RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis.

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

Potentiating the Anti-Tuberculosis Efficacy of Peptide Nucleic Acids through Combinations with Permeabilizing Drugs.

The emergence of antimicrobial resistance warrants for the development of improved treatment approaches. In this regard, peptide nucleic acids (PNAs) have shown great promise, exhibiting antibiotic properties through the targeting of cellular nucleic acids. We aimed to study the efficacy of PNA as an anti-tuberculosis agent. Since the efficacy of PNA is limited by its low penetration into the cell, we also investigated combinatorial treatments using permeabilizing drugs to improve PNA efficacy. Various concentrations of anti-inhA PNA, permeabilizing drugs, and their combinations were screened against extracellular and intracellular mycobacteria.0.625 to 5 µM anti-inhA PNA was observed to merely inhibit the growth of extracellular M. smegmatis, while low intracellular bacterial load was reduced by 2 or 2.5 log-fold when treated with 2.5 or 5 µM PNA, respectively. Anti-inhA PNA against M. tuberculosis H37Ra exhibited bactericidal properties at 2.5 and 5 µM and enabled a slight reduction in intracellular M. tuberculosis at concentrations from 2.5 to 20 µM. Of the permeabilizing drugs tested, ethambutol showed the most permeabilizing potential and ultimately potentiated anti-inhA PNA to the greatest extent, reducing its efficacious concentration to 1.25 µM against both M. smegmatis and M. tuberculosis. Furthermore, an enhanced clearance of 1.3 log-fold was observed for ethambutol-anti-inhA PNA combinations against intracellular M. tuberculosis. Thus, permeabilizing drug-PNA combinations indeed exhibit improved efficacies. We therefore propose that anti-inhA PNA could improve therapy even when applied in minute doses as an addition to the current anti-tuberculosis drug regimen. IMPORTANCE Peptide nucleic acids have great potential in therapeutics as anti-gene/anti-sense agents. However, their limited uptake in cells has curtailed their widespread application. Through this study, we explore a PNA-drug combinatorial strategy to improve the efficacy of PNAs and reduce their effective concentrations. This work also focuses on improving tuberculosis treatment, which is hindered by the emergence of antimicrobial-resistant strains of Mycobacterium tuberculosis. It is observed that the antibacterial efficacy of anti-inhA PNA is enhanced when it is combined with permeabilizing drugs, particularly ethambutol. This indicates that the addition of even small concentrations of anti-inhA PNA to the current TB regimen could potentiate their therapeutic efficiency. We hypothesize that this system would also overcome isoniazid resistance, since the resistance mutations lie outside the designed anti-inhA PNA target site.

Kupyaphores are zinc homeostatic metallophores required for colonization of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) endures a combination of metal scarcity and toxicity throughout the human infection cycle, contributing to complex clinical manifestations. Pathogens counteract this paradoxical dysmetallostasis by producing specialized metal trafficking systems. Capture of extracellular metal by siderophores is a widely accepted mode of iron acquisition, and Mtb iron-chelating siderophores, mycobactin, have been known since 1965. Currently, it is not known whether Mtb produces zinc scavenging molecules. Here, we characterize low-molecular-weight zinc-binding compounds secreted and imported by Mtb for zinc acquisition. These molecules, termed kupyaphores, are produced by a 10.8 kbp biosynthetic cluster and consists of a dipeptide core of ornithine and phenylalaninol, where amino groups are acylated with isonitrile-containing fatty acyl chains. Kupyaphores are stringently regulated and support Mtb survival under both nutritional deprivation and intoxication conditions. A kupyaphore-deficient Mtb strain is unable to mobilize sufficient zinc and shows reduced fitness upon infection. We observed early induction of kupyaphores in Mtb-infected mice lungs after infection, and these metabolites disappeared after 2 wk. Furthermore, we identify an Mtb-encoded isonitrile hydratase, which can possibly mediate intracellular zinc release through covalent modification of the isonitrile group of kupyaphores. Mtb clinical strains also produce kupyaphores during early passages. Our study thus uncovers a previously unknown zinc acquisition strategy of Mtb that could modulate host-pathogen interactions and disease outcome.

CD4 and CD8 co-receptors modulate functional avidity of CD1b-restricted T cells.

