Systematic Evaluation of Mycobacterium tuberculosis Proteins for Antigenic Properties Identifies Rv1485 and Rv1705c as Potential Protective Subunit Vaccine Candidates.

The lack of efficacious vaccines against Mycobacterium tuberculosis (MTB) infection is a limiting factor in the prevention and control of tuberculosis (TB), the leading cause of death from an infectious agent. Improvement or replacement of the BCG vaccine with one that reliably protects all age groups is urgent. Concerns exist that antigens currently being evaluated are too homogeneous. To identify new protective antigens, we screened 1,781 proteins from a high-throughput proteome-wide protein purification study for antigenic activity. Forty-nine antigens (34 previously unreported) induced antigen-specific gamma interferon (IFN-γ) release from peripheral blood mononuclear cells (PBMCs) derived from 4,452 TB and suspected TB patients and 167 healthy donors. Three (Rv1485, Rv1705c, and Rv1802) of the 20 antigens evaluated in a BALB/c mouse challenge model showed protective efficacy, reducing lung CFU counts by 66.2%, 75.8%, and 60%, respectively. Evaluation of IgG2a/IgG1 ratios and cytokine release indicated that Rv1485 and Rv1705c induce a protective Th1 immune response. Epitope analysis of PE/PPE protein Rv1705c, the strongest candidate, identified a dominant epitope in its extreme N-terminal domain accounting for 90% of its immune response. Systematic preclinical assessment of antigens Rv1485 and Rv1705c is warranted.

Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III-A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA-seq approaches, we show it is active in invader defense and has features atypical of type III-A systems: mature CRISPR RNA (crRNA) in its crRNA-CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3'-end processing after Cas6 cleavage of repeat RNA 8 nt from its 3' end. crRNAs generated resemble mature crRNA in type I systems, having both 5' (8 nt) and 3' (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3' hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis, providing a foundation for investigating the potential of a type III-A-based genome editing system.-Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.-E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.

Retrospective Analysis of False-Positive and Disputed Rifampin Resistance Xpert MTB/RIF Assay Results in Clinical Samples from a Referral Hospital in Hunan, China.

Concerns about the specificity of the Xpert MTB/RIF (Xpert) assay have arisen, as false-positive errors in the determination of Mycobacterium tuberculosis complex (MTBC) infection and rifampin (RIF) resistance in clinical practice have been reported. Here, we investigated 33 cases where patients were determined to be RIF susceptible using the Bactec MGIT 960 (MGIT) culture system but RIF resistant using the Xpert assay. Isolates from two of these patients were found not to have any mutations in the rifampin resistance determining region (RRDR) region of rpoB and had good treatment outcomes with first-line antituberculosis (anti-TB) drugs. The remaining 31 patients included 5 new cases and 26 previously treated patients. A large number of well-documented disputed mutations, including Leu511Pro, Asp516Tyr, His526Asn, His526Leu, His526Cys, and Leu533Pro, were detected, and mutations, including a 508 to 509 deletion and His526Gly, were described here as disputed mutations for the first time. Twenty-one (81%) of the 26 previously treated patients had poor treatment outcomes, and isolates from 19 (90%) of these 21 patients were resistant to isoniazid (INH) as determined using the MGIT culture system. Twenty-seven of the 31 isolates with disputed rpoB mutations were phenotypically resistant to INH, 21 (78%) being predicted by GenoType MTBDRplus to have a high level of INH resistance. Most (77.4%) of the isolates with disputed mutations were of the Beijing lineage. These findings have implications for the interpretation of false-positive and disputed rifampin resistance Xpert MTB/RIF results in clinical samples and provide guidance on how clinicians should manage patients carrying isolates with disputed rpoB mutations.

An automated approach for global identification of sRNA-encoding regions in RNA-Seq data from Mycobacterium tuberculosis.

Deep-sequencing of bacterial transcriptomes using RNA-Seq technology has made it possible to identify small non-coding RNAs, RNA molecules which regulate gene expression in response to changing environments, on a genome-wide scale in an ever-increasing range of prokaryotes. However, a simple and reliable automated method for identifying sRNA candidates in these large datasets is lacking. Here, after generating a transcriptome from an exponential phase culture of Mycobacterium tuberculosis H37Rv, we developed and validated an automated method for the genome-wide identification of sRNA candidate-containing regions within RNA-Seq datasets based on the analysis of the characteristics of reads coverage maps. We identified 192 novel candidate sRNA-encoding regions in intergenic regions and 664 RNA transcripts transcribed from regions antisense (as) to open reading frames (ORF), which bear the characteristics of asRNAs, and validated 28 of these novel sRNA-encoding regions by northern blotting. Our work has not only provided a simple automated method for genome-wide identification of candidate sRNA-encoding regions in RNA-Seq data, but has also uncovered many novel candidate sRNA-encoding regions in M. tuberculosis, reinforcing the view that the control of gene expression in bacteria is more complex than previously anticipated.

