The studies were funded by the Wellcome Trust and Meningitis UK. HBMEC cell line was provided by Dr K.S. Kim. Confocal imaging and electron microscopy were performed in the Wolfson Bioimaging Facility, University of Bristol. We would also like to thank Mr Alan Leard, Dr. Mark Jepson (University of Bristol), and Mr. Alan Tilley (PerkinElmer) for their help and advice.

1. Immunofluorescence Protocol
Seeding, Infection &amp; immuno-staining
The following procedures require suitable safety level tissue culture and microbiological laboratory facilities.
DAY 1
A.Preparation of target cells for infection
<ol>
	<li>Seed HBMECs at 50% confluence (~1.5x104 cells/cm2) on glass coverslips (16 mm diameter) placed into a 12-well plate (3.8 cm2 growth area/well). Growth medium: RPMI 1640 supplemented with 15% heat-inactivated (56 &deg;C, 30min) FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin/streptomycin, 1% (v/v) MEM non-essential amino acids solution and 1% MEM vitamins solution.</li>
	<li>Culture over night (O/N) at 37&deg;C, in a 5% CO2 incubator.</li>
</ol>
B. Bacterial culture
<ol>
	<li>Grow the strain of interest O/N (16-18 h) on the required medium e.g. Brain Heart Infusion (BHI) agar plates supplemented with 10% heated horse blood at 37&deg;C in 5% CO2 atmosphere4.</li>
</ol>
DAY 2
A. Preparation Of Bacterial (N. meningitidis) Suspension
<ol>
	<li>Using a 10 &mu;L culture loop, make a suspension of the overnight bacterial culture in 2 mL PBSB (PBS with calcium and magnesium).</li>
	<li>Allow large bacterial aggregates to settle by leaving the suspension to stand for 5 min at room temperature (RT).</li>
	<li>Without disturbing the pellet, transfer the top 1 mL of the suspension (stock inoculum) into a sterile tube.</li>
	<li>To estimate bacterial numbers in the stock inoculum, add aliquots (20-50 &mu;L) to tubes containing 1mL volumes of 1% SDS in 0.1M NaOH and mix gently to solubilise. </li>
	<li>Measure the nucleic acid content of the solution by determining the absorbance at 260nm (A260). In our hands, A260 of 1 corresponds to 5x108 colony forming units/ml (cfu/mL). This can be verified by preparing a series of dilutions of the stock inoculum in PBSB, plating out on agar plates and counting colonies after O/N incubation.</li>
	<li>Dilute an aliquot of the stock inoculum in the infection medium [(M199 supplemented with 2% decomplemented (56&deg;C, 30min) normal human serum (NHS)] to obtain the required bacterial density for the infection of target cells.
 <ol>
 	<li>In our laboratory, an infection ratio of 200-300 bacteria per target cell is routinely used.</li>
	</ol>
 </li>
</ol>
B. Cell Culture Infection
<ol>
	<li>Wash the coverslips with cultured target cells 3 times with Hank's medium to remove any trace of antibiotics. </li>
	<li>Infect cells with freshly prepared bacterial suspensions (described above) for 3 h to 8 h at 37&deg;C, in 5%CO2. </li>
	<li>At the end of infection period, wash cells three times with PBS and fix in 500 &mu;L of 2% paraformaldehyde (PFA) for 30-45 min at RT or O/N at 4&deg;C.
 <ol>
		<li>Paraformaldehyde fixation at the concentration and time shown above was found to be suitable for preservation of bacterial and cellular morphology.</li>
	</ol>
 </li>
	<li>After washing off paraformaldehyde, permeabilise the paraformaldehyde-fixed cells by incubating in 0.1%Triton X-100 diluted in PBS for 10 min. </li>
	<li>Wash the samples 3 times with PBS. </li>
	<li>Proceed to immuno-staining or, alternatively, leave samples in 500 &mu;L of 1%BSA/PBST O/N at 4&deg;C.</li>
</ol>
DAY 3
Immuno-staining
Staining of intracellular bacteria and &alpha;-actinin can be performed sequentially or simultaneously by the use of appropriate primary and secondary antibodies as follows; all procedures can be carried out in 12-well plates.
<ol>
	<li>Block the coverslips containing the permeabilised cells with 500 &mu;L of 3% BSA/PBST (PBS containing 0.05% Triton X-100) for 30-60 min at RT</li>
	<li>After washing with PBS, transfer each coverslip to a new dry well in the 12-well plate. </li>
	<li>Add the primary antibodies against bacteria and &alpha;-actinin; eighty to 100 &mu;L of antibody per coverslip is sufficient if added carefully to cover the surface of the coverslip. Incubate for 1h at room temperature.
 <ol>
 	<li>We used rabbit polyclonal antiserum against Neisseria meningitidis (laboratory-raised) and mouse monoclonal antibody (mAb) against &alpha;-actinin (Abcam) simultaneously. In some experiments, in place of anti &alpha;-actinin, mAbs against actin or vimentin were used (Sigma).</li>
 </ol>
 </li>
	<li>At the end of the incubation, add 200 &mu;L of PBS to the well, lift the coverslip and place in a new well containing 500 &mu;L PBS. </li>
	<li>After 5 min, remove PBS by pipetting and then add fresh PBS. Repeat twice. Transfer coverslips to a new dry well.</li>
	<li>Add the appropriate secondary antibodies conjugated to different fluorochromes diluted in 1% BSA/PBST; incubate at RT for 1h in the dark.
