Novel Protein Kinase Signaling Systems Regulating Lifespan Identified by Small Molecule Library Screening Using Drosophila

Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity

Enterohaemorrhagic Escherichia coli and Shigella dysenteriae type 1-induced haemolytic uraemic syndrome

Haemolytic uraemic syndrome (HUS) can be classified according to the aetiology of the different disorders from which it is composed. The most prevalent form is that induced by shigatoxin producing Escherichia coli (STEC) and, in some tropical regions, by Shigella dysenteriae type 1. STEC cause a zoonosis, are widely distributed in nature, enter the food chain in different ways, and show regional differences. Not all STEC are human pathogens. Enterohaemorrhagic E. coli usually cause attachment and effacing lesions in the intestine. This is not essential, but production of a shigatoxin (Stx) is. Because Stx are encoded by a bacteriophage, this property is transferable to naïve strains. Laboratory methods have improved by identifying STEC either via the toxin or its bacteriophage. Shigella dysenteriae type 1 produces shigatoxin, identical to Stx-1, but also has entero-invasive properties that enterohaemorrhagic Escherichia coli (EHEC) do not. Shigella patients risk bacteremia and benefit from early antibiotic treatment, unlike those with EHEC

Active Suppression of Early Immune Response in Tobacco by the Human Pathogen Salmonella Typhimurium

The persistence of enteric pathogens on plants has been studied extensively, mainly due to the potential hazard of human pathogens such as Salmonella enterica being able to invade and survive in/on plants. Factors involved in the interactions between enteric bacteria and plants have been identified and consequently it was hypothesized that plants may be vectors or alternative hosts for enteric pathogens. To survive, endophytic bacteria have to escape the plant immune systems, which function at different levels through the plant-bacteria interactions. To understand how S. enterica survives endophyticaly we conducted a detailed analysis on its ability to elicit or evade the plant immune response. The models of this study were Nicotiana tabacum plants and cells suspension exposed to S. enterica serovar Typhimurium. The plant immune response was analyzed by looking at tissue damage and by testing oxidative burst and pH changes. It was found that S. Typhimurium did not promote disease symptoms in the contaminated plants. Live S. Typhimurium did not trigger the production of an oxidative burst and pH changes by the plant cells, while heat killed or chloramphenicol treated S. Typhimurium and purified LPS of Salmonella were significant elicitors, indicating that S. Typhimurium actively suppress the plant response. By looking at the plant response to mutants defective in virulence factors we showed that the suppression depends on secreted factors. Deletion of invA reduced the ability of S. Typhimurium to suppress oxidative burst and pH changes, indicating that a functional SPI1 TTSS is required for the suppression. This study demonstrates that plant colonization by S. Typhimurium is indeed an active process. S. Typhimurium utilizes adaptive strategies of altering innate plant perception systems to improve its fitness in the plant habitat. All together these results suggest a complex mechanism for perception of S. Typhimurium by plants

Hmgcr in the Corpus Allatum Controls Sexual Dimorphism of Locomotor Activity and Body Size via the Insulin Pathway in Drosophila

The insulin signaling pathway has been implicated in several physiological and developmental processes. In mammals, it controls expression of 3-Hydroxy-3-Methylglutaryl CoA Reductase (HMGCR), a key enzyme in cholesterol biosynthesis. In insects, which can not synthesize cholesterol de novo, the HMGCR is implicated in the biosynthesis of juvenile hormone (JH). However, the link between the insulin pathway and JH has not been established. In Drosophila, mutations in the insulin receptor (InR) decrease the rate of JH synthesis. It is also known that both the insulin pathway and JH play a role in the control of sexual dimorphism in locomotor activity. In studies here, to demonstrate that the insulin pathway and HMGCR are functionally linked in Drosophila, we first show that hmgcr mutation also disrupts the sexual dimorphism. Similarly to the InR, HMGCR is expressed in the corpus allatum (ca), which is the gland where JH biosynthesis occurs. Two p[hmgcr-GAL4] lines were therefore generated where RNAi was targeted specifically against the HMGCR or the InR in the ca. We found that RNAi-HMGCR blocked HMGCR expression, while the RNAi-InR blocked both InR and HMGCR expression. Each RNAi caused disruption of sexual dimorphism and produced dwarf flies at specific rearing temperatures. These results provide evidence: (i) that HMGCR expression is controlled by the InR and (ii) that InR and HMGCR specifically in the ca, are involved in the control of body size and sexual dimorphism of locomotor activity

A Functional Misexpression Screen Uncovers a Role for Enabled in Progressive Neurodegeneration

Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism

Isolation and Characterization of Cytotoxic, Aggregative Citrobacter freundii

Citrobacter freundii is an infrequent but established cause of diarrhea in humans. However, little is known of its genetic diversity and potential for virulence. We analyzed 26 isolates, including 12 from human diarrheal patients, 2 from human fecal samples of unknown diarrheal status, and 12 from animals, insects, and other sources. Pulsed field gel electrophoresis using XbaI allowed us to divide the 26 isolates into 20 pulse types, while multi-locus sequence typing using 7 housekeeping genes allowed us to divide the 26 isolates into 6 sequence types (STs) with the majority belonging to 4 STs. We analyzed adhesion and cytotoxicity to HEp-2 cells in these 26 strains. All were found to adhere to HEp-2 cells. One strain, CF74, which had been isolated from a goat, showed the strongest aggregative adhesion pattern. Lactate dehydrogenase (LDH) released from HEp-2 cells was evaluated as a measure of cytotoxicity, averaging 7.46%. Strain CF74 induced the highest level of LDH, 24.3%, and caused >50% cell rounding, detachment, and death. We named strain CF74 “cytotoxic and aggregative C. freundii.” Genome sequencing of CF74 revealed that it had acquired 7 genomic islands, including 2 fimbriae islands and a type VI secretion system island, all of which are potential virulence factors. Our results show that aggregative adherence and cytotoxicity play an important role in the pathogenesis of C. freundii

Induction of <i>Salmonella </i>enterotoxin (<i>stn</i>)<i> </i>gene expression by epithelial cells (IEC-6)

1101-1104Prevalence of Salmonella enterotoxin (stn) gene among Salmonella enterica and S. bongori was investigated by polymerase chain react ion (PCR) and gene probe and its status of phenotypic expression was examined on chinese hamster ovary cells by cultivating the strains with conventional method for enterotoxin production and by cultivating the organisms in contact with intestinal epithelial cells of rats (IEC-6). All the 19 strains and serovars of S. enterica such as Typhimurium, Enteritidis, Newport, Weltevreden, Indiana, Gallinarum and Kentucky were found to carry stn gene as examined by PCR and gene probe but only a limited number of strains (13 out of 19) expressed phenotypically the enterotoxin when cultured by conventional method. Cultivation of organisms in contact with epithelial cells induced expression of situ gene phenotypically in all the 19 strains. In contrast to S. enterica, strains of S. bongori were found neither genotypically (stn) nor phenotypically (stn) positive

Effect of norepinephrine on growth of <i>Salmonella </i>and its enterotoxin production

285-286Salmonella typhimurium was cultured in presence or absence of norepinephrine in conditioned media. Two conditioned media containing bovine and pig serum were prepared. Supplementation of fresh cultures with norepinephrine (5 × 10-5 M per mL of medium) resulted in ten-fold increase in growth as compared to controls. No significant difference in growth of organisms in media containing bovine and pig serum was observed. Growth was more in culture incubated under shaking condition than in non-shaking condition. Enterotoxin production increased by two to eight-folds in the medium supplemented with norepinephrine