Name: a visual assay to monitor t6ss-mediated bacterial competition
Uploaded: 2013-03-20T12:00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about A Visual Assay to Monitor T6SS-mediated Bacterial Competition at JoVE.com

This work was funded by the Wellcome trust grant WT091939MA. Alain Filloux is supported by the Royal Society.

1. Bacterial Strains and Cultures
<ol>
 <li>Engineer an Escherichia coli recipient cell (Prey, P) by transforming (using standard CaCl2 treatment or electroporation)15 E. coli DH5&alpha; cells with a plasmid allowing the a-complementation of the lacZ gene (Table 1). Plate the transformed cells on Luria-Bertani agar plates (LBA, 1.5% agar) containing 5-bromo-4-chloro-indolyl-&beta;-D-galactopyranoside (X-gal) at 40 mg/ml final concentration and appropriate antibiotic. Incubate at 37 &deg;C overnight and the following morning select for blue transformants (see &sect;1.3).</li>
 <li>Grow the Pseudomonas aeruginosa donor cells that are T6SS-active (D+) or T6SS-inactive (D-) (Table 1) on LBA overnight at 37 &deg;C. In the example presented here we used a P. aeruginosa retS strain possessing a constitutively active H1-T6SS16 and an isogenic mutant with a deletion of the H1-T6SS gene cluster (Table 1).</li>
 <li>The next day, prepare an overnight liquid culture in aerated flask by inoculating a bright blue clone among the E. coli transformants (P) from the plate described in &sect;1.1, in 5 ml of tryptic soy broth (TSB) supplemented with the appropriate antibiotic. Grow under agitation at 37 &deg;C. Proceed equally with a single colony of (D+) and of (D-) from the plate described in &sect;1.2.</li>
</ol>
2. Competition Assay
<ol>
 <li>Prepare LBA plates for the next day experiment. Make sure these plates are properly dried (either near the Bunsen burner or in laminar flow cabinet). Prepare an "Assay-input" (A-Input) plate for the strains (D+), (D-), and (P). Prepare an "Assay-output" (A-output) plate for the strains (D-+P) and (D++P). Divide and label the plates accordingly.</li>
 <li>After overnight growth (see &sect;1.3), measure the optical density (OD600nm) of the input bacterial culture (D-, D+ and P) and calculate the volume required to obtain a cell density equivalent to 1 unit OD600nm of each strain. Each "input" cell culture (D-, D+ and P) is initially collected in a sterile 1.5 ml Eppendorf tubes.</li>
 <li>Centrifuge the bacterial samples at 13,000 rpm during 1 min at room temperature and discard the supernatant.</li>
 <li>Resuspend the pellets of the D-, D+, and P cultures, in 100 &mu;l of fresh TSB by gentle pipetting and inoculate 10 ml of each corresponding strain as a single spot on the "A-input" plate (prepared in &sect;2.1).</li>
 <li>Inoculate the "A-output" plate (prepared in &sect;2.1). More precisely, mix gently 30 &mu;l of (D+) with 30 &mu;l (P) and 30 &mu;l of (D-) with 30 ml (P) in two separate Eppendorf tubes (Note that the cultures used are those described in &sect;2.4). Inoculate 20 &mu;l of the mixes (D+/P) and (D-/P) as individual spots on the "A-output" plate. Allow the spots to dry nearby a Bunsen burner and place the plate in an incubator at 37 &deg;C for an appropriate period of time during which the bacterial killing is taking place. In the case of P. aeruginosa, an efficient bacterial killing is observed after 5 hr incubation when comparing a T6SS active with a T6SS defective strain.</li>
