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Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci
接合实验检测葡萄球菌CRISPR-Cas的抗质粒免疫功能   

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Abstract

CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. Testing the ability of CRISPR-Cas systems to block plasmid transfer in various conditions or with CRISPR-Cas mutants provides key insights into the functionality and mechanisms of CRISPR-Cas as well as how antibiotic resistance spreads within bacterial communities. Here, we describe a method for quantifying the impact of CRISPR-Cas on the efficiency of plasmid transfer by conjugation. While this method is presented in Staphylococcus species, it could be more broadly used for any conjugative prokaryote.

Keywords: Conjugation(接合), CRISPR-Cas(CRISPR-Cas), Plasmids(质粒), Staphylococcus(葡萄球菌), Antibiotic resistance(抗生素耐药), Horizontal gene transfer(水平基因转移)

Background

CRISPR-Cas (Clustered, regularly interspaced, short palindromic repeats-CRISPR associated) is a prokaryotic adaptive immune system found in almost 90% of sequenced archaea and about 40% of bacteria (Makarova et al., 2015). These systems recognize and destroy nucleic acid invaders in a sequence-specific manner (van der Oost et al., 2014). A CRISPR locus typically contains an array of short DNA repeats (~35 nucleotides in length) separated by equally-short unique sequences called spacers, which are often derived from mobile genetic elements. The repeats and spacers are transcribed and processed into small CRISPR RNAs (crRNAs) that each specify a single target. crRNAs assemble with one or more Cas proteins to form an effector complex that recognizes and degrades nucleic acids that bear a sequence, called a protospacer, complementary to the crRNA. Depending on the CRISPR-Cas type present in an organism, other limitations exist on target recognition, such as the presence of a short protospacer-adjacent motif (PAM) required for targeting by Type I and II systems (Mojica et al., 2009) or the requirement in Type III systems for specific base-pair mismatches between the crRNA and the target (Marraffini and Sontheimer, 2010). Nucleic acid invaders targeted by CRISPR-Cas are typically bacteriophages or mobile genetic elements such as plasmids or transposons that often carry virulence factors, including antibiotic resistance genes and toxins (Novick, 2003; Liu et al., 2016). CRISPR-Cas is thus a potential major barrier to the spread of virulence factors within prokaryotes and has been shown to prevent conjugative transfer of antibiotic resistance plasmids within human clinical isolates of Staphylococcus species (Marraffini and Sontheimer, 2008). Some staphylococci, including human pathogenic isolates of S. epidermidis and S. aureus, the two Staphylococcus species most commonly found in human infections, carry CRISPR-Cas systems (Cao et al., 2016; Li et al., 2016). Within these strains, multiple CRISPR spacers have been found that naturally bear homology to mobile staphylococcal plasmids, indicating that these organisms are capable of using CRISPR-Cas to limit the spread of virulence factors (Samai et al., 2015; Li et al., 2016). Investigations of the effect of mutations within the cas genes and the repeat-spacer array in Staphylococcus on anti-plasmid immunity have provided key insights into the function and mechanisms of Type III-A CRISPR-Cas in S. epidermidis RP62a (Hatoum-Aslan et al., 2011 and 2014; Maniv et al., 2016). Notably, similar assays have been used for mechanistic CRISPR-Cas studies in other organisms, including Enterococcus faecalis, Escherichia coli, and Listeria monocytogenes (Richter et al., 2014; Sesto et al., 2014; Price et al., 2016). Other methods of quantifying CRISPR anti-plasmid immunity, namely transformation via electroporation, have been used (Cao et al., 2016); however, this method cannot be used in non-competent or weakly/selectively competent organisms such as L. monocytogenes and many strains of Staphylococcus (Monk et al., 2012; Sesto et al., 2014). In these organisms, conjugation assays provide not only a viable alternative, but also a more physiologically relevant means of testing CRISPR-Cas anti-plasmid immunity. Described below is a quantitative method to determine the efficacy of CRISPR-mediated interference of the transfer of conjugative plasmid pG0400 between S. aureus and S. epidermidis. While this protocol focuses on staphylococci, it could be adapted for any prokaryotes capable of conjugation.

Materials and Reagents

  1. 15 ml centrifuge tubes (VWR, catalog number: 21008-216 )
  2. 1.7 ml microcentrifuge tubes (VWR, catalog number: 87003-294 )
  3. 0.45 μm membrane filters, 25 mm (EMD Millipore, catalog number: HAWP02500 )
  4. 50 ml centrifuge tubes (VWR, catalog number: 21008-242 )
  5. Pipette tips with filter, 100-1,000 μl (VWR, catalog number: 89003-060 )
  6. Pipette tips with filter, 1-200 μl (VWR, catalog number: 89003-056 )
  7. Pipette tips with filter, 1-40 μl (VWR, catalog number: 89003-048 )
  8. 100 x 15 mm Petri dishes (VWR, catalog number: 25384-088 )
  9. 0.2 ml PCR strip tubes (VWR, catalog number: 20170-004 )
  10. 1 mm path length cuvettes (VWR, catalog number: 97000-586 )
  11. Recipient S. epidermidis RP62a (Christensen et al., 1985), bearing a CRISPR-Cas system (see Note 1) (ATCC, catalog number: 35984 )
  12. Donor S. aureus RN4220 (Kreiswirth et al., 1983), bearing the conjugative plasmid pG0400 (Morton et al., 1995) (see Note 2) (ATCC, BEI Resources, catalog number: NR-45913 )
  13. Negative control S. epidermidis LAM104 (Marraffini and Sontheimer, 2008), lacking the CRISPR repeat-spacer array (see Note 3)
  14. Mupirocin (The United States Pharmacopeial Convention, catalog number: 1448901 )
  15. Brain-heart infusion broth (BD, BBL, catalog number: 211060 )
  16. Brain-heart infusion agar (BD, Difco, catalog number: 241830 )
  17. Tryptic soy broth (BD, BactoTM, catalog number: 211822 )
  18. Neomycin sulfate (AMRESCO, catalog number: 0558-25G )
  19. Brain-heart infusion broth (see Recipes)
  20. Brain-heart infusion agar (see Recipes)
  21. Tryptic soy broth (see Recipes)

