1H-MRS Quantitation of Age-Dependent Taurine Changes in Mouse Brain.

PURPOSE: A mouse model of Alzheimer's disease demonstrates reduced beta-amyloid levels in the whole brain, associated with a gain of hippocampal memory, after drinking taurine-enriched water; this suggests that a taurine supplement could be a promising treatment for cognitive deficit. The objective of this study is to establish a methodology for quantifying taurine in the whole brain, taking advantage of the rapid development of non-invasive imaging techniques such as magnetic resonance imaging and magnetic resonance spectroscopy (MRS). PROCEDURES: Single-voxel proton MRS was used to obtain quantifiable taurine peaks at 3.25 and 3.43 ppm. Quantitative MRS results were obtained in C57BL/6 mice of various age groups: 4, 11, 18, and 27 months old. RESULTS: Compared with the 4-month-old group, taurine levels dropped significantly only at 27 months of age (p = 0.03). However, a significant decrease of N-acetyl-aspartate (NAA) in the brain was observed at both 18 and 27 months (p = 0.03 and p = 0.02). In addition, MRS-measured taurine level is highly correlated with hippocampal volume (r = 0.95). CONCLUSIONS: These results suggest that decreased taurine levels in the brain could be used as biomarkers for hippocampal changes and are fully translatable into putative cognitive loss in both animal models and human studies without the ex vivo approach.

Francisella-arthropod vector interaction and its role in patho-adaptation to infect mammals.

Francisella tularensis is a Gram-negative, intracellular, zoonotic bacterium, and is the causative agent of tularemia with a broad host range. Arthropods such as ticks, mosquitoes, and flies maintain F. tularensis in nature by transmitting the bacteria among small mammals. While the tick is largely believed to be a biological vector of F. tularensis, transmission by mosquitoes and flies is largely believed to be mechanical on the mouthpart through interrupted feedings. However, the mechanism of infection of the vectors by F. tularensis is not well understood. Since F. tularensis has not been localized in the salivary gland of the primary human biting ticks, it is thought that bacterial transmission by ticks is through mechanical inoculation of tick feces containing F. tularensis into the skin wound. Drosophila melanogaster is an established good arthropod model for arthropod vectors of tularemia, where F. tularensis infects hemocytes, and is found in hemolymph, as seen in ticks. In addition, phagosome biogenesis and robust intracellular proliferation of F. tularensis in arthropod-derived cells are similar to that in mammalian macrophages. Furthermore, bacterial factors required for infectivity of mammals are often required for infectivity of the fly by F. tularensis. Several host factors that contribute to F. tularensis intracellular pathogenesis in D. melanogaster have been identified, and F. tularensis targets some of the evolutionarily conserved eukaryotic processes to enable intracellular survival and proliferation in evolutionarily distant hosts.

Host factors required for modulation of phagosome biogenesis and proliferation of Francisella tularensis within the cytosol.

Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of approximately 21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals.

Molecular bases of proliferation of Francisella tularensis in arthropod vectors.

Arthropod vectors are important vehicles for transmission of Francisella tularensis between mammals, but very little is known about the F. tularensis-arthropod vector interaction. Drosophila melanogaster has been recently developed as an arthropod vector model for F. tularensis. We have shown that intracellular trafficking of F. tularensis within human monocytes-derived macrophages and D. melanogaster-derived S2 cells is very similar. Within both evolutionarily distant host cells, the Francisella-containing phagosome matures to a late endosome-like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol where the bacterial proliferate. To decipher the molecular bases of intracellular proliferation of F. tularensis within arthropod-derived cells, we screened a comprehensive library of mutants of F. tularensis ssp. novicida for their defect in intracellular proliferation within D. melanogaster-derived S2 cells. Our data show that 394 genes, representing 22% of the genome, are required for intracellular proliferation within D. melanogaster-derived S2 cells, including many of the Francisella Pathogenicity Island (FPI) genes that are also required for proliferation within mammalian macrophages. Functional gene classes that exhibit growth defect include metabolic (25%), FPI (2%), type IV pili (1%), transport (16%) and DNA modification (5%). Among 168 most defective mutants in intracellular proliferation in S2 cells, 80 are defective in lethality and proliferation within adult D. melanogaster. The observation that only 135 of the 394 mutants that are defective in S2 cells are also defective in human macrophages indicates that F. tularensis utilize common as well as distinct mechanisms to proliferate within mammalian and arthropod cells. Our studies will facilitate deciphering the molecular aspects of F. tularensis-arthropod vector interaction and its patho-adaptation to infect mammals.

