Impact of Genomic Deletion RD16 on the Expression of the Mycobacterium bovis BCG Moreau VapBC47 Toxin-Antitoxin System.

Mycobacterium bovis BCG is the only vaccine against tuberculosis. The variable forms of cultivation throughout the years, before seed-lots were developed, allowed in vitro evolution of the original strain, generating a family of vaccines with different phenotypic and genotypic characteristics. Molecular studies revealed regions of difference (RDs) in the genomes of the various BCG strains. This work aims to characterize the gene pair rv3407-rv3408 (vapB47-vapC47), coding for a toxin-antitoxin system of the VapBC family, and to evaluate possible transcriptional effects due to the adjacent BCG Moreau-specific genomic deletion RD16. We show that these genes are co-transcribed in BCG strains Moreau and Pasteur, and that the inactivation of an upstream transcriptional repressor (Rv3405c) due to RD16 has a polar effect, leading to increased vapBC47 expression. Furthermore, we detect VapB47 DNA binding in vitro, dependent on a 5' vapB47 sequence that contributes to a palindrome, spanning the promoter and coding region. Our data shed light on the regulation of VapBC systems and on the impact of the BCG Moreau RD16 deletion in the expression of adjacent genes, contributing to a better understanding of BCG Moreau physiology.

Pharmacokinetics and Efficacy of the Benzothiazinone BTZ-043 against Tuberculous Mycobacteria inside Granulomas in the Guinea Pig Model.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's leading cause of mortality from a single bacterial pathogen. With increasing frequency, emergence of drug-resistant mycobacteria leads to failures of standard TB treatment regimens. Therefore, new anti-TB drugs are urgently required. BTZ-043 belongs to a novel class of nitrobenzothiazinones, which inhibit mycobacterial cell wall formation by covalent binding of an essential cysteine in the catalytic pocket of decaprenylphosphoryl-ß-d-ribose oxidase (DprE1). Thus, the compound blocks the formation of decaprenylphosphoryl-ß-d-arabinose, a precursor for the synthesis of arabinans. An excellent in vitro efficacy against M. tuberculosis has been demonstrated. Guinea pigs are an important small-animal model to study anti-TB drugs, as they are naturally susceptible to M. tuberculosis and develop human-like granulomas after infection. In the current study, dose-finding experiments were conducted to establish the appropriate oral dose of BTZ-043 for the guinea pig. Subsequently, it could be shown that the active compound was present at high concentrations in Mycobacterium bovis BCG-induced granulomas. To evaluate its therapeutic effect, guinea pigs were subcutaneously infected with virulent M. tuberculosis and treated with BTZ-043 for 4 weeks. BTZ-043-treated guinea pigs had reduced and less necrotic granulomas than vehicle-treated controls. In comparison to the vehicle controls a highly significant reduction of the bacterial burden was observed after BTZ-043 treatment at the site of infection and in the draining lymph node and spleen. Together, these findings indicate that BTZ-043 holds great promise as a new antimycobacterial drug.

Detection of a target protein (GroEl2) in Mycobacterium tuberculosis using a derivative of 1,2,4-triazolethiols.

Herein, we identified a potent lead compound RRA2, within a series of 54 derivatives of 1,2,4-triazolethiols (exhibit good potency as an anti-mycobacterial agents) against intracellular Mycobacterium tuberculosis (Mtb). Compound RRA2 showed significant mycobactericidal activity against active stage Mycobacterium bovis BCG and Mtb with minimum inhibitory concentration (MIC) values of 2.3 and 2.0 µg/mL, respectively. At MIC value, RRA2 compound yielded 0.82 log reduction of colony-forming unit (cfu) against non-replicating Mtb. Furthermore, RRA2 compound was selected for further target identification due to the presence of alkyne group, showing higher selectivity index (> 66.66 ± 0.22, in non-replicating stage). Using "click" chemistry, we synthesized the biotin linker-RRA2 conjugate, purified with HPLC method and confirmed the conjugation of biotin linker-RRA2 complex by HR-MS analysis. Furthermore, we successfully pulled down and identified a specific target protein GroEl2, from Mtb whole-cell extract. Furthermore, computational molecular modeling indicated RRA2 could interact with GroEl2, which explains the structure-activity relationship observed in this study. GroEL-2 identified a potent and specific target protein for RRA 2 compound in whole cell extract of Mtb H37Ra.

