Иммуно-флуоресценции Проба лептоспирозный поверхности, подвергшихся воздействию белки
Summary
Эффективным методом для оценки поверхностной воздействия лептоспирозный белков описывается. Метод разработан специально, чтобы избежать разрушения хрупкой внешней мембраны лептоспирозный клеток. Этот метод требует применения нескольких отрицательных контролей для оценки целостности наружной мембраны и специфичность реакции антител.
Abstract
Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity.
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic β-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.
Protocol
Discussion
Наша поверхности IFA метод похож на что используется для демонстрации поверхностного воздействия различных borrelial 15, 16 и лептоспирозный 12, 17 белков. Детали метода поверхностных IFA, описанные здесь, чтобы свести к минимуму манипуляции клеток в попытке сохранить целостность вне…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Это исследование было поддержано Общественной службы здравоохранения грант АИ-34 431 (на ПРЗ) из Национального института аллергии и инфекционных заболеваний, а также В. А. Медицинский научно-исследовательские фонды (на ПРЗ).
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Two-well chamber glass slides | Lab-Tek | 177380 | |
| Rabbit serum | Rockland Immunochemicals | D209-00-0100 | |
| Leptospira Enrichment EMJH | BD Difco | 279510 | |
| Alexa Fluor 488 goat anti-rabbit IgG (H+L) | Invitrogen/Molecular Probes | A-11034 | |
| Alexa Fluor 488 goat anti-mouse IgG (H+L) | Invitrogen/Molecular Probes | A-11029 | |
| 4’6-diamidino-2-phenyl-indole dihydrochloride (DAPI) | Invitrogen/Molecular Probes | D1306 | |
| ProLong Gold anti-fade mounting medium | Invitrogen/Molecular Probes | P36930 | Equilibrate to room temperature before use |
| Fisherfinest Premium Cover Glass | Fisher | 12-544-14 | |
| Fluorescence microscope | Zeiss | Axioskop 40 |
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