Quantitative analysis of the lysine acetylome reveals the role of SIRT3-mediated HSP60 deacetylation in suppressing intracellular Mycobacterium tuberculosis survival.

Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with Mycobacterium tuberculosis (M. tb), these processes are essential for host-pathogen interactions and immune responses. However, the specific effects of acetylation and deacetylation on cellular functions during M. tb infection are not fully understood. This study employed Tandem Mass Tag (TMT) labeling for quantitative proteomic profiling to examine the acetylproteome (acetylome) profiles of noninfected and M. tb-infected macrophages. We identified 715 acetylated peptides from 1,072 proteins and quantified 544 lysine acetylation sites (Kac) in 402 proteins in noninfected and M. tb-infected macrophages. Our research revealed a link between acetylation events and metabolic changes during M. tb infection. Notably, the deacetylation of heat shock protein 60 (HSP60), a key chaperone protein, was significantly associated with this process. Specifically, the deacetylation of HSP60 at K96 by sirtuin3 (SIRT3) enhances macrophage apoptosis, leading to the elimination of intracellular M. tb. These findings underscore the pivotal role of the SIRT3-HSP60 axis in the host immune response to M. tb. This study offers a new perspective on host protein acetylation and suggests that targeting host-directed therapies could be a promising approach for tuberculosis immunotherapy. IMPORTANCE: Protein acetylation is crucial for the onset, development, and outcome of tuberculosis (TB). Our study comprehensively investigated the dynamics of lysine acetylation during M. tb infection, shedding light on the intricate host-pathogen interactions that underlie the pathogenesis of tuberculosis. Using an advanced quantitative lysine proteomics approach, different profiles of acetylation sites and proteins in macrophages infected with M. tb were identified. Functional enrichment and protein-protein network analyses revealed significant associations between acetylated proteins and key cellular pathways, highlighting their critical role in the host response to M. tb infection. Furthermore, the deacetylation of HSP60 and its influence on macrophage-mediated clearance of M. tb underscore the functional significance of acetylation in tuberculosis pathogenesis. In conclusion, this study provides valuable insights into the regulatory mechanisms governing host immune responses to M. tb infection and offers promising avenues for developing novel therapeutic interventions against TB.

Comparative analysis of genomic characteristics and immune response between Mycobacterium tuberculosis strains cultured continuously for 25 years and H37Rv.

Tuberculosis (TB) continues to pose a significant global health challenge, emphasizing the critical need for effective preventive measures. Although many studies have tried to develop new attenuated vaccines, there is no effective TB vaccine. In this study, we report a novel attenuated Mycobacterium tuberculosis (M. tb) strain, CHVAC-25, cultured continuously for 25 years in the laboratory. CHVAC-25 exhibited significantly reduced virulence compared to both the virulent H37Rv strain in C57BL/6J and severe combined immunodeficiency disease mice. The comparative genomic analysis identified 93 potential absent genomic segments and 65 single nucleotide polymorphic sites across 47 coding genes. Notably, the deletion mutation of ppsC (Rv2933) involved in phthiocerol dimycocerosate synthesis likely contributes to CHVAC-25 virulence attenuation. Furthermore, the comparative analysis of immune responses between H37Rv- and CHVAC-25-infected macrophages showed that CHVAC-25 triggered a robust upregulation of 173 genes, particularly cytokines crucial for combating M. tb infection. Additionally, the survival of CHVAC-25 was significantly reduced compared to H37Rv in macrophages. These findings reiterate the possibility of obtaining attenuated M. tb strains through prolonged laboratory cultivation, echoing the initial conception of H37Ra nearly a century ago. Additionally, the similarity of CHVAC-25 to genotypes associated with attenuated M. tb vaccine positions it as a promising candidate for TB vaccine development.

The incremental value of Mycobacterium tuberculosis trace nucleic acid detection in CT-guided percutaneous biopsy needle rinse solutions for the diagnosis of tuberculosis.

