Regulation and transcript analysis of the ceroperon responsible for quorum sensing in Rhodobacter sphaeroides 2.4.1

Rhodobacter sphaeroides 2.4.1 is a Gram negative facultative photoheterotrophic bacterium that has been shown to have an N-acyl homoserine lactone-based quorum sensing system called cer for c&barbelow;ommunity e&barbelow;scape r&barbelow;esponse. The cer ORFs are cerR, the transcriptional regulator, cerI, the autoinducer synthase and cerA , whose function is unknown. The autoinducer molecule, 7,8- cis-N-(tetradecenoyl) homoserine lactone, has been characterized. The objective of this study was to identify an environmental stimulus that influences the regulation of cerRAI and, to characterize transcription of the cer operon. ^ A cerR::lacZ transcriptional fusion was made and β-Galactosidase assays were performed in R. sphaeroides 2.4.1 strains, wild type, AP3 (CerI−) and AP4 (CerR−). The cerR::lacZ β-Galactosidase assays were used as an initial survey of the mode of regulation of the Cer system. A cerA::lacZ translational fusion was created and was used to show that cerA can be translated. The presence of 7,8-cis-N-(tetradecenoyl) homoserine lactone was detected from R. sphaeroides strains wild type and AP4 (CerR−) using a lasR::lacZ translational fusion autoinducer bioassay. The cerR::lacZ transcriptional fusion in R. sphaeroides 2.4.1 wild type was tested under different environmental stimuli, such as various carbon sources, oxygen tensions, light intensities and culture media to determine if they influence transcription of the cer ORFs. Although lacZ assay data implicated high light intensity at 100 W/m2 to stimulate cer transcription, quantitative Northern RNA data of the cerR transcript showed that low light intensity at 3 W/m2 is at least one environmental stimulus that induces cer transcription. This finding was supported by DNA microarray analysis. Northern analysis of the cerRAI transcript provided evidence that the cer ORFs are co-transcribed, and that the cer operon contains two additional genes. Bioinformatics was used to identify genes that may be regulated by the Cer system by identifying putative lux box homologue sequences in the presumed promoter region of these genes. Genes that were identified were fliQ, celB and calsymin, all implicated in interacting with plants. Primer extension was used to help localize cis-elements in the promoter region. The cerR::lacZ transcriptional fusion was monitored in a subset of different global DNA binding transcriptional regulator mutant strains of R. sphaeroides 2.4.1. Those regulators involved in maintaining an anaerobic photosynthetic lifestyle appeared to have an effect. Collectively, the data imply that R. sphaeroides 2.4.1 activates the Cer system when grown anaerobic photosynthetically at low light intensity, 3 W/m2, and it may be involved in an interaction with plants. ^

Comparison of OG1RF and an isogenic fsrB deletion mutant by transcriptional analysis: the Fsr system of Enterococcus faecalis is more than the activator of gelatinase and serine protease.

The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus the fsrB deletion mutant, expression of ef1617 to -1634 (eut-pdu orthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects.

REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections.

Mechanism of damage sensing and cell killing following the inhibition of nucleotide excision repair by fludarabine in quiescent cells

Inhibition of DNA repair by the nucleoside of fludarabine (F-ara-A) induces toxicity in quiescent human cells. The sensing and signaling mechanisms following DNA repair inhibition by F-ara-A are unknown. The central hypothesis of this project was that the mechanistic interaction of a DNA repair initiating agent and a nucleoside analog initiates an apoptotic signal in quiescent cells. The purpose of this research was to identify the sensing and signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A. Lymphocytes were treated with F-ara-A, to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Pre-incubation of lymphocytes with 3 μM F-ara-A inhibited DNA repair initiated by 2 J/m2 UV and induced greater than additive apoptosis after 24 h. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 μM aphidicolin considerably lowered the apoptotic response. ^ Wild-type quiescent cells showed a significant loss in viability than did cells lacking functional sensor kinase DNA-PKcs or p53 as measured by colony formation assays. The functional status of ATM did not appear to affect the apoptotic outcome. Immunoprecipitation studies showed an interaction between the catalytic sub-unit of DNA-PK and p53 following DNA repair inhibition. Confocal fluorescence microscopy studies have indicated the localization pattern of p53, DNA-PK and γ-H2AX in the nucleus following DNA damage. Foci formation by γ-H2AX was seen as an early event that is followed by interaction with DNA-PKcs. p53 serine-15 phosphorylation and accumulation were detected 2 h after treatment. Fas/Fas ligand expression increased significantly after repair inhibition and was dependent on the functional status of p53. Blocking the interaction between Fas and Fas ligand by neutralizing antibodies significantly rescued the apoptotic fraction of cells. ^ Collectively, these results suggest that incorporation of the nucleoside analog into repair patches is critical for cytotoxicity and that the DNA damage, while being sensed by DNA-PK, may induce apoptosis by a p53-mediated signaling mechanism. Based on the results, a model is proposed for the sensing of F-ara-A-induced DNA damage that includes γ-H2AX, DNA-PKcs, and p53. Targeting the cellular DNA repair mechanism can be a potential means of producing cytotoxicity in a quiescent population of neoplastic cells. These results also provide mechanistic support for the success of nucleoside analogs with cyclophosphamide or other agents that initiate excision repair processes, in the clinic. ^

The molecular mechanisms of sensing nucleoside analogue-induced DNA damage

Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematological malignancies. Upon being metabolized to their active triphosphate form, these agents are incorporated into DNA during replication or excision repair synthesis. Because DNA polymerases have a greatly decreased affinity for primers terminated by most nucleoside analogues, their incorporation causes stalling of replication forks. The molecular mechanisms that recognize blocked replication may contribute to drug resistance but have not yet been elucidated. Here, several molecules involved in sensing nucleoside analogue-induced stalled replication forks have been identified and examined for their contribution to drug resistance. ^ The phosphorylation of the DNA damage sensor, H2AX, was characterized in response to nucleoside analogues and found to be dependent on both time and drug concentration. This response was most evident in the S-phase fraction and was associated with an inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1-Cdc25A-Cdk2). Exposure of the Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), to cultures previously treated with nucleoside analogues caused increased apoptosis, clonogenic death, and a further log-order increase in H2AX phosphorylation, suggesting enhanced DNA damage. Ataxia-telangiectasia mutated (ATM) has been identified as a key DNA damage signaling kinase for initiating cell cycle arrest, DNA repair, and apoptosis while the Mre11-Rad50-Nbs1 (MRN) complex is known for its functions in double-strand break repair. Activated ATM and the MRN complex formed distinct nuclear foci that colocalized with phosphorylated H2AX after inhibition of DNA synthesis by the nucleoside analogues, gemcitabine, ara-C, and troxacitabine. Since double-strand breaks were undetectable, this response was likely due to stalling of replication forks. A similar DNA damage response was observed in human lymphocytes after exposure to ionizing radiation and in acute myelogenous leukemia blasts during therapy with the ara-C prodrug, CP-4055. Deficiencies in ATM, Mre11, and Rad50 led to a two- to five-fold increase in gemcitabine sensitivity, suggesting that these molecules contribute to drug resistance. Based on these results, a model is proposed for the sensing of nucleoside analogue-induced stalled replication forks that includes H2AX, ATM, and the Mre11-Rad50-Nbs1 complex. ^

A role for p53 in sensing DNA damage and triggering apoptotic responses to anticancer agents

The p53 tumor suppressor protein plays a major role in cellular responses to anticancer agents that target DNA. DNA damage triggers the accumulation of p53, resulting in the transactivation of genes, which induce cell cycle arrest to allow for repair of the damaged DNA, or signal apoptosis. The exact role that p53 plays in sensing DNA damage and the functional consequences remain to be investigated. The main goal of this project was to determine if p53 is directly involved in sensing DNA damage induced by anticancer agents and in mediating down-stream cellular responses. This was tested in two experimental models of DNA damage: (1) DNA strand termination caused by anticancer nucleoside analogs and (2) oxidative DNA damage induced by reactive oxygen species (ROS). Mobility shift assays demonstrated that p53 and DNA-PK/Ku form a complex that binds DNA containing the anticancer nucleoside analog gemcitabine monophosphate in vitro. Binding of the p53-DNA-PK/Ku complex to the analog-containing DNA inhibited DNA strand elongation. Furthermore, treatment of cells with gemcitabine resulted in the induction of apoptosis, which was associated with the accumulation of p53 protein, its phosphorylation, and nuclear localization, suggesting the activation of p53 to trigger apoptosis following gemcitabine induced DNA strand termination. The role of p53 as a DNA damage sensor was further demonstrated in response to oxidative DNA damage. Protein pull-down assays demonstrated that p53 complexes with OGG1 and APE, and binds DNA containing the oxidized DNA base 8-oxoG. Importantly, p53 enhances the activities of APE and OGG1 in excising the 8-oxoG residue as shown by functional assays in vitro. This correlated with the more rapid removal of 8-oxoG from DNA in intact cells with wild-type p53 exposed to exogenous ROS stress. Interestingly, persistent exposure to ROS resulted in the accelerated onset of apoptosis in cells with wild-type p53 when compared to isogenic cells lacking p53. Apoptosis in p53+/+ cells was associated with accumulation and phosphorylation of p53 and its nuclear localization. Taken together, these results indicate that p53 plays a key role in sensing DNA damage induced by anticancer nucleoside analogs and ROS, and in triggering down-stream apoptotic responses. This study provides new mechanistic insights into the functions of p53 in cellular responses to anticancer agents. ^