Pre‐adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates

Recent years have seen renewed interest in phage therapy – the use of viruses to specifically kill disease‐causing bacteria – because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre‐adapted all phage strains against all bacterial strains and then compared the efficacy of pre‐adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre‐adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.     

A web services choreography scenario for interoperating bioinformatics applications

Background  

Very often genome-wide data analysis requires the interoperation of multiple databases and analytic tools. A large number of genome databases and bioinformatics applications are available through the web, but it is difficult to automate interoperation because: 1) the platforms on which the applications run are heterogeneous, 2) their web interface is not machine-friendly, 3) they use a non-standard format for data input and output, 4) they do not exploit standards to define application interface and message exchange, and 5) existing protocols for remote messaging are often not firewall-friendly. To overcome these issues, web services have emerged as a standard XML-based model for message exchange between heterogeneous applications. Web services engines have been developed to manage the configuration and execution of a web services workflow.     

A relational database for the discovery of genes encoding amino acid biosynthetic enzymes in pathogenic fungi

Fungal phytopathogens continue to cause major economic impact, either directly, through crop losses, or due to the costs of fungicide application. Attempts to understand these organisms are hampered by a lack of fungal genome sequence data. A need exists, however, to develop specific bioinformatics tools to collate and analyse the sequence data that currently is available. A web-accessible gene discovery database (http://cogeme.ex.ac.uk/biosynthesis.html) was developed as a demonstration tool for the analysis of metabolic and signal transduction pathways in pathogenic fungi using incomplete gene inventories. Using Bayesian probability to analyse the currently available gene information from pathogenic fungi, we provide evidence that the obligate pathogen Blumeria graminis possesses all amino acid biosynthetic pathways found in free-living fungi, such as Saccharomyces cerevisiae. Phylogenetic analysis was also used to deduce a gene history of succinate-semialdehyde dehydrogenase, an enzyme in the glutamate and lysine biosynthesis pathways. The database provides a tool and methodology to researchers to direct experimentation towards predicting pathway conservation in pathogenic microorganisms.     

Functional analysis of lipid metabolism in Magnaporthe grisea reveals a requirement for peroxisomal fatty acid beta-oxidation during appressorium-mediated plant infection

The rice blast fungus Magnaporthe grisea infects plants by means of specialized infection structures known as appressoria. Turgor generated in the appressorium provides the invasive force that allows the fungus to breach the leaf cuticle with a narrow-penetration hypha gaining entry to the underlying epidermal cell. Appressorium maturation in M. grisea involves mass transfer of lipid bodies to the developing appressorium, coupled to autophagic cell death in the conidium and rapid lipolysis at the onset of appressorial turgor generation. Here, we report identification of the principal components of lipid metabolism in M. grisea based on genome sequence analysis. We show that deletion of any of the eight putative intracellular triacylglycerol lipase-encoding genes from the fungus is insufficient to prevent plant infection, highlighting the complexity and redundancy associated with appressorial lipolysis. In contrast, we demonstrate that a peroxisomally located multifunctional, fatty acid beta-oxidation enzyme is critical to appressorium physiology, and blocking peroxisomal biogenesis prevents plant infection. Taken together, our results indicate that, although triacylglycerol breakdown in the appressorium involves the concerted action of several lipases, fatty acid metabolism and consequent generation of acetyl CoA are necessary for M. grisea to complete its prepenetration phase of development and enter the host plant.     

Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

Introduction  

The vast difference in the abundance of different proteins in biological samples limits the determination of the complete proteome of a cell type, requiring fractionation of proteins and peptides before MS analysis.     

Infection-associated nuclear degeneration in the rice blast fungus Magnaporthe oryzae requires non-selective macro-autophagy

Background

The rice blast fungus Magnaporthe oryzae elaborates a specialized infection structure called an appressorium to breach the rice leaf surface and gain access to plant tissue. Appressorium development is controlled by cell cycle progression, and a single round of nuclear division occurs prior to appressorium formation. Mitosis is always followed by programmed cell death of the spore from which the appressorium develops. Nuclear degeneration in the spore is known to be essential for plant infection, but the precise mechanism by which it occurs is not known.

Methodology/Principal Findings

In yeast, nuclear breakdown requires a specific form of autophagy, known as piecemeal microautophagy of the nucleus (PMN), and we therefore investigated whether this process occurs in the rice blast fungus. Here, we report that M. oryzae possesses two conserved components of a putative PMN pathway, MoVac8 and MoTsc13, but that both are dispensable for nuclear breakdown during plant infection. MoVAC8 encodes a vacuolar membrane protein and MoTSC13 a peri-nuclear and peripheral ER protein.

Conclusions/Significance

We show that MoVAC8 is necessary for caffeine resistance, but dispensable for pathogenicity of M. oryzae, while MoTSC13 is involved in cell wall stress responses and is an important virulence determinant. By functional analysis of ΔMoatg1 and ΔMoatg4 mutants, we demonstrate that infection-associated nuclear degeneration in M. oryzae instead occurs by non-selective macroautophagy, which is necessary for rice blast disease.