Name: Invasion of Human Cells by a Bacterial Pathogen (Video) | JoVE Journal
Uploaded: 2011-03-21T11:00:00.000+00:00
Duration: 7 min 15 s
Description: Learn to measure bacterial invasion of human endothelial cells using Staphylococcus aureus and Gentamycin protection assays. This protocol enables quantification of intracellular bacteria and identification of key virulence factors.

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5 minutes

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°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°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°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 μl 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 × 105 cells per well (in 500 μl 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 μl 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 μM cytochalasin D can be added; or for the role of caveolae, 5 mM methyl-β-cyclodextrin can be added.</li>
</ol>
3. Invasion Assay
<ol>
 <li>Add 10 μl of washed bacteria (resulting in approximately 2 × 107 cfu ml-1 S. aureus ) to each well containing a washed coverslip with a confluent layer of endothelial cells in 490 μl DMEM containing 10% FBS.</li>
 <li>Incubate for 15-90 minutes at 37°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 μl 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 μl DMEM/10% FBS supplemented with 200 μg ml−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°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 μg ml−1) instead of gentamicin to physically destroy the extracellular bacteria.</li>
 <li>Incubate coverslips for 20min at 37°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 (Δfnb ) 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 ± 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="Bacterial internalization graph; S. aureus, L. lactis comparison; experimental results; bar chart." data-altupdatedat="1788304762000" data-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/pubmed?term=((Permanent+cell+line+expressing+human+factor+VIII-related+antigen+established+by+hybridization%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">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/pubmed?term=((Phase+variation+of+the+opacity+outer+membrane+protein+controls+invasion+by+Neisseria+gonorrhoeae+into+human+epithelial+cells%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((Pathogenic+bacteria+attach+to+human+fibronectin+through+a+tandem+beta-zipper%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((The+protection+of+intracellular+Brucella+against+streptomycin+alone+and+in+combination+with+other+antibiotics%5BTitle%5D)+AND+%22J.+Lab.+Clin.+Med%22%5BJournal%5D)">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/pubmed?term=((Interaction+of+intraleukocytic+bacteria%5BTitle%5D)+AND+%22Antimicrob.+Agents+Chemother%22%5BJournal%5D)">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/pubmed?term=((Gentamicin+antibacterial+activity+in+the+presence+of+human+polymorphonuclear+leukocytes%5BTitle%5D)+AND+%22+Antimicrob.+Agents+Chemother%22%5BJournal%5D)">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/pubmed?term=((Effect+of+mucin+on+Campylobacter+jejuni+association+and+invasion+on+HEp-2+cells%5BTitle%5D)+AND+%22Microb.+Pathog%22%5BJournal%5D)">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/pubmed?term=((Model+for+invasion+of+human+tissue+culture+cells+by+Neisseria+gonorrhoeae%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">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/pubmed?term=((Staphylococcus+aureus+Host+Cell+Invasion+and+Virulence+in+Sepsis+is+Facilitated+by+the+Multiple+Repeats+within+FnBPA%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">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/pubmed?term=((Evidence+for+invasion+of+a+human+oral+cell+line+by+Actinobacillus+actinomycetemcomitans%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fusobacterium+nucleatum+transports+noninvasive+Streptococcus+cristatus+into+human+epithelial+cells%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Fusobacterium nucleatum transports noninvasive Streptococcus cristatus into human epithelial cells.</a> Infect. Immun. 74, 654-662 (2006).</li>
<li>Menzies, B. E., Kourteva, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Internalization+of+Staphylococcus+aureus+by+Endothelial+Cells+Induces+Apoptosis%5BTitle%5D)+AND+%22Infect.+Immun.%22%5BJournal%5D)">Internalization of Staphylococcus aureus by Endothelial Cells Induces Apoptosis.</a> Infect. Immun.. 66, 5994-5998 (1998).</li>
<li>Schröder, A., Schröder, B., Roppenser, B., Linder, S., Sinha, B., Fässler, R., Aepfelbacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus+fibronectin+binding+protein-A+induces+motile+attachment+sites+and+complex+actin+remodeling+in+living+endothelial+cells%5BTitle%5D)+AND+%22Mol.+Biol.+Cell%22%5BJournal%5D)">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/pubmed?term=((The+role+played+by+the+group+A+streptococcal+negative+regulator+Nra+on+bacterial+interactions+with+epithelial+cells%5BTitle%5D)+AND+%22Mol.+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Streptococcus+pyogenes+serotype+M1+encodes+multiple+pathways+for+entry+into+human+epithelial+cells%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells.</a> Infect. Immun. 66, 4593-4601 (1998).</li>
<li>Eiff, C. von <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus+small+colony+variants%3A+a+challenge+to+microbiologists+and+clinicians%5BTitle%5D)+AND+%22Int+J.+Antimicrob.+Agents%22%5BJournal%5D)">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/pubmed?term=((Recent+findings+on+the+viable+but+nonculturable+state+in+pathogenic+bacteria%5BTitle%5D)+AND+%22FEMS+Microbiol.+Rev%22%5BJournal%5D)">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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrin-linked+kinase+is+an+essential+link+between+integrins+and+uptake+of+bacterial+pathogens+by+epithelial+cells%5BTitle%5D)+AND+%22Cell.+Microbiol%22%5BJournal%5D)">Integrin-linked kinase is an essential link between integrins and uptake of bacterial pathogens by epithelial cells.</a> Cell. Microbiol. 8, 257-266 (2006).</li>
</ol>

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.

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>31885-023</td><td></td></tr><tr><td>Brain heart infusion</td><td>BioChemika</td><td>53286</td><td></td></tr><tr><td>F&amp;#8211;tal bovine serum</td><td>Invitrogen</td><td>10091-148</td><td></td></tr><tr><td>HUVECs</td><td>Lonza Inc.</td><td>CC-2519</td><td></td></tr><tr><td>Endothelial basal medium</td><td>Lonza Inc.</td><td>CC-3121</td><td></td></tr><tr><td>Bovine brain extract (including heparin)</td><td>Lonza Inc.</td><td>CC-4092</td><td></td></tr><tr><td>Human endothelial growth factor</td><td>Lonza Inc.</td><td>CC-4017C</td><td></td></tr><tr><td>Hydrocortisone</td><td>Lonza Inc.</td><td>CC-4035C</td><td></td></tr><tr><td>GA-1000</td><td>Lonza Inc.</td><td>CC-4092C</td><td></td></tr><tr><td>Gentamicin</td><td>Invitrogen</td><td>15710</td><td></td></tr><tr><td>Lysostaphin</td><td>AMBI</td><td>LSPN-50</td><td></td></tr><tr><td>Thermanox coverslips</td><td>Thermo Fisher Scientific, Inc.</td><td>TKT-210-330P</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Cytopath</td><td>TCS Biosciences</td><td>HC8595</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma-Aldrich</td><td>C3886</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>T75 flasks</td><td>Thermo Fisher Scientific, Inc.</td><td>430641</td><td></td></tr><tr><td>Cytochalasin D</td><td>Sigma-Aldrich</td><td>C2618</td><td></td></tr><tr><td>Methyl-B-cyclodextrin</td><td>Sigma-Aldrich</td><td>332615</td><td></td></tr></tbody></table>

invasion of human cells by a bacterial pathogen

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.

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 ...