T cells recognize mycobacterial glycolipid (mycolipid) antigens presented by CD1b molecules, but the role of CD4 and CD8 co-receptors in mycolipid recognition is unknown. Here we show CD1b-mycolipid tetramers reveal a hierarchy in which circulating T cells expressing CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity than CD4-CD8- T cells. CD4+ primary T cells transduced with mycolipid-specific T cell receptors bind CD1b-mycolipid tetramer with a higher fluorescence intensity than CD8+ primary T cells. The presence of either CD4 or CD8 also decreases the threshold for interferon-γ secretion. Co-receptor expression increases surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Targeted transcriptional profiling of mycolipid-specific T cells from individuals with active tuberculosis reveals canonical markers associated with cytotoxicity among CD8+ compared to CD4+ T cells. Thus, expression of co-receptors modulates T cell receptor avidity for mycobacterial lipids, leading to in vivo functional diversity during tuberculosis disease.

New silver(I) phosphino complexes: Evaluation of their potential as prospective agents against Mycobacterium tuberculosis.

Despite being a preventable and curable disease, Tuberculosis (TB) is the world's top infectious killer. Development of new drugs is urgently needed. In this work, the synthesis and characterization of new silver(I) complexes, that include N'-[(E)-(pyridine-2-ylmethylene)pyrazine-2-carbohydrazide, HPCPH, as main ligand and substituted aryl-phosphines as auxiliary ligands, is reported. HPCPH was synthesized from pyrazinoic acid, the active metabolite of the first-line antimycobacterial drug pyrazinamide. Complexes [Ag(HPCPH)(PPh3)2]OTf (1), [Ag(HPCPH)((P(p-tolyl)3)2]OTf (2) and [Ag(HPCPH)(P(p-anisyl)3)2]OTf (3) were characterized in solid state and in solution by elemental analysis and FTIR and NMR spectroscopies (OTftriflate). Crystal structures of (1,2) were determined by XRD. The Ag atom is coordinated to azomethine and pyridine nitrogen atoms of HPCPH ligand and to the phosphorous atom of each aryl-phosphine co-ligand. Although HPCPH did not show activity, the Ag(I) compounds demonstrated activity against Mycobacterium tuberculosis (MTB), H37Rv strain, and multi-drug resistant clinical isolates (MDR-TB). Globally, results showed that the compounds are not only effective against the sensitive strain, but are more potent against MDR-TB than antimycobacterial drugs used in therapy. The compounds showed low to moderate selectivity index values (SI) towards the bacteria, using MRC-5 cells (ATCC CCL-171) as mammalian cell model. Interaction with DNA was explored to get insight into the potential mechanism of action against the pathogen. No significant interaction was detected, allowing to discard this biomolecule as a potential molecular target. Compound 1 was identified as a hit compound (MIC90 2.23 µM; SI 4.4) to develop further chemical modifications in the search for new drugs.

Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis.

Studies involving the pathogenic organism Mycobacterium tuberculosis routinely require advanced biosafety laboratory facilities, which might not be readily available in rural areas where tuberculosis burdens are high. Attempts to adapt heat inactivation techniques have led to inconsistent conclusions, and the risk of protein denaturation due to extensive heating is impractical for subsequent mass spectrometry (MS)-based protein analyses. In this study, 240 specimens with one or two loops of M. tuberculosis strain H37Rv biomass and specific inactivated solutions were proportionally assigned to six heat inactivation methods in a thermal block at 80°C and 95°C for 20, 30, and 90 min. Twenty untreated specimens served as a positive control, and bacterial growth was followed up for 12 weeks. Our results showed that 90 min of heat inactivation was necessary for samples with two loops of biomass. Further protein extraction and a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS assay demonstrated adequate scores for bacterial identification (≥1.7), with the highest score achieved in the 80°C/90 min and 95°C/30 min treatment groups. A proteomics study also confidently identified 648 proteins with ∼93% to 96% consistent protein abundances following heating at 95°C for 20, 30, and 90 min. Heat inactivation at 95°C for 90 min yielded the most quantifiable proteins, and a functional analysis revealed proteins located in the ribosomal subunit. In summary, we proposed a heat inactivation method for the M. tuberculosis strain H37Rv and studied the preservation of protein components for subsequent bacterial identification and protein-related assays. IMPORTANCE Inactivation of Mycobacterium tuberculosis is an important step to guarantee biosafety for subsequent M. tuberculosis identification and related research, notably in areas of endemicity with minimal resources. However, certain biomolecules might be denatured or hydrolyzed because of the harsh inactivation process, and a standardized protocol is yet to be determined. We evaluated distinct heating conditions to report the inactivation efficiency and performed downstream mass spectrometry-based M. tuberculosis identification and proteomics study. The results are important and useful for both basic and clinical M. tuberculosis studies.

