A genome-wide overexpression screen reveals Mycobacterium smegmatis growth inhibitors encoded by mycobacteriophage Hammy.

During infection, bacteriophages produce diverse gene products to overcome bacterial antiphage defenses, to outcompete other phages, and to take over cellular processes. Even in the best-studied model phages, the roles of most phage-encoded gene products are unknown, and the phage population represents a largely untapped reservoir of novel gene functions. Considering the sheer size of this population, experimental screening methods are needed to sort through the enormous collection of available sequences and identify gene products that can modulate bacterial behavior for downstream functional characterization. Here, we describe the construction of a plasmid-based overexpression library of 94 genes encoded by Hammy, a Cluster K mycobacteriophage closely related to those infecting clinically important mycobacteria. The arrayed library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 24 gene products (representing ∼25% of the Hammy genome) capable of inhibiting growth of the host bacterium Mycobacterium smegmatis. Half of these are related to growth inhibitors previously identified in related phage Waterfoul, supporting their functional conservation; the other genes represent novel additions to the list of known antimycobacterial growth inhibitors. This work, conducted as part of the HHMI-supported Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists (SEA-GENES) project, highlights the value of parallel, comprehensive overexpression screens in exploring genome-wide patterns of phage gene function and novel interactions between phages and their hosts.

Connexin 43 confers chemoresistance through activating PI3K.

Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1-a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide-inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit ß (PIK3CB, also called PI3Kß or p110ß), suggesting that Cx43 activates PIK3CB/p110ß independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110ß, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110ß-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110ß together is an effective therapeutic approach for overcoming chemoresistance.

Probing the Conformation-Dependent Preferential Binding of Ethanol to Cationic Glycylalanylglycine in Water/Ethanol by Vibrational and NMR Spectroscopy.

The conformational propensity of amino acid residues is determined by an intricate balance of peptide-solvent and solvent-solvent interactions. To explore how the systematic replacement of water by a cosolvent affects the solvation of both the amino acid backbone and side chains, we performed a combined vibrational spectroscopy and NMR study of cationic glycylalanylglycine (GAG) in different ethanol/water mixtures of between 0 and 42 mol percent ethanol. Classical model peptide N'-methylacetamide was used as a reference system to probe solvent-induced spectroscopic changes. The alanine residue of GAG in water is known to exhibit a very high propensity for polyproline II (pPII). Adding up to 30 mol % ethanol at room temperature leads only to minor changes in the Ramachandran distribution of alanine, which mostly changes within the individual conformational subspaces. A further increase in the ethanol fractions leads to a destabilization of pPII and a stabilization of ß-strand conformations. At higher temperatures, different degrees of enthalpy-entropy compensations lead to a much stronger influence of ethanol on the peptide's conformational distribution. Ethanol-induced changes in chemical shifts and amide I wavenumbers strongly suggest that ethanol replaces water preferentially in the solvation shell of the polar C-terminal peptide group and of the alanine side chain, whereas the N-terminal group remains mostly hydrated. Furthermore, we found that ethanol interacts more strongly with the peptide if the latter adopts ß-strand conformations. This leads to an unusual positive temperature coefficient for the chemical shift of the C-terminal amide proton. Our data suggests a picture in which GAG eventually accumulates at water-ethanol interfaces if the ethanol fractions exceed 0.3, which explains why the further addition of ethanol eventually causes self-aggregation and the subsequent formation of a hydrogel.