Safety and Immunogenicity of Recombinant Bacille Calmette-Guérin Strain VPM1002 and Its Derivatives in a Goat Model.

A more effective vaccine against tuberculosis than Bacille Calmette-Guérin (BCG) is urgently needed. BCG derived recombinant VPM1002 has been found to be more efficacious and safer than the parental strain in mice models. Newer candidates, such as VPM1002 Δpdx1 (PDX) and VPM1002 ΔnuoG (NUOG), were generated to further improve the safety profile or efficacy of the vaccine. Herein, we assessed the safety and immunogenicity of VPM1002 and its derivatives, PDX and NUOG, in juvenile goats. Vaccination did not affect the goats' health in regards to clinical/hematological features. However, all three tested vaccine candidates and BCG induced granulomas at the site of injection, with some of the nodules developing ulcerations approximately one month post-vaccination. Viable vaccine strains were cultured from the injection site wounds in a few NUOG- and PDX- vaccinated animals. At necropsy (127 days post-vaccination), BCG, VPM1002, and NUOG, but not PDX, still persisted at the injection granulomas. All strains, apart from NUOG, induced granuloma formation only in the lymph nodes draining the injection site. In one animal, the administered BCG strain was recovered from the mediastinal lymph nodes. Interferon gamma (IFN-γ) release assay showed that VPM1002 and NUOG induced a strong antigen-specific response comparable to that elicited by BCG, while the response to PDX was delayed. Flow cytometry analysis of IFN-γ production by CD4+, CD8+, and γδ T cells showed that CD4+ T cells of VPM1002- and NUOG-vaccinated goats produced more IFN-γ compared to BCG-vaccinated and mock-treated animals. In summary, the subcutaneous application of VPM1002 and NUOG induced anti-tuberculous immunity, while exhibiting a comparable safety profile to BCG in goats.

Vaccine-Induced Subcutaneous Granulomas in Goats Reflect Differences in Host-Mycobacterium Interactions between BCG- and Recombinant BCG-Derivative Vaccines.

Tuberculous granulomas are highly dynamic structures reflecting the complex host-mycobacterium interactions. The objective of this study was to compare granuloma development at the site of vaccination with BCG and its recombinant derivatives in goats. To characterize the host response, epithelioid cells, multinucleated giant cells (MNGC), T cell subsets, B cells, plasma cells, dendritic cells and mycobacterial antigen were labelled by immunohistochemistry, and lipids and acid-fast bacteria (AFB) were labelled by specific staining. Granulomas with central caseous necrosis developed at the injection site of most goats though lesion size and extent of necrosis differed between vaccine strains. CD4+ T and B cells were more scarce and CD8+ cells were more numerous in granulomas induced by recombinant derivatives compared to their parental BCG strain. Further, the numbers of MNGCs and cells with lipid bodies were markedly lower in groups administered with recombinant BCG strains. Microscopic detection of AFB and mycobacterial antigen was rather frequent in the area of central necrosis, however, the isolation of bacteria in culture was rarely successful. In summary, BCG and its recombinant derivatives induced reproducibly subcutaneous caseous granulomas in goats that can be easily monitored and surgically removed for further studies. The granulomas reflected the genetic modifications of the recombinant BCG-derivatives and are therefore suitable models to compare reactions to different mycobacteria or TB vaccines.

Role of Premycofactocin Synthase in Growth, Microaerophilic Adaptation, and Metabolism of Mycobacterium tuberculosis.

