Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs.

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M. smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M. tuberculosis transcriptome.

Arginine methylation sites on SepIVA help balance elongation and septation in Mycobacterium smegmatis.

The growth of mycobacterial cells requires successful coordination between elongation and septation. However, it is not clear which factors mediate this coordination. Here, we studied the function and post-translational modification of an essential division factor, SepIVA, in Mycobacterium smegmatis. We find that SepIVA is arginine methylated, and that alteration of its methylation sites affects both septation and polar elongation of Msmeg. Furthermore, we show that SepIVA regulates the localization of MurG and that this regulation may impact polar elongation. Finally, we map SepIVA's two regulatory functions to different ends of the protein: the N-terminus regulates elongation while the C-terminus regulates division. These results establish SepIVA as a regulator of both elongation and division and characterize a physiological role for protein arginine methylation sites for the first time in mycobacteria.

ClpX Is Essential and Activated by Single-Strand DNA Binding Protein in Mycobacteria.

The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX's essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting that the ClpX-SSB complex may play a role in DNA replication in mycobacteria.

Correction: TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities.

[This corrects the article DOI: 10.1371/journal.ppat.1006939.].

Small RNA profiling in Mycobacterium tuberculosis identifies MrsI as necessary for an anticipatory iron sparing response.

One key to the success of Mycobacterium tuberculosis as a pathogen is its ability to reside in the hostile environment of the human macrophage. Bacteria adapt to stress through a variety of mechanisms, including the use of small regulatory RNAs (sRNAs), which posttranscriptionally regulate bacterial gene expression. However, very little is currently known about mycobacterial sRNA-mediated riboregulation. To date, mycobacterial sRNA discovery has been performed primarily in log-phase growth, and no direct interaction between any mycobacterial sRNA and its targets has been validated. Here, we performed large-scale sRNA discovery and expression profiling in M. tuberculosis during exposure to five pathogenically relevant stresses. From these data, we identified a subset of sRNAs that are highly induced in multiple stress conditions. We focused on one of these sRNAs, ncRv11846, here renamed mycobacterial regulatory sRNA in iron (MrsI). We characterized the regulon of MrsI and showed in mycobacteria that it regulates one of its targets, bfrA, through a direct binding interaction. MrsI mediates an iron-sparing response that is required for optimal survival of M. tuberculosis under iron-limiting conditions. However, MrsI is induced by multiple host-like stressors, which appear to trigger MrsI as part of an anticipatory response to impending iron deprivation in the macrophage environment.

Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132.

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based ß-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure-activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

TnSeq of Mycobacterium tuberculosis clinical isolates reveals strain-specific antibiotic liabilities.

Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among Mycobacterium tuberculosis (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for in vitro growth across these strains. We developed an analytic approach to identify quantitative differences in genetic requirements between these genetically diverse strains, which vary in genomic structure and gene content. Using this methodology, we found differences between strains in their requirements for genes involved in fundamental cellular processes, including redox homeostasis and central carbon metabolism. Among the genes with differential requirements were katG, which encodes the activator of the first-line antitubercular agent isoniazid, and glcB, which encodes malate synthase, the target of a novel small-molecule inhibitor. Differences among strains in their requirement for katG and glcB predicted differences in their response to these antimicrobial agents. Importantly, these strain-specific differences in antibiotic response could not be predicted by genetic variants identified through whole genome sequencing or by gene expression analysis. Our results provide novel insight into the basis of variation among Mtb strains and demonstrate that TnSeq is a scalable method to predict clinically important phenotypic differences among Mtb strains.

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

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 Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium 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 cis-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.

Neocortical 40 Hz oscillations during carbachol-induced rapid eye movement sleep and cataplexy.

