Chromatographic profiling and identification of two new iridoid-indole alkaloids by UPLC–MS and HPLC-SPE-NMR analysis of an antimalarial extract from Nauclea pobeguinii

The total 80% EtOH extract of stem bark of Nauclea pobeguinii (Rubiaceae), which is active against uncomplicated falciparum malaria as shown in previous clinical studies, was analysed by means of UPLC–MS and HPLC-SPE-NMR. Apart from the main constituent, strictosamide, a series of minor constituents was identified, including two new iridoid-indole alkaloids, i.e. naucleidinic acid and 19-O-methyl-3,14-dihydroangustoline, together with 8 known iridoid-indole alkaloids, i.e. naucleidinal, magniflorine, naucleofficine D, two diastereoisomers of 3,14-dihydroangustoline, strictosidine, desoxycordifoline, 3α,5α-tetrahydrodeoxycordifoline lactam, and a phenol glycoside 3,4,5-trimethoxyphenol β-d-apiofuranosyl-(1-6)-β-d-glucopyranoside (kelampayoside A).     

Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation

Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This Phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin-antitoxin locus for its action in vivo.     

An experimental type II mixed cryoglobulinemia with renal glomerulopathy in ICR mice triggered by Capillaria hepatica infection

Type II mixed cryoglobulinemia is characterized by systemic vasculitis with deposition of cryoprecipitatable-immunoglobulins containing rheumatoid factor. Pathogenesis of type II mixed cryoglobulinemia has not yet been completely clarified because of the lack of an experimental animal. Here, we report an animal model of type II mixed cryoglobulinemia that is induced by experimental infection with Capillaria hepatica in ICR mice. Capillaria hepatica is a nematode that causes necrotic hepatitis in several mammals. In this study, mice experimentally infected with C. hepatica eggs developed cryoglobulinemia at 20 and 30 days post injection. Using immunological analysis, cryoglobulinemia in infected mice was classified as type II mixed cryoglobulinemia by detection of monoclonal IgM rheumatoid factor and IgA in the cryoprecipitate of serum. Using immunofluorescence, we observed an increase in the number of double-positive cells for μ heavy and κ light chains of immunoglobulin in the spleens of infected mice. Histopathologically, this model was characterized by glomerulopathy associated with intense deposition of IgM and IgA filling in capillary lumina. Ultrastructural analysis showed that glomerular deposits consisted of stacks of twisted microtubular structures. These serological and histological features resembled those of type II mixed cryoglobulinemia in human. This is the first experimental animal model of type II mixed cryoglobulinemia that will enable detailed studies on the pathogenesis of cryoglobulinemia.     

Novel coiled-coil cell division factor ZapB stimulates Z ring assembly and cell division

Formation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring are regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals, whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI, and ZapB interacted with FtsZ in a bacterial two-hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB–GFP is present at mid-cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84 ^ts allele exhibited a synthetic sick phenotype and aberrant cell divisions. The crystal structure showed that ZapB exists as a dimer that is 100% coiled-coil. In vitro , ZapB self-assembled into long filaments and bundles. These results raise the possibility that ZapB stimulates Z ring formation directly via its capacity to self-assemble into larger structures.     

The RES domain toxins of RES‐Xre toxin‐antitoxin modules induce cell stasis by degrading NAD+

Type II toxin‐antitoxin (TA) modules, which are important cellular regulators in prokaryotes, usually encode two proteins, a toxin that inhibits cell growth and a nontoxic and labile inhibitor (antitoxin) that binds to and neutralizes the toxin. Here, we demonstrate that the res‐xre locus from Photorhabdus luminescens and other bacterial species function as bona fide TA modules in Escherichia coli. The 2.2 Å crystal structure of the intact Pseudomonas putida RES‐Xre TA complex reveals an unusual 2:4 stoichiometry in which a central RES toxin dimer binds two Xre antitoxin dimers. The antitoxin dimers each expose two helix‐turn‐helix DNA‐binding domains of the Cro repressor type, suggesting the TA complex is capable of binding the upstream promoter sequence on DNA. The toxin core domain shows structural similarity to ADP‐ribosylating enzymes such as diphtheria toxin but has an atypical NAD⁺‐binding pocket suggesting an alternative function. We show that activation of the toxin in vivo causes a depletion of intracellular NAD⁺ levels eventually leading to inhibition of cell growth in E. coli and inhibition of global macromolecular biosynthesis. Both structure and activity are unprecedented among bacterial TA systems, suggesting the functional scope of bacterial TA toxins is much wider than previously appreciated.     

Prokaryotic DNA segregation by an actin-like filament

The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments with properties expected for a force-generating protein. Filament formation depended on the other components encoded by par, ParR and the centromere-like parC region to which ParR binds. Mutants defective in ParM ATPase exhibited hyperfilamentation and did not support plasmid partitioning. ParM polymerization was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR-parC complex functions as a nucleation point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.     

The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex

MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.