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>
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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. 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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.

Watch this Scientific Journal Video about Invasion of Human Cells by a Bacterial Pathogen at JoVE.com

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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Immunology and Infection

34.7K Aufrufe.  University of Bath. Ein allgemeines Protokoll für die Untersuchung der Invasion von Wirtszellen durch eine bakterielle Erreger, die sich auf Staphylococcus aureus Und humanen Endothelzellen.

Serie: Invasion of Human Cells by a Bacterial Pathogen

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 ...

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 ...

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 ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

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.

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

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

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

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.

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

Medicine

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.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016)
This video demonstrates a method to establish an infection model in placental tissues to study infection propagation. An ex vivo placental organ culture is infected with Listeria monocytogenes, a facultative intracellular parasite. The bacteria invade extravillous trophoblasts (EVTs) to enter the placental tissue and then propagate to other cells via cell-to-cell spread.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

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

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
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.

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...

Immunodiagnostics

Specialized Assays

A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins

Source: Flaherty, R. A., et al. <a href="https://app.jove.com/v/54406">Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells.</a> J. Vis. Exp. (2016).
This video demonstrates a permeable membrane insert-based infection system to assess the impact of secreted bacterial toxins. By eliminating direct contact between the bacterial pathogen and the host cells using a permeable membrane, this technique allows for studying the effect of toxins secreted by pathogenic bacteria on mammalian host cells.

1. Bacterial Culture

	Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest.
	Note: The GAS (Group A ...