Comparison of targeted next-generation sequencing and the Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in clinical isolates and sputum specimens.

Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.

Identification of a Diagnostic Multiomics-Based Biomarker Cluster in a Mouse Model of Pulmonary Tuberculosis.

BACKGROUND: Tuberculosis (TB) stands as the second most prevalent infectious agent-related cause of death worldwide in 2022, trailing only COVID-19. With 1.13 million reported deaths, this figure is more than half of the mortality associated with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), which accounted for 0.63 million deaths. Diagnosing Mycobacterium tuberculosis (MTB) infection remains a formidable challenge due to the inability to isolate and detect MTB in sputum and within the human body. The absence of universally reliable diagnostic criteria for MTB infection globally poses a significant obstacle to preventing the progression of tuberculosis from the MTB infection stage. METHODS: In this study, our objective was to formulate a diagnostic biomarker cluster capable of discerning the progression of MTB infection and disease. This was achieved through a comprehensive joint multiomics analysis, encompassing transcriptome, proteome, and metabolome, conducted on lung tissue samples obtained from both normal control mice and those infected with MTB. RESULTS: A total of 1690 differentially expressed genes and 94 differentially expressed proteins were systematically screened. From this pool, 10 core genes were singled out. Additionally, eight long non-coding ribonucleic acids and eight metabolites linked to these core genes were identified to establish a cohesive cluster of biomarkers. This multiomics-based biomarker cluster demonstrated its capability to differentiate uninfected samples from MTB-infected samples effectively in both principle component analysis and the construction of a random forest model. CONCLUSION: The outcomes of our study strongly suggest that the multiomics-based biomarker cluster holds significant potential for enhancing the diagnosis of MTB infection.

A study on factors influencing delayed sputum conversion in newly diagnosed pulmonary tuberculosis based on bacteriology and genomics.

Conversion of sputum from positive to negative is one of the indicators to evaluate the efficacy of anti-tuberculosis treatment (ATT). We investigate the factors associated with delayed sputum conversion after 2 or 5 months of ATT from the perspectives of bacteriology and genomics. A retrospective study of sputum conversion in sputum positive 1782 pulmonary tuberculosis (PTB) was conducted from 2021 to 2022 in Beijing, China. We also designed a case-matched study including 24 pairs of delayed-sputum-conversion patients (DSCPs) and timely-sputum-conversion patients (TSCPs), and collect clinical isolates from DSCPs before and after ATT and initial isolates of TSCPs who successfully achieved sputum conversion to negative after 2 months of ATT. A total of 75 strains were conducted drug sensitivity testing (DST) of 13 anti-TB drugs and whole-genome sequencing (WGS) to analyze the risk factors of delayed conversion and the dynamics changes of drug resistance and genomics of Mycobacterium tuberculosis (MTB) during ATT. We found TSCPs have better treatment outcomes and whose initial isolates show lower levels of drug resistance. Clinical isolates of DSCPs showed dynamically changing of resistance phenotypes and intra-host heterogeneity. Single nucleotide polymorphism (SNP) profiles showed large differences between groups. The study provided insight into the bacteriological and genomic variation of delayed sputum conversion. It would be helpful for early indication of sputum conversion and guidance on ATT.

Type-III-A structure of mycobacteria CRISPR-Csm complexes involving atypical crRNAs.

Tuberculosis (TB), a leading cause of mortality globally, is a chronic infectious disease caused by Mycobacterium tuberculosis that primarily infiltrates the lung. The mature crRNAs in M. tuberculosis transcribed from the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus exhibit an atypical structure featured with 5' and 3' repeat tags at both ends of the intact crRNA, in contrast to typical Type-III-A crRNAs that possess 5' repeat tags and partial crRNA sequences. However, this structural peculiarity particularly concerning the specific binding characteristics of the 3' repeat end within the mature crRNA within the Csm complex, has not been comprehensively elucidated. Here, our Mycobacteria CRISPR-Csm complexes structure represents the largest Csm complex reported to date. It incorporates an atypical Type-III-A CRISPR RNA (crRNA) (46 nt) with 5' 8-nt and 3' 4-nt repeat sequences in the stoichiometry of Mycobacteria Csm1125364151. The PAM-independent single-stranded RNAs (ssRNAs) are the most suitable substrate for the Csm complex. The 3'-repeat end trimming of mature crRNA was not necessary for its cleavage activity in Type-III-A Csm complex. Our work broadens our understanding of the Type-III-A Csm complex and identifies another mature crRNA processing mechanism in the Type-III-A CRISPR-Cas system based on structural biology.

