This work was funded by the Wellcome Trust (WT 0795880).

The following protocol will describe the study of endothelial cell invasion by S. aureus but can theoretically be used to study cellular invasion by any culturable bacterium. Stages specific to S. aureus and endothelial cells are indicated.
1. Preparation of Bacteria
<ol>
 <li>Culture S. aureus strains for 4-16 h (depending on growth phase required) in 10 ml Brain-Heart Infusion (BHI) broth at 37&deg;C in air with shaking at 200 rpm. These growth conditions are specific for S. aureus and may need to be adapted for other bacteria.</li>
 <li>Wash bacteria three times in Dulbecco's Modified Eagle's medium (DMEM; Invitrogen) by alternate rounds of centrifugation at room temperature (5,000 x g , 10 min), removal of the culture supernatant and resuspension of the bacterial pellet in an equivalent volume of DMEM. Measure the optical density of the resulting suspension of bacteria which can then be adjusted as required. For S. aureus, we prepare a suspension at OD600 = 1, which corresponds to ~109 cfu ml-1.</li>
</ol>
2. Endothelial Cell Culture
<ol>
 <li>Culture the endothelial cell line EA.hy926 1 in DMEM supplemented with foetal bovine serum (FBS; 10%) and L-glutamine (2 mM) at 37&deg;C in 5% CO2. Alternatively, pooled primary human umbilical vein endothelial cells (HUVECs) can be purchased from Lonza (Basel, Switzerland) and cultured in endothelial basal medium supplemented with 2% FBS, bovine brain extract (including heparin), human endothelial growth factor and hydrocortisone at 37&deg;C in 5% CO2 according to manufacturer's instructions (Lonza). These growth conditions are specific for these cells and may need to be adapted for other host cell types.</li>
 <li>Grow endothelial cells in T75 flasks to complete confluency, verified by eye.</li>
 <li>Prepare the 24-well plates for the insertion of the coverslips. Fine forceps (flame sterilised) are needed to move the coverslips, which have one opaque and one shiny surface. Place coverslips in the wells of the 24-well plate with the opaque surface facing upwards to allow cell attachment.</li>
 <li>Liberate cells from the T75 flask with 3 ml trypsin-EDTA (0.25%) and add to 10 ml of the relevant culture medium.</li>
 <li>Add 500 &mu;l of resuspended cells to 24-well plates containing Thermanox glass coverslips. One T75 confluent flask of cells provided sufficient cells for two 24-well plates, resulting in approximately 5 &times; 105 cells per well (in 500 &mu;l medium).</li>
 <li>Incubate plates for 48 h as described above, and verify 100% cell confluency by inverted light microscopy.</li>
 <li>Dip-wash coverslips in PBS and add to new 24-well plates containing 490 &mu;l DMEM containing 10% FBS in each well.</li>
 <li>To examine the role of specific metabolic processes in cell invasion, inhibitors can be added to the cultured cells 1 h prior to the addition of bacteria and concentrations maintained during the assay. For example, to determine the role of actin rearrangement in S. aureus invasion of endothelial cells, 50 &mu;M cytochalasin D can be added; or for the role of caveolae, 5 mM methyl-&beta;-cyclodextrin can be added.</li>
</ol>
3. Invasion Assay
<ol>
 <li>Add 10 &mu;l of washed bacteria (resulting in approximately 2 &times; 107 cfu ml-1 S. aureus ) to each well containing a washed coverslip with a confluent layer of endothelial cells in 490 &mu;l DMEM containing 10% FBS.</li>
 <li>Incubate for 15-90 minutes at 37&deg;C in 5% CO2.</li>
 <li>To measure the total number of bacteria associated with the cells (adherent and internalised), dip-wash the coverslips three times in PBS and add to fresh wells containing 500 &mu;l 0.5% Triton X-100 in PBS. To ensure the cells fully lyse and release all the internalised bacteria, pipette the several times, pointing the tip of the pipette directly at the surface of the coverslip.</li>
 <li>Enumerate the bacteria by plating the liquid suspension (or dilutions of this where necessary) onto the surface of TSA plates. Since TX-100 will lyse many Gram-negative bacteria, saponin can be used instead 2.</li>
 <li>To measure the number of internalised bacteria, remove the culture supernatant from each well containing unbound bacteria and replace with 500 &mu;l DMEM/10% FBS supplemented with 200 &mu;g ml&minus;1 gentamicin. We routinely use gentamicin rather than lysostaphin here as it is cheaper and allows us to switch between experiments using different types of bacteria (e.g. Staphylococci and Lactococci) without having to change the experimental protocol.</li>
 <li>Incubate plates at 37&deg;C in 5% CO2 for 60 min to kill all extracellular bacteria.</li>
 <li>Wash coverslips 3 times in PBS, lyse and enumerate by plating onto TSA as described for the adhesion assay above.</li>
 <li>In some cases it may be preferable to visually count the number of bacteria using light microscopy. In this instance, and specific to S. aureus cell invasion, use lysostaphin (10 &mu;g ml&minus;1) instead of gentamicin to physically destroy the extracellular bacteria.</li>
 <li>Incubate coverslips for 20min at 37&deg;C in CO2 in the lysostaphin solution, then rinse and fix with Cytopath (Cellpath).</li>
 <li>Flood the coverslips with crystal violet (0.5%, w/v) for 5min.</li>
 <li>Dip-rinse in water, air-dry and mount onto glass slides. The number of bacteria per mm2 of confluent endothelial cells can be quantified using light microscopy.</li>
</ol>
4. Representative Results:
Expression of fibronectin binding proteins (FnBPA and FnBPB) on the surface of S. aureus confers the ability to invade endothelial cells. Recent work has redefined the fibronectin-binding domain of FnBPA 3. Wild-type (WT) S. aureus 8325.4 invades endothelial cells with high efficiency, whilst a strain lacking both FnBPA and B (&Delta;fnb ) showed significantly reduced levels of internalisation (Figure 1A). Complementation of the mutant with a plasmid encodingfnb A minus the fibronectin-binding domain (pFnR0) did not promote invasion (Figure 1A). By contrast, complementation of the mutant with a plasmid encoding the entire fnb A gene (pFnBA4) restored invasion to WT levels (Figure 1A).
The role of FnBPA in endothelial cell invasion can also be demonstrated using the heterologous expression host Lactococcus lactis . Expression of plasmid-encoded FnBPA in L. lactis (pRM9 9) did not significantly enhance adhesion to endothelial cells compared to bacteria expressing no FnBPA (CTL) (open bars, Figure 1B). By contrast, FnBPA-expressing L. lactis invaded endothelial cells at significantly higher levels than the non-expressing strain (closed bars, Figure 1B).
Experiments were performed four times in duplicate and the mean &plusmn; standard deviation is presented. * represent data that are statistically significantly different (p = &lt;0.05) from WT or CTL values.
<img src="/files/ftp_upload/2693/2693fig1.jpg" alt="Figure 1" /> 
Figure 1. Invasion of EA. Hy926 endothelial cells by S. aureus (A) or L. lactis (B).