Introduction: Tuberculosis (TB) diagnosis still faces challenges with high proportion of bacteriologic test negative incidences worldwide. We assessed the diagnostic value of digital PCR (dPCR) analysis of ultramicro Mycobacterium tuberculosis (M.tb) nucleic acid in CT-guided percutaneous biopsy needle rinse solution (BNRS) for TB. Methods: BNRS specimens were consecutively collected and total DNA was purified. The concentrations of M.tb-specific IS6110 and IS1081 were quantified using droplet dPCR. The diagnostic performances of BNRS-dPCR and its sensitivity in comparison with conventional tests were analyzed. Results: A total of 106 patients were enrolled, 63 of whom were TB (48 definite and 15 clinically suspected TB) and 43 were non-TB. The sensitivity of BNRS IS6110 OR IS1081-dPCR for total, confirmed and clinically suspected TB was 66.7%, 68.8% and 60.0%, respectively, with a specificity of 97.7%. Its sensitivity was higher than that of conventional etiological tests, including smear microscopy, mycobacterial culture and Xpert using sputum and BALF samples. The positive detection rate in TB patients increased from 39.3% for biopsy AFB test alone to 73.2% when combined with BNRS-dPCR, and from 71.4% for biopsy M.tb molecular detection alone to 85.7% when combined with BNRS-dPCR. Conclusion: Our results preliminarily indicated that BNRS IS6110 OR IS1081-dPCR is a feasible etiological test, which has the potential to be used as a supplementary method to augment the diagnostic yield of biopsy and improve TB diagnosis.

Use of T-SPOT.TB for the diagnosis of unconventional pleural tuberculosis is superior to ADA in high prevalence areas: a prospective analysis of 601 cases.

BACKGROUND: Tuberculous pleural effusion (TPE) is the most common extrapulmonary manifestation and may have lasting effect on lung function. However conventional diagnostic tests for TPE register multiple limitations. This study estimates diagnostic efficacy of the interferon gamma release assay (IGRA: T-SPOT.TB) in TPE patients of different characteristics. METHODS: We performed a prospective, single-centre study including all suspected pleural effusion patients consecutively enrolled from June 2015 to October 2018. Through receiver operating characteristic (ROC) curves, technical cut-offs and the utility of T-SPOT on pleural fluid (PF) were determined and analysed. Logistic regression analysis was performed to obtain the independent risk factors for TPE, and evaluated the performance of the T-SPOT assay stratified by risk factors in comparison to ADA. RESULTS: A total of 601 individuals were consecutively recruited. The maximum spot-forming cells (SFCs) of early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) in the PF T-SPOT assay had the best diagnostic efficiency in our study, which was equal to ADA (0.885 vs 0.887, P = 0.957) and superior to peripheral blood (PB), with a sensitivity of 83.0% and a specificity of 83.1% (The cut-off value was 466 SFCs/106 mononuclear cells). Among the TPE patients with low ADA (< 40 IU/L), the sensitivity and specificity of PF T-SPOT were still 87.9 and 90.5%, respectively. The utility of ADA was negatively related to increasing age, but the PF T-SPOT test had a steady performance at all ages. Age (< 45 yrs.; odds ratio (OR) = 5.61, 95% confidence interval (CI) 3.59-8.78; P < 0.001), gender (male; OR = 2.68, 95% CI 1.75-2.88; P < 0.001) and body mass index (BMI) (< 22; OR = 1.93, 95% CI 1.30-2.88; P = 0.001) were independently associated with the risk of TB by multivariate logistic regression analysis. Notably, when stratified by risk factor, the sensitivity of PF T-SPOT was superior to the sensitivity for ADA (76.5% vs. 23.5%, P = 0.016) and had noninferior specificity (84.4% vs. 96.9%, P = 0.370). CONCLUSIONS: In conclusion, the PF T-SPOT assay can effectively discriminate TPE patients whose ADA is lower than 40 IU/L and is superior to ADA in unconventional TPE patients (age ≥ 45 yrs., female or BMI ≥ 22). The PF T-SPOT assay is an excellent choice to supplement ADA to diagnose TPE.

