Genetic Engineering Presentation

Mycobacteria in early endosome

HIV in phagosome

By :

Supriyo Ray

Checklist of keywords

p  Mycobacterial lipids- ManLAB, PIM, p  Vesicular trafficking markers- Rab, LBPA, LAMP, EEA1 p  Vesicular maturation- PI-3K (hVPS34),PI(3)P p  Vesicles - Phagosome, phagolysosome, early and late endosomes, p  Vesicular maturation inhibitors – wortmannin,LY20049

Overview- Mycobacterium

p  There are more than 50 species in the genus Mycobacterium. p  Majority are non pathogenic and are closely related to soil bacteria

Streptomyces & Actinomyces p  Few are highly successful pathogens eg. M. tuberculosis, M.

leprae, M. ulcerans causing tuberculosis ,leprosy and Buruli ulcers respectively.

p  The complex immune response (innate & adaptive) evoked by the host sequesters the organism in structures known as granulomas

p  Over one-third of the world's population has been exposed to the TB bacterium

p  It latently infects 2 billion people worldwide and each year there are 8 million new cases and causes 2 million deaths.

Tuberculosis : an Overview p  Is a severe and contagious airborne disease caused by Mycobacterium tuberculosis p  More than one-third of the world’s population has the TB bacterium in their bodies, - only a fraction of people infected with Mtb develops active TB disease. Latent TB -Those who do not get sick are known to have non-contagious latent TB, so-

called because the bacteria are inactive - TB bacteria can remain in this dormant state for months, years, and even

decades without increasing in number and without making the person sick. - Most people with latent Mtb infection will test positive on the tuberculin skin

test, or their chest X-ray will show signs of latent TB. p  A person gets active TB, means the TB bacteria are multiplying and attacking the

lung(s), or other parts of the body such as the lymph nodes, bones, kidney, brain, spine, and even the skin. From the lungs, the TB bacteria move through the blood to different parts of the body.

Multidrug-Resistant Tuberculosis (MDR TB) p  MDR TB is a form of drug-resistant TB in which the TB bacteria can no longer be

killed by at least the two best antibiotics, isoniazid (INH) and rifampin (RIF), commonly used to cure TB. As a result, this form of the disease is more difficult to treat than ordinary TB and requires up to two years of multidrug treatment.

p  “HIV is the main reason for failure to meet Tuberculosis (TB) control targets. TB is

a major cause of death among people living with HIV/AIDS.

Multidrug- Resistant Tuberculosis

41 COUNTRIES WITH MDR-TB TO DATE

Mycobacterium Uptake

Important slides relevant to this paper from the last 2 lectures

Phagocytosis

Macrophage pix Plus three pathways of survival

Survival in Macrophages 1.  Escape from phagosome 2.  Inhibition of lysosome fusion 3.  Survival in phagolysosome

Isolated Vacuoles: Mycobacterium tuberculosis

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Dec. 2007, p. 636–652

Fidelity in Vesicle Trafficking

rab4 rab11

Rab 5 clathrin-dependent endocytosis homotypic fusion of early endosomes early endosome motility on microtubules Rab 4 early endosome to surface recycling Rab 11 recycling endosome to surface recycling recycling endosome to TGN transport Rab 7 ECV/MVB to late endosome transport homotypic fusion of late endosomes late endosome motility on microtubules Rab 9 late endosome to TGN transport SNARE proteins also display organelle-specific distributions

ECV/MVB

EEA-1

Annu Rev Cell Dev Biol. 1996;12:575-625

Rab Function

Rab effectors contribute to rab specificity and rab function EEA-1: rab5 effector involved with vesicle docking and fusion SNARE complex disassembled by NSF and alpha-SNAP SNAP: soluble NSF attachment protein SNARE: SNAP receptor NSF: N-ethylmaleimide-sensitive factor Figure 13-14

Phago-Lysosome Maturation

Alberts Figure 13-49

Lysosome Overview

Lumen contains hydrolytic enzymes

PI-3Kinase p  Secretion stimulated by hormones

accompanies change in intracellular PIP pool had been known and studied for many years.

p  PIPs role in constitutive membrane traffic arose when mammalian PI-3Kinase was cloned and was found to have a sequence homology with Vps34p, a yeast protein homolog essential for delivery of proteins.

p  Like PI-3Kinases (PI-3K) there are PI-4Kinases (PI-4K) and PI-5Kinases (PI-5K).

p  (PI-3K) phosphorylates at D3 position, (PI-4K) at D4 position and (PI-5K) at D5 position of the substrate respectively. Hence, the nomenclature of these enzymes.

p  hVPS34 is a type of type III PI-3K p  EEA1 (Early Endosome Antigen1) -

Binds to phospholipid rich early endosome vesicles containing Phosphotidyl inositol 3 Phosphate, -- Necessary for endosomal trafficking.

p  It binds to lipids through its FYVE domain and forms a homodimer through a coiled coil domain

Phosphorylated at D-3 position PI

Phosphorylated at D-4,D-5 position

Phosphorylated at D-3, D-4,D-5 position

Intracellular distribution of Phosphoinositides for membrane traffic

Various proteins with FYVE domain & involved in vesicular trafficking

Interaction of PIP2 in the FYVE domain of EEA1

p  Hypothesis n  Rab5 interacting effectors must be involved in the early

endosomal arrest of Mycobacteria. p  Goal

n  Identify the Rab 5 effectors involved n  Determine the mechanism by which the inhibition is

brought about n  Identify the probable factors involved in the above

process p  Strategy

n  Compare the effects in presence of model and mycobacterial phagosome

n  Use Inhibitors, antibodies to suppress the effect n  Use or establish markers to study the effect in presence

and absence of inhibitors and antibodies.

