Design and proof of concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain.

Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these "dual-display" phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.

Adjunctive Host-Directed Therapy With Statins Improves Tuberculosis-Related Outcomes in Mice.

BACKGROUND: Tuberculosis (TB) treatment is lengthy and complicated and patients often develop chronic lung disease. Recent attention has focused on host-directed therapies aimed at optimizing immune responses to Mycobacterium tuberculosis (Mtb), as adjunctive treatment given with antitubercular drugs. In addition to their cholesterol-lowering properties, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have broad anti-inflammatory and immunomodulatory activities. METHODS: In the current study, we screened 8 commercially available statins for cytotoxic effect, anti-TB activity, synergy with first-line drugs in macrophages, pharmacokinetics and adjunctive bactericidal activity, and, in 2 different mouse models, as adjunctive therapy to first-line TB drugs. RESULTS: Pravastatin showed the least toxicity in THP-1 and Vero cells. At nontoxic doses, atorvastatin and mevastatin were unable to inhibit Mtb growth in THP-1 cells. Simvastatin, fluvastatin, and pravastatin showed the most favorable therapeutic index and enhanced the antitubercular activity of the first-line drugs isoniazid, rifampin, and pyrazinamide in THP-1 cells. Pravastatin modulated phagosomal maturation characteristics in macrophages, phenocopying macrophage activation, and exhibited potent adjunctive activity in the standard mouse model of TB chemotherapy and in a mouse model of human-like necrotic TB lung granulomas. CONCLUSIONS: These data provide compelling evidence for clinical evaluation of pravastatin as adjunctive, host-directed therapy for TB.

The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Diagnostic Performance of Tuberculosis-Specific IgG Antibody Profiles in Patients with Presumptive Tuberculosis from Two Continents.

BACKGROUND: Development of rapid diagnostic tests for tuberculosis is a global priority. A whole proteome screen identified Mycobacterium tuberculosis antigens associated with serological responses in tuberculosis patients. We used World Health Organization (WHO) target product profile (TPP) criteria for a detection test and triage test to evaluate these antigens. METHODS: Consecutive patients presenting to microscopy centers and district hospitals in Peru and to outpatient clinics at a tuberculosis reference center in Vietnam were recruited. We tested blood samples from 755 HIV-uninfected adults with presumptive pulmonary tuberculosis to measure IgG antibody responses to 57 M. tuberculosis antigens using a field-based multiplexed serological assay and a 132-antigen bead-based reference assay. We evaluated single antigen performance and models of all possible 3-antigen combinations and multiantigen combinations. RESULTS: Three-antigen and multiantigen models performed similarly and were superior to single antigens. With specificity set at 90% for a detection test, the best sensitivity of a 3-antigen model was 35% (95% confidence interval [CI], 31-40). With sensitivity set at 85% for a triage test, the specificity of the best 3-antigen model was 34% (95% CI, 29-40). The reference assay also did not meet study targets. Antigen performance differed significantly between the study sites for 7/22 of the best-performing antigens. CONCLUSIONS: Although M. tuberculosis antigens were recognized by the IgG response during tuberculosis, no single antigen or multiantigen set performance approached WHO TPP criteria for clinical utility among HIV-uninfected adults with presumed tuberculosis in high-volume, urban settings in tuberculosis-endemic countries.

Non-monotonic Response to Monotonic Stimulus: Regulation of Glyoxylate Shunt Gene-Expression Dynamics in Mycobacterium tuberculosis.

Understanding how dynamical responses of biological networks are constrained by underlying network topology is one of the fundamental goals of systems biology. Here we employ monotone systems theory to formulate a theorem stating necessary conditions for non-monotonic time-response of a biochemical network to a monotonic stimulus. We apply this theorem to analyze the non-monotonic dynamics of the σB-regulated glyoxylate shunt gene expression in Mycobacterium tuberculosis cells exposed to hypoxia. We first demonstrate that the known network structure is inconsistent with observed dynamics. To resolve this inconsistency we employ the formulated theorem, modeling simulations and optimization along with follow-up dynamic experimental measurements. We show a requirement for post-translational modulation of σB activity in order to reconcile the network dynamics with its topology. The results of this analysis make testable experimental predictions and demonstrate wider applicability of the developed methodology to a wide class of biological systems.

Statin adjunctive therapy shortens the duration of TB treatment in mice.

BACKGROUND: The repurposing of existing agents may accelerate TB drug development. Recently, we reported that the lipid-lowering drug simvastatin, when added to the first-line antitubercular regimen, reduces the lung bacillary burden in chronically infected mice. OBJECTIVES: We investigated whether the addition of simvastatin to the first-line regimen (isoniazid/rifampicin/pyrazinamide) shortens the duration of curative TB treatment in mice. METHODS: Mycobacterium tuberculosis-infected THP-1 cells were exposed to simvastatin to determine the effect of statins on the activity of first-line anti-TB drug activity and intracellular rifampicin concentration. Single-dose and steady-state pharmacokinetic studies guided optimized simvastatin dosing in vivo. BALB/c mice were aerosol-infected with M. tuberculosis H37Rv and drug treatment was initiated 6 weeks post-infection. Separate groups of mice received standard TB treatment with or without simvastatin. Relapse rates were assessed 3 months after discontinuation of each treatment regimen. MALDI-MS imaging was used to image the cholesterol content of mouse lung lesions. RESULTS: Simvastatin significantly enhanced the bactericidal activity of first-line drugs against intracellular M. tuberculosis without altering intracellular rifampicin concentrations. Adjunctive treatment with 60 mg/kg simvastatin shortened the time required to achieve culture-negative lungs from 4.5 to 3.5 months. Following 2.5, 3.5 and 4.5 months of treatment, relapse rates were 100%, 50% and 0%, respectively, in the control group and 50% (P = 0.03), 20% and 0%, respectively, in the statin group. Simvastatin did not alter plasma or lung lesion cholesterol levels. CONCLUSIONS: Statins are attractive candidates for host-directed, adjunctive TB therapy. Further preclinical studies are needed to define the optimal statin and dosing.

Simvastatin increases the in vivo activity of the first-line tuberculosis regimen.

BACKGROUND: The need to develop new, improved treatments for tuberculosis (TB) remains urgent, and the repurposing of existing drugs represents a possible shortcut to market. Recently, there has been significant interest in host-directed adjuvant therapy to enhance bacillary killing. HMG-CoA reductase inhibitors (statins), which are among the most commonly prescribed drugs, have immunomodulatory properties and improve the clinical outcomes of bacterial infections. METHODS: We studied the tuberculocidal activity of simvastatin alone and in combination with first-line anti-TB drugs in J774 macrophages and during chronic TB infection. RESULTS: Exposure to 5 µM simvastatin significantly increased the tuberculocidal activity of isoniazid in J774 macrophages at Day 3 after infection versus isoniazid alone (P=0.02). Similarly, relative to the standard oral regimen of rifampicin (10 mg/kg), isoniazid (10 mg/kg) and pyrazinamide (150 mg/kg) given five times weekly, the addition of 25 mg/kg simvastatin enhanced bacillary killing, reducing the number of lung cfu by an additional 1 log10 at Day 28 (P<0.01) and by a further 1.25 log10 at Day 56 (P<0.01). CONCLUSIONS: The potential additive activity of simvastatin to first-line TB treatment holds promise. However, further studies to identify the optimal statin and dosing are required. In addition the ability of combination treatment with statins to accelerate the time required to achieve a stable cure remains to be explored.

Dynamic antibody responses to the Mycobacterium tuberculosis proteome.

Considerable effort has been directed toward controlling tuberculosis, which kills almost two million people yearly. High on the research agenda is the discovery of biomarkers of active tuberculosis (TB) for diagnosis and for monitoring treatment outcome. Rational biomarker discovery requires understanding host-pathogen interactions leading to biomarker expression. Here we report a systems immunology approach integrating clinical data and bacterial metabolic and regulatory information with high-throughput detection in human serum of antibodies to the entire Mycobacterium tuberculosis proteome. Sera from worldwide TB suspects recognized approximately 10% of the bacterial proteome. This result defines the M. tuberculosis immunoproteome, which is rich in membrane-associated and extracellular proteins. Additional analyses revealed that during active tuberculosis (i) antibody responses focused on an approximately 0.5% of the proteome enriched for extracellular proteins, (ii) relative target preference varied among patients, and (iii) responses correlated with bacillary burden. These results indicate that the B cell response tracks the evolution of infection and the pathogen burden and replicative state and suggest functions associated with B cell-rich foci seen in tuberculous lung granulomas. Our integrated proteome-scale approach is applicable to other chronic infections characterized by diverse antibody target recognition.

Proteome-scale antibody responses and outcome of Mycobacterium tuberculosis infection in nonhuman primates and in tuberculosis patients.

BACKGROUND: Biomarkers of progression from latent Mycobacterium tuberculosis infection to active tuberculosis are needed. We assessed correlations between infection outcome and antibody responses in macaques and humans by high-throughput, proteome-scale serological studies. METHODS: Mycobacterium tuberculosis proteome microarrays were probed with serial sera from macaques representing various infection outcomes and with single-point human sera from tuberculosis suspects. Fluorescence intensity data were analyzed by calculating Z scores and associated P values. Temporal changes in macaque antibody responses were analyzed by polynomial regression. Correlations between human responses and sputum bacillary burden were assessed by quantile and hurdle regression. RESULTS: Macaque outcome groups exhibited distinct antibody profiles: early, transient responses in latent infection and stable antibody increase in active and reactivation disease. In humans, antibody levels and reactive protein numbers increased with bacillary burden. Responses to a subset of 10 proteins were more tightly associated with disease state than reactivity to the broader reactive proteome. CONCLUSIONS: Integration of macaque and human data reveals dynamic properties of antibody responses in relation to outcome and leads to actionable findings for translational research. These include the potential of antibody responses to detect acute infection and preclinical tuberculosis and to identify serodiagnostic proteins for the spectrum of bacillary burden in tuberculosis.

Prevalence and Correlates of SARS CoV-2 Among a Community-Based Sample Recruited Using Randomized Venue-Based Sampling. Essex County, NJ, 2020.

Disparities in morbidity and mortality related to COVID-19 based on race and ethnicity have been documented in the USA. However, it is unclear if these disparities also exist at the exposure stage. To determine this, studies are needed to document the underlying burden of disease, potential disparities through serologic surveillance. Additionally, such studies can help identify where along the disease spectrum (e.g., exposure, infection, diagnosis, treatment, death) and with regard to the structural factors that necessitate public health and/or clinical interventions. Our objectives in this study were to estimate the true burden of SARS CoV-2 in the community of Essex County, NJ, an early and hard hit area, to determine the correlates of SARS CoV-2 prevalence and to determine if COVID-19 disparities seen by race/ethnicity were also reflected in SARS CoV-2 burden. We utilized venue-based-sampling (VBS) to sample members of the community in Essex County. Participants completed a short electronic survey and provided finger stick blood samples for testing. We sampled 924 residents of Essex County, New Jersey. Testing conducted in this study identified 83 (9.0%) participants as positive for SARS-CoV-2 antibodies. Importantly, our findings suggest that the true burden of SARS-Cov-2 and the pool of persons potentially spreading the virus are slightly more than six times than that suggested by PCR testing Notably, there were no significant differences in odds of testing positive for SARS CoV-2 antibodies in terms of race/ethnicity where we compared Black and Latinx participants to other race participants. Our study suggests that disparities in COVID-19 outcomes stem from potential upstream issues such as underlying conditions, access to testing, and access to care rather than disparities in exposure to the virus.

Carbon flux rerouting during Mycobacterium tuberculosis growth arrest.

