MYCOBACTERIAL DORMANCY AND PERSISTENCE: MOLECULAR MECHANISMS CONTROLLING STRESS RESPONSE, SURVIVAL AND ADAPTATION

Ph.DDOCTOR OF PHILOSOPH

Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria.Singapore-MIT Alliance for Research and TechnologyNational Institute of Environmental Health Sciences (ES017010)National Institute of Environmental Health Sciences (ES002109)Singapore-MIT Alliance (Graduate Fellowship)Singapore. National Research Foundatio

tRNA epitranscriptomics and biased codon are linked to proteome expression in

Among components of the translational machinery, ribonucleoside modifications on tRNAs are emerging as critical regulators of cell physiology and stress response. Here, we demonstrate highly coordinated behavior of the repertoire of tRNA modifications of Plasmodium falciparum throughout the intra-erythrocytic developmental cycle (IDC). We observed both a synchronized increase in 22 of 28 modifications from ring to trophozoite stage, consistent with tRNA maturation during translational up-regulation, and asynchronous changes in six modifications. Quantitative analysis of ~2,100 proteins across the IDC revealed that up- and down-regulated proteins in late but not early stages have a marked codon bias that directly correlates with parallel changes in tRNA modifications and enhanced translational efficiency. We thus propose a model in which tRNA modifications modulate the abundance of stage-specific proteins by enhancing translation efficiency of codon-biased transcripts for critical genes. These findings reveal novel epitranscriptomic and translational control mechanisms in the development and pathogenesis of Plasmodium parasites.Singapore. National Research FoundationSingapore-MIT Alliance (Graduate Fellowship

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria—which models tuberculous granulomas—are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo[superscript 5]U in tRNA[superscript Thr(UGU)], which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria.Singapore-MIT Alliance for Research and Technology (SMART)National Institute of Environmental Health Sciences (grants ES017010 and ES002109)National Science Foundation (U.S.) (grant CHE-1308839)Singapore-MIT Alliance for Research and Technology (SMART) (SMA3 Graduate Fellowship

Use of age-dependent FRAX-based intervention thresholds for Singapore

Summary Assessment and treatment pathways based on age-specific intervention thresholds in Singapore using FRAX paths can be used to identify patients at high risk of fracture and avoid unnecessary treatment in those at low risk. Purpose Intervention thresholds for the treatment of osteoporosis have been based historically on the measurement of bone mineral density. The development of FRAX® has permitted a more accurate assessment of fracture risk. The aim of the present study was to explore treatment paths and characteristics of women selected for treatment in Singapore based on FRAX. Methods The approach to the setting of intervention and assessment thresholds used the methodology adopted by the National Osteoporosis Guideline Group for FRAX-based guidelines in the UK but based on the epidemiology of fracture and death in Singapore. The methodology was applied to women age 50 years or more drawn from the population-based Singapore Chinese Health Study (SCHS) cohort. Missing data for the calculation of FRAX was simulated using data from Chinese cohorts from Hong Kong. Results Intervention thresholds expressed as a 10-year probability of a major osteoporotic fracture ranged from 2.9% at the age of 50 years increasing to 32% at the age of 90 years. A total of 1927 of 29,323 women (7%) had a prior fragility fracture and would be eligible for treatment for this reason. An additional 3019 women (10.3%) would be eligible for treatment on the basis of age-dependent thresholds. The mean BMD T-score of women so selected was −2.94. Conclusion Probability-based assessment of fracture risk using age-specific intervention thresholds was developed for Singapore to help guide decisions about treatment

Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa

Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNA[superscript Met(CAU)] and tRNA[superscript Trp(CCA)] are substrates for Cm formation, tRNA[superscript Gln(UUG)], tRNA[superscript Pro(UGG)], tRNA[superscript Pro(CGG)] and tRNA[superscript His(GUG)] for Um, and tRNA[superscript Pro(GGG)] for Am. tRNA[superscript Ser(UGA)], previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNA[superscriptPro(GGG)] and Um in tRNA[superscript Gln(UUG)] by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE. These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.National Science Foundation (U.S.) (CHE-1308839)Agilent TechnologiesSingapore-MIT Alliance for Research and Technology (SMART

Pyruvate Kinase regulates the Pentose-Phosphate pathway in Response to Hypoxia in Mycobacterium tuberculosis

In response to the stress of infection, Mycobacterium tuberculosis (Mtb) reprograms its metabolism to accommodate nutrient and energetic demands in a changing environment. Pyruvate kinase (PYK) is an essential glycolytic enzyme in the phosphoenolpyruvate–pyruvate–oxaloacetate node that is a central switch point for carbon flux distribution. Here we show that the competitive binding of pentose monophosphate inhibitors or the activator glucose 6-phosphate (G6P) to MtbPYK tightly regulates the metabolic flux. Intriguingly, pentose monophosphates were found to share the same binding site with G6P. The determination of a crystal structure of MtbPYK with bound ribose 5-phosphate (R5P), combined with biochemical analyses and molecular dynamic simulations, revealed that the allosteric inhibitor pentose monophosphate increases PYK structural dynamics, weakens the structural network communication, and impairs substrate binding. G6P, on the other hand, primes and activates the tetramer by decreasing protein flexibility and strengthening allosteric coupling. Therefore, we propose that MtbPYK uses these differences in conformational dynamics to up- and down-regulate enzymic activity. Importantly, metabolome profiling in mycobacteria reveals a significant increase in the levels of pentose monophosphate during hypoxia, which provides insights into how PYK uses dynamics of the tetramer as a competitive allosteric mechanism to retard glycolysis and facilitate metabolic reprogramming toward the pentose-phosphate pathway for achieving redox balance and an anticipatory metabolic response in Mtb

A clinician’s guide to management of intra-abdominal hypertension and abdominal compartment syndrome in critically ill patients

This article is one of ten reviews selected from the Annual Update in Intensive Care and Emergency Medicine 2020. Other selected articles can be found online at . Further information about the Annual Update in Intensive Care and Emergency Medicine is available from http://www.springer.com/series/8901

Reciprocal regulation of TORC signaling and tRNA modifications by Elongator enforces nutrient-dependent cell fate

Nutrient availability has a profound impact on cell fate. Upon nitrogen starvation, wild-type fission yeast cells uncouple cell growth from cell division to generate small, round-shaped cells that are competent for sexual differentiation. The TORC1 (TOR complex 1) and TORC2 complexes exert opposite controls on cell growth and cell differentiation, but little is known about how their activity is coordinated. We show that transfer RNA (tRNA) modifications by Elongator are critical for this regulation by promoting the translation of both key components of TORC2 and repressors of TORC1. We further identified the TORC2 pathway as an activator of Elongator by down-regulating a Gsk3 (glycogen synthase kinase 3)-dependent inhibitory phosphorylation of Elongator. Therefore, a feedback control is operating between TOR complex (TORC) signaling and tRNA modification by Elongator to enforce the advancement of mitosis that precedes cell differentiation

Loss of gastrokine-2 drives premalignant gastric inflammation and tumor progression

Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130[superscript F/F] GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori–infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130[superscript F/F] mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression