Mtb Proteins Research Articles

Functional genomic analysis of the 68-1 RhCMV-Mycobacteria tuberculosis vaccine reveals an IL-15 response signature that is conserved with vector attenuation.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is a deadly infectious disease having a major impact on global health. Using the CMV vector for development of novel vaccines is a promising new strategy that elicits strong and durable, high frequency memory T cell responses against heterologous immunogens. We conducted functional transcriptomic analysis of whole blood samples collected from cohorts of rhesus (Rh) macaques that were administered RhCMV/TB vector using a prime-boost strategy. Two modified CMV vectors were used in this study, including 68-1 RhCMV/TB-6Ag (encoding 6 Mtb protein immunogens, including Ag85A, ESAT-6, Rv3407, Rv2626, Rpf A, and Rpf D) and its attenuated variant, 68-1 RhCMV/Δpp71-TB-6Ag (a cell-to-cell spread-deficient vaccine vector lacking the Rh110 gene encoding the pp71 tegument protein). Bulk mRNA sequencing, differential gene expression, and functional enrichment analyses showed that these RhCMV/TB vaccines induce the innate and adaptive immune responses with specific transcriptomic signatures, including the IL-15-induced protective gene signature previously defined to be linked with protection against simian immunodeficiency virus (SIV) by the 68-1 RhCMV/SIV vaccine. While both vectors exhibited a transcriptomic response of the IL-15 protective signature in whole blood, we show that lack of pp71 does not maintain induction of the protective signature for the full duration of the study compared to the parental non-attenuated vector. Our observations indicate that RhCMV vector vaccines induce a transcriptomic response in whole blood that include a conserved IL-15 signature of which vector-encoded pp71 is an important component of response durability that upon future Mtb challenge may define specific vaccine protection outcomes against Mtb infection.

Differential Abundance of Protein Acylation in Mycobacterium tuberculosis Under Exposure to Nitrosative Stress.

Human macrophages generate antimicrobial reactive nitrogen species in response to infection by Mycobacterium tuberculosis (Mtb). Exposure to these redox-reactive compounds induces stress response in Mtb, which can affect posttranslational modifications (PTM). Here, we present the global analysis of the PTM acylation of Mtb proteins in response to a sublethal dose of nitrosative stress in the form of nitric oxide (NO) using label free quantification. A total of 6437 acylation events were identified on 1496 Mtb proteins, and O-acylation accounted for 92.2% of the events identified, while 7.8% were N-acylation events. About 22% of the sites identified were found to be acylated by more than one acyl-group. Furthermore, the abundance of each acyl-group decreased as their molecular weight increased. Quantitative PTM analysis revealed differential abundance of acylation in proteins involved in stress response, iron ion homeostasis, growth, energy metabolism, and antimicrobial resistance (AMR) induced by nitrosative stress over time. The results reveal a potential role of Mtb protein acylation in the bacterial stress responses and AMR. To our knowledge, this is the first report on global O-acylation profile of Mtb in response to NO. This will significantly improve our understanding of the changes in Mtb acylation under nitrosative stress, highly relevant for global health.

Protease shaving of Mycobacterium tuberculosis facilitates vaccine antigen discovery and delivery of novel cargoes to the Mtb surface.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of infectious disease death and lacks a vaccine capable of protecting adults from pulmonary TB. Studies have shown that Mtb uses a variety of mechanisms to evade host immunity. Secreted Mtb proteins such as Type VII secretion system substrates have been characterized for their ability to modulate anti-Mtb immunity; however, studies of other pathogens such as Salmonella Typhi and Staphylococcus aureus have revealed that outer membrane proteins can also interact with the innate and adaptive immune system. The Mtb outer membrane proteome has received relatively less attention due to limited techniques available to interrogate this compartment. We filled this gap by deploying protease shaving and quantitative mass spectrometry to identify Mtb outer membrane proteins which serve as nodes in the Mtb-host interaction network. These analyses revealed several novel Mtb proteins on the Mtb surface largely derived from the PE/PPE class of Mtb proteins, including PPE18, a component of a leading Mtb vaccine candidate. We next exploited the localization of PPE18 to decorate the Mtb surface with heterologous proteins and deliver these surface-engineered Mtb to the phagosome. Together, these studies reveal potential novel targets for new Mtb vaccines as well as facilitate new approaches to study difficult to study cellular compartments during infection.

