Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.

Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the clp gene regulator (ClgR) of Mycobacterium tuberculosis activates the transcription of at least ten genes, including four that encode protease systems (ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, ClpB and the chaperonin Rv3269). Thus, M. tuberculosis ClgR controls a larger network of protein homeostatic and regulatory systems than ClgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for M. tuberculosis to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as M. tuberculosis lacking ClgR is deficient in the ability to control phagosome pH 1 h post-phagocytosis

Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca2+ overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca2+ activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca2+, stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.

BackgroundAn important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission.MethodsWe examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and M.tuberculosis strains including H37Rv, and isogenic virulent and avirulent strains of the Beijing lineage isolate GC1237.ResultsWe show that bacterial virulence influences the dynamics of caspase activation and the total level of cytotoxicity. We show that the powerful ability of M.tuberculosis to inhibit exogenously stimulated apoptosis is abrogated by loss of virulence. However, loss of virulence did not influence the balance of macrophage apoptosis and necrosis – both virulent and avirulent isogenic strains of GC1237 induced predominantly necrotic cell death compared to H37Rv which induced a higher relative level of apoptosis.ConclusionsThis reveals that macrophage necrosis and apoptosis are independently regulated during M. tuberculosis infection of macrophages. Virulence affects the level of host cell death and ability to inhibit apoptosis but other strain-specific characteristics influence the ultimate mode of host cell death and alter the balance of apoptosis and necrosis.

ADP-ribosyltransferases (ARTs) utilise NAD + to catalyse substrate ADP-ribosylation 1 , thereby regulating cellular pathways or contributing to toxin-mediated pathogenicity of bacteria [2][3][4] . Reversible ADP-ribosylation has traditionally been considered a protein-specific modification 5 , but recent in vitro studies have suggested nucleic acids as targets [6][7][8][9] . Here, we present evidence that specific reversible DNA ADP-ribosylation on thymidine bases occurs in cellulo through the DarT/DarG toxin/antitoxin system which is found in a variety of bacteria including global pathogens such as Mycobacterium tuberculosis, EPEC and Pseudomonas aeruginosa 10 . We report the first DarT structure which identifies this protein as a diverged member of the PARP family. Moreover, a set of high-resolution structures in ligand-free, pre-and post-reaction states reveals a specialised mechanism of catalysis that includes a key active-site arginine, extending the canonical ART toolkit. Comparison with the well-established DNA-repair protein ADP-ribosylation complex, PARP/HPF1, offers insights into how the DarT class of ARTs evolved into specific DNA-modifying enzymes. Together, the structural and mechanistic data provide unprecedented detail for a PARP family member and contribute to fundamental understanding of nucleic acid ADP-ribosylation. We furthermore show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin, functioning as non-canonical DNA-repair factor, are utilised not only for targeted DNA damage to induce toxicity but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar we show that DarTG regulates growth by DNA ADP-ribosylation at the origin of chromosome replication.

As a clinical setting in which local live biological therapy is already well established, non-muscle invasive bladder cancer (NMIBC) presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)-targeted immunotherapeutic virus. This study investigated CVA21-induced cytotoxicity in a panel of human bladder cancer cell lines, revealing a range of sensitivities largely correlating with expression of the viral receptor ICAM-1. CVA21 in combination with low doses of mitomycin-C enhanced CVA21 viral replication and oncolysis by increasing surface expression levels of ICAM-1. This was further confirmed using 300-μm precision slices of NMIBC where levels of virus protein expression and induction of apoptosis were enhanced with prior exposure to mitomycin-C. Given the importance of the immunogenicity of dying cancer cells for triggering tumor-specific responses and long-term therapeutic success, the ability of CVA21 to induce immunogenic cell death was investigated. CVA21 induced immunogenic apoptosis in bladder cancer cell lines, as evidenced by expression of the immunogenic cell death (ICD) determinant calreticulin, and HMGB-1 release and the ability to reject MB49 tumors in syngeneic mice after vaccination with MB49 cells undergoing CVA21 induced ICD. Such CVA21 immunotherapy could offer a potentially less toxic, more effective option for the treatment of bladder cancer.

Background BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG’s efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host’s immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria. Results To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. Conclusion These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host’s innate immune system to allow it to persist, a property that is important for its protective efficacy. Electronic supplementary material The online version of this article (10.1186/s12864-019-5791-1) contains supplementary material, which is available to authorized users.

Whenever a genetically homogenous population of bacterial cells is exposed to antibiotics, a tiny fraction of cells survives the treatment, the phenomenon known as bacterial persistence [G.L. Hobby et al., Exp. Biol. Med. 50, 281–285 (1942); J. Bigger, The Lancet 244, 497–500 (1944)]. Despite its biomedical relevance, the origin of the phenomenon is still unknown, and as a rare, phenotypically resistant subpopulation, persisters are notoriously hard to study and define. Using computerized tracking we show that persisters are small at birth and slowly replicating. We also determine that the high-persister mutant strain of Escherichia coli, HipQ, is associated with the phenotype of reduced phenotypic inheritance (RPI). We identify the gene responsible for RPI, ydcI, which encodes a transcription factor, and propose a mechanism whereby loss of phenotypic inheritance causes increased frequency of persisters. These results provide insight into the generation and maintenance of phenotypic variation and provide potential targets for the development of therapeutic strategies that tackle persistence in bacterial infections.