Sterilization of granulomas is common in both active and latent tuberculosis despite extensive within-host variability in bacterial killing

Over 30% of the world’s population is infected with Mycobacterium tuberculosis (Mtb), yet only ~5–10% will develop clinical disease1. Despite considerable effort, we understand little about what distinguishes individuals who progress to active tuberculosis (TB) from those who remain latent for decades. The variable course of disease is recapitulated in cynomolgus macaques infected with Mtb2. Active disease in macaques is defined by clinical, microbiologic and immunologic signs and occurs in ~45% of animals, while the remaining are clinically asymptomatic2,3. Here, we use barcoded Mtb isolates and quantitative measures of culturable and cumulative bacterial burden to show that most lesions are likely founded by a single bacterium and reach similar maximum burdens. Despite common origins, the fate of individual lesions varies substantially within the same host. Strikingly, in active disease, the host sterilizes some lesions even while others progress. Our data suggest that lesional heterogeneity arises, in part, through differential killing of bacteria after the onset of adaptive immunity. Thus, individual lesions follow diverse and overlapping trajectories, suggesting critical responses occur at a lesional level to ultimately determine the clinical outcome of infection. Defining the local factors that dictate outcome will be important in developing effective interventions to prevent active TB

Mindfulness and Behavior Change

Initiating and maintaining behavior change is key to the prevention and treatment of most preventable chronic medical and psychiatric illnesses. The cultivation of mindfulness, involving acceptance and nonjudgment of present-moment experience, often results in transformative health behavior change. Neural systems involved in motivation and learning have an important role to play. A theoretical model of mindfulness that integrates these mechanisms with the cognitive, emotional, and self-related processes commonly described, while applying an integrated model to health behavior change, is needed. This integrative review (1) defines mindfulness and describes the mindfulness-based intervention movement, (2) synthesizes the neuroscience of mindfulness and integrates motivation and learning mechanisms within a mindful self-regulation model for understanding the complex effects of mindfulness on behavior change, and (3) synthesizes current clinical research evaluating the effects of mindfulness-based interventions targeting health behaviors relevant to psychiatric care. The review provides insight into the limitations of current research and proposes potential mechanisms to be tested in future research and targeted in clinical practice to enhance the impact of mindfulness on behavior change

Mindfulness training for primary care for portuguese-speaking immigrants : a pilot study

Background: Portuguese-speaking immigrants are a growing underserved population in the Unites States who experience high levels of psychological distress and increased vulnerability to mental health disorders such as depression and anxiety. Current evidence shows that mindfulness-based interventions (MBIs) are effective to promote physical and mental health among educated English speakers; nonetheless, the lack of diversity in the mindfulness literature is a considerable limitation. To our knowledge, the feasibility and acceptability of MBIs among Portuguese-speaking immigrants have not yet been investigated. Methods: This single-arm pilot study (N = 30) explored the feasibility, acceptability, and cultural aspects of Mindfulness Training for Primary Care (MTPC)-Portuguese among Portuguese-speaking immigrants in the Boston area. MTPC is an 8-week, primary care-adapted, referral-based, insurance-reimbursable, trauma-informed MBI that is fully integrated into a healthcare system. The study also examined intervention preliminary effectiveness on mental health outcomes (depression and anxiety symptoms) and self-regulation (emotional regulation, mindfulness, self-compassion, interoceptive awareness), and initiation of health behavior was explored. Results: Primary care providers referred 129 patients from 2018 to 2020. Main DSM-5 primary diagnoses were depression (76.3%) and anxiety disorders (6.7%). Participants (N = 30) attended a mean of 6.1 (SD 1.92) sessions and reported a mean of 213.7 (SD = 124.3) min of practice per week. All survey finishers would recommend the program to a friend, found the program helpful, and rated the overall program as “very good” or “excellent,” and 93% would participate again, with satisfaction mean scores between 4.6 and 5 (Likert scale 0– 5). Participants and group leaders provided feedback to refine MTPC-Portuguese culturally responsiveness regarding materials language, settings, time, food, and community building. Patients exhibited reductions in depression (d = 0.67; p < 0.001) and anxiety (d = 0.48; p = 0.011) symptoms, as well as enhanced emotional regulation (d = 0.45; p = 0.009), and among survey finishers, 50% initiated health behavior change through action plan initiation. Conclusion: This pilot study suggests that MTPC-Portuguese is feasible, acceptable, and culturally appropriate among Portuguese-speaking patients in the Boston area. Furthermore, the intervention might potentially decrease depression and anxiety symptoms, facilitate health behavior change, and improve emotional regulation. MTPC-Portuguese investigation with larger samples in controlled studies is warranted to support its dissemination and implementation in the healthcare system

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader

The DNA replication machinery is an important target for antibiotic development for increasingly drug resistant bacteria including Mycobacterium tuberculosis1. While blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In E. coli, the proofreading subunit of the replisome, the ε-exonuclease, is essential for high fidelity DNA replication2; however, we find that it is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase, DnaE1, encodes a novel editing function that proofreads DNA replication, mediated by an intrinsic 3′-5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by greater than 3,000 fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP-domain mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader

Investigating the Rates and Drivers of Drug Resistance in Mycobacterium Tuberculosis

