Characterization of the inhibitor-resistant SHV β-lactamase SHV-107 in a clinical Klebsiella pneumoniae strain co-producing GES-7 enzyme

The clinical Klebsiella pneumoniae INSRA6884 strain exhibited nonsusceptibility to all penicillins tested (MICs of 64 to>2,048 g/ml). The MICs of penicillins were weakly reduced by clavulanate (from 2,048 to 512 g/ml), and tazobactam restored piperacillin susceptibility. Molecular characterization identified the genes blaGES-7 and a new -lactamase gene, blaSHV-107, which encoded an enzyme that differed from SHV-1 by the amino acid substitutions Leu35Gln and Thr235Ala. The SHV-107-producing Escherichia coli strain exhibited only a -lactam resistance phenotype with respect to amoxicillin, ticarcillin, and amoxicillinclavulanate combination. The kinetic parameters of the purified SHV-107 enzyme revealed a high affinity for penicillins. However, catalytic efficiency for these antibiotics was lower for SHV-107 than for SHV-1. No hydrolysis was detected against oxyimino- -lactams. The 50% inhibitory concentration (IC50) for clavulanic acid was 9-fold higher for SHV-107 than for SHV-1, but the inhibitory effects of tazobactam were unchanged. Molecular dynamics simulation suggested that the Thr235Ala substitution affects the accommodation of clavulanate in the binding site and therefore its inhibitory activity.This work was supported financially by the project POCTI/ESP/43037 from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal, awarded to M. Caniça, and by a grant from INRA and Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (Paris, France), awarded to R. Bonnet. V. Manageiro was supported by grant SFRH/BD/32578/2006 from Fundação para a Ciência e a Tecnologia, Lisbon,Portugal

Escherichia coli colibactin producers and tumor growth, from mechanism to prevention.

La colibactine est une toxine largement distribuée chez Escherichia coli. Sa synthèse est assurée par des enzymes codés par un îlot génomique appelé pks. Elle provoque des cassures double brin de l'ADN, des mutations, d'important remaniements chromosomiques et favorise l'émergence de tumeurs intestinales en modèle murin. Par ailleurs, les E. coli producteurs colonisent fréquemment les tumeurs de patients atteints de cancer colorectal. Nos travaux montrent que les bactéries productrices de colibactine induisent la sénescence cellulaire et stimulent de façon indirect la prolifération cellulaire in vitro et la croissance tumorale in vivo. L'action pro-proliférative des cellules rendues sénescentes par les E. coli producteurs de colibactine est liée à la production du facteur de croissance HGF. L'étude de la signalisation cellulaire responsable montre l'implication du facteur de transcription c-Myc, l'activation de la transcription d'un microARN qui en ciblent la peptidase SENP1, et une modification de la SUMOylation des protéines de l'hôte, notamment p53, un effecteur connu de la sénescence cellulaire. Cette voie de signalisation et les transcripts codant HGF ont été analysés dans des tumeurs de patients atteints de cancer colorectal colonisés ou non par des E. coli producteurs de colibactine. Les résultats obtenus soutiennent les résultats obtenus in vitro et dans le modèle murin. L'ensemble suggère que les bactéries productrices de colibactine favorisent l'émergence d'un micro-environnement tumoral sénescent susceptible de favoriser la croissance tumorale via la sécrétion de HGF. En parallèle, nous avons caractérisé sur le plan structural et fonctionnel la protéine ClbP de l'îlot pks. Les résultats obtenus montrent que ClbP est une peptidase à serine active dont le site actif est extracytoplasmique et indispensable à l'activité biologique de l'îlot pks. Des inhibiteurs « drug-like » de ClbP ont été identifiés à l'aide d'approches structurales, biochimiques, cellulaires et microbiologiques. Ces molécules se lient au site actif de ClbP avec une affinité nanomolaire et bloquent les activités génotoxiques et pro-tumorales des E. coli producteurs de colibactine. ClbP constitue donc une cible thérapeutique potentielle permettant de bloquer les effets délétères des E. coli producteurs de colibactine.The colibactin toxin is widely distributed in Escherichia coli. Its synthesis is performed by enzymes encoded by the genomic island pks. It causes DNA double-strand breaks, mutations, chromosomal rearrangements in host cells and contributes to tumorigenesis in a mouse model. In addition, colibactin-producing E. coli are frequently isolated from tumors of patients with colorectal cancer. Our work shows that colibactin-producing bacteria induce cellular senescence and, consequently, can indirectly stimulate cell proliferation in vitro and tumor growth in vivo. The pro-proliferative effect mediated by these senescent cells is due to the secretion of growth factors, in particular HGF. The cell signaling responsible for cellular senescence shows the involvement of the transcription factor c-Myc, the transcription of a microRNA targeting the peptidase SENP1, and a modification of protein SUMOylation, including p53, a well-known effector of cellular senescence. This signaling pathway and HGF-encoding transcripts were analyzed in tumors of patients with colorectal cancer colonized or not by colibactin-producing E. coli. The results support the findings obtained in vitro and in the mouse model. Taken together, the results suggest that, in tumors, colibactin-producing bacteria promote the emergence of a senescent microenvironment, which can stimulate tumor growth via the secretion of HGF. In parallel, we determined the structure and function of the pks-encoded protein ClbP. The results show that ClbP is an active serine peptidase, whose active site is extracytoplasmic and essential to the biological activity of pks island. "Drug-like" inhibitors of ClbP were identified using structural, biochemical, cellular and microbiological approaches. These molecules bind to the active site of ClbP with nanomolar affinity and block the genotoxic and pro-tumoral activities of colibactin-producing E. coli. ClbP is therefore a potential therapeutic target to block the deleterious effects of bacteria-producing colibactin

