Immunology and Infection
Published: March 20th, 2013
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 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 DH5α) 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.
1. Bacterial Strains and Cultures
2. Competition Assay
3. Qualitative Observation of the Bacterial Killing
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.
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 β-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.
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 α-complementation of the lacZ gene and thus produced β-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. Click here to view larger figure.
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.
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 β-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.
We have nothing to disclose.
This work was funded by the Wellcome trust grant WT091939MA. Alain Filloux is supported by the Royal Society.
|Name of Reagent/Material
|Described in Reference 16
|P. aeruginosa PAKΔretSΔH1-T6SS (D-)(inactive T6SS)
|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'CGAGGCCGATCAGGCCTTCAGAACTGA-3'. The Down fragment primers :
|F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) phoA supE44 λ- thi-1 gyrA96 relA1
|pBluescript II SK(+)
|This vector expresses the α peptide of β-galactosidase used for α-complementation.
|Use at 40 μg/ml
|Luria Bertani agar
|TSB (casein soya bean broth)
|Vortex shaker Genius 3
|CO8000 Cell Density Meter
Table 1. Strain, plasmid, material and reagent used.
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