 <ol>
		<li>For bacterial detection, we used anti-rabbit Ig conjugated to TRITC and for &alpha;-actinin and other cytoskeletal protein detection, we used anti-mouse Ig conjugated to either FITC- (Sigma) or Alexa Fluor 488- (Invitrogen).</li>
 </ol>
 </li>
	<li>At the end of the incubation, wash as in steps 4 and 5.</li>
	<li>Counter stain with 0.6 &mu;g/mL DAPI in PBS (DNA stain) for 5 min at RT in the dark.</li>
	<li>Rinse with PBS.</li>
	<li>Mount coverslips (cells facing down) on slides with a drop of mounting medium (e,g. Mowiol or Vectashield)</li>
	<li> Store in the dark at 4&deg;C.</li>
	<li>Samples are ready for observation under the microscope.</li>
</ol>
2. Confocal Laser Scanning Microscopy (CLSM)
To observe and capture images of intracellular bacteria and cytoskeletal elements, we used immunolabelled samples and captured images using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope. All images were collected using a 63x NA 1.4 oil immersion objective and process with Leica software.
CLSM procedure:
<ol>
	<li>To begin the CLSM procedure, add a drop of immersion oil to the objective and place the specimen slide, coverslip-side down, on the microscope stage.</li>
	<li>Set the microscope to a visual mode and find the area of interest using the eye pieces of the microscope. </li>
	<li>Using Leica Software, select the xyz acquisition mode. </li>
	<li>Select the 512 x 512 format (frame size). For colocalisation studies, the higher resolution, the more accurate the image; keeping in mind the resolution limit of the microscope. Then select the bidirectional X mode, which will increase the scanning speed and help reduce photo-bleaching.</li>
	<li>Set up sequential scanning settings. Click in the "seq" function and choose one of the scanning modes. We use "between lines" mode.</li>
	<li>Select laser beams according to the fluorochromes conjugated to the secondary antibodies: 405 nm for DAPI (blue), 488 nm for Alexa Fluor 488 (green) and 561 nm for TRITC (red). Activate Photomultiplier Tube (PMT) 1, 2, and 3, respectively. Adjust PMT settings to detect the correct emission wavelength. </li>
	<li>Set up top ("begin") and bottom ("end") of z-stacks or series. Next, set the required "z-step size". 
	<ol>
		<li>Neisseria meningitidis is a diplococcus (Figure 1G) and since each coccus has an approximate diameter of 0.5 &mu;m, the z-step size of 0.20 &mu;m was chosen to enhance the probability of scanning each coccus at least twice. It is also within the step size for optimum resolution of 0.1 - 0.2 &mu;m.</li>
 </ol>
 </li>
	<li>Set the final scan parameters by selecting line averaging of 3 to improve the signal to noise ratio.</li>
	<li>By clicking "start" double- or triple-stained images are obtained by sequential scanning at different wavelengths to eliminate cross-talk between different chromophores.</li>
	<li>For an indication of colocalisation of two fluorochromes, select the "overlay" function to merge selected channels into a single image; for example, when both Alexa 488 (green) and TRITC (red) fluorochromes colocalise, yellow color will appear in the overlaid image. </li>
	<li>Compile z-stacks or series using the "maximum projection" function for the construction of a 2D image required for visualising possible colocalisation. More detailed analysis of colocalisation can be obtained by analysis of each optical section.</li>
	<li>After acquiring the z-stacks or series, process your data to obtain an orthogonal image for visualisation of intracellular localisation of various elements (Figure 1E).</li>
</ol>
3. Quantification of Colocalisation
Statistical analyses of the confocal scanning microscope images are performed with Volocity software (Improvision, PerkinElmer). This software provides a tool designed specifically for colocalisation analysis as described by Manders et al.(1993)5. Colocalisation in the context of digital fluorescence imaging can be described as the detection of a signal at the same voxel (pixel volume) location in each channel. The two channels are made up of images of two different fluorochromes taken from the same sample area (Volocity user guide). Statistical analyses are performed with Volocity software (Improvision, PerkinElmer) using Quantitative Colocalisation Analysis described below.
Quantitative Colocalisation Analysis
<ol>
	<li>Create a library with CLSM images using Volocity software. </li>
	<li>Select "extended focus" from image in the top bar. This tool will compile z-stacks in a 2D image to be analyzed.</li>
	<li>Select "Colocalization" tool. The two channels to be analyzed should have the same color depth.</li>
	<li>Select the area that it is going to be quantified. Set the threshold to remove any background.</li>
	<li>Create the colocalisation output by selecting "generate colocalization". Colocalisation statistics are generated for regions of interest selected previously.</li>
	<li>Select Manders' coefficients R (overlap coefficient) and My (colocalisation coefficient). 