Equipment

  1. Micropipettes set (Eppendorf, model: Research® Plus )
  2. 37 °C incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HerathermTM Advanced Protocol Microbiological Incubators )
  3. 37 °C incubated shaker capable of 160 rpm (Eppendorf, model: I26 )
  4. Autoclave (Getinge)
  5. Spectrophotometer (GE Healthcare, model: UltroSpec 10 )
  6. Microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 )
  7. Digital vortex mixer (VWR, model: Advanced Heavy-Duty Vortex Mixer )
  8. Inoculating wire loop
  9. Forceps
  10. Magnetic stir plate

Procedure

  1. Day 1. Preparing strains
    1. Streak a brain-heart infusion (BHI) agar plate containing 15 μg/ml neomycin with S. epidermidis RP62a (from here called ‘recipient’) from a -80 °C freezer stock to obtain single colonies. Incubate overnight at 37 °C.
      Note: In the same manner, streak out the negative control strain, S. epidermidis LAM104.
    2. Inoculate S. aureus carrying pG0400 (here onward called ‘donor’) from a freezer stock into 2 ml of tryptic soy broth (TSB) containing 5 μg/ml mupirocin. Incubate with shaking at 37 °C overnight.
      Note: While both S. epidermidis and S. aureus strains may be propagated in either BHI or TSB, the media indicated for each strain in this protocol are consistent with the growth media used in the original protocol demonstrating anti-plasmid immunity by the Type III-A CRISPR-Cas system in S. epidermidis RP62a (Marraffini and Sontheimer, 2008).
  2. Day 2. Growing colonies and filter mating
    1. After at least 16 h of incubation, large (> 1 mm in diameter) individual recipient colonies should be seen. Inoculate the entirety of 3 separate colonies into 15-ml centrifuge tubes containing 2 ml of BHI with 15 μg/ml neomycin.
      Notes:
      1. Also, inoculate any controls included in the experiment in triplicate.
      2. At the same time, add 50 μl of the overnight donor culture to 5 ml of TSB with 5 μg/ml mupirocin in a 15-ml centrifuge tube.
    2. Grow both the donor and recipient at 37 °C with shaking.
      Note: At the same time, place one BHI plate per 3 growing recipient cultures into the 30 °C incubator to pre-warm (see Note 4).
    3. After ~6 h, check the OD600 of both the recipient and donor strains.
      1. Add 900 μl of BHI broth into a spectrophotometer cuvette. Add 100 μl of growing culture of one of the recipient cultures. Mix well.
      2. After blanking with BHI, measure and record the OD600 of this 1:10 dilution of the growing culture.
      3. Repeat steps 2c i and 2c ii with the remaining recipient cultures.
      4. Repeat steps 2c i and 2c ii for the donor culture, substituting TSB for BHI.
      5. OD600 of all culture dilutions needs to be greater than 0.20 before proceeding. If the OD600 are not yet high enough, return to the shaker and let grow a while before checking the OD600 again.
    4. Calculate the amount of donor and recipient to combine to achieve a 1:4 ratio (recipients:donors) for filter mating, as in the example in Table 1.
      Note: Divide 200 by the measured OD600 found in step 2c for each culture. At an OD600 of 1, there are approximately 8 x 108 cfu/ml of the donor and 2 x 108 cfu/ml of the recipient. This calculation allows for standard numbers of donors (approximately 1.6 x 109 cfu) and recipients (approximately 4 x 108 cfu) to be combined in all samples that are tested.

      Table 1. Sample calculations to determine volumes of cultures to combine for filter mating
      Culture
      OD600 (1:10 dilution)
      200/OD600 (Volume to add, μl)
      RP62a (1)
      0.42
      476
      RP62a (2)
      0.42
      476
      RP62a (3)
      0.41
      488
      LAM104 (1)
      0.44
      455
      LAM104 (2)
      0.39
      513
      LAM104 (3)
      0.44
      455
      RN4220/pG0400
      0.56
      357

    5. Into a 1.7 ml microcentrifuge tube, combine the above-calculated volume of the first, undiluted, recipient culture with the calculated volume of the undiluted donor.
      Note: Repeat until all donor-recipient pairs are combined into separate microcentrifuge tubes.
    6. Wash the combined cultures to remove the antibiotics by centrifuging the microcentrifuge tubes at 6,200 x g for 2 min in a table-top centrifuge. Pipette off the supernatant and resuspend the pellets in 1 ml of BHI without antibiotics. Repeat the centrifugation.
    7. Using sterile forceps, place one 0.45 μm membrane filter for each donor-recipient pair onto a BHI plate containing no antibiotics, with up to 3 filters on a single plate (Figure 1).


