Virulence, drug sensitivity and transmission success in the rodent malaria, Plasmodium chabaudi

Here, we test the hypothesis that virulent malaria parasites are less susceptible to drug treatment than less virulent parasites. If true, drug treatment might promote the evolution of more virulent parasites (defined here as those doing more harm to hosts). Drug-resistance mechanisms that protect parasites through interactions with drug molecules at the sub-cellular level are well known. However, parasite phenotypes associated with virulence might also help parasites survive in the presence of drugs. For example, rapidly replicating parasites might be better able to recover in the host if drug treatment fails to eliminate parasites. We quantified the effects of drug treatment on the in-host survival and between-host transmission of rodent malaria (Plasmodium chabaudi) parasites which differed in virulence and had never been previously exposed to drugs. In all our treatment regimens and in single- and mixed-genotype infections, virulent parasites were less sensitive to pyrimethamine and artemisinin, the two antimalarial drugs we tested. Virulent parasites also achieved disproportionately greater transmission when exposed to pyrimethamine. Overall, our data suggest that drug treatment can select for more virulent parasites. Drugs targeting transmission stages (such as artemisinin) may minimize the evolutionary advantage of virulence in drug-treated infections.     

Benzo[a]pyrene potentiates the pathogenesis of abdominal aortic aneurysms in apolipoprotein E knockout mice

The objective of this study was to determine the effect of benzo[a]pyrene (BaP), an abundant environmental polycyclic aromatic hydrocarbon compound, on the pathogenesis of abdominal aortic aneurysms (AAA). Earlier studies have shown that BaP promotes vasculopathy, including atherosclerosis, a predisposing factor for AAA development. In two experimental arms, 203 apolipoprotein E knockout (ApoE-/-) mice were evaluated in 4 groups: BaP, angiotensin II (AngII), BaP+AngII and control. Mice in the first arm were exposed to 5mg/kg/week of BaP for 42 days, and in the second arm to 0.71mg/kg daily for 60 days. In arm one, AAA incidence was higher in the BaP+AngII (14/28) versus AngII (8/27) group (p < 0.05), rupture (n=3) was observed only in BaP+AngII treated mice (p < 0.05). In the second arm, AAA incidence did not differ between AngII (17/30) and BaP+AngII (16/29) groups. However, intact AAA diameter was larger in the BaP+AngII (2.3 ± 0.1mm) versus AngII (1.9 ± 0.1mm) group (p < 0.05), but AAA rupture did not differ (p=NS). In both experimental arms, BaP+AngII mice showed increased expression of tumor necrosis factor alpha (TNF-α), cyclophilin A (Cyp A), and matrix metalloproteinase-9 (MMP9) (p < 0.05). No AAA occurred in control or BaP groups. These findings suggest the role of BaP exposure in potentiating AAA pathogenesis, which may have potential public health significance.     

The importance of weathered crude oil as a source of hydrocarbonoclastic microorganisms in contaminated seawater

This study investigated the hydrocarbonoclastic microbial community present on weathered crude oil and their ability to degrade weathered oil in seawater obtained from the Gulf St. Vincent (SA, Australia). Examination of the native seawater communities capable of utilizing hydrocarbon as the sole carbon source identified a maximum recovery of just 6.6 × 10(1) CFU/ml, with these values dramatically increased in the weathered oil, reaching 4.1 × 10(4) CFU/ml. The weathered oil (dominated by >C30 fractions; 750,000 +/- 150,000 mg/l) was subject to an 8 week laboratory-based degradation microcosm study. By day 56, the natural inoculums degraded the soluble hydrocarbons (initial concentrations 3,400 +/- 700 mg/l and 1,700 +/- 340 mg/l for the control and seawater, respectively) to below detectable levels, and biodegradation of the residual oil reached 62% (254,000 +/- 40,000 mg/l) and 66% (285,000 +/- 45,000 mg/l) in the control and seawater sources, respectively. In addition, the residual oil gas chromatogram profiles changed with the presence of short and intermediate hydrocarbon chains. 16S rDNA DGGE sequence analysis revealed species affiliated with the genera Roseobacter, Alteromonas, Yeosuana aromativorans, and Pseudomonas, renowned oil-degrading organisms previously thought to be associated with the environment where the oil contaminated rather than also being present in the contaminating oil. This study highlights the importance of microbiological techniques for isolation and characterisation, coupled with molecular techniques for identification, in understanding the role and function of native oil communities.     

Identification of fungal sphingolipid C9-methyltransferases by phylogenetic profiling

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24-71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Delta4,8-desaturated ceramide as substrates.     