	<ol>
		<li>Manders' coefficients are not sensitive to the intensity of staining as they are normalized against total pixel intensity; thus they can be employed when the staining of one antigen is stronger than the other.</li>
		<li>Overlap coefficient R according to Manders5,6 represents the true degree of colocalisation, i.e. the number of pixels that colocalise compared with the total number of pixels.</li>
		<li>On the other hand, the colocalisation coefficient, My, describes the fluorescence contribution of the more abundant moiety (in this case &alpha;-actinin, green) to the less abundant moiety (in this case bacteria, red), i.e. the number of red pixels that overlap with green pixels compared with the total number of red pixels.</li>
		<li>Manders' coefficients range between 1 and 0, with 1 being high colocalisation, 0 being none; but they can be expressed as percentage for easier interpretation.</li>
	</ol>
 </li>
	<li>Export statistics values to an Excel document for data presentation.</li>
</ol>
4. Representative Results
Intracellular localisation of Opc-expressing Neisseria meningitidis and &alpha;-actinin
Confocal imaging of human brain microvascular endothelial cells infected with Nm for 3 and 8 hours as described above indicated colocalisation of &alpha;-actinin and Nm which appeared to be less frequent in 3 h infection experiments (not shown) compared with cultures infected for 8 h (Figure 1 A-F). A demonstrable colocalisation of &alpha;-actinin with Opc-expressing meningococci was observed each time in &gt;5 replicate experiments. Statistical analysis of colocalisation using several confocal images was carried out as described above. Overall, in HBMEC infected with Opc-expressing meningococci, >25% overlap of the green (&alpha;-actinin) and red (Nm) pixels was obtained (Figure 2B, Overlap coefficient R). In contrast to &alpha;-actinin, experiments in which labeling of internalised bacteria and either actin or vimentin was performed, occasional colocalisation was observed with actin but that with vimentin was rare (Figure. 2B).
The data were also analyzed using the coefficient My, which takes into account the relative abundance of each moiety. My is a measure of the frequency of occurrence of the more abundant signal (in this case green, &alpha;-actinin) each time the less abundant signal (in this case red, bacteria) occurs. This measure shows a striking level of occurrence of &alpha;-actinin in the vicinity of internalised meningococci (Figure 2A and C).
<img src="/files/ftp_upload/2045/2045fig1.jpg" alt="Figure 1" /> Figure 1. Confocal laser scanning microscopy to assess intracellular interactions of N. meningitidis with &alpha;-actinin. A-H. Confluent endothelial monolayers grown on coverslips were infected with the Opc-expressing (A-F) N. meningitidis. After 8 h, non-adherent bacteria were washed off, cells fixed with paraformaldehyde and permeabilised with 0.1% Triton X-100. Subsequently, bacteria and &alpha;-actinin were stained as described above (&alpha;-actinin, green; bacteria, red).
A-C. One field showing x-y images of the location of Nm (A) or &alpha;-actinin (B). The overlay image in (C) indicates several regions in which yellow-orange color appears suggesting colocalisation. Arrows in (A) and (B) show regions where a high degree of &alpha;-actinin accumulation appears to have occurred around bacteria.
D. Optical dissection of an infected HBMEC monolayer indicating colocalisation around intracellular bacteria located at the base of a cell.
Again, this colocalisation is not due to accidental proximity of &alpha;-actinin, as the general stain of &alpha;-actinin in this region is low.
E and F. Three-dimensional images of infected HBMEC monolayers processed as above. An oblique view of the apical surface (E) shows adherent bacteria stained red (red arrow) whereas several bacteria located towards the basal surfaces of endothelial cells (yellow arrow) are distinctly orange/yellow in color. Basal location can be more clearly seen in (F) which is an end-on X-Z cross-section.
G. A negatively stained electron microscope image of N. meningitidis showing its predominant diplococcal from. Each coccus is approximately 0.5 &mu;m in diameter.
<img src="/files/ftp_upload/2045/2045fig2.jpg" alt="Figure 2" /> Figure 2. Localisation and distribution of &alpha;-actinin, actin and vimentin in HBMEC cells.
A. Infected monolayers of HBMEC were treated as described in the legend above but in addition to &alpha;-actinin, some coverslips were used for the detection of actin or vimentin by procedure similar to that for &alpha;-actinin. As above, &alpha;-actinin concentrated around several internalised bacteria (white arrows). Vimentin and actin did not colocalise with bacteria to appreciable levels. Bar represents 20 &mu;m.
B. &amp; C. The values for the coefficients R and My were obtained from more than three experiments using Volocity software as described above.