Polypharmacology-Driven Discovery and Design of Highly Selective, Dual and Multitargeting Inhibitors of Mycobacterium tuberculosis - A Review

: The increasing demand for novel antitubercular agents has been the main 'force' of many TB research efforts due to the uncontrolled growing number of drug-resistant strains of M. tuberculosis in the clinical setting. Many strategies have been employed to address the drug-resistant issue, including a trend that is gaining attention, which is the design and discovery of Mtb inhibitors that are either dual- or multitargeting. The multiple-target design concept is not new in medicinal chemistry. With a growing number of newly discovered Mtb proteins, numerous targets are now available for developing new biochemical/cell-based assays and computer-aided drug design (CADD) protocols. To describe the achievements and overarching picture of this field in anti- infective drug discovery, we provide in this review small molecules that exhibit profound inhibitory activity against the tubercle bacilli and are identified to trace two or more Mtb targets. This review also presents emerging design methodologies for developing new anti-TB agents, particularly tailored to structure-based CADD. Dedicated on the special occasion of the 70th birthday of Prof. Dr. Ma. Alicia Aguinaldo, whose scientific efforts elevated antituberculosis drug discovery in the Philippines.

Screening of bioactive compounds from selected mushroom species against putative drug targets in Mycobacterium tuberculosis: a multi-target approach

Mycobacterium tuberculosis (Mtb) is a notorious pathogen that causes one of the highest mortalities globally. Due to a pressing demand to identify novel therapeutic alternatives, the present study aims to focus on screening the putative drug targets and prioritizing their role in antibacterial drug development. The most vital proteins involved in the Biotin biosynthesis pathway and the Lipoarabinomannan (LAM) pathway such as biotin synthase (bioB) and alpha-(1->6)-mannopyranosyltransferase A (mptA) respectively, along with other essential virulence proteins of Mtb were selected as drug targets. Among these, the ones without native structures were modelled and validated using standard bioinformatics tools. Further, the interactions were performed with naturally available lead molecules present in selected mushroom species such as Agaricus bisporus, Pleurotus djamor, Hypsizygus ulmarius. Through Gas Chromatography-Mass Spectrometry (GC-MS), 15 bioactive compounds from the methanolic extract of mushrooms were identified. Further, 4 were selected based on drug-likeness and pharmacokinetic screening for molecular docking analysis against our prioritized targets wherein Benz[e]azulene from Pleurotus djamor illustrated a good binding affinity with a LF rank score of −9.036 kcal mol −1 against nuoM (NADH quinone oxidoreductase subunit M) and could be used as a prospective candidate in order to combat Tuberculosis (TB). Furthermore, the stability of the complex are validated using MD Simulations and subsequently, the binding free energy was calculated using MM-GBSA analysis. Thus, the current in silico analysis suggests a promising role of compounds extracted from mushrooms in tackling the TB burden. Communicated by Ramaswamy H. Sarma

Genomic and phenotypic characterization of Mycobacterium tuberculosis' closest-related non-tuberculous mycobacteria.

Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis (Mtb) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb. We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb, was present only in M. decipiens, which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis (Mtb) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum. We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb.

Artemisinin derivatives as potential drug candidates against Mycobacterium tuberculosis: insights from molecular docking, MD simulations, PCA, MM/GBSA and ADMET analysis

ABSTRACT Tuberculosis, one of the most ancient and formidable infectious diseases, primarily arises from the pathogenic bacterium Mycobacterium tuberculosis (Mtb). Phosphoenolpyruvate carboxykinase (Pck), a potential drug target, is essential for the growth of Mtb that involves in the pathway of gluconeogenesis at the centre of phosphoenolpyruvate-pyruvate-oxaloacetate node. This study aims to understand drug-like properties of the compounds derived from artemisinin, and to investigate their inhibitory roles against Mtb. Molecular docking was performed on a set of 56 artemisinin compounds to identify their binding efficacy to the Mtb protein target. Subsequently, the selected top three complexes were subjected to molecular dynamics simulations. By the trajectory analysis, root mean square deviation and radius of gyration indicated the compactness of the systems without much fluctuation. The principal component analysis revealed that the complexes were less dynamic and energetically more favourable, and Gibbs energy landscape revealed the favourable energetic transitions between conformations. The artemisinin dimer primary alcohol holding good bioavailability scores appears highly stable in complex with Pck (MM/GBSA of −37.67 kcal/mol) that stands as a potential inhibitor of the Mtb target. However, further preclinical experiments and investigations are necessary to evaluate the intrinsic properties and to confirm effectiveness of the drug candidate.

