Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A₂, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (V_max∼15.5 μmol/min/mg; K_m∼83 μM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall–deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.     

Flow cytometric method for quantifying viable Mycoplasma agassizii,an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii)

Aims: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. Methods and Results: Here, we demonstrate that the nonfluorescent molecule 5‐carboxyfluorescein (5‐CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5‐CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml^?1 can be labelled and counted in less than 1 h. Experiments using temperature‐induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. Conclusions: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. Significance and Impact of the Study: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.     

High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F

Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.     

Translocation of amino acids in the xylem of apple (Malus domestica Borkh.) trees in spring as a consequence of both N remobilization and root uptake

Nitrogen is remobilized from storage for the growth of Malus domestica leaves each spring. Seasonal patterns of N translocation in the xylem sap as a consequence of remobilization were determined in 2-year-old 'Golden delicious' trees grafted on M9 rootstocks. The trees were grown in sand culture and (15)NH(4)(15)NO(3) at 10.4 atom% abundance supplied during August-September. The following year no further N was supplied and destructive harvests were taken during bud burst and leaf growth to determine the patterns of N remobilization together with the isolation of xylem sap for an analysis of their amino acid profiles and (15)N enrichments by GC-MS. The concentration of amino acids in the xylem sap rose following bud burst, peaked at full bloom and then fell again during petal fall and fruit set. The peak in amino acid concentration corresponded with the period when the rate of N remobilization was the fastest. The majority of labelled N was recovered in Asn, Gln + Glu and Asp demonstrating that they were being translocated as a consequence of remobilization. In a second experiment, 8-year-old trees growing in an orchard were fertilized with N either in the autumn or spring. Xylem sap samples were collected in the spring and early summer and, by comparison with the amino acid profiles recovered in trees from both treatments, Asn was identified as the main compound translocated as a consequence of both remobilization and root uptake of N, although there was evidence that root uptake of N occurred later. The data are discussed in relation to quantifying the internal cycling of N in trees.     

The mitotic centromeric protein MEI-S332 and its role in sister-chromatid cohesion

Faithful segregation of sister chromatids during cell division requires properly regulated cohesion between the sister centromeres. The sister chromatids are attached along their lengths, but particularly tightly in the centromeric regions. Therefore specific cohesion proteins may be needed at the centromere. Here we show that Drosophila MEI-S332 protein localizes to mitotic metaphase centromeres. Both overexpression and mutation of MEI-S332 increase the number of apoptotic cells. In mei-S332 mutants the ratio of metaphase to anaphase figures is lower than wild type, but it is higher if MEI-S332 is overexpressed. In chromosomal squashes centromeric attachments appear weaker in mei-S332 mutants than wild type and tighter when MEI-S332 is overexpressed. These results are consistent with MEI-S332 contributing to centromeric sister-chromatid cohesion in a dose-dependent manner. MEI-S332 is the first member identified of a predicted class of centromeric proteins that maintain centromeric cohesion. Received: 11 December 1998; in revised form: 4 August 1999 / Accepted: 13 August 1999     

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.