The overall goal of this experiment is to measure the ability of bacterial strains to invade host cells. Host cells in culture are transferred onto glass cover slips in a 24 well plate and incubated until they reach co fluency. The cover slips are then washed and transferred to a new plate.Bacteria harvested from a standard broth culture are washed by centrifugation and then added to the host cell culture. Once invasion has occurred, extracellular bacteria are killed using Gentamycin and the cover slips are washed and transferred to a new plate. Finally, the host cells are lysed and the lysates plated on agar plates to determine the number of intracellular bacteria.Ultimately, results can be obtained that show the invasive ability of specific bacteria. This method can help answer key questions in the field of microbial pathogenesis, such as how bacterial invasion of host cells causes disease. It can also facilitate the identification and characterization of bacterial proteins involved in host pathogen interactions.Demonstrating this procedure will be Dr.Andrew Edwards, a postdoc from a laboratory. For this assay we use human endothelial EAHY 9 26 cells cultured in T 75, flasks at 37 degrees Celsius. Harvest the cells once they've reached about 95%Co fluency.Wash the cells with 10 milliliters of PBS To remove the growth media. Detach the cells by incubating in trypsin EDTA solution for five minutes. Then transfer to a chronicle tube.Next centrifuge cells and Resus, suspend them in about 25 milliliters of complete DMEM medium or at a concentration of 1 million cells per milliliter using sterile forceps. Place a thermon cover slip in each well of a 24 well tissue culture plate. Be sure to place the opaque side of the cover slip on which cells can adhere facing upwards.Next, add 500 microliters of cells to each well and incubate the plate at 37 degrees Celsius for 48 hours or until the cells have reached co fluency. Following the incubation, use clean forceps to carefully remove each cover slip and dip them in DMEM to remove soluble secreted proteins. Transfer the washed cover slips to individual wells of a fresh 24 well plate containing 490 microliters per well of complete DMEM.Taking care to maintain the orientation of the cover slips so that the cells are facing upward. Prepare a 10 milliliter culture of staphylococcus aureus in brain heart infusion broth. Transfer the bacterial culture into a conical tube and centrifuge the bacteria 5, 000 times G for 10 minutes.Once the cells have been pelleted, remove the supinate and Reese. Bend the pellet in 10 milliliters of sterile DMEM to wash the cells, centrifuge the cells once more and repeat the wash three times. Reese bend the bacteria in DMEM and measure the optical density of the suspension.Adjust the concentration to obtain an OD 600 of 1.0, which corresponds to approximately 10 to the nine colony forming units per milliliter. Once the bacterial suspension is ready, add 10 microliters of bacteria to each well. Of the 24 well plate containing the cover slips.Incubate the plate at 37 degrees Celsius for 15 to 90 minutes to allow the bacteria to attach to and invade the cells. Following this incubation, aspirate the supinate from each well containing the unbound bacteria and replace it with 10%F-B-S-D-M-E-M supplemented with Gentamycin. Incubate the plate at 37 degrees Celsius for 60 minutes to kill the extracellular bacteria.Following this incubation dip. Wash each cover slip in DMEM three times to remove antibiotic. Then transfer them to a new 24 well plate containing 500 microliters of 0.5%Triton X 100 in PBS per well.As previously, take care to keep the cell covered side facing up vigorously rinse each cover slip with Triton solution by pipetting the liquid in the well up and down directly onto the cover slip so as to ensure that all the cells will be lysed and the intracellular bacteria released. Perform serial dilutions of the lysate to quantify the number of infecting bacteria. Collect the lysate and plate serial dilutions on TSA plates.Colony forming units can be determined on these plates after 24 hours of incubation at 37 degrees Celsius. This graph shows the number of wild type or fibrin connecting binding protein deficient staphylococcus aureus invading human endothelial cells compared to wild type bacteria. Fibronectin binding protein deficient, staphylococcus aureus are significantly impaired in their ability to invade endothelial cells.Complementation of the mutant with a plasmid inod in fibronectin binding protein, A minus the fibronectin binding domain did not promote invasion. In contrast, complementation of the mutant with a plasmid encoding the entire fibronectin binding protein, aging restored invasion to wild type levels. In this experiment, lactococcus Lactus expressing the fibrin necking binding protein was shown to be able to invade host cells.Whereas bacteria not expressing the protein were not the ability to adhere to cells was the same for both strains. Once mastered, this technique can be performed within three hours. After watching this video, you should have a good understanding of how to prepare bacteria and host cells to study cell invasion.You should also be able to identify the stages of which additional questions can be asked to increase your understanding of this aspect of disease.

invasion-of-human-cells-by-a-bacterial-pathogen

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JoVE Journal

Immunology and Infection

This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

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.

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

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.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen 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.

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

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T Cells Capture Bacteria by Transinfection from Dendritic Cells

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

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

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

The study presents a method for in vitro investigation of bacterial infection using tissue stem cell-derived intestinal organoids. The protocol describes how to culture and differentiate organoids in two dimensions, infect them with adherent-invasive Escherichia coli, and assess key readouts, including epithelial barrier breach, infection imaging, and epithelial RNA response.

A Two-Dimensional Organoid Culture on Invasin or Basement Membrane Extract to Study Host-Bacteria Interactions

M cells in a specialized follicle-associated epithelium covering Peyer’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.

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

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage: 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.

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

Learn to measure bacterial invasion of human endothelial cells using Staphylococcus aureus and Gentamycin protection assays. This protocol enables quantification of intracellular bacteria and identification of key virulence factors.

Invasion of Human Cells by a Bacterial Pathogen

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