Mycobacterium tuberculosis PE_PGRS38 Enhances Intracellular Survival of Mycobacteria by Inhibiting TLR4/NF-κB-Dependent Inflammation and Apoptosis of the Host.

Mycobacterium tuberculosis (Mtb) ranks as the most lethal human pathogen, able to fend off repeated attacks by the immune system or medications. PE_PGRS proteins are hallmarks of the pathogenicity of Mtb and contribute to its antigenic diversity, virulence, and persistence during infection. M. smegmatis is a nonpathogenic mycobacterium that naturally lacks PE_PGRS and is used as a model to express Mtb proteins. PE_PGRS has the capability to evade host immune responses and enhance the intracellular survival of M. smegmatis. Despite the intense investigations into PE_PGRS proteins, their role in tuberculosis remains elusive. We engineered the recombinant M. smegmatis strain Ms-PE_PGRS38. The result shows that PE_PGRS38 is expressed in the cell wall of M. smegmatis. PE_PGRS38 contributes to biofilm formation, confers permeability to the cell wall, and shows variable responses to exogenous stresses. PE_PGRS38 downregulated TLR4/NF-κB signaling in RAW264.7 macrophages and lung tissues of infected mice. In addition, PE_PGRS38 decreased NLRP3-dependent IL-1β release and limited pathogen-mediated inflammasome activity during infection. Moreover, PE_PGRS38 inhibited the apoptosis of RAW264.7 cells by downregulating the expression of apoptotic markers including Bax, cytochrome c, caspase-3, and caspase-9. In a nutshell, our findings demonstrate that PE_PGRS38 is a virulence factor for Mtb that enables recombinant M. smegmatis to survive by resisting and evading the host's immune responses during infection.

Genome mining of Mycobacterium tuberculosis: targeting SufD as a novel drug candidate through in silico characterization and inhibitor screening.

Tuberculosis (TB) stands as the second most fatal infectious disease globally, causing 1.3 million deaths in 2022. The resurgence of TB and the alarming rise of antibiotic resistance demand urgent call to develop novel antituberculosis drugs. Despite concerted efforts to control TB, the disease persists and spreads rapidly on a global scale. Targeting stress response pathways in Mycobacterium tuberculosis (Mtb) has become imperative to achieve complete eradication. This study employs subtractive genomics to identify and prioritize potential drug targets among the hypothetical proteins of Mtb, focusing on indispensable pathways. Amongst 177 essential hypothetical proteins, 152 were nonhomologous to human. These proteins participated in 34 pathways, and a 20-fold enrichment of SUF pathway genes led to its selection as a target pathway. Fe-S clusters are fundamental, widely distributed protein cofactors involved in vital cellular processes. The survival of Mtb in a hypoxic environment relies on the iron-sulfur (Fe-S) cluster biogenesis pathway for the repair of damaged Fe-S clusters. It also protects pathogen against drugs, ensuring controlled iron utilization and contributing to drug resistance. In Mtb, six proteins of Fe-S cluster assembly pathway are encoded by the suf operon. The present study was focused on SufD because of its role in iron acquisition and prevention of Fenton reaction. The research further delves into the in silico characterization of SufD, utilizing bioinformatics tools for sequence and structure based analysis. The protein's structural features, including the identification of conserved regions, motifs, and 3D structure prediction enhanced functional annotation. Target based virtual screening of compounds from the ChEMBL database resulted in 12 inhibitors with best binding affinities. Drug likeness and ADMET profiling of potential inhibitors identified promising compounds with favorable drug-like properties. The study also involved cloning in SUMO-pRSF-Duet1 expression vector, overexpression, and purification of recombinant SufD from E. coli BL21 (DE3) cells. Optimization of expression conditions resulted in soluble production, and subsequent purification highlighting the efficacy of the SUMO fusion system for challenging Mtb proteins in E. coli. These findings provide valuable insights into pharmacological targets for future experimental studies, holding promise for the development of targeted therapy against Mtb.