<ol>
	<li>Edgell, C. J., McDonald, C. C., Graham, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization.">Permanent cell line expressing human factor VIII-related antigen established by hybridization.</a> Proc. Natl. Acad. Sci. U. S. A. 80, 3734-3737 (1983).</li><li>Makino, S., van Putten, J. P., Meyer, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+variation+of+the+opacity+outer+membrane+protein+controls+invasion+by+Neisseria+gonorrhoeae+into+human+epithelial+cells.">Phase variation of the opacity outer membrane protein controls invasion by Neisseria gonorrhoeae into human epithelial cells.</a> EMBO J. 10, 1307-1315 (1991).</li><li>Schwarz-Linek, U., Werner, J. M., Pickford, A. R., Gurusiddappa, S., Kim, J. H., Pilka, E. S., Briggs, J. A., Gough, T. S., Höök, M., Campbell, I. D., Potts, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+bacteria+attach+to+human+fibronectin+through+a+tandem+beta-zipper.">Pathogenic bacteria attach to human fibronectin through a tandem beta-zipper.</a> Nature. 423, 177-181 (2003).</li><li>Magoffin, R. L., Spink, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protection+of+intracellular+Brucella+against+streptomycin+alone+and+in+combination+with+other+antibiotics.">The protection of intracellular Brucella against streptomycin alone and in combination with other antibiotics.</a> J. Lab. Clin. Med. 37, 924-930 (1951).</li><li>Mandell, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+intraleukocytic+bacteria.">Interaction of intraleukocytic bacteria.</a> Antimicrob. Agents Chemother. 52, 1673-1679 (1973).</li><li>Vaudaux, P., Waldvogel, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gentamicin+antibacterial+activity+in+the+presence+of+human+polymorphonuclear+leukocytes.">Gentamicin antibacterial activity in the presence of human polymorphonuclear leukocytes.</a> Antimicrob. Agents Chemother. 16, 743-749 (1979).</li><li>De Melo, M. A., Pechere, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+mucin+on+Campylobacter+jejuni+association+and+invasion+on+HEp-2+cells.">Effect of mucin on Campylobacter jejuni association and invasion on HEp-2 cells.</a> Microb. Pathog. 5, 71-76 (1988).</li><li>Shaw, J. H., Falkow, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+for+invasion+of+human+tissue+culture+cells+by+Neisseria+gonorrhoeae.">Model for invasion of human tissue culture cells by Neisseria gonorrhoeae.</a> Infect. Immun. 56, 1625-1632 (1988).</li><li>Edwards, A. M., Potts, J. R., Josefsson, E., Massey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+Host+Cell+Invasion+and+Virulence+in+Sepsis+is+Facilitated+by+the+Multiple+Repeats+within+FnBPA.">Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis is Facilitated by the Multiple Repeats within FnBPA.</a> PLoS Pathog. 6 (6), e1000964-e1000964 (2010).</li><li>Meyer, D. H., Sreenivasan, P. K., Fives-Taylor, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+invasion+of+a+human+oral+cell+line+by+Actinobacillus+actinomycetemcomitans.">Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans.</a> Infect. Immun. 59, 2719-2726 (1991).</li><li>Edwards, A. M., Grossman, T. J., Rudney, J. 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Immun.. 66, 5994-5998 (1998).</li><li>Schr&#246;der, A., Schröder, B., Roppenser, B., Linder, S., Sinha, B., Fässler, R., Aepfelbacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+fibronectin+binding+protein-A+induces+motile+attachment+sites+and+complex+actin+remodeling+in+living+endothelial+cells.">Staphylococcus aureus fibronectin binding protein-A induces motile attachment sites and complex actin remodeling in living endothelial cells.</a> Mol. Biol. Cell. 17, 5198-5210 (2006).</li><li>Molinari, G., Rohde, M., Talay, S. R., Chhatwal, G. S., Beckert, S., Podbielski, A. <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+played+by+the+group+A+streptococcal+negative+regulator+Nra+on+bacterial+interactions+with+epithelial+cells.">The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.</a> Mol. Microbiol. 40, 99-114 (2001).</li><li>Cue, D., Dombek, P. E., Lam, H., Cleary, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pyogenes+serotype+M1+encodes+multiple+pathways+for+entry+into+human+epithelial+cells.">Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells.</a> Infect. Immun. 66, 4593-4601 (1998).</li><li>Eiff, C. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+small+colony+variants:+a+challenge+to+microbiologists+and+clinicians.">Staphylococcus aureus small colony variants: a challenge to microbiologists and clinicians.</a> Int J. Antimicrob. Agents. 31, 507-510 (2008).</li><li>Oliver, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+findings+on+the+viable+but+nonculturable+state+in+pathogenic+bacteria.">Recent findings on the viable but nonculturable state in pathogenic bacteria.</a> FEMS Microbiol. Rev. 34, 415-425 (2010).</li><li>Wang, B., Yurecko, R. S., Dedhar, S., Cleary, P. 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No conflicts of interest declared.

