Mycobacterium tuberculosis Rv2179c Protein Establishes a New Exoribonuclease Family with Broad Phylogenetic Distribution

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3′ single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom.     

The Mycobacterium tuberculosis Rv2745c Plays an Important Role in Responding to Redox Stress

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death from an infectious disease worldwide. Over the course of its life cycle in vivo, Mtb is exposed to a plethora of environmental stress conditions. Temporal regulation of genes involved in sensing and responding to such conditions is therefore crucial for Mtb to establish an infection. The Rv2745c (clgR) gene encodes a Clp protease gene regulator that is induced in response to a variety of stress conditions and potentially plays a role in Mtb pathogenesis. Our isogenic mutant, Mtb:ΔRv2745c, is significantly more sensitive to in vitro redox stress generated by diamide, relative to wild-type Mtb as well as to a complemented strain. Together with the fact that the expression of Rv2745c is strongly induced in response to redox stress, these results strongly implicate a role for ClgR in the management of intraphagosomal redox stress. Additionally, we observed that redox stress led to the dysregulation of the expression of the σ^H/σ^E regulon in the isogenic mutant, Mtb:ΔRv2745c. Furthermore, induction of clgR in Mtb and Mtb:ΔRv2745c (comp) did not lead to Clp protease induction, indicating that clgR has additional functions that need to be elucidated. Our data, when taken together with that obtained by other groups, indicates that ClgR plays diverse roles in multiple regulatory networks in response to different stress conditions. In addition to redox stress, the expression of Rv2745c correlates with the expression of genes involved in sulfate assimilation as well as in response to hypoxia and reaeration. Clearly, the Mtb Rv2745c-encoded ClgR performs different functions during stress response and is important for the pathogenicity of Mtb in-vivo, regardless of its induction of the Clp proteolytic pathway.     

Unexpected Role for IL-17 in Protective Immunity against Hypervirulent Mycobacterium tuberculosis HN878 Infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world''s population. Among these infections, clinical isolates belonging to the W-Beijing appear to be emerging, representing about 50% of Mtb isolates in East Asia, and about 13% of all Mtb isolates worldwide. In animal models, infection with W-Beijing strain, Mtb HN878, is considered “hypervirulent” as it results in increased mortality and causes exacerbated immunopathology in infected animals. We had previously shown the Interleukin (IL) -17 pathway is dispensable for primary immunity against infection with the lab adapted Mtb H37Rv strain. However, it is not known whether IL-17 has any role to play in protective immunity against infection with clinical Mtb isolates. We report here that lab adapted Mtb strains, such as H37Rv, or less virulent Mtb clinical isolates, such as Mtb CDC1551, do not require IL-17 for protective immunity against infection while infection with Mtb HN878 requires IL-17 for early protective immunity. Unexpectedly, Mtb HN878 induces robust production of IL-1β through a TLR-2-dependent mechanism, which supports potent IL-17 responses. We also show that the role for IL-17 in mediating protective immunity against Mtb HN878 is through IL-17 Receptor signaling in non-hematopoietic cells, mediating the induction of the chemokine, CXCL-13, which is required for localization of T cells within lung lymphoid follicles. Correct T cell localization within lymphoid follicles in the lung is required for maximal macrophage activation and Mtb control. Since IL-17 has a critical role in vaccine-induced immunity against TB, our results have far reaching implications for the design of vaccines and therapies to prevent and treat emerging Mtb strains. In addition, our data changes the existing paradigm that IL-17 is dispensable for primary immunity against Mtb infection, and instead suggests a differential role for IL-17 in early protective immunity against emerging Mtb strains.     