Catalytic importance of acidic amino acids on subunit NuoB of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN and contains one FMN and 9 iron-sulfur clusters as redox groups. Electron transfer from NADH to ubiquinone is coupled with the translocation of protons across the membrane by a yet unknown mechanism. Redox-induced Fourier transform infrared difference spectroscopy showed that the oxidation of iron-sulfur cluster N2 located on NuoB is accompanied by the protonation of acidic amino acid(s). Here, we describe the effect of mutating the conserved acidic amino acids on NuoB. The complex was assembled in all mutants but the electron transfer activity was completely abolished in the mutants E67Q, D77N, and D94N. The complex isolated from these mutants contained N2 although in diminished amounts. The protonation of acidic amino acid(s) coupled with the oxidation of N2 was not detectable in the complex from the mutant E67Q. However, the conservative mutations E67D and D77E did not disturb the enzymatic activity, and the signals because of the protonation of acidic amino acid(s) were detectable in the E67D mutant. We discuss the possible participation of Glu(67) in a proton pathway coupled with the redox reaction of N2.     

Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor

Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.     

Characterization of a novel Photosystem I-LHCI supercomplex isolated from Chlamydomonas reinhardtii under anaerobic (State II) conditions

A novel supercomplex of Photosystem I (PSI) with light harvesting complex I (LHCI) was isolated from the green alga Chlamydomonas reinhardtii. This novel supercomplex is unique as it is the first stable supercomplex of PSI together with its external antenna. The supercomplex contains 256 chlorophylls per reaction center. The supercomplex was isolated under anaerobic conditions and may represent the State II form of the photosynthetic unit. In contrast to previously reported supercomplexes isolated in State I, which contain only 4 LHC I proteins, this supercomplex contains 10-11 LHC I proteins tightly bound to the PSI core. In contrast to plants, no LHC II is tightly bound to the PSI-LHCI supercomplex in State II. Investigation of the energy transfer from the antenna system to the reaction center core shows that the LHC supercomplexes are tightly coupled to the PSI core, not only structurally but also energetically. The excitation energy transfer kinetics are completely dominated by the fast phase, with a near-complete lack of long-lived fluorescence. This tight coupling is in contrast to all reports of energy transfer in PSI-LHCI supercomplexes (in State I), which have so far been described as weakly coupled supercomplexes with low efficiency for excitation energy transfer. These results indicate that there are large and dynamic changes of the PSI-LHCI supercomplex during the acclimation from aerobic (State I) to anaerobic (State II) conditions in Chlamydomonas.     

Annexin A5 inhibits engulfment through internalization of PS-expressing cell membrane patches

Apoptosis and subsequent clearance of apoptotic cells are important for the prevention of diseases. Therefore, it is essential to understand the mechanisms underlying the biology of phagocytic clearance of apoptotic cells. The best characterized "eat me" signal on the surface of apoptotic cells is phosphatidylserine (PS). Recently, we demonstrated that annexin A5 mediates the internalization of PS-expressing membrane patches and down regulates surface expression of tissue factor. Here, we investigated the role of PS in the phagocytosis of apoptotic cells using annexin A5. Using a novel flow cytometric-based phagocytosis assay, we observed that engulfment was inhibited with 20% if annexin A5 was added to PS-expressing cells that had completed apoptosis. The inhibition increased to more than 50% if annexin A5 was added during the apoptotic process. This inhibition is specific for annexin A5, since the mutant M23 and annexin A1 did not further increase the inhibition of phagocytosis when added during the apoptotic process. Interestingly, cells with internalized annexin A5 still express PS at their surface. We conclude that other ligands within the PS-expressing membrane patch act together with PS as an "eat me" signal.     

Overexpression of Kelch domain containing-2 (mKlhdc2) inhibits differentiation and directed migration of C2C12 myoblasts

Targeted migration of muscle precursor cells to the anlagen of limb muscles is a complex process, which is only partially understood. We have used Lbx1 mutant mice, which are unable to establish correct migration paths of muscle precursor cells into the limbs to identify new genes involved in the accurate placement of myogenic cells in developing muscles. We found that mKlhdc2 (Kelch domain containing-2), a novel member of the family of Kelch domain containing proteins, is significantly downregulated in Lbx1 homozygous mutant embryos. Functional characterization of mKlhdc2 by targeted overexpression in 10T1/2 fibroblasts and C2C12 muscle cells rendered these cells unable to respond to chemoattractants such as HGF. Furthermore, C2C12 myoblasts overexpressing mKlhdc2 display altered cellular morphology and are unable to differentiate into mature myotubes. Our results suggest that a tightly controlled expression of mKlhdc2 is essential for a faithful execution of the myogenic differentiation and migration program.