Effects of increasing the affinity of CarD for RNA polymerase on Mycobacterium tuberculosis growth, rRNA transcription, and virulence

CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis. In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro. Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and broaden our understanding of prokaryotic transcription

Interaction of CarD with RNA polymerase mediates Mycobacterium tuberculosis viability, rifampin resistance, and pathogenesis

Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP β subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP β interactions demonstrates that the CarD/RNAP β association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP β interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP β interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding

Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction

The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the β-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP β-subunit. We determined the 2.9-Å resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-β1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP

Acceptability, feasibility and preliminary efficacy of low-moderate intensity Constraint Induced Aphasia Therapy and Multi-Modality Aphasia Therapy in chronic aphasia after stroke

Background: High-intensity Constraint-Induced Aphasia Therapy Plus (CIAT-Plus) and Multi-Modality Aphasia Therapy (M-MAT) are effective interventions for chronic post-stroke aphasia but challenging to provide in clinical practice. Providing these interventions may be more feasible at lower intensities, but comparative evidence is lacking. We therefore explored feasibility, acceptability, and preliminary efficacy of the treatments at a lower intensity. Methods: A multisite, single-blinded, randomized Phase II trial was conducted within the Phase III COMPARE trial. Groups of participants with chronic aphasia from the usual care arm of the COMPARE trial were randomized to M-MAT or CIAT-Plus, delivered at the same dose as the COMPARE trial but at lower intensity (6 hours/week × 5 weeks rather than 15 hours/week × 2 weeks). Blinded assessors measured aphasia severity (Western Aphasia Battery-Revised Aphasia Quotient), word retrieval, connected speech, multimodal communication, functional communication, and quality of life immediately post interventions and after 12 weeks. Feasibility and acceptability were explored. Results: Of 70 eligible participants, 77% consented to the trial; 78% of randomized participants completed intervention and 98% of assessment visits were conducted. Fatigue and distress ratings were low with no related withdrawals. Adverse events related to the trial (n = 4) were mild in severity. Statistically significant treatment effects were demonstrated on word retrieval and functional communication and both interventions were equally effective. Conclusions: Low–moderateintensity CIAT-Plus and M-MAT were feasible and acceptable. Both interventions show preliminary efficacy at a low–moderate intensity. These results support a powered trial investigating these interventions at a low–moderate intensity

No evidence for an association of plasma homocysteine levels and refractive error - Results from the population-based Gutenberg Health Study (GHS)

Purpose There is a strong association between severe hyperhomocysteinemia and myopia. Thus we studied the hypothesis that even moderately increased levels of homocysteine (Hcy) might be a potentially treatable risk factor for myopia. Methods The Gutenberg Health Study (GHS) is a population-based, prospective, observational cohort study in Germany, including 15,010 participants aged between 35 and 74 at recruitment. The baseline examination was conducted from 2007-2012. Refraction was measured using autorefraction (HARK 599, Carl Zeiss AG, Jena, Germany). Hcy was measured by an immunoassay. We included only phakic participants without a history of corneal surgery or corneal laser treatment. We used linear regression models to evaluate the potential association between Hcy and refraction at baseline, and between Hcy and change in refraction between baseline and 5-year-follow-up examination. We used generalized estimating equation models to account for the correlation between fellow eyes. Results We included 13,749 participants, categorized as having no myopia (spherical equivalent > -0.75 D, 65.2%), low myopia (-0.75 D-2.75 D, 21.5%), moderate myopia (-3.00 D- 5.75 D, 9.8%) and high myopia (≤ -6

Not all SCN1A epileptic encephalopathies are Dravet syndrome: Early profound Thr226Met phenotype.

