F-Type Pyocins Are Diverse Noncontractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa.

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3' end of the F-type pyocin cluster are divergent in sequence and likely determine bactericidal specificity. We use sequence similarities among these proteins to define eleven distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly reassorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins may have emerged earlier. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as antibacterial therapeutics. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes antibiotic-resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great need for the development of alternative therapeutics. In this study, we investigate one such potential therapeutic: F-type pyocins, which are bacteriocins naturally produced by P. aeruginosa that resemble noncontractile phage tails. We show that they are potent killers of P. aeruginosa and identify their probable bactericidal specificity determinants, which opens up the possibility of engineering them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well-characterized phage tails will greatly facilitate their development into effective antibacterials.

c-Src Phosphorylates and Inhibits the Function of the CIC Tumor Suppressor Protein.

Capicua (CIC) is a transcriptional repressor that counteracts activation of genes in response to receptor tyrosine kinase (RTK)/Ras/ERK signaling. Following activation of RTK, ERK enters the nucleus and serine-phosphorylates CIC, releasing it from its targets to permit gene expression. We recently showed that ERK triggers ubiquitin-mediated degradation of CIC in glioblastoma (GBM). In this study, we examined whether another important downstream effector of RTK/EGFR, the non-RTK c-Src, affects CIC repressor function in GBM. We found that c-Src binds and tyrosine-phosphorylates CIC on residue 1455 to promote nuclear export of CIC. On the other hand, CIC-mutant allele (CIC-Y1455F), that escapes c-Src-mediated tyrosine phosphorylation, remains localized to the nucleus and retains strong repressor function against CIC targets, the oncogenic transcription factors ETV1 and ETV5. Furthermore, we show that the orally available Src family kinase inhibitor, dasatinib, which prevents EGF-mediated tyrosine phosphorylation of CIC and attenuates elevated ETV1 and ETV5 levels, reduces viability of GBM cells and glioma stem cells (GSC), but not of their control cells with undetectable c-Src activity. In fact, GBM cells and GSC expressing the tyrosine-defective CIC mutant (Y1455F) lose sensitivity to dasatinib, further endorsing the effect of dasatinib on Src-mediated tyrosine phosphorylation of CIC. These findings elucidate important mechanisms of CIC regulation and provide the rationale to target c-Src alongside ERK pathway inhibitors as a way to fully restore CIC tumor suppressor function in neoplasms such as GBM. IMPLICATIONS: c-Src tyrosine-phosphorylates CIC exports to cytoplasm and inactivates its repressor function in GBM.

CIC protein instability contributes to tumorigenesis in glioblastoma.

Capicua (CIC) is a transcriptional repressor that counteracts activation of genes downstream of receptor tyrosine kinase (RTK)/Ras/ERK signaling. It is well-established that tumorigenesis, especially in glioblastoma (GBM), is attributed to hyperactive RTK/Ras/ERK signaling. While CIC is mutated in other tumors, here we show that CIC has a tumor suppressive function in GBM through an alternative mechanism. We find that CIC protein levels are negligible in GBM due to continuous proteasome-mediated degradation, which is mediated by the E3 ligase PJA1 and show that this occurs through binding of CIC to its DNA target and phosphorylation on residue S173. PJA1 knockdown increased CIC stability and extended survival using in-vivo models of GBM. Deletion of the ERK binding site resulted in stabilization of CIC and increased therapeutic efficacy of ERK inhibition in GBM models. Our results provide a rationale to target CIC degradation in Ras/ERK-driven tumors, including GBM, to increase efficacy of ERK inhibitors.