sepharose and Tuberculosis

Modern drug discovery is a target-driven approach in which a particular protein such as an enzyme is implicated in the disease process. Commonly, small-molecule drugs are identified using screening, rational design, and structural biology approaches. Drug screening, testing and optimization is typically conducted in vitro, and copious amounts of protein are required. The advent of recombinant DNA technologies has resulted in a rise in proteins purified by affinity techniques, typically by incorporating an "affinity tag" at the N- or C-terminus. Use of these tagged proteins and affinity techniques comes with a host of issues. This chapter describes the production of an untagged enzyme, α-methylacyl-CoA racemase (MCR) from Mycobacterium tuberculosis, using a recombinant E. coli system. Purification of the enzyme on a 100 mg scale using tandem anion-exchange chromatographies (DEAE-sepharose and RESOURCE-Q columns), and size-exclusion chromatographies is described. A modified protocol allowing the purification of cationic proteins is also described, based on tandem cation-exchange chromatographies (using CM-sepharose and RESOURCE-S columns) and size-exclusion chromatographies. The resulting MCR protein is suitable for biochemical and structural biology applications. The described protocols have wide applicability to the purification of other recombinant proteins and enzymes without using affinity chromatography.

Topics: Chromatography, Affinity; Escherichia coli; Humans; Kinetics; Mycobacterium tuberculosis; Recombinant Proteins; Sepharose; Tuberculosis

The QuantaMatrix Microfluidic Agarose Channel (QMAC) system was used for rapid drug susceptibility testing (DST). Here, we performed DST using QMAC integrated with the mycobacteria growth indicator tube (MGIT) liquid culture employing a specially designed cross agarose channel for the tuberculosis chip. MGIT-, QMAC-, and Löwenstein-Jensen (LJ)-DSTs were performed using 13 drugs. The protocol for QMAC-DST was optimized using the inoculum obtained after the disaggregation of Mycobacterium tuberculosis clumps in MGIT culture. The completion times of QMAC-DST and MGIT-DST were analyzed, and the results of all three DSTs were compared. Discrepant results were analyzed using line probe assays and DNA sequencing. Nontuberculous mycobacteria were distinguished using the ρ-nitrobenzoic acid inhibition test. The overall agreement rate of QMAT-DST and LJ-DST was 97.0% and that of QMAT-DST and MGIT-DST was 86.3%. An average turnaround time for DST was 5.4 days, which was considerably less than the time required for MGIT-DST. The overall time required to obtain DST results using QMAC-DST integrated with MGIT culture was an average of 18.6 days: 13.2 days for culture and identification and 5.4 days for DST. Hence, QMAC-DST integrated with liquid culture can be used to perform DSTs with short turnaround times and effective detection. KEY POINTS: • QMAC system can simultaneously perform phenotypic DST with 13 anti-TB drugs and PNB. • An optimized DST protocol led to a marked decrease in clumping in MGIT culture. • QMAC system integrated with MGIT liquid culture system reduced the turnaround time.

Topics: Bacteriological Techniques; Culture Media; Humans; Microbial Sensitivity Tests; Microfluidics; Mycobacterium tuberculosis; Sepharose; Tuberculosis

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.

Topics: Apoptosis; Caspase 8; Chromatography, Affinity; Concanavalin A; Extracellular Space; Humans; Kinetics; Monocytes; Mycobacterium tuberculosis; RNA, Bacterial; Sepharose; Tuberculosis; Tumor Necrosis Factor-alpha

In the majority of individuals infected with Mycobacterium tuberculosis, the bacilli cause a long-term asymptomatic infection called latent tuberculosis, a state during which the bacilli reside within granulomas. Latently infected individuals have around 10% risk of progression to clinical disease at a later stage. Determining the state of the mycobacteria and the host cells during this latent phase, i.e. within the granulomas, would greatly improve our understanding of the physiopathology of tuberculosis, and thus enable the development of new therapeutic means to treat the one-third of the world's population who are latently infected. We have developed an in vitro model of human mycobacterial granulomas, enabling the cellular and molecular analysis of the very first steps in the host granulomatous response to either mycobacterial compounds or live mycobacterial species. In vitro mycobacterial granulomas mimic natural granulomas very well, with the progressive recruitment of macrophages around live bacilli or mycobacterial antigen-coated beads, their differentiation into multinucleated giant cells and epithelioid cells, and the final recruitment of a ring of activated lymphocytes. Besides morphological similarities, in vitro granulomas also functionally resemble natural ones, with the development of intense cellular co-operation and intracellular mycobactericidal activities.

Topics: Animals; Antigens, Bacterial; BCG Vaccine; Cells, Cultured; Disease Progression; Granuloma; Humans; In Vitro Techniques; Leukocytes, Mononuclear; Lymphocyte Activation; Mycobacterium tuberculosis; Sepharose; Tuberculosis