Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells.

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.

Selective Inhibition of Neisseria gonorrhoeae by a Dithiazoline in Mixed Infections with Lactobacillus gasseri.

The Gram-negative human pathogen Neisseria gonorrhoeae has progressively developed resistance to antibiotic monotherapies, and recent failures of dual-drug therapy have heightened concerns that strains resistant to all available antibiotics will begin circulating globally. Targeting bacterial cell wall assembly has historically been effective at treating infections with N. gonorrhoeae, but as the effectiveness of ß-lactams (including cephalosporins) is challenged by increasing resistance, research has expanded into compounds that target the numerous other enzymes with roles in peptidoglycan metabolism. One example is the dithiazoline compound JNJ-853346 (DTZ), which inhibits the activity of an Escherichia coli serine protease l,d-carboxypeptidase (LdcA). Recently, the characterization of an LdcA homolog in N. gonorrhoeae revealed localization and activity differences from the characterized E. coli LdcA, prompting us to explore the effectiveness of DTZ against N. gonorrhoeae We found that DTZ is effective at inhibiting N. gonorrhoeae in all growth phases, unlike the specific stationary-phase inhibition seen in E. coli Surprisingly, DTZ does not inhibit gonococcal LdcA enzyme activity, and DTZ sensitivity is not significantly decreased in ldcA mutants. While effective against numerous N. gonorrhoeae strains, including recent multidrug-resistant isolates, DTZ is much less effective at inhibiting growth of the commensal species Lactobacillus gasseri DTZ treatment during coinfections of epithelial cells resulted in significant lowering of gonococcal burden and interleukin-8 secretion without significantly impacting recovery of viable L. gasseri This selective toxicity presents a possible pathway for the use of DTZ as an effective antigonococcal agent at concentrations that do not impact vaginal commensals.

Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion.

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady-state levels of two T4SS structural proteins were affected by a homolog of tail-specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem-loop structures contained within the 5' untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post-translational mechanisms controlling T4SS gene expression and DNA secretion.