The assay we describe is based on the gentamicin protection assay, which has been used widely to study the invasion of host cells by bacteria. Observations of the inability of gentamicin and other antibiotics to kill intracellular bacteria were utilised in early studies on the invasion of host cells by bacteria 4-8. The use of 24, 48 or even 96-well plates allows the generation of large amounts of quantitative, reproducible data in a short period of time and at relatively low cost. The assay is also attractive because it requires no specialist equipment, it can be tailored to work with various bacteria and cells, and can be used to study the role of both bacterial and host cell processes in invasion 9. Indeed, since the earliest experiments, the gentamicin protection assay has been widely employed to measure host cell invasion by a range of different bacteria or even combinations of bacteria 10,11. Whilst the core principles are the same, many subtle variations in the method have been reported. In this article we have described our version and indicated where modifications may be necessary for other bacteria or host cells. When designing an experiment to measure host cell invasion using this approach there are a number of important points to consider:
Bacterial sensitivity to gentamicin. This may seem obvious, but it is important to ensure that the bacterium being studied is susceptible to gentamicin at the concentration, temperature and over the time duration to be used. In the case of insensitivity, it may be possible to use other antibiotics 5. An alternative approach can be to use lytic enzymes (lysostaphin, mutanolysin, lysozyme) 12.
Incubation and inoculum. When performing this assay for the first time it is important to establish the optimal incubation times and bacterial inoculum to be used. Therefore, initial experiments should examine both adhesion and invasion over time (5 min to up to 6 h). It is also important to assess how invasion is affected by the multiplicity of infection (MOI; number of bacteria per cell). In general it is best to use the fewest number of bacteria possible as this will reduce damage to the cultured cells. Important differences between strains may be missed if extended incubation periods or large inocula are used 9,13.
Cellular damage. It is important to wash bacteria prior to use. However, cellular damage is not restricted to toxin production. Bacterial invasion, induction of apoptosis and disruption of normal cellular functions can all cause cellular damage. This can lead to penetration of gentamicin into the cell, killing internalised bacteria and giving the impression that invasion has not occurred 14.
Incubation conditions. The temperature and composition of the culture medium can have a significant impact on invasion. For example, invasion of HeLa cells by Streptococcus pyogenes is dependent upon the presence of soluble fibronectin, which is typically present in serum 15. Another consideration is bacterial growth in the culture medium during the course of the experiment.
Culture and quantification of intracellular bacteria. Although TX-100 may not kill intracellular bacteria, it may inhibit their growth. As such, it is important to check bacterial replication on solid media in the presence of the detergent. Where affected, it may be possible to use lower concentrations of the detergent or to use an alternative such as saponin. A benefit of the gentamicin protection assay over the crystal violet staining and microscopy assay is that it is less labor intensive, and it allows the detection and differentiation of sub-populations of internalised bacteria. For example, S. aureus can produce either a normal colony type (NCT) or the small colony variant (SCV) 16, which would not be distinguishable by microscopy of individual bacterial cells. A benefit of the crystal violet staining and microscopy assay over the gentamicin protection assay is cell clumping can be accounted for and that intracellular bacteria that enter a 'viable but non-culturable' state will be detected 17.
Examining the role of host cell processes in bacterial invasion. Many studies have employed inhibitors of host cell function such as cytochalasin D, which interferes with actin rearrangement. Such studies can also include antibodies that target cell-surface receptors and siRNA knockdown of specific genes 18. It is vitally important to ensure that these treatments do not affect the viability or attachment of cultured cells or have toxic effects on bacteria.
Future directions:
Because this assay is typically performed in multi-well cell culture plates, it is theoretically possible to adapt it to a high-throughput screening operation, which might involve the examination of libraries of potential cell-function inhibitors, anti-infectives or antibiotics designed to target intracellular bacteria. Alternatively, such an approach could be used to screen mutant libraries to identify genes involved in adhesion, invasion or intracellular survival. In some cases it may be desirable to include shear forces (flow) in the model system, for example when modeling the endothelium, to more closely mimic the in vivo environment. Current advances in the design and manufacture of flow cells and microfluidic cell culture systems provide the possibility of employing the gentamicin protection assay in host-pathogen models that incorporate shear forces.
In summary, the protocol described here is based upon similar approaches used over many years. It is widely applicable to many types of cultured cells and bacterial species and can generate large amounts of data quickly and at relatively little cost.