Sirtuin 7 Regulates Nitric Oxide Production and Apoptosis to Promote Mycobacterial Clearance in Macrophages.

The host immune system plays a pivotal role in the containment of Mycobacterium tuberculosis (Mtb) infection, and host-directed therapy (HDT) is emerging as an effective strategy to treat tuberculosis (TB), especially drug-resistant TB. Previous studies revealed that expression of sirtuin 7 (SIRT7), a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, was downregulated in macrophages after Mycobacterial infection. Inhibition of SIRT7 with the pan-sirtuin family inhibitor nicotinamide (NAM), or by silencing SIRT7 expression, promoted intracellular growth of Mtb and restricted the generation of nitric oxide (NO). Addition of the exogenous NO donor SNAP abrogated the increased bacterial burden in NAM-treated or SIRT7-silenced macrophages. Furthermore, SIRT7-silenced macrophages displayed a lower frequency of early apoptotic cells after Mycobacterial infection, and this could be reversed by providing exogenous NO. Overall, this study clarified a SIRT7-mediated protective mechanism against Mycobacterial infection through regulation of NO production and apoptosis. SIRT7 therefore has potential to be exploited as a novel effective target for HDT of TB.

Novel Assay Platform to Evaluate Intracellular Killing of Mycobacterium tuberculosis: In Vitro and In Vivo Validation.

One of the main hallmarks of tuberculosis (TB) is the ability of the causative agent to transform into a stage of dormancy and the capability of long persistence in the host phagocytes. It is believed that approximately one-third of the population of the world is latently infected with Mycobacterium tuberculosis (Mtb), and 5%-10% of these individuals can develop clinical manifestations of active TB even decades after the initial infection. In this latent, intracellular form, the bacillus is shielded by an extremely robust cell wall and becomes phenotypically resistant to most antituberculars. Therefore, there is a clear rationale to develop novel compounds or carrier-conjugated constructs of existing drugs that are effective against the intracellular form of the bacilli. In this paper, we describe an experimental road map to define optimal candidates against intracellular Mtb and potential compounds effective in the therapy of latent TB. To validate our approach, isoniazid, a first-line antitubercular drug was employed, which is active against extracellular Mtb in the submicromolar range, but ineffective against the intracellular form of the bacteria. Cationic peptide conjugates of isoniazid were synthesized and employed to study the host-directed drug delivery. To measure the intracellular killing activity of the compounds, Mtb-infected MonoMac-6 human monocytic cells were utilized. We have assessed the antitubercular activity, cytotoxicity, membrane interactions in combination with internalization efficacy, localization, and penetration ability on interface and tissue-mimicking 3D models. Based on these in vitro data, most active compounds were further evaluated in vivo in a murine model of TB. Intraperitoneal infectious route was employed to induce a course of slowly progressive and systemic disease. The well-being of the animals, monitored by the body weight, allows a prolonged experimental setup and provides a great opportunity to test the long-term activity of the drug candidates. Having shown the great potency of this simple and suitable experimental design for antimicrobial research, the proposed novel assay platform could be used in the future to develop further innovative and highly effective antituberculars.

Evaluation of Berberine as an Adjunct to TB Treatment.

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.

The threat of persistent bacteria and fungi contamination in tuberculosis sputum cultures.

