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Endocytosis can be of three forms:

1. Pinocytosis
(literally, cell drinking) is the invagination of the cell membrane to form a pocket filled with extracellular fluid.
Caption: Endocytosis (pinocytosis) by a capillary endothelial cell. Magnification*: x12,880 Type: TEM

2. Receptor-mediated endocytosis is

similar to pinocytosis, except it is prompted by the binding of a large extracellular molecule - such as a protein - to a receptor on the cell membrane. is the process by which cells ingest large objects, such as prey cells or large chunks of dead organic matter. These are sealed off into large vacuoles: phagosomes. Macrophages are cells of the immune system that specialize in the destruction of bacteria by phagocytosis.

3. Phagocytosis

Three pathways leading to lysosomes


1. Phagocytosis ("cell eating"): 2. Autophagy 3. Endocytosis (RME)

Ilya Ilyich Mechnikov awarded the Nobel Prize for Physiology or Medicine for discovering the role of phagocytosis in higher animals

Endocytosis is the opposite of exocytosis

http://www.sp.uconn.edu/~terry/Common/phago053.html

Lysosomes in Phagocytosis and Autophagy

Russell et al,JBC,2002,158:421-26

invasion

1. RECONHECIMENTO: RECEPTORES 2. FAGOCITOSE: PODER BACTERICIDA 3. MATURAO DO FAGOSSOMA: Kiss and Run 4. SOBREVIVNCIA INTRACELULAR DE PATOGENES 5. SOBREVIVNCIA INTRACELULAR DE MICOBACTRIAS

Phagocytic cells
Can engulf (phagocytose) foreign substances. Phagocytosis involves: 1. chemotaxis, where phagocytic cells are attracted to microorganisms by means of chemotactic chemicals like microbial products; 2. adhesion, where phagocytes sticked to microorganisms. are

3. ingestion, where phagocytes extends their projections, forming pseudopods that engulf the organism. Finally the bacterium would be digested by the enzymes in the lysosome.

FAGOCITOSE
Reconhecimento via receptores Sinalizao pr vs antiinflamatria
Captura com reorganizao do

citosqueleto de actina microtbulos Libertao de espcies Reactivas de oxignio e de Nitrognio (ROS; RNS)

Induo da apoptose vs necrose

Apresentao de antignios ao sist. Imunitrio sist. Resposta Humoral e/ou Celular

Acidificao e Digesto com enzimas lisossomais

Patogenes intracelulares foram a fagocitose

Outras bactrias sobrevivem por conseguirem evitar a fagocitose

TUBERCULOSIS
1/3 of the world population is infected 10% of the infected develop the disease

Receptores FC Receptores C3R

RNS ROS
Radicais livres matam em segundos os bacilos fagocitados

A quantidade de RNS libertada aquando do contgio no suficiente para matar 100% dos bacilos. A no ser que os macrfagos estejam pr activados pelo IFN

M. tuberculosis produz catalase e peroxidase resiste a ROIs

Receptores FC

Receptores de acares

Man-6-fosfato

CR3

Listeria

Salmonella

Mycobacterium

Shigella

Zipper
COILING
Legionella

Trigger

Phagosome Maturation
F-Actin assembly
Pseudopododia extensions

Oxidative burst

EE

Early Phagosome

Kiss and Run model

LE

Lysosome

Phagosome Maturation
F-Actin assembly
Pseudopododia extensions

Oxidative burst

EE

Early Phagosome

Kiss and Run model

LE
Lysosome

Phagolysosome

Rafts

Bomba ATPsica fuso

Enzimas hidrolticos

Ligao actina

1000 protenas 50 lpidos Direccionamento trfego

Ligao microtbulos

Kiss and Run model for phagosome maturation

LE

EE

LE

EE

LE

EE

Markers for phagosome maturation


Rab5 V-ATPase LE EE
Cathepsin

TfR

H+ V-Atpase H+ H+
Cathepsin

H+

Rab7

Rab7

Proteases lipases

H+

Ly LE

H+ H+

Lamp1

Early Markers
Rab5 EEA1 TfR transferrin

Rab7 LAMP1/CI-M6PR V-ATPase


Hydrolases (cathepsins)

Late Markers

CD63

Envolvimento do RE na fagocitose

Michell Desjardins Fenmeno global na fagocitose

RE

LAMP1

CALNEXINA

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Parasitas que sobrevivem intracelularmente


Bloqueiam maturao Micobactrias Salmonella Leishmania (promastigotas)

Ficam num vacuolo like RE Clamdia Brucella Legionella Toxoplasma gondii RE Escapam do fagossoma Shigella Listeria vaccinia

Resistem ao lisossoma Coxiella Leishmania (amastigotas)

Bombas protnicas

Bomba protnica ATPsica do tipo V

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Bomba protnica tipo V fraco V0

Fagossoma micobacteriano

EE

EPh

Funde activamente com EE E com vesculas transportadoras De TfrR e transferrina

no adquire ou est limitado na aquisio da bomba protnica ATPdependente e de receptor man-6-P-CI

LE Caractersticas de fagosoma precoce. Ly Bactria bloqueia a maturao em fagolisosoma

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Tuberculosis vs acidification
Latex BeadsPhagosomes or heat killed bugs

EE pH 4.5 LE/Lys

Mycobacterium tuberculosis phagosome

EE

pH~6.3

LE/Lys

Lysotracker-red

M.tb-GFP Vivo

M.tb-GFP morto pelo calor

Fagosoma pH~6,2