Interception of host fatty acid metabolism by mycobacteria under hypoxia to suppress anti-TB immunity

Pathogenic mycobacteria induce the formation of hypoxic granulomas during latent tuberculosis (TB) infection, in which the immune system contains, but fails to eliminate the mycobacteria. Fatty acid metabolism-related genes are relatively overrepresented in the mycobacterial genome and mycobacteria favor host-derived fatty acids as nutrient sources. However, whether and how mycobacteria modulate host fatty acid metabolism to drive granuloma progression remains unknown. Here, we report that mycobacteria under hypoxia markedly secrete the protein Rv0859/MMAR_4677 (Fatty-acid degradation A, FadA), which is also enriched in tuberculous granulomas. FadA acts as an acetyltransferase that converts host acetyl-CoA to acetoacetyl-CoA. The reduced acetyl-CoA level suppresses H3K9Ac-mediated expression of the host proinflammatory cytokine Il6, thus promoting granuloma progression. Moreover, supplementation of acetate increases the level of acetyl-CoA and inhibits the formation of granulomas. Our findings suggest an unexpected mechanism of a hypoxia-induced mycobacterial protein suppressing host immunity via modulation of host fatty acid metabolism and raise the possibility of a novel therapeutic strategy for TB infection.

Replacement of S14 protein in ribosomes of zinc-starved mycobacteria reduces spectinamide sensitivity

Zinc is an essential micronutrient for mycobacteria and its depletion induces multiple adaptive changes in cellular physiology, the most remarkable of which are remodeling and hibernation of ribosomes. Ribosome remodeling, induced upon relatively moderate depletion of zinc, involves replacement of multiple ribosomal proteins containing the zinc-binding CXXC motif (called C+ r-proteins) by their motif-free C- paralogs. Acute zinc depletion induces binding of mycobacterial protein Y (Mpy) to the 70S C- ribosome, thereby stabilizing the ribosome in an inactive state that is also resistant to kanamycin and streptomycin. Because the Mpy binding region on the ribosome is proximal to the binding pocket of spectinamides (Spa), the preclinical drug candidates for tuberculosis, we addressed the impact of remodeling and hibernation of ribosomes on Spa sensitivity. We report here that while Mpy binding has no significant effect on Spa sensitivity to the ribosome, replacement of S14c+ with its C- counterpart reduces the binding affinity of the drug by ∼2-fold, causing increased Spa tolerance in Mycobacterium smegmatis and Mycobacterium tuberculosis cells harboring the C- ribosome. The altered interaction between Spa and ribosomes likely results from new contact points for D67 and R83 residues of S14c- with U1138 and C1184 of 16S rRNA helix 34, respectively. Given that M. tuberculosis induces ribosome remodeling during progression from acute to chronic phase of a lung infection, our findings highlight new considerations in the development of Spa as effective drugs against TB.

Recent Advancement in Predicting Subcellular Localization of Mycobacterial Protein with Machine Learning Methods

Mycobacterium tuberculosis (MTB) can cause the terrible tuberculosis (TB), which is reported as one of the most dreadful epidemics. Although many biochemical molecular drugs have been developed to cope with this disease, the drug resistance—especially the multidrug-resistant (MDR) and extensively drug-resistance (XDR)—poses a huge threat to the treatment. However, traditional biochemical experimental method to tackle TB is time-consuming and costly. Benefited by the appearance of the enormous genomic and proteomic sequence data, TB can be treated via sequence-based biological computational approach-bioinformatics. Studies on predicting subcellular localization of mycobacterial protein (MBP) with high precision and efficiency may help figure out the biological function of these proteins and then provide useful insights for protein function annotation as well as drug design. In this review, we reported the progress that has been made in computational prediction of subcellular localization of MBP including the following aspects: 1) Construction of benchmark datasets. 2) Methods of feature extraction. 3) Techniques of feature selection. 4) Application of several published prediction algorithms. 5) The published results. 6) The further study on prediction of subcellular localization of MBP.