Genetic factors associated with acquired phenotypic drug resistance and its compensatory evolution during tuberculosis treatment.

OBJECTIVES: We elucidated the factors, evolution, and compensation of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (MTB) isolates under dual pressure from the intra-host environment and anti-tuberculosis (anti-TB) drugs. METHODS: This retrospective case-control study included 337 patients with pulmonary tuberculosis from 15 clinics in Tianjin, China, with phenotypic drug susceptibility testing results available for at least two time points between January 1, 2009 and December 31, 2016. Patients in the case group exhibited acquired AMR to isoniazid (INH) or rifampicin (RIF), while those in the control group lacked acquired AMR. The whole-genome sequencing (WGS) was conducted on 149 serial longitudinal MTB isolates from 46 patients who acquired or reversed phenotypic INH/RIF-resistance during treatment. The genetic basis, associated factors, and intra-host evolution of acquired phenotypic INH/RIF-resistance were elucidated using a combined analysis. RESULTS: Anti-TB interruption duration of ≥30 days showed association with acquired phenotypic INH/RIF resistance (aOR = 2·2, 95% CI, 1·0-5·1) and new rpoB mutations (p = 0·024). The MTB evolution was 1·2 (95% CI, 1·02-1·38) single nucleotide polymorphisms per genome per year under dual pressure from the intra-host environment and anti-TB drugs. AMR-associated mutations occurred before phenotypic AMR appearance in cases with acquired phenotypic INH (10 of 16) and RIF (9 of 22) resistances. DISCUSSION: Compensatory evolution may promote the fixation of INH/RIF-resistance mutations and affect phenotypic AMR. The TB treatment should be adjusted based on gene sequencing results, especially in persistent culture positivity during treatment, which highlights the clinical importance of WGS in identifying reinfection and AMR acquisition before phenotypic drug susceptibility testing.

A novel approach has been developed to produce pure plant-based gel soy yogurt by combining soy proteins (7S/11S), high pressure homogenization, and glycation reaction.

This research sought to examine how the physicochemical characteristics of soy globulins and different processing techniques influence the gel properties of soy yogurt. The goal was to improve these gel properties and rectify any texture issues in soy yogurt, ultimately aiming to produce premium-quality plant-based soy yogurt. In this research study, the investigation focused on examining the impact of 7S/11S, homogenization pressure, and glycation modified with glucose on the gel properties of soy yogurt. A plant-based soy yogurt with superior gel and texture properties was successfully developed using a 7S/11S globulin-glucose conjugate at a 1:3 ratio and a homogenization pressure of 110 MPa. Compared to soy yogurt supplemented with pectin or gelatin, this yogurt demonstrated enhanced characteristics. These findings provide valuable insights into advancing plant protein gels and serve as a reference for cultivating new soybean varieties by soybean breeding experts.

The dimer state of GyrB is an active form: implications for the initial complex assembly and processive strand passage.

In a previous study, we presented the dimer structure of DNA gyrase B' domain (GyrB C-terminal domain) from Mycobacterium tuberculosis and proposed a 'sluice-like' model for T-segment transport. However, the role of the dimer structure is still not well understood. Cross-linking and analytical ultracentrifugation experiments showed that the dimer structure exists both in the B' protein and in the full-length GyrB in solution. The cross-linked dimer of GyrB bound GyrA very weakly, but bound dsDNA with a much higher affinity than that of the monomer state. Using cross-linking and far-western analyses, the dimer state of GyrB was found to be involved in the ternary GyrA-GyrB-DNA complex. The results of mutational studies reveal that the dimer structure represents a state before DNA cleavage. Additionally, these results suggest that the dimer might also be present between the cleavage and reunion steps during processive transport.

Hagnifinder: Recovering magnification information of digital histological images using deep learning.

