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 listeriolyin O gene LLO to enter macrophages and TDMH to kill mycobacteria. Additionally we thought about how we could deliver the E. coli to the lungs and decided that the most effective way would be through an inhaler.

  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 describes how we performed the setup of our experience by making a coculture of M.smegmatis and E.coli.

Figure 2(below) 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.

  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 3 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 mycobacteria. We reason that this killing may be even more effective inside macrophages, where constrained volumes will increase the effective TDMH concentration.

Figure 2 TDMH-expressing E.coli kill mycobacteria in culture.

  We investigated further the mechanism of TDMH-mediated cell killing. The TDMH enzyme 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 4.

Figure 4A shows that inducing TDMH-expressing E. coli with IPTG rapidly kills the E. coli. In figure 4B, 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 4 Induction of TDMH kills E. coli and releases protein to the media.

  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.

We hypothesized that a bacterial delivery system could be efficient and specific in targeting mycobacterial-infected macrophages. In effect, we might take advantage of the natural tendency of macrophages to phagocytose E. coli and their enzymatic payload.

Because LLO expression is known to be associated with Listeria pathogenicity, we took extra precautions in our cloning and experiments. These measures are detailed on our team safety page. Briefly, we worked in a Biosafety Level 2 lab under the supervision of two full-time lab managers. We researched and complied with French national and insitutional biosafety rules as they apply to this gene.

  Figure 6 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 7 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.

Figure 6 Coinfection of J774 macrophages with mycobacteria and E. coli expressing TDMH and LLO

  We wanted to study the pH influence on M. smegmatis growth and on TDMH enzyme activity. We know that the phagosomal pH is usually acid (around 6) but can reach 4,5. We wondered if TDMH enzyme was working at this range of pH.

We studied 7 different buffers : 3 ; 4,5 ; 5,1 ; 6,4 ; 7,35 ; 8,2 and 8,8.

Figure 8 shows the activity of TDMH enzyme at various pH levels.

Mycobacteria was unable to grow at pH 3 with or without the presence of TDMH. Therefore we are unable to make any conclusions regarding the activity of the enzyme at very low pH values.

TDMH activity was active across a large range of pH from 4.5 to 8.8, though there was a small decrease in efficiency observed at pH 8.2. This is a promising result due to the difference in pH between the phagosome and the cytosol. Additionally, the high activity of TDMH even at low pH is promising as it will be required to function at low pH inside the phagosome.

  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.

Figure 9 The effect of TDMH-induced E. coli on macrophage infection rates after 3 hours.