<ol>
	<li>Virji, M., Makepeace, K., Moxon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mechanisms+of+interactions+of+Opc-expressing+meningococci+at+apical+and+basolateral+surfaces+of+human+endothelial+cells;+the+role+of+integrins+in+apical+interactions.">Distinct mechanisms of interactions of Opc-expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions.</a> Molecular Microbiology. 14, 173-184 (1994).</li><li>Virji, M., Makepeace, K., Peak, I. R., Ferguson, D. J., Jennings, M. P., Moxon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opc-+and+pilus-+dependent+interactions+of+meningococci+with+human+endothelial+cells:+molecular+mechanisms+and+modulation+by+surface+polysaccharides.">Opc- and pilus- dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides.</a> Molecular Microbiology. 18, 741-754 (1995).</li><li>Sa E Cunha, C., Griffiths, N. J., Murillo, I., Virji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+Opc+invasin+binds+to+the+cytoskeletal+protein+alpha-actinin.">Neisseria meningitidis Opc invasin binds to the cytoskeletal protein alpha-actinin.</a> Cellular Microbiology. 11, 384-405 (2009).</li><li>Virji, M., Kayhty, H., Ferguson, D. J., Alexandrescu, C., Alexandrescu, J. E., Heckels, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pili+in+the+interactions+of+pathogenic+Neisseria+with+cultured+human+endothelial+cells.">The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells.</a> Molecular Microbiology. 5, 1831-1841 (1991).</li><li>Manders, E. M. M., Verbeek, F. J., Aten, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+colocalization+of+objects+in+dual+color+confocal+images.">Measurement of colocalization of objects in dual color confocal images.</a> Journal of Microscopy. 6, 357-382 (1993).</li><li>Zinchuk, V., Zinchuk, O., Okada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+multicolor+confocal+immunofluorescence+microscopy+images:+pushing+pixels+to+explore+biological+phenomena.">Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena.</a> Acta Histochemica et Cytochemica. 40, 101-111 (2007).</li></ol>