Techniques Used

Macrophage membrane

Fluorescent dye

Latex Beads

- LysoTracker Red is a dye which fluoresces in acidic compartments,

- Similarly, there are MitoTracker dyes which fluoresces in presence of ROS

Syntaxins involved in Endosome fusions

Is arrest of MPC through alteration of Rab 5 interacting component?

Ab Ab

Time dependent Co-localization Study

MPC GFP labeled LBC auto florescence

LBC

MPC EEA1

Rab 5 effector molecule EEA1 is efficiently recruited by normal phagosomes whereas M. tuberculosis phagosomes fail to do so.

Rab5,NSF, αSNAP, Syntaxin 13 are present on both MPC & LBC

Probable Pathway ???

Does inhibiting the PI-3K activity diminish EEA1 recruitment to model phagosomes?

Wortmannin is an irreversible inhibitor of PI-3K; also affects other lipid kinases; covalently modifies Lys 802 of p110 catalytic subunit

Wortmannin causes decrease in EEA1 recruitment and it is not because of inhibition of latex bead uptake

Are you sure its PI-3K inhibition only? If yes, then can its effect be reversed thereafter?

LY294002 is a reversible specific inhibitor of PI-3K; competes with ATP for the ATP binding site of PI-3K

EEA1 recruitment was restored in model phagosomes after washing off the specific inhibitor for PI-3K, LY294002

Does PI-3K inhibition prevent phagosome acidification?

Lysotracker (-ve)

Lysotracker (+ve)

PI-3K inhibition prevent phagosome maturation by stalling acidification.

Does acquisition of LBPA (a late endosomal marker) occur ?

LBPA starts accumulating with time around model phagosomes symbolizing its presence as a marker for phagosomal maturation

Does PI-3K inhibition reduce acquisition?

- Washing of LY294002 restores LBPA to that of control.

- LBPA acquisition is very much reduced in Mycobacterial phagosome

Mycobacterial phagosomes prevent phagosomal maturity by inhibiting PI-3K

Short Summary

p  Rab 5 effector EEA1 was identified- using BCG phagosome and latex beads

p  PI-3K inhibition was responsible for scarcity of EEA1 -using latex beads

p  PI-3K inhibition was also responsible for decreased acidity in vesicle- using latex beads

p  Scarcity of Late Endosome Marker post PI-3K inhibition - using latex beads

p  So, now they asked what happens if we add directly inhibt using antibodies instead of synthetic inhibitors?

p  Further experiments were conducted using latex beads with macrophage membrane and mycobacterial membrane

Is LB Phagosome maturation Inhibited by anti hVPS34?

-Anti hVPS34 reduces localization of late endosomal marker and the reduction is comparable with that of anti EEA1.

- Nonspecific Anti IgG did not have any effect.

Latex Bead Phagosome maturation was inhibited by PI-3K inhibitor affirming its vital role played by it in the endosomal maturation

Mycobacterium Surface Membrane

Does ManLAM or PIM effect EEA1 recruitment?

-ManLAM could effect because its structure is analogous to PI3P.

-Earlier it had been shown that ManLAM & its precursor PIM are released by mycobacteria and found to be localized in endosomal compartments

-Latex beads were coated with ManLAM & its precursor PIM & phagocytosed by J774 cells

- If ManLAM or PIM effect EEA1 recruitment then can the effect be reversed with the removal of these membrane with TX-100?

- EEA1 reduction reduces LBPA accumulation.

EEA1

ManLAM should be considered as a toxin interfering with early endosomal trafficking

ManLAM in Phagolysosome arrest (Summary of Events )

ManLAM

Phagosome

ManLAM hVPS34 PI3P EEA1 Early Endosome Fusion

Conclusion

p  Mycobacterium tuberculosis prevents Early endosome maturation by inhibiting PI-3Kinase.

p  Its membrane lipid can act as competitive inhibitor for PI-3K as it bears close resemblance to phosphotidyl inositol.

p  Once PI-3K is inhibited, EEA1 fails to assemble on the early endosome membrane preventing vesicle fusion.

p  Why n  Tuberculosis is one of the most potent diseases n  To understand the intracellular survival of Mycobacterium n  Identify and understand other proteins/factors involved in the endosomal

trafficking

p  How n  Co-localization studies using GFP labeled latex beads & dye tagged

antibodies , (+/-) inhibitor treatment and Tracking dye followed by Epifluorescence & confocal microscopy

p  Result n  PI-3Kinase inhibition diminishes EEA1 recruitment whereby it prevents

phagosome and lysosome fusion and this effect is brought about by mycobacterial lipid toxin.

p  Importance

n  Identified EEA1 (Rab5 effector) as a key factor involved in early endosome endosome arrest of Mycobacteria

n  Identified PI-3K and PI3P formation as key factor in the EEA1 recruitment and early endosome maturation

n  Identified ManLAM, a Mycobacterial lipid as a toxin which has the capacity to bring about this effect

Mechanisms for Vesicle Fusion Arrest

CaMKII

(Ca2+ )

(Recruitment of hVPS34)

(EEA1)

(p38 MAPK)

GDP Dissociation Inhibitor (GDI)

Rab5

(arrest in GDP state)

MLAM

(Active) phosphorylation

Summary of Events + Update

90˚

GTP GD P

Rab-5

hVPS34 FYVE

PIP PIP3

EEA1

GTP GTP GTP

Rab-5

LAMP2

Rabaptin5

G TP

G

TP

G TP

90˚

Early Endosome

Late Endosome

H+

Synaptobrevin

Syntaxin13 NSF

Rabex5

ManLAM, Wortmannin, LY20049

ManLAM ?

Thank You !!!