      Figure 1. Sample plate for filter mating showing a plate with three membrane filters covered in overnight growth in the center of each filter

    8. Remove the suspension from each microcentrifuge tube containing the donor and recipient combination. Resuspend the pellet in 100 μl of BHI broth and carefully pipet it onto the center of the membrane filter, being careful to not let any of the suspension fall off the membrane, as in Figure 1. It is crucial to keep all of the bacterial suspension on the filter, as this assay is quantitative and requires total recovery of all the cells.
    9. Repeat this with each microcentrifuge tube, placing the resuspension of each culture onto its own filter.
    10. Cover the plates and leave on the benchtop until the cell suspension has fully dried.
    11. Incubate the plates upside-down in a 30 °C incubator overnight.
  3. Day 3. Serially diluting and plating recipients and transconjugants
    1. Warm one BHI agar plate containing neomycin (15 μg/ml) and one BHI agar plate containing both neomycin (15 μg/ml) and mupirocin (5 μg/ml) for each filter from the previous night.
    2. In 50-ml centrifuge tubes (one for each filter), add 3 ml of BHI broth without antibiotics.
    3. Using sterile forceps, pick each filter off the plate and place it into a 50-ml tube prepared in step 3b.
    4. Vortex each tube until the cells on the filter have been fully resuspended and no growth is visible on the filter.
    5. Serially dilute the cell suspension
      1. Into each of the 8 tubes in a strip of PCR tubes, add 90 μl BHI broth.
      2. Remove 10 μl of the bacterial suspension from step 3d and add it to the first PCR strip tube, pipetting up and down repeatedly to resuspend (this is the 10-1 dilution).
      3. Using a new pipette tip, remove 10 μl from the 10-1 dilution and add it to the second PCR tube, pipetting up and down repeatedly to resuspend (this is the 10-2 dilution).
      4. Repeat step 3e iii to create dilutions all the way out to 10-8.
        Note: Alternative methods of performing a series of 10-fold serial dilutions, such as using a multichannel pipette in a 96-well plate may be used instead.
    6. Spot 10 μl of each dilution onto a pre-warmed BHI plate containing neomycin and another containing neomycin and mupirocin (see Note 5). All dilutions to be plated for recipient colony counting should be plated on the same plate, as in Figure 2A, and all dilutions to be plated for transconjugant colony counting should be plated on an additional plate, as in Figure 2B.
      Note: Tilt the plate at a 45° angle until the drop of the dilution almost reaches the edge of the plate, then set it flat on the benchtop and let dry.


      Figure 2. Sample plates from a conjugation assay using a strain of RP62a, showing recipients (A. 1.5 x 109 cfu/ml) and transconjugants (B. 0 cfu/ml)

    7. Repeat steps 3e and 3f for each filter.
    8. Let the plates dry while covered on the benchtop, then incubate at 37 °C overnight.
  4. Day 4. Recipient and transconjugant counting
    1. Count the number of recipients (colonies on plates containing only neomycin) for each filter from Day 2, as shown in Figure 2A.
      1. Determine the number of colonies in the highest dilution where the colonies are still countable.
      2. Multiply the number of colonies by 100 and by the dilution factor (101 for the 10-1 dilution, 102 for the 10-2 dilution, etc.) to obtain the number of colony forming units per milliliter.
    2. Count the number of transconjugants (colonies on plates containing neomycin and mupirocin) for each filter from Day 2, as in step 4a and shown in Figure 2B.
    3. Calculate the conjugation efficiency (see Data analysis).

Data analysis

  1. Calculation of conjugation efficiency
    1. Conjugation efficiency is a measure of the number of transconjugants produced per each initial recipient cell. It can be simply calculated as follows, with an example shown in Table 2:



      Table 2. Calculation of conjugation efficiency of a sample conjugation assay
      Strain
      Transconjugants (cfu/ml)
      Recipients (cfu/ml)
      Conjugation Efficiency
      RP62a (1)
      1 x 103
      7 x 109
      1 x 10-7
      RP62a (2)
      2 x 102
      1 x 109
      2 x 10-7
      RP62a (3)
      0
      4 x 109
      0
      LAM104 (1)
      1.3 x 105
      3 x 109
      4 x 10-5
      LAM104 (2)
      9 x 104
      3 x 109
      3 x 10-5
      LAM104 (3)
      9 x 104
      4 x 109
      2 x 10-5

    2. A high efficiency indicates an inactive CRISPR system, while a lowered efficiency indicates active CRISPR defense.
      1. A 100 to 1,000-fold decrease in conjugation efficiency is typically seen in the presence of a CRISPR system as compared to a strain without a CRISPR system.
  2. Data presentation
    Data is typically shown in a bar graph showing the number of both recipients and transconjugants in cfu/ml (colony-forming units per milliliter) on a logarithmic scale, with the calculated conjugation efficiencies accompanying the graph, as in Figure 3.


    Figure 3. Sample graph of data from a conjugation assay using S. epidermidis RP62a and LAM104 as recipients. Error bars indicate the standard deviation of triplicate measurements. 