No conflicts of interest declared.

The possibility of binding of internalised Opc-expressing Neisseria meningitidis to &alpha;-actinin was explored using HBMEC by the examination of the colocalisation of bacteria and the cytoskeletal protein in infected cells after 3 and 8 h incubation period. By confocal microscopy, colocalisation of Neisseria meningitidis with &alpha;-actinin could be demonstrated. Notably, although bacteria were internalised at 3 h, there was little colocalisation with &alpha;-actinin at this time point. Bacterial association with the cytoskeletal protein appeared to require a longer period of intracellular residence as after 8 h infection period, significant numbers of bacteria had &alpha;-actinin apparently in close association. Alpha-actinin is a multifunctional protein, and bacterial interactions with the cytoskeletal element could have significant influence on the target cell function which is a subject of current studies.
Quantification of colocalisation as described above requires meticulous specimen preparation. Particular attention should be given to specimen fixation, blocking period and antibody dilutions. For the best signal to noise ratio, each primary and secondary antibody should be titrated in preliminary experiments to determine the optimum concentrations. In our experience, the mounting medium Mowiol produced better images.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>12 well plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>BC311</td>
<td>&nbsp;</td>
<tr>
<td>Glass Coverslips</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>631-0152</td>
<td>&nbsp;</td>
<tr>
<td>BHI AGAR</td>
<td>&nbsp;</td>
<td>LAB M</td>
<td>LAB048</td>
<td>&nbsp;</td>
<tr>
<td>M199</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2154</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>BE12-167E</td>
<td>&nbsp;</td>
<tr>
<td>MEM Non Essential Aminoacids</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7145</td>
<td>&nbsp;</td>
<tr>
<td>HANK&#x2019;S balanced salt solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H9269</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Biowhittaker</td>
<td>DE14-801F</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P0781</td>
<td>&nbsp;</td>
<tr>
<td>MEM Vitamin solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M6895</td>
<td>&nbsp;</td>
<tr>
<td>PBSB</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>BE17-513F</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9418</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P/0840/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>X-100</td>
<td>&nbsp;</td>
<tr>
<td>DAPI</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-9564</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>H-1000</td>
<td>&nbsp;</td>
<tr>
<td>Mowiol 4-88</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>475904</td>
<td>&nbsp;</td>
<tr>
<td>Anti&#x2011;Nm antiserum</td>
<td>&nbsp;</td>
<td>Laboratory raised</td>
<td>&nbsp;</td>
<td>Rabbit serum</td>
</tr>
<tr>
<td>TRITC anti-rabbit Ig</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T6778</td>
<td>&nbsp;</td>
<tr>
<td>Anti &alpha;&#x2011;actinin mAb [7H6]</td>
<td>&nbsp;</td>
<td>Abcam</td>
<td>Ab32816</td>
<td>IgG</td>
</tr>
<tr>
<td>Anti actin mAb</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4700</td>
<td>IgG2a</td>
</tr>
<tr>
<td>Anti vimentin mAb</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>V6630</td>
<td>IgG1</td>
</tr>
<tr>
<td>ALEXA FLUOR 488 anti&#x2011;mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A11001</td>
<td>&nbsp;</td>
<tr>
<td>FITC anti-mouse IgG</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F-2012</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			1. Confocal Laser Scanning Microscopy (CLSM):
Leica SP5 confocal imaging system: This system, by using a combination of AOTF (acousto-optical tuneable filter) and an AOBS (acousto-optical beam splitter), simplifies excitation with specific wave lengths.
 2-Software:
 Leica confocal software LCS, Leica Microsystems, Germany. Volocity 5, Improvision, PerkinElmer, USA.		</div>

visualisation and quantification of intracellular interactions of neisseria meningitidis and human &alpha;-actinin by confocal imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Watch this Scientific Journal Video about Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human Î±-actinin by Confocal Imaging at JoVE.com

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

visualisation-quantification-intracellular-interactions-neisseria

Research

JoVE Journal

Immunology and Infection

18.0K Views.  University of Bristol, UK. Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human α-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular α-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs). 

Video: Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...