Label-Free Quantitative Proteomics Identifies Novel Biomarkers for Distinguishing Tuberculosis Pleural Effusion from Malignant Pleural Effusion.

PURPOSE: To identify potential protein biomarkers for distinguishing tuberculosis plural effusion (TBPE) from malignant plural effusion (MPE). EXPERIMENTAL DESIGN: Five independent samples from each group (TBPE and MPE) are enrolled for label-free quantitative proteomics analyses. The differentially expressed proteins are validated by western blot and ELISA. Logistic regression analysis is used to obtain the optimal diagnostic model. RESULTS: In total, 14 proteins with significant difference are identified between TBPE and MPE. Seven differentially expressed proteins are validated using western blot, and the expression patterns of these seven proteins are similar with those in proteomics analysis. Statistically significant differences in four proteins (AGP1, ORM2, C9, and SERPING1) are noted between TBPE and MPE in the training set (n = 230). Logistic regression analysis shows the combination of AGP1-ORM2-C9 presents a sensitivity of 73.0% (92/126) and specificity of 89.4% (93/104) in discriminating TBPE from MPE. Additional validation is performed to evaluate the diagnostic model in an independent blind testing set (n = 80), and yielded a sensitivity of 74.4% (32/43) and specificity of 91.9% (34/37) in discriminating TBPE from MPE. CONCLUSION: The study uncovers the proteomic profiles of TBPE and MPE, and provides new potential diagnostic biomarkers for distinguishing TBPE from MPE.

Label-Free Quantitative Proteomics Identifies Novel Plasma Biomarkers for Distinguishing Pulmonary Tuberculosis and Latent Infection.

The lack of effective differential diagnostic methods for active tuberculosis (TB) and latent infection (LTBI) is still an obstacle for TB control. Furthermore, the molecular mechanism behind the progression from LTBI to active TB has been not elucidated. Therefore, we performed label-free quantitative proteomics to identify plasma biomarkers for discriminating pulmonary TB (PTB) from LTBI. A total of 31 overlapping proteins with significant difference in expression level were identified in PTB patients (n = 15), compared with LTBI individuals (n = 15) and healthy controls (HCs, n = 15). Eight differentially expressed proteins were verified using western blot analysis, which was 100% consistent with the proteomics results. Statistically significant differences of six proteins were further validated in the PTB group compared with the LTBI and HC groups in the training set (n = 240), using ELISA. Classification and regression tree (CART) analysis was employed to determine the ideal protein combination for discriminating PTB from LTBI and HC. A diagnostic model consisting of alpha-1-antichymotrypsin (ACT), alpha-1-acid glycoprotein 1 (AGP1), and E-cadherin (CDH1) was established and presented a sensitivity of 81.2% (69/85) and a specificity of 95.2% (80/84) in discriminating PTB from LTBI, and a sensitivity of 81.2% (69/85) and a specificity of 90.1% (64/81) in discriminating PTB from HCs. Additional validation was performed by evaluating the diagnostic model in blind testing set (n = 113), which yielded a sensitivity of 75.0% (21/28) and specificity of 96.1% (25/26) in PTB vs. LTBI, 75.0% (21/28) and 92.3% (24/26) in PTB vs. HCs, and 75.0% (21/28) and 81.8% (27/33) in PTB vs. lung cancer (LC), respectively. This study obtained the plasma proteomic profiles of different M.TB infection statuses, which contribute to a better understanding of the pathogenesis involved in the transition from latent infection to TB activation and provide new potential diagnostic biomarkers for distinguishing PTB and LTBI.

[Comparison of interferon-gamma release assays and adenosine deaminase of pleural fluid for the diagnosis of pleural tuberculosis].