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 Å), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Pro- and Anti-Inflammatory Cytokines against Rv2031 Are Elevated during Latent Tuberculosis: A Study in Cohorts of Tuberculosis Patients,Household Contacts and Community Controls in an Endemic Setting

Tuberculosis (TB) is among the leading causes of morbidity and mortality. The causative agent, Mycobacterium tuberculosis (Mtb), has evolved virulent factors for entry, survival, multiplication and immune evasion. Rv2031 (also called alpha crystallin, hspX, 16-kDa antigen), one of the most immunogenic latency antigens, is believed to play a key role in long-term viability of Mtb. Here, we report the dynamics of pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokines against Rv2031 using whole blood assay in human cohorts in a TB endemic setting. Cytokine responses to ESAT-6-CFP-10 were also measured for comparison. Blood samples were collected from smear positive pulmonary TB patients and their contacts at baseline, 6 and 12 months, and from community controls at entry. At baseline, 54.4% of controls and 73.2% of contacts were QFT-GIT test positive. Baseline IFN-γ, TNF-α and IL-10 responses to Rv2031 were significantly higher in controls compared to contacts and untreated patients (p<0.001). Furthermore, untreated patients had significantly higher TNF-α and IL-10 responses to Rv2031 compared to contacts (p<0.001). In contacts and treated patients, IFN-γ, TNF-α and IL-10 responses to Rv2031 significantly increased over 12 months (p<0.0001) and became comparable with the corresponding levels in controls. There was a positive and significant correlation between Rv2031 and ESAT-6-CFP-10 specific cytokine responses in each study group. The fact that the levels of IFN-γ, TNF-α and IL-10 against Rv2031 were highest during latent TB infection may indicate their potential as markers of protection against TB. Taken together, the findings of this study suggest the potential of IFN-γ, TNF-α and IL-10 against Rv2031 as biomarkers of the host response to Mtb during convalescence from, and the absence of, active tuberculosis.     

Mycobacterium tuberculosis: the honey badger of pathogens

Mycobacterium tuberculosis is a fascinating object of study: it is one of the deadliest pathogens of humankind, able to fend off persistent attacks by the immune system or drugs Subject Categories: Immunology, Microbiology, Virology & Host Pathogen Interaction, Chemical Biology

