PemK Toxin of Bacillus anthracis Is a Ribonuclease

Abstract

Bacillus anthracis genome harbors a toxin-antitoxin (TA) module encoding pemI (antitoxin) and pemK (toxin). This study describes the rPemK as a potent ribonuclease with a preference for pyrimidines (C/U), which is consistent with our previous study that demonstrated it as a translational attenuator. The in silico structural modeling of the PemK in conjunction with the site-directed mutagenesis confirmed the role of His-59 and Glu-78 as an acid-base couple in mediating the ribonuclease activity. The rPemK is shown to form a complex with the rPemI, which is in line with its function as a TA module. This rPemIrPemK complex becomes catalytically inactive when both the proteins interact in a molar stoichiometry of 1. The rPemI displays vulnerability to proteolysis but attains conformational stability only upon rPemK interaction. The pemI-pemK transcript is shown to be up-regulated upon stress induction with a concomitant increase in the amount of PemK and a decline in the PemI levels, establishing the role of these modules in stress. The artificial perturbation of TA interaction could unleash the toxin, executing bacterial cell death. Toward this end, synthetic peptides are designed to disrupt the TA interaction. The peptides are shown to be effective in abrogating TA interaction in micromolar range in vitro. This approach can be harnessed as a potential antibacterial strategy against anthrax in the future. Toxin-antitoxin (TA)3 modules in prokaryotes consist of two adjacent open reading frames encoding a stable toxin and a cognate labile antitoxin (1, 2). The vulnerability of antitoxin to proteolysis demands a constant de novo synthesis of antitoxin to maintain a steady-state level that can sequester the toxin and circumvent its deleterious activity (3). A negative feedback homeostatic loop also exists, wherein antitoxin acts as a transcriptional repressor in conjunction with the toxin as a co-repressor (4). A multitude of functions have been ascribed to the chromosome-encoded TA loci, primarily as stress managers (5-7). It has been envisaged that these modules respond to intracellular stress facilitating phenotypic switching of the bacterial cell to a quasidormant stage amenable to survival under extreme conditions (8 -12). However, a recent study demonstrates that TA loci do not impart selective advantage to the cells grown in stress, indicating that these genes can innocuously be deleted from the genome (13).Several families of two component TA loci have been identified on bacterial chromosomes, namely relBE, higBA, mazEF, ccdAB, vapBC, parDE, phd-doc, and yoeB-yefM. The toxins of these families share sequence and structural similarity to some extent, but the corresponding antidotes do not, thus indicating their distinct evolutionary origin (2). However, despite their structural similarity and common ancestral origin, their downstream targets are quite different. CcdB and ParE function as gyrase poison, whereas MazF, Kid, YdcE, and YoeB are potent ribonucleases (14 -22). VapC toxins represent a structurally dist...

Structure, Biology, and Therapeutic Application of Toxin–Antitoxin Systems in Pathogenic Bacteria

“…For example, peptide inhibitors of the B. anthracis PemIK interaction were designed to mimic the C -terminal toxin-binding region of the antitoxin [193]. The toxin PemK cleaves single-stranded RNAs, and toxicity is inhibited by the antitoxin PemI.…”

“…Deletion mutant studies showed that the C -terminal fragment of PemI is responsible for PemK binding [194]. The two peptides (LLFQHLTE and RRGYIEMG) block the PemIK interaction and inhibit in vitro ribonuclease activity of the PemK toxin, whereas peptides derived from the N -terminal fragment of PemI do not influence the TA interaction [193]. A rationally designed octapeptide (SKIGAWAS) is predicted to form an α-helix and to occupy the TA binding interface [194].…”

Artificial activation of toxin–antitoxin systems as an antibacterial strategy

“…PemK, of the B. anthracis PemIK TA module, cleaves single-stranded RNAs and is inhibited by the binding of antitoxin PemI [64]. Analysis of PemI deletion variants indicated that the C-terminus is required to bind to PemK.…”

“…Analysis of PemI deletion variants indicated that the C-terminus is required to bind to PemK. Based on this information, six hepta- and octa-peptides, representing fragments of the antitoxin located in a predicted helical region within the TA binding interface, were analyzed for their ability to inhibit the PemI-PemK interaction [64]. ELISA results revealed that each designed peptide was capable of preventing the PemI-PemK interaction to a certain extent, whereas nonspecific 15- and 9-residue peptides based on the N-terminus of PemI did not affect the PemI-PemK interaction [64].…”

“…The authors then examined the effect of the peptides on PemK ribonuclease activity using a fluorogenic chimeric DNA-RNA substrate or a fluorogenic rC substrate [64]. The two peptides that prevented the PemI-PemK interaction to the greatest extent, LLFQHLTE (35% prevention) and RRGYIEMG (30% prevention), inhibited the PemK ribonuclease activity in vitro , while the remaining four peptides did not inhibit PemK ribonuclease activity.…”