Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), and remains a leading public health problem. Previous studies have identified host genetic factors that contribute to Mtb infection outcomes. However, much of the heritability in TB remains unaccounted for and additional susceptibility loci most likely exist. We perform a multistage genome-wide association study on 2949 pulmonary TB patients and 5090 healthy controls (833 cases and 1220 controls were genome-wide genotyped) from Han Chinese population. We discover two risk loci: 14q24.3 (rs12437118, Pcombined = 1.72 × 10−11, OR = 1.277, ESRRB) and 20p13 (rs6114027, Pcombined = 2.37 × 10−11, OR = 1.339, TGM6). Moreover, we determine that the rs6114027 risk allele is related to decreased TGM6 transcripts in PBMCs from pulmonary TB patients and severer pulmonary TB disease. Furthermore, we find that tgm6-deficient mice are more susceptible to Mtb infection. Our results provide new insights into the genetic etiology of TB.

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Tuberculosis caused by Mycobacterium tuberculosis (Mtb) infection remains a large global public health problem. One striking characteristic of Mtb is its ability to adapt to hypoxia and trigger the ensuing transition to a dormant state for persistent infection, but how the hypoxia response of Mtb is regulated remains largely unknown. Here we performed a quantitative acetylome analysis to compare the acetylation profile of Mtb under aeration and hypoxia, and showed that 377 acetylation sites in 269 Mtb proteins were significantly changed under hypoxia. In particular, deacetylation of dormancy survival regulator (DosR) at K182 promoted the hypoxia response in Mtb and enhanced the transcription of DosR-targeted genes. Mechanistically, recombinant DosRK182R protein demonstrated enhanced DNA-binding activity in comparison with DosRK182Q protein. Moreover, Rv0998 was identified as an acetyltransferase that mediates the acetylation of DosR at K182. Deletion of Rv0998 also promoted the adaptation of Mtb to hypoxia and the transcription of DosR-targeted genes. Mice infected with an Mtb strain containing acetylation-defective DosRK182R had much lower bacterial counts and less severe histopathological impairments compared with those infected with the wild-type strain. Our findings suggest that hypoxia induces the deacetylation of DosR, which in turn increases its DNA-binding ability to promote the transcription of target genes, allowing Mtb to shift to dormancy under hypoxia.

Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is an important receptor for Mycobacterium tuberculosis on human dendritic cells. Previous studies have shown that the variation, especially the -871A/G and -336A/G in DC-SIGN promoter influenced the susceptibility to tuberculosis. We therefore investigated whether polymorphisms in the DC-SIGN gene were associated with susceptibility to tuberculosis in an eastern Chinese population. A total of 237 culture-positive pulmonary tuberculosis case patients and 244 controls were genotyped for -871A/G and -336A/G by pyrosequencing. Our results suggested that the 2 promoter variants of DC-SIGN gene were not associated with susceptibility to tuberculosis in Chinese. Further analysis showed that the allele -336G was associated with a protective effect against fever in pulmonary tuberculosis patients, but not against cavity formation. In addition, we compared the allelic frequencies of -871A/G and -336A/G in African, Caucasian, and Asian groups. The results showed that the tw forms of allelic frequencies detected Chinese individuals in our study were similar to the reported frequencies in other Asian populations but differed significantly from those in the African and Caucasian groups studied.

Tuberculosis, caused by Mycobacterium tuberculosis infection, remains a global threat to human health, but knowledge of the molecular mechanisms underlying the pathogenesis of tuberculosis is still limited. Although Notch4, a member of the Notch receptor family, is involved in the initiation of mammary tumors, its function in M. tuberculosis infection remains unclear. In this study, we found that Notch4-deficient mice were more resistant to M. tuberculosis infection, with a much lower bacterial burden and fewer pathological changes in the lungs. Notch4 inhibited M. tuberculosis-induced production of proinflammatory cytokines by interaction with TAK1 and inhibition of its activation. Furthermore, we found that Notch intracellular domain 4 prevented TRAF6 autoubiquitination and suppressed TRAF6-mediated TAK1 polyubiquitination. Finally, Notch inhibitors made mice more resistant to M. tuberculosis infection. These results suggest that Notch4 is a negative regulator of M. tuberculosis-induced inflammatory response, and treatment with a Notch inhibitor could serve as a new therapeutic strategy for tuberculosis.

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and biosynthetical target for anti-tuberculosis (TB) drug development.Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin-9 with high affinity, and galectin-9 associates with transforming growth factor b-activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal-regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin-9 or inhibition of MMPs blocks AG-induced pathological impairments in the lung, and the AG-galectin-9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin-9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl–Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 10 3 colony forming units (CFU)/mL H 37 R v was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 10 3 CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples.

BackgroundPresently, tuberculosis (TB) poses a global threat to human health. The development of reliable laboratory tools is vital to the diagnosis and treatment of TB. MPT64, a protein secreted by Mycobacterium tuberculosis complex, is highly specific for TB, making antibody to MPT64 a reagent specific for the diagnosis of TB.MethodAntibody to MPT64 was obtained by a combination of genetic engineering and immunization by the system evolution of ligands by exponential enrichment. A high-affinity aptamer of antibody to MPT64 was selected from a random single-stranded DNA library, and a sandwich ELISA method based on this aptamer was developed. This ELISA method was used to detect TB in 328 serum samples, 160 from patients with pulmonary TB (PTB) and 168 from non-tuberculous controls.ResultsThe minimum limit of detection of the ELISA method was 2.5 mg/L, and its linear range varied from 10 mg/L to 800 mg/L. Its sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and area under the curve, with 95 % confidence intervals, were 64.4 % (56.7 %–71.4 %), 99.4 % (96.7 %–99.9 %), 108.2 (15.3–765.9), 0.350 (0.291–0.442) and 0.819 (0.770–0.868), respectively. No significant difference in sensitivity was observed between sputum smear positive (73/112, 65.2 %) and negative (30/48, 62.5 %) individuals.ConclusionsThis sandwich ELISA based on an MPT64 antibody aptamer may be useful for the serological diagnosis of PTB, both in sputum smear positive and negative patients.