'Small' talk: Opa proteins as mediators of Neisseria–host-cell communication

Hauck, Christof R.; Meyer, Thomas F.

doi:10.1016/s1369-5274(03)00004-3

Cited by 105 publications

(92 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…25 Different peptide sequences were selected based on the biological relevance (two regions in loops 2 and 3 are identified to be important in engaging host receptors 26 ), solubility, and amino acid composition. There are several hydrophobic residues in the loop regions, which dramatically affected the GRAVY value 27 and likely the solubility of many peptides that were considered.…”

Section: Facilitating Loop Assignments: Synthetic Peptidementioning

confidence: 99%

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Fox

Columbus

2013

Protein Science

View full text Add to dashboard Cite

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of b-barrel membrane proteins using Opa 60 from Neisseria gonorrhoeae as a model system. Opa 60 is an eight-stranded b-barrel with long extracellular loops (~63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa 60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the b-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized b-barrel. The removal of the loop resonances significantly improved the assignment of the Opa 60 b-barrel region (97% of the resonances corresponding to the b-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of b-barrel membrane proteins.

show abstract

Section: Facilitating Loop Assignments: Synthetic Peptidementioning

confidence: 99%

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Fox

Columbus

2013

Protein Science

View full text Add to dashboard Cite

show abstract

“…To analyse the potential of this flow cytometry approach to quantify intracellular bacteria, we took advantage of the well characterized uptake of Opa CEAexpressing gonococci by specific members of the human CEACAM family (see (Hauck and Meyer, 2003)). Previous investigations have established an essential role of the Opa CEA -CEACAM interaction for uptake into epithelial cells in vitro ( (Virji et al, 1996;Bos et al, 1997;Chen et al, 1997;Gray-Owen et al, 1997;Schmitter et al, 2004).…”

Section: Flow Cytometry To Detect Neisseria Gonorrhoeae Within Epithementioning

confidence: 99%

“…Listeria monocytogenes targets host E-cadherin by means of the InlA protein to enter epithelial cells (Mengaud et al, 1996;Lecuit et al, 1999). Variants of N. gonorrhoeae expressing distinct colony opacity-associated (Opa) proteins bind to receptors of the carcinoembyronic antigen-related cell adhesion molecule (CEACAM) family to direct their uptake by human cells (for review see (Hauck and Meyer, 2003)). Clearly, each pathogen seems to have evolved a specific set of virulence determinants which together orchestrate the sequence of events guiding the uptake by the host cell.…”

Section: Introductionmentioning

confidence: 99%

Quantification of bacterial invasion into adherent cells by flow cytometry

Pils

Schmitter

Neske

et al. 2006

Journal of Microbiological Methods

View full text Add to dashboard Cite

Quantification of invasive, intracellular bacteria is critical in many areas of cellular microbiology and immunology. We describe a novel and fast approach to determine invasion of bacterial pathogens in adherent cell types such as epithelial cells or fibroblasts based on flow cytometry. Using the CEACAM-mediated uptake of Opa-expressing Neisseria gonorrhoeae as a well-characterized model of bacterial invasion, we demonstrate that the flow cytometry-based method yields results comparable to a standard antibiotic protection assay. Furthermore, the quantification of intracellular bacteria by the novel approach is not biased by intracellular killing of the microbes and correctly discriminates between cell-associated extracellular and bona fide intracellular bacteria. As flow cytometry-based quantification is also applicable to other pathogen-host interactions such as the integrinmediated internalization of Staphylococcus aureus, this approach provides a fast and convenient alternative for the quantification of bacterial uptake and should be particularly useful in elucidating the molecular mechanisms of pathogen-triggered host cell invasion. D

show abstract

“…In many instances, invasive bacteria, including Listeria monocytogenes, Staphylococcus aureus, Shigella flexneri, Neisseria meningitidis and Neisseria gonorrhoeae exploit cellular adhesion molecules, such as cadherins, integrins, or members of the immunoglobulin superfamily of cell adhesion molecules (IgCAMs), to gain entry into the cell . In the case of the humanspecific pathogens N. gonorrhoeae (Ngo) and N. meningitidis (Nme), invasion can be mediated by colony opacity associated (Opa) proteins that are integral membrane proteins of the outer membrane of these Gram-negative bacteria (Hauck and Meyer, 2003). Whereas the meningococcal genome encodes up to 4 distinct Opa proteins, gonococci harbour up to 11 copies of Opa genes (Bhat et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

“…Besides a few Opa protein variants that recognize cell surface expressed heparansulfate proteoglycans (Opa HSPG ) (Chen et al, 1995;van Putten and Paul, 1995), most Opa proteins of diverse strains of Nme and Ngo recognize one or more members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family (Chen and Gotschlich, 1996;Gray-Owen et al, 1997a;Virji et al, 1996b). These Opa proteins bind to the non-glycosylated face of the immunoglobulin-variable (IgV)-like amino-terminal domain of CEACAMs and have been collectively referred to as Opa CEA (Hauck and Meyer, 2003). CEACAMs are composed of a single IgV-like amino-terminal domain followed by up to 6 Ig-constant (IgC2)-like domains (Kuespert et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Profiling of bacterial adhesin — host receptor recognition by soluble immunoglobulin superfamily domains

Kuespert¹,

Weibel²,

Hauck³

2007

Journal of Microbiological Methods

View full text Add to dashboard Cite

Several Gram-negative human pathogens recognize members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Pathogenic Neisseriae employ distinct isoforms of the colony opacity-associated proteins (Opa CEA proteins) to bind to the amino-terminal domains of CEACAMs. Here we present a novel approach to rapidly determine the CEACAM-binding properties of single bacteria. Expression of the isolated amino-terminal domains of various CEACAMs in eukaryotic cells yields soluble probes that selectively recognize Opa CEA -expressing bacteria in a pull-down assay format. Furthermore, by expressing soluble CEACAMs as fusions to green-fluorescent protein (CEACAM-N-GFP), CEACAM-binding bacteria can be decorated with a fluorescent label and analysed by flow cytometry allowing the specific detection of receptor binding events on the level of single bacteria. Besides its potential for rapid and quantitative analysis of pathogen-receptor interactions, this novel approach allows the detection of receptor recognition in heterogeneous bacterial populations and might represent a valuable tool for profiling the host binding capabilities of various microorganisms.

show abstract

'Small' talk: Opa proteins as mediators of Neisseria–host-cell communication

Cited by 105 publications

References 68 publications

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Quantification of bacterial invasion into adherent cells by flow cytometry

Profiling of bacterial adhesin — host receptor recognition by soluble immunoglobulin superfamily domains

Contact Info

Product

Resources

About