Rab6b localizes to the Golgi complex in murine macrophages and promotes tumor necrosis factor release in response to mycobacterial infection.

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in the host control of mycobacterial infections. Expression and release of TNF are tightly regulated, yet the molecular mechanisms that control the release of TNF by mycobacteria-infected host cells, in particular macrophages, are incompletely understood. Rab GTPases direct the transport of intracellular membrane-enclosed vesicles and are important regulators of macrophage cytokine secretion. Rab6b is known to be predominantly expressed in the brain where it functions in retrograde transport and anterograde vesicle transport for exocytosis. Whether it executes similar functions in the context of immune responses is unknown. Here we show that Rab6b is expressed by primary mouse macrophages, where it localized to the Golgi complex. Infection with Mycobacterium bovis bacille Calmette-Guérin (BCG) resulted in dynamic changes in Rab6b expression in primary mouse macrophages in vitro as well as in organs from infected mice in vivo. We further show that Rab6b facilitated TNF release by M. bovis BCG-infected macrophages, in the absence of discernible impact on Tnf messenger RNA and intracellular TNF protein expression. Our observations identify Rab6b as a positive regulator of M. bovis BCG-induced TNF trafficking and secretion by macrophages and positions Rab6b among the molecular machinery that orchestrates inflammatory cytokine responses by macrophages.

3D QSAR studies on amphiphilic indoles for antimycobacterial activity.

A persistent infection prolongs treatment duration and also enhances the chance of resistance development against antibiotics. Recently, a class of amphiphilic indole derivatives was discovered exhibiting bactericidal activity against both growing and nongrowing Mycobacterium bovis BCG (M. bovis BCG). These antibacterials are suggested to disturb the integrity and functioning of the cell membrane, a property that can help eradicate persistent organisms. This study article describes field-based three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of 79 amphiphilic indole derivatives. The aim of this QSAR study is to optimize this class of compounds for the development of more potent antimycobacterial agents. The results obtained indicate that steric interactions are crucial for antimycobacterial activity, while hydrogen bond donor groups participate negligibly in activity. The derived 3D-QSAR models showed acceptable r2 (0.91) and q2 (0.91) with a root mean squared error (RMSE) of 0.08. The models were cross-validated using the leave-one-out method. Applying the same QSAR model to another congeneric series of amphiphilic indoles externally validated the QSAR model. The model could appreciably predict the activity (pMIC50 ) of this congeneric series of amphiphilic indoles, with an RMSE of 0.49, indicating the robustness of the model and its efficiency in predicting the potentially active compounds.

Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle.

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.

Bacillus Calmette-Guérin Cases Reported to the National Tuberculosis Surveillance System, United States, 2004-2015.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is used as a vaccine to protect against disseminated tuberculosis (TB) and as a treatment for bladder cancer. We describe characteristics of US TB patients reported to the National Tuberculosis Surveillance System (NTSS) whose disease was attributed to BCG. We identified 118 BCG cases and 91,065 TB cases reported to NTSS during 2004-2015. Most patients with BCG were US-born (86%), older (median age 75 years), and non-Hispanic white (81%). Only 17% of BCG cases had pulmonary involvement, in contrast with 84% of TB cases. Epidemiologic features of BCG cases differed from TB cases. Clinicians can use clinical history to discern probable BCG cases from TB cases, enabling optimal clinical management. Public health agencies can use this information to quickly identify probable BCG cases to avoid inappropriately reporting BCG cases to NTSS or expending resources on unnecessary public health interventions.

Primary Role of Suppressor of Cytokine Signaling 1 in Mycobacterium bovis BCG Infection.

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of JAK/STAT signaling and is induced by mycobacterial infection. To understand the major function of SOCS1 during infection, we established a novel system in which recombinant Mycobacterium bovis bacillus Calmette-Guérin expressed dominant-negative SOCS1 (rBCG-SOCS1DN) because it would not affect the function of SOCS1 in uninfected cells. When C57BL/6 mice and RAG1-/- mice were intratracheally inoculated with rBCG-SOCS1DN, the amount of rBCG-SOCS1DN in the lungs was significantly reduced compared to that in the lungs of mice inoculated with a vector control counterpart and wild-type BCG. However, these significant differences were not observed in NOS2-/- mice and RAG1-/- NOS2-/- double-knockout mice. These findings demonstrated that SOCS1 inhibits nitric oxide (NO) production to establish mycobacterial infection and that rBCG-SOCS1DN has the potential to be a powerful tool for studying the primary function of SOCS1 in mycobacterial infection.

An Iron-Rich Diet Decreases the Mycobacterial Burden and Correlates With Hepcidin Upregulation, Lower Levels of Proinflammatory Mediators, and Increased T-Cell Recruitment in a Model of Mycobacterium bovis Bacille Calmette-Guerin Infection.

Background: Recent evidence indicates a robust competition between the host and mycobacteria for iron acquisition during mycobacterial infection. Variable effects of iron supplementation on the susceptibility to mycobacterial infection have been reported. In this study, we revisited the effects of an experimental iron-enriched diet on Mycobacterium bovis bacille Calmette-Guerin (BCG) infection. Methods: Mice fed a standard diet or a diet moderately enriched with iron were infected with M. bovis BCG expressing green fluorescent protein. Colony-forming unit numbers, host myeloid cell counts, cell recruitment, cytokine production, and iron gene expression were determined at different stages of infection. Bone marrow-derived macrophages incubated with or without iron were also used to measure bacterial uptake, levels of inflammation markers, and iron gene expression. Results: In vivo analysis of BCG-infected mice revealed that moderate iron supplementation reduced inflammation, as measured by decreased proinflammatory cytokine levels and neutrophil recruitment and enhanced T-cell recruitment in granulomas, and decreased the bacterial load. Enhanced bacterial clearance in the liver correlated with upregulation of the gene encoding hepcidin, which is known to have antimicrobial proprieties, and with sequestration of iron in tissues. In cultured macrophages, iron supplementation induced reactive oxygen species and reduced uptake and intracellular growth of BCG. Conclusion: Moderate iron diet supplementation diminished inflammation and growth of M. bovis BCG via enhanced reactive oxygen species production, immune cell activation, and local hepcidin expression.

Mouse Bone Marrow Sca-1+ CD44+ Mesenchymal Stem Cells Kill Avirulent Mycobacteria but Not Mycobacterium tuberculosis through Modulation of Cathelicidin Expression via the p38 Mitogen-Activated Protein Kinase-Dependent Pathway.

Mycobacterium tuberculosis primarily infects lung macrophages. However, a recent study showed that M. tuberculosis also infects and persists in a dormant form inside bone marrow mesenchymal stem cells (BM-MSCs) even after successful antibiotic therapy. However, the mechanism(s) by which M. tuberculosis survives in BM-MSCs is still not known. Like macrophages, BM-MSCs do not contain a well-defined endocytic pathway, which is known to play a central role in the clearance of internalized mycobacteria. Here, we studied the fate of virulent and avirulent mycobacteria in Sca-1+ CD44+ BM-MSCs. We found that BM-MSCs were able to kill avirulent Mycobacterium smegmatis and Mycobacterium bovis BCG but not the pathogenic species M. tuberculosis Further mechanistic studies revealed that pathogenic M. tuberculosis dampens the antibacterial response of BM-MSCs by downregulating the expression of the cationic antimicrobial peptide cathelicidin. In contrast, avirulent mycobacteria were effectively killed by inducing the Toll-like receptor 2/4 (TLR2/4) pathway-dependent expression of cathelicidin, while small interfering RNA (siRNA)-mediated cathelicidin silencing increased the survival of M. bovis BCG in BM-MSCs. We also showed that M. bovis BCG infection caused increased expression levels of MyD88, phospho-interleukin-1 receptor-associated kinase 4 (pIRAK-4), and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Further downstream investigations demonstrated that IRAK-4-p38 activation increased the nuclear translocation of NF-κB, which subsequently induced the expression of cathelicidin and the cytokine interleukin-1ß (IL-1ß), resulting in the decreased survival of M. bovis BCG. On the other hand, inhibition of TLR2/4, pIRAK-4, p38, and NF-κB nuclear translocation decreased cathelicidin and IL-1ß expression levels and therefore increased the survival of avirulent mycobacteria. This is the first report that demonstrates that virulent mycobacteria manipulate the TLR2/4-MyD88-IRAK-4-p38-NF-κB-Camp-IL-1ß pathway to survive inside bone marrow stem cells.

Mycobacterial receptor, Clec4d (CLECSF8, MCL), is coregulated with Mincle and upregulated on mouse myeloid cells following microbial challenge.

The C-type lectin receptor (CTLR), Clec4d (MCL, CLECSF8), is a member of the Dectin-2 cluster of CTLRs, which also includes the related receptors Mincle and Dectin-2. Like Mincle, Clec4d recognizes mycobacterial cord factor, trehalose dimycolate, and we recently demonstrated its key role in anti-mycobacterial immunity in mouse and man. Here, we characterized receptor expression in naïve mice, under inflammatory conditions, and during Mycobacterium bovis BCG infection using newly generated monoclonal antibodies. In naïve mice, Clec4d was predominantly expressed on myeloid cells within the peritoneal cavity, blood, and bone marrow. Unexpectedly, basal expression of Clec4d was very low on leukocytes in the lung. However, receptor expression was significantly upregulated on pulmonary myeloid cells during M. bovis BCG infection. Moreover, Clec4d expression could be strongly induced in vitro and in vivo by various microbial stimuli, including TLR agonists, but not exogenous cytokines. Notably, we show that Clec4d requires association with the signaling adaptor FcRγ and Mincle, but not Dectin-2, for surface expression. In addition, we provide evidence that Clec4d and Mincle, but not Dectin-2, are interdependently coregulated during inflammation and infection. These data show that Clec4d is an inducible myeloid-expressed CTLR in mice, whose expression is tightly linked to that of Mincle.

Enhanced expression of PD-L1 and IFN-γ on dendritic cells is associated with BCG-induced Th2 inhibition.

Accumulating evidence indicates that the exposure to Mycobacterium bovis bacillus Calmette-Guérin (BCG) prevents the development of allergy and the airway dendritic cells (DCs) may be involved in this protective effect. However, studies to better characterize the specific interactions between BCG and DCs and their role in this mycobacteria-mediated Th2 cell suppression are still ongoing. This study aimed to evaluate the effect of the neonatal BCG vaccination in the innate immune response in a mouse model of ovalbumin (OVA)-induced airway inflammation. BCG treated neonatal BALB/c mice were sensitized and challenged with aerosolized OVA. Twenty-four hours after the last challenge, samples were collected for analysis. The intranasal BCG treatment inhibited the allergic Th2-response by decreasing the allergen-induced eosinophilic inflammation, EPO activity, CCL11, IL-25, TSLP, IL-4 and IL-5 lung levels, and serum levels of IgE. Mycobacteria treatment increased lung levels of IL-10 and TGF-ß, and the TLR2 and TLR4 expressions by pulmonary CD11c+CD103+CD8α+ DCs. Additionally an enhanced expression of PD-L1 was observed besides an increased production of IFN-γ by these cells. These results indicated that neonatal BCG vaccination inhibits key features of allergic airway inflammation, probably by promoting T regulatory immune response via an enhanced expression of TLR2, TLR4 and PD-L1 on DCs.

Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction.

BACKGROUND: The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans. METHODS: Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages. RESULTS: Immunoglobulin G (IgG) responses to AM increased significantly 4-8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera. CONCLUSIONS: Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans.

Potassium ion channel Kir2.1 negatively regulates protective responses to Mycobacterium bovis BCG.

Tuberculosis caused by the pathogen Mycobacterium tuberculosis leads to increased mortality and morbidity worldwide. The prevalence of highly drug-resistant strains has reinforced the need for greater understanding of host-pathogen interactions at the cellular and molecular levels. Our previous work demonstrated critical roles of calcium ion channels in regulating protective responses to mycobacteria. In this report, we deciphered the roles of inwardly rectifying K+ ion channel Kir2.1 in epithelial cells. Data showed that infection of epithelial cells (and macrophages) increases the surface expression of Kir2.1. This increased expression of Kir2.1 results in higher intracellular mycobacterial survival, as either inhibiting or knocking down Kir2.1 results in mounting of a higher oxidative burst leading to a significant attenuation of mycobacterial survival. Further, inhibiting Kir2.1 also led to increased expression of T cell costimulatory molecules accompanied with increased activation of MAP kinases and transcription factors nuclear factor κB and phosphorylated CREB. Furthermore, inhibiting Kir2.1 induced increased autophagy and apoptosis that could also contribute to decreased bacterial survival. Interestingly, an increased association of heat shock protein 70 kDa with Kir2.1 was observed. These results showed that mycobacteria modulate the expression and function of Kir2.1 in epithelial cells to its advantage.

A viral vaccine design harnessing prior BCG immunization confers protection against Ebola virus.

Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4+ helper T (Th) cells induced by Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions. Furthermore, BCG vaccination also upregulated FcγR expression to potentially maximize antibody-dependent effector activities. Using a mouse model of Ebola virus (EBOV) infection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and protection against lethal challenge. This general approach can be easily adapted to other viruses, and may be a rapid and effective method of immunization against emerging pandemics in populations that routinely receive BCG vaccination.

Evaluation of protein extraction protocols for MALDI-TOF Biotyper analysis of mycobacteria.

Infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria represent a significant global threat and medical concern. Therefore, accurate and reliable methods must be employed to identify mycobacteria rapidly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that compares the cellular protein profiles of unknown isolates with reference mass spectra in a database to identify microorganisms. However, the thick and waxy lipid layer, which is rich in mycolic acids and is present in mycobacterial cells, makes protein extraction challenging. To identify the optimal protocol for correctly identifying bacilli using MALDI-TOF mass spectrometry, this study compared four different cellular protein extraction methods. Four strains of M. bovis BCG were selected as representatives of slow-growing mycobacteria, while three strains of fast-growing mycobacteria were also included: M. peregrinum, M. smegmatis, and M. farcinogenes. The extraction method that proved most effective was the extraction of inactivated cells with chloroform and methanol, which partially delipidates the cells. These cells were then extracted with formic acid, as is standard practice for protein extraction. The advantage of this method is that it allows the parallel analysis of cellular lipids and proteins from a single sample. It is therefore important to optimize mycobacterial protein extraction for MALDI-TOF MS analysis in clinical microbiology laboratories.

Engineering a dual vaccine against COVID-19 and tuberculosis.

The COVID-19 pandemic, caused by SARS-CoV-2 virus, has been one of the top public health threats across the world over the past three years. Mycobacterium bovis BCG is currently the only licensed vaccine for tuberculosis, one of the deadliest infectious diseases in the world, that is caused by Mycobacterium tuberculosis. In the past decades, recombinant M.bovis BCG has been studied as a novel vaccine vector for other infectious diseases in humans besides tuberculosis, such as viral infections. In the current study, we generated a recombinant M. bovis BCG strain AspikeRBD that expresses a fusion protein consisting of M. tb Ag85A protein and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using synthetic biology technique. Our results show that the recombinant M. bovis BCG strain successfully expressed this fusion protein. Interestingly, the recombinant M. bovis BCG strain AspikeRBD significantly induced SARS-CoV-2 spike-specific T cell activation and IgG production in mice when compared to the parental M.bovis BCG strain, and was more potent than the recombinant M.bovis BCG strain expressing SARS-CoV-2 spike RBD alone. As expected, the recombinant M. bovis BCG strain AspikeRBD activated an increased number of M. tb Ag85A-specific IFNγ-releasing T cells and enhanced IgG production in mice when compared to the parental M.bovis BCG strain or the BCG strain expressing SARS-CoV-2 spike RBD alone. Taken together, our results indicate a potential application of the recombinant M. bovis BCG strain AspikeRBD as a novel dual vaccine against SARS-CoV-2 and M. tb in humans.

DosR's multifaceted role on Mycobacterium bovis BCG revealed through multi-omics.

Mycobacterium tuberculosis (Mtb) is an intracellular bacterium that causes a highly contagious and potentially lethal tuberculosis (TB) in humans. It can maintain a dormant TB infection within the host. DosR (dormancy survival regulator) (Rv3133c) has been recognized as one of the key transcriptional proteins regulating bacterial dormancy and participating in various metabolic processes. In this study, we extensively investigate the still not well-comprehended role and mechanism of DosR in Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) through a combined omics analysis. Our study finds that deleting DosR significantly affects the transcriptional levels of 104 genes and 179 proteins. Targeted metabolomics data for amino acids indicate that DosR knockout significantly upregulates L-Aspartic acid and serine synthesis, while downregulating seven other amino acids, including L-histidine and lysine. This suggests that DosR regulates amino acid synthesis and metabolism. Taken together, these findings provide molecular and metabolic bases for DosR effects, suggesting that DosR may be a novel regulatory target.

Telacebec Interferes with Virulence Lipid Biosynthesis Protein Expression and Sensitizes to Other Antibiotics.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a public health issue, particularly due to multi-drug-resistant Mtb. The bacillus is wrapped in a waxy envelope containing lipids acting as essential virulence factors, accounting for the natural antibiotic resistance of mycobacteria. Telacebec (previously known as Q203) is a promising new anti-TB agent inhibiting the cytochrome bc1 complex of a mycobacterial electron transport chain (ETC). Here, we show that the telacebec-challenged M. bovis BCG exhibited a reduced expression of proteins involved in the synthesis of phthiocerol dimycocerosates (PDIMs)/phenolic glycolipids (PGLs), lipid virulence factors associated with cell envelope impermeability. Consistently, telacebec, at concentrations lower than its MIC, downregulated the transcription of a PDIM/PGL-synthesizing operon, suggesting a metabolic vulnerability triggered by the drug. The drug was able to synergize on BCG with rifampicin or vancomycin, the latter being a drug exerting a marginal effect on PDIM-bearing bacilli. Telacebec at a concentration higher than its MIC had no detectable effect on cell wall PDIMs, as shown by TLC analysis, a finding potentially explained by the retaining of previously synthesized PDIMs due to the inhibition of growth. The study extends the potential of telacebec, demonstrating an effect on mycobacterial virulence lipids, allowing for the development of new anti-TB strategies.

Vascular graft infections caused by Mycobacterium bovis BCG after BCG immunotherapy for non-muscle-invasive bladder cancer: Case report and review of literature.

Bacillus Calmette-Guerin (BCG) immunotherapy (i.e., intravesical instillation of live attenuated strain of Mycobacterium bovis) is a standard of care for non-muscle-invasive bladder cancer (NMIBC). The risk of infective adverse events is generally low as studies have reported an incidence of systemic BCG infections between 3% and 7%. In the majority of cases, BCG infections are disseminated (34.4%), genitourinary (23.4%), osteomuscular (19.9%), or vascular (6.7%). Regarding vascular involvement, mycotic aortic aneurysm, aorto-enteric fistula and vascular bypass graft infections have been described. A 73-year-old man with a prosthetic femoral-popliteal bypass was treated with BCG immunotherapy for a relapsed NMIBC. Two months later, the patient developed fever and hyporexia. PET-CT and CT scans of the abdomen showed an abscess surrounding the superficial femoral artery, while blood cultures yielded M. bovis BCG, and antitubercular therapy (with RMP + EMB + INH) was started. The prosthetic graft was removed and its cultures tested positive for M. bovis as well. A total of 14 cases of vascular prosthesis infections caused by M. bovis BCG following BCG instillation are so far reported. All the cases occurred in adult symptomatic men. Abdominal aorta was involved in the majority of cases. CT scan played a pivotal role in the diagnostic process. Mycobacterium bovis BCG was isolated from several different sources. Treatment required surgery and medical therapy, the latter showing wide variability. Previous BCG immunotherapy must be considered in the differential diagnosis in patients with infected vascular grafts. These infectious complications are rare and, while the infected grafts should be removed, there are no definite recommendations regarding the type of regimen and duration of treatment.