its activation by Unr suggesting a strong dependency of the IRES on Unr. Unr stimulation effect is furthermore counteracted by NCp7, but not by Gag overexpression, which increases the IRES activity in an additive manner to Unr suggesting a differential Unr effect on the early and late phases of the infection. Finally, knockdown of Unr in HeLa cells leads to a decline in infection by a non-replicative lentivector proving its functional implication in the early phase

Upstream of N-Ras (Unr/CSDE1) Interacts with NCp7 and Gag, Modulating HIV-1 IRES-Mediated Translation Initiation

10.3390/v14081798VIRUSES-BASEL14

男子體操競賽簡介

<div><p>The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection.</p></div

FRAP-based estimation of diffusion coefficient values (A) and mobile fraction (B) of eGFP and NCp7-eGFP in the cytoplasm and in the nucleus.

<p>FRAP-based estimation of diffusion coefficient values (A) and mobile fraction (B) of eGFP and NCp7-eGFP in the cytoplasm and in the nucleus.</p

Intracellular distribution of NCp7-eGFP.

<p>(A) Amino acid sequence of NCp7. Confocal images of HeLa cells expressing transiently eGFP (B) or NCp7-eGFP (C, D). Comparison with the localization of DNA labeled by 1.6 μM Hoechst 33342, (C) and RNA labeled by 1 μM Pyronin Y. The cyan color of the merge panel in (C) indicates colocalization of NCp7 with DNA in the nucleus. The nearly uniform yellow color of the merge panel in (D) indicates a strong colocalization of NCp7 with RNA all over the cell.</p

Diffusion coefficients (D) and anomalous coefficients (α) inferred from FCS and RICS measurements of eGFP and NCp7-eGFP expressing cells.

<p>The D and α values are given as means +/- SD for 800 correlation curves in 16 cells (FCS) and 40 measurements in 10 cells (RICS).</p><p>Diffusion coefficients (D) and anomalous coefficients (α) inferred from FCS and RICS measurements of eGFP and NCp7-eGFP expressing cells.</p