Escherichia Coli producteurs de colibactine et croissance tumorale, du mécanisme à la prévention.

The colibactin toxin is widely distributed in Escherichia coli. Its synthesis is performed by enzymes encoded by the genomic island pks. It causes DNA double-strand breaks, mutations, chromosomal rearrangements in host cells and contributes to tumorigenesis in a mouse model. In addition, colibactin-producing E. coli are frequently isolated from tumors of patients with colorectal cancer. Our work shows that colibactin-producing bacteria induce cellular senescence and, consequently, can indirectly stimulate cell proliferation in vitro and tumor growth in vivo. The pro-proliferative effect mediated by these senescent cells is due to the secretion of growth factors, in particular HGF. The cell signaling responsible for cellular senescence shows the involvement of the transcription factor c-Myc, the transcription of a microRNA targeting the peptidase SENP1, and a modification of protein SUMOylation, including p53, a well-known effector of cellular senescence. This signaling pathway and HGF-encoding transcripts were analyzed in tumors of patients with colorectal cancer colonized or not by colibactin-producing E. coli. The results support the findings obtained in vitro and in the mouse model. Taken together, the results suggest that, in tumors, colibactin-producing bacteria promote the emergence of a senescent microenvironment, which can stimulate tumor growth via the secretion of HGF. In parallel, we determined the structure and function of the pks-encoded protein ClbP. The results show that ClbP is an active serine peptidase, whose active site is extracytoplasmic and essential to the biological activity of pks island. "Drug-like" inhibitors of ClbP were identified using structural, biochemical, cellular and microbiological approaches. These molecules bind to the active site of ClbP with nanomolar affinity and block the genotoxic and pro-tumoral activities of colibactin-producing E. coli. ClbP is therefore a potential therapeutic target to block the deleterious effects of bacteria-producing colibactin.La colibactine est une toxine largement distribuée chez Escherichia coli. Sa synthèse est assurée par des enzymes codés par un îlot génomique appelé pks. Elle provoque des cassures double brin de l'ADN, des mutations, d'important remaniements chromosomiques et favorise l'émergence de tumeurs intestinales en modèle murin. Par ailleurs, les E. coli producteurs colonisent fréquemment les tumeurs de patients atteints de cancer colorectal. Nos travaux montrent que les bactéries productrices de colibactine induisent la sénescence cellulaire et stimulent de façon indirect la prolifération cellulaire in vitro et la croissance tumorale in vivo. L'action pro-proliférative des cellules rendues sénescentes par les E. coli producteurs de colibactine est liée à la production du facteur de croissance HGF. L'étude de la signalisation cellulaire responsable montre l'implication du facteur de transcription c-Myc, l'activation de la transcription d'un microARN qui en ciblent la peptidase SENP1, et une modification de la SUMOylation des protéines de l'hôte, notamment p53, un effecteur connu de la sénescence cellulaire. Cette voie de signalisation et les transcripts codant HGF ont été analysés dans des tumeurs de patients atteints de cancer colorectal colonisés ou non par des E. coli producteurs de colibactine. Les résultats obtenus soutiennent les résultats obtenus in vitro et dans le modèle murin. L'ensemble suggère que les bactéries productrices de colibactine favorisent l'émergence d'un micro-environnement tumoral sénescent susceptible de favoriser la croissance tumorale via la sécrétion de HGF. En parallèle, nous avons caractérisé sur le plan structural et fonctionnel la protéine ClbP de l'îlot pks. Les résultats obtenus montrent que ClbP est une peptidase à serine active dont le site actif est extracytoplasmique et indispensable à l'activité biologique de l'îlot pks. Des inhibiteurs « drug-like » de ClbP ont été identifiés à l'aide d'approches structurales, biochimiques, cellulaires et microbiologiques. Ces molécules se lient au site actif de ClbP avec une affinité nanomolaire et bloquent les activités génotoxiques et pro-tumorales des E. coli producteurs de colibactine. ClbP constitue donc une cible thérapeutique potentielle permettant de bloquer les effets délétères des E. coli producteurs de colibactine