Mycofactocin is a new class of peptide-derived redox cofactors present in a selected group of bacteria including Mycobacterium tuberculosis. Mycofactocin biosynthesis requires at least six genes, including mftD, encoding putative lactate dehydrogenase, which catalyzes the penultimate biosynthetic step. Cellular functions remained unknown until recent reports on the significance of mycofactocin in primary alcohol metabolism. Here, we show that mftD transcript levels were increased in hypoxia-adapted M. tuberculosis; however, mftD functionality was found likely dispensable for l-lactate metabolism. Targeted deletion of mftD reduced the survival of M. tuberculosis in in vitro and in vivo hypoxia models but increased the bacterial growth in glucose-containing broth as well as in the lungs and spleens, albeit modestly, of aerosol-infected C57BL/6J mice. The cause of this growth advantage remains unestablished; however, the mftD-deficient M. tuberculosis strain had reduced NAD(H)/NADP(H) levels and glucose-6-phosphate dehydrogenase activity with no impairment in phthiocerol dimycocerosate lipid synthesis. An ultrastructural examination of parental and mycofactocin biosynthesis gene mutants in M. tuberculosis, M. marinum, and M. smegmatis showed no altered cell morphology and size except the presence of outer membrane-bound fibril-like features only in a mutant subpopulation. A cell surface-protein analysis of M. smegmatis mycofactocin biosynthesis mutants with trypsin revealed differential abundances of a subset of proteins that are known to interact with mycofactocin and their homologs that can enhance protein aggregation or amyloid-like fibrils in riboflavin-starved eukaryotic cells. In sum, phenotypic analyses of the mutant strain implicate the significance of MftD/mycofactocin in M. tuberculosis growth and persistence in its host. IMPORTANCE Characterization of proteins with unknown functions is a critical research priority as the intracellular growth and metabolic state of Mycobacterium tuberculosis, the causative agent of tuberculosis, remain poorly understood. Mycofactocin is a peptide-derived redox cofactor present in almost all mycobacterial species; however, its functional relevance in M. tuberculosis pathogenesis and host survival has never been studied experimentally. In this study, we examine the phenotypes of an M. tuberculosis mutant strain lacking a key mycofactocin biosynthesis gene in in vitro and disease-relevant mouse models. Our results pinpoint the multifaceted role of mycofactocin in M. tuberculosis growth, hypoxia adaptation, glucose metabolism, and redox homeostasis. This evidence strongly implies that mycofactocin could fulfill specialized biochemical functions that increase the survival fitness of mycobacteria within their specific niche.

ESX-5-targeted export of ESAT-6 in BCG combines enhanced immunogenicity & efficacy against murine tuberculosis with low virulence and reduced persistence.

Tuberculosis (TB) is the leading infectious cause of death globally. The only licensed TB vaccine, Bacille Calmette-Guérin (BCG), has low efficacy against TB in adults and is not recommended in people with impaired immunity. The incorporation of the Mycobacterium tuberculosis (Mtb) secretion system ESX-1 into BCG improves immunogenicity and protection against TB in animal models, which is associated with the secretion of the ESX-1-dependent protein ESAT-6. However, the resulting strain, BCG::ESX1Mtb, has been deemed unsafe as a human vaccine, due to prolonged persistence and increased virulence in immunocompromised mice. In this study, we describe a new recombinant BCG strain that uncouples the beneficial aspects of ESAT-6 secretion from the detrimental ESX-1effects on virulence and persistence. The strain was constructed by fusing the ESAT-6-encoding gene esxA to the general secretion signal for the mycobacterial type VII secretion pathway protein PE25. This new strain, BCG::ESAT6-PE25SS, secretes full-length ESAT-6 via the ESX-5 secretion system, which in contrast to ESX-1 is also present in BCG. In vivo testing revealed that ESX-5-targeted ESAT-6 export, induces cytosolic contact, generates ESAT-6-specific T cells and enhances the protective efficacy against TB disease, but is associated with low virulence and reduced persistence in immunocompetent and immunocompromised mice. Additionally, compared to BCG::ESX1Mtb and parental BCG, mucosal administration of BCG::ESAT6-PE25SS is associated with more rapid clearance from the lung. These results warrant further studies to evaluate BCG::ESAT6-PE25SS as a potential live attenuated vaccine candidate for TB.

Preclinical Assessment of Bacteriophage Therapy against Experimental Acinetobacter baumannii Lung Infection.

Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages.

FX11 limits Mycobacterium tuberculosis growth and potentiates bactericidal activity of isoniazid through host-directed activity.

Lactate dehydrogenase A (LDHA) mediates interconversion of pyruvate and lactate, and increased lactate turnover is exhibited by malignant and infected immune cells. Hypoxic lung granuloma in Mycobacterium tuberculosis-infected animals present elevated levels of Ldha and lactate. Such alterations in the metabolic milieu could influence the outcome of host-M. tuberculosis interactions. Given the central role of LDHA for tumorigenicity, targeting lactate metabolism is a promising approach for cancer therapy. Here, we sought to determine the importance of LDHA for tuberculosis (TB) disease progression and its potential as a target for host-directed therapy. To this end, we orally administered FX11, a known small-molecule NADH-competitive LDHA inhibitor, to M. tuberculosis-infected C57BL/6J mice and Nos2-/- mice with hypoxic necrotizing lung TB lesions. FX11 did not inhibit M. tuberculosis growth in aerobic/hypoxic liquid culture, but modestly reduced the pulmonary bacterial burden in C57BL/6J mice. Intriguingly, FX11 administration limited M. tuberculosis replication and onset of necrotic lung lesions in Nos2-/- mice. In this model, isoniazid (INH) monotherapy has been known to exhibit biphasic killing kinetics owing to the probable selection of an INH-tolerant bacterial subpopulation. However, adjunct FX11 treatment corrected this adverse effect and resulted in sustained bactericidal activity of INH against M. tuberculosis As a limitation, LDHA inhibition as an underlying cause of FX11-mediated effect could not be established as the on-target effect of FX11 in vivo was unconfirmed. Nevertheless, this proof-of-concept study encourages further investigation on the underlying mechanisms of LDHA inhibition and its significance in TB pathogenesis.

