Team:Paris Bettencourt/Project/Infiltrate

   Mycobacterium tuberculosis (Mtb), the bacterium responsible for tuberculosis (TB), spreads by aerosol and infects its host through the airways. The bacterium is phagocytosed by macrophages in the lung, yet often evades death in the lysosome. Mtb can persist for years or even decades inside macrophages by inhibiting phagosome/lysosome fusion and supressing the normal acidification of the lysosome.

An efficient treatment for persistent TB must enter infected macrophages and kill the pathogen there. In our system, E. coli is both the vector and the therapeutic agent agent by expressing the gene LLO to enter macrophages and TDMH to kill mycobacteria.

  Mycobacterium species share a characteristic cell wall: thick, waxy, hydrophobic, and rich in mycolic acids. The low permeability of the envelope to hydrophilic solutes contributes to the intrinsic drug tolerance in mycobacteria.

  Trehalose Dimycolate Hydrolase (TDMH) is a cutinase-like serine esterase that triggers rapid lysis of the mycobacterial cell wall by degrading the mycolate layer. The enzyme was first isolated from Mycobacterium smegmatis and subsequently shown to hydrolyze purified TDM from various mycobacterial species. Exposure to TDMH triggers an immediate release of free mycolic acids, ultimately leading to lysis of many mycobacteria including Mtb (Yang et al. 2012).

  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.

  Listeria monocytogenes is a bacterial pathogen that replicates in the cytosol of mammalian cells. The pathogen is initially internalized in host phagosomes, then lyses them to obtain access to the cytosol. A single gene, LLO, is responsible for this activity and is sufficient to convey the phenotype to E. coli.

The LLO protein is activated by the low pH of the lysosme. By forming large pores in the lysosomal membrane, it allows protein delivery to the cytosol of macrophages (Higgins et al. 1999). Using E. coli as a protein vector means we don't need to isolate and purify the TDMH enzyme.

  Figure 4 displays J774 macrophages one hour after coinfection with E. coli and M. smegmatis. As described in the methods, the E. coli express TDMH and LLO and fluorescent mRFP as a label. The M. smegmatis express GFP as a label, but are otherwise wild type.

The two movies displayed in figure 5 are time-lapse microscopy of live coinfected macrophages collected over a period of 6 hours. Fluorescence patterns revealing the localization of E. coli and M. smegmatis within the macrophages are relatively stable over this time period.

We next applied quantitative microscopy to characterize the behavior of our system inside live macrophages. We imaged macrophage populations under several conditions and time points and scored their rates of infection with E. coli, M. smegmatis or both. In all we scored over 13,000 individual cells collected from more than 100 separate images.

The results, presented in figure 6, showed no clear effect of our TDMH-expressing E. coli on mycobacterial infection rates over a period of 3 hours. Unexpectedly, we observed an increase in infection rates of both E. coli and M. smegmatis, regardless of the TDMH induction state. These results are preliminary and not fully understood, but we tentatively attribute them to continuing phagocytosis of free bacteria that were not removed from the media by repeated washing. In figure 5 the replication of free contaminating bacteria is apparent near the end of the movie, supporting this hypothesis. We therefore expect that longer time courses and improved sterile technique will allow us to investigate the time evolution of our system in greater detail.

  We have shown that E. coli can effectively kill mycobacteria in culture by expressing the TDMH enzyme, now a BioBrick. We have further shown that E. coli and mycobateria may co-localize within macrophages, where LLO-based systems can effectively deliver protein payloads.

In the coming weeks, we will continue to refine our cell culture techniques to precisely characterize the effectiveness of our system in situ, within the living macrophage.