OBJECTIVE: To define a distinct SCN1A developmental and epileptic encephalopathy with early onset, profound impairment, and movement disorder. METHODS: A case series of 9 children were identified with a profound developmental and epileptic encephalopathy and SCN1A mutation. RESULTS: We identified 9 children 3 to 12 years of age; 7 were male. Seizure onset was at 6 to 12 weeks with hemiclonic seizures, bilateral tonic-clonic seizures, or spasms. All children had profound developmental impairment and were nonverbal and nonambulatory, and 7 of 9 required a gastrostomy. A hyperkinetic movement disorder occurred in all and was characterized by dystonia and choreoathetosis with prominent oral dyskinesia and onset from 2 to 20 months of age. Eight had a recurrent missense SCN1A mutation, p.Thr226Met. The remaining child had the missense mutation p.Pro1345Ser. The mutation arose de novo in 8 of 9; for the remaining case, the mother was negative and the father was unavailable. CONCLUSIONS: Here, we present a phenotype-genotype correlation for SCN1A. We describe a distinct SCN1A phenotype, early infantile SCN1A encephalopathy, which is readily distinguishable from the well-recognized entities of Dravet syndrome and genetic epilepsy with febrile seizures plus. This disorder has an earlier age at onset, profound developmental impairment, and a distinctive hyperkinetic movement disorder, setting it apart from Dravet syndrome. Remarkably, 8 of 9 children had the recurrent missense mutation p.Thr226Met

tRNA is a new target for cleavage by a MazF toxin

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNAPro14 D-loop or within the tRNALys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth

Municipal water quantities and health in Nunavut households: an exploratory case study in Coral Harbour, Nunavut, Canada

Background: Access to adequate quantities of water has a protective effect on human health and well-being. Despite this, public health research and interventions are frequently focused solely on water quality, and international standards for domestic water supply minimums are often overlooked or unspecified. This trend is evident in Inuit and other Arctic communities even though numerous transmissible diseases and bacterium infections associated with inadequate domestic water quantities are prevalent. Objectives: Our objective was to explore the pathways by which the trucked water distribution systems being used in remote northern communities are impacting health at the household level, with consideration given to the underlying social and environmental determinants shaping health in the region. Methods: Using a qualitative case study design, we conducted 37 interviews (28 residents, 9 key informants) and a review of government water documents to investigate water usage practices and perspectives. These data were thematically analysed to understand potential health risks in Arctic communities and households. Results: Each resident receives an average of 110 litres of municipal water per day. Fifteen of 28 households reported experiencing water shortages at least once per month. Of those 15, most were larger households (5 people or more) with standard sized water storage tanks. Water shortages and service interruptions limit the ability of some households to adhere to public health advice. The households most resilient, or able to cope with domestic water supply shortages, were those capable of retrieving their own drinking water directly from lake and river sources. Residents with extended family and neighbours, whom they can rely on during shortages, were also less vulnerable to municipal water delays. Conclusions: The relatively low in-home water quantities observed in Coral Harbour, Nunavut, appear adequate for some families. Those living in overcrowded households, however, are accessing water in quantities more typically seen in water insecure developing countries. We recommend several practical interventions and revisions to municipal water supply systems

Progress On Compare: A Randomised Controlled Trial Comparing Constraint-Induced And Multi-Modal Aphasia Therapy To Usual Care In People With Chronic Aphasia

Background/Aims: Chronic post-stroke aphasia impacts 30% of stroke survivors. The aim of this trial is to determine whether two contrasting, intensive treatments, Constraint Induced Aphasia Therapy (CIAT) and Multi-Modal Aphasia Therapy (M-MAT), are superior and cost-effective than usual care (UC). The variable response to these therapies may be accounted for by aphasia severity and co-occurring cognitive problems. Methods: 3-arm prospective, single-blinded, randomised controlled trial. Primary end point at 3 months post-treatment. Participants (n = 198) are randomised to CIAT, M-MAT or UC. CIAT and M-MAT both focus on intensive speech practice (30 hours over 2 weeks) using interactive game formats. However, while M-MAT involves gesture, writing, and drawing cues, CIAT focuses on speech alone. UC is usual health service-based aphasia therapy. Primary outcome immediately post treatment is the Aphasia Quotient of the Western Aphasia Battery. Secondary outcomes at 3-month follow-up include measures of connected speech, multi-modal communication, and quality of life. Participant cognitive and linguistic predictors of treatment response are identified. Re-randomised UC participants will undertake a less intense schedule (30 hours over 5 weeks) in a nested sub-study exploring the impact of treatment intensity on outcomes. A full cost-effectiveness analysis will be undertaken. Results: The trial is underway: ethics approval, trial registration, and database are established. Recruitment from five Australian states has commenced. Conclusions: This trial will determine if there is a significant therapeutic effect of, and variable response to treatment types in chronic aphasia. Essential economic evaluation information for service delivery standards for aphasia rehabilitation will be described.1 page(s