Higher cognitive functions require the integration and coordination of large populations of neurons in cortical and subcortical regions. Oscillations in the gamma band (30-45 Hz) of the electroencephalogram (EEG) have been involved in these cognitive functions. In previous studies, we analysed the extent of functional connectivity between cortical areas employing the 'mean squared coherence' analysis of the EEG gamma band. We demonstrated that gamma coherence is maximal during alert wakefulness and is almost absent during rapid eye movement (REM) sleep. The nucleus pontis oralis (NPO) is critical for REM sleep generation. The NPO is considered to exert executive control over the initiation and maintenance of REM sleep. In the cat, depending on the previous state of the animal, a single microinjection of carbachol (a cholinergic agonist) into the NPO can produce either REM sleep [REM sleep induced by carbachol (REMc)] or a waking state with muscle atonia, i.e. cataplexy [cataplexy induced by carbachol (CA)]. In the present study, in cats that were implanted with electrodes in different cortical areas to record polysomnographic activity, we compared the degree of gamma (30-45 Hz) coherence during REMc, CA and naturally-occurring behavioural states. Gamma coherence was maximal during CA and alert wakefulness. In contrast, gamma coherence was almost absent during REMc as in naturally-occurring REM sleep. We conclude that, in spite of the presence of somatic muscle paralysis, there are remarkable differences in cortical activity between REMc and CA, which confirm that EEG gamma (≈40 Hz) coherence is a trait that differentiates wakefulness from REM sleep.

Control of hypoglossal motoneurones during naturally occurring sleep and wakefulness in the intact, unanaesthetized cat: a field potential study.

The present electrophysiological study was designed to determine the discharge threshold of hypoglossal motoneurones during naturally occurring states of sleep and wakefulness in the intact, unanaesthetized cat. The antidromic field potential, which reflects the net level of membrane excitability of motoneurones and therefore their discharge threshold, was recorded in the hypoglossal nucleus following stimulation of the hypoglossal nerve. The amplitude of the antidromic field potential was larger during wakefulness and non-rapid eye movement (NREM) sleep compared with REM sleep. There was no significant difference in the amplitude of the field potential when wakefulness was compared with NREM sleep (P = 0.103, df = 3, t = 2.324). However, there was a 46% reduction in amplitude during REM sleep compared with NREM sleep (P < 0.001, df = 10, t = 6.421) or wakefulness (P < 0.01, df = 4, t = -4.598). These findings indicate that whereas the excitability of motoneurones that comprise the hypoglossal motor pool is relatively constant during wakefulness and NREM sleep, their excitability is significantly reduced during REM sleep. This state-dependent pattern of control of hypoglossal motoneurones during REM sleep is similar to that reported for motoneurones in other motor nuclei at all levels of the neuraxis. The decrease in the evoked response of hypoglossal motoneurones, which reflects a significant increase in the discharge threshold of individual motoneurones, results in atonia of the lingual and related muscles during REM sleep.

A genome-wide cytotoxicity screen of cluster F1 mycobacteriophage Girr reveals novel inhibitors of Mycobacterium smegmatis growth.

Over the past decade, thousands of bacteriophage genomes have been sequenced and annotated. A striking observation from this work is that known structural features and functions cannot be assigned for >65% of the encoded proteins. One approach to begin experimentally elucidating the function of these uncharacterized gene products is genome-wide screening to identify phage genes that confer phenotypes of interest like inhibition of host growth. This study describes the results of a screen evaluating the effects of overexpressing each gene encoded by the temperate Cluster F1 mycobacteriophage Girr on the growth of the host bacterium Mycobacterium smegmatis. Overexpression of 29 of the 102 Girr genes (~28% of the genome) resulted in mild to severe cytotoxicity. Of the 29 toxic genes described, 12 have no known function and are predominately small proteins of <125 amino acids. Overexpression of the majority of these 12 cytotoxic no known functions proteins resulted in moderate to severe growth reduction and represent novel antimicrobial products. The remaining 17 toxic genes have predicted functions, encoding products involved in phage structure, DNA replication/modification, DNA binding/gene regulation, or other enzymatic activity. Comparison of this dataset with prior genome-wide cytotoxicity screens of mycobacteriophages Waterfoul and Hammy reveals some common functional themes, though several of the predicted Girr functions associated with cytotoxicity in our report, including genes involved in lysogeny, have not been described previously. This study, completed as part of the HHMI-supported SEA-GENES project, highlights the power of parallel, genome-wide overexpression screens to identify novel interactions between phages and their hosts.