Discovery of a Natural Product That Binds to the Mycobacterium tuberculosis Protein Rv1466 Using Native Mass Spectrometry

Elucidation of the mechanism of action of compounds with cellular bioactivity is important for progressing compounds into future drug development. In recent years, phenotype-based drug discovery has become the dominant approach to drug discovery over target-based drug discovery, which relies on the knowledge of a specific drug target of a disease. Still, when targeting an infectious disease via a high throughput phenotypic assay it is highly advantageous to identifying the compound’s cellular activity. A fraction derived from the plant Polyalthia sp. showed activity against Mycobacterium tuberculosis at 62.5 μge/μL. A known compound, altholactone, was identified from this fraction that showed activity towards M. tuberculosis at an minimum inhibitory concentration (MIC) of 64 μM. Retrospective analysis of a target-based screen against a TB proteome panel using native mass spectrometry established that the active fraction was bound to the mycobacterial protein Rv1466 with an estimated pseudo-Kd of 42.0 ± 6.1 µM. Our findings established Rv1466 as the potential molecular target of altholactone, which is responsible for the observed in vivo toxicity towards M. tuberculosis.

Structural investigation of Mycobacterial protein complexes involved in the stationary phase stress response

Large protein complexes play key roles in mediating biological processes in the cell. Little structural information is known on the protein complex mediators governing persistence in the host for Mycobacterium tuberculosis (Mtb). We applied the ‘shotgun EM’ method for the structural characterisation of protein complexes produced after exposure to stationary phase stress for the model Mycobacterium, M smegmatis (Msm). We identified glutamine synthetase I, essential for Mtb virulence, in addition to bacterioferritin, critical for Mtb iron regulation, and encapsulin, which produces a cage-like structure to enclose target proteins. Further investigation found that encapsulin carries dye-decolourising peroxidase (DyP), a potent protein antioxidant, as the primary cargo during stationary phase stress. Our ‘proof-of-concept’ application of this method offers insight into identifying potential key-mediators in Mtb persistence.

The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

Vector vaccine Flu/ESAT-6 application in the regimen of complex therapy of drug-sensitive and drug-resistant experimental tuberculosis estimation of the efficiency

The steady growth of drug-resistant tuberculosis, charac-terized by low therapeutic efficacy, requires the develop-ment of innovative approaches to the treatment of this infection. One of such approaches is vaccine therapy that has immunostimulating activity and is capable to correct dysfunction of the immune system in this infection. In this study, the authors aimed to estimate the efficacy of the mucosal vector vaccine Flu/ESAT-6 included into the complex therapy of experimental pulmonary tuberculosis. C57BL/6 mice were intravenously infected with drug-sus-ceptible (DS) (M. tuberculosis H37Rv) or drug- resistant (DR) (M. tuberculosis Beijing genotype) MBT strains. DS-in-fection was treated with two anti-TB drugs (HR), and DR-infection with four (AETppBq). Flu/ESAT-6 vaccine based on the attenuated influenza A virus strain expres-sing the mycobacterial protein ESAT-6 was administered intranasally at a dose of 6 lg/mouse. Efficacy of vari-ous therapeutic regimens was assessed with biometric, bacteriological, and histological methods. Two optimal therapeutic regimens of Flu/ESAT-6 administration were chosen: twice (with a 3 week interval) at the beginning of chemotherapy in case of DS infection; and thrice (with a 4 week interval) 2 weeks after the beginning of chemo-therapy in case of DR infection. Flu/ESAT-6 addition to the anti-tuberculosis treatment led to a significant increase in the efficacy of therapy for both forms of infection which manifested in (i) the pulmonary bacterial load decrease at the end of a 2-months therapy of DS tuberculosis by 1.83 lg CFU; and after 4-months therapy of DR tuberculosis by 0.93 lg CFU; (ii) decrease in the prevalence of specific and the severity of exudative inflammation, and reduction of alternative components in case of DR tuberculosis; (iii) stimulation of the lymphoid component of the cellular im-mune response in lungs, demonstrated by reorganization of granulomas’ cellular composition to lymphoid type, and in the formation of peribronchial lymphohistiocytic infiltrates. Thus, therapeutic immunization with Flu/ESAT-6 vaccine significantly increases the efficacy of experimen-tal tuberculosis therapy and can shorten the duration of anti-tuberculosis therapy which is especially important in case of DR tuberculosis characterized by a slow involution of specific inflammation.