Measurement of the cellular deacetylase activity of SIRT1 on p53 via LanthaScreen® technology.

Upon genomic insult, the tumor suppressor p53 is phosphorylated and acetylated at specific serine and lysine residues, increasing its stability and transactivation function. Deacetylases, including the type III histone deacetylase SIRT1, remove acetyl groups from p53 and counterbalance acetyltransferase activity during a DNA damage response. This report describes a series of high-throughput LanthaScreen® time-resolved Förster resonance energy transfer (TR-FRET) immunoassays for detection of intracellular p53 phosphorylation of Ser15 and acetylation of Lys382 upon treatment with DNA damage agents, such as etoposide. These assays were used to measure the deacetylase activity of SIRT1 and/or Type I/II Histone deacetylases (HDACs). First, BacMam-mediated overexpression of SIRT1 resulted in dose-dependent deacetylation of GFP-p53 following etoposide treatment of U-2 OS cells, confirming that GFP-p53 serves as a SIRT1 substrate in this assay format. Further, overexpression of the acetyltransferase p300 via BacMam increased the acetylation of GFP-p53 at Lys382. Next, siRNA-mediated knockdown of SIRT1 resulted in increased GFP-p53 acetylation, indicating that endogenous SIRT1 activity can also be measured in U-2 OS cells. Consistent with these results, GFP-p53 acetylation was also increased upon treatment of cells with a small-molecule inhibitor of SIRT1, EX-527. The effect of this compound was dramatically increased when used in combination with chemotherapeutic drug and/or the HDAC inhibitor Trichostatin A, confirming a proposed synergistic mechanism of p53 deacetylation by SIRT1 and Type I/II HDACs. Taken together, the cellular assays described here can be used as high-throughput alternatives to traditional immunoassays such as western blotting for identifying pharmacological modulators of specific p53-modifying enzymes.

Development of the predictor HERG fluorescence polarization assay using a membrane protein enrichment approach.

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.

The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.

CesT is a multi-effector chaperone and recruitment factor required for the efficient type III secretion of both LEE- and non-LEE-encoded effectors of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is an intestinal attaching and effacing pathogen that utilizes a type III secretion system (T3SS) for the delivery of effectors into host cells. The chaperone CesT has been shown to bind and stabilize the type III translocated effectors Tir and Map in the bacterial cytoplasm prior to their delivery into host cells. In this study we demonstrate a role for CesT in effector recruitment to the membrane embedded T3SS. CesT-mediated effector recruitment was dependent on the presence of the T3SS membrane-associated ATPase EscN. EPEC DeltacesT carrying a C-terminal CesT variant, CesT(E142G), exhibited normal cytoplasmic Tir stability function, but was less efficient in secreting Tir, further implicating CesT in type III secretion. In vivo co-immunoprecipitation studies using CesT-FLAG containing EPEC lysates demonstrated that CesT interacts with Tir and EscN, consistent with the notion of CesT recruiting Tir to the T3SS. CesT was also shown to be required for the efficient secretion of several type III effectors encoded within and outside the locus of enterocyte effacement (LEE) in addition to Tir and Map. Furthermore, a CesT affinity column was shown to specifically retain multiple effector proteins from EPEC culture supernatants. These findings indicate that CesT is centrally involved in recruiting multiple type III effectors to the T3SS via EscN for efficient secretion, and functionally redefine the role of CesT in multiple type III effector interactions.

Structural characterization of the molecular platform for type III secretion system assembly.

Type III secretion systems (TTSSs) are multi-protein macromolecular 'machines' that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross 'needle-shaped' morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-A crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit 'ring' superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.

Regulation of type III secretion hierarchy of translocators and effectors in attaching and effacing bacterial pathogens.

Human enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and the mouse pathogen Citrobacter rodentium (CR) belong to the family of attaching and effacing (A/E) bacterial pathogens. They possess the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system. These pathogens secrete a number of proteins into culture media, including type III effector proteins and translocators that are required for the translocation of effectors into host cells. Preliminary evidence indicated that the LEE-encoded SepL and Rorf6/SepD may form a molecular switch that controls the secretion of translocators and effectors in CR. Here, we show that SepL and SepD indeed perform this function in A/E pathogens such as EHEC and EPEC. Their sepL and sepD mutants do not secrete translocators but exhibit enhanced secretion of effectors. We demonstrate that SepL and SepD interact with each other and that both SepL and SepD are localized to the bacterial membranes. Furthermore, we demonstrate that culture media influence the type III secretion profile of EHEC, EPEC, and CR and that low-calcium concentrations inhibit secretion of translocators but promote the secretion of effectors, similar to effects on type III secretion by mutations in sepL and sepD. However, the secretion profile of the sepD and sepL mutants is not affected by these culture conditions. Collectively, our results suggest that SepL and SepD not only are necessary for efficient translocator secretion in A/E pathogens but also control a switch from translocator to effector secretion by sensing certain environmental signals such as low calcium.

Structure and biochemical analysis of a secretin pilot protein.

The ability to translocate virulence proteins into host cells through a type III secretion apparatus (TTSS) is a hallmark of several Gram-negative pathogens including Shigella, Salmonella, Yersinia, Pseudomonas, and enteropathogenic Escherichia coli. In common with other types of bacterial secretion apparatus, the assembly of the TTSS complex requires the preceding formation of its integral outer membrane secretin ring component. We have determined at 1.5 A the structure of MxiM28-142, the Shigella pilot protein that is essential for the assembly and membrane association of the Shigella secretin, MxiD. This represents the first atomic structure of a secretin pilot protein from the several bacterial secretion systems containing an orthologous secretin component. A deep hydrophobic cavity is observed in the novel 'cracked barrel' structure of MxiM, providing a specific binding domain for the acyl chains of bacterial lipids, a proposal that is supported by our various lipid/MxiM complex structures. Isothermal titration analysis shows that the C-terminal domain of the secretin, MxiD525-570, hinders lipid binding to MxiM.

Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.