A hallmark of the Mycobacterium tuberculosis life cycle is the pathogen's ability to switch between replicative and non-replicative states in response to host immunity. Transcriptional profiling by qPCR of ∼ 50 M. tuberculosis genes involved in central and lipid metabolism revealed a re-routing of carbon flow associated with bacterial growth arrest during mouse lung infection. Carbon rerouting was marked by a switch from metabolic pathways generating energy and biosynthetic precursors in growing bacilli to pathways for storage compound synthesis during growth arrest. Results of flux balance analysis using an in silico metabolic network were consistent with the transcript abundance data obtained in vivo. Similar transcriptional changes were seen in vitro when M. tuberculosis cultures were treated with bacteriostatic stressors under different nutritional conditions. Thus, altered expression of key metabolic genes reflects growth rate changes rather than changes in substrate availability. A model describing carbon flux rerouting was formulated that (i) provides a coherent interpretation of the adaptation of M. tuberculosis metabolism to immunity-induced stress and (ii) identifies features common to mycobacterial dormancy and stress responses of other organisms.

Design and proof-of-concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain.

Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. SIGNIFICANCE: The ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.

Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Is Comparable in Clinical Samples Preserved in Saline or Viral Transport Medium.

As the coronavirus disease 2019 (COVID-19) pandemic sweeps across the world, the availability of viral transport medium (VTM) has become severely limited, contributing to delays in diagnosis and rationing of diagnostic testing. Given that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA has demonstrated stability, we posited that phosphate-buffered saline (PBS) may be a viable transport medium, as an alternative to VTM, for clinical real-time quantitative PCR (qPCR) testing. The intra-individual reliability and interindividual reliability of SARS-CoV-2 qPCR were assessed in clinical endotracheal secretion samples transported in VTM or PBS to evaluate the stability of the qPCR signal for three viral targets (N gene, ORF1ab, and S gene) when samples were stored in these media at room temperature for up to 18 hours. We report that the use of PBS as a transport medium allows high intra-individual and interindividual reliability, maintains viral stability, and compares with VTM in the detection of the three SARS-CoV-2 genes through 18 hours of storage. This study establishes PBS as a clinically useful medium that can be readily deployed for transporting and short-term preservation of specimens containing SARS-CoV-2. Use of PBS as a transport medium has the potential to increase testing capacity for SARS-CoV-2, aiding more widespread screening and early diagnosis of COVID-19.

The temporal response of the Mycobacterium tuberculosis gene regulatory network during growth arrest.

The virulence of Mycobacterium tuberculosis depends on the ability of the bacilli to switch between replicative (growth) and non-replicative (dormancy) states in response to host immunity. However, the gene regulatory events associated with transition to dormancy are largely unknown. To address this question, we have assembled the largest M. tuberculosis transcriptional-regulatory network to date, and characterized the temporal response of this network during adaptation to stationary phase and hypoxia, using published microarray data. Distinct sets of transcriptional subnetworks (origons) were responsive at various stages of adaptation, showing a gradual progression of network response under both conditions. Most of the responsive origons were in common between the two conditions and may help define a general transcriptional signature of M. tuberculosis growth arrest. These results open the door for a systems-level understanding of transition to non-replicative persistence, a phenotypic state that prevents sterilization of infection by the host immune response and promotes the establishment of latent M. tuberculosis infection, a condition found in two billion people worldwide.

Transcriptional characterization of the antioxidant response of Mycobacterium tuberculosis in vivo and during adaptation to hypoxia in vitro.

Transcriptional profiling of antioxidant genes of Mycobacterium tuberculosis was performed by real-time RT-PCR during mouse lung infection and during adaptation to gradual oxygen depletion in vitro. M. tuberculosis genes involved in major detoxification pathways of oxidative stress were not up-regulated during chronic mouse lung infection, which is established in response to expression of host adaptive immunity. This result suggests that a major function of bacterial antioxidant enzymes is to protect from oxidants generated during the early, acute phase of infection. In vivo transcription profiles of bacterial antioxidant enzymes differed from those seen under adaptation to low oxygen in vitro, indicating differences between growth arrest in vivo and that induced by hypoxia in vitro.

Role of Alanine Dehydrogenase of Mycobacterium tuberculosis during Recovery from Hypoxic Nonreplicating Persistence.

