Kinetic Features of L,D-Transpeptidase Inactivation Critical for β-Lactam Antibacterial Activity

Active-site serine D,D-transpeptidases belonging to the penicillin-binding protein family (PBPs) have been considered for a long time as essential for peptidoglycan cross-linking in all bacteria. However, bypass of the PBPs by an L,D-transpeptidase (Ldt_fm) conveys high-level resistance to β-lactams of the penam class in Enterococcus faecium with a minimal inhibitory concentration (MIC) of ampicillin >2,000 µg/ml. Unexpectedly, Ldt_fm does not confer resistance to β-lactams of the carbapenem class (imipenem MIC = 0.5 µg/ml) whereas cephems display residual activity (ceftriaxone MIC = 128 µg/ml). Mass spectrometry, fluorescence kinetics, and NMR chemical shift perturbation experiments were performed to explore the basis for this specificity and identify β-lactam features that are critical for efficient L,D-transpeptidase inactivation. We show that imipenem, ceftriaxone, and ampicillin acylate Ldt_fm by formation of a thioester bond between the active-site cysteine and the β-lactam-ring carbonyl. However, slow acylation and slow acylenzyme hydrolysis resulted in partial Ldt_fm inactivation by ampicillin and ceftriaxone. For ampicillin, Ldt_fm acylation was followed by rupture of the C⁵–C⁶ bond of the β-lactam ring and formation of a secondary acylenzyme prone to hydrolysis. The saturable step of the catalytic cycle was the reversible formation of a tetrahedral intermediate (oxyanion) without significant accumulation of a non-covalent complex. In agreement, a derivative of Ldt_fm blocked in acylation bound ertapenem (a carbapenem), ceftriaxone, and ampicillin with similar low affinities. Thus, oxyanion and acylenzyme stabilization are both critical for rapid L,D-transpeptidase inactivation and antibacterial activity. These results pave the way for optimization of the β-lactam scaffold for L,D-transpeptidase-inactivation.     

Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis

During male germ cell postmeiotic maturation, dramatic chromatin reorganization occurs, which is driven by completely unknown mechanisms. For the first time, we describe a specific reprogramming of mouse pericentric heterochromatin. Initiated when histones undergo global acetylation in early elongating spermatids, this process leads to the establishment of new DNA packaging structures organizing the pericentric regions in condensing spermatids. Five new histone variants were discovered, which are expressed in late spermiogenic cells. Two of them, which we named H2AL1 and H2AL2, specifically mark the pericentric regions in condensing spermatids and participate in the formation of new nucleoprotein structures. Moreover, our investigations also suggest that TH2B, an already identified testis-specific H2B variant of unknown function, could provide a platform for the structural transitions accompanying the incorporation of these new histone variants.     

Function and Regulation of the Na+-Ca2+ Exchanger NCX3 Splice Variants in Brain and Skeletal Muscle

Isoform 3 of the Na⁺-Ca²⁺ exchanger (NCX3) is crucial for maintaining intracellular calcium ([Ca²⁺]_i) homeostasis in excitable tissues. In this sense NCX3 plays a key role in neuronal excitotoxicity and Ca²⁺ extrusion during skeletal muscle relaxation. Alternative splicing generates two variants (NCX3-AC and NCX3-B). Here, we demonstrated that NCX3 variants display a tissue-specific distribution in mice, with NCX3-B as mostly expressed in brain and NCX-AC as predominant in skeletal muscle. Using Fura-2-based Ca²⁺ imaging, we measured the capacity and regulation of the two variants during Ca²⁺ extrusion and uptake in different conditions. Functional studies revealed that, although both variants are activated by intracellular sodium ([Na⁺]_i), NCX3-AC has a higher [Na⁺]_i sensitivity, as Ca²⁺ influx is observed in the presence of extracellular Na⁺. This effect could be partially mimicked for NCX3-B by mutating several glutamate residues in its cytoplasmic loop. In addition, NCX3-AC displayed a higher capacity of both Ca²⁺ extrusion and uptake compared with NCX3-B, together with an increased sensitivity to intracellular Ca²⁺. Strikingly, substitution of Glu⁵⁸⁰ in NCX3-B with its NCX3-AC equivalent Lys⁵⁸⁰ recapitulated the functional properties of NCX3-AC regarding Ca²⁺ sensitivity, Lys⁵⁸⁰ presumably acting through a structure stabilization of the Ca²⁺ binding site. The higher Ca²⁺ uptake capacity of NCX3-AC compared with NCX3-B is in line with the necessity to restore Ca²⁺ levels in the sarcoplasmic reticulum during prolonged exercise. The latter result, consistent with the high expression in the slow-twitch muscle, suggests that this variant may contribute to the Ca²⁺ handling beyond that of extruding Ca²⁺.     

Quantification of raltegravir (MK0518) in human plasma by high-performance liquid chromatography with photodiode array detection

A precise and accurate high-performance liquid chromatography (HPLC) method with photodiode array detection has been developed and validated for raltegravir, a human immunodeficiency virus integrase strand transfer inhibitor (HIV-1 INSTI). Plasma (300 μL) was extracted with dichloromethane/hexane 50:50 (v/v) after addition of the internal standard, 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline. The compounds were separated using a dC18 column and detected with ultraviolet detection at 320 nm. The limit of quantification was 10 ng/mL for raltegravir. The method was linear and validated over a concentration range of 0–10,000 ng/mL. The intra-day precision ranged from 3.1 to 12.3%, while the intra-day accuracy ranged from ?15.0 to ?0.5%, the inter-day precision and accuracy were less than 7%. The mean recovery was 76.8%. Application to clinical samples taken from patients treated with raltegravir indicated that the method is suitable for measuring plasma concentrations of raltegravir in pharmacokinetic studies of clinical trials.     

Goat SRY induces testis development in XX transgenic mice

The testis-determining gene SRY is not well-conserved among mammals, particularly between mouse and other mammals, both in terms of protein structure and of expression regulation. To evaluate SRY phylogenic conservation in regards to its function, we expressed the goat gene (gSRY) in XX transgenic mouse gonads. Here, we show that gSRY induces testis formation, despite a goat expression profile. Our results demonstrate that sex-reversal can be induced in XX-mice by a non-mouse SRY thus suggesting a conserved molecular mechanism of action of this testis-determining gene across mammalian species.     

Identification of the centromeres of Leishmania major: revealing the hidden pieces

Leishmania affects millions of people worldwide. Its genome undergoes constitutive mosaic aneuploidy, a type of genomic plasticity that may serve as an adaptive strategy to survive distinct host environments. We previously found high rates of asymmetric chromosome allotments during mitosis that lead to the generation of such ploidy. However, the underlying molecular events remain elusive. Centromeres and kinetochores most likely play a key role in this process, yet their identification has failed using classical methods. Our analysis of the unconventional kinetochore complex recently discovered in Trypanosoma brucei (KKTs) leads to the identification of a Leishmania KKT gene candidate (LmKKT1). The GFP‐tagged LmKKT1 displays “kinetochore‐like” dynamics of intranuclear localization throughout the cell cycle. By ChIP‐Seq assay, one major peak per chromosome is revealed, covering a region of 4 ±2 kb. We find two largely conserved motifs mapping to 14 of 36 chromosomes while a higher density of retroposons are observed in 27 of 36 centromeres. The identification of centromeres and of a kinetochore component of Leishmania chromosomes opens avenues to explore their role in mosaic aneuploidy.     

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNA^Gly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNA^Gly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these ‘non-proteogenic’ tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of ‘proteogenic’ aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNA^Gly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.