The emergence and transmission of drug resistant strains of Mycobacterium tuberculosis (Mtb) calls for urgency in understanding the barriers to effective treatment. Here we developed a chemostat cultivation system to measure the mycobacterial mutation rate during periods of slow growth versus fast growth. We found that mutants accumulated at the same rate per unit time during slow growth and during fast growth, suggesting that mutation rate does not vary in accordance with growth rate. By competing genetically barcoded mutant strains at fast and slow growth rates and using a mathematical model to estimate mutant fitness, we confirmed that there is not aggravated loss of mutants at fast growth rates, but that equivalence in mutation rate per unit time reflects time-based mutation rather than replication-based mutation. To investigate a possible driver of replication-independent mutation, we developed a mass-spectrometry-based analytic method to detect DNA damage during metabolism of fatty acids, a major component of the bacterial diet in vivo. We discovered that a novel glyoxylate-dG adduct forms when cells use the glyoxylate shunt to metabolize fatty acids, and that the levels of this adduct increase in the absence of nucleotide excision repair. Using fluctuation analysis to measure the mutation rate in these conditions, we found that fatty acid metabolism is mutagenic in nucleotide repair deficient cells. Finally, cholesterol metabolism was mutagenic in both wild-type and repair-deficient backgrounds. These findings demonstrate that metabolic state can drive both mutation and DNA damage. Many patients develop recurrent tuberculosis (TB) despite receiving adequate treatment for TB, yet the causes of recurrent disease are poorly understood. We used whole-genome sequencing (WGS) and MIRU-VNTR typing to investigate the cause of recurrent infection in 13 HIV-infected individuals who had been successfully treated as part of their enrollment in a prospective cohort study in KwaZulu Natal, South Africa. By comparing the genetic relatedness in the presenting and recurrent strains, we found that 7 of the 13 recurrent episodes appeared to represent relapse of the primary infection, despite the force of TB transmission in KwaZulu Natal and the successful HIV and TB treatment history. Results from MIRU-VNTR typing were concordant with WGS for highly related and highly divergent strains, but only WGS analysis could resolve intermediate genetic distances. Patients presented with relapsed infection and reinfection up to 3 years after completion of treatment, and no difference was found between the timing of relapse and reinfection cases. Strains causing relapsed infection were more likely to harbor genetic polymorphisms associated with changes in INH susceptibility or acquisition of INH resistance despite being phenotypically drug sensitive, suggesting a possible role for low-level or undiagnosed drug resistance in tuberculosis relapse.Biological Sciences in Public Healt

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader

The DNA replication machinery is an important target for antibiotic development for increasingly drug resistant bacteria including Mycobacterium tuberculosis1. While blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In E. coli, the proofreading subunit of the replisome, the ε-exonuclease, is essential for high fidelity DNA replication2; however, we find that it is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase, DnaE1, encodes a novel editing function that proofreads DNA replication, mediated by an intrinsic 3′-5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by greater than 3,000 fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP-domain mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

<div><p>RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in <i>Mycobacterium smegmatis</i>, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in <i>Mycobacterium tuberculosis</i>, provide complementary, empirical datasets to examine the congruence of transcription and translation in the <i>Mycobacterium</i> genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5’ untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the <i>cis</i>-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5’ end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5’ ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5’ UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.</p></div

Small protein ORFs are frequently coupled to the ORF downstream.

<p>(A) <i>M</i>. <i>tuberculosis</i> leaderless transcripts initiate unannotated small protein ORFs that terminate at the start of the annotated gene downstream more often than expected. All small protein ORF stop codons within 100 nucleotides of an annotated gene start are shown relative to that start codon (0 = coupled RTGA overlap). Three structural classes are identified: uORFs (the small ORF terminates upstream of the annotated start), coupled ORFs (linked by an RTGA tetramer), and overlapping ORFs. The y-axis shows the fraction of small ORFs that terminate a specified distance (x-axis) from the annotated start codon of the downstream gene. (B) One example of a coupled small protein in <i>M</i>. <i>tuberculosis</i> and <i>M</i>. <i>smegmatis</i>, upstream of orthologous genes. The primary sequence of the encoded small protein is not conserved, but the leaderless initiation and coupled linkage is maintained.</p

Leaderless gene architectures bring promoters and ORFs together.

<p>(A) Logo plot of TSS and proximal promoter region of traditional leadered genes. A purine (A or G) is favored at the +1 nucleotide, and an AT rich -10 element appears upstream. The 5’ UTR downstream of the transcription start site shows no sequence constraints or enrichment. (B) A Logo plot of the 5’ UTR from the translation initiation codon shows a Shine-Dalgarno-like AGGAGG sequence enrichment, centered 9–10 nt upstream (positions 10–11). From the initiation codon, the coding sequence downstream shows the wobble bias of the G-C rich mycobacterial genome. (C) The proximal promoter regions of leaderless genes have a -10 sequence of similar composition and spacing to that of leadered genes (compare to 2A). The TSS is also the first nucleotide of the translation initiation codon. There is no evidence of Shine-Dalgarno sequence enrichment upstream. The ORF initiated by leaderless codons shows the same wobble bias as seen in leadered ORFs.</p