Antibiotic Production by Myxobacteria Plays a Role in Predation

Myxobacteria are predatory and are prolific producers of secondary metabolites. Here, we tested a hypothesized role that secondary metabolite antibiotics function as weapons in predation. To test this, a Myxococcus xanthus Δta1 mutant, blocked in antibiotic TA (myxovirescin) production, was constructed. This TA⁻ mutant was defective in producing a zone of inhibition (ZOI) against Escherichia coli. This shows that TA is the major M. xanthus-diffusible antibacterial agent against E. coli. Correspondingly, the TA⁻ mutant was defective in E. coli killing. Separately, an engineered E. coli strain resistant to TA was shown to be resistant toward predation. Exogenous addition of spectinomycin, a bacteriostatic antibiotic, rescued the predation defect of the TA⁻ mutant. In contrast, against Micrococcus luteus the TA⁻ mutant exhibited no defect in ZOI or killing. Thus, TA plays a selective role on prey species. To extend these studies to other myxobacteria, the role of antibiotic corallopyronin production in predation was tested and also found to be required for Corallococcus coralloides killing on E. coli. Next, a role of TA production in myxobacterial fitness was assessed by measuring swarm expansion. Here, the TA⁻ mutant had a specific swarm rate reduction on prey lawns, and thus reduced fitness, compared to an isogenic TA⁺ strain. Based on these observations, we conclude that myxobacterial antibiotic production can function as a predatory weapon. To our knowledge, this is the first report to directly show a link between secondary metabolite production and predation.     

Major Role for Cysteine Proteases during the Early Phase of Acanthamoeba castellanii Encystment

Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in A. castellanii is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.The bacteriovorous Acanthamoeba spp. occur ubiquitously in the environment (27) and have a two-stage life cycle consisting of the replicating and feeding trophozoite stage and the dormant, double-walled, cyst stage (16). Cysts are formed in order to survive in an inhospitable environment and are able to persist in a wide variety of habitats (4, 17). Indeed, the ubiquity of Acanthamoeba is made possible by the extreme resistance of the cyst against desiccation, temperature changes, chemicals, radiation, and prolonged starvation. Also, various antiamoebic agents, such as benzalkonium chloride and propamidine isethionate, have no effect on cysts (9, 13, 29). Since acanthamoebae are facultative pathogens that can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE), encystment is also of medical relevance (16). An often occurring complication in the treatment of AK is the presence of viable cysts that remain in the corneal stroma after initial successful therapy, as these can eventually excyst again and lead to recurrent infections (23).According to Weisman (31), the encystment process comprises three phases: induction, wall synthesis, and dormancy. During the induction phase, trophozoites begin to lose their amoeboid appearance and become round. The first wall that is formed gives rise to the exocyst; this wall is 0.3 to 0.5 μm thick and consists mostly of acid-insoluble proteins. The endocyst is formed after the appearance of a well-defined layer whose major component is cellulose (31). Cell wall synthesis is usually accompanied by a decrease in cytoplasmic mass of approximately 80% through a gradual dehydration of the amoeba, thereby causing retraction of the protoplast from the cell wall (2). Rather early, autolysosomes appear and remain in the cytoplasm throughout the whole encystment process. In light of these dramatic changes in the cell''s physiology, it is surprising that the encysting cell can stop and revert the process until 15 h after induction (30). Afterwards, however, cells become committed to the completion of the encystment process.At the molecular level, a number of factors involved in the encystment process have been characterized thus far. For example, cyst-specific protein 21 (Csp21) is a cyst wall protein found in group II acanthamoebae and was reported to be synthesized approximately 12 h after induction (6). The expression of the respective gene is repressed under normal growth conditions via one or more repressor elements between the TATA box and nucleotide (nt) +63 (3). Furthermore, encystment requires serine protease activity (5, 20) and autophagy proteins (22), all of which are suggested to be involved in autolytic processes, and glycogen phosphorylase, which is necessary for the breakdown of glycogen (14). The glucose-1-phosphate that is thereby liberated is subsequently used for the buildup of cellulose in the cyst wall.In the search for additional factors, there have been several successful attempts in the past years to screen encysting Acanthamoeba castellanii for genes specifically expressed during encystment at the mRNA level (19, 21) as well as at the protein level (1, 24). However, there is still a lack of information on the extent of cellular reorganization during the encystment process at the protein level. In this study, we therefore aimed to monitor the encystment process in PAT06, a new clinical isolate of A. castellanii (10), by using two-dimensional gel electrophoresis (2DE) and to analyze the developmental and molecular processes at the proteomic level.     

In Silico Prediction of Mutant HIV-1 Proteases Cleaving a Target Sequence

HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636–1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidates for specificity and cleavability towards the target sequence.     

Potential of Tissue Culture for Breeding Root-Knot Nematode Resistance into Vegetables

Plant protoplast technology is being investigated as a means of transferring root-knot nematode resistance factors from Solanum sisymbriifolium into the susceptible S. melongena. Solanum sisymbriifolium plants regenerated from callus lost resistance to Meloidogyne javanica but retained resistance to M. incognita. Tomato plants cloned from leaf discs of the root-knot nematode resistant ''Patriot'' were completely susceptible to M. incognita, while sections of stems and leaves rooted in sand in the absence of growth hormones retained resistance. Changes in resistance persisted for three generations. It is postulated that the exogenous hormonal constituents of the culture medium are modifying the expression of genetic resistance.     

Riboswitch Control of Aminoglycoside Antibiotic Resistance

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Highlights? The 5′ leader RNA of aminoglycoside antibiotic-resistance genes is conserved ? Aminoglycosides induce reporter gene expression by interacting with the leader RNA ? The aminoglycosides bind to the leader RNA and induce a change in the RNA structure ? Induction is independent of leader peptide stalling or drug-ribosome interactions     

Schistosoma japonicum Eggs Induce a Proinflammatory,Anti-Fibrogenic Phenotype in Hepatic Stellate Cells

Hepatic fibrosis induced by egg deposition is the most serious pathology associated with chronic schistosomiasis, in which the hepatic stellate cell (HSC) plays a central role. While the effect of Schistosoma mansoni eggs on the fibrogenic phenotype of HSCs has been investigated, studies determining the effect of eggs of S . japonicum on HSCs are lacking. Disease caused by S . japonicum is much more severe than that resulting from S. mansoni infection so it is important to compare the pathologies caused by these two parasites, to determine whether this phenotype is due to the species interacting differently with the mammalian host. Accordingly, we investigated the effect of S . japonicum eggs on the human HSC cell line, LX-2, with and without TGF-β (Transforming Growth Factor beta) co-treatment, so as to determine the impact on genes associated with fibrogenesis, inflammation and matrix re-organisation. Activation status of HSCs was assessed by αSMA (Alpha Smooth Muscle Actin) immunofluorescence, accumulation of Oil Red O-stained lipid droplets and the relative expression of selected genes associated with activation. The fibrogenic phenotype of HSCs was inhibited by the presence of eggs both with or without TGF-β treatment, as evidenced by a lack of αSMA staining and reduced gene expression of αSMA and Col1A1 (Collagen 1A1). Unlike S. mansoni-treated cells, however, expression of the quiescent HSC marker PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) was not increased, nor was there accumulation of lipid droplets. In contrast, S . japonicum eggs induced the mRNA expression of MMP-9 (Matrix Metalloproteinase 9), CCL2 (Chemokine (C-C motif) Ligand 2) and IL-6 (Interleukin 6) in HSCs indicating that rather than inducing complete HSC quiescence, the eggs induced a proinflammatory phenotype. These results suggest HSCs in close proximity to S . japonicum eggs in the liver may play a role in the proinflammatory regulation of hepatic granuloma formation.     

Control of Globodera tabacum solanacearum by Alternating Host Resistance and Nematicide

The feasibility of alternating use of resistant vs. susceptible flue-cured tobacco cultivars to improve control of Globodera tabacum subsp, solanacearum (TCN) was investigated at two Virginia locations in 1984-86. Post-harvest TCN population densities were reduced in each year of the study when fenamiphos was used with a TCN-resistant cultivar (NC 567), relative to susceptible cultivars (K 326 or Mc 944). Using NC 567 with fenamipbos also reduced preplant TCN population densities in the next growing season. Egg population densities before planting in 1986 were significantly lower in plots planted with NC 567 in 1984, even when a susceptible cultivar had been planted in 1985. Use of fenamiphos with NC 567 in 1984 and 1985 further reduced preplant egg population densities in 1986. Economic returns were significantly greater in 1984 when NC 567 was used with fenamiphos, rather than a susceptible cultivar. Treatments involving fenamiphos and (or) NC 567 in 1984 and 1985 resulted in higher economic returns in 1986 than did treatments using a susceptible cultivar without fenamiphos in both previous years. Economic returns were highest in 1986 when fenamiphos and NC 567 were used in 1984 and 1985 and a susceptible cultivar was planted in 1986.