BACKGROUND: Tuberculosis (TB) sputum culture contaminants make it difficult to obtain pure TB isolates.We aimed to study and identify persistent TB sputum culture contaminants post the standard laboratory pre-culture sample decontamination techniques. METHODS: This was a longitudinal study of TB sputum culture contamination for a cohort of TB patients on standard treatment at: baseline, during TB treatment and post TB treatment. Sputum samples were decontaminated with 1.5%NaOH and neutralized using 6.8 Phosphate buffer solution.Sputum was then inoculated into MGIT (mycobactrial growth indicator tube) supplemented with 0.8ml PANTA. A drop of each positive MGIT culture was sub cultured onto blood agar and incubated for 48 hours at 35 -37OC.Any growth was identified using growth characteristics and colony morphology. RESULTS: From October 2017 through May 2019;we collected 8645 sputum samples of which 8624(99.8%) were eligible and inoculated into MGIT where 2444(28.3%)samples were TB culture positive and 255(10.4%)were positive for contaminants: 237 none-tuberculosis bacteria, 12 fungi and 6 mixed(none-tuberculous bacteria+fungi). There was no statistically significant difference between none tuberculosis bacteria and fungi in the treatment (OR=1.4,95%CI:0.26-7.47,p=0.690) and the post treatment TB phases(OR=2.02,95%CI:0.38-10.79,p=0.411)Vs baseline. CONCLUSION: None-tuberculous bacteria and fungi dominate the plethora of TB sputum culture contamination and persist beyond the standard laboratory pre-culture decontamination algorithm.

Synthetic Sansanmycin Analogues as Potent Mycobacterium tuberculosis Translocase I Inhibitors.

Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.

Characterisation of a putative M23-domain containing protein in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis remains a global health concern, further compounded by the high rates of HIV-TB co-infection and emergence of multi- and extensive drug resistant TB, all of which have hampered efforts to eradicate this disease. As a result, novel anti-tubercular interventions are urgently required, with the peptidoglycan component of the M. tuberculosis cell wall emerging as an attractive drug target. Peptidoglycan M23 endopeptidases can function as active cell wall hydrolases or degenerate activators of hydrolases in a variety of bacteria, contributing to important processes such as bacterial growth, division and virulence. Herein, we investigate the function of the Rv0950-encoded putative M23 endopeptidase in M. tuberculosis. In silico analysis revealed that this protein is conserved in mycobacteria, with a zinc-binding catalytic site predictive of hydrolytic activity. Transcript analysis indicated that expression of Rv0950c was elevated during lag and log phases of growth and reduced in stationary phase. Deletion of Rv0950c yielded no defects in growth, colony morphology, antibiotic susceptibility or intracellular survival but caused a reduction in cell length. Staining with a monopeptide-derived fluorescent D-amino acid, which spatially reports on sites of active PG biosynthesis or repair, revealed an overall reduction in uptake of the probe in ΔRv0950c. When stained with a dipeptide probe in the presence of cell wall damaging agents, the ΔRv0950c mutant displayed reduced sidewall labelling. As bacterial peptidoglycan metabolism is important for survival and pathogenesis, the role of Rv0950c and other putative M23 endopeptidases in M. tuberculosis should be explored further.

Therapeutic Deep Eutectic Systems towards the Treatment of Tuberculosis and Colorectal Cancer: Opportunities and Challenges.

What if a new technology based on therapeutic deep eutectic systems would disrupt the current treatment of major economic and socially burden diseases? The classical definition of eutectic systems is that they are the combination of two or more compounds that interact via hydrogen bonds, from which results a melting temperature depression in comparison with that of its individual components. Therapeutic deep eutectic systems are defined as eutectic systems in which at least one of the individual components is an active pharmaceutical ingredient, or a eutectic system in which the active pharmaceutical ingredient is dissolved. Current literature reports on tuberculosis have been mostly based on the most common anti-tuberculosis drugs prescribed. Using eutectic systems based on naturally occurring molecules known for their anti-microbial activity may also present a promising therapeutic strategy able to cope with the prevalence of Mycobacterium tuberculosis and prevent the appearance of multidrug resistance strains. With regards to colorectal cancer, literature has been unravelling combinations of terpenes with anti-inflammatory drugs that are selectively cytotoxic towards colorectal cancer cells and do not compromise the viability of normal intestinal cells. This technology could contribute to preventing tumor growth and metastasis while providing a patient compliance therapeutics, which will be crucial to the success of overcoming the challenges presented by cancers.

Variation of tuberculosis prevalence across diagnostic approaches and geographical areas of Indonesia.

BACKGROUND: Tuberculosis (TB) has contributed to a significant disease burden and economic loss worldwide. Given no gold standard for diagnosis, early identification of TB infection has been challenging. This study aimed to comparatively investigate the prevalence of TB across diagnostic approaches (sputum AFB, sputum culture, sputum genetic test, and chest x-ray) and geographical areas of Indonesia. METHODS: Participant demographic variables and TB screening test results were obtained from the Tuberculosis Unit, Health Research and Development Agency, Ministry of Health (HRDA-MoH). The prevalence of pulmonary TB in populations aged 15 years and over was calculated using TB cases as a numerator and populations aged 15 years and over as a denominator. Variations across geographical areas and diagnostic approaches were expressed as prevalence and 95% confidence interval (CI). RESULTS: A total of 67,944 records were reviewed. Based on bacteriological evidence, the prevalence of TB per 100,000 in Indonesia was 759 (95% CI: 589.7-960.8) with variations across areas: 913 (95% CI 696.7-1,176.7; Sumatra), 593 (95% CI 447.2-770.6; Java-Bali), and 842 (95% CI 634.7-1,091.8; other islands). Also, the prevalence of TB varied across diagnostic approaches: 256.5 (sputum AFB), 545 (sputum culture), 752.2 (chest x-ray), and 894.9 (sputum genetic test). Based on sputum AFB, the TB prevalence varied from 216.6 (95% CI 146.5-286.8; Java-Bali), 259.9 (95% CI 184.2-335.6; other islands) to 307.4 (95% CI 208.3-406.5; Sumatra). Based on sputum culture, the TB prevalence ranged from 487.9 (95% CI 433.6-548.6; Java-Bali), 635.9 (95% CI 564.9-715.1; Sumatra), to 2,129.8 (95% CI 1,664.0-2,735.6; other islands). Based on chest x-ray, the TB prevalence varied from 152.1 (95% CI 147.9-156.3; Java-Bali), 159.2 (95% CI 154.1-164.3; Sumatra), to 864 (95% CI 809-921.4; other islands). Based on sputum genetic test, the TB prevalence ranged from 838.7 (95% CI 748.4-900.8; Java-Bali), 875 (95% CI 775.4-934.2; Sumatra), to 941.2 (95% CI 663.6-992.3; other islands). CONCLUSIONS: The variation of TB prevalence across geographical regions could be confounded by the diagnostic approaches. TRIAL REGISTRATION: This study was approved by the Institutional Review Board of Chulalongkorn University (IRB No. 684/63).

Designing new antitubercular isoniazid derivatives with improved reactivity and membrane trafficking abilities.

Isoniazid (INH) is one of the two most effective first-line antitubercular drugs and is still used at the present time as a scaffold for developing new compounds to fight TB. In a previous study, we have observed that an INH derivative, an hydrazide N'-substituted with a C10acyl chain, was able to counterbalance its smaller reactivity with a higher membrane permeability. This resulted in an improved performance against the most prevalent Mycobacterium tuberculosis (Mtb) resistant strain (S315T), compared to INH. In this work, we have designed two new series of INH derivatives (alkyl hydrazides and hydrazones) with promising in silico properties, namely membrane permeabilities and spontaneous IN* radical formation. The kinetics, cytotoxicity, and biological activity evaluations confirmed the in silico predictions regarding the very high reactivity of the alkyl hydrazides. The hydrazones, on the other hand, showed very similar behavior compared to INH, particularly in biological tests that take longer to complete, indicating that these compounds are being hydrolyzed back to INH. Despite their improved membrane permeabilities, the reactivities of these two series are too high, impairing their overall performance. Nevertheless, the systematic data gathered about these compounds have showed us the need to find a balance between lipophilicity and reactivity, which is paramount to devise better INH-based derivatives aimed at circumventing Mtb resistance.

Anti-Tuberculosis Activity of Pediococcus acidilactici Isolated from Young Radish Kimchi against Mycobacterium tuberculosis.

