Sunday, July 20, 2014

Bacterial CRISPRs: Not Just For Targeting Foreign Nucleic Acids

The CRISPR-Cas system has been found to play roles
in the antibiotic resistance of some bacteria. <Source>
In recent years, CRISPRs (Clustered, regularly interspaced, short palindromic repeats) have been gaining popularity in the microbiology field.  Briefly, CRISPRs serve as an adaptive immune system for bacteria, meaning that they are able to remember what viruses (bacteriophages) or other entities have infected them and mount a targeted defensive response the next time they are infected with the same entity (think of it as an analog to our adaptive immune response which uses antibodies and other agents to target invading microbes).  More specifically, the CRISPR-Cas (Cas are the CRISPR associated genes) system facilitates the integration of a small section of the foreign genomic DNA into the CRISPR array within the bacterial genome (see left side of the detailed diagram below).  While in the array, this section of foreign DNA will serve as a template for recognizing the invading genome again if another infection occurs, and the template will be used for targeting that invading genome for rapid destruction.  As can be seen in the figure below, this system is similar to the Eukaryotic RNA-interference system found in organisms including humans.  I've already gotten pretty technical here, and anything more in-depth would be beyond the scope of this post, so please check out reference [1] for further reading.

There is a lot to this figure, so please read more about it here.

So far the CRISPR-Cas bacterial defense system has been primarily considered an adaptive immune targeting system, as described above.  A couple of days ago a research group, consisting of Sampson, TR et al, reported their evidence for new CRISPR-Cas functions that are involved in antibiotic resistance and host (human) immune system evasion [2].  This is important because it suggests that, in some cases, CRISPR-Cas systems can be used as a defense against infecting agents (including bacteriophages), as well as environmental stressors (including antibiotics and host inflammation).  As Sampson et al describes, this seems to be facilitated by CRISPR-Cas mediated changes in bacterial membrane integrity.

Cartoon of a macrophage engulfing (eating)
a bacterial cell. <Source>
In this report, the research group studied the intracellular human pathogen Francisella novicida, which is able to grow in human macrophages.  To start, the group showed that the bacteria was normally able to grow with the membrane targeting antibiotic polymyxin B, but was unable to grow when the CRISPR-Cas gene cas9 was mutated (therefore non-functional).  When the mutant was allowed to express the cas9 gene again, the bacteria was once again able to grow with the antibiotic.  This result was also observed with other antibiotics.  This very strongly suggests that the cas9 gene is involved in resistance against the polymyxin B antibiotic.  They further showed that the absence of the functional cas9 gene resulted in bacteria with more permeable membranes (chemicals were more easily able to penetrate the bacterial outer membrane), despite no bacterial growth change in stress-free growth medium.

In addition to being more susceptible to antibiotic activities when the cas9 regulatory system is mutated and the membranes made more permeable, the increased permeability also increases the likelihood that they will be detected by the human immune system.  This means that, as the bacteria are living in their macrophage hosts, the increased permeability increases the chances that they are detected, which results in the death of their host macrophage, and consequently the death of themselves.  Therefore a permeable membrane inhibits the bacteria's ability to successfully infect a macrophage.

The group went on to show these macrophage-related finding are relevant in the mouse animal model.  Sampson, TR et al showed that cas9 deficient F. novicida were able to successfully infect mice who were themselves deficient in ASC (mediator of inflammation) and TLR2 (immune detection of the bacteria).  In contrast, the cas9 mutant bacteria were unable to infect mice who were only deficient in one system, or deficient in neither system.  The wild type bacteria (the unaltered, normal bacteria) were able to infect all of these mice.  Together this strongly suggests that the cas9 regulatory pathway is needed for evasion of the mammalian immune pathways ASC and TLR2.

Microscope image of bacterial cells (red) that are infecting
a macrophage (green). <Source>
Together this paper suggests that the CRISPR-Cas system, and more specifically the cas9 regulatory system, is important for facilitating bacterial membrane permeability.  This effect on permeability has implications in the modulation of the bacteria's susceptibility to certain antibiotics and to detection by host mammalian immune systems.  As the authors of the paper suggest, it seems feasible that this increased detection of the immune system could be caused by increased release of the bacterial nucleic acid as a result of increased membrane permeability.  This would especially make sense since TLR2 detects this bacterial nucleic acid.

If you find this summary interesting, I encourage you to check out the paper (reference [2] below).  It is a nice study and a straightforward read.  This is of course only a short post and I skimmed over some of the details of this paper, so please check read the paper to get the whole story.  Finally, please let me know if you have and questions, comments, or concerns.  You can reach me in the comments below, on Twitter, or by email.

Works Cited

Wiedenheft B, Sternberg SH, & Doudna JA (2012). RNA-guided genetic silencing systems in bacteria and archaea. Nature, 482 (7385), 331-8 PMID: 22337052

Sampson TR, Napier BA, Schroeder MR, Louwen R, Zhao J, Chin CY, Ratner HK, Llewellyn AC, Jones CL, Laroui H, Merlin D, Zhou P, Endtz HP, & Weiss DS (2014). A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion. Proceedings of the National Academy of Sciences of the United States of America PMID: 25024199

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