Identification of bacterial determinants of tuberculosis infection and treatment outcomes: a phenogenomic analysis of clinical strains.

BACKGROUND: Bacterial diversity could contribute to the diversity of tuberculosis infection and treatment outcomes observed clinically, but the biological basis of this association is poorly understood. The aim of this study was to identify associations between phenogenomic variation in Mycobacterium tuberculosis and tuberculosis clinical features. METHODS: We developed a high-throughput platform to define phenotype-genotype relationships in M tuberculosis clinical isolates, which we tested on a set of 158 drug-sensitive M tuberculosis strains sampled from a large tuberculosis clinical study in Ho Chi Minh City, Viet Nam. We tagged the strains with unique genetic barcodes in multiplicate, allowing us to pool the strains for in-vitro competitive fitness assays across 16 host-relevant antibiotic and metabolic conditions. Relative fitness was quantified by deep sequencing, enumerating output barcode read counts relative to input normalised values. We performed a genome-wide association study to identify phylogenetically linked and monogenic mutations associated with the in-vitro fitness phenotypes. These genetic determinants were further associated with relevant clinical outcomes (cavitary disease and treatment failure) by calculating odds ratios (ORs) with binomial logistic regressions. We also assessed the population-level transmission of strains associated with cavitary disease and treatment failure using terminal branch length analysis of the phylogenetic data. FINDINGS: M tuberculosis clinical strains had diverse growth characteristics in host-like metabolic and drug conditions. These fitness phenotypes were highly heritable, and we identified monogenic and phylogenetically linked variants associated with the fitness phenotypes. These data enabled us to define two genetic features that were associated with clinical outcomes. First, mutations in Rv1339, a phosphodiesterase, which were associated with slow growth in glycerol, were further associated with treatment failure (OR 5·34, 95% CI 1·21-23·58, p=0·027). Second, we identified a phenotypically distinct slow-growing subclade of lineage 1 strains (L1.1.1.1) that was associated with cavitary disease (OR 2·49, 1·11-5·59, p=0·027) and treatment failure (OR 4·76, 1·53-14·78, p=0·0069), and which had shorter terminal branch lengths on the phylogenetic tree, suggesting increased transmission. INTERPRETATION: Slow growth under various antibiotic and metabolic conditions served as in-vitro intermediate phenotypes underlying the association between M tuberculosis monogenic and phylogenetically linked mutations and outcomes such as cavitary disease, treatment failure, and transmission potential. These data suggest that M tuberculosis growth regulation is an adaptive advantage for bacterial success in human populations, at least in some circumstances. These data further suggest markers for the underlying bacterial processes that contribute to these clinical outcomes. FUNDING: National Health and Medical Research Council/A∗STAR, National Institutes of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the Wellcome Trust Fellowship in Public Health and Tropical Medicine.

CD4 T cells and CD8α+ lymphocytes are necessary for intravenous BCG-induced protection against tuberculosis in macaques.

Tuberculosis (TB) is a major cause of morbidity and mortality worldwide despite widespread intradermal (ID) BCG vaccination in newborns. We previously demonstrated that changing the route and dose of BCG vaccination from 5×105 CFU ID to 5×107 CFU intravenous (IV) resulted in prevention of infection and disease in a rigorous, highly susceptible non-human primate model of TB. Identifying the immune mechanisms of protection for IV BCG will facilitate development of more effective vaccines against TB. Here, we depleted select lymphocyte subsets in IV BCG vaccinated macaques prior to Mtb challenge to determine the cell types necessary for that protection. Depletion of CD4 T cells or all CD8α expressing lymphoycytes (both innate and adaptive) resulted in loss of protection in most macaques, concomitant with increased bacterial burdens (~4-5 log10 thoracic CFU) and dissemination of infection. In contrast, depletion of only adaptive CD8αß+ T cells did not significantly reduce protection against disease. Our results demonstrate that CD4 T cells and innate CD8α+ lymphocytes are critical for IV BCG-induced protection, supporting investigation of how eliciting these cells and their functions can improve future TB vaccines.

The mutation rate of mycobacterial repetitive unit loci in strains of M. tuberculosis from cynomolgus macaque infection.