Notes

  1. Other strains can be used as the recipient strain in this assay. S. epidermidis RP62a naturally carries a CRISPR spacer that targets the nickase gene found on conjugative staphylococcal plasmids such as pG0400 (Marraffini and Sontheimer, 2008). Other CRISPR-containing prokaryotes can be used if they are amenable to receiving conjugative plasmids. An additional important consideration in recipient selection is to make sure that the recipient has a selectable marker that differentiates it from the donor strain. In the case of conjugation assays with S. epidermidis, S. epidermidis RP62a is resistant to neomycin, so this resistance can be used to select only for transconjugants and not the donor strain itself on Day 3. Recipient strains that do not have differing antibiotic susceptibilities naturally can be transformed with a plasmid harbouring an additional resistance marker (such as pC194 [Ehrlich, 1977] for Staphylococcus). Whichever plasmid is chosen, ensure its origin is compatible with the conjugative plasmid to allow for maintenance and selection of both plasmids in the transconjugants. If using different selective markers, change antibiotics as appropriate for culturing and plating.
  2. Other strains of S. aureus can be used as the donor strain in this assay, as can additional conjugative plasmids. The donor strain needs to be able to perform conjugation with the recipient strain, and RN4220 is very convenient for this conjugation assay as it can perform conjugation with S. epidermidis RP62a and lacks a CRISPR system, allowing for ready uptake and maintenance of plasmids such as pG0400 that could be degraded in a CRISPR-bearing strain. Additional conjugative plasmids can also be used, depending on the CRISPR spacers found within the recipient strain. If testing to see if CRISPR functions in anti-plasmid activity in the recipient strain, a conjugative plasmid that bears a protospacer complementary to a spacer found within the CRISPR array of the recipient must be used. In the case of RP62a, pG0400 naturally has this protospacer, but in many systems, it will likely be necessary to engineer a conjugative plasmid with a protospacer. If using a different conjugative plasmid, change antibiotics as appropriate for culturing and plating. Additionally, if engineering a conjugative plasmid, be sure to follow the rules required for CRISPR targeting in the specific system to be tested, such as following the PAM requirement for Types I and II systems (Mojica et al., 2009) and the required mismatches that permit interference by Type III-A systems (Marraffini and Sontheimer, 2010).
  3. LAM104 is an engineered derivative of RP62a that lacks the repeat-spacer array, preventing CRISPR-Cas from functioning (Marraffini and Sontheimer, 2008). If using a strain other than RP62a, it is recommended to use a negative control in which CRISPR-Cas does not function. Suitable negative controls include 1) the use of a recipient strain that lacks a crucial component of CRISPR-Cas or 2) the use of a donor strain that carries a conjugative plasmid lacking an appropriate protospacer. S. epidermidis LAM104 is not available commercially, but can be made available from the Hatoum-Aslan laboratory on request.
  4. With S. epidermidis RP62a, increased background counts of recipients have been observed on Day 4 if filter mating occurs at 37 °C. To obtain a cleaner result, a 30 °C incubation is routinely used, although this can be modified to suit the growth requirements of any organism.
  5. With S. epidermidis strains as recipients, transconjugants are typically seen in a concentration from 100-105 cfu/ml, and therefore only the 100 to 10-3 dilutions are typically plated on the BHI plates containing neomycin and mupirocin to obtain colony counts. Recipients are usually around 109 cfu/ml and thus usually only the 10-5 to 10-8 dilutions are plated on the BHI plates containing neomycin to obtain colony counts.

Recipes

  1. Brain-heart infusion broth
    1. Dissolve 19.5 g brain-heart infusion in 500 ml deionized (DI) water
    2. Autoclave at 121 °C for 30 min and store at room temperature
  2. Brain-Heart infusion agar
    1. Dissolve 29 g brain-heart infusion agar in 500 ml DI water
    2. Autoclave at 121 °C for 30 min and stir on a magnetic stir plate until it has cooled enough to be comfortable to the touch, then add antibiotics and pour plates immediately (20 plates per 500 ml)
      Note: Media can also be stored at room temperature and later heated in the microwave to liquefy and pour.
  3. Tryptic soy broth
    1. Dissolve 15 g of tryptic soy broth in 500 ml DI water
    2. Autoclave at 121 °C for 30 min and store at room temperature

Acknowledgments

A.-H-A. is supported by the University of Alabama (UA) College of Arts and Sciences; a grant from the UA College Academy of Research, Scholarship, and Creative Activity (CARSCA); and the National Institutes of Health [5K22AI113106-02]. This protocol was adapted from that published in Marraffini and Sontheimer (2008).

