Team:Paris Bettencourt/Project/Infiltrate

Bacterial vectors offer a biological route to gene and protein delivery to cells such as macrophages. We want to investigate the potential use of Escherichia coli as a drug delivery system to kill Mycobacerium tuberculosis inside of the infected macrophages. In our system E. coli contains two expression systems enabeling Listeriolysin O (LLO) and Trechalose Dimycolate Hydrolase (TDMH) production. Subsequent to phagocytosis the E. coli are degraded within the phagosomes, causing the release of LLO and TDMH from the bacteria. LLO acts by forming large pores in the phagosomal membrane, thus releasing TDMH into the cytosol. TDMH can then trigger lysis of the mycobacterial cell wall.

Mycobacterium tuberculosis (Mtb) is the bacterium responsible for tuberculosis (TB). For decades, it was believed that TB was a disease of the past, but the onset of the HIV epidemic, the emergence of antibiotic resistant Mtb, and the relative ineffectiveness of the BCG vaccine have put TB back on the agenda.

One of the main problems in treating TB is reaching its causer, which could evade the drug in use and survive in the cells of the immune system. Mtb infects its host through the airways. The bacterium is phagocytosed by resident macrophages in the lung and is able to replicate inside them. Its able to evade macrophage responses in part by inhibiting the fusion between the phagosome in which it resides and bactericidal lysosomes, as well as by dampening the acidification of the vacuole.

A more efficient treatment of TB should contain a drug which rapidly kills mycobacteria and a delivery system for such drug, which could enable the killing of mycobacteria inside the infected macrophages. In our system the rapid drug is Trehalose Dimycolate Hydrolase (TDMH), while the delivery system is LLO carrying strain of E. coli.

Background

Why is it so difficult to treat tuberculosis?

This is due to several parameters linked to the nature of the pathogen, Mycobacterium tuberculosis:
•This bacterium has the power to replicate or remain dormant for years inside the macrophages. It happens by many complex mechanisms including prevention of the phagosomal maturation.
•The cell wall of M.tuberculosis is very complex and it's difficult for drugs to get inside.
•The growth of the bacteria is very slow and majority of drugs can reach only bacteria which are dividing.

Trehalose Dimycolate Hydrolase

Mycobacterium species share a characteristic cell wall, which consists of the hydrophobic mycolate layer and a peptidoglycan layer held together by a polysaccharide arabinogalactan. The hydrophobicity of the envelope contributes to a high level of intrinsic drug tolerance in mycobacteria. All this makes mycolic components of the mycobacterial cell wall important as targets in drug design, where one such component is trehalose-6,6-dimycolate (TDM).

TDM molecule comprises trehalose sugar esterified into two mycolic acid residues which range from 20–80 carbons in length. It’s the main virulence factor for the M. tuberculosis that makes it resistance to anti-tuberculosis medications, blocks macrophage activation by interferones and induces secretion of tumor necrosis factors.

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 shown 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).

In our system a gene which encodes TDMH is driven under the T7 promoter in the pET21b(+) expression system. The plasmid was kindly provided by Dr. Anil Ojha from the University of Pittsburgh. a.

E. coli expressing LLO as a drug delivery system

Listeria monocytogenes is a bacterial pathogen that replicates within the cytosol of mammalian cells. After internalization into host cells, bacteria are initially contained within host phagosomes which they subsequently lyse to gain access to the cytosol. The ability of L. monocytogenes to lyse the phagosome is primarily mediated by listeriolysin O (LLO). This protein is activated by the low pH in the lysosme. By causing large pore formation in the lysosomal membrane, it could provide protein delivery to the cytosol of macrophages. Higgins et al. reported that E.coli expressing cytoplasmic LLO can be used to efficiently and rapidly deliver b-galactosidase and chicken ovalbumin to the cytosol of macrophages. In our system we use the LLO carrying plasmid described in this paper - a pACYC184 vector where the LLO encoding gene, hly , is driven under the tetracycline promoter.

The advantages of using E. coli as a protein delivery system is that no TDMH isolation and purification is needed. As well, the delivery system is efficient and specific, because of the natural tendency of the macrophages to phagocytose the bacteria and the drug which they contain.

However, dangerous factors are involved in using live bacteria as a drug delivery system. The act of releasing the drug from the burst cell also involves releasing the rest of intracellular contents. There are also complications arising from the possible interactions between the the bacteria and the immune system.

Experimental design

The killing assay in liquid media

At first we wanted to investigate how TDMH producing E. coli is efficient in killing mycobacteria. E. coli BL21 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 m while the growth of E. coli was followed on kanamycin plates. 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 .

The killing assay inside the macrophages

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 plasmids: 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. E. coli were resuspended in RPMI where the final O.D. was set to 0.1. Then the infected cells were incubated for 1 h (37 oC, 5% CO2), washed 10 times with PBS and fresh RPMI media was added. Macrophages were then observed under the fluorescent microscope and after 3 h of 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.

Main results

Killing assay outside the macrophages

2 ratios were successful regarding the killing of M.smegmatis:
1 to 10 and 1 to 1.Whereas in the 1 to 100 ratio we observed a recovery of M.smegmatis.
In the figure 1 you can see in blue the behavior of M.smegmatis alone, in red the behavior of M.smegmatis when we add E.coli uninduced and in green the behavior of M.smegmatis when we add E.coli and we induced to produce TDMH.
We decided that we will use the 1/1 ratio for the rest of our experiments.

Killing assay inside the macrophages: microscopy and video movie

Discussion

Our results show an indeniable role of the protein TDMH in the lysis of M.smegmatis. This target is independant of the Mycobacteria’s growth making it a molecule of choice in the war against tuberculosis.

Using E.coli as a vector allows us to target the macrophages using a natural process: the phagocytosis. We showed that E.coli can enter a macrophage where there is already M.smegmatis. They didn’t seem to be in the same phagosome but the TDMH produced by E.coli can reach M.smegmatis. This is possible thanks to the lysteriolysin that lysed E.coli’s phagosome.

Perspectives

It will be interesting to study the stability of TDMH at the phagosome’s pH and to imagine a protein delivery system that can target the macrophages without using a living organism (for example like they do with imiglucerase). The problem is our protein is not glycosylated so it will not be that easy.

Bibliography

1. Yang Y, Bhatti A, Ke D, Gonzalez-Juarrero M, Lenaerts A, Kremer L, Guerardel Y, Zhang P, Ojha AK (2012) : Exposure to a cutinase-like serine esterase triggers rapid lysis of multiple mycobacterial species. J Biol Chem. 2013 Jan 4;288(1):382-92.
2. Rajesh Jayachandran, Varadharajan Sundaramurthy, Benoit Combaluzier , Philipp Mueller, Hannelie Korf, Kris Huygen, Toru Miyazaki, Imke Albrecht, Jan Massner, Jean Pieters (2007) : Survival of Mycobacteria in Macrophages Is Mediated by Coronin 1-Dependent Activation of Calcineurin. Cell, Volume 130, Issue 1, 13 July 2007, Pages 12-14