Background and objective: Training a robust cancer diagnostic or prognostic artificial intelligent model using histology images requires a large number of representative cases with labels or annotations, which are difficult to obtain. The histology snapshots available in published papers or case reports can be used to enrich the training dataset. However, the magnifications of these invaluable snapshots are generally unknown, which limits their usage. Therefore, a robust magnification predictor is required for utilizing those diverse snapshot repositories consisting of different diseases. This paper presents a magnification prediction model named Hagnifinder for H&E-stained histological images. Methods: Hagnifinder is a regression model based on a modified convolutional neural network (CNN) that contains 3 modules: Feature Extraction Module, Regression Module, and Adaptive Scaling Module (ASM). In the training phase, the Feature Extraction Module first extracts the image features. Secondly, the ASM is proposed to address the learned feature values uneven distribution problem. Finally, the Regression Module estimates the mapping between the regularized extracted features and the magnifications. We construct a new dataset for training a robust model, named Hagni40, consisting of 94 643 H&E-stained histology image patches at 40 different magnifications of 13 types of cancer based on The Cancer Genome Atlas. To verify the performance of the Hagnifinder, we measure the accuracy of the predictions by setting the maximum allowable difference values (0.5, 1, and 5) between the predicted magnification and the actual magnification. We compare Hagnifinder with state-of-the-art methods on a public dataset BreakHis and the Hagni40. Results: The Hagnifinder provides consistent prediction accuracy, with a mean accuracy of 98.9%, across 40 different magnifications and 13 different cancer types when Resnet50 is used as the feature extractor. Compared with the state-of-the-art methods focusing on 4-5 levels of magnification classification, the Hagnifinder achieves the best and most comparable performance in the BreakHis and Hagni40 datasets. Conclusions: The experimental results suggest that Hagnifinder can be a valuable tool for predicting the associated magnification of any given histology image.

EspB and HtpG interact with the type III-A CRISPR/Cas system of Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis (MTB) has a type III-A clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system consisting of a Csm1-5 and CRISPR RNA (crRNA) complex involved in the defense against invading nucleic acids. However, CRISPR/Cas system in the MTB still is clearly unknown and needs to be further explored. Methods: In our work, two non-Cas system proteins EspB and HtpG protein were found and identified by LC-MS/MS. The effect of EspB and HtpG on Type III-A CRISPR/Cas System of M. tuberculosis was examined by using Plasmid interference assay and Co-immunoprecipitation analyses. We explored that EspB could interact with the crRNA RNP complex, but HtpG could inhibit the accumulation of the MTB Csm proteins and defense the mechanism of CRISPR/Cas system. Results: The proteins ESAT-6 secretion system-1(Esx-1) secreted protein B (EspB) and high-temperature protein G (HtpG), which were not previously associated with CRISPR/Cas systems, are involved in mycobacterial CRISPR/Cas systems with distinct functions. Conclusion: EspB is a novel crRNA-binding protein that interacts directly with the MTB crRNP complex. Meanwhile, HtpG influences the accumulation of MTB Csm proteins and EspB and interferes with the defense mechanism of the crRNP complex against foreign DNA in vivo. Thereby, our study not only leads to developing more precise clinical diagnostic tool to quickly detect for MTB infection, but also knows these proteins merits for TB biomarkers/vaccine candidates.

Omics analysis of Mycobacterium tuberculosis isolates uncovers Rv3094c, an ethionamide metabolism-associated gene.

Global control of the tuberculosis epidemic is threatened by increasing prevalence of drug resistant M. tuberculosis isolates. Many genome-wide studies focus on SNP-associated drug resistance mechanisms, but drug resistance in 5-30% of M. tuberculosis isolates (varying with antibiotic) appears unrelated to reported SNPs, and alternative drug resistance mechanisms involving variation in gene/protein expression are not well-studied. Here, using an omics approach, we identify 388 genes with lineage-related differential expression and 68 candidate drug resistance-associated gene pairs/clusters in 11 M. tuberculosis isolates (variable lineage/drug resistance profiles). Structural, mutagenesis, biochemical and bioinformatic studies on Rv3094c from the Rv3093c-Rv3095 gene cluster, a gene cluster selected for further investigation as it contains a putative monooxygenase/repressor pair and is associated with ethionamide resistance, provide insights on its involvement in ethionamide sulfoxidation, the initial step in its activation. Analysis of the structure of Rv3094c and its complex with ethionamide and flavin mononucleotide, to the best of our knowledge the first structures of an enzyme involved in ethionamide activation, identify key residues in the flavin mononucleotide and ethionamide binding pockets of Rv3094c, and F221, a gate between flavin mononucleotide and ethionamide allowing their interaction to complete the sulfoxidation reaction. Our work broadens understanding of both lineage- and drug resistance-associated gene/protein expression perturbations and identifies another player in mycobacterial ethionamide metabolism.