Intracellular fate of Francisella tularensis within arthropod-derived cells.

Since transmission of Francisella tularensis into the mammalian host occurs via arthropod vectors such as ticks, mosquitoes, horseflies and deerflies, recent studies have established Drosophila melanogaster as an arthropod vector model system. Nothing is known about the intracellular fate of F. tularensis within arthropod-derived cells, and the role of this host-parasite adaptation in the evolution of this pathogen to infect mammals. In this report, we explored intracellular trafficking of F. tularensis ssp. novicida in D. melanogaster-derived S2 cells. First, we show that similar to the F. tularensis ssp. holarctica-derived LVS strain, F. tularensis ssp. novicida is highly infectious, replicates exponentially within S2 cells and within adult flies, and is fatal to adult fruit flies in a dose-dependent manner, while the iglC, iglD and mglA mutants are defective. Using electron and fluorescence microscopy-based phagosome integrity assays, we show that the wild-type strain escapes into the cytosol of S2 cells within 30-60 min post infection and by 6 h, 90% were cytosolic. In contrast, approximately 40-50% of the iglC and iglD mutants escape into the cytosol by 6 h while the other subpopulation becomes enclosed within multilamellar vesicles (MLVs). Pre-treatment of S2 cells with the autophagy inhibitor methyl adenine blocks formation of the MLVs and all the vacuolar subpopulation of the iglC and iglD mutant bacteria become enclosed within single membrane-surrounded vacuoles. Endocytic trafficking studies of F. tularensis within S2 cells show transient colocalization of the bacterial phagosome with D. melanogaster LAMP2-GFP fusion but not with lysosomes pre-loaded with fluorescent dextran. Our data show that MLVs harbouring the iglC mutant acquire Lamp2 and dextran while MLVs harbouring the iglD mutant exclude these late endosomal and lysosomal markers. Our data indicate crucial differences in the role of the pathogenicity island-encoded proteins in modulating intracellular trafficking within human macrophages and arthropod vector-derived cells.

The Moraxella catarrhalis outer membrane protein CD contains two distinct domains specifying adherence to human lung cells.

Most Moraxella catarrhalis isolates express a highly-conserved outer membrane protein of 453 residues designated OMPCD, which has been previously shown to mediate binding to A549 human lung cells. Here, it is reported that two distinct domains of the M. catarrhalis strain O35E OMPCD protein specify adherence. Truncated proteins were expressed in Escherichia coli to demonstrate that OMPCD residues 1-240 as well as 241-400 are important for attachment to A549 cells, and database searches indicated that amino acids 285-299 resemble an adhesive motif found in eukaryotic proteins termed thrombospondin-type 3 repeat (TT3R). Cellular enzyme-linked immunosorbent assay using His-tagged proteins demonstrated that residues 236-300 of OMPCD, containing the TT3R motif, specify adhesive properties. Furthermore, these assays revealed that a purified protein encompassing residues 16-236 binds to A549 cells. The two cell-binding domains of OMPCD were further defined to amino acids 16-150 and 261-300 by utilizing a surface-display system, which was constructed from the M. catarrhalis autotransporter protein McaP, to express foreign peptides on the surface of recombinant bacteria.

The Moraxella catarrhalis autotransporter McaP is a conserved surface protein that mediates adherence to human epithelial cells through its N-terminal passenger domain.

The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded beta-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen.