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Tuberculosis—the world’s leading cause of death by infectious disease—is increasingly resistant to current first-line antibiotics1. The bacterium Mycobacterium tuberculosis (which causes tuberculosis) can survive low-energy conditions, allowing infections to remain dormant and decreasing their susceptibility to many antibiotics2. Bedaquiline was developed in 2005 from a lead compound identified in a phenotypic screen against Mycobacterium smegmatis3. This drug can sterilize even latent M. tuberculosis infections4 and has become a cornerstone of treatment for multidrug-resistant and extensively drug-resistant tuberculosis1,5,6. Bedaquiline targets the mycobacterial ATP synthase3, which is an essential enzyme in the obligate aerobic Mycobacterium genus3,7, but how it binds the intact enzyme is unknown. Here we determined cryo-electron microscopy structures of M. smegmatis ATP synthase alone and in complex with bedaquiline. The drug-free structure suggests that hook-like extensions from the α-subunits prevent the enzyme from running in reverse, inhibiting ATP hydrolysis and preserving energy in hypoxic conditions. Bedaquiline binding induces large conformational changes in the ATP synthase, creating tight binding pockets at the interface of subunits a and c that explain the potency of this drug as an antibiotic for tuberculosis. Structures of Mycobacterium smegmatis ATP synthase provide insights into how the enzyme conserves energy by autoinhibition of ATP hydrolysis and the mechanism of action of bedaquiline, a drug used in treatment of multidrug-resistant tuberculosis.

Summary The type VII secretion system (T7SS) of Mycobacterium tuberculosis secretes three substrate classes: Esx, Esp, and PE/PPE proteins, that play important roles in bacterial physiology and host interaction. Five subtypes of T7SS, namely ESX-1 to ESX-5, are present in M. tb. ESX-4 is the progenitor of T7SS but its function is not understood. We investigated the ESX-4 system in Mycobacterium marinum. We show that ESX-4 of M. marinum does not secrete its cognate substrates, EsxT and EsxU, under the conditions tested. Paradoxically, the deletion of eccC4, an essential component of ESX-4, resulted in elevated secretion of protein substrates of ESX-1 and ESX-5. Consequently, the ΔeccC4 mutant was more efficient in inducing actin cytoskeleton rearrangement, which led to enhanced phagocytosis by macrophages. Our results reveal an intimate crosstalk between the progenitor of T7SS and its more recent duplication and expansion, and provide new insight into the evolution of T7SS in mycobacteria., Graphical abstract, Highlights • ESX-4 of M. marinum does not constitute an active secretion system • Deletion of eccC4 of ESX-4 increases secretion of ESX-1 and ESX-5 substrates • ΔeccC4 is more efficient in inducing cytoskeletal rearrangement of macrophages • Deletion of eccC4 does not affect the virulence of M. marinum in zebrafish, Molecular biology; Microbiology

Bacterial small regulatory RNAs (sRNAs) are the most abundant class of post-transcriptional regulators and have been well studied in Gram-negative bacteria. Little is known about the functions and mechanisms of sRNAs in high GC Gram-positive bacteria including Mycobacterium and Streptomyces. Here, we performed an in-depth study of 6C sRNA of Mycobacterium tuberculosis, which is conserved among high GC Gram-positive bacteria. Forty-seven genes were identified as possible direct targets of 6C sRNA and 15 of them were validated using an in vivo translational lacZ fusion system. We found that 6C sRNA plays a pleotropic role and regulates genes involved in various cellular processes, including DNA replication and protein secretion. Mapping the interactions of 6C sRNA with mRNA targets panD and dnaB revealed that the C-rich loops of 6C sRNA act as direct binding sites to mRNA targets. Unlike in Gram-negative bacteria where RNA binding proteins Hfq and ProQ are required, the interactions of 6C sRNA with mRNAs appear to be independent of RNA chaperones. Our findings suggest that the multiple G–C pairings between single stranded regions are sufficient to establish stable interactions between 6C sRNA and mRNA targets, providing a mechanism for sRNAs in high GC Gram-positive bacteria.

The COVID-19 pandemic has affected more than 120 million people and resulted in over 2.8 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all of the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Here, we describe intranasal immunization of a COVID-19 vaccine delivered by a novel platform, the helper-dependent adenoviral (HD-Ad) vector. Since HD-Ad vectors are devoid of adenoviral coding sequences, they have a superior safety profile and a large cloning capacity for transgenes. The vaccine (HD-Ad_RBD) codes for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and intranasal immunization induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. As such, intranasal immunization based on the HD-Ad vector promises to provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

SummaryTuberculosis (TB), the leading cause of death by infectious disease worldwide, is increasingly resistant to first line antibiotics. Developed from a screen againstMycobacterium smegmatis, bedaquiline can sterilize even latentM. tuberculosisinfections that may otherwise persist for decades and has become a cornerstone of treatment for multidrug resistant and extensively-drug resistant TB. Bedaquiline targets mycobacterial ATP synthase, an essential enzyme in the obligate aerobicMycobacteriumgenus. However, how the drug binds the intact enzyme is unknown. We determined the structure ofM. smegmatisATP synthase with and without bedaquiline. The drug-free structure reveals hook-like extensions from the enzyme’s α subunits that inhibit ATP hydrolysis in low-energy conditions, such as during latent infections. Bedaquiline binding induces global conformational changes in ATP synthase, creating tight binding pockets at the interface of subunits a and c. These binding sites explain the drug’s structure-activity relationship and its potency as an antibiotic for TB.

Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development.