Tuberculosis is a highly contagious disease caused by Mycobacterium tuberculosis. It affects about 10 million people each year and is still one of the leading causes of death worldwide. About 2 to 3 billion people (equivalent to 1 in 3 people in the world) are infected with latent tuberculosis. Moreover, as the number of multidrug-resistant, extensively drug-resistant, and totally drug-resistant strains of M. tuberculosis continues to increase, there is an urgent need to develop new anti-tuberculosis drugs that are different from existing drugs to combat antibiotic-resistant M. tuberculosis. Against this background, we aimed to develop new anti-tuberculosis drugs using probiotics. Here, we report the anti-tuberculosis effect of Pediococcus acidilactici PMC202 isolated from young radish kimchi, a traditional Korean fermented food. Under coculture conditions, PMC202 inhibited the growth of M. tuberculosis. In addition, PMC202 inhibited the growth of drug-sensitive and -resistant M. tuberculosis- infected macrophages at a concentration that did not show cytotoxicity and showed a synergistic effect with isoniazid. In a 2-week, repeated oral administration toxicity study using mice, PMC202 did not cause weight change or specific clinical symptoms. Furthermore, the results of 16S rRNA-based metagenomics analysis confirmed that dysbiosis was not induced in bronchoalveolar lavage fluid after oral administration of PMC202. The anti-tuberculosis effect of PMC202 was found to be related to the reduction of nitric oxide. Our findings indicate that PMC202 could be used as an anti-tuberculosis drug candidate with the potential to replace current chemicalbased drugs. However, more extensive toxicity, mechanism of action, and animal efficacy studies with clinical trials are needed.

Association between 9-month isoniazid prophylaxis of latent tuberculosis and severe hepatitis in patients treated with TNF inhibitors.

To investigate associations between isoniazid for latent tuberculosis and risk of severe hepatitis, affecting patients with rheumatoid arthritis or ankylosing spondylitis whose treatment includes tumor necrosis factor inhibitors. Our self-controlled case series study analyzed Taiwan's National Health Insurance Database from 2003 to 2015 to identify RA or AS patients, aged ≥ 20 years, receiving TNF inhibitors and a 9-month single isoniazid treatment. The outcome of interest was hospitalization due to severe hepatitis. We defined risk periods by isoniazid exposure (days): 1-28, 29-56, 57-84, 85-168, 169-252, and 253-280. To compare risk of severe hepatitis in exposed and non-exposed periods, we performed conditional Poisson regressions to generate incidence rate ratios (IRR) and 95% confidence intervals, with adjustment of patients' baseline covariates including age, sex, HBV, HCV and related medication. Of 54,267 RA patients and 137,889 AS patients identified between 2000 and 2015, 11,221 (20.7%) RA and 4,208 (3.1%) AS patients underwent TNFi therapy, with 722 (5%) receiving isoniazid for latent tuberculosis. We identified 31 incident cases (4.3%) of hospitalization due to severe hepatitis. Of these hospitalization events, 5 occurred in the exposed periods, 25 occurred in the INH unexposed periods, and 1 occurred in the pre-exposure period. Compared with non-exposure, the risk of severe hepatitis was higher in exposed periods (incidence rate ratio [IRR]: 5.1, 95% CI: 1.57-16.55), especially 57-84 days (IRR: 17.29, 95% CI: 3.11-96.25) and 85-168 days (IRR:10.55, 95% CI: 1.90-58.51). The INH related fatal hepatotoxicity was not identified in our study. Our findings suggest an association between risk of severe hepatitis and exposure to isoniazid in patients with RA or AS under TNFi therapy, particularly within the exposed period 57-168 days. A close monitoring of liver function is mandatory to minimize the risk, especially within the first 6 months after initiation of 9 months isoniazid.

6-Fluorophenylbenzohydrazides inhibit Mycobacterium tuberculosis growth through alteration of tryptophan biosynthesis.

A major constraint in reducing tuberculosis epidemic is the emergence of strains resistant to one or more of clinically approved antibiotics, which emphasizes the need of novel drugs with novel targets. Genetic knockout strains of Mycobacterium tuberculosis (Mtb) have established that tryptophan (Trp) biosynthesis is essential for the bacterium to survive in vivo and cause disease in animal models. An anthranilate-like compound, 6-FABA, was previously shown to synergize with the host immune response to Mtb infection in vivo. Herein, we present a class of anthranilate-like compounds endowed with good antimycobacterial activity and low cytotoxicity. We show how replacing the carboxylic moiety with a hydrazide led to a significant improvement in both activity and cytotoxicity relative to the parent compound 6-FABA. Several new benzohydrazides (compounds 20-31, 33, 34, 36, 38 and 39) showed good activities against Mtb (0.625 ≤ MIC≤6.25 µM) and demonstrated no detectable cytotoxicity against Vero cell assay (CC50 ≥ 1360 µM). The target preliminary studies confirmed the hypothesis that this new class of compounds inhibits Trp biosynthesis. Taken together, these findings indicate that fluorophenylbenzohydrazides represent good candidates to be assessed for drug discovery.

