This study was supported by Public Health Service grant AI-34431 (to D.A.H.) from the National Institute of Allergy and Infectious Diseases and by VA Medical Research Funds (to D.A.H.).

1. Fixation of Leptospira to glass slides
<ol>
<li>Leptospira interrogans is a class BSL2 pathogen. Working with live cells it requires appropriate handling, such as wearing gloves, lab-coat and pipetting steps in a sterile-hood.</li>
<li>Grow Leptospira in EMJH medium11, supplemented with 1% rabbit serum at 30&deg; C until they reach mid- to late-log phase (density of 5 x 107 to 5 x 108 cells/ml) for approximately 6 days.</li>
<li>Harvest the culture by centrifugation at &tilde;2000 x g ...

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Our surface IFA technique is similar to that used to demonstrate the surface exposure of various borrelial 15, 16 and leptospiral 12, 17 proteins. The details of the surface IFA method described here are designed to minimize the manipulation of cells in an effort to maintain outer membrane integrity while taking advantage of the tendency of Leptospira cells to adhere to glass slides. The method involves a lower concentration (2% versus 4%) of paraformaldehyde, washing with outer-membrane stabilizin...

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>Two-well chamber glass slides</td>
 <td>Lab-Tek</td>
 <td>177380

</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Rabbit serum</td>
 <td>Rockland Immunochemicals</td>
 <td>D209-00-0100</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Leptospira Enrichment EMJH</td>
 <td>BD Difco</td>
 <td>279510</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>A-11034</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>A-11029</td>
 <td>&nbsp;</td>
 </tr>

 <tr>
 	<td>4&#x2019;6-diamidino-2-phenyl-indole dihydrochloride (DAPI)</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>D1306</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>ProLong Gold anti-fade mounting medium</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>P36930</td>
 <td>Equilibrate to room temperature before use</td>
 </tr>
 <tr>
 	<td>Fisherfinest Premium Cover Glass</td>
 <td>Fisher</td>
 <td>12-544-14</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Fluorescence microscope</td>
 <td>Zeiss</td>
 <td>Axioskop 40</td>
 <td>&nbsp;</td>
 </tr>
 

 </tbody>
</table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

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 &beta;-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.

Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

immuno-fluorescence-assay-of-leptospiral-surface-exposed-proteins

Research

JoVE Journal

Immunology and Infection

15.6K 조회수.  University of California, Los Angeles. leptospiral 단백질의 표면 노출을 평가하기위한 효율적인 방법이 설명되어 있습니다. 방법은 특별히 leptospiral 세포의 연약한 바깥 막의 중단을 방지하도록 설계되었습니다. 이 기술은 몇 가지 부정적인 컨트롤의 고용은 바깥 막과 항체 반응의 특이성의 무결성을 평가해야합니다.