I have always been interested in infectious diseases since I began to study biology. As a graduate student, my pathogen of choice was Salmonella typhimurium, which typically causes diarrhea that can potentially lead to death. Salmonella''s rapid doubling time, and the availability of elegant genetic tools, a wealth of reagents, and a robust animal infection model put this bug at the apex of ideal host–pathogen systems to study. After I finished my PhD studies—and for reasons to be told another day—my career took an unexpected detour into an area of research I never thought I would be interested in: I went from the sublime to the ridiculous, from Salmonella to Mycobacterium tuberculosis (Mtb), an excruciatingly slow‐growing bacillus with few genetic tools, a paucity of reagents, and an animal model in which an experiment can take a year or longer. Having said all of that, I love working on this pathogen.For those of you who do not know much about Mtb, it is the world''s deadliest bacterium that causes the disease tuberculosis (TB). As Mtb is spread in aerosol droplets coughed up by infected individuals, TB is highly contagious, and about one‐third of the world''s population may be infected with Mtb, although this number has been reasonably challenged (Behr et al, 2021). Even if the numbers of latent or asymptomatic infections are debated, there are some back‐of‐the‐envelope estimates that Mtb has killed more than a billion humans over the millennia. Although TB is often treatable with antibiotics and most Mtb‐infected healthy individuals are asymptomatic, the appearance of multi‐drug‐resistant Mtb and HIV/AIDS has further increased the number of deaths caused by this pathogen.How has Mtb become such a successful pathogen? For one, we lack an effective vaccine to prevent infection. Many readers may point out that they have themselves been given a TB vaccine; known as “BCG” for bacille Calmette–Guérin, this is a laboratory‐attenuated strain of a species related to Mtb called Mycobacterium bovis. While BCG does provide some protection for children against TB, BCG is essentially ineffective against pulmonary TB in adults. For this reason, it is not used in the USA and many other countries.Another major challenge to treating TB has been a lack of antimicrobials that can access Mtb bacilli in privileged sites known as granulomas, which are cell‐fortified structures our immune system builds to contain microbial growth. In addition to the granuloma walls, Mtb has a highly complex cell envelope that protects it from many small molecules. I imagine that antimicrobial molecules have the challenging task of reaching an enemy shielded in armor, hiding deep inside a castle keep, and surrounded by a vast moat, and an army of orcs.On top of these therapeutic barriers, most antimicrobials work on metabolically active or growing bacteria. Mtb, however, grows very slowly, with a doubling time under optimal laboratory conditions of about 20 h—compared with 20 min for Salmonella. Moreover, Mtb is believed to enter a “persistent” or “latent” state in its natural host with limited cell divisions. This extremely slow growth makes treatment a long and tedious prospect: 6–12 months of antibiotic treatment are generally required, during which time one cannot drink alcohol due to the potential liver toxicity of the drugs. Believe it or not, there are people who would rather refuse TB treatment than give up alcohol for a few months. Additionally, the perception of “feeling cured” after a few weeks of TB therapy can also lead to a lapse in compliance. The consequence of failing to clear a partially treated infection is the emergence of drug resistance, which has created strains that are extensively resistant to most frontline TB drugs.When thinking about the difficulty of curing Mtb infections, I am reminded of the fierce and fearless honey badger, which came to fame through a viral YouTube video. The narrator points out how honey badgers “don''t care” about battling vicious predators in order to get food: venomous snakes, stinging bees—you name it. I once saw a photo of a honey badger that looked more like a pin cushion, harpooned with numerous porcupine quills. This battle royale of the wilderness is a perfect analogy of Mtb versus the immune system: Like the honey badger, Mtb really don''t care.Vaccines primarily work by coaxing our immune system to make antibodies that neutralize foreign invaders, most typically viruses, but also bacteria, some of which have evolved mechanisms to evade detection by antibodies or otherwise render them useless. In most cases, phagocytes then gobble up and kill invading bacteria. While phagocytes are critical in controlling Mtb infections, it is unclear which of their molecules or “effectors” act as executioners of Mtb. For example, nitric oxide and copper play roles in controlling Mtb in a mouse model, but it is unknown how these molecules exert their host‐protective activity, and whether or not they play a similar role in humans. Furthermore, despite the production of these antibacterial effectors—the “porcupine quills”—Mtb often persists due to intrinsic resistance mechanisms. Thus, while our immune system may have the tools to keep Mtb under control, it falls short of eradicating it from our bodies and, in many cases, fails to prevent the progression of the disease. Perhaps a most worrying observation is that prior infection, which is generally considered the most effective path to immunity for many infectious diseases, does not consistently protect against reinfection with Mtb.The above facts have left the TB field scrambling to identify new ways to fight this disease. Much of this work requires that researchers understand both the fundamental processes of the bacterium and its host. Studies of human populations around the globe have revealed differences in susceptibility to infection, the genetic and immunological bases of which are being investigated (Bellamy et al, 2000; Berry et al, 2010; Möller et al, 2010). These studies have made researchers increasingly aware that how the immune system responds to Mtb may play a critical role in disease control. For example, understanding why some individuals or populations are more or less susceptible to TB may help in the development of better vaccines. Also, the more we understand what makes this pathogen so resilient to the immune system could facilitate the development of new antibacterial drugs or host‐directed therapies. These questions can only be answered once we fully understand how the host combats Mtb infections, and how the bacteria counteract these host defenses. While it is a daunting endeavor, my hope is that the efforts of many laboratories around the world will get a better understanding of the host–Mtb interface and ultimately help to eradicate this disease for good.     

Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.     

Genetic Incorporation of Unnatural Amino Acids into Proteins in Mycobacterium tuberculosis

New tools are needed to study the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), to facilitate new drug discovery and vaccine development. We have developed methodology to genetically incorporate unnatural amino acids into proteins in Mycobacterium smegmatis, BCG and Mtb, grown both extracellularly in culture and inside host cells. Orthogonal mutant tRNA^Tyr/tyrosyl-tRNA synthetase pairs derived from Methanococcus jannaschii and evolved in Escherichia coli incorporate a variety of unnatural amino acids (including photocrosslinking, chemically reactive, heavy atom containing, and immunogenic amino acids) into proteins in response to the amber nonsense codon. By taking advantage of the fidelity and suppression efficiency of the MjtRNA/pIpaRS pair in mycobacteria, we are also able to use p-iodophenylalanine to induce the expression of proteins in mycobacteria both extracellularly in culture and inside of mammalian host cells. This provides a new approach to regulate the expression of reporter genes or mycobacteria endogenous genes of interest. The establishment of the unnatural amino acid expression system in Mtb, an intracellular pathogen, should facilitate studies of TB biology and vaccine development.     