Genistein protects against hyperglycemia and fatty liver disease in diet-induced prediabetes mice via activating hepatic insulin signaling pathway.

Introduction: Insulin signaling via the insulin receptor (IR) may be associated with the amelioration of diet-induced metabolic syndrome. Genistein, a soy isoflavone, has been suggested to play a role in the amelioration of high-fat diet-induced metabolic disorders. Methods: Here, we aimed to explore whether genistein regulates glucose and hepatic lipid by activating the insulin signaling pathway in diet-induced obesity mice. Results: We showed that treatment of western-style diet-fed mice with genistein (60 mg/kg) significantly improved insulin resistance with decreased hyperglycemia and HOMA-IR index. These effects were linked to activating hepatic IRß/PI3K/Akt signaling. Furthermore, genistein suppressed gluconeogenesis and promoted glycogen synthesis to maintain glucose homeostasis by increasing the phosphorylation of hepatic FOXO1/GSK3ß in vivo and in vitro. The reduced level of insulin and upregulation of insulin signaling in genistein-treated mice also lead to an increase in hepatic energy status by inducing energy-sensing AMPK, reducing hepatic SREBP1c/ACC/FAS without affecting ß-oxidation to prevent hepatic lipid accumulation. The protective effect of genistein on hepatic lipid accumulation was also validated in vitro. Besides, genistein had little effect on improvements in intestinal function and liver inflammation. Conclusion: Taken together, our results showed that genistein prevents insulin resistance and hyperglycemia through improvements in hepatic function. This study provides new insight into the mechanisms of genistein mediating glucose metabolism and suggests that genistein may be a promising diet ingredient for preventing prediabetes and hepatic lipid accumulation.

CD84 is a Suppressor of T and B Cell Activation during Mycobacterium tuberculosis Pathogenesis.

Interest in host-directed therapies as alternatives/adjuncts to antibiotic treatment has resurged with the increasing prevalence of antibiotic-resistant tuberculosis (TB). Immunotherapies that reinvigorate immune responses by targeting immune checkpoints like PD-1/PD-L1 have proved successful in cancer therapy. Immune cell inhibitory receptors that trigger Mycobacterium tuberculosis-specific immunosuppression, however, are unknown. Here, we show that the levels of CD84, a SLAM family receptor, increase in T and B cells in lung tissues from M. tuberculosis-infected C57BL/6 mice and in peripheral blood mononuclear cells (PBMCs) from pulmonary TB patients. M. tuberculosis challenge experiments using CD84-deficient C57BL/6 mice suggest that CD84 expression likely leads to T and B cell immunosuppression during M. tuberculosis pathogenesis and also plays an inhibitory role in B cell activation. Importantly, CD84-deficient mice showed improved M. tuberculosis clearance and longer survival than M. tuberculosis-infected wild-type (WT) mice. That CD84 is a putative M. tuberculosis infection-specific inhibitory receptor suggests it may be a suitable target for the development of TB-specific checkpoint immunotherapies. IMPORTANCE Immune checkpoint therapies, such as targeting checkpoints like PD-1/PD-L1, have proved successful in cancer therapy and can reinvigorate immune responses. The potential of this approach for treating chronic infectious diseases like TB has been recognized, but a lack of suitable immunotherapeutic targets, i.e., immune cell inhibitory receptors that trigger immunosuppression specifically during Mycobacterium tuberculosis pathogenesis, has limited the application of this strategy in the development of new TB therapies. Our focus in this study was to address this gap and search for an M. tuberculosis-specific checkpoint target. Our results suggest that CD84 is a putative inhibitory receptor that may be a suitable target for the development of TB-specific checkpoint immunotherapies.