Comparison of LAMP, GeneXpert, Mycobacterial Culture, Smear Microscopy, TSPOT.TB, TBAg/PHA Ratio for Diagnosis of Pulmonary Tuberculosis.

OBJECTIVE: To investigate the application value of loop-mediated isothermal amplification (LAMP), GeneXpert, mycobacterial culture, smear microscopy, TSPOT.TB (TSPOT), ratio of TB-specific antigen to phytohemagglutinin (TBAg/PHA ratio) in the detection of mycobacterium tuberculosis in the bronchoalveolar lavage fluid. METHODS: A retrospective analysis was performed on the patients who underwent bronchoscopy from December 2018 to November 2019 in Tongji Hospital. The patients with positive tuberculosis culture or positive GeneXpert in bronchoalveolar lavage fluid were selected as the case group, and those without tuberculosis served as the control group. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of LAMP, GeneXpert, culture, smear microscopy, TSPOT, and TBAg/PHA ratio. RESULTS: For the patients with positive cultures as case, the sensitivity of LAMP, GeneXpert, smear microscopy, TSPOT and TBAg/PHA ratio was 73.49%, 89.16%, 25.30%, 80.00%, 33.85%, respectively, the specificity was 99.00%, 100.00%, 99.00%, 86.00%, 100.00%, respectively, the area under the ROC curve (AUC) was 0.849, 0.938, 0.633, 0.830, 0.669, respectively. For the patients with positive GeneXpert as case, the sensitivity of LAMP, mycobacterial culture, smear microscopy, TSPOT and TBAg/PHA ratio was 73.20%, 74.23%, 22.68%, 68.92%, 29.73%, respectively, the specificity was 99.00%, 100.00%, 99.00%, 86.00%, 100.00%, respectively, the AUC was 0.853, 0.878, 0.623, 0.775, 0.649, respectively. CONCLUSION: The sensitivity of GeneXpert was best. The sensitivity and diagnostic value of LAMP were slightly lower than those of GeneXpert, and were similar to tuberculosis culture. The sensitivity of smear microscopy was low. The specificity of TSPOT was low. When TBAg/PHA ratio >0.2 was used as a diagnostic index, the specificity was improved, but the sensitivity was low.

PGRS Domain of Rv0297 of Mycobacterium tuberculosis Functions in A Calcium Dependent Manner.

Mycobacterium tuberculosis (M.tb), the pathogen causing tuberculosis, is a major threat to human health worldwide. Nearly 10% of M.tb genome encodes for a unique family of PE/PPE/PGRS proteins present exclusively in the genus Mycobacterium. The functions of most of these proteins are yet unexplored. The PGRS domains of these proteins have been hypothesized to consist of Ca2+ binding motifs that help these intrinsically disordered proteins to modulate the host cellular responses. Ca2+ is an important secondary messenger that is involved in the pathogenesis of tuberculosis in diverse ways. This study presents the calcium-dependent function of the PGRS domain of Rv0297 (PE_PGRS5) in M.tb virulence and pathogenesis. Tandem repeat search revealed the presence of repetitive Ca2+ binding motifs in the PGRS domain of the Rv0297 protein (Rv0297PGRS). Molecular Dynamics simulations and fluorescence spectroscopy revealed Ca2+ dependent stabilization of the Rv0297PGRS protein. Calcium stabilized Rv0297PGRS enhances the interaction of Rv0297PGRS with surface localized Toll like receptor 4 (TLR4) of macrophages. The Ca2+ stabilized binding of Rv0297PGRS with the surface receptor of macrophages enhances its downstream consequences in terms of Nitric Oxide (NO) production and cytokine release. Thus, this study points to hitherto unidentified roles of calcium-modulated PE_PGRS proteins in the virulence of M.tb. Understanding the pathogenic potential of Ca2+ dependent PE_PGRS proteins can aid in targeting these proteins for therapeutic interventions.