A new paper published in PLoS Biology characterizes two bacterial death pathways
Programmed cell death (PCD) in eukaryotes is a well-studied process that is used by organisms to maintain homeostasis. The mechanisms of PCD are under intense study because altered regulation leads to excessive cell death (inflammatory diseases) or excessive cell growth (cancer). The classic pathway in eukaryotes is apoptosis. Likewise, some bacteria also contain PCD pathways, but the reactions are less well characterized. In bacterial PCD, it is also not clear how a death program in single-celled organisms provides an evolutionary advantage. It is important to note that PCD refers to any form of cell death mediated by an intracellular program, regardless of whether the program displays all of the hallmarks of eukaryotic apoptosis.
In bacteria, PCD is mediated through modules that consist of a pair of genes, called “addiction modules” or toxin-antitoxin (TA) systems. One gene of the module encodes for a toxin, and the second gene encodes for a labile antitoxin. Under normal growth conditions (that is, non-stressed), the continual production of the antitoxin inhibits the activity of the toxin.
One such TA system in E. coli is the mazEF module, studied for a number of years by the Engelberg-Kulka group. The MazF protein is an endoribonuclease that cleaves single-stranded mRNA at ACA sequences, resulting in leaderless mRNA transcripts. The MazE protein binds to MazF and inhibits MazF activity. MazE is labile because it is cleaved by the ClpPA system; removal of MazE by proteolysis results in activation of MazF.
To complicate matters further, the system is tied to quorum sensing through a small pentapeptide called Extracellular Death Factor (EDF). High population density of cells results in high concentrations of EDF. Under stress conditions, such as DNA damage, the presence of EDF results in MazF-induced death.
In an earlier paper (Engelberg-Kulka et al., 2009), the Engelberg-Kulka group showed that when EDF was added in trans to a strain of E. coli that did not naturally produce EDF, the antibiotic action of rifampicin switched from bacteriostatic, it’s normal mode of action, to bacteriocidal. Importantly, the results showed cross-talk between the two modes and established that the signaling was more complex than two independent mechanisms.
EDF provides a unique mechanism of quorum sensing – as a post-transcriptional sensor of gene expression. In contrast, most known quorum sensing molecules function at the level of gene transcription. Biochemical and modeling studies showed that EDF binds to MazF and that the binding site overlaps that of MazE (Belitsky et al., 2011). So when EDF is present, MazE is released from the MazEF complex, thus activating MazF. The Engelberg-Kulka group has examined each of the positions of the pentapeptide and described key interactions between the binding pocket on MazF and the peptide (or MazE).
When MazF is activated, it cleaves mRNA to produce leaderless transcripts. In addition, it also cleaves the 3′-terminus of the 16S rRNA subunit, which removes the anti-Shine Dalgarno region. The cleavage of 16S rRNA has the effect of producing a subpopulation of ribosomes that translates leaderless mRNAs. The modification results in a significant reduction in protein synthesis (~90%), but the subpopulation of ribosomes selectively translates ~10% of the mRNAs. This is important because some of the proteins synthesized from this mechanism permit survival of a subpopulation of cells under stressful conditions (Vesper et al., 2011).
In the current paper (Erental et al., 2012), the group characterized EDF-MazF-mediated cell death under various stress conditions and examined two hallmarks of eukaryotic apoptosis – DNA cleavage and membrane depolarization. Using a dye (DiBAC4) and flow cytometry to measure membrane depolarization, they found that depolarization occurred only upon DNA damage, as opposed to other stress conditions, and when the mazEF genes were deleted (ΔmazEF).
When MazF was present, such that cells underwent MazF-mediated death, there was no membrane depolarization. Taken together, the data suggest that E. coli contains two death pathways, the previously characterized pathway mediated by EDF-MazF, and a newly proposed pathway that exhibits characteristics similar to eukaryotic apoptosis – namely membrane depolarization and DNA fragmentation. The new pathway is named “apoptosis-like death,” or ALD.
In further studies, the authors showed that the ALD pathway is linked to the SOS DNA damage response in bacteria through RecA. In this mechanism, RecA acts to inactivate LexA, a transcriptional repressor of SOS genes. In essence, the cleavage of LexA activates the SOS response. Likwise, the MazEF-mediated cell death pathway inhibits the ALD pathway by decreasing the levels of RecA mRNA; this is due to a product of a downstream gene rather than to MazF itself.
So, what is the evolutionary advantage for a unicellular organism to have these pathways? The MazEF pathway provides a subpopulation of cells that survive under stressful conditions. The surviving cells can then ensure the growth of a new population of cells upon return to optimal conditions. As such, the MazEF cell death pathway is referred to as “an altruistic mechanism for bacterial survival under stressful conditions.”
In contrast, the newly described ALD pathway may function at the level of the individual cell (as opposed to a population of cells) since survival appears to be due to repair of individual damaged cells. It may also provide a backup if the MazEF pathway is inactivated. Because the ALD pathway responds to DNA damage and not other stresses, the surviving cells may be less responsive to other future stress conditions.
The evolutionary connection between bacterial ALD and eukaryotic PCD, if any, remains to be determined.
Erental, A., Sharon, I., & Engelberg-Kulka, H. (2012). Two Programmed Cell Death Systems in Escherichia coli: An Apoptotic-Like Death Is Inhibited by the mazEF-Mediated Death Pathway PLoS Biology, 10 (3) DOI: 10.1371/journal.pbio.1001281
Vesper, O., Amitai, S., Belitsky, M., Byrgazov, K., Kaberdina, A., Engelberg-Kulka, H., & Moll, I. (2011). Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli Cell, 147 (1), 147-157 DOI: 10.1016/j.cell.2011.07.047
Belitsky, M., Avshalom, H., Erental, A., Yelin, I., Kumar, S., London, N., Sperber, M., Schueler-Furman, O., & Engelberg Kulka, H. (2011). The Escherichia coli Extracellular Death Factor EDF Induces the Endoribonucleolytic Activities of the Toxins MazF and ChpBK Molecular Cell, 41 (6), 625-635 DOI: 10.1016/j.molcel.2011.02.023
Engelberg-Kulka, H., Yelin, I., & Kolodkin-Gal, I. (2009) Activation of a built-in bacterial programmed cell death system as a novel mechanism of action of some antibiotics. Communicative & Integrative Biology 2:3, 211-212. [Previously published online as a Communicative & Integrative Biology E-publication: http://www.landesbioscience.com/journals/cib/article/7876 Addendum to: Kolodkin-Gal I, Sat B, Keshet A, Engelberg Kulka H. The communication factor EDF and the toxin antitoxin module mazEF determine the mode of action of antibiotics. PLoS Biol 2008; 6:319; PMID: 19090622; DOI: 10.1371/journal. pbio.0060319.