KPC1-mediated ubiquitination and proteasomal processing of NF-κB1 p105 to p50 restricts tumor growth.

NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.

Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to intracellular S. Typhimurium.

Although Salmonella Typhimurium (STM) and Salmonella Paratyphi A (SPA) belong to the same phylogenetic species, share large portions of their genome and express many common virulence factors, they differ vastly in their host specificity, the immune response they elicit, and the clinical manifestations they cause. In this work, we compared their intracellular transcriptomic architecture and cellular phenotypes during human epithelial cell infection. While transcription induction of many metal transport systems, purines, biotin, PhoPQ and SPI-2 regulons was similar in both intracellular SPA and STM, we identified 234 differentially expressed genes that showed distinct expression patterns in intracellular SPA vs. STM. Surprisingly, clear expression differences were found in SPI-1, motility and chemotaxis, and carbon (mainly citrate, galactonate and ethanolamine) utilization pathways, indicating that these pathways are regulated differently during their intracellular phase. Concurring, on the cellular level, we show that while the majority of STM are non-motile and reside within Salmonella-Containing Vacuoles (SCV), a significant proportion of intracellular SPA cells are motile and compartmentalized in the cytosol. Moreover, we found that the elevated expression of SPI-1 and motility genes by intracellular SPA results in increased invasiveness of SPA, following exit from host cells. These findings demonstrate unexpected flagellum-dependent intracellular motility of a typhoidal Salmonella serovar and intriguing differences in intracellular localization between typhoidal and non-typhoidal salmonellae. We propose that these differences facilitate new cycles of host cell infection by SPA and may contribute to the ability of SPA to disseminate beyond the intestinal lamina propria of the human host during enteric fever.

The Typhi colonization factor (Tcf) is encoded by multiple non-typhoidal Salmonella serovars but exhibits a varying expression profile and interchanging contribution to intestinal colonization.

Salmonella enterica serovars Typhi and Paratyphi A are human-restricted pathogens and the leading causative agents of enteric fever. The Typhi colonization factor (Tcf) is a chaperone-usher fimbria, thought to play a role in the host-specificity of typhoidal serovars. Here we show that the tcf cluster (tcfABCD tinR tioA) is present in at least 25 non-typhoidal Salmonella (NTS) serovars and demonstrate its native expression in clinically-important serovars including Schwarzengrund, 9,12:l,v:-, Choleraesuis, Bredeney, Heidelberg, Montevideo, Virchow and Infantis. Although the genetic organization of the tcf cluster is well conserved, the N-terminal half of the fimbrial adhesin, TcfD is highly diverse, suggesting different binding properties of distinct tcfD variants. Comparison of tcfA expression in typhoidal and NTS serovars demonstrated unexpected differences in its expression profiles, with the highest transcription levels in S. Typhi, S. Choleraesuis and S. Infantis. In the latter, tcf is induced in rich broth and under microaerobic conditions, characterizing the intestines of warm blooded animals. Furthermore, Tcf is negatively regulated by the ancestral leucine-responsive transcriptional regulator (Lrp). Using the colitis mouse model, we demonstrate that during mice infection tcfA is expressed at higher levels by S. Infantis than S. Schwarzengrund or S. Heidelberg. Moreover, while Tcf is dispensable for S. Schwarzengrund and S. Heidelberg mouse colonization, Tcf is involved in cecum and colon colonization by S. Infantis. Taken together, our results establish that Tcf is broadly encoded by multiple NTS serovars, but presents variable expression profiles and contributes differently to their virulence.

Genetic and Phenotypic Characterization of a Salmonella enterica serovar Enteritidis Emerging Strain with Superior Intra-macrophage Replication Phenotype.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the ubiquitous Salmonella serovars worldwide and a major cause of food-born outbreaks, which are often associated with poultry and poultry derivatives. Here we report a nation-wide S. Enteritidis clonal outbreak that occurred in Israel during the last third of 2015. Pulsed field gel electrophoresis and whole genome sequencing identified genetically related strains that were circulating in Israel as early as 2008. Global comparison linked this outbreak strain to several clinical and marine environmental isolates that were previously isolated in California and Canada, indicating that similar strains are prevalent outside of Israel. Phenotypic comparison between the 2015 outbreak strain and other clinical and reference S. Enteritidis strains showed only limited intra-serovar phenotypic variation in growth in rich medium, invasion into Caco-2 cells, uptake by J774.1A macrophages, and host cell cytotoxicity. In contrast, significant phenotypic variation was shown among different S. Enteritidis isolates when biofilm-formation, motility, invasion into HeLa cells and uptake by THP-1 human macrophages were studied. Interestingly, the 2015 outbreak clone was found to possess superior intra-macrophage replication ability within both murine and human macrophages in comparison to the other S. Enteritidis strains studied. This phenotype is likely to play a role in the virulence and host-pathogen interactions of this emerging clone.