A seven-sex species recognizes self and non-self mating-type via a novel protein complex

Although most species have two sexes, multisexual (or multi-mating type) species are also widespread. However, it is unclear how mating-type recognition is achieved at the molecular level in multisexual species. The unicellular ciliate Tetrahymena thermophila has seven mating types, which are determined by the MTA and MTB proteins. In this study, we found that both proteins are essential for cells to send or receive complete mating-type information, and transmission of the mating-type signal requires both proteins to be expressed in the same cell. We found that MTA and MTB form a mating-type recognition complex that localizes to the plasma membrane, but not to the cilia. Stimulation experiments showed that the mating-type-specific regions of MTA and MTB mediate both self- and non-self-recognition, indicating that T. thermophila uses a dual approach to achieve mating-type recognition. Our results suggest that MTA and MTB form an elaborate multifunctional protein complex that can identify cells of both self and non-self mating types in order to inhibit or activate mating, respectively.

A seven-sex species recognizes self and non-self mating-type via a novel protein complex.

Although most species have two sexes, multisexual (or multi-mating type) species are also widespread. However, it is unclear how mating-type recognition is achieved at the molecular level in multisexual species. The unicellular ciliate Tetrahymena thermophila has seven mating types, which are determined by the MTA and MTB proteins. In this study, we found that both proteins are essential for cells to send or receive complete mating-type information, and transmission of the mating-type signal requires both proteins to be expressed in the same cell. We found that MTA and MTB form a mating-type recognition complex that localizes to the plasma membrane, but not to the cilia. Stimulation experiments showed that the mating-type-specific regions of MTA and MTB mediate both self- and non-self-recognition, indicating that T. thermophila uses a dual approach to achieve mating-type recognition. Our results suggest that MTA and MTB form an elaborate multifunctional protein complex that can identify cells of both self and non-self mating types in order to inhibit or activate mating, respectively.

Effect of glycosylation on the affinity of the MTB protein Ag85B for specific antibodies: towards the design of a dual-acting vaccine against tuberculosis

BackgroundTo create a dual-acting vaccine that can fight against tuberculosis, we combined antigenic arabino-mannan analogues with the Ag85B protein. To start the process, we studied the impact of modifying different parts of the Ag85B protein on its ability to be recognized by antibodies.ResultsThrough our research, we discovered that three modified versions of the protein, rAg85B-K30R, rAg85B-K282R, and rAg85B-K30R/K282R, retained their antibody reactivity in healthy individuals and those with tuberculosis. To further test the specificity of the sugar AraMan for AraMan antibodies, we used Human Serum Albumin glycosylated with AraMan-IME and Ara3Man-IME. Our findings showed that this specific sugar was fully and specifically modified. Bio-panning experiments revealed that patients with active tuberculosis exhibited a higher antibody response to Ara3Man, a sugar found in lipoarabinomannan (LAM), which is a major component of the mycobacterial cell wall. Bio-panning with anti-LAM plates could eliminate this increased response, suggesting that the enhanced Ara3Man response was primarily driven by antibodies targeting LAM. These findings highlight the importance of Ara3Man as an immunodominant epitope in LAM and support its role in eliciting protective immunity against tuberculosis. Further studies evaluated the effects of glycosylation on the antibody affinity of recombinant Ag85B and its variants. The results indicated that rAg85B-K30R/K282R, when conjugated with Ara3Man-IME, demonstrated enhanced antibody recognition compared to unconjugated or non-glycosylated versions.ConclusionsCoupling Ara3Man to rAg85B-K30R/K282R could lead to the development of effective dual-acting vaccines against tuberculosis, stimulating protective antibodies against both AraMan and Ag85B, two key tuberculosis antigens.

Застосування інформаційної технології BLAST для пошуку мікроорганізмів, потенційних продуцентів біогенних магнітних наночасток, які призводять до серцевих захворювань

With the help of BLAST information technology, the alignment of MTB proteins with the proteins of pathogenic and conditionally pathogenic microorganisms, causative agents of heart diseases in humans, was carried out using the methods of comparative genomics.

Mucosal and systemic antigen-specific antibody responses correlate with protection against active tuberculosis in nonhuman primates

Increasing evidence supports that antibodies can protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially in the airways, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) versus uncontrolled infection (TB) in nonhuman primates. In a case-control design, using non-parametric group comparisons with false discovery rate adjustments, we assessed antibodies in 57 cynomolgus macaques which, following low-dose airway Mtb infection, developed either LTBI or TB. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to, two-, and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays. Macaques that developed LTBI (n=36) had significantly increased airway and plasma IgA reactivities to specific arabinomannan (AM) motifs prior to Mtb infection compared to those that developed TB (n=21; p<0.01, q<0.05). Furthermore, LTBI macaques had higher plasma IgG reactivity to protein MTB32A (Rv0125) early post Mtb infection (p<0.05) and increasing airway IgG responses to some proteins over time. Our results support a protective role of pre-existing mucosal (lung) and systemic IgA to specific Mtb glycan motifs, suggesting that prior exposure to nontuberculous mycobacteria could be protective against TB. They further suggest that IgG to Mtb proteins early post infection could provide an additional protective mechanism. These findings could inform TB vaccine development strategies. NIH/NIAID AI117927, AI146329, and AI127173 to JMA.

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.

Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells.

Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses.

Non-clinical evaluation of local and systemic immunity induced by different vaccination strategies of the candidate tuberculosis vaccine M72/AS01

A new efficacious tuberculosis vaccine targeting adolescents/adults represents an urgent medical need. The M72/AS01E vaccine candidate protected half of the latently-infected adults against progression to pulmonary tuberculosis in a Phase IIb trial (NCT01755598). We report that three immunizations of mice, two weeks apart, with AS01-adjuvanted M72 induced polyfunctional, Th1-cytokine-expressing M72-specific CD4+/CD8+ T cells in blood and lungs, with the highest frequencies in lungs. Antigen-dose reductions across the three vaccinations skewed pulmonary CD4+ T-cell profiles towards IL-17 expression. In blood, reducing antigen and adjuvant doses of only the third injection (to 1/5th or 1/25th of those of the first injections) did not significantly alter CD4+ T-cell/antibody responses; applying a 10-week delay for the fractional third dose enhanced antibody titers. Delaying a full-dose booster enhanced systemic CD4+ T-cell and antibody responses. Cross-reactivity with PPE and non-PPE proteins was assessed, as Mycobacterium tuberculosis (Mtb) virulence factors and evasion mechanisms are often associated with PE/PPE proteins, to which Mtb39a (contained in M72) belongs. In silico/in vivo analyses revealed that M72/AS01 induced cross-reactive systemic CD4+ T-cell responses to epitopes in a non-vaccine antigen (putative latency-associated Mtb protein PPE24/Rv1753c). These preclinical data describing novel mechanisms of M72/AS01-induced immunity could guide future clinical development of the vaccine.

The heparin-binding hemagglutinin protein of Mycobacterium tuberculosis is a nucleoid-associated protein

Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor.

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps.

Neutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence. Here, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy. For the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation. These findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens.

Design, Synthesis, and in vitro Biological Evaluation of ROS‐Generating Phenanthridin‐trione‐Epoxide Conjugates as Agents against Mycobacterium tuberculosis

AbstractTuberculosis (TB) is one of the devasting infectious diseases and continues to spread among people despite having several specific drugs. Total eradication of TB is one of the shared interests of both the World Health Organization (WHO) and India globally. A library of antitubercular 6‐((1‐(aryl/heteroaryl)‐1H‐1,2,3‐triazol‐4‐yl)methyl)oxireno[2,3‐b] phenanthridine‐5,7,9(6H,7aH,8aH)‐trione (6 a–e) has been prepared in five steps including click chemistry and tested against active and dormant strains of Mycobacterium tuberculosis H37Ra using XRMA protocol. The result showed the inhibitory potential of 6 d IC50 at 0.74 μg/mL concentration against active strain and at 0.9 μg/mL against the dormant strain of Mtb. ROS generating ability of the compounds has been confirmed by luminol, H2O2, and glutathione assays. The molecular docking with the thioredoxin protein of Mtb showed a docking score of −9.6 Kcal/mol. To understand the mechanism with the thioredoxin protein of Mtb, the adduct formation of compounds with cystine was confirmed with HPLC. The involvement of lead molecules with existing drugs can be helpful in the eradication of tuberculosis.