References

  1. Cao, L., Gao, C. H., Zhu, J., Zhao, L., Wu, Q., Li, M. and Sun, B. (2016). Identification and functional study of type III-A CRISPR-Cas systems in clinical isolates of Staphylococcus aureus. Int J Med Microbiol 306(8): 686-696.
  2. Christensen, G. D., Simpson, W. A., Younger, J. J., Baddour, L. M., Barrett, F. F., Melton, D. M. and Beachey, E. H. (1985). Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22(6): 996-1006.
  3. Ehrlich, S. D. (1977). Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc Natl Acad Sci U S A 74(4): 1680-1682.
  4. Hatoum-Aslan, A., Maniv, I. and Marraffini, L. A. (2011). Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc Natl Acad Sci U S A 108(52): 21218-21222.
  5. Hatoum-Aslan, A., Maniv, I., Samai, P. and Marraffini, L. A. (2014). Genetic characterization of antiplasmid immunity through a type III-A CRISPR-Cas system. J Bacteriol 196(2): 310-317.
  6. Kreiswirth, B. N., Lofdahl, S., Betley, M. J., O'Reilly, M., Schlievert, P. M., Bergdoll, M. S. and Novick, R. P. (1983). The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305(5936): 709-712.
  7. Li, Q., Xie, X., Yin, K., Tang, Y., Zhou, X., Chen, Y., Xia, J., Hu, Y., Ingmer, H., Li, Y. and Jiao, X. (2016). Characterization of CRISPR-Cas system in clinical Staphylococcus epidermidis strains revealed its potential association with bacterial infection sites. Microbiol Res 193: 103-110.
  8. Liu, J., Chen, D., Peters, B. M., Li, L., Li, B., Xu, Z. and Shirliff, M. E. (2016). Staphylococcal chromosomal cassettes mec (SCCmec): A mobile genetic element in methicillin-resistant Staphylococcus aureus. Microb Pathog 101: 56-67.
  9. Makarova, K. S., Wolf, Y. I., Alkhnbashi, O. S., Costa, F., Shah, S. A., Saunders, S. J., Barrangou, R., Brouns, S. J., Charpentier, E., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J., Terns, R. M., Terns, M. P., White, M. F., Yakunin, A. F., Garrett, R. A., van der Oost, J., Backofen, R. and Koonin, E. V. (2015). An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 13(11): 722-736.
  10. Maniv, I., Jiang, W., Bikard, D. and Marraffini, L. A. (2016). Impact of different target sequences on type III CRISPR-Cas immunity. J Bacteriol 198(6): 941-950.
  11. Marraffini, L. A. and Sontheimer, E. J. (2008). CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322(5909): 1843-1845.
  12. Marraffini, L. A. and Sontheimer, E. J. (2010). Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463(7280): 568-571.
  13. Mojica, F. J., Díez-Villasenor, C., Garcia-Martinez, J. and Almendros, C. (2009). Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155(Pt 3): 733-740.
  14. Monk, I. R., Shah, I. M., Xu, M., Tan, M. W. and Foster, T. J. (2012). Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. MBio 3(2).
  15. Morton, T. M., Johnston, J. L., Patterson, J. and Archer, G. L. (1995). Characterization of a conjugative staphylococcal mupirocin resistance plasmid. Antimicrob Agents Chemother 39(6): 1272-1280.
  16. Novick, R. P. (2003). Mobile genetic elements and bacterial toxinoses: the superantigen-encoding pathogenicity islands of Staphylococcus aureus. Plasmid 49(2): 93-105.
  17. Price, V. J., Huo, W., Sharifi, A. and Palmer, K. L. (2016). CRISPR-Cas and restriction-modification act additively against conjugative antibiotic resistance plasmid transfer in Enterococcus faecalis. mSphere 1(3).
  18. Richter, C., Dy, R. L., McKenzie, R. E., Watson, B. N., Taylor, C., Chang, J. T., McNeil, M. B., Staals, R. H. and Fineran, P. C. (2014). Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer. Nucleic Acids Res 42(13): 8516-8526.
  19. Samai, P., Pyenson, N., Jiang, W., Goldberg, G. W., Hatoum-Aslan, A. and Marraffini, L. A. (2015). Co-transcriptional DNA and RNA cleavage during type III CRISPR-Cas immunity. Cell 161(5): 1164-1174.
  20. Sesto, N., Touchon, M., Andrade, J. M., Kondo, J., Rocha, E. P., Arraiano, C. M., Archambaud, C., Westhof, E., Romby, P. and Cossart, P. (2014). A PNPase dependent CRISPR system in Listeria. PLoS Genet 10(1): e1004065.
  21. van der Oost, J., Westra, E. R., Jackson, R. N. and Wiedenheft, B. (2014). Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nat Rev Microbiol 12(7): 479-492.

简介

CRISPR-Cas是一种原核适应性免疫系统,可防止移植遗传因子如噬菌体和质粒的摄取。细菌之间的质粒转移是特别临床关注的,因为由于在物种内和物种之间转移抗性质粒而在人体中发现的抗生素抗性病原体的量增加。测试CRISPR-Cas系统在各种条件下或使用CRISPR-Cas突变体阻断质粒转移的能力,为CRISPR-Cas的功能和机制以及抗菌素抗性如何在细菌群落中传播提供了重要的见解。在这里,我们描述了一种量化CRISPR-Cas对通过共轭进行质粒转移效率的影响的方法。虽然这种方法在葡萄球菌属物种中呈现,但它可以更广泛地用于任何共轭原核生物。

背景 CRISPR-Cas(聚集的,定期间隔的,短的回文重复CRISPR相关联)是几乎90%的测序古菌和约40%的细菌中发现的原核适应性免疫系统(Makarova等人,2015) )。这些系统以序列特异性方式识别和破坏核酸侵入体(van der Oost等人,2014)。 CRISPR基因座通常包含短的DNA重复序列(长度为约35个核苷酸),其通过称为间隔区的等短的唯一序列分开,其通常来自移动遗传元件。重复序列和间隔区被转录并处理成小的CRISPR RNA(crRNA),每个指定单个靶标。 crRNA与一种或多种Cas蛋白质组装形成效应物复合物,其识别和降解与crRNA互补的称为原始序列的序列的核酸。根据存在于生物体中的CRISPR-Cas类型,目标识别存在其他限制,例如由I型和II型系统靶向所需的短原型相邻基序(PAM)的存在(Mojica等,2009)或III型系统对crRNA和靶标之间特异性碱基对错配的要求(Marraffini和Sontheimer,2010)。 CRISPR-Cas靶向的核酸侵入物通常是噬菌体或移动遗传元件,例如通常携带毒力因子的质粒或转座子,包括抗生素抗性基因和毒素(Novick,2003; Liu等人,2016年) )。因此,CRISPR-Cas是原核生物中毒力因子扩散的潜在主要障碍,已被证明可以预防人类金黄色葡萄球菌种临床分离株(Marraffini和Sontheimer,2008)中抗生素抗性质粒的共转移, 。一些葡萄球菌,包括人类致病性分离株。表皮细胞和 S。金黄色葡萄球菌,人类感染中最常见的两种葡萄球菌属携带CRISPR-Cas系统(Cao等人,2016; Li等人。,2016)。在这些菌株中,已经发现多个CRISPR间隔物自然地与移动葡萄球菌质粒具有同源性,表明这些生物体能够使用CRISPR-Cas来限制毒力因子的扩散(Samai等人, 2015; Li等人,2016)。研究了cas基因突变和葡萄球菌重复间隔序列对抗质粒免疫的影响,为III型A CRISPR-Cas的功能和机制提供了重要的见解。上。表皮炎 RP62a(Hatoum-Aslan等人,2011和2014; Maniv等人,2016)。值得注意的是,类似的测定已被用于其他生物体的机械CRISPR-Cas研究,包括粪肠球菌,大肠杆菌和单核细胞增生利斯特氏菌(Richter) ,2014; Sesto等人,2014; Price& et al。,2016)。已经使用其他量化CRISPR抗质粒免疫的方法,即通过电穿孔进行转化(Cao等人,2016);然而,该方法不能用于非能力或弱/有选择性的生物体,例如L。单核细胞增生李斯特氏菌和许多葡萄球菌菌株(Monk等人,2012; Sesto等人,2014)。在这些生物体中,缀合测定不仅提供了可行的替代方法,而且提供了更具生理学意义的测试CRISPR-Cas抗质粒免疫的方法。下面描述的是确定RISPR介导的干扰转移宿主质粒pG0400在功能之间的定量方法。金黄色葡萄球菌和 S。表皮。虽然这个协议集中在葡萄球菌,它可以适应任何能够共轭的原核生物。