Discovery of a unique Mycobacterium tuberculosis protein through proteomic analysis of urine from patients with active tuberculosis

Identification of pathogen-specific biomarkers present in patients' serum or urine samples can be a useful diagnostic approach. In efforts to discover Mycobacterium tuberculosis (Mtb) biomarkers we identified by mass spectroscopy a unique 21-mer Mtb peptide sequence (VVLGLTVPGGVELLPGVALPR) present in the urines of TB patients from Zimbabwe. This peptide has 100% sequence homology with the protein TBCG_03312 from the C strain of Mtb (a clinical isolate identified in New York, NY, USA) and 95% sequence homology with Mtb oxidoreductase (MRGA423_21210) from the clinical isolate MTB423 (identified in Kerala, India). Alignment of the genes coding for these proteins show an insertion point mutation relative to Rv3368c of the reference H37Rv strain, which generated a unique C-terminus with no sequence homology with any other described protein. Phylogenetic analysis utilizing public sequence data shows that the insertion mutation is apparently a rare event. However, sera from TB patients from distinct geographical areas of the world (Peru, Vietnam, and South Africa) contain antibodies that recognize a purified recombinant C-terminus of the protein, thus suggesting a wider distribution of isolates that produce this protein.     

HspX protein as a candidate vaccine against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: an overview

Background

Tuberculosis (TB) is a contagious infectious disease caused by Mycobacterium tuberculosis (Mtb). This disease with two million deaths per year has the highest mortality rate among bacterial infections. The only available vaccine against TB is BCG vaccine. BCG is an effective vaccine against TB in childhood, however, due to some limitations, has not proper efficiency in adults. Also, BCG cannot produce an adequately protective response against reactivation of latent infections.

Objective

In the present study we will review the most recent findings about contribution of HspX protein in the vaccines against tuberculosis.

Methods

Therefore, many attempts have been made to improve BCG or to find its replacement. Most of the subunit vaccines for TB in various phases of clinical trials were constructed as prophylactic vaccines using Mtb proteins expressed in the replicating stage. These vaccines might prevent active TB but not reactivation of latent tuberculosis infection (LTBI). A literature search was performed on various online databases (PubMed, Scopus, and Google Scholar) regarding the roles of HspX protein in tuberculosis vaccines.

Results

Ideal subunit post-exposure vaccines should target all forms of TB infection, including active symptomatic and dormant (latent) asymptomatic forms. Among these subunit vaccines, HspX is the most important latent phase antigen of M. tuberculosis with a strong immunological response. There are many studies that have evaluated the immunogenicity of this protein to improve TB vaccine.

Conclusion

According to the studies, HspX protein is a good candidate for development of subunit vaccines against TB infection.

     

A Novel Tuberculosis Antigen Identified from Human Tuberculosis Granulomas

Tuberculosis is a global infectious disease caused by Mycobacterium tuberculosis (Mtb). Although novel Mtb biomarkers from both the pathogen and host have been studied, more breakthroughs are still needed to meet different clinic requirements. In an effort to identify Mtb antigens, chaperone-peptide complexes were purified from TB infected lungs using free-solution isoelectric focusing combined with high resolution LTQ Orbitrap Velos mass spectrometry. Antigen specific cellular immune responses in vitro were then examined. Those efforts led to the identification of six Mtb peptides only identified in Tuberculosis lung samples and that were not found in the control samples. Additionally, antigen-specific IFN-γ secretion, T-cell proliferation, cytokine expression, and a cytotoxic assay were also evaluated. Among the peptides isolated, we identified a 34 amino acid peptide named PKA_p belonging to a serine/threonine–protein kinase, as being able to generate Mtb-specific cellular immune responses as noted by elevated antigen-specific cytokine secretion levels, increased CD8⁺ T-cell proliferation and a strong cytotoxic lymphocyte (CTL) response. Moreover, the immune stimulating abilities of PKA_p were further validated in vivo, with target peptide immunized mice showing an increased cellular IFN-γ in both the lungs and spleen without causing immunopathogenesis. In conclusion, we identified novel functional Mtb antigens directly from the granulomatous lesions of Tuberculosis patients, inducing not only significant antigen-specific IFN-γ secretion but also a marked cytotoxic lymphocyte functional response. These findings indicated that PKA_p has potential as a novel antigen biomarker for vaccine development.Mycobacterium tuberculosis (Mtb),¹ the infectious agent that causes tuberculosis, is associated with an estimated 1.4 million deaths per year and remains a major global health concern (1). Current research and diagnostics have focused on antigen screening and biomarker discovery, with most antigen screening methods focused on the bacterial pathogen itself, with less focus on the Mtb infected host (2). The pathogenic progression of TB occurs in the lungs, making the characterization of any functional antigens existing in the lungs during infection potentially useful for immunotherapy or vaccine development. The immune response to an Mtb infection results in the formation of a granuloma that initially contains bacterial expansion, but may fail to eliminate the pathogen (3, 4). This immune response brings with the possibility of identifying Mtb functional antigens in the lung tissue and to gain a clearer understanding of the immune mechanisms (5, 6). Although it has been well studied that a T-cell mediated adaptive immune response plays a central role during Mtb infection and is crucial in both protection and pathogenesis, a better understanding of the antigen induced immune response and correlations to pathogenicity is necessary (2, 7).It has been reported that heat shock proteins (such as the HSP70 family members) and others chaperones such as Gp96 can specifically bind many hydrophobic sequences, enabling them to bind foreign peptides associated with intracellular bacterial or viral challenge (8), such as Gp96 associating with a HBV-specific peptide (9). Previous studies have shown that chaperone-peptide complexes can induce a disease-specific immune response (10–12), with the gp96-peptide complex from H37Rv infected cells able to induce a protective antigen specific immune response (13). Currently, no Mtb chaperone-associated peptides have been isolated directly from patients, thus the present study explores the possible existence of these complexes in TB lung tissue.To achieve this objective, the free-solution isoelectric focusing (FS-IEF) technique, which has been reported to enrich chaperones in cell lysates or tissue samples, was combined with Linear Trap Quadrupole (LTQ) OrbitrapVelos mass spectrometry, which was used to identify the associated Mtb peptides. Using these techniques, we obtained chaperone-rich cell lysates from the granulomatous lung lesions of active TB patients and identified six Mtb-associated peptides not noted in the control samples. Among them, a peptide (PKA_p) derived from Mtb Protein Kinase A not only contributed to significant antigen-specific IFN-γ secretion, but also contributed to CTL function and T-cell proliferation. Importantly, murine immunization with PKA_p derived peptides elicited an antigen-specific cellular activation without the occurrence of immune pathogenesis.     

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

Background

Novel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB.

Methodology/Principal Findings

We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σ^B (the σ^B regulon) was turned on by THZ treatment. σ^B has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σ^B regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σ^H and σ^E, two alternate stress response sigma factors that induce the expression of σ^B, exhibited higher sensitivity to THZ, indicating that the presence and expression of σ^B allows Mtb to resist the impact of THZ. Conditional induction of σ^B levels increased the survival of Mtb in the presence of THZ.

Conclusions/Significance

THZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σ^H, σ^E and σ^B plays a crucial role in protecting the pathogen against cell-envelope damage.     

DRAM1 promotes the targeting of mycobacteria to selective autophagy

Autophagy provides an important defense mechanism against intracellular bacteria, such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease (TB). We recently reported that pathogen recognition and antibacterial autophagy are connected by the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor (TLR)-MYD88-NFKB innate immunity signaling pathway. Having shown that DRAM1 colocalizes with Mtb in human macrophages, we took advantage of a zebrafish model for TB to investigate the function of DRAM1 in autophagic host defense in vivo. We found that DRAM1 protects the zebrafish host from infection with Mycobacterium marinum (Mm), a close relative of Mtb. Overexpression of DRAM1 increases autophagosome formation and promotes autophagic flux by a mechanism dependent on the cytosolic DNA sensor TMEM173/STING and the ubiquitin receptor SQSTM1/p62. Here we summarize and discuss the implications of these findings.     

Evaluation of Rv0220, Rv2958c,Rv2994 and Rv3347c of Mycobacterium tuberculosis for serodiagnosis of tuberculosis

Tuberculosis (TB), the leading cause of death among infectious diseases worldwide, is caused by Mycobacterium tuberculosis (M. tuberculosis). Early accurate diagnosis means earlier prevention, treatment and control of TB. To confirm efficient diagnostic antigens for M. tuberculosis, the serodiagnosis value of four recombinant proteins including Rv0220, Rv2958c, Rv2994 and Rv3347c was evaluated in this study. The specificities and sensitivities of four recombinant proteins were determined based on enzyme‐linked immunosorbent assay (ELISA) by screening sera from smear‐positive pulmonary TB patients (n = 92), uninfected individuals (n = 60) and patients with Mycoplasma pneumoniae (n = 32) that potentially cross‐react with M. tuberculosis. The ELISAs showed that Rv0220, Rv2958c, Rv2994 and Rv3347c exhibited high specificities and sensitivities in detecting immunoglobulin G (IgG) antibody, with 98.3/91.3%, 91.7/85.9%, 93.3/89.1% and 93.3/80.4% respectively. According to the receiver‐operating characteristic (ROC) analysis, the area under the ROC of the target proteins was 0.988, 0.969, 0.929 and 0.945 respectively. Western blot was established to evaluate the immunoreactivities of target proteins to mice and human sera. Results demonstrated that Rv0220, Rv2958c, Rv2994 and Rv3347c could specifically recognize TB‐positive sera and the sera of mice immunized with the corresponding protein. Thus, Rv0220, Rv2958c, Rv2994 and Rv3347c were valuable potential diagnostic antigens for M. tuberculosis.     

Crystal structure of the arginine repressor protein in complex with the DNA operator from Mycobacterium tuberculosis

The arginine repressor (ArgR) from Mycobacterium tuberculosis (Mtb) is a gene product encoded by the open reading frame Rv1657. It regulates the l-arginine concentration in cells by interacting with ARG boxes in the promoter regions of the arginine biosynthesis and catabolism operons. Here we present a 2.5-Å structure of MtbArgR in complex with a 16-bp DNA operator in the absence of arginine. A biological trimer of the protein-DNA complex is formed via the crystallographic 3-fold symmetry axis. The N-terminal domain of MtbArgR has a winged helix-turn-helix motif that binds to the major groove of the DNA. This structure shows that, in the absence of arginine, the ArgR trimer can bind three ARG box half-sites. It also reveals the structure of the whole MtbArgR molecule itself containing both N-terminal and C-terminal domains.     

DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters.     

Self-clearance of Mycobacterium tuberculosis infection: implications for lifetime risk and population at-risk of tuberculosis disease

Background: it is widely assumed that individuals with Mycobacterium tuberculosis (Mtb) infection remain at lifelong risk of tuberculosis (TB) disease. However, there is substantial evidence that self-clearance of Mtb infection can occur. We infer a curve of self-clearance by time since infection and explore its implications for TB epidemiology. Methods and findings: data for self-clearance were inferred using post-mortem and tuberculin-skin-test reversion studies. A cohort model allowing for self-clearance was fitted in a Bayesian framework before estimating the lifetime risk of TB disease and the population infected with Mtb in India, China and Japan in 2019. We estimated that 24.4% (17.8–32.6%, 95% uncertainty interval (UI)) of individuals self-clear within 10 years of infection, and 73.1% (64.6–81.7%) over a lifetime. The lifetime risk of TB disease was 17.0% (10.9–22.5%), compared to 12.6% (10.1–15.0%) assuming lifelong infection. The population at risk of TB disease in India, China and Japan was 35–80% (95% UI) smaller in the self-clearance scenario. Conclusions: the population with a viable Mtb infection may be markedly smaller than generally assumed, with such individuals at greater risk of TB disease. The ability to identify these individuals could dramatically improve the targeting of preventive programmes and inform TB vaccine development, bringing TB elimination within reach of feasibility.