</ol>
3. Qualitative Observation of the Bacterial Killing
<ol>
 <li>Prepare LBA plates containing 40 &mu;g/ml X-gal for the readout of the assay. The plates will be called "Readout input" or "R-input" to spot isolated bacteria or "Readout output" or "R-output" to spot mixed bacterial cultures and thus read the killing performance. These plates need also to be properly dried. Divide the plate in four equal parts on its back and annotate these parts 0, 10-1, 10-2 and 10-3, designating the dilutions of bacterial culture to spot on the agar plates (see &sect;3.3). The dilution will allow a semi-quantitative evaluation of the blue/white bacteria ratio within the same spot.</li>
 <li>Collect with a sterile loop the individual bacterial spots from the "A-input" plate (see &sect;2.4) and "A-output" plate (see &sect;2.5) and resuspend each spot in distinct 1.5 ml Eppendorf tubes containing 1 ml of TSB. Place in an Eppendorf shaker block during 30 min to resuspend efficiently the bacteria.</li>
 <li>Prepare a series of 5 times 3 Eppendorf tubes containing 900 &mu;l of TSB and proceed for each resuspended spot to ten fold serial dilutions up to 10-3. Make sure to change pipette tips and to vortex 5 seconds between each dilution step.</li>
 <li>Proceed to the spotting of your bacterial dilutions prepared in &sect;3.3 on well-dried LBA plates containing 40 &mu;g/ml X-gal by starting with the most diluted to the undiluted suspension, making these the "R-input" plates for the "A-input" spots and the "R-output" plates for the "A-output" spots (Figure 2). Vortex briefly each tube before proceeding to the spotting to keep a homogenous bacterial suspension. For reproducibility of the results, spot 20 &mu;l in triplicate within a quadrant (Figure 2). The relative dryness of your plate is important at this stage since the spots need to remain individually separated on the plate and not glide towards each other. 
 A quantification of the competition assay is also possible at this stage of the experiment. Following the step 3.3, spread 100 &mu;l of the dilution 10-3 on LBA plates containing 40 &mu;g/ml X-gal. For reproducibility of the results, proceed to a plating in triplicate for each spot from the "A-output" plate. Place the dilution plates in a 37 &deg;C incubator overnight (corresponding to 16 hr incubation). Proceed to the counting of the blue colonies (the P cells that remained alive). Typical results obtained after 5 hr of competition are shown in Figure 3.
 </li>
 <li>Leave the plate open (no lid) within a sterile zone to allow the absorption of the liquid in excess within each spot.</li>
 <li>Place the plate in a 37 &deg;C incubator overnight. During this incubation time, the killing is still occurring in the mix (D+/P) since both strains are still in contact.</li>
 <li>Take a picture or scan your plates for the output analysis (Figure 2). Where spots remain largely blue it indicates that E. coli has not been killed by P. aeruginosa. That is the case when E. coli is mixed with a T6SS-defective P. aeruginosa strain (D-) (Figure 2, plate at the bottom right).</li>
</ol>