关键字:接合, CRISPR-Cas, 质粒, 葡萄球菌, 抗生素耐药, 水平基因转移

材料和试剂

  1. 15ml离心管(VWR,目录号:21008-216)
  2. 1.7ml微量离心管(VWR,目录号:87003-294)
  3. 0.45μm膜过滤器,25mm(EMD Millipore,目录号:HAWP02500)
  4. 50ml离心管(VWR,目录号:21008-242)
  5. 带过滤器的移液器吸头,100-1,000μl(VWR,目录号:89003-060)
  6. 移液器吸头带过滤器,1-200μl(VWR,目录号:89003-056)
  7. 移液器吸头带过滤器,1-40μl(VWR,目录号:89003-048)
  8. 100 x 15毫米培养皿(VWR,目录号:25384-088)
  9. 0.2毫升PCR条形管(VWR,目录号:20170-004)
  10. 1 mm路径长度比色皿(VWR,目录号:97000-586)
  11. 收件人表皮葡萄球菌RP62a(Christensen等人,1985),携带CRISPR-Cas系统(见注1)(ATCC,目录号:35984)
  12. 捐助者具有缀合质粒pG0400(Morton等人,1995)(见附注2)的ATCC420(Kreiswirth等人,1983)(ATCC, BEI资源,目录号:NR-45913)
  13. 负面控制表皮炎 LAM104(Marraffini和Sontheimer,2008),缺少CRISPR重复间隔阵列(见注3)
  14. 莫匹罗星(美国药典,目录号:1448901)
  15. 脑心脏输液汤(BD,BBL,目录号:211060)
  16. 脑心浸液琼脂(BD,Difco,目录号:241830)
  17. 胰蛋白酶大豆肉汤(BD,Bacto TM ,目录号:211822)
  18. 硫酸新霉素(AMRESCO,目录号:0558-25G)
  19. 脑心脏输液汤(见食谱)
  20. 脑心浸液琼脂(见食谱)
  21. 胰蛋白酶大豆肉汤(见食谱)

设备

  1. 微型移液器组(Eppendorf,型号:Research ® Plus)
  2. 37℃培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heratherm TM高级微生物培养箱)
  3. 37℃孵育的振荡器能够以160rpm(Eppendorf,型号:I26)
  4. 高压灭菌器(Getinge)
  5. 分光光度计(GE Healthcare,型号:UltroSpec 10)
  6. 微量离心机(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Heraeus TM Pico TM 17)
  7. 数字涡街搅拌机(VWR,型号:Advanced Heavy-Duty Vortex Mixer)
  8. 接线线圈
  9. 镊子
  10. 磁力搅拌板

程序

  1. 第一天准备品种
    1. 使用含有15μg/ml新霉素的脑 - 心脏输注(BHI)琼脂平板条纹。表皮炎 RP62a(从这里称为"受体")从-80°C冷冻库存中获得单个菌落。在37°C孵育过夜。
      注意:以相同的方式,染色阴性对照菌株,表皮葡萄球菌LAM104。
    2. 接种S。金黄色葡萄球菌将pG0400(这里称为"供体")从冷冻库中加入到含有5μg/ml莫匹罗星的2ml胰蛋白酶大豆肉汤(TSB)中。在37℃下摇荡过夜过夜。
      注意:虽然表皮葡萄球菌和金黄色葡萄球菌菌株都可以在BHI或TSB中繁殖,但是在本方案中针对每个菌株表达的培养基与原始方案中使用的生长培养基一致,证明了抗质粒免疫表皮葡萄球菌RP62a中的III-A型CRISPR-Cas系统(Marraffini和Sontheimer,2008)。
  2. 第二天生长的殖民地和过滤器交配
    1. 在孵育至少16小时后,应该看到大的(> 1毫米直径)个体受体菌落。将3个单独菌落的全部接种到含有2μgBHI的15ml离心管中,其中含有15μg/ml新霉素。
      注意:
      1. 此外,将实验中包含的任何对照一式三份接种。
      2. 同时,在15 ml离心管中加入50μl的过夜供体培养物至5ml TSP与5μg/ml莫匹罗星。
    2. 在37°C振荡下,供体和受体均可生长。
      注意:同时,将每3个生长的受体培养物中的一个BHI板置于30℃的孵育器中以预热(参见注4)。
    3. 约6小时后,检查受体和供体菌株的OD <600>
      1. 将900μlBHI肉汤加入分光光度计比色杯中。加入100μl培养物种之一的受体培养物。混合好。
      2. 在用BHI消隐后,测量和记录培养物的1:10稀释度的OD 600。
      3. 用剩余的受体培养物重复步骤2c i和2c ii。
      4. 对供体培养物重复步骤2c i和2c ii,用TSB代替BHI
      5. 在进行之前,所有培养稀释液的OD 600必须大于0.20。如果OD 600还不够高,则返回振荡器,并在再次检查OD 600之前生长一段时间。
    4. 计算供体和受体结合以达到1:4比例(受体:供体)用于过滤器交配的量,如表1中的示例所示。
      注意:对于每个培养物,将200除以步骤2c中所测量的OD 600。在1 000的OD 600下,有约8×10 8 cfu/ml的供体和2×10 8个/cfc/ml的供体收件人。该计算允许标准数量的供体(大约1.6×10 9个/cfu)和接受者(约4×10 8个/cfu)组合在所有被测试的样品中。

      表1.确定用于过滤器交配的组合c 的体积的样本计算
      文化
      OD 600 (1:10稀释)
      200/OD 600 (要添加的体积,μl)
      RP62a(1)
      0.42
      476
      RP62a(2)
      0.42
      476
      RP62a(3)
      0.41
      488
      LAM104(1)
      0.44
      455
      LAM104(2)
      0.39
      513
      LAM104(3)
      0.44
      455
      RN4220/pG0400
      0.56
      357

    5. 将1.7ml微量离心管中的第一,未稀释的受体培养物的计算体积与未稀释的供体的计算体积相结合。
      注意:重复上述步骤,直到所有供体 - 受体对组合成分离的微量离心管。
    6. 洗涤合并的培养物以通过将离心管以6,200×g离心在台式离心机中离心2分钟来除去抗生素。取出上清液,将颗粒重悬于1 ml BHI中,无抗生素。重复离心。
    7. 使用无菌镊子,将一个0.45μm的膜过滤器放置在不含抗生素的BHI板上,每个供体 - 受体对上,在单个板上放置多达3个过滤器(图1)。


      Fi gure 1。用于过滤器配合的样品板,显示具有三个膜过滤器的板 在每个过滤器中心的过夜生长中覆盖

    8. 从含有供体和受体组合的每个微量离心管中取出悬浮液。将沉淀重悬于100μlBHI肉汤中,并小心地将其移至膜过滤器的中心,小心不要让任何悬浮液从膜中脱落,如图1所示。保持所有细菌悬浮液至关重要在过滤器上,因为该测定是定量的,并且需要所有细胞的完全恢复。
    9. 每个微量离心管重复此操作,将每种培养物的重悬浮物置于其自身的过滤器上
    10. 盖上板子,放在台面上,直到细胞悬浮液完全干燥
    11. 在30℃的培养箱中将板倒置倒置过夜。
  3. 第3天。连续稀释和电镀接收者和转接合子
    1. 将含有新霉素(15μg/ml)的一个BHI琼脂平板和含有来自前一夜的每个过滤器的新霉素(15μg/ml)和莫匹罗星(5μg/ml)的一个BHI琼脂平板。
    2. 在50ml离心管(每个过滤器一个)中,加入3ml不含抗生素的BHI肉汤。
    3. 使用无菌镊子,将每个过滤器从板上取下,并将其放入步骤3b中制备的50ml管中
    4. 旋转每个管,直到过滤器上的细胞完全重新悬浮,并且在过滤器上不可见生长
    5. 连续稀释细胞悬浮液
      1. 在一条PCR管中的每个8个管中,加入90μlBHI肉汤。
      2. 从步骤3d中取出10μl细菌悬浮液,并将其加入第一个PCR条形管中,重复上下移液重悬(这是10次/sup的稀释)。
      3. 使用新的移液管吸头,从10μL/sup稀释液中取出10μl,并将其加入第二个PCR管中,反复进行上下移液以重悬浮(这是10次/sup>稀释)
      4. 重复步骤3e iii以创建稀释液至10 -8
        注意:可以使用进行一系列10倍连续稀释的替代方法,例如在96孔板中使用多通道移液管。
    6. 将每个稀释液10μl点样到含有新霉素的预热BHI板上,另一个含有新霉素和莫匹罗星(参见附注5)。要接受接受者菌落计数的所有稀释液应铺板在同一块板上,如图2A所示,所有要进行转结合菌落计数的稀释液应镀在额外的板上,如图2B所示。
      注意:以45°的角度倾斜板,直到稀释液的下降几乎到达板的边缘,然后将其平放在台面上并让其干燥。


      图2.来自使用RP62a菌株的缀合测定的样品平板,显示接受者(A. 1.5×10 9 cfu/ml)和转导结合物(B.0cfu/ml)强>

    7. 对每个过滤器重复步骤3e和3f。
    8. 让台板在台面上覆盖时干燥,然后在37℃下孵育过夜。
  4. 第4天。接收者和转运结合计数
    1. 从第2天计算每个过滤器的收件人数(仅含有新霉素的平板上的菌落数),如图2A所示。
      1. 确定最高稀释度的殖民地数量,其中殖民地仍然可以计数。
      2. 将菌落数乘以100,通过稀释因子(10 1 稀释10倍,将10 稀释10倍。 > -2 稀释, e c )以获得每毫升的菌落形成单位数。
    2. 计数第2天每个过滤器的转导结合物(含有新霉素和莫匹罗星的平板上的菌落数),如步骤4a所示,并显示在图2B中。
    3. 计算共轭效率(见数据分析)

数据分析

  1. 共轭效率的计算
    1. 共轭效率是每个初始受体细胞产生的转导结合物的数量的量度。它可以简单地计算如下,示例如表2所示:



      表2.计算 共轭度 n 样本共轭测定的效率
      St rain
      Transconj ugants (cfu/ml)
      收件人 s (cfu/ml)
      共轭 Ef ficiency
      RP62a(1)
      1 x 10 3
      7 x 10 9
      1 x 10 -7
      RP62a(2)
      2 x 10 2
      1 x 10 9
      2 x 10 -7
      RP62a(3)
      0
      4 x 10 9
      0
      LAM104(1)
      1.3 x 10 5
      3 x 10 9
      4 x 10 -5
      LAM104(2)
      9 x 10 4
      3 x 10 9
      3 x 10 -5
      LAM104(3)
      9 x 10 4
      4 x 10 9
      2 x 10 -5

    2. 高效率表示无效的CRISPR系统,而降低的效率表示活动的CRISPR防御。
      1. 与没有CRISPR系统的应变相比,CRISPR系统存在时通常可以看到缀合效率降低100至1,000倍。
  2. 数据呈现
    数据通常以条形图显示,以对数标度显示cfu/ml(菌落形成单位/毫升)中的接受者和转录结合体的数目,并伴随图3计算的共轭效率,如图3所示。 >

    Fi gure 3。来自使用S的共轭测定的数据的样本图。表皮细胞 RP62a和LAM104作为收件人 错误栏表示一式三份测量的标准差。 

笔记

  1. 本试验中可以使用其他菌株作为受体菌株。 S上。表皮细胞RP62a自然携带CRISPR间隔区,其靶向在共轭葡萄球菌质粒如pG0400(Marraffini和Sontheimer,2008)上发现的 基因。其他含CRISPR的原核生物可以被使用,如果它们适合接受共轭质粒。接受者选择中的另一个重要考虑是确保接受者具有将其与供体菌株区分开的选择标记。在与S共轭测定的情况下。表皮细胞, S 。表皮葡萄球菌RP62a对新霉素具有抗性,因此这种抗性可用于仅在第3天仅选择转导结合体而不是选择供体菌株本身。天然不具有不同抗生素敏感性的受体菌株可以用含有附加电阻标记(例如pC194 [Ehrlich,1977] for /em>的)。选择任何质粒,确保其来源与缀合质粒相容,以允许在转导结合体中维持和选择两种质粒。如果使用不同的选择性标记,请更换适合培养和电镀的抗生素
  2. 其他菌株 可以用作该测定中的供体菌株,以及额外的共轭质粒也可以使用。供体菌株需要能够与受体菌株进行缀合,并且RN4220对于这种共轭测定是非常方便的,因为它可以与 进行共轭。 pidermidis RP62a,并且缺少CRISPR系统,允许即时摄取和维持质粒,例如可能在携带CRISPR的菌株中降解的pG0400质粒。也可以使用另外的共轭质粒,这取决于受体菌株内发现的CRISPR间隔区。如果测试CRISPR在受体菌株中是否具有抗质粒活性的功能,则必须使用与接受者的CRISPR阵列内发现的间隔物相互补充的原始质粒的共轭质粒。在RP62a的情况下,pG0400自然具有该原始样品,但是在许多系统中,可能需要用原始样品技术设计共轭质粒。如果使用不同的共轭质粒,请适当选择抗生素进行培养和铺板。另外,如果工程化共轭质粒,请务必按照要测试的特定系统中CRISPR靶向所需的规则,例如遵循I类和II类系统的PAM要求(Mojica e ,以及允许Type III-A系统干扰的所需不匹配(Marraffini和Sontheimer,2010) >
  3. LAM104是RP62a的工程衍生产品,缺乏重复间隔阵列,可防止CRISPR-Cas发挥作用(Marraffini和Sontheimer,2008)。如果使用RP62a以外的应变,建议使用CRISPR-Cas不起作用的阴性对照。合适的阴性对照包括1)使用缺乏CRISPR-Cas的关键组分的受体菌株,或2)使用携带缺乏合适原始分离物的共轭质粒的供体菌株。 S上。表皮炎 LAM104无法在商业上可用,但可根据要求由Hatoum-Aslan实验室提供。
  4. S。表皮葡萄球菌RP62a,如果在37℃下进行过滤器交配,则在第4天观察到受试者的背景计数增加。为了获得更清洁的效果,常规使用30°C的孵育,尽管可以对其进行修改以适应任何生物体的生长要求。
  5. S。作为收件人的菌株,transconjugants通常以10个浓度的浓度观察到,其中10μg/ 0 -10 5 cfu/ml,因此通常仅将10℃至10℃-3小时稀释在含有新霉素和莫匹罗星的BHI平板上获得菌落数。接受者通常为约10 cfu/ml,因此通常将仅有10℃-5℃至10℃-8小时的稀释液涂覆在含有新霉素获得菌落数。

食谱

  1. 脑心输液汤
    1. 将19.5 g脑心脏输注溶解于500 ml去离子水(DI)水中
    2. 在121°C高压灭菌30分钟,并在室温下储存
  2. 脑 - 心输液琼脂
    1. 将29克脑心脏输注琼脂溶解于500毫升去离子水中
    2. 在121℃高压灭菌30分钟,然后在磁力搅拌板上搅拌,直至冷却至足以舒适,然后立即加入抗生素并倒入盘中(每500毫升20片)
      注意:介质也可以在室温下储存,然后在微波炉中加热,液化并倒入。

  3. 胰蛋白酶大豆肉汤
    1. 将15g胰蛋白酶大豆肉汤溶于500ml去离子水中
    2. 在121℃高压灭菌30分钟,并在室温下储存

致谢

A.-H-A。由阿拉巴马大学(UA)艺术与科学学院支持; UA学院研究,奖学金和创意活动研究所(CARSCA)的资助;和国立卫生研究院[5K22AI113106-02]。该协议改编自Marraffini和Sontheimer(2008)发表。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Walker, F. C. and Hatoum-Aslan, A. (2017). Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci. Bio-protocol 7(9): e2293. DOI: 10.21769/BioProtoc.2293.
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