The use of Mycobacterium tuberculosis H37Ra-infected immunocompetent mice as an in vivo model of persisters.

Persistence of Mycobacterium tuberculosis (Mtb) is one of the challenges to successful treatment of tuberculosis (TB). In vitro models of non-replicating Mtb are used to test the efficacy of new molecules against Mtb persisters. The H37Ra strain is attenuated for growth in macrophages and mice. We validated H37Ra-infected immunocompetent mice for testing anti-TB molecules against slow/non-replicating Mtb in vivo. Swiss mice were infected intravenously with H37Ra and monitored for CFU burden and histopathology for a period of 12 weeks. The bacteria multiplied at a slow pace reaching a maximum load of ∼106 in 8-12 weeks depending on the infection dose, accompanied by time and dose-dependent histopathological changes in the lungs. Surprisingly, four-weeks of treatment with isoniazid-rifampicin-ethambutol-pyrazinamide combination caused only 0.4 log10 and 1 log10 reduction in CFUs in lungs and spleen respectively. The results show that ∼40 % of the H37Ra bacilli in lungs are persisters after 4 weeks of anti-TB therapy. Isoniazid/rifampicin monotherapy also showed similar results. A combination of bedaquiline and isoniazid reduced the CFU counts to <200 (limit of detection), compared to ∼5000 CFUs by isoniazid alone. The study demonstrates an in vivo model of Mtb persisters for testing new leads using a BSL-2 strain.

Design, synthesis and biological evaluation of (Quinazoline 4-yloxy)acetamide and (4-oxoquinazoline-3(4H)-yl)acetamide derivatives as inhibitors of Mycobacterium tuberculosis bd oxidase.

New chemical scaffolds with novel mechanism of action are urgently needed for the treatment of drug resistant tuberculosis. The oxidative phosphorylation pathway of Mycobacterium tuberculosis consists of multiple clinically validated drug targets. This pathway can function through any one of the two terminal oxidases-the proton pumping cytochrome bc1-aa3 supercomplex, or the less energy efficient but high affinity cytochrome bd oxidase. Inhibiting the bc1 complex alone has been found bacteriostatic and not bactericidal. On the other hand, inhibition of both these oxidases turns lethal to the pathogen. In the present study, we used a bc1 complex mutant of M. tuberculosis to screen (Quinazoline 4-yloxy)acetamide and (4-oxoquinazoline-3(4H)-yl)acetamide derivatives against the alternate oxidase, i.e., cytochrome bd oxidase. Two molecules, S-021-0601 and S-021-0607 were found to inhibit the mutant with MICs 8 and 16 µM respectively, compared to MICs of 128 and 256 µM against the wild type M. tuberculosis. In the wild type, one of the compounds showed synergism with Q203, an inhibitor of bc1 complex, in inhibiting growth under aerobic conditions. Both compounds showed synergism with Q203 in depleting bacterial ATP and inhibiting oxygen consumption. Both the compounds at 32 µM (one-fourth or one-eighth of their MICs for wild type) were bactericidal to wild type bacteria under hypoxic condition, causing ∼1.9 log10 reduction in viable counts which increased to ∼4-log10 when combined with Q203.

Anti-mycobacterial activity evaluation of designed peptides: cryptic and database filtering based approach.

Worldwide, TB is one of the deadly airborne diseases, which accounts for 10.4 million deaths annually. Serious toxicity issue, prolonged treatment regimens of the current drugs, rise in multidrug-resistant strains, and the unique defensive mechanism makes the development of novel therapeutic molecules against Mycobacterium tuberculosis (MT) an urgent need. As MT has a lengthy latent phase and unique cell wall architecture, a reasonable approach is needed to find molecules having a different killing mechanism rather than traditional approaches. Host defence peptides (HDPs) will be the most promising alternative, potential therapeutic candidates as they target the microbial membrane in particular and are an essential part of the innate immunity of humans. This works demonstrates the utility of "Database filtering" and three-dimensional (3D) modelling approach in finding novel AMPs with appreciable activity towards MT. Results of this study indicate that peptides with 70% hydrophobicity, but without hydrophobicity patches (> 4 hydrophobic amino acids in series) and charge of + 4 or + 5 are most likely to be good anti-tubercular candidates.

Triacylglycerols: Fuelling the Hibernating Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) has the remarkable ability to persist with a modified metabolic status and phenotypic drug tolerance for long periods in the host without producing symptoms of active tuberculosis. These persisters may reactivate to cause active disease when the immune system becomes disrupted or compromised. Thus, the infected hosts with the persisters serve as natural reservoir of the deadly pathogen. Understanding the host and bacterial factors contributing to Mtb persistence is important to devise strategies to tackle the Mtb persisters. Host lipids act as the major source of carbon and energy for Mtb. Fatty acids derived from the host cells are converted to triacylglycerols (triglycerides or TAG) and stored in the bacterial cytoplasm. TAG serves as a dependable, long-term energy source of lesser molecular mass than other storage molecules like glycogen. TAG are found in substantial amounts in the mycobacterial cell wall. This review discusses the production, accumulation and possible roles of TAG in mycobacteria, pointing out the aspects that remain to be explored. Finally, the essentiality of TAG synthesis for Mtb is discussed with implications for identification of intervention strategies.

The diacylglycerol acyltransferase Rv3371 of Mycobacterium tuberculosis is required for growth arrest and involved in stress-induced cell wall alterations.

Triacylglycerol (TAG) is important to mycobacteria both as cell envelope component and energy reservoir. Mycobacterium tuberculosis (Mtb) genome encodes at least 15 putative TAG synthase (tgs)s. We report that one of these genes, Rv3371, specific to pathogenic mycobacteria, when expressed in M. smegmatis leads to modifications in colony morphotype, bacterial architecture, cell surface properties and elevated TAG levels. Rv3371 was found to largely localize in the cell membrane. The Rv3371 promoter is minimally active during exponential growth in vitro, however, is up-regulated under stationary phase, hypoxia, nutrient starvation, nitrosative stress, low iron, in IFN-γ activated macrophages and infected mice. The low iron-induced expression of Rv3371 is likely due to the de-repression by Rv1404, which is probably activated by ideR. An Rv3371 deletion mutant of Mtb showed impaired non-replicating persistence in vitro and altered sensitivity to anti-mycobacterial drugs. In low iron medium, the Rv3371 deletion mutant showed reduced formation of TAG containing extracellular vesicles. Therefore Rv3371 is likely involved in Mtb growth arrest and cell wall alterations during persistence.

Down-regulation of PE11, a cell wall associated esterase, enhances the biofilm growth of Mycobacterium tuberculosis and reduces cell wall virulence lipid levels.

PE11 (Rv1169c or LipX) is a cell wall associated esterase/lipase of Mycobacterium tuberculosis (Mtb). Evidences suggest that PE11 is expressed by Mtb both in vitro and in vivo. Previous studies have shown that PE11 leads to modification in cell wall lipid content and enhanced virulence when expressed in the non-pathogenic surrogate Mycobacterium smegmatis. Since cell wall lipids often play different roles in pathogenic and non-pathogenic mycobacteria, we investigated the role of PE11 in its host, Mtb. Mtb with lowered expression of PE11 (PE11 knock-down) displayed significant changes in colony morphology and cell wall lipid profile, confirming the role of PE11 in cell wall architecture. In addition, the levels of phthiocerol dimycocerosates, a cell wall virulence factor, were decreased. Levels of trehalose esters and free mycolic acids were increased. In contrast to M. smegmatis expressing Mtb PE11, a role reversal was observed in Mtb with respect to pellicle/biofilm formation. The PE11 knock-down Mtb strain showed significantly enhanced aggregation and early biofilm growth in detergent-free medium, compared to the wild-type. Knock-down strain also showed nearly 27-fold up-regulation of a fibronectin attachment protein (Rv1759c), linking biofilm growth with over-expression of bacterial proteins that help in aggregation and/or binding to host extracellular matrix. The knock-down also resulted in poor virulence of Mtb in PMA (phorbol 12-myristate 13-acetate) treated and PMA+IFN-γ treated THP-1 macrophages. Therefore, the study not only links PE11 to cell wall virulence lipids but also reveals the involvement of this cell wall associated esterase in down-regulation of biofilm in Mtb.

Use of an adipocyte model to study the transcriptional adaptation of Mycobacterium tuberculosis to store and degrade host fat.

During its persistence in the infected host, Mycobacterium tuberculosis (Mtb) accumulates host-derived fatty acids in intracytoplasmic lipid inclusions as triacylglycerols which serve primarily as carbon and energy reserves. The Mtb genome codes for more than 15 triacylglycerol synthases, 24 lipase/esterases, and seven cutinase-like proteins. Hence, we looked at the expression of the corresponding genes in intracellular bacilli persisting amidst the host triacylglycerols. We used the Mtb infected murine adipocyte model to ensure persistence and transcripts were quantified using real-time reverse transcriptase polymerase chain reaction. Dormancy and glyoxylate metabolism was confirmed by the upregulated expression of dosR and icl, respectively, by intra-adipocyte bacilli compared with in vitro growing bacilli. The study revealed that tgs1, tgs2, Rv3371, and mycolyltransferase Ag85A are the predominant triacylglycerol synthases, while lipF, lipH, lipJ, lipK, lipN, lipV, lipX, lipY, culp5, culp7, and culp6 are the predominant lipases/esterases used by Mtb for the storage and degradation of host-derived fat. Moreover, it was observed that many of these enzymes are used by Mtb during active replication rather than during nonreplicating persistence, indicating their probable function in cell wall synthesis.

Mycobacterium tuberculosis can gain access to adipose depots of mice infected via the intra-nasal route and to lungs of mice with an infected subcutaneous fat implant.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis has the remarkable ability to persist as non-replicating forms in the host. These persisters are tolerant to drugs targeting actively replicating bacilli and hence are responsible for the need of an extended duration of anti-tubercular therapy. The anatomical locations and cell types housing Mtb persisters are being investigated in the recent times. Adipose tissue and the adipocytes are proposed niches of Mtb persisters. In the present study, we carried out experiments in the immunocompetent Swiss mice to see the dissemination of Mtb from lungs to adipose tissue and vice versa. Mice infected intra-nasally with ∼ 10(6), 10(4) or 10(2) bacilli harboured Mtb in various adipose depots distal to the lungs such as the visceral, subcutaneous and peri-renal depots. The dissemination was minimal at two weeks post-infection, as evident from culture negative adipose tissue samples. But at seven weeks post-infection, viable Mtb could be detected in 78%, 66% and 66% of the samples from high, moderate and low dose-infection groups respectively. In a separate experiment, Mtb-infected pre-adipocytes were implanted subcutaneously to un-infected mice. At five weeks post-implantation, the intact implants had a mean 7 ± 0.53 log10 CFUs/100 mg tissue, while the lungs had a mean 3.25 ± 0.32 log10 CFUs/100 mg tissue. In conclusion, the study shows that Mtb can disseminate from lungs to distant adipose depots and vice versa.

Primary mouse lung fibroblasts help macrophages to tackle Mycobacterium tuberculosis more efficiently and differentiate into myofibroblasts up on bacterial stimulation.

Keeping with their classical role in wound healing, fibroblasts of the lung take part in the resolution of tubercular granulomas. They are totally absent in nascent granulomas, but surround necrotizing granulomas, and are the majority of cells in healed granulomas. Lung fibroblasts may become infected with Mycobacterium tuberculosis (Mtb). Two previous studies suggested an immunomodulatory effect of fibroblasts on infected macrophages. In the present study, we looked at the role of primary mouse lung fibroblasts on naive or activated mouse bone marrow macrophages infected with Mtb and the effect of infection on fibroblast properties. We observed that with fibroblasts in the vicinity, infected naive macrophages restricted the bacterial growth, while activated macrophages turned more bactericidal with concomitant increase in nitrite production. Neutralizing IL-1α in fibroblast supernatant reduced the nitrite production by infected macrophages. Secretion of IL-6 and MCP-1 was down-regulated, while TNF-α was up-regulated in infected naive macrophages. In infected activated macrophages, the secretion of IL-6 was up-regulated, while that of MCP-1 and TNF-α was unaffected. The 'fibroblast effects' were enhanced when the fibroblasts too were infected. Mtb induced IL-1 secretion and pro-fibrotic responses by fibroblasts. Mtb-induced myofibroblast conversion was blocked by rapamycin suggesting cell signalling via mTOR.

Mycobacterium tuberculosis persistence in various adipose depots of infected mice and the effect of anti-tubercular therapy.

The adipocytes are one of the non-professional phagocytes postulated to be a haven for Mycobacterium tuberculosis during persistence in the human host. The adipocyte - M. tuberculosis interaction data available to date are ex vivo. The present study was primarily aimed to investigate M. tuberculosis infection of adipocytes in course of infection of mouse model. Using primary murine adipocytes, the study first confirmed the infection and immunomodulation of natural adipocytes by M. tuberculosis. The bacilli could be isolated form visceral, subcutaneous, peri renal and mesenteric adipose depots of immunocompetent mice infected with M. tuberculosis intravenously. The bacilli could be isolated from adipocytes and the stromal vascular fraction, even though the numbers were significantly higher in the latter. The bacterial burden in the adipose depots was comparable to those in lungs in the early phase of infection. But with time, the burden in the adipose depots was either decreased or kept under control, despite the increasing burden in the lungs. Infected mice treated with standard anti tubercular drugs, despite effective elimination of bacterial loads in the lungs, continued to harbour M. tuberculosis in adipose depots at loads similar to untreated mice in the late infection phase.

In vitro antimicrobial activities of capuramycin analogues against non-tuberculous mycobacteria.

OBJECTIVES: To determine antibacterial activity of capuramycin analogues SQ997, SQ922, SQ641 and RKS2244 against several non-tuberculous mycobacteria (NTM). METHODS: In vitro antibiotic activities, i.e. MIC, MBC, rate of killing and synergistic interaction with other antibiotics, were evaluated. RESULTS: SQ641 was the most active compound against all the NTM species studied. The MIC of SQ641 was ≤0.06-4 mg/L for Mycobacterium avium complex (MAC; n = 20), 0.125-2 mg/L for M. avium paratuberculosis (MAP; n = 9), 0.125-2 mg/L for Mycobacterium kansasii (MKN;n = 2), 0.25-1 mg/L for Mycobacterium abscessus (MAB; n = 11), 4 mg/L for Mycobacterium smegmatis (MSMG; n = 1), and 1 and 8 mg/L for Mycobacterium ulcerans (MUL; n = 1), by microdilution and agar dilution methods, respectively. SQ641 was bactericidal against NTM, with an MBC/MIC ratio of 1 to 32, and killed all mycobacteria faster than positive control drugs for each strain. In chequerboard titrations, SQ641 was synergistic with ethambutol against both MAC and MSMG, and was synergistic with streptomycin and rifabutin against MAB. CONCLUSIONS: In vitro, SQ641 was the most potent of the capuramycin analogues against all NTM tested, both laboratory and clinical strains.

Effects of interactions of antibacterial drugs with each other and with 6-mercaptopurine on in vitro growth of Mycobacterium avium subspecies paratuberculosis.

OBJECTIVES: Mycobacterium avium subspecies paratuberculosis (MAP) has been targeted for treatment with clarithromycin and rifamycin derivatives in numerous cases of Crohn's disease (CD). 6-Mercaptopurine and its pro-drug azathioprine are widely used as immunomodulators in the treatment of CD and have recently been shown to have anti-MAP activity in vitro. The objectives of the study were to evaluate the in vitro effects on MAP of (i) 6-mercaptopurine when combined with each of eight conventional antibacterial agents with in vitro anti-MAP activity and (ii) antibacterial combinations consisting of two drugs (clarithromycin combined with amikacin, rifampicin, ciprofloxacin or ethambutol) and three drugs (clarithromycin, rifabutin and clofazimine). METHODS: The drug interaction effects on nine human isolates of MAP were determined by the chequerboard method adapted for the BACTECMGIT960 culture system and by calculation of the fractional inhibitory concentration index (FICI) for drug combinations. RESULTS: Synergism (FICI < or = 0.5) was observed between 6-mercaptopurine and azithromycin (seven isolates), clarithromycin, rifampicin, rifabutin (four isolates each) and ethambutol (two isolates). 6-Mercaptopurine was not antagonistic with any of the antibacterial agents tested. Among the combinations of two and three antibacterials tested, the clarithromycin/rifampicin combination was synergistic against four isolates, while all other combinations showed no interaction. CONCLUSIONS: This in vitro study suggests that 6-mercaptopurine may be synergistic with macrolides and rifamycin derivatives against MAP. The activity of clarithromycin against MAP seems to be enhanced by rifampicin.

Comparison of three methods for susceptibility testing of Mycobacterium avium subsp. paratuberculosis to 11 antimicrobial drugs.

OBJECTIVES: To evaluate the BACTEC(TM) MGIT(TM) 960/MGIT Para TB (MGIT) system for drug susceptibility testing of Mycobacterium avium subsp. paratuberculosis (MAP), a pathogen implicated in some forms of Crohn's disease. METHODS: MICs of 11 drugs for 10 MAP strains were determined using the MGIT system, the BACTEC(TM)460TB system (BACTEC) and conventional agar dilution methods. RESULTS: MICs determined by MGIT methods showed 80%-100% agreement (+/-1 log(2) dilution) with those determined by the BACTEC and agar dilution methods for ciprofloxacin, levofloxacin, azithromycin and clofazimine. The MGIT and BACTEC methods showed 70%, 80% and 90% agreement (+/-1 log(2) dilution) for MICs of ethambutol, rifabutin and rifampicin; agreement for all drugs increased to 100% at 2 log(2) dilution differences. For clarithromycin, the MGIT method had greater agreement with the agar dilution method (70% at the same dilution) than the BACTEC method (60% at +/-1 log(2) dilution); agreement increased to 100% at +/-2 log(2) dilutions in both cases. The MGIT and agar dilution methods agreed 60% and 100% for amikacin MICs at +/-1 log(2) dilution and +/-2 log(2) dilutions, respectively. By all methods MICs were higher than achievable serum concentrations for isoniazid and dapsone. There was 100% agreement between all three methods for azithromycin, clarithromycin and ciprofloxacin, and 80% agreement for rifampicin using published MIC thresholds available for M. avium complex strains. CONCLUSIONS: This study shows that the MGIT system can be used for rapid and reliable drug susceptibility testing of MAP.

Combined use of Amplified Fragment Length Polymorphism and IS6110-RFLP in fingerprinting clinical isolates of Mycobacterium tuberculosis from Kerala, South India.

BACKGROUND: DNA fingerprinting by IS6110-RFLP has shown a high incidence of Mycobacterium tuberculosis isolates having no and low copies of the insertion sequence in Kerala, South India. Amplified Fragment Length Polymorphism (AFLP) would scan the entire genome rather than a few repetitive elements, we thought that this technique would help us in differentiating the large reservoir of isolates from an endemic region. Here we evaluate the ability of Amplified Fragment Length Polymorphism (AFLP) to type clinical isolates. METHODS: Fifty clinical isolates of M. tuberculosis were analysed by conventional radioactive AFLP and IS6110- RFLP. M. bovis, M. bovis BCG and two non tuberculous mycobacteria were also analysed to see species specific differences generated by AFLP. Cluster analysis was performed using the AFLP profile that showed the maximum polymorphism within M. tuberculosis and this was compared to the number of copies of IS6110 insertions. RESULTS: For AFLP, out of ten primer pairs tested, the EO/MC pair generated maximum polymorphism among the clinical isolates of M. tuberculosis. The similarity between the isolates ranged between 88 and 99.5%. Majority (nearly 85%) of the 'low copy' IS6110 isolates clustered together, while the rest clustered irrespective of the copy numbers. AFLP could show rare differences between isolates of M. tuberculosis, M. bovis and M. bovis BCG. The AFLP profiles for non-tuberculous mycobacteria were highly different from those of M. tuberculosis. CONCLUSION: Polymorphism generated by AFLP within the M. tuberculosis species is limited and hence AFLP alone seems to have limited use in fingerprinting the isolates in Kerala. The combined use of AFLP and IS6110-RFLP showed relatively better differentiation of 'high copy' IS6110 isolates, but failed to differentiate the 'low copy' isolates. However, the technique may be efficient in inter-species differentiation, and hence potentially useful in identifying and developing species-specific markers.