Characterization of mRNA Interferases from Mycobacterium tuberculosis

Abstract

mRNA interferases are sequence-specific endoribonucleases encoded by the toxin-antitoxin systems in the bacterial genomes. MazF from Escherichia coli has been shown to be an mRNA interferase that specifically cleaves at ACA sequences in single-stranded RNAs. It has been shown that MazF induction in E. coli effectively inhibits protein synthesis leading to cell growth arrest in the quasidormant state. Here we have demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7), four of which (MazF-mt1, -mt3, -mt4, and -mt6) caused cell growth arrest when induced in E. coli. MazFmt1 and MazF-mt6 were purified and characterized for their mRNA interferase specificities. We showed that MazF-mt1 preferentially cleaves the era mRNA between U and A in UAC triplet sequences, whereas MazF-mt6 preferentially cleaves U-rich regions in the era mRNA both in vivo and in vitro. These results indicate that M. tuberculosis contains sequence-specific mRNA interferases, which may play a role in the persistent dormancy of this devastating pathogen in human tissues.Most bacteria contain "suicidal" or "toxic" genes whose expression leads to growth arrest and eventual death upon exposure to stress. These toxin genes are usually coexpressed with their cognate antitoxin genes present in the same operon (1, 2). The Escherichia coli chromosome contains six such operons called toxin-antitoxin (TA) 2 systems. Of these, the MazE (antitoxin)/MazF (toxin) system is one of the most extensively characterized TA systems, and MazF has been shown to be a sequence-specific endoribonuclease that cleaves at ACA sequences in mRNAs (3, 4). Thus, MazF is an mRNA interferase, and its induction causes the effective inhibition of protein synthesis leading to cell growth arrest. However, MazF-induced cells fully retain cellular metabolic activities, including ATP production and amino acid and nucleotide synthesis, as well as RNA and protein synthesis. Therefore, MazF-induced cells are capable of synthesizing a protein, if the gene encoding this protein is devoid of ACA sequences (4). This metabolically active dormant state caused by mRNA interferase induction is called "quasidormancy" (4) and has important implications in the physiology of various pathogenic bacteria, including persistent multidrug resistance.Notably, Mycobacterium tuberculosis, one of the most devastating human pathogens, contains nearly forty TA systems on its genome, among which nine have been shown to be homologous to E. coli MazF (5). It has been proposed that the TA modules play important roles in bacterial survival and formation of stable persisters in adverse conditions. These phenomena are essential for the persistence of M. tuberculosis. Persisters are the few rare pre-existing bacterial cells in a culture at any growth phase that are not growing and are intrinsically resistant to antibiotics by virtue of their subdued metabolism (6, 7).In the present paper, we have characterized two of the MazF homologues from this pathogen, wh...

MazF ribonucleases promote Mycobacterium tuberculosis drug tolerance and virulence in guinea pigs

“…We observed that the overexpression of MazF3, MazF6 or MazF9 in Mtb induced a state of reversible bacteriostasis, consistent with the results of previous studies (Fig. 1a,b) 24,26,48 . The mycobacterial strains overexpressing these MazF toxins were viable with intact membranes under aerobic conditions.…”

“…Our results are consistent with those of previous studies demonstrating that the overexpression of MazF toxins induces bacteriostasis in Staphylococcus aureus and E. coli 49,50 . However, in contrast to previously published studies in E. coli and Msm, the overexpression of MazF2, MazF5 or MazF7 did not affect the viability of M. bovis BCG 24,26,48 . Such variations could be due to the use of (i) different inducer-based expression systems, (ii) different host strains or (iii) vectors with different translation signals.…”

“…MazF and RelE homologues from Mtb have been extensively characterized biochemically and possess ribonuclease activity. MazF homologues from Mtb exhibit varying substrate specificities; Rv1991c (MazF6), Rv2801c (MazF9) and Rv1495 (MazF4) cleave single-stranded RNA at U k CCUU, U k AC and U k CGCU, respectively [25][26][27] . In addition to mRNA, Rv1102c (MazF3) also cleaves 23S rRNA at the UU k CCU site and contributes to the formation of both kanamycin-and gentamycin-tolerant persisters of Mycobacterium smegmatis (Msm) 28,29 .…”

A regulatory role for Staphylococcus aureus toxin–antitoxin system PemIKSa

“…Multiple chromosome-encoded TA systems have been identified 32 , and many of these contain site-specific ribonucleases. The low-specificity mycobacterial toxins MazFmt1 (UkAC) and MazF-mt6 (cleaves preferentially in U-rich regions) cause growth inhibition when heterologously expressed in E. coli, whereas MazF-mt3 (UUkCCU, CUkCCU) and MazFmt7 (UkCGCU) show almost no effect 16,33 . PemK Sa (UkAUU) induces an intermediate effect when expressed in E. coli, inhibiting growth in liquid, but not on solid media.…”

Biochemical characterization of a chromosomal toxin–antitoxin system in Mycobacterium tuberculosis

“…We have reported that seven M. tuberculosis genes encode the proteins homologous to MazF, the toxin in Escherichia coli mazEF TA system. Four of the MazF homologues, Rv2801, Rv1991c, Rv0659c and Rv1102c, were identified as toxins, among which Rv2801c and Rv1102c were characterized as ribonucleases cleaving RNA in a sequence-specific manner [8]. An open reading frame (ORF) locates immediately upstream of Rv199c and overlaps with Rv1991c by 7 base pairs.…”