Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem

The beta-lactam antibiotics mimic the D-alanyl(4)-D-alanine(5) extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the dd-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high level resistance to ampicillin. Ldt(fm) is specific for the L-lysyl(3)-D-alanine(4) bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine(5). This specificity was proposed to account for resistance, because the substrate of Ldt(fm) does not mimic beta-lactams in contrast to the D-alanyl(4)-D-alanine(5) extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a beta-lactam of the carbapenem class, totally inhibited Ldt(fm) at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt(fm) is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt(fm) by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt(fm) containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt(fm) is likely to involve rupture of the beta-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of beta-lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.     

The intra-annual variability of soft-bottom macrobenthos abundance patterns in the North Channel of the Seine estuary

Temporal and spatial variability of the Abra alba–Pectinaria koreni and Macoma balthica communities was examined in the northern part of the Seine estuary (North Channel) over different space and time scales in order to assess the role that the hydrologic regime and/or anthropogenic influences play in defining benthic communities over time. Sediment in the North Channel displayed strong spatial and temporal variability, sustained by intense sediment transport episodes. Total macrobenthic abundances ranged widely on the course of the year and there was no evidence of a seasonal signal for the density fluctuations, whatever the spatial scale considered. The bio-sedimentary dynamics can be divided into two periods: the first corresponds to the high flow rate period (January–May) during which fauna is influenced by fine silt/clay deposition, and the second to the low flow rate period (June–December) during which sandy deposits prevail. Despite the absence of significant correlations between sediment composition and abundance, episodes of sediment transport seem to be an important structuring mechanism in the Seine estuary. As a consequence, the faunal composition varied throughout the year. The winter and spring fauna, characterised by species living on muddy fine-sands or muds, were enriched during the summer and autumn by species living in clean fine sand, such as Donax vittatus, Nephtys cirrosa or Spio decoratus, mainly represented by adult individuals. Secondary settlement of drifters may explain the rapid structuration of assemblages a few days after the sandy deposits. Our results suggest the importance of the bentho-pelagic coupling, primarily induced by the sedimentary instability, on the macrobenthic fauna dynamics. The intra-annual variability of assemblages at the mouth of the Seine river and the silted situation of the North Channel might simply be the result of the silting up and alteration of the inner estuary, generated by several decades of man-made modifications and natural processes.     

Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan

The L,D-transpeptidase Ldt(fm) catalyzes peptidoglycan cross-linking in beta-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldt(fm) homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the L,D-transpeptidation reaction.     

Heterocyclic alpha-helix mimetics for targeting protein-protein interactions

The design and synthesis of alpha-helix peptidomimetics using inverse electron demand Diels-Alder reactions is described. The potency of the resulting pyridazine-based library to disrupt the Bak/Bcl-X(L) interaction was tested using an in vitro fluorescence polarization assay.     

Antibody response against saliva antigens of Anopheles gambiae and Aedes aegypti in travellers in tropical Africa

Exposure to vectors of infectious diseases has been associated with antibody responses against salivary antigens of arthropods among people living in endemic areas. This immune response has been proposed as a surrogate marker of exposure to vectors appropriate for evaluating the protective efficacy of antivectorial devices. The existence and potential use of such antibody responses in travellers transiently exposed to Plasmodium or arbovirus vectors in tropical areas has never been investigated. The IgM and IgG antibody responses of 88 French soldiers against the saliva of Anopheles gambiae and Aedes aegypti were evaluated before and after a 5-month journey in tropical Africa. Antibody responses against Anopheles and Aedes saliva increased significantly in 41% and 15% of the individuals, respectively, and appeared to be specific to the mosquito genus. A proteomic and immunoproteomic analysis of anopheles and Aedes saliva allowed for the identification of some antigens that were recognized by most of the exposed individuals. These results suggest that antibody responses to the saliva of mosquitoes could be considered as specific surrogate markers of exposure of travellers to mosquito vectors that transmit arthropod borne infections.     

Differentiating megakaryocytes in myelodysplastic syndromes succumb to mitochondrial derangement without caspase activation

Myelodysplastic syndromes (MDS) constitute a preneoplastic condition in which potentially malignant cancer stem cells continuously die during differentiation. This MDS-associated cell death often involves caspase-3 activation, yet can also occur without caspase activation, for instance in differentiating megakaryocytes (MK). We investigated, the mechanisms through which MK from MDS patients undergo premature cell death. While polyploid, mature MK from healthy subjects or MDS patients manifested caspase-3 activation during terminal differentiation, freshly isolated, immature MK from MDS died without caspase-3 activation. Similarly, purified bone marrow CD34⁺ cells from MDS patients that were driven into MK differentiation in vitro died without caspase-3 activation at an immature stage, before polyploidization. The premature death of MDS MK was accompanied by the mitochondrial release of cytochrome c, Smac/DIABLO and endonuclease G, a caspase-independent death effector, as well loss of the mitochondrial membrane potential and plasma membrane phosphatidylserine exposure before definitive loss of viability. Thus, a stereotyped pattern of mitochondrial alterations accompanies differentiation-associated MK death in MDS. T. Braun and G. Carvalho contributed equally to this paper.     

Persistence of long-term memory storage requires a late protein synthesis- and BDNF- dependent phase in the hippocampus

Persistence is the most characteristic attribute of long-term memory (LTM). To understand LTM, we must understand how memory traces persist over time despite the short-lived nature and rapid turnover of their molecular substrates. It is widely accepted that LTM formation is dependent upon hippocampal de novo protein synthesis and Brain-Derived Neurotrophic Factor (BDNF) signaling during or early after acquisition. Here we show that 12 hr after acquisition of a one-trial associative learning task, there is a novel protein synthesis and BDNF-dependent phase in the rat hippocampus that is critical for the persistence of LTM storage. Our findings indicate that a delayed stabilization phase is specifically required for maintenance, but not formation, of the memory trace. We propose that memory formation and memory persistence share some of the same molecular mechanisms and that recurrent rounds of consolidation-like events take place in the hippocampus for maintenance of the memory trace.     

Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod- driven motility and cell-cycle progression

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Highlights? Rac1 and the Scar/WAVE complex drive pseudopod-based motility of melanoblasts ? Rac1-depleted melanoblasts move using unique actin-based stubs and not blebs ? Rac1 controls pseudopod frequency but is dispensable for pseudopod formation ? Loss of Rac1 delays melanoblast cell-cycle progression and cytokinesis