Manju Y. Krishnan

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 Crohns 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.

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 Crohns 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.

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.

Insight into the structural flexibility and function of Mycobacterium tuberculosis isocitrate lyase.

Isocitrate lyase (ICL), is a key enzyme of the glyoxylate shunt crucial for the survival of Mycobacterium tuberculosis (Mtb) in macrophages during persistent infection. MtbICL catalyses the first step of this carbon anaplerosis cycle and is considered as a potential anti-tubercular drug target. The MtbICL is a tetramer with 222 symmetry, and each subunit of the enzymeis composed of 14 α-helices and 14 β-strands. We studied the conformational flexibility of the enzyme to get a deeper insight into its stability and function. Our studies show that the mutation of His180, close to the MtbICL signature sequence (K193KCGH197) completely abolishes the oligomeric conformation and function of the enzyme. Molecular dynamics studies suggest that the loss of interaction between His180 and Tyr89 most likely alters the orientation of Tyr89 side chain, thereby causing the movement of helices α6, α12, α13 and α14 in the vicinity and affecting the tetrameric assembly. We further show that the oligomerization of MtbICL is primarily mediated by the inter subunit interactions, and strengthened by the helix swapping of α12-α13 between adjacent subunits. Furthermore, the enzyme activity is influenced by the interactions between the residues of lid region (P411NSSTTALTGSTEEGQFH428) and the loop region (T391KHQREV397). Mutation of glutamates of the lid region to non homologous residues (E423A or E424A) or basic residues (E423K or E424K) inactivates the enzyme, whereas the activity is not much compromised in case of homologous mutations (E423D or E424D).

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.

Isocitrate lyase of Mycobacterium tuberculosis is inhibited by quercetin through binding at N-terminus

Combating tuberculosis requires new therapeutic strategies that not only target the actively dividing bacilli but also the dormant bacilli during persistent infection. Isocitrate lyase (ICL) is a key enzyme of the glyoxylate shunt, crucial for the survival of bacteria in macrophages and mice. MtbICL is considered as one of the potential and attractive drug targets against persistent infection. We report the inhibition of MtbICL by quercetin with IC50 of 3.57 μM. In addition, quercetin strongly inhibited the growth of Mtb H37Rv utilizing acetate, rather than glucose as the sole carbon source, suggesting the inhibition of glyoxylate shunt. Quercetin binds at the N-terminus of MtbICL (Kd - 6.68 μM).

Biological evaluation of novel substituted chloroquinolines targeting mycobacterial ATP synthase

The ATP synthase of Mycobacterium tuberculosis is a validated drug target against which a diarylquinoline drug is under clinical trials. The enzyme is crucial for the viability both of actively replicating and non-replicating/dormant M. tuberculosis. Enzyme levels drop drastically as the bacilli enter dormancy and hence an inhibitor would make the dormant bacilli even more vulnerable. In this study, a set of 18 novel substituted chloroquinolines were screened against Mycobacterium smegmatis ATP synthase; 6 compounds with the lowest 50% inhibitory concentration (IC(50)) values (0.36-1.83 μM) were selected for further in vitro studies. All six compounds inhibited the growth of M. tuberculosis H37Rv in vitro, with minimum inhibitory concentrations (MICs) of 3.12 μg/mL (two compounds) or 6.25 μg/mL (four compounds). All of them were bactericidal to non-replicating M. tuberculosis H37Rv in hypoxic culture; three compounds caused a >2 log(10) reduction in CFU counts in 4 days at concentrations of 16× or 32× their MICs, compared with a 0.2 log(10) reduction by isoniazid and a >4 log(10) reduction by rifampicin at 100× their MICs. The compounds also contributed to a greater reduction in total cellular ATP of the bacilli compared with isoniazid and rifampicin during an exposure time of 18 h. The compounds at 100 μM caused only 5-35% inhibition of mouse liver mitochondrial ATP synthase, leading to selectivity indices ranging from >55-fold to >278-fold. In vitro cytotoxicity to the Vero cell line measured as the 50% cytotoxic concentration (CC(50)) of the compounds ranged between 55 μg/mL and >300 μg/mL.

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.

Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.

The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45μM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300μg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51μM, mammalian ATPase IC50>100μM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12μg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100μg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5μg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173μmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains.

Insights into the Mechanism of Action of Hyaluronate Lyase ROLE OF C-TERMINAL DOMAIN AND Ca2+ IN THE FUNCTIONAL REGULATION OF ENZYME

Hyaluronate lyases (HLs) cleave hyaluronan and certain other chondroitin/chondroitin sulfates. Although native HL from Streptococcus agalactiae is composed of four domains, it finally stabilizes after autocatalytic conversion as a 92-kDa enzyme composed of the N-terminal spacer, middle α-, and C-terminal domains. These three domains are independent folding/unfolding units of the enzyme. Comparative structural and functional studies using the enzyme and its various fragments/domains suggest a relatively insignificant role of the N-terminal spacer domain in the 92-kDa enzyme. Functional studies demonstrate that the α-domain is the catalytic domain. However, independently it has a maximum of only about 10% of the activity of the 92-kDa enzyme, whereas its complex with the C-terminal domain in vitro shows a significant enhancement (about 6-fold) in the activity. It has been previously proposed that the C-terminal domain modulates the enzymatic activity of HLs. In addition, one of the possible roles for calcium ions was suggested to induce conformational changes in the enzyme loops, making HL more suitable for catalysis. However, we observed that calcium ions do not interact with the enzyme, and its role actually is in modulating the hyaluronan conformation and not in the functional regulation of enzyme.