BACKGROUND: Mycobacterial interspersed repetitive units (MIRUs) are minisatellites within the Mycobacterium tuberculosis (Mtb) genome. Copy number variation (CNV) in MIRU loci is used for epidemiological typing, making the rate of variation important for tracking the transmission of Mtb strains. In this study, we developed and assessed a whole-genome sequencing (WGS) approach to detect MIRU CNV in Mtb. We applied this methodology to a panel of Mtb strains isolated from the macaque model of tuberculosis (TB), the animal model that best mimics human disease. From these data, we have estimated the rate of MIRU variation in the host environment, providing a benchmark rate for future epidemiologic work. RESULTS: We assessed variation at the 24 MIRU loci used for typing in a set of Mtb strains isolated from infected cynomolgus macaques. We previously performed WGS of these strains and here have applied both read depth (RD) and paired-end mapping (PEM) metrics to identify putative copy number variants. To assess the relative power of these approaches, all MIRU loci were resequenced using Sanger sequencing. We detected two insertion/deletion events both of which could be identified as candidates by PEM criteria. With these data, we estimate a MIRU mutation rate of 2.70 × 10-03 (95% CI: 3.30 × 10-04- 9.80 × 10-03) per locus, per year. CONCLUSION: Our results represent the first experimental estimate of the MIRU mutation rate in Mtb. This rate is comparable to the highest previous estimates gathered from epidemiologic data and meta-analyses. Our findings allow for a more rigorous interpretation of data gathered from MIRU typing.

Coherent neocortical 40-Hz oscillations are not present during REM sleep.

During cognitive processes there are extensive interactions between various regions of the cerebral cortex. Oscillations in the gamma frequency band (≈40 Hz) of the electroencephalogram (EEG) are involved in the binding of spatially separated but temporally correlated neural events, which results in a unified perceptual experience. The extent of these interactions can be examined by means of a mathematical algorithm called 'coherence', which reflects the 'strength' of functional interactions between cortical areas. The present study was conducted to analyse EEG coherence in the gamma frequency band of the cat during alert wakefulness (AW), quiet wakefulness (QW), non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep. Cats were implanted with electrodes in the frontal, parietal and occipital cortices to monitor EEG activity. Coherence values within the gamma frequency (30-100 Hz) from pairs of EEG recordings were analysed. A large increase in coherence occurred between all cortical regions in the 30-45 Hz frequency band during AW compared with the other behavioral states. As the animal transitioned from AW to QW and from QW to NREM sleep, coherence decreased to a moderate level. Remarkably, there was practically no EEG coherence in the entire gamma band spectrum (30-100 Hz) during REM sleep. We conclude that functional interactions between cortical areas are radically different during sleep compared with wakefulness. The virtual absence of gamma frequency coherence during REM sleep may underlie the unique cognitive processing that occurs during dreams, which is principally a REM sleep-related phenomenon.

High-throughput phenogenotyping of Mycobacteria tuberculosis clinical strains reveals bacterial determinants of treatment outcomes.