Colorectal cancer associated-E. coli induce the emergence of chemotherapy-resistant stem cell-like phenotype of host cells

International audienc

Differential Proteomics Reveals miR-155 as a Novel Indicator of Liver and Spleen Pathology in the Symptomatic Niemann-Pick Disease, Type C1 Mouse Model

Niemann-Pick disease, type C1 (NPC1) is a rare, autosomal recessive, lipid storage disorder caused by mutations in NPC1. As a result, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system. Clinically, patients can present with splenomegaly and hepatomegaly. In the current study, we analyzed the differential proteome of the spleen in symptomatic Npc1−/− mice to complement previous studies focused on the differential proteome of the liver, and then evaluated biomolecules that may serve as tissue biomarkers. The proteomic analysis revealed altered pathways in NPC1 representing different functional categories including heme synthesis, cellular regulation and phosphoinositide metabolism in both tissues. Differential proteins included several activators of the ubiquitous and critical protein, Akt, a major kinase involved in multiple cellular processes. Evaluation of Akt revealed decreased expression in both the liver and spleen tissues of symptomatic Npc1−/− mice. Upstream regulation analysis also suggested that miR-155 may modulate the differences of known downstream protein targets observed in our dataset. Upon evaluation of miR-155, we observed an increased expression in the liver and decreased expression in the spleen of symptomatic Npc1−/− mice. Here, we propose that miR-155 may be a novel indicator of spleen and liver pathology in NPC1

Abnormal LAMP1 glycosylation may play a role in Niemann-Pick disease, type C pathology.

A hallmark of Niemann-Pick disease, type C (NPC) is the progressive degeneration of Purkinje neurons in the cerebellum caused by the accumulation of free cholesterol and glycosphingolipids in the lysosome. Recent studies suggest that the state of glycosylation of lysosomal membrane proteins may play a role in disease progression. Our study has identified the presence of a highly glycosylated form of Lysosome Associated Membrane Protein 1 (LAMP1) that correlated spatiotemporally with Purkinje neuron loss. This form of LAMP1 was predominantly localized to activated microglia; showing a ~5-fold increase in surface labeling by FACS analysis. This suggests a potential role for LAMP1 in the neuro-inflammatory process in these mice during disease progression. Analysis of other mouse models of neurodegeneration that exhibit neuro-inflammation showed little or no presence of this glycosylated form of LAMP1, suggesting this observation for LAMP1 is specific to NPC disease. Furthermore, early treatment of Npc1-/- mice with 2-hydroxypropyl-β-cyclodextrin (HPβCD), significantly prevented the appearance of the glycosylated LAMP1 in the cerebellum of Npc1-/- mice at 7 weeks, consistent with the prevention of neuro-inflammation in mice treated with this drug. Treatment of Npc1-/- mice with HPβCD at 7 weeks, after disease onset, did not reverse or prevent further appearance of the hyperglycosylated LAMP1, demonstrating that once this aspect of neuro-inflammation began, it continued despite the HPβCD treatment. Analysis of LAMP1 in cerebellar tissue of NPC1 patients showed a small level of hyperglycosylated LAMP1 in the tissue, however, this was not seen in the CSF of patients

Chromosome-mediated OXA-48 carbapenemase in highly virulent Escherichia coli

International audienceBacteria multiresistant to antibiotics are widely supposed to be weakly virulent. However, the virulence traits of carbapenem-resistant Enterobacteriaceae have not been investigated. In this work, we investigated the virulence and resistance mechanism of an extraintestinal pathogenic Escherichia coli (ExPEC) strain (LEB15) that exhibited decreased susceptibility to carbapenems. The MICs were determined by a microdilution method. The -lactamase-encoding gene was identified by PCR and sequencing, and the genetic environment was analysed by PFGE and PCR mapping. The genetic background was investigated by multilocus sequence typing (MLST). Virulence-factor-encoding genes and pathogenic islands (PAIs) were detected by multiplex PCR. Virulence was assessed in a mouse sepsis model. Strain LEB15 produced a chromosomal OXA-48 carbapenemase. The complete bla(OXA-48)-encoding Tn1999.2 transposon was inserted in the LEB15 chromosome. The strain belonged to an MLST cluster of emerging ExPEC strains (ST-127/ST-22). It had a high pathogenic score and eight PAIs (I-536, II536, III536, IV536, VI536, I-CFT073, IICFT073 and IIJ96) and induced an unusually high lethality in the mouse sepsis model. Strain LEB15 combines both an atypical broad accumulation of virulence factors, which confers a strong killer phenotype, and a decrease in susceptibility to carbapenems following the chromosomal acquisition of bla(OXA-48). This association of virulence and carbapenemase in E. coli strains might pose major problems in the future for E. coli infection management