OBJECTIVE: To compare the diagnostic performance of interferon gamma releasing assays (T-SPOT. TB) and adenosine deaminase (ADA) in pleural tuberculosis, and therefore to evaluate the value of T-SPOT. TB in a high tuberculosis burden country. METHODS: From June 2011 to November 2012, 111 patients with pleural fluid in Beijing Chest Hospital, Capital Medical University were enrolled prospectively and categorized as culture/biopsy-confirmed pleural tuberculosis group (n = 59) and non-pleural tuberculosis group (n = 52). Patients with uncertain diagnosis and clinically diagnosed pleural tuberculosis were excluded from the study. Pleural fluid T-SPOT. TB and ADA measurements were performed, in addition to other routine laboratory tests. Continuous variables (spot forming cells, SFCs) were compared using nonparametric Mann-Whitney U test. Comparisons between proportions were performed using Chi-squared test. RESULTS: The receiver operating characteristic (ROC) curve and cut-off value of pleural fluid T-SPOT. TB were established according to spot forming cells (SFC) between culture/biopsy-confirmed pleural tuberculosis group and non-pleural tuberculosis group (216 SFC/10(6) pleural fluid mononuclear cells). The sensitivity of pleural fluid T-SPOT. TB and ADA was 91.5% (54/59) and 71.2% (42/59), respectively. The specificity was 90.4% (47/52) and 92.0% (46/50), respectively. The sensitivity of pleural fluid T-SPOT. TB was significantly higher than that of ADA (χ(2) = 8.045, P < 0.01). There was no significant difference of specificity between pleural fluid T-SPOT. TB and ADA (χ(2) = 0.000, P > 0.05). The area under the ROC curve was 0.912 for pleural fluid T-SPOT. TB and 0.903 for ADA. The sensitivity of combination diagnosis of ADA and pleural fluid T-SPOT. TB decreased to 67.8% (40/59), but the specificity increased to 100.0% (50/50). CONCLUSIONS: Pleural fluid T-SPOT. TB are relatively accurate supplementary assays for the diagnosis of pleural tuberculosis in this high tuberculosis burden country, and the combination of pleural fluid ADA and T-SPOT. TB is of diagnostic value.

[Screening and analysis of in vivo induced genes of Mycobacterium tuberculosis].

OBJECTIVE: To screen in vivo induced genes of Mycobacterium tuberculosis and search possible molecular targets of new drugs, vaccines, and early diagnostic methods. METHODS: In vivo induced antigen technology (IVIAT) was used in this study. Genomic DNA from M. tuberculosis of the strain H37Rv was extracted. The DNA was partially digested with Sau3A I and the purified fragments were inserted into the pET30a (+), pET30b (+) and pET30c (+) expression vectors to construct a genomic library. The library was induced with IPTG and then was screened with pooled tuberculosis patient sera preabsorbed with in vitro grown M. tuberculosis of the strain H37Rv and Escherichia coli of the strain BL21 (DE3). The inserts of positive clones were sequenced with primer T7 promoter. The sequences were aligned in the genomic database of M. tuberculosis strain H37Rv (http://genolist.pasteur.fr/Tuberculose) to identify the open reading frame (ORF). RESULTS: The genomic expression library included 4.3 x 10(4) clones, and more than eighty percent were recombinant plasmids. The library reached the theoretic requirement. The successive adsorptions significantly decreased the anti-M. tuberculosis antibody titer of sera, and no significant difference was found between the last two adsorption groups, suggesting that the antibodies reactive against the M. tuberculosis H37Rv antigens expressed in vitro were removed. After screening of the genomic expression library and searching in the genome database, 51 ORFs were identified and they were classified into 8 categories according to the classification criterion on the website, including 1 virulence gene, 13 cell wall and cell processes genes, 11 intermediary metabolism and respiration genes, 7 lipid metabolism genes, 2 information pathways genes, 3 PE/PPE genes, 12 conserved hypotheticals, and 2 conserved hypotheticals with an orthologue in M. bovis. CONCLUSION: Genes expressed specially during human M. tuberculosis infections can be identified with in vivo induced antigen technology. Analysis of these genes identified using IVIAT shows that some genes are related to virulence, some are essential genes for M. tuberculosis, and some encoded proteins have strong immunogenicity, suggesting that some of them can be used as molecular targets of anti-tuberculosis drugs, vaccines, and tuberculosis early diagnosis.