Aryl Hydrocarbon Receptor Modulation by Tuberculosis Drugs Impairs Host Defense and Treatment Outcomes.

Antimicrobial resistance in tuberculosis (TB) is a public health threat of global dimension, worsened by increasing drug resistance. Host-directed therapy (HDT) is an emerging concept currently explored as an adjunct therapeutic strategy for TB. One potential host target is the ligand-activated transcription factor aryl hydrocarbon receptor (AhR), which binds TB virulence factors and controls antibacterial responses. Here, we demonstrate that in the context of therapy, the AhR binds several TB drugs, including front line drugs rifampicin (RIF) and rifabutin (RFB), resulting in altered host defense and drug metabolism. AhR sensing of TB drugs modulates host defense mechanisms, notably impairs phagocytosis, and increases TB drug metabolism. Targeting AhR in vivo with a small-molecule inhibitor increases RFB-treatment efficacy. Thus, the AhR markedly impacts TB outcome by affecting both host defense and drug metabolism. As a corollary, we propose the AhR as a potential target for HDT in TB in adjunct to canonical chemotherapy.

Mycofactocin Is Associated with Ethanol Metabolism in Mycobacteria.

Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many ActinobacteriaMycobacterium tuberculosis assimilates cholesterol during chronic infection, and its in vitro growth in the presence of cholesterol requires most of the MFT biosynthesis genes (mftA, mftB, mftC, mftD, mftE, and mftF), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in Mycobacterium smegmatis, M. marinum, and M. tuberculosis We found that the growth deficit of mft deletion mutants in medium containing cholesterol-a phenotypic basis for gene essentiality prediction-depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, MSMEG_6242 was indispensable for M. smegmatis ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in M. tuberculosis During ethanol treatment, mftC mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mftC mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems.IMPORTANCE Tuberculosis is caused by Mycobacterium tuberculosis, and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways-e.g., cholesterol assimilation-that are critical for persistence and for pathogenesis of M. tuberculosis In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for M. tuberculosisin vitro growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.

Humanized Mouse Model Mimicking Pathology of Human Tuberculosis for in vivo Evaluation of Drug Regimens.

Human immune system mice are highly valuable for in vivo dissection of human immune responses. Although they were employed for analyzing tuberculosis (TB) disease, there is little data on the spatial organization and cellular composition of human immune cells in TB granuloma pathology in this model. We demonstrate that human immune system mice, generated by transplanted human fetal liver derived hematopoietic stem cells develop a continuum of pulmonary lesions upon Mycobacterium tuberculosis aerosol infection. In particular, caseous necrotic granulomas, which contribute to prolonged TB treatment time, developed, and had cellular phenotypic spatial-organization similar to TB patients. By comparing two recommended drug regimens, we confirmed observations made in clinical settings: Adding Moxifloxacin to a classical chemotherapy regimen had no beneficial effects on bacterial eradication. We consider this model instrumental for deeper understanding of human specific features of TB pathogenesis and of particular value for the pre-clinical drug development pipeline.

Phagosomal Copper-Promoted Oxidative Attack on Intracellular Mycobacterium tuberculosis.

Copper (Cu) ions are critical in controlling bacterial infections, and successful pathogens like Mycobacterium tuberculosis (Mtb) possess multiple Cu resistance mechanisms. We report, as proof of concept, that a novel Cu hypersensitivity phenotype can be generated in mycobacteria, including Mtb, through a peptide, DAB-10, that is able to form reactive oxygen species (ROS) following Cu-binding. DAB-10 induces intramycobacterial oxidative stress in a Cu-dependent manner in vitro and during infection. DAB-10 penetrates murine macrophages and encounters intracellular mycobacteria. Significant intracellular Cu-dependent protection was observed when Mtb-infected macrophages were treated with DAB-10 alongside a cell-permeable Cu chelator. Treatment with the Cu chelator reversed the intramycobacterial oxidative shift induced by DAB-10. We conclude that DAB-10 utilizes the pool of phagosomal Cu ions in the host-Mtb interface to augment the mycobactericidal activity of macrophages while simultaneously exploiting the susceptibility of Mtb to ROS. DAB-10 serves as a model with which to develop next-generation, multifunctional antimicrobials.