M. tuberculosis CRISPR/Cas proteins are secreted virulence factors that trigger cellular immune responses.

The role of prokaryotic CRISPR/Cas system proteins as a defensive shield against invasive nucleic acids has been studied extensively. Non-canonical roles in pathogenesis involving intracellular targeting of certain virulence-associated endogenous mRNA have also been reported for some Type I and Type II CRISPR/Cas proteins, but no such roles have yet been established for Type III system proteins. Here, we demonstrate that M. tuberculosis (Type III-A system) CRISPR/Cas proteins Csm1, Csm3, Csm5, Csm6, and Cas6 are secreted and induce host immune responses. Using cell and animal experiments, we show that Cas6, in particular, provokes IFN-γ release from PBMCs from active tuberculosis (TB) patients, and its deletion markedly attenuates virulence in a murine M. tuberculosis challenge model. Recombinant MTBCas6 induces apoptosis of macrophages and lung fibroblasts, and interacts with the surface of cells in a caspase and TLR-2 independent manner. Transcriptomic and signal pathway studies using THP-1 macrophages stimulated with MTBCas6 indicated that MTBCas6 upregulates expression of genes associated with the NF-κB pathway leading to higher levels of IL-6, IL-1ß, and TNF-α release, cytokines known to activate immune system cells in response to M. tuberculosis infection. Our findings suggest that, in addition to their intracellular shielding role, M. tuberculosis CRISPR/Cas proteins have non-canonical extracellular roles, functioning like a virulent sword, and activating host immune responses.

Mycobacterium tuberculosis CRISPR/Cas system Csm1 holds clues to the evolutionary relationship between DNA polymerase and cyclase activity.

Prokaryotic CRISPR/Cas systems confer immunity against invading nucleic acids through effector complexes. Csm1, the signature protein of Type III effector complexes, catalyses cyclic oligoadenylate synthesis when in the effector complex, but not when alone, activating the Csm6 nuclease and switching on the antiviral response. Here, we provide biochemical evidence that M. tuberculosis Csm1 (MtbCsm1) has ion-dependent polymerase activity when independent of the effector complex. Structural studies provide supporting evidence that the catalytic core of the MtbCsm1 palm2 domain is almost identical to that of classical DNA polymerase Pol IV, and that the palm1 and B domains function as the other structural elements required (thumb and fingers) for DNA polymerase activity. MtbCsm1 polymerase activity is relatively weak in vitro and its functional relevance in vivo is unknown. Our structural and mutagenesis data suggest that residue K692 in the palm2 domain has been significant in the evolution of Csm1 from a polymerase to a cyclase, and support the notion that the cyclase activity of Csm1 requires the presence of other elements provided by the CRISPR/Cas effector complex. This structural rationale for Csm1 polymerase (alone) and cyclase (within the effector complex) activity should benefit future functional investigations and engineering.

Functional interaction between MutL and 3'-5' exonuclease X in Escherichia coli.

Exonuclease X is a 3'-5' distributive exonuclease that functions in DNA recombination and repair. It undergoes multiple rounds of binding, hydrolysis, and release to degrade long substrate molecules and thus is very inefficient. In order to identify a cofactor that elevates the excision activity of ExoX, we screened many proteins involved in repair and recombination. We observed that MutL greatly promoted the exonuclease activity of ExoX, and then verified the interaction between MutL and ExoX using SPR and Far-Western analysis. This promotion is independent of ATP and the DNA-binding activity of MutL. We constructed two deletion mutants to analyze this interaction and its regulation of ExoX activity, and found that this functional interaction with ExoX is mainly due to ionic interactions with the N-terminus of MutL. This adds a new role to MutL and gives a clue to MutL's possible regulation on other DnaQ family exonuclease members.

Kinetic analysis of DNA compaction by mycobacterial integration host factor at the single-molecule level.

Nucleoid-associated proteins (NAPs) play an important role on chromosome condensation and organization. Mycobacterial integration host factor (mIHF) is one of the few mycobacterial NAPs identified so far. mIHF has the ability to stimulate mycobacteriophage L5 integration and compact DNA into nucleoid-like or higher order filamentous structures by atomic force microscopy observation. In this study, M. smegmatis IHF (MsIHF), which possesses the sequence essential for mIHF's functions, binds 30-bp dsDNA fragments in a sequence-independent manner and displays sensitivity to ion strength in bio-layer interferometry (BLI) experiments. The DNA compaction process of MsIHF was observed at the single-molecule level using the total internal reflection fluorescence microscopy (TIRFM). MsIHF efficiently compacted λ DNA into a highly condensed structure with the concentration of 0.25 and 1.0 µM, and the packing ratios were higher than 10. Further kinetic analysis revealed MsIHF compacts DNA in a three-step mechanism, which consists of two compaction steps with different compacting rates separated by a lag step. This study would help us better understand the mechanisms of chromosomal DNA organization in mycobacteria.