Mycobacterium tuberculosis can maintain a nonreplicating persistent state in the host for decades, but must maintain the ability to efficiently reactivate and produce active disease to survive and spread in a population. Among the enzymes expressed during this dormancy is alanine dehydrogenase, which converts pyruvate to alanine, and glyoxylate to glycine concurrent with the oxidation of NADH to NAD. It is involved in the metabolic remodeling of M. tuberculosis through its possible interactions with both the glyoxylate and methylcitrate cycle. Both mRNA levels and enzymatic activities of isocitrate lyase, the first enzyme of the glyoxylate cycle, and alanine dehydrogenase increased during entry into nonreplicating persistence, while the gene and activity for the second enzyme of the glyoxylate cycle, malate synthase were not. This could suggest a shift in carbon flow away from the glyoxylate cycle and instead through alanine dehydrogenase. Expression of ald was also induced in vitro by other persistence-inducing stresses such as nitric oxide, and was expressed at high levels in vivo during the initial lung infection in mice. Enzyme activity was maintained during extended hypoxia even after transcription levels decreased. An ald knockout mutant of M. tuberculosis showed no reduction in anaerobic survival in vitro, but resulted in a significant lag in the resumption of growth after reoxygenation. During reactivation the ald mutant had an altered NADH/NAD ratio, and alanine dehydrogenase is proposed to maintain the optimal NADH/NAD ratio during anaerobiosis in preparation of eventual regrowth, and during the initial response during reoxygenation.

Infection with Mycobacterium tuberculosis induces the Warburg effect in mouse lungs.

To elucidate the little-known bioenergetic pathways of host immune cells in tuberculosis, a granulomatous disease caused by the intracellular pathogen Mycobacterium tuberculosis, we characterized infected murine lung tissue by transcriptomic profiling and confocal imaging. Transcriptomic analysis revealed changes of host energy metabolism during the course of infection that are characterized by upregulation of key glycolytic enzymes and transporters for glucose uptake, and downregulation of enzymes participating in the tricarboxylic acid cycle and oxidative phosphorylation. Consistent with elevated glycolysis, we also observed upregulation of a transporter for lactate secretion and a V type H(+) -ATPase involved in cytosolic pH homeostasis. Transcription profiling results were corroborated by immunofluorescence microscopy showing increased expression of key glycolytic enzymes in macrophages and T cells in granulomatous lesions. Moreover, we found increased mRNA and protein levels in macrophages and T cells of hypoxia inducible factor 1 alpha (HIF-1α), the regulatory subunit of HIF-1, a master transcriptional regulator. Thus, our findings suggest that immune cells predominantly utilize aerobic glycolysis in response to M. tuberculosis infection. This bioenergetic shift is similar to the Warburg effect, the metabolic signature of cancer cells. Finding immunometabolic changes during M. tuberculosis infection opens the way to new strategies for immunotherapy against tuberculosis.

Genome-wide discovery of small RNAs in Mycobacterium tuberculosis.

Only few small RNAs (sRNAs) have been characterized in Mycobacterium tuberculosis and their role in regulatory networks is still poorly understood. Here we report a genome-wide characterization of sRNAs in M. tuberculosis integrating experimental and computational analyses. Global RNA-seq analysis of exponentially growing cultures of M. tuberculosis H37Rv had previously identified 1373 sRNA species. In the present report we show that 258 (19%) of these were also identified by microarray expression. This set included 22 intergenic sRNAs, 84 sRNAs mapping within 5'/3' UTRs, and 152 antisense sRNAs. Analysis of promoter and terminator consensus sequences identified sigma A promoter consensus sequences for 121 sRNAs (47%), terminator consensus motifs for 22 sRNAs (8.5%), and both motifs for 35 sRNAs (14%). Additionally, 20/23 candidates were visualized by Northern blot analysis and 5' end mapping by primer extension confirmed the RNA-seq data. We also used a computational approach utilizing functional enrichment to identify the pathways targeted by sRNA regulation. We found that antisense sRNAs preferentially regulated transcription of membrane-bound proteins. Genes putatively regulated by novel cis-encoded sRNAs were enriched for two-component systems and for functional pathways involved in hydrogen transport on the membrane.