BCG Strain Research Articles

DBUH][HSO4]-catalyzed Solvent-free Synthesis of 1,5-benzodiazepine Derivatives: Bioevaluation and In silico Molecular Docking Study

: Here, we report the solvent-free one-pot multicomponent synthesis of 4- substituted-1,5-benzodiazepine derivatives from O-phenylenediamine, aromatic aldehydes, and dimedone using [DBUH][HSO4] as a catalyst in excellent yields. This process was carried out in search of a reusable, easily accessible, affordable, and efficient catalyst. 1,5- Benzodiazepines demonstrate a new family of good inhibitors with potent antimycobacterial properties. The most promising compounds in the present series are 4c, 4i, and 4l which showed excellent activity and inhibited the growth of both MTB H37Ra and M. bovis BCG strains with lower MICs. The most active compounds were further studied for their cytotoxicity against cell lines MCF-7, A549, HCT116, and THP-1 by MTT assays and the compounds were found to be non-toxic. The fact that none of these compounds work against either Gram-positive or Gram-negative bacteria suggests that they are only effective against MTB. The in silico docking of the molecules against mycobacterial enoyl reductase, InhA enzyme could provide well-clustered solutions and have given valuable insights into the thermodynamic elements governing the binding affinities. The findings of this investigation unmistakably point to the discovery of extremely specific and selective MTB inhibitors, which can now be investigated further in search of possible anti-tubercular drugs.

Mycobacterium manipulate glutaminase 1 mediated glutaminolysis to regulate macrophage autophagy for bacteria intracellular survival

Autophagy plays a vital role in eliminating intracellular mycobacterium. It is regulated by multiple metabolic processes including glutaminolysis. Glutaminase 1 (GLS1) is the rate-limiting enzyme of glutaminolysis and has been reported to control intracellular Gln content. However, its function on regulating autophagy in mycobacterium infected macrophage is still obscure. Hence, the current study hired mycobacterium virulent strain H37Rv or attenuated strain BCG to infect macrophage and detected the changes in cell glutaminolysis. The function of GLS1 on regulating autophagy in mycobacterium infected macrophages was further investigated. The results showed that BCG infection promoted macrophage autophagy, enhanced glutaminolysis, reduced intracellular Gln content, accompanied with the up-regulation of GLS1. Conversely, H37Rv infection resulted in completely opposite effects. Meanwhile, knockdown of GLS1 increased Gln content and attenuated autophagy in BCG infected macrophages. In addition, the deprivation of Gln not only promoted the autophagy of H37Rv infected macrophages, but also abolished the effect of knockdown GLS1 on regulating BCG infection-induced mTOR activation or autophagy. To sum up, our study suggested that different virulent strains of mycobacterium infection have totally opposite effects on glutaminolysis and the expression of GLS1. Specifically, mycobacterium virulent strain reduced GLS1 expression and decreased Gln content but mycobacterium attenuated strain promoted GLS1 expression and enhanced Gln content. Furthermore, GLS1 inhibits the activation of the mTOR signaling pathway and promotes autophagy by decreasing Gln content.

A "suicide" BCG strain provides enhanced immunogenicity and robust protection against Mycobacterium tuberculosis in macaques.

Intravenous (IV) BCG delivery provides robust protection against Mycobacterium tuberculosis (Mtb) in macaques but poses safety challenges. Here, we constructed two BCG strains (BCG-TetON-DL and BCG-TetOFF-DL) in which tetracyclines regulate two phage lysin operons. Once the lysins are expressed, these strains are killed in immunocompetent and immunocompromised mice yet induced similar immune responses and provided similar protection against Mtb challenge as wild type BCG. Lysin induction resulted in release of intracellular BCG antigens and enhanced cytokine production by macrophages. In macaques, cessation of doxycycline administration resulted in rapid elimination of BCG-TetOFF-DL. However, IV BCG-TetOFF-DL induced increased pulmonary CD4 T cell responses compared to WT BCG and provided robust protection against Mtb challenge, with sterilizing immunity in 6 of 8 macaques, compared to 2 of 8 macaques immunized with WT BCG. Thus, a "suicide" BCG strain provides an additional measure of safety when delivered intravenously and robust protection against Mtb infection.

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance

ObjectivesHeteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has...

