Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.

For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection.

Allosteric competitive inhibitors of the glucose-1-phosphate thymidylyltransferase (RmlA) from Pseudomonas aeruginosa.

Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.

Tetrahydrodipicolinate N-succinyltransferase and dihydrodipicolinate synthase from Pseudomonas aeruginosa: structure analysis and gene deletion.

The diaminopimelic acid pathway of lysine biosynthesis has been suggested to provide attractive targets for the development of novel antibacterial drugs. Here we report the characterization of two enzymes from this pathway in the human pathogen Pseudomonas aeruginosa, utilizing structural biology, biochemistry and genetics. We show that tetrahydrodipicolinate N-succinyltransferase (DapD) from P. aeruginosa is specific for the L-stereoisomer of the amino substrate L-2-aminopimelate, and its D-enantiomer acts as a weak inhibitor. The crystal structures of this enzyme with L-2-aminopimelate and D-2-aminopimelate, respectively, reveal that both compounds bind at the same site of the enzyme. Comparison of the binding interactions of these ligands in the enzyme active site suggests misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition. P. aeruginosa mutants where the dapA gene had been deleted were viable and able to grow in a mouse lung infection model, suggesting that DapA is not an optimal target for drug development against this organism. Structure-based sequence alignments, based on the DapA crystal structure determined to 1.6 Å resolution revealed the presence of two homologues, PA0223 and PA4188, in P. aeruginosa that could substitute for DapA in the P. aeruginosa PAO1ΔdapA mutant. In vitro experiments using recombinant PA0223 protein could however not detect any DapA activity.

Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system.

BACKGROUND: Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors. METHODS: To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)). The resulting fusion proteins were individually tested for their ability to repress transcription of the constitutively active hypoxanthine phosphoribosyltransferase (HPRT) promoter. In addition, compatibility with the commonly used reverse tetracycline-controlled transactivator system (rtTA-system) and responsiveness to the pharmacological effector doxycycline (DOX) were evaluated. Finally, inducibility, effector-dependent promoter activity and the modification of histone H3 and H4 of the active versus the repressed target promoter were determined. RESULTS: Fusion of the human deacetylase 4 (HDAC4) carboxy-terminal silencing domain to TetR (B/E) resulted in a functional transcriptional repressor. This novel repressor, termed tTS-H4, efficiently reduced the activity of the murine HPRT promoter and a constitutively active human cytomegalovirus (hCMV) minimal promoter. Furthermore, combining tTS-H4 with the rtTA transcriptional activator allowed for grading, turning off and resuming target gene expression over several orders of magnitude without background. CONCLUSIONS: The tTS-H4 repressor is compatible with the commonly used rtTA transcriptional activation system and is a versatile new tool for tightly and adjustably regulating conditional gene expression.

Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes, and c-kit-expressing lineage-negative hematopoietic cells.

The stem cell leukemia gene SCL, also known as TAL-1, encodes a basic helix-loop-helix transcription factor expressed in erythroid, myeloid, megakaryocytic, and hematopoietic stem cells. To be able to make use of the unique tissue-restricted and spatio-temporal expression pattern of the SCL gene, we have generated a knock-in mouse line containing the tTA-2S tetracycline transactivator under the control of SCL regulatory elements. Analysis of this mouse using different tetracycline-dependent reporter strains demonstrated that switchable transgene expression was restricted to erythrocytes, megakaryocytes, granulocytes, and, importantly, to the c-kit-expressing and lineage-negative cell fraction of the bone marrow. In addition, conditional transgene activation also was detected in a very minor population of endothelial cells and in the kidney. However, no activation of the reporter transgene was found in the brain of adult mice. These findings suggested that the expression of tetracycline-responsive reporter genes recapitulated the known endogenous expression pattern of SCL. Our data therefore demonstrate that exogenously inducible and reversible expression of selected transgenes in myeloid, megakaryocytic, erythroid, and c-kit-expressing lineage-negative bone marrow cells can be directed through SCL regulatory elements. The SCL knock-in mouse presented here represents a powerful tool for studying normal and malignant hematopoiesis in vivo.

Unaltered prion protein cleavage in plasminogen-deficient mice.

In normal brains and cultured cells, cellular prion protein (PrP) is partially found as N-terminally truncated fragments, designated C1 and C2. The cleavage of recombinant PrP to a fragment corresponding to C1 can be mediated by the protease plasmin (Pln) in vitro, suggesting that plasmin might be responsible for the generation of the C1 fragment in vivo as well. The cleavage pattern of PrP found in both brain lysates and other tissues of plasminogen knock-out mice, however, is unaltered. The presence of C1 fragment in homogenates from plasminogen-deficient mice in a comparable ratio with full-length PrP as can be found in wild-type animals indicates that other proteases in addition to plasmin are responsible for PrP cleavage in vivo.

Premature senescence is a primary fail-safe mechanism of ERBB2-driven tumorigenesis in breast carcinoma cells.

The receptor tyrosine kinase ERBB2 plays a central role in the development of breast cancer and other epithelial malignancies. Elevated ERBB2 activity is believed to transform cells by transmitting mitogenic and antiapoptotic signals. Here we show that tightly regulated overexpression of oncogenic ERBB2 in human breast carcinoma cells does not stimulate proliferation but provokes premature senescence, accompanied by up-regulation of the cyclin-dependent kinase inhibitor P21(WAF1/CIP1). A similar effect was caused by retrovirus-mediated overexpression of oncogenic ERBB2 in low-passage murine embryonic fibroblasts. In contrast to previous observations based on constitutively overexpressing cell lines, P21 induced by tetracycline-regulated ERBB2 localizes to the nucleus in arrested cells. P21 up-regulation seems to be independent of the P53 tumor suppressor protein, and senescence-associated phenotypic alterations are reversed by specific inhibition of P38 mitogen-activated protein kinases. Functional inactivation of P21 by antisense oligonucleotides is sufficient to prevent cell cycle arrest as well as the senescent phenotype, thereby identifying the P21 protein as the key mediator of hypermitogenic cell cycle arrest and premature senescence in breast carcinoma cells. Our results may thus indicate that premature senescence represents an inherent anticarcinogenic program during ERBB2-driven mammary tumorigenesis. We propose a multistep model for the process of malignant transformation by ERBB2 wherein secondary lesions either target P21 or downstream effectors of senescence to bypass this primary fail-safe mechanism.

Of mice and models: improved animal models for biomedical research.

The ability to engineer the mouse genome has profoundly transformed biomedical research. During the last decade, conventional transgenic and gene knockout technologies have become invaluable experimental tools for modeling genetic disorders, assigning functions to genes, evaluating drugs and toxins, and by and large helping to answer fundamental questions in basic and applied research. In addition, the growing demand for more sophisticated murine models has also become increasingly evident. Good state-of-principle knowledge about the enormous potential of second-generation conditional mouse technology will be beneficial for any researcher interested in using these experimental tools. In this review we will focus on practice, pivotal principles, and progress in the rapidly expanding area of conditional mouse technology. The review will also present an internet compilation of available tetracycline-inducible mouse models as tools for biomedical research (http://www.zmg.uni-mainz.de/tetmouse/).