Importance: Risk factors associated with COVID-19, the viral pneumonia originating in Wuhan, China, in Dec 2019, require clarification so that medical resources can be prioritized for those at highest risk of severe COVID-19 complications. Infection with M. tuberculosis (MTB), the pathogen that causes TB and latently infects ∼25% of the global population, may be a risk factor for SARS-CoV-2 infection and severe COVID-19 pneumonia. Objective: To determine if latent or active TB increase susceptibility to SARS-COV-19 infection and disease severity, and lead to more rapid development of COVID-19 pneumonia. Design: An observational case-control study of 36 confirmed COVID-19 cases from Shenyang, China, conducted in Feb 2020. Final date of follow-up: Feb 29, 2020. Cases were grouped according to COVID-19 pneumonia severity (mild/moderate, severe/critical), and MTB infection status compared. Comparisons were made with MTB infection data from another case-control study on bacterial/viral pneumonia at Shenyang Chest Hospital. Setting: Multi-center study involving three primary care hospitals in Shenyang, China. Participants: 86 suspected COVID-19 cases from participating primary-care hospitals in Shenyang. All 36 SARS-CoV-2 +ve cases (based on RT-PCR assay) were included. Disease severity was assessed using the Diagnostic and Treatment Guidelines of the National Health Commission of China (v6). Mean age, 47 years (range: 25-79), gender ratio, 1:1. Exposures: Confirmed COVID-19 pneumonia. Interferon-gamma Release Assays (IGRA) were performed using peripheral blood to determine MTB infection. Main Outcome and Measures: Epidemiological, demographic, clinical, radiological, and laboratory data were collected. Comparison of MTB infection status between patients with mild/moderate and severe/critical COVID-19 pneumonia. Results: Mean age of 36 COVID-19 patients: 47 (range: 25-79); M/F: 18/18; Wuhan/Hubei connection: 42%. Mild/moderate cases: 27 (75%); severe/critical: 9 (25%). MTB infection (IGRA+ve): 13 cases (36.11%), including 7 of 9 severe/critical cases. MTB infection rate: higher in COVID-19 (36.11%) than bacterial pneumonia (20%; p=0.0047) and viral pneumonia patients (16.13%; p=0.024). MTB infection more common than other co-morbidities (36.11% vs diabetes: 25%; hypertension: 22.2%; coronary heart disease: 8.33%; COPD: 5.56%). MTB co-infection linked with disease severity (severe/critical 78% vs mild/moderate cases 22%; p=0.0049), and rate of disease progression: infection to development of symptoms (MTB+SARS-CoV-2: 6.5±4.2 days vs SARS-COV-2: 8.9±5.2 days; p=0.073); from symptom development to diagnosed as severe (MTB+SARS-CoV-2: 3.4±2.0 days vs SARS-COV-2: 7.5±0.5 days; p=0.075). Conclusions and Relevance: MTB infection likely increases susceptibility to SARS-CoV-2, and increases COVID-19 severity, but this requires validation in a larger study. MTB infection status of COVID-19 patients should be checked routinely at hospital admission. Key Points: Question: Is latent or active tuberculosis (TB) a risk factor for SARS-CoV-19 infection and progression to severe COVID-19 pneumonia? Findings: In this observational case-control study of 36 COVID-19 cases from Shenyang, China, we found tuberculosis history (both of active TB and latent TB) to be an important risk factor for SARS-CoV-2 infection. Patients with active or latent TB were more susceptible to SARS-CoV-2, and COVID-19 symptom development and progression were more rapid and severe. Meaning: Tuberculosis status should be assessed carefully at patient admission and management and therapeutic strategies adjusted accordingly to prevent rapid development of severe COVID-19 complications.

Tuberculosis (TB) remains a significant human health issue. More effective biomarkers for use in tuberculosis prevention, diagnosis, and treatment, including markers that can discriminate between healthy individuals and those with latent infection, are urgently needed. To identify a set of such markers, we used Solexa sequencing to examine microRNA expression in the serum of patients with active disease, healthy individuals with latent TB, and those with or without prior BCG inoculation. We identified 24 microRNAs that are up-regulated (2.85–1285.93 fold) and 6 microRNAs that are down-regulated (0.003–0.11 fold) (P<0.05) in patients with active TB relative to the three groups of healthy controls. In addition, 75 microRNAs were up-regulated (2.05–2454.58 fold) and 11 were down-regulated (0.001–0.42 fold) (P<0.05) in latent-TB infected individuals relative to BCG- inoculated individuals. Of interest, 134 microRNAs were differentially-expressed in BCG-inoculated relative to un-inoculated individuals (18 up-regulated 2.9–499.29 fold, 116 down-regulated 0.0002–0.5 fold), providing insights into the effects of BCG inoculation at the microRNA level. Target prediction of differentially-expressed microRNAs by microRNA-Gene Network analysis and analysis of pathways affected suggest that regulation of the host immune system by microRNAs is likely to be one of the main factors in the pathogenesis of tuberculosis. qRT-PCR validation indicated that hsa-miR-196b and hsa-miR-376c have potential as markers for active TB disease. The microRNA differential-expression profiles generated in this study provide a good foundation for the development of markers for TB diagnosis, and for investigations on the role of microRNAs in BCG-inoculated and latent-infected individuals.

Crystal structure of L,D-transpeptidase Ldt_Mt2 in complex with meropenem reveals the mechanism of carbapenem against Mycobacterium tuberculosis

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis , the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III‐A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA‐seq approaches, we show it is active in invader defense and has features atypical of type III‐A systems: mature CRISPR RNA (crRNA) in its crRNA‐CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3′‐end processing after Cas6 cleavage of repeat RNA 8 nt from its 3′ end. crRNAs generated resemble mature crRNA in type I systems, having both 5′ (8 nt) and 3′ (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3′ hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis , providing a foundation for investigating the potential of a type III‐A–based genome editing system.—Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.‐E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III‐A CRISPR/Cas system crRNA and its maturation have atypical features. FASEB J. 33, 1496–1509 (2019). www.fasebj.org

Pathogenic mycobacteria transport virulence factors across their complex cell wall via a type VII secretion system (T7SS)/early secreted antigenic target‐6 of kDa secretion system (ESX). ESX conserved component (Ecc) B, a core component of the T7SS architecture, is predicted to be a membrane bound protein, but little is known about its structure and function. Here, we characterize EccB1, showing that it is an ATPase with no sequence or structural homology to other ATPases located in the cell envelope of Mycobacterium tuberculosis H37Rv. We obtained the crystal structure of an EccB1‐ΔN72 truncated transmembrane helix and performed modeling and ATP docking studies, showing that EccB1 likely exists as a hexamer. Sequence alignment and ATPase activity determination of EccB1 homologues indicated the presence of 3 conserved motifs in the N‐ and C‐terminals of EccB1‐ΔN72 that assemble together between 2 membrane proximal domains of the EccB1‐ΔN72 monomer. Models of the EccB1 hexamer show that 2 of the conserved motifs are involved in ATPase activity and form an ATP binding pocket located on the surface of 2 adjacent molecules. Our results suggest that EccB may act as the energy provider in the transport of T7SS virulence factors and may be involved in the formation of a channel across the mycomembrane.—Zhang, X.‐L., Li, D.‐F., Fleming, J., Wang, L.‐W., Zhou, Y., Wang, D.‐C., Zhang, X.‐E., Bi, L.‐J. Core component EccB1 of the Mycobacterium tuberculosis type VII secretion system is a periplasmic ATPase. FASEB J. 29, 4804–4814 (2015). www.fasebj.org

MarR family proteins are transcriptional regulators that control expression of bacterial proteins involved in metabolism, virulence, stress responses and multi-drug resistance, mainly via ligand-mediated attenuation of DNA binding. Greater understanding of their underlying regulatory mechanism may open up new avenues for the effective treatment of bacterial infections. To gain molecular insight into the mechanism of Rv2887, a MarR family protein in M. tuberculosis, we first showed that it binds salicylate (SA) and para-aminosalicylic acid (PAS), its structural analogue and an antitubercular drug, in a 1:1 stoichiometry with high affinity. Subsequent determination and analysis of Rv2887 crystal structures in apo form, and in complex with SA, PAS and DNA showed that SA and PAS bind to Rv2887 at similar sites, and that Rv2887 interacts with DNA mainly by insertion of helix α4 into the major groove. Ligand binding triggers rotation of the wHTH domain of Rv2887 toward the dimerization domain, causing changes in protein conformation such that it can no longer bind to a 27 bp recognition sequence in the upstream region of gene Rv0560c. The structures provided here lay a foundation for the design of small molecules that target Rv2887, a potential new approach for the development of anti-mycobacterials.