The Sole Mycobacterium smegmatis MazF Toxin Targets tRNALys to Impart Highly Selective, Codon-Dependent Proteome Reprogramming.

Survival of mycobacteria, both free-living and host-dependent pathogenic species, is dependent on their ability to evade being killed by the stresses they routinely encounter. Toxin-antitoxin (TA) systems are unique to bacteria and archaea and are thought to function as stress survival proteins. Here, we study the activity of the endoribonuclease toxin derived from the MazEF TA system in Mycobacterium smegmatis, designated MazEF-ms. We first enlisted a specialized RNA-seq method, 5' RNA-seq, to identify the primary RNA target(s) of the MazF-ms toxin. Just two tRNA species, tRNALys-UUU and tRNALys-CUU, were targeted for cleavage by MazF-ms at a single site within their anticodon sequence (UU↓U and CU↓U) to render these tRNAs nonfunctional for protein synthesis. The 5' RNA-seq dataset also revealed hallmarks of ribosome stalling predominantly at Lys AAA codons even though both Lys tRNAs were cleaved by MazF-ms. Stalled ribosomes were then cleaved on their 5' side by one or more RNases, resulting in very selective degradation of only those mRNAs harboring ribosomes stalled at Lys codons. This highly surgical, codon-dependent degradation of mRNA transcripts was validated using quantitative mass spectrometry of proteins that were newly synthesized during MazF-ms expression. The M. smegmatis proteome was altered as predicted, Lys AAA codon-rich proteins was downregulated while Lys AAA codon deficient proteins were upregulated. Analysis of specific subsets of proteins that were upregulated or downregulated was consistent with the growth-arrested phenotype of MazF-ms expressing cells. Curiously, the tRNA target and mechanism of action of MazF-ms paralleled that of one atypical MazF toxin in M. tuberculosis, suggesting manipulation of the levels of lysine tRNAs as the preferred conduit for reprogramming the proteomes via ribosome stalling at rare AAA codons in these GC-rich mycobacteria.

Heterogeneous persister cells formation in Acinetobacter baumannii.

Bacterial persistence is a feature that allows susceptible bacteria to survive extreme concentrations of antibiotics and it has been verified in a number of species, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp., Mycobacterium spp. However, even though Acinetobacter baumannii is an important nosocomial pathogen, data regarding its persistence phenotype are still lacking. Therefore, the aim of this study was to evaluate the persistence phenotype in A. baumannii strains, as well as its variation among strains after treatment with polymyxin B and tobramycin. Stationary cultures of 37 polymyxin B-susceptible clinical strains of A. baumannii were analyzed for surviving cells after exposure to 15 µg/mL of polymyxin B for 6 h, by serial dilutions and colony counting. Among these, the 30 tobramycin-susceptible isolates also underwent tobramycin treatment at a concentration of 160 µg/mL and persister cells occurrence was evaluated equally. A high heterogeneity of persister cells formation patterns among isolates was observed. Polymyxin B-treated cultures presented persister cells corresponding from 0.0007% to 10.1% of the initial population and two isolates failed to produce detectable persister cells under this condition. A high variability could also be observed when cells were treated with tobramycin: the persister fraction corresponded to 0.0003%-11.84% of the pre-treatment population. Moreover, no correlation was found between persister subpopulations comparing both antibiotics among isolates, indicating that different mechanisms underlie the internal control of this phenotype. This is the first report of persister cells occurrence in A. baumannii. Our data suggest that distinct factors regulate the tolerance for unrelated antibiotics in this species, contrasting the multi-drug tolerance observed in other species (eg. dormancy-mediated tolerance). Supporting this observation, polymyxin B--an antibiotic that is believed to act on non-dividing cells as well--failed to eradicate persister cells in the majority of the isolates, possibly reflecting a disconnection between persistence and dormancy.

Effectiveness of the proton pump inhibitor omeprazole associated with calcium hydroxide as intracanal medication: an in vivo study.

INTRODUCTION: The objective of this study was to evaluate the efficacy of the association of a proton pump inhibitor (omeprazole) with Ca(OH)(2) as intracanal medication in a rat model of periapical lesions. METHODS: Periapical lesions were induced on the first right mandibular molar tooth of 36 male Wistar rats (6 per group). After 28 days, the distal canal of each tooth was prepared, filled with the respective dressing (negative control group, PEG 400; positive control group, Ca(OH)(2) + PEG400; test group, Ca(OH)(2) + omeprazole + PEG 400), and sealed with amalgam for 15 or 28 days. Microbiological samples were taken in 3 periods: S1, after 28 days of lesion induction; S2, after the biomechanical preparation; and S3, after the medication (15 and 28 days). RESULTS: The radiographic and histologic analysis revealed that either Ca(OH)(2) or Ca(OH)(2) plus omeprazole dressings produced a reduction of periapical lesions at 28 days, when compared with the negative control group. The reduction of periapical lesions and inflammatory cell infiltration was visibly improved by associating omeprazole with Ca(OH)(2), with an increase of reparative bone areas. The microbiological assessment showed a significant decrease of colony-forming units count from S1 to S2 or S3 collecting times, but no differences were observed between the S2 and the S3 time-periods or among the experimental groups within the S3 period. Further bacterial characterization showed a possible selective activity of the medications. CONCLUSIONS: Our data showed that association of omeprazole with Ca(OH)(2) favored a superior repair of rat periapical lesions and seemed to display different selective activity over endodontic microbiota, in comparison with the conventional Ca(OH)(2) dressing.