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. 2018 May 1;9(3):e00685-18.
doi: 10.1128/mBio.00685-18.

Quorum Sensing Extracellular Death Peptides Enhance the Endoribonucleolytic Activities of Mycobacterium tuberculosis MazF Toxins

Akanksha Nigam  1 , Sathish Kumar  1 , Hanna Engelberg-Kulka  2
Affiliations

Quorum Sensing Extracellular Death Peptides Enhance the Endoribonucleolytic Activities of Mycobacterium tuberculosis MazF Toxins

Akanksha Nigam et al. mBio. .

Abstract

mazEF is a toxin-antitoxin module located on chromosomes of most bacteria. MazF toxins are endoribonucleases antagonized by MazE antitoxins. Previously, we characterized several quorum sensing peptides called "extracellular death factors" (EDFs). When secreted from bacterial cultures, EDFs induce interspecies cell death. EDFs also enhance the endoribonucleolytic activity of Escherichia coli MazF. Mycobacterium tuberculosis carries several mazEF modules. Among them, the endoribonucleolytic activities of MazF proteins mt-1, mt-3, and mt-6 were identified. MazF-mt6 and MazF-mt-3 cleave M. tuberculosis rRNAs. Here we report the in vitro effects of EDFs on the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF (EcEDF) and the three Pseudomonas aeruginosa EDFs (PaEDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3 and overcome the inhibitory effect of MazE-mt3 or MazE-mt6 on the endoribonucleolytic activities of the respective toxins. We propose that these EDFs can serve as a basis for a novel class of antibiotics against M. tuberculosisIMPORTANCEMycobacterium tuberculosis is one of the leading causes of death from infectious disease. M. tuberculosis is highly drug resistant, and drug delivery to the infected site is very difficult. In previous studies, we showed that extracellular death factors (EDFs) can work as quorum sensing molecules which participate in interspecies bacterial cell death. In this study, we demonstrated the role of different EDFs in the endoribonucleolytic activities of M. tuberculosis MazFs. Escherichia coli EDF (EcEDF) and the three Pseudomonas aeruginosa EDFs (PaEDFs) individually enhance the endoribonucleolytic activities of MazF-mt6 and MazF-mt3. The current report provides a basis for the use of the EDF peptides EcEDF and PaEDF as novel antibiotics against M. tuberculosis.

Keywords: Mycobacterium tuberculosis; extracellular death peptides; quorum sensing.