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		<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>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31885-023</td>
<td>&nbsp;</td>
<tr>
<td>Brain heart infusion</td>
<td>&nbsp;</td>
<td>BioChemika</td>
<td>53286</td>
<td>&nbsp;</td>
<tr>
<td>Foetal bovine serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>HUVECs</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-2519</td>
<td>&nbsp;</td>
<tr>
<td>Endothelial basal medium</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-3121</td>
<td>&nbsp;</td>
<tr>
<td>Bovine brain extract (including heparin)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092</td>
<td>&nbsp;</td>
<tr>
<td>Human endothelial growth factor</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4017C</td>
<td>&nbsp;</td>
<tr>
<td>Hydrocortisone</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4035C</td>
<td>&nbsp;</td>
<tr>
<td>GA-1000</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-4092C</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Lysostaphin</td>
<td>&nbsp;</td>
<td>AMBI</td>
<td>LSPN-50</td>
<td>&nbsp;</td>
<tr>
<td>Thermanox coverslips</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>TKT-210-330P</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Cytopath</td>
<td>&nbsp;</td>
<td>TCS Biosciences</td>
<td>HC8595</td>
<td>&nbsp;</td>
<tr>
<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3886</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4049</td>
<td>&nbsp;</td>
<tr>
<td>T75 flasks</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>430641</td>
<td>&nbsp;</td>
<tr>
<td>Cytochalasin D</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2618</td>
<td>&nbsp;</td>
<tr>
<td>Methyl-B-cyclodextrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>332615</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

invasion of human cells by a bacterial pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

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Invasion of Human Cells by a Bacterial Pathogen

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

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34.6K Views.  University of Bath. A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Video: Invasion of Human Cells by a Bacterial Pathogen

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

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 ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.