PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 16554829

Imaging specific cell-surface proteolytic activity in single living cells.

John P Hobson·Shihui Liu·Birgitte Rønø·Stephen H Leppla·Thomas H Bugge
View original source ↗Export metadata ↓

Abstract

We describe a simple, sensitive and noninvasive assay that uses nontoxic, reengineered anthrax toxin-beta-lactamase fusion proteins with altered protease cleavage specificity to visualize specific cell-surface proteolytic activity in single living cells. The assay could be used to specifically image endogenous cell-surface furin, urokinase plasminogen activator and metalloprotease activity. We have adapted the assay for fluorescence microscopy, flow cytometry and fluorescent plate reader formats, and it is amenable for automation and high-throughput analysis.

Explore related subjects

Antigens, BacterialAutomationBacterial ProteinsBacterial ToxinsBiological AssayCell MembraneCells, CulturedFlow CytometryFurinMetalloproteasesMicroscopy, FluorescenceReceptors, Cell SurfaceReceptors, Urokinase Plasminogen ActivatorRecombinant Fusion ProteinsSubstrate Specificitybeta-LactamasesJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, N.I.H., IntramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

John P Hobson, Shihui Liu, Birgitte Rønø, Stephen H Leppla, Thomas H Bugge. 2006. Imaging specific cell-surface proteolytic activity in single living cells.. https://doi.org/10.1038/nmeth862

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2010

Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved.

Mycobacterium tuberculosis is an obligate human pathogen capable of persisting in individual hosts for decades. We sequenced the genomes of 21 strains representative of the global diversity and six major lineages of the M. tuberculosis complex (MTBC) at 40- to 90-fold coverage using Illumina next-generation DNA sequencing. We constructed a genome-wide phylogeny based on these genome sequences. Comparative analyses of the sequences showed, as expected, that essential genes in MTBC were more evolutionarily conserved than nonessential genes. Notably, however, most of the 491 experimentally confirmed human T cell epitopes showed little sequence variation and had a lower ratio of nonsynonymous to synonymous changes than seen in essential and nonessential genes. We confirmed these findings in an additional data set consisting of 16 antigens in 99 MTBC strains. These findings are consistent with strong purifying selection acting on these epitopes, implying that MTBC might benefit from recognition by human T cells.

Antigens, Bacterial↗
PubMed2007

Enterohaemorrhagic Escherichia coli O157 and non-O157 serovars differ in their mechanisms for iron supply.

Clinical isolates of enterohaemorrhagic Escherichia coli, both O157 and non-O157 serotypes, were investigated for siderophore production, for growth promotion by haem and esculetin in iron-restricted conditions, for production of enterohaemolysin and esculin hydrolase, and for the presence of the chuA and ehx genes by PCR. As expected, all the strains produced enterobactin, but the prevalence of other factors varied among the serovars tested. None of the O157 and O26 strains produced aerobactin or "colibactin", whereas among other enterohaemorrhagic E. coli non-O157 serovars the frequencies of aerobactin and "colibactin" production were similar to those of commensal E. coli strains. The ability to use ferric esculetin for growth in iron-limited media was markedly more prevalent among non-O157 serovars and less prevalent among O157 strains compared with commensal E. coli strains. Almost all O157, O26 and O103 strains expressed enterohaemolysin, compared with only 50% of other non-O157 strains. Similarly, almost all O157 and O26 strains utilised haem as a host iron source; the frequency of haem use by other non-O157 strains was generally lower and variable among serovars, such that none of the O103:H2 isolates tested used haem as an iron source. The gene chuA, which encodes the haem transport protein ChuA and which is prevalent in O157:H7 strains, was only rarely noted among non-O157 serovars of enterohaemorrhagic E. coli, even among isolates that could use haem as an iron source. Overall our data demonstrate that O157:H7 and non-O157 serovars, in particular O26:H(-)/H11 and O103:H2, use distinctly different strategies for obtaining iron, and suggest two evolutionary distinct lines of enterhaemorrhagic E. coli.

Antigens, Bacterial↗
PubMed2007

Disinfection of Legionella pneumophila by photocatalytic oxidation.

Photocatalytic oxidation (PCO) was proven to be efficacious in the inactivation of Legionella pneumophila serogroup 1 Strains 977, 1009, 1014 and ATCC 33153. The local (Strains 997, 1009 and 1014) and ATCC (Strain 33153) strains showed sensitivity differences towards PCO. The inactivation mechanisms of PCO were investigated by transmission and scanning electron microscopy by which PCO was found to disintegrate the cells eventually. Before the disintegration, there was lipid peroxidation of outer and cytoplasmic membrane causing holes formation and leading to the entry of OH into the cells to oxidize the intracellular components. Fatty acid profile analysis found that the amount of saturated, 16-carbon branched-chain fatty acid, which is predominant in Legionella, decreased in the surviving populations from PCO. A relationship between the amount of this fatty acid and the PCO sensitivity of the tested strains was also observed. Mineralization of cells by PCO was proven by total organic carbon analysis.

Antigens, Bacterial↗