Two plasmid-encoded genes of enteropathogenic Escherichia coli strain K798 promote invasion and survival within HEp-2 cells.

Enteropathogenic Escherichia coli (EPEC) are considered to be extracellular pathogens, inducing attaching and effacing lesions following their attachment to the surface of eukaryotic cells; however, in vitro and in vivo invasion by EPEC has been reported in several studies. A cloned 4.6 kb fragment of EPEC plasmid pLV501 has been shown to facilitate invasion of E. coli K-12, and here we further investigate the nature of this process. Two of the three complete open reading frames contained within the plasmid fragment have been cloned to E. coli, and in HEp-2 adherence assays both tniA2 and pecM were shown to be expressed during the first 3 h of infection from a plac promoter. Escherichia coli transformants carrying pecM alone or in combination with tniA2 were able to both survive intracellularly and escape eukaryotic cells to re-establish themselves within the medium, whereas those bacterial cells carrying tniA2 alone could not be isolated from within HEp-2 cells after 24 h of infection, but were present in the previously sterile medium surrounding the cells. Bacteria carrying pecM and tniA2 adhered to HEp-2 cells with sites of adhesion characterized by underlying actin polymerization. The invasive potential conferred by these genes may give EPEC strains a survival advantage during prolonged infection.

Deubiquitinating enzyme Usp12 is a novel co-activator of the androgen receptor.

The androgen receptor (AR), a member of the nuclear receptor family, is a transcription factor involved in prostate cell growth, homeostasis, and transformation. AR is a key protein in growth and development of both normal and malignant prostate, making it a common therapeutic target in prostate cancer. AR is regulated by an interplay of multiple post-translational modifications including ubiquitination. We and others have shown that the AR is ubiquitinated by a number of E3 ubiquitin ligases, including MDM2, CHIP, and NEDD4, which can result in its proteosomal degradation or enhanced transcriptional activity. As ubiquitination of AR causes a change in AR activity or stability and impacts both survival and growth of prostate cancer cells, deubiquitination of these sites has an equally important role. Hence, deubiquitinating enzymes could offer novel therapeutic targets. We performed an siRNA screen to identify deubiquitinating enzymes that regulate AR; in that screen ubiquitin-specific protease 12 (Usp12) was identified as a novel positive regulator of AR. Usp12 is a poorly characterized protein with few known functions and requires the interaction with two cofactors, Uaf-1 and WDR20, for its enzymatic activity. In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, deubiquitinates the AR to enhance receptor stability and transcriptional activity. Our data show that Usp12 acts in a pro-proliferative manner by stabilizing AR and enhancing its cellular function.