ABSTRACT
Mycobacterium tuberculosis (Mtb) defends itself against host immunity and chemotherapy at several levels, including the repair or degradation of irreversibly oxidized proteins (IOPs). To investigate how Mtb deals with IOPs that can neither be repaired nor degraded, we used new chemical and biochemical probes and improved image analysis algorithms for time-lapse microscopy to reveal a defense against stationary phase stress, oxidants, and antibiotics--the sequestration of IOPs into aggregates in association with the chaperone ClpB, followed by the asymmetric distribution of aggregates within bacteria and between their progeny. Progeny born with minimal IOPs grew faster and better survived a subsequent antibiotic stress than their IOP-burdened sibs. ClpB-deficient Mtb had a marked recovery defect from stationary phase or antibiotic exposure and survived poorly in mice. Treatment of tuberculosis might be assisted by drugs that cripple the pathway by which Mtb buffers, sequesters, and asymmetrically distributes IOPs.
Subject(s)
Bacterial Proteins/metabolism , Endopeptidase Clp/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Oxidative Stress , Animals , Anti-Bacterial Agents/toxicity , Endopeptidase Clp/genetics , Mice , Microbial Viability/drug effects , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/physiology , Oxidants/toxicity , Oxidation-Reduction , Protein Aggregates , Protein Multimerization , Protein Processing, Post-Translational , Protein TransportABSTRACT
CONTEXT: The circadian clock represents the body's molecular time-keeping system. Recent findings revealed strong changes of clock gene expression in various types of human cancers. OBJECTIVE: Due to emerging evidence on the connection between the circadian oscillator, cell cycle, and oncogenic transformation, we aimed to characterize the circadian clockwork in human benign and malignant thyroid nodules. DESIGN: Clock transcript levels were assessed by quantitative RT-PCR in thyroid tissues. To provide molecular characteristics of human thyroid clockwork, primary thyrocytes established from normal or nodular thyroid tissue biopsies were subjected to in vitro synchronization with subsequent clock gene expression analysis by circadian bioluminescence reporter assay and by quantitative RT-PCR. RESULTS: The expression levels of the Bmal1 were up-regulated in tissue samples of follicular thyroid carcinoma (FTC), and in papillary thyroid carcinoma (PTC), as compared with normal thyroid and benign nodules, whereas Cry2 was down-regulated in FTC and PTC. Human thyrocytes derived from normal thyroid tissue exhibited high-amplitude circadian oscillations of Bmal1-luciferase reporter expression and endogenous clock transcripts. Thyrocytes established from FTC and PTC exhibited clock transcript oscillations similar to those of normal thyroid tissue and benign nodules (except for Per2 altered in PTC), whereas cells derived from poorly differentiated thyroid carcinoma exhibited altered circadian oscillations. CONCLUSIONS: This is the first study demonstrating a molecular makeup of the human thyroid circadian clock. Characterization of the thyroid clock machinery alterations upon thyroid nodule malignant transformation contributes to understanding the connections between circadian clocks and oncogenic transformation. Moreover, it might help in improving the thyroid nodule preoperative diagnostics.
