A novel tankyrase inhibitor, MSC2504877, enhances the effects of clinical CDK4/6 inhibitors.

Inhibition of the PARP superfamily tankyrase enzymes suppresses Wnt/β-catenin signalling in tumour cells. Here, we describe here a novel, drug-like small molecule inhibitor of tankyrase MSC2504877 that inhibits the growth of APC mutant colorectal tumour cells. Parallel siRNA and drug sensitivity screens showed that the clinical CDK4/6 inhibitor palbociclib, causes enhanced sensitivity to MSC2504877. This tankyrase inhibitor-CDK4/6 inhibitor combinatorial effect is not limited to palbociclib and MSC2504877 and is elicited with other CDK4/6 inhibitors and toolbox tankyrase inhibitors. The addition of MSC2504877 to palbociclib enhances G 1 cell cycle arrest and cellular senescence in tumour cells. MSC2504877 exposure suppresses the upregulation of Cyclin D2 and Cyclin E2 caused by palbociclib and enhances the suppression of phospho-Rb, providing a mechanistic explanation for these effects. The combination of MSC2504877 and palbociclib was also effective in suppressing the cellular hyperproliferative phenotype seen in Apc defective intestinal stem cells in vivo. However, the presence of an oncogenic Kras p.G12D mutation in mice reversed the effects of the MSC2504877/palbociclib combination, suggesting one molecular route that could lead to drug resistance

Absence of repellents in Ustilago maydis induces genes encoding small secreted proteins

The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae

Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens

Comparative Genome Analysis of Filamentous Fungi Reveals Gene Family Expansions Associated with Fungal Pathogenesis

Fungi and oomycetes are the causal agents of many of the most serious diseases of plants. Here we report a detailed comparative analysis of the genome sequences of thirty-six species of fungi and oomycetes, including seven plant pathogenic species, that aims to explore the common genetic features associated with plant disease-causing species. The predicted translational products of each genome have been clustered into groups of potential orthologues using Markov Chain Clustering and the data integrated into the e-Fungi object-oriented data warehouse (http://www.e-fungi.org.uk/). Analysis of the species distribution of members of these clusters has identified proteins that are specific to filamentous fungal species and a group of proteins found only in plant pathogens. By comparing the gene inventories of filamentous, ascomycetous phytopathogenic and free-living species of fungi, we have identified a set of gene families that appear to have expanded during the evolution of phytopathogens and may therefore serve important roles in plant disease. We have also characterised the predicted set of secreted proteins encoded by each genome and identified a set of protein families which are significantly over-represented in the secretomes of plant pathogenic fungi, including putative effector proteins that might perturb host cell biology during plant infection. The results demonstrate the potential of comparative genome analysis for exploring the evolution of eukaryotic microbial pathogenesis

Cryptococcus gattii Virulence Composite: Candidate Genes Revealed by Microarray Analysis of High and Less Virulent Vancouver Island Outbreak Strains

Human and animal cryptococcosis due to an unusual molecular type of Cryptococcus gattii (VGII) emerged recently on Vancouver Island, Canada. Unlike C. neoformans, C. gattii causes disease mainly in immunocompetent hosts, despite producing a similar suite of virulence determinants. To investigate a potential relationship between the regulation of expression of a virulence gene composite and virulence, we took advantage of two subtypes of VGII (a and b), one highly virulent (R265) and one less virulent (R272), that were identified from the Vancouver outbreak. By expression microarray analysis, 202 genes showed at least a 2-fold difference in expression with 108 being up- and 94 being down-regulated in strain R265 compared with strain R272. Specifically, expression levels of genes encoding putative virulence factors (e.g. LAC1, LAC2, CAS3 and MPK1) and genes encoding proteins involved in cell wall assembly, carbohydrate and lipid metabolism were increased in strain R265, whereas genes involved in the regulation of mitosis and ergosterol biosynthesis were suppressed. In vitro phenotypic studies and transcription analysis confirmed the microarray results. Gene disruption of LAC1 and MPK1 revealed defects in melanin synthesis and cell wall integrity, respectively, where CAS3 was not essential for capsule production. Moreover, MPK1 also controls melanin and capsule production and causes a severe attenuation of the virulence in a murine inhalational model. Overall, this study provides the basis for further genetic studies to characterize the differences in the virulence composite of strains with minor evolutionary divergences in gene expression in the primary pathogen C. gattii, that have led to a major invasive fungal infection outbreak

Steering Evolution with Sequential Therapy to Prevent the Emergence of Bacterial Antibiotic Resistance

The increasing rate of antibiotic resistance and slowing discovery of novel antibiotic treatments presents a growing threat to public health. Here, we consider a simple model of evolution in asexually reproducing populations which considers adaptation as a biased random walk on a fitness landscape. This model associates the global properties of the fitness landscape with the algebraic properties of a Markov chain transition matrix and allows us to derive general results on the non-commutativity and irreversibility of natural selection as well as antibiotic cycling strategies. Using this formalism, we analyze 15 empirical fitness landscapes of E. coli under selection by different β-lactam antibiotics and demonstrate that the emergence of resistance to a given antibiotic can be either hindered or promoted by different sequences of drug application. Specifically, we demonstrate that the majority, approximately 70%, of sequential drug treatments with 2–4 drugs promote resistance to the final antibiotic. Further, we derive optimal drug application sequences with which we can probabilistically ‘steer’ the population through genotype space to avoid the emergence of resistance. This suggests a new strategy in the war against antibiotic–resistant organisms: drug sequencing to shepherd evolution through genotype space to states from which resistance cannot emerge and by which to maximize the chance of successful therapy

Discovery of enzymes for toluene synthesis from anoxic microbial communities

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum