Designed by: Matthew Deyell   Group: iGEM13_Paris_Bettencourt   (2013-09-23)


Cutinase-like serine esterase. Triggers rapid lysis of mycobacteria such as M. smegmatis and M. tuberculosis.

Sequence and Features

Assembly Compatibility:
  • 10
  • 12
    Illegal NotI site found at 530
  • 21
    Illegal BamHI site found at 389
    Illegal XhoI site found at 655
  • 23
  • 25
    Illegal NgoMIV site found at 18
  • 1000
    Illegal BsaI site found at 652

Usage and Biology

Trehalose Dimycolate Hydrolase (TDMH) is a cutinase-like serine esterase that triggers rapid lysis of the mycobacterial cell wall. This enzyme was first isolated form M. smegmatis mutant strain and it could hydrolyze purified TDM from various mycobacterial species. It was show that exposure to TDMH triggers an immediate release of free mycolic acids from noncovalently associated mycolyl-containing glycolipids, ultimately leading to rapid and extensive lysis of pathogenic species, such as M. tuberculosis, M. bovis, and M. marinum, as well as to a lesser extent of M. smegmatis and M. avium (Yang et al. 2012).

Characterization of TDMH

We wanted to investigate how TDMH producing E. coli is efficient in killing mycobacteria first outside the macrophage and then inside.

Experimental setup

In our system Msmeg_1529, a gene which encodes TDMH, is driven under the T7 promoter in the pET21b(+) expression system. The plasmid was transformed in E. coli BL21 strain and the expression was induced with 10 µM IPTG. For the overexpression of the Msmeg_1529 gene, followed by E. coli BL21 lysis and protein release, 10 mM IPTG was used.

E. coli BL21 used in this test was transformed with the TDMH carrying plasmid (pET21b) and with the pColA Duet plasmid carrying an RFP cassette with a constitutive Anderson promoter (BBa_J23102). The killing efficiency was tested on Mycobacterium smegmatis MC2, a non-phatogenic rapidly growing strain of mycobacteria. We cultivated E. coli BL21 and M. smegmatis MC2 in LB media, with different ratios of the two strains:

1 E. coli for 1 M. smegmatis
1 E. coli for 10 M. smegmatis
1 E. coli for 100 M. smegmatis

In these cultures E. coli was induced with 10 mM IPTG, which led to overexpression and release of TDMH in the media. The growth of both strains was measured for different time points (0, 1, 2, 3 and 6 h) by standard dilution series procedure and was expressed in CFUs/mL. Nalidixic acid plates (3 µg/mL) were used for following the growth of M. smegmatis, where the nalidixic acid acts as a selective agent for growing only mycobacteria. The growth of E. coli was followed on kanamycin plates, where the kanamycin resistance was carried on the pColA vector. For each time point the total amount of proteins was followed by the Bradford assay. Different control groups were also designed – a group where M. smgematis is grown without E. coli, a group where E. coli is induced and grown without M. smegmatis, and a group where M. smegmatis is was cultivated with the un-induced strain of E. coli.

To test if E. coli carrying TDMH could kill mycobacteria which are inside of the macrophages a J774 macrophage cell line was used. M. smegmatis MC2 was transformed with pMyco-gfp plasmid (Alibaud et al. 2011). E. coli BL21 was transformed with 3 plasmid: pET21b + TDMH, pACYC184 + hly and pColA + BBa_J23102. The macrophages were grown in 24 well plates in RPMI 1640 media supplemented with 1 % glutamine, 10% of fetal bovine serum, 1% HEPES, 1% sodium pyruvate. When around 90 % of confluence was reached, the cells were infected with M. smegmatis. Mycobacteria from an overnight culture were resuspended in RPMI and set to the final O.D. of 0.1. After infection, cells were incubated for 1 h (37 oC, 5% CO2) and then washed 10 times with PBS. After washing the cells new RPMI media was added and the cells were infected with an overnight culture of E. coli which was induced with 10 µM IPTG. Same as for mycobacteria, E. coli were resuspended in RPMI where the final O.D. was set to 0.1. Then the cells were incubated for 1 h (37 oC, 5% CO2), washed 10 times with PBS and fresh RPMI media was added. Cells were observed under the fluorescent microscope right after the final infection step (time 0 h on our graphs) and after 3 h long incubation. We counted and compared the percentage of the macrophages which were infected with mycobacteria for these two time points. We also formed control groups: macrophages which were infected only with mycobacteria or only with E. coli so we could see the infection rate for these two strains; macrophages which were infected with M. smegmatis and with E. coli which wasn’t induced with IPTG. We also performed the same experiment where in the first infection step the cells were infected with E. coli and in the second one they were infected with mycobacteria.


Figure 1 shows the killing of M. smegmatis with TDMH expressed by E. coli. We mixed liquid cultures of E. coli and M. smegmatis at equal cell densities as determined by plating assays. When expression of TMDH was induced with IPTG, nearly 99% of the M. smegmatis were killed within six hours. We saw no change in viability in cultures of M. smegmatis alone or when mixed with uninduced E. coli.


Figure 1 TDMH-expressing E. coli kill mycobacteria in culture. We mixed TDMH-expressing E. coli and WT M. smegmatis in LB media at an initial cell density of 107 cells/ml each. Plating assays were used to count specifically M. smegmatis after the indicated times. When TDMH-expression was fully induced with 1 mM IPTG, more than 98% of mycobacteria were killed after 6 hours (red line). Populations of mycobacteria alone (black line) and mycobacteria mixed with uninduced E. coli (blue line) were stable.


Figure 2 Dose-dependence in killing of mycobacteria by E. coli. We mixed M. smegmatis at a density of 10^7 cells/ml with E. coli at densities of 10^5 cells/ml (blue line) 10^6 cells/ml (green line) or 10^7 cells/ml to produce the ratios above. Even low densities of E. coli produced significant killing. Uninduced E. coli produced no significant killing.

We next sought to quantify the effectiveness of TDMH killing. We mixed induced E. coli and mycobacteria in different ratios and used plating assays to measure viability. As shown in figure 2 mycobacterial killing displayed dose dependence on the E. coli cell density. Small numbers of E. coli could kill many mycobacteria. For example, in mixed populations with 100 mycobactera for each E. coli, we still observed >50% mycobacterial killing after 2 hours. This indicates that, on average, each E. coli produced enough TDMH to kill 50 mycobacterial. We reason that this killing may be even more effective inside macrophages, where constrained volumes will increase the effective TDMH concentration.

We investigated further the mechanism of TDMH-mediated cell killing. The TDMH enyme in our system does not carry a secretion tag. Therefore, lysis of the E. coli membrane is probably required for the protein to reach (and lyse) M. smegmatis. We used plating assays to investigate the effect of TDMH induction on E. coli viability and Bradford assays to measure released protein. The results are presented in figure 2.

Figure 2A shows that inducing TDMH-expressing E. coli with IPTG rapidly kills the E. coli. In figure 2B, we show that inducing TDMH expression increases the concentration of free protein in the media. In our model, TDMH induction leads to spontaneous lysis of TDMH-expressing E. coli. This could be simply due to the extremely high protein expression levels driven by the T7 promoter, or it may be partially due to the esterase activities of the TDMH enzyme. Once the TDMH is released from the E. coli it is free to act on mycobacterial membranes, causing lysis and the release of additional proteins.


Figure 3 Induction of TDMH kills E. coli and relases protein to the media.A)Plating assays were used to determine E. coli viability in LB media after the indicated times. TDMH-induced E. coli rapidly lost viability (red line) Uninduced E. coli grew normally following a lag phase (black line).B)Bradford assays were used to measure free protein concentrations in mixed and induced bacterial cultures. When E. coli and mycobacteria are mixed and TDMH-expression is induced, free protein concentrations increase. This is consistent with a model in which TDMH induction leads to the lysis of the E. coli membrane. Action of TDMH on mycobacteria leads to further lysis and protein release.

TDMH modulates mycomembrane and induces nutrient acquisition and stress sensitivity: NYU Abu Dhabi


(A) Simplified model for mycomembrane remodeling (B) Biosynthesis and breakdown of TDM. (Holmes et. al., 2019)

TDMH breaks TDM down in nutrient limiting environments, modulating the mycomembrane to enhance nutrient acquisition. It was shown that the concentration of TDMH is tightly regulated in the mycobacteria to balance nutrient absorption and susceptibility to stress from the host’s immune response (Holmes et. al., 2019). While exposure to high levels of exogenously introduced TDMH has been shown to trigger cell lysis in mycobacterium, these pathogens endogenously produce TDMH in response to nutrient depravation. The breakdown of TDM in the mycomembrane increases the membrane’s permeability to nutrient intake, but concurrently sensitizes it to stress of the host. It was shown TDMH confers a growth advantage to intracellular Mtb in MyD88−/− mice hosts. A study showed Rv3451 is the primary TDMH of mycobacterium tuberculosis (TdmhMtb). TdmhMtb responds to the host’s immunity by regulating Mtb growth, providing a growth advantage to Mtb in an immunocompromised host (Yang et. al., 2014).


  • Holmes, N. J., Kavunja, H. W., Yang, Y., Vannest, B. D., Ramsey, C. N., Gepford, D. M., Banahene, N., Poston, A. W., Piligian, B. F., Ronning, D. R., Ojha, A. K., & Swarts, B. M. (2019). A FRET-Based Fluorogenic Trehalose Dimycolate Analogue for Probing Mycomembrane-Remodeling Enzymes of Mycobacteria. ACS omega, 4(2), 4348–4359.
  • Yang, Y., Kulka, K., Montelaro, R. C., Reinhart, T. A., Sissons, J., Aderem, A., & Ojha, A. K. (2014). A hydrolase of trehalose dimycolate induces nutrient influx and stress sensitivity to balance intracellular growth of Mycobacterium tuberculosis. Cell host & microbe, 15(2), 153–163.

This contribution was added by the 2020 NYU Abu Dhabi team.