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+VI+secretion+apparatus+and+phage+tail-associated+protein+complexes+share+a+common+evolutionary+origin.">Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (11), 4154 (2009).</li><li>Pukatzki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+VI+secretion+system+translocates+a+phage+tail+spike-like+protein+into+target+cells+where+it+cross-links+actin.">Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (39), 15508 (2007).</li><li>Ma, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+a+Vibrio+cholerae+type+VI+secretion+effector+requires+bacterial+endocytosis+by+host+cells.">Translocation of a Vibrio cholerae type VI secretion effector requires bacterial endocytosis by host cells.</a> Cell Host Microbe. 5 (3), 234 (2009).</li><li>Hood, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+type+VI+secretion+system+of+Pseudomonas+aeruginosa+targets+a+toxin+to+bacteria.">A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria.</a> Cell Host Microbe. 7 (1), 25 (2010).</li><li>Russell, A. 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We have nothing to disclose.

The method presented in this article allows a visual observation of T6SS-mediated bactericidal/bacteriostasis activity. The assay is performed on the surface of an agar plate. It has been previously shown that T6SS-dependent killing assay performed with mixed bacterial liquid culture is not efficient, likely because of the lack of steady contact between the two bacteria8. The T6SS is believed to operate with a mechanism akin to the one used by bacteriophages to inject DNA into target cells17. In liquid culture, the tube-like structure of the T6SS may break more easily, inter-bacterial contact can be lost, and the toxins are not efficiently delivered.
In terms of incubation times, the 5 initial hours of contact that we describe between the donor strain and the prey are sufficient to observe bacterial killing between P. aeruginosa and E. coli, as illustrated in Figure 3. Nevertheless, it is advisable to adjust the incubation time by performing a kinetic in order to optimize the experimental conditions.
Since this method is a color based technique, the output results can be compromised by the pigmentation of the donor strain. For instance, in the case of P. aeruginosa, some strains produce high levels of colored pigments such as pyocyanin and pyoverdine, which can interfere with the assay readout, making the distinction from the prey relatively difficult. Other chromogenic &beta;-galactosidase substrates, such as the magenta-gal or the red-gal, can be used instead of the X-gal (Table 1).
The competition assay can make use of other reporter genes for the readout. For instance, similar assay has also been performed by using green fluorescent protein-labeled preys12.
Our assay, while not quantitative, gives a good indication of the T6SS activity since it is based on the survival or the killing of a reporter prey. This technique presents the advantage of being easy and convenient to evaluate the bactericidal/bacteriostasis activity of T6SSs from any bacterial species. So far, the activity of the T6SS has been shown against Gram-negative bacteria and no clear example of T6SS-sensitive Gram-positive bacteria has been reported yet12. It is also obvious that incompatibility in the culture of the different bacterial species to test (e.g. growth temperature, oxygenation, specific media) is to be considered.
Our assay can also be used to evaluate which of the T6SS components are absolutely essential since even traces of a secreted toxin might be sufficient to kill the prey. Even weak activity of the T6SS could then clearly be detected by our assay as compared to standard procedure testing T6SS-dependent secretion using culture supernatant and western blot analysis. However, a proper colony-forming unit (CFU) counting is still required for accurate quantification of this T6SS activity.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>P. aeruginosa PAK&Delta;retS (D+) (active T6SS)</td>
 <td>Lab strain</td>
 <td></td>
 <td>Described in Reference 16</td>
 </tr>
 <tr>
 <td>P. aeruginosa PAK&Delta;retS&Delta;H1-T6SS (D-)(inactive T6SS)</td>
 <td>This study</td>
 <td></td>
 <td>The H1-T6SS cluster (encompassing the genes PA0070 to PA0095) has been deleted by allelic exchange following the procedure described in Reference 18. The mutator fragment was generated with the following set of primers: The Up fragment primers: 
 5'-ATGGTCAACGACATGGAGCTGGAG-3', and 
 5'CGAGGCCGATCAGGCCTTCAGAACTGA-3'. The Down fragment primers : 
 5'-TCAGGCCTTCAGAACTGAAGCGGCGCA-3', 
 <pre>5'-GGTGGCGTTCAACAGTTCCATGTC-3' </pre>
 </td>
 </tr>
 <tr>
 <td>E.coli DH5&alpha;</td>
 <td>Invitrogen</td>
 <td>18258-012</td>
 <td>F- &phi;80lacZ&Delta;M15 &Delta;(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 &lambda;- thi-1 gyrA96 relA1 </td>
 </tr>
 <tr>
 <td>pBluescript II SK(+)</td>
 <td>Agilent</td>
 <td>212205</td>
 <td>This vector expresses the &alpha; peptide of &beta;-galactosidase used for &alpha;-complementation.</td>
 </tr>
 <tr>
 <td>X-gal</td>
 <td>Invitrogen</td>
 <td>15520-018</td>
 <td>Use at 40 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Luria Bertani agar</td>
 <td>Merck-chemicals</td>
 <td>1.10283.0500</td>
 <td></td>
 </tr>
 <tr>
 <td>TSB (casein soya bean broth)</td>
 <td>Oxoid</td>
 <td>CM109</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex shaker Genius 3</td>
 <td>IKA</td>
 <td>3340000</td>
 <td></td>
 </tr>
 <tr>
 <td>Scanner </td>
 <td>Epson</td>
 <td>V700</td>
 <td></td>
 </tr>
 <tr>
 <td>Spectrophotometer</td>
 <td>WPA Biowave</td>
 <td>CO8000 Cell Density Meter</td>
 <td></td>
 </tr>
 <tr>
 <td>Magenta-gal</td>
 <td>Bioworld</td>
 <td>30350001-1 (715241)</td>
 <td></td>
 </tr>
 <tr>
 <td>Red-gal</td>
 <td>Research organics</td>
 <td>1364c</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 1. Strain, plasmid, material and reagent used.</td>
 </tr>
 </tbody>
</table>

a visual assay to monitor t6ss-mediated bacterial competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

Typical results are shown in Figure 1 with the strains and reagents described in Table 1. The plates shown in this figure were scanned after an overnight incubation. The "Readout-Input" plates show a serial dilution pattern for the strains used in this assay. As expected, the E. coli prey spots (P) overexpressing the lacZ gene appear blue on media supplemented with X-gal, while the donor P. aeruginosa strains (D+, T6SS active) and (D-, T6SS inactive) remain white. The "Readout-output" plates on which the mix between the prey and a T6SS active strain (D+/P) has been spotted show the disappearance of the blue prey thus indicating it has been killed. This demonstrates the ability of the donor to outcompete the prey. The persistence of the blue color on the (D-/P) plate demonstrates the inability of an inactive T6SS donor to kill the blue prey.
<img src="/files/ftp_upload/50103/50103fig1.jpg" alt="Figure 1"/> 
Figure 1. Killing of E. coli by T6SS-proficient P. aeruginosa. P. aeruginosa injects toxins into the E. coli target cell in a T6SS-dependent manner (shown by the white arrow). Two toxins Tse1 and Tse3 (orange and red circles) are injected into the E. coli periplasm and degrade the peptidoglycan9. The Tse2 toxin (yellow circle) is injected into the E. coli cytoplasm and has a bacteriostatic activity8,10. The combined action of the toxins kills the target cells (flash of lightning and skull). The survival of target cells can be detected by monitoring the activity of the produced &beta;-galactosidase (see also Figure 2). P. aeruginosa is protected against the activity of the toxins by the immunity proteins Tsi1, Tsi2 and Tsi3 (orange, yellow and red squares, respectively)8,9,10.
<img src="/files/ftp_upload/50103/50103fig2.jpg" alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50103/50103fig2highres.jpg"/> 
Figure 2. Agar plate assay to monitor T6SS-dependent bacterial killing. In this figure are shown on the upper part the "Readout-Input" plates consisting of the serial dilutions of the D-, D+, and P input cells. The P input cells are blue due to the &alpha;-complementation of the lacZ gene and thus produced &beta;-galactosidase that cleaves X-gal. In the lower part is shown the "Readout-Output" plates consisting of serial dilutions of the bacterial mix between an active (D+/P),or an inactive (D-/P), T6SS donor P. aeruginosa strain with the E. coli prey. <a href="https://www.jove.com/files/ftp_upload/50103/50103fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/50103/50103fig3.jpg" alt="Figure 3"/> 
Figure 3. Quantification of the killing of E. coli by P. aeruginosa after 5 hr incubation. The graph presents the CFU counting of E. coli described in the 3.4 step. The results presented here show a 3-fold difference between the T6SS+ and the T6SS- strains, suggesting that most of the killing is taking place during the 5 initial hours of contact.

Watch this Scientific Journal Video about A Visual Assay to Monitor T6SS-mediated Bacterial Competition at JoVE.com

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

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Immunology and Infection

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

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&#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 ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...