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Figures

FIG 1
FIG 1
EcEDF amplified the in vitro endoribonucleolytic activity of MazF-mt6. (a) Illustration of the reaction used for studies on the endoribonucleolytic activity of MazF-mt6. Cleavage of the chimeric fluorescent oligonucleotide by MazF was measured as an increase of fluorescence emission of the fluorophore FAM. The cleavage site was UU↓CCU; “r” represents an RNA base. (b) (Left) Addition of (4 µM or 8 µM) wild-type EcEDF (NNWNN) led to an increase in the in vitro activity of MazF-mt6. (Right) The relative (percent) increase of MazF-mt6 activity caused by the addition of EcEDF. MazF-mt6 activity without the addition of EcEDF was defined as 100%. The data represent means ± standard deviations of results from three experiments performed in triplicate. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (statistical significance compared to the control data).
FIG 2
FIG 2
The importance of EcEDF sequence for increasing MazF-mt6 endoribonucleolytic activity in vitro. (a) The third residue of EcEDF, tryptophan (W), was replaced with glycine (G) as follows: NNGNN. (b) The first residue of EcEDF, asparagine (N), was replaced with (G) as follows: GNWNN. (c) The second residue of EcEDF, (N), was replaced with (G) as follows: NGWNN. (d) The fourth residue of EcEDF, (N), was replaced with (G) as follows: NNWGN. (e) The fifth residue of EcEDF, (N), was replaced with (G) as follows: NNWNG. (f) Wild-type (Wt) EcEDF was lengthened by the addition of a new “N” residue at each end as follows: NNNWNNN. (g) Wt EcEDF was shortened by the removal of both external “N” residues at each end as follows: NWN. The data represent means ± standard deviations of results from three experiments performed in triplicate. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (statistical significance compared to the control data).
FIG 3
FIG 3
EcEDF overcomes the inhibitory effect of MazE-mt6 on MazF-mt6 endoribonucleolytic activity in vitro. (a) A mixture of (0.5 µM) MazE-mt6 and (4 µM or 8 µM) EcEDF (NNWNN) was added to preparations of (0.5 µM) MazF-mt6. (Left) The in vitro activity of MazF-mt6 was inhibited in the presence of MazE-mt6. (Right) Percent (%) increase in MazF-mt6 activity after the addition of EcEDF to wells containing both MazF-mt6 and MazE-mt6. (b) Data were determined as described for panel a, but instead of NNWNN, NNGNN was added. (c) Data were determined as described for panel a, but instead of NNWNN, GNWNN was added. (d) Data were determined as described for panel a, but instead of NNWNN, NGWNN was added. (e) Data were determined as described for panel a, but instead of NNWNN, NNWGN was added. (f) Data were determined as described for panel a, but instead of NNWNN, NNWNG was added. (g) Data were determined as described for panel a, but instead of NNWNN, a lengthened, modified EDF to which a new N residue was attached at each end (NNNWNNN) was added. (h) Data were determined as described for panel a, but instead of NNWNN, a shortened, modified EDF from which an N residue has been removed from each end (NWN) was added. The data represent means ± standard deviations of results from three experiments performed in triplicate. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (statistical significance compared to the control data).
FIG 4
FIG 4
The effects of the addition of each of the EDFs of P. aeruginosa, i.e., PaEDF-1, PaEDF-2, or PaEDF-3, on the endoribonucleolytic activities of M. tuberculosis MazF toxins MazF-mt6 and MazF-mt3. (a) (Left) PaEDF-1 added to MazF-mt6. (Right) The relative (percent) increase of MazF-mt6 activity in the presence of PaEDF-1. (b) (Left) PaEDF-2 added to MazF-mt6. (Right) The relative (percent) increase of MazF-mt6 activity in the presence of PaEDF-2. (c) (Left) PaEDF-3 added to MazF-mt6. (Right) The relative (percent) increase of MazF-mt6 activity in the presence of PaEDF-3. (d) (Left) Addition of PaEDF-1 to MazF-mt3. (Right) The relative (percent) increase of MazF-mt3 activity in the presence of PaEDF-1. (e) (Left) Addition of PaEDF-2 to MazF-mt3. (Right) The relative (percent) increase of MazF-mt3 activity in the presence of PaEDF-2. (f) (Left) Addition of PaEDF-3 to MazF-mt3. (Right) The relative (percent) increase of MazF-mt3 activity in the presence of PaEDF-3. MazF activity without the addition of EDF was defined as 100%. The data represent means ± standard deviations of results from three experiments performed in triplicate. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (statistical significance compared to the control data).
FIG 5
FIG 5
PaEDFs partially overcomes the inhibitory effect of MazE-mt6 on MazF-mt6 and of MazE-mt3 on MazF-mt3 endoribonucleolytic activity in vitro. (a) A mixture of (0.5 µM) MazE-mt6 and (4 µM or 8 µM) PaEDF-1 was added to preparations of (0.5 µM) MazF-mt6. (Left) The in vitro activity of MazF-mt6 was inhibited in the presence of MazE-mt6, and PaEDF-1 overcomes this inhibitory effect. (Right) Percent (%) increase in MazF-mt6 activity after the addition of PaEDF-1 to wells containing both MazF-mt6 and MazE-mt6. (b) Data were determined as described for panel a, but instead of PaEDF-1, PaEDF-2 was added. (c) Data were determined as described for panel a, but instead of PaEDF-1, PaEDF-3 was added. (d) A mixture of (0.5 µM) MazE-mt3 and (4 µM or 8 µM) PaEDF-1 was added to preparations of (0.5 µM) MazF-mt3. (Left) The in vitro activity of MazF-mt3 was inhibitedin the presence of MazE-mt3 and PaEDF-1 overcomes this inhibitory effect. (Right) Percent (%) increase in MazF-mt3 activity after the addition of PaEDF-1 to wells containing both MazF-mt3and MazE-mt3. (e) Data were determined as described for panel d, but instead of PaEDF-1, PaEDF-2 was added. (f) Data were determined as described for panel d, but instead of PaEDF-1, PaEDF-3 was added. The data represent means ± standard deviations of results from three experiments performed in triplicate. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 (statistical significance compared to the control data).
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