Novel Targets for the Development of Tuberculosis Vaccine

Abstract: In underdeveloped nations, tuberculosis (TB) continues to be a major source of morbidity and mortality. The currently available vaccine against tuberculosis in endemic areas is mainly ineffective, which triggers the need for a clinically effective vaccine against tuberculosis. In the present review, we emphasized the impact of genetic variations in the BCG strains, which influence the efficacy of BCG vaccines. We also discussed the current status of BCG vaccines and their potential mechanisms on the modulation of B cells and, thereby, humoral immunity, which trigger immune responses against various intracellular pathogens. Further, we also elaborated upon the pre-clinical and clinical studies demonstrating the efficacy and safety of the vaccines. Moreover, we also presented the putative novel targets such as polysaccharide-induced antibodies for the protection against Mtb, PGRS domain as an important target for Humoral immunity, HLA-E pathway-Target strategy for new TB vaccine, Coronin-1a - Novel player for Mycobacterial survival, IRGM, IFN-I3, an autophagy inducer with Irgm1 serving as a core part in the Tuberculosis vaccine development.

Enhanced Glycosylation Caused by Overexpression of Rv1002c in a Recombinant BCG Promotes Immune Response and Protects against Mycobacterium tuberculosis Infection.

Tuberculosis (TB) is a major global health threat despite its virtual elimination in developed countries. Issues such as drug accessibility, emergence of multidrug-resistant strains, and limitations of the current BCG vaccine highlight the urgent need for more effective TB control measures. This study constructed BCG strains overexpressing Rv1002c and found that the rBCG-Rv1002c strain secreted more glycosylated proteins, significantly enhancing macrophage activation and immune protection against Mycobacterium tuberculosis (M. tb). These results indicate that Rv1002c overexpression promotes elevated levels of O-glycosylation in BCG bacteriophages, enhancing their phagocytic and antigenic presentation functions. Moreover, rBCG-Rv1002c significantly upregulated immune regulatory molecules on the macrophage surface, activated the NF-κB pathway, and facilitated the release of large amounts of NO and H2O2, thereby enhancing bacterial control. In mice, rBCG-Rv1002c immunization induced greater innate and adaptive immune responses, including increased production of multifunctional and long-term memory T cells. Furthermore, rBCG-Rv1002c-immunized mice exhibited reduced lung bacterial load and histological damage upon M. tb infection. This result shows that it has the potential to be an excellent candidate for a preventive vaccine against TB.

Multi-omics analysis reveals that linoleic acid metabolism is associated with variations of trained immunity induced by distinct BCG strains.

Trained immunity is one of the mechanisms by which BCG vaccination confers persistent nonspecific protection against diverse diseases. Genomic differences between the different BCG vaccine strains that are in global use could result in variable protection against tuberculosis and therapeutic effects on bladder cancer. In this study, we found that four representative BCG strains (BCG-Russia, BCG-Sweden, BCG-China, and BCG-Pasteur) covering all four genetic clusters differed in their ability to induce trained immunity and nonspecific protection. The trained immunity induced by BCG was associated with the Akt-mTOR-HIF1α axis, glycolysis, and NOD-like receptor signaling pathway. Multi-omics analysis (epigenomics, transcriptomics, and metabolomics) showed that linoleic acid metabolism was correlated with the trained immunity-inducing capacity of different BCG strains. Linoleic acid participated in the induction of trained immunity and could act as adjuvants to enhance BCG-induced trained immunity, revealing a trained immunity-inducing signaling pathway that could be used in the adjuvant development.

Evaluation of the viability of the Bulgarian BCG vaccine by the adenosine triphosphate assay

We applied a modified assay for the measurement of the intracellular ATP content as an alternative biochemical method for estimating the viability of the BCG vaccine instead of the standard cultural microbiological assay. Eight lyophilized BCG ampoule samples (Lot 287-2 and Lot 254-2, Bulgarian BCG strain SL222 Sofia) were tested on different days. Each calibration curve was measured for the following range of ATP concentrations: 100, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.1562 pmol. The obtained results were compared to the traditional cultural colony counting method for evaluating the viability of the BCG vaccine. The NCP of Lot 287-2 (min 2.325–max 3.300) was higher in comparison to Lot 254-2 (min 1.438–max 3.330). That result corresponded to higher ATP-content registered for Lot 287-2 (min 0.409–max 1.338) than Lot 254-2 (min 0.18–max 1.104). Our results confirmed the observation made by other researchers that the modified ATP assay is a reliable, simple, and quick method for the evaluation of the viability of the BCG vaccine. Additional compiled data about all BCG strains are needed to perform studies on the correlation between the luciferin-luciferase bioluminescent ATP assay and the cultural method for viability and enumeration of the units in the lyophilized BCG vaccine.

PCR Development for Analysis of Some Type II Toxin-Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis.

Toxin-Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64M. tuberculosis and 26M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

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.

Transcriptional Profiling of Homologous Recombination Pathway Genes in Mycobacterium bovis BCG Moreau.

Mycobacterium bovis BCG Moreau is the main Brazilian strain for vaccination against tuberculosis. It is considered an early strain, more like the original BCG, whereas BCG Pasteur, largely used as a reference, belongs to the late strain clade. BCG Moreau, contrary to Pasteur, is naturally deficient in homologous recombination (HR). In this work, using a UV exposure test, we aimed to detect differences in the survival of various BCG strains after DNA damage. Transcription of core and regulatory HR genes was further analyzed using RT-qPCR, aiming to identify the molecular agent responsible for this phenotype. We show that early strains share the Moreau low survival rate after UV exposure, whereas late strains mimic the Pasteur phenotype, indicating that this increase in HR efficiency is linked to the evolutionary clade history. Additionally, RT-qPCR shows that BCG Moreau has an overall lower level of these transcripts than Pasteur, indicating a correlation between this gene expression profile and HR efficiency. Further assays should be performed to fully identify the molecular mechanism that may explain this differential phenotype between early and late BCG strains.

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.

Attenuated but immunostimulatory Mycobacterium tuberculosis variant bovis strain Ravenel shows variation in T cell epitopes

Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC) organisms, affects a range of humans and animals globally. Mycobacterial pathogenesis involves manipulation of the host immune system, partially through antigen presentation. Epitope sequences across the MTBC are evolutionarily hyperconserved, suggesting their recognition is advantageous for the bacterium. Mycobacterium tuberculosis var. bovis (MBO) strain Ravenel is an isolate known to provoke a robust immune response in cattle, but typically fails to produce lesions and persist. Unlike attenuated MBO BCG strains that lack the critical RD1 genomic region, Ravenel is classic-type MBO structurally, suggesting genetic variation is responsible for defective pathogenesis. This work explores variation in epitope sequences in MBO Ravenel by whole genome sequencing, and contrasts such variation against a fully virulent clinical isolate, MBO strain 10-7428. Validated MTBC epitopes (n = 4818) from the Immune Epitope Database were compared to their sequences in MBO Ravenel and MBO 10-7428. Ravenel yielded 3 modified T cell epitopes, in genes rpfB, argC, and rpoA. These modifications were predicted to have little effect on protein stability. In contrast, T cells epitopes in 10-7428 were all WT. Considering T cell epitope hyperconservation across MTBC variants, these altered MBO Ravenel epitopes support their potential contribution to overall strain attenuation. The affected genes may provide clues on basic pathogenesis, and if so, be feasible targets for reverse vaccinology.

Recombinant BCG expressing the LTAK63 adjuvant increased memory T cells and induced long-lasting protection against Mycobacterium tuberculosis challenge in mice.

Vaccine-induced protection against Mycobacterium tuberculosis (Mtb) is usually ascribed to the induction of Th1, Th17, and CD8+ T cells. However, protective immune responses should also involve other immune cell subsets, such as memory T cells. We have previously shown improved protection against Mtb challenge using the rBCG-LTAK63 vaccine (a recombinant BCG strain expressing the LTAK63 adjuvant, a genetically detoxified derivative of the A subunit from E. coli heat-labile toxin). Here we show that mice immunized with rBCG-LTAK63 exhibit a long-term (at least until 6 months) polyfunctional Th1/Th17 response in the draining lymph nodes and in the lungs. This response was accompanied by the increased presence of a diverse set of memory T cells, including central memory, effector memory and tissue-resident memory T cells. After the challenge, the T cell phenotype in the lymph nodes and lungs were characterized by a decrease in central memory T cells, and an increase in effector memory T cells and effector T cells. More importantly, when challenged 6 months after the immunization, this group demonstrated increased protection in comparison to BCG. In conclusion, this work provides experimental evidence in mice that the rBCG-LTAK63 vaccine induces a persistent increase in memory and effector T cell numbers until at least 6 months after immunization, which correlates with increased protection against Mtb. This improved immune response may contribute to enhance the long-term protection.

Frequency of absent BCG Scar in Babies Vaccinated with BCG

Objective; In a TB endemic country find the frequency and associations of absent BCG scar in babies vaccinated with BCG.
 Study Design; Interventional prospective cohort study.
 Palace and Duration of Study; Immunization centre at Paediatric department of Fauji Foundation Hospital, Lahore from Mar 2021 to Aug 2021
 Methodology; Infants with age ranging from 1day-1month, born at term with no antenatal, natal or post natal complications were selected by non-purposive convenient sampling and vaccinated by a single vaccinator using only one strain of BCG. Presence or absence of BCG scar was checked on their following visits for vaccinations at 6, 10 and 14 weeks of age and evaluated against different variables.
 Result; 540 infants were included in this study. 384 had a complete follow-up, of which 35(9.1%) did not develop a scar after vaccination. This absence of scar was evaluated against gender , birth weight , day of vaccination, type of feed and socio economic status and there was no statistically significant association as p-value was >0.05
 Conclusion; Probability of developing a BCG scar is independent of gender , birth weight , day of vaccination , type of feed and socioeconomic status. We suggest that babies who do not develop a scar should be revaccinated if they show poor response to TST after 6 months of ageas it can give rise to cross-protection against micro-organisms that might be unrelated totarget disease.

Pd(II)dipyrrinato complexes: Synthesis, crystal structure and their antibacterial applications

The synthesis and biological studies of a series of Pd(II)dipyrrinato complexes are reported. The thiogalactosyl and/or oligoethyleneglycol moieties were linked with themeso-aryl rings of the Pd(II)dipyrrinato complexes. The Pd(II)dipyrrinato complexes were characterized by MASS, IR, UV-Vis, and NMR spectroscopic techniques. Single crystal X-ray structure of the homoleptic Pd(II)dipyrrinato complex (Pd1) revealed typical square planar geometry around the metal center. The anti-bacterial studies of Pd(II) dipyrrinato complexes were performed on theM. Tuberculosis H37Rv and M. Bovis BCG strains and complex Pd4 showed excellent anti-TB activity with MIC values of 25 [Formula: see text]M and 12.5 [Formula: see text]M, respectively. The ease of synthesis and good MIC values of Pd(II)dipyrrinato complexes make them promising candidates for anti-TB activity.

Pathogenicity and Genomic Characterization of a Novel Genospecies, Bacillus shihchuchen, of the Bacillus cereus Group Isolated from Chinese Softshell Turtle (Pelodiscus sinensis).

The Chinese softshell turtle (CST; Pelodiscus sinensis) is a freshwater aquaculture species of substantial economic importance that is commercially farmed across Asia, particularly in Taiwan. Although diseases caused by the Bacillus cereus group (Bcg) pose a major threat to commercial CST farming systems, information regarding its pathogenicity and genome remains limited. Here, we investigated the pathogenicity of Bcg strains isolated in a previous study and performed whole-genome sequencing. Pathogenicity analysis indicated that QF108-045 isolated from CSTs caused the highest mortality rate, and whole-genome sequencing revealed that it was an independent group distinct from other known Bcg genospecies. The average nucleotide identity compared to other known Bcg genospecies was below 95%, suggesting that QF108-045 belongs to a new genospecies, which we named Bacillus shihchuchen. Furthermore, genes annotation revealed the presence of anthrax toxins, such as edema factor and protective antigen, in QF108-045. Therefore, the biovar anthracis was assigned, and the full name of QF108-045 was Bacillus shihchuchen biovar anthracis. In addition to possessing multiple drug-resistant genes, QF108-045 demonstrated resistance to various types of antibiotics, including penicillins (amoxicillin and ampicillin), cephalosporins (ceftifour, cephalexin, and cephazolin), and polypeptides, such as vancomycin.

BCG∆BCG1419c and BCG differ in induction of autophagy, c-di-GMP content, proteome, and progression of lung pathology in Mycobacterium tuberculosis HN878-infected male BALB/c mice

The efficacy of BCG vaccines against Mycobacterium tuberculosis (Mtb) strains of lineage 2 (Beijing) in preclinical models and humans has been questioned. We have developed BCG∆BCG1419c, by deletion of BCG1419c in BCG Pasteur, which improved control of tuberculosis (TB) in preclinical models. Here, we compared the capacity of BCG and BCG∆BCG1419c to induce autophagy in murine macrophages, modify c-di-GMP content and transcript levels of BCG1416c, encoding the enzyme responsible for c-di-GMP synthesis/degradation, and of BCG1419c, encoding the phosphodiesterase involved in c-di-GMP degradation. Furthermore, we evaluated proteomic differences in vitro and compared protection against TB produced by a low dose of the HN878-Beijing strain at 3- and 6-months post-infection. We found that BCG∆BCG1419c induced more autophagy and produced different levels of c-di-GMP as well as different transcription of BCG1416c with no expression of BCG1419c. BCG∆BCG1419c differentially produced several proteins, including some involved in interaction with host cells. Vaccination with either BCG strain led to control of bacillary burden in lungs and spleen at 3- but not 6-months post-infection, whereas it reduced pneumonic areas compared with unvaccinated controls at 6 months post-infection. Vaccination with BCG∆BCG1419c delayed progression of lung necrosis as this was observed only at 6 months post-infection. Taken together, compared with BCG, BCG∆BCG1419c increased autophagy, presented different levels of c-di-GMP and transcription of BCG1416c in vitro in a growth-phase dependent manner, modified its proteome and delayed progression of lung pathology produced by a highly virulent Beijing strain.

Does intravenous vaccination with self-killing BCG lead to development of an immune response that protects against Mycobacterium tuberculosis?

Abstract The use of intradermal BCG offers limited protection against tuberculosis. Intravenous (IV) administration of BCG has shown to produce robust protection against infection in a non-human primate model of tuberculosis. However, the duration of exposure and time required to elicit an efficacious, protective response is unknown and safety of IV administration of live BCG remains a concern. Here we investigated whether a self-limiting strain of BCG (killswitchBCG; ksBCG) could induce protective responses in macaques. ksBCG uses two transcriptionally repressible lysins; the inducer doxycycline is required for repression, with the absence of inducer leading to BCG death. Our first study demonstrated the robust in vivo killing of ksBCG once doxycycline is stopped IV ksBCG elicited a robust, multicellular immune response in airways and lung up to 8 weeks, similar to standard BCG. In the current study we are assessing protection conferred against Mycobacterium tuberculosis with IV ksBCG. Mauritian cynomolgus macaques (MCM) were vaccinated intravenously with ksBCG. 5 months post vaccination, animals were challenged with M. tuberculosis and later euthanized 12 weeks post infection. Bronchoalveolar lavages were routinely taken throughout the study, and alongside tissue samples, analyzed using spectral flow cytometry. The presence of multiple cell types (CD4, CD8, and gd T cells, B cells, NK cells), cytokines (IFNg, IL-2, IL-17, TNF) and cytotoxic molecules (granzymes B and K, perforin, granulysin), as well as M. tuberculosis bacterial burden will be assessed to determine the level of protection provided by IV ksBCG. NIH R01 AI143788

In vitro and in silico evaluations of actinomycin X2and actinomycin D as potent anti-tuberculosis agents.

Multidrug-resistant tuberculosis (MDR-TB) is one of the world's most devastating contagious diseases and is caused by the MDR-Mycobacterium tuberculosis (MDR-Mtb) bacteria. It is therefore essential to identify novel anti-TB drug candidates and target proteins to treat MDR-TB. Here, in vitro and in silico studies were used to investigate the anti-TB potential of two newly sourced actinomycins, actinomycin-X2 (act-X2) and actinomycin-D (act-D), from the Streptomyces smyrnaeus strain UKAQ_23 (isolated from the Jubail industrial city of Saudi Arabia). The anti-TB activity of the isolated actinomycins was assessed in vitro using the Mtb H37Ra, Mycobacterium bovis (BCG), and Mtb H37Rv bacterial strains, using the Microplate Alamar Blue Assay (MABA) method. In silico molecular docking studies were conducted using sixteen anti-TB drug target proteins using the AutoDock Vina 1.1.2 tool. The molecular dynamics (MD) simulations for both actinomycins were then performed with the most suitable target proteins, using the GROningen MAchine For Chemical Simulations (GROMACS) simulation software (GROMACS 2020.4), with the Chemistry at HARvard Macromolecular Mechanics 36m (CHARMM36m) forcefield for proteins and the CHARMM General Force Field (CGenFF) for ligands. In vitro results for the Mtb H37Ra, BCG, and Mtb H37Rv strains showed that act-X2 had minimum inhibitory concentration (MIC) values of 1.56 ±0.0, 1.56 ±0.0, and 2.64 ±0.07 µg/mL and act-D had MIC values of 1.56 ±0.0, 1.56 ±0.0, and 1.80 ±0.24 µg/mL respectively. The in silico molecular docking results showed that protein kinase PknB was the preferred target for both actinomycins, while KasA and pantothenate synthetase were the least preferred targets for act-X2and act-D respectively. The molecular dynamics (MD) results demonstrated that act-X2 and act-D remained stable inside the binding region of PknB throughout the simulation period. The MM/GBSA (Molecular Mechanics/Generalized Born Surface Area) binding energy calculations showed that act-X2 was more potent than act-D. In conclusion, our results suggest that both actinomycins X2 and D are highly potent anti-TB drug candidates. We show that act-X2is better able to antagonistically interact with the protein kinase PknB target than act-D, and thus has more potential as a new anti-TB drug candidate.