Background: Combatting the tuberculosis (TB) epidemic caused by Mycobacterium tuberculosis ( Mtb ) necessitates a better understanding of the factors contributing to patient clinical outcomes and transmission. While host and environmental factors have been evaluated, the impact of Mtb genetic background and phenotypic diversity is underexplored. Previous work has made associations between Mtb genetic lineages and some clinical and epidemiological features, but the bacterial traits underlying these connections are largely unknown. Methods: We developed a high-throughput functional genomics platform for defining genotype-phenotype relationships across a panel of Mtb clinical isolates. These phenotypic fitness profiles function as intermediate traits which can be linked to Mtb genetic variants and associated with clinical and epidemiological outcomes. We applied this approach to a collection of 158 Mtb strains from a study of Mtb transmission in Ho Chi Minh City, Vietnam. Mtb strains were genetically tagged in multiplicate, which allowed us to pool the strains and assess in vitro competitive fitness using deep sequencing across a set of 14 host-relevant antibiotic and metabolic conditions. Phylogenetic and monogenic associations with these intermediate traits were identified and then associated with clinical outcomes. Findings: Mtb clinical strains have a broad range of growth and drug response dynamics that can be clustered by their phylogenetic relationships. We identified novel monogenic associations with Mtb fitness in various metabolic and antibiotic conditions. Among these, we find that mutations in Rv1339 , a phosphodiesterase, which were identified through their association with slow growth in glycerol, are further associated with treatment failure. We also identify a previously uncharacterized subclade of Lineage 1 strains (L1.1.1.1) that is phenotypically distinguished by slow growth under most antibiotic and metabolic stress conditions in vitro . This clade is associated with cavitary disease, treatment failure, and demonstrates increased transmission potential. Interpretation: High-throughput phenogenotyping of Mtb clinical strains enabled bacterial intermediate trait identification that can provide a mechanistic link between Mtb genetic variation and patient clinical outcomes. Mtb strains associated with cavitary disease, treatment failure, and transmission potential display intermediate phenotypes distinguished by slow growth under various antibiotic and metabolic conditions. These data suggest that Mtb growth regulation is an adaptive advantage for host bacterial success in human populations, in at least some circumstances. These data further suggest markers for the underlying bacterial processes that govern these clinical outcomes. Funding: National Institutes of Allergy and Infectious Diseases: P01 AI132130 (SS, SMF); P01 AI143575 (XW, SMF); U19 AI142793 (QL, SMF); 5T32AI132120-03 (SS); 5T32AI132120-04 (SS); 5T32AI049928-17 (SS) Wellcome Trust Fellowship in Public Health and Tropical Medicine: 097124/Z/11/Z (NTTT) National Health and Medical Research Council (NHMRC)/A*STAR joint call: APP1056689 (SJD) The funding sources had no involvement in study methodology, data collection, analysis, and interpretation nor in the writing or submission of the manuscript. Research in context: Evidence before this study: We used different combinations of the words mycobacterium tuberculosis, tuberculosis, clinical strains, intermediate phenotypes, genetic barcoding, phenogenomics, cavitary disease, treatment failure, and transmission to search the PubMed database for all studies published up until January 20 th , 2022. We only considered English language publications, which biases our search. Previous work linking Mtb determinants to clinical or epidemiological data has made associations between bacterial lineage, or less frequently, genetic polymorphisms to in vitro or in vivo models of pathogenesis, transmission, and clinical outcomes such as cavitary disease, treatment failure, delayed culture conversion, and severity. Many of these studies focus on the global pandemic Lineage 2 and Lineage 4 Mtb strains due in part to a deletion in a polyketide synthase implicated in host-pathogen interactions. There are a number of Mtb GWAS studies that have led to novel genetic determinants of in vitro drug resistance and tolerance. Previous Mtb GWAS analyses with clinical outcomes did not experimentally test any predicted phenotypes of the clinical strains. Published laboratory-based studies of Mtb clinical strains involve relatively small numbers of strains, do not identify the genetic basis of relevant phenotypes, or link findings to the corresponding clinical outcomes. There are two recent studies of other pathogens that describe phenogenomic analyses. One study of 331 M. abscessus clinical strains performed one-by-one phenotyping to identify bacterial features associated with clearance of infection and another details a competition experiment utilizing three barcoded Plasmodium falciparum clinical isolates to assay antimalarial fitness and resistance. Added value of this study: We developed a functional genomics platform to perform high-throughput phenotyping of Mtb clinical strains. We then used these phenotypes as intermediate traits to identify novel bacterial genetic features associated with clinical outcomes. We leveraged this platform with a sample of 158 Mtb clinical strains from a cross sectional study of Mtb transmission in Ho Chi Minh City, Vietnam. To enable high-throughput phenotyping of large numbers of Mtb clinical isolates, we applied a DNA barcoding approach that has not been previously utilized for the high-throughput analysis of Mtb clinical strains. This approach allowed us to perform pooled competitive fitness assays, tracking strain fitness using deep sequencing. We measured the replicative fitness of the clinical strains in multiplicate under 14 metabolic and antibiotic stress condition. To our knowledge, this is the largest phenotypic screen of Mtb clinical isolates to date. We performed bacterial GWAS to delineate the Mtb genetic variants associated with each fitness phenotype, identifying monogenic associations with several conditions. We then defined Mtb phenotypic and genetic features associated with clinical outcomes. We find that a subclade of Mtb strains, defined by variants largely involved in fatty acid metabolic pathways, share a universal slow growth phenotype that is associated with cavitary disease, treatment failure and increased transmission potential in Vietnam. We also find that mutations in Rv1339 , a poorly characterized phosphodiesterase, also associate with slow growth in vitro and with treatment failure in patients. Implications of all the available evidence: Phenogenomic profiling demonstrates that Mtb strains exhibit distinct growth characteristics under metabolic and antibiotic stress conditions. These fitness profiles can serve as intermediate traits for GWAS and association with clinical outcomes. Intermediate phenotyping allows us to examine potential processes by which bacterial strain differences contribute to clinical outcomes. Our study identifies clinical strains with slow growth phenotypes under in vitro models of antibiotic and host-like metabolic conditions that are associated with adverse clinical outcomes. It is possible that the bacterial intermediate phenotypes we identified are directly related to the mechanisms of these outcomes, or they may serve as markers for the causal yet unidentified bacterial determinants. Via the intermediate phenotyping, we also discovered a surprising diversity in Mtb responses to the new anti-mycobacterial drugs that target central metabolic processes, which will be important in considering roll-out of these new agents. Our study and others that have identified Mtb determinants of TB clinical and epidemiological phenotypes should inform efforts to improve diagnostics and drug regimen design.

CD8+ lymphocytes are critical for early control of tuberculosis in macaques.

The functional role of CD8+ lymphocytes in tuberculosis remains poorly understood. We depleted innate and/or adaptive CD8+ lymphocytes in macaques and showed that loss of all CD8α+ cells (using anti-CD8α antibody) significantly impaired early control of Mycobacterium tuberculosis (Mtb) infection, leading to increased granulomas, lung inflammation, and bacterial burden. Analysis of barcoded Mtb from infected macaques demonstrated that depletion of all CD8+ lymphocytes allowed increased establishment of Mtb in lungs and dissemination within lungs and to lymph nodes, while depletion of only adaptive CD8+ T cells (with anti-CD8ß antibody) worsened bacterial control in lymph nodes. Flow cytometry and single-cell RNA sequencing revealed polyfunctional cytotoxic CD8+ lymphocytes in control granulomas, while CD8-depleted animals were unexpectedly enriched in CD4 and γδ T cells adopting incomplete cytotoxic signatures. Ligand-receptor analyses identified IL-15 signaling in granulomas as a driver of cytotoxic T cells. These data support that CD8+ lymphocytes are required for early protection against Mtb and suggest polyfunctional cytotoxic responses as a vaccine target.

Annotation of Secretariat and Hydrus, two DJ cluster phages isolated on Gordonia rubripertincta.

Secretariat and Hydrus are phages grouped into the DJ cluster that were isolated on Gordonia rubripertincta NRRL B-16540. The phages have 75% nucleotide identity and share 73% gene content. Secretariat has a genome with 84 predicted genes, while Hydrus has 91 predicted genes and can also infect Gordonia terrae 3612.

Behavioral responses of terrestrial mammals to COVID-19 lockdowns.

COVID-19 lockdowns in early 2020 reduced human mobility, providing an opportunity to disentangle its effects on animals from those of landscape modifications. Using GPS data, we compared movements and road avoidance of 2300 terrestrial mammals (43 species) during the lockdowns to the same period in 2019. Individual responses were variable with no change in average movements or road avoidance behavior, likely due to variable lockdown conditions. However, under strict lockdowns 10-day 95th percentile displacements increased by 73%, suggesting increased landscape permeability. Animals' 1-hour 95th percentile displacements declined by 12% and animals were 36% closer to roads in areas of high human footprint, indicating reduced avoidance during lockdowns. Overall, lockdowns rapidly altered some spatial behaviors, highlighting variable but substantial impacts of human mobility on wildlife worldwide.

EspA acts as a critical mediator of ESX1-dependent virulence in Mycobacterium tuberculosis by affecting bacterial cell wall integrity.

Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall.