Bicarbonate enhances expression of the endocarditis and biofilm associated pilus locus, ebpR-ebpABC, in Enterococcus faecalis.

BACKGROUND: We previously identified ebpR, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the Enterococcus faecalis endocarditis and biofilm associated pilus operon, ebpABC. Although ebpR is not absolutely essential for ebpABC expression (100-fold reduction), its deletion led to phenotypes similar to those of an ebpABC mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated ebpABC mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the ebpR-ebpABC locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the ebpR-ebpABC locus. RESULTS: In this study, we examined the effect of CO2/HCO3(-), pH, and the Fsr system on the ebpR-ebpABC locus expression. The presence of 5% CO2/0.1 M HCO3(-) increased ebpR-ebpABC expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the ebpR-ebpABC locus expression appears independent of the effects of CO2(-) bicarbonate. Furthermore, by using an ebpA::lacZ fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO2, induced ebpA expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, P < 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with ebpA mRNA levels under the conditions tested. CONCLUSIONS: This study reports that the ebp locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, E. faecalis joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO2.

Interaction between cell division proteins FtsE and FtsZ.

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.

A trilocus sequence typing scheme for hospital epidemiology and subspecies differentiation of an important nosocomial pathogen, Enterococcus faecalis.

In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes, ace and salA (encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance, lsa (encoding a putative ABC transporter), for subspecies differentiation of Enterococcus faecalis. Each of the alleles was analyzed using 50 E. faecalis isolates representing 42 diverse multilocus sequence types (ST(M); based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis [PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.

Post-translational regulation of an Aplysia glutamate transporter during long-term facilitation.

Regulation of glutamate transporters accompanies plasticity of some glutamatergic synapses. The regulation of glutamate uptake at the Aplysia sensorimotor synapse during long-term facilitation (LTF) was investigated. Previously, increases in levels of ApGT1 (Aplysia glutamate transporter 1) in synaptic membranes were found to be related to long-term increases in glutamate uptake. In this study, we found that regulation of ApGT1 during LTF appears to occur post-translationally. Serotonin (5-HT) a transmitter that induces LTF did not increase synthesis of ApGT1. A pool of ApGT1 appears to exist in sensory neuron somata, which is transported to the terminals by axonal transport. Blocking the rough endoplasmic reticulum-Golgi-trans-Golgi network (TGN) pathway with Brefeldin A prevented the 5-HT-induced increase of ApGT1 in terminals. Also, 5-HT produced changes in post-translational modifications of ApGT1 as well as changes in the levels of an ApGT1-co-precipitating protein. These results suggest that regulation of trafficking of ApGT1 from the vesicular trafficking system (rough endoplasmic reticulum-Golgi-TGN) in the sensory neuron somata to the terminals by post-translational modifications and protein interactions appears to be the mechanism underlying the increase in ApGT1, and thus, glutamate uptake during memory formation.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

NOVEL AMINO ACID TRANSPORTER-TARGETED RADIOTRACERS FOR BREAST CANCER IMAGING

Breast cancer is the most common malignancy among women in the world. Its 5-year survival rate ranges from 23.4% in patients with stage IV to 98% in stage I disease, highlighting the importance of early detection and diagnosis. 18F-2-Fluoro-2-deoxy-glucose (18F-FDG), using positron emission tomography (PET), is the most common functional imaging tool for breast cancer diagnosis currently. Unfortunately, 18F-FDG-PET has several limitations such as poorly differentiating tumor tissues from inflammatory and normal brain tissues. Therefore, 18F-labeled amino acid-based radiotracers have been reported as an alternative, which is based on the fact that tumor cells uptake and consume more amino acids to sustain their uncontrolled growth. Among those radiotracers, 18F-labeled tyrosine and its derivatives have shown high tumor uptake and great ability to differentiate tumor tissue from inflammatory sites in brain tumors and squamous cell carcinoma. They enter the tumor cells via L-type amino acid transporters (LAT), which were reported to be highly expressed in many cancer cell lines and correlate positively with tumor growth. Nevertheless, the low radiosynthesis yield and demand of an on-site cyclotron limit the use of 18F-labeled tyrosine analogues. In this study, four Technetium-99m (99mTc) labeled tyrosine/ AMT (α-methyl tyrosine)-based radiotracers were successfully synthesized and evaluated for their potentials in breast cancer imaging. In order to radiolabel tyrosine and AMT, the chelators N,N’-ethylene-di-L-cysteine (EC) and 1,4,8,11-tetra-azacyclotetradecane (N4 cyclam) were selected to coordinate 99mTc. These chelators have been reported to provide stable chelation ability with 99mTc. By using the chelator technology, the same target ligand could be labeled with different radioisotopes for various imaging modalities for tumor diagnosis, or for internal radionuclide therapy in future. Based on the in vitro and in vivo evaluation using the rat mammary tumor models, 99mTc-EC-AMT is considered as the most suitable radiotracer for breast cancer imaging overall, however, 99mTc-EC-Tyrosine will be more preferred for differential diagnosis of tumor from inflammation.

Design, Synthesis and Development of Transporter Targeting Agents for Image-guided Therapy and Drug Delivery

The purpose of this study was to design, synthesize and develop novel transporter targeting agents for image-guided therapy and drug delivery. Two novel agents, N4-guanine (N4amG) and glycopeptide (GP) were synthesized for tumor cell proliferation assessment and cancer theranostic platform, respectively. N4amG and GP were synthesized and radiolabeled with 99mTc and 68Ga. The chemical and radiochemical purities as well as radiochemical stabilities of radiolabeled N4amG and GP were tested. In vitro stability assessment showed both 99mTc-N4amG and 99mTc-GP were stable up to 6 hours, whereas 68Ga-GP was stable up to 2 hours. Cell culture studies confirmed radiolabeled N4amG and GP could penetrate the cell membrane through nucleoside transporters and amino acid transporters, respectively. Up to 40% of intracellular 99mTc-N4amG and 99mTc-GP was found within cell nucleus following 2 hours of incubation. Flow cytometry analysis revealed 99mTc-N4amG was a cell cycle S phase-specific agent. There was a significant difference of the uptake of 99mTc-GP between pre- and post- paclitaxel-treated cells, which suggests that 99mTc-GP may be useful in chemotherapy treatment monitoring. Moreover, radiolabeled N4amG and GP were tested in vivo using tumor-bearing animal models. 99mTc-N4amG showed an increase in tumor-to-muscle count density ratios up to 5 at 4 hour imaging. Both 99mTc-labeled agents showed decreased tumor uptake after paclitaxel treatment. Immunohistochemistry analysis demonstrated that the uptake of 99mTc-N4amG was correlated with Ki-67 expression. Both 99mTc-N4amG and 99mTc-GP could differentiate between tumor and inflammation in animal studies. Furthermore, 68Ga-GP was compared to 18F-FDG in rabbit PET imaging studies. 68Ga-GP had lower tumor standardized uptake values (SUV), but similar uptake dynamics, and different biodistribution compared with 18F-FDG. Finally, to demonstrate that GP can be a potential drug carrier for cancer theranostics, several drugs, including doxorubicin, were selected to be conjugated to GP. Imaging studies demonstrated that tumor uptake of GP-drug conjugates was increased as a function of time. GP-doxorubicin (GP-DOX) showed a slow-release pattern in in vitro cytotoxicity assay and exhibited anti-cancer efficacy with reduced toxicity in in vivo tumor growth delay study. In conclusion, both N4amG and GP are transporter-based targeting agents. Radiolabeled N4amG can be used for tumor cell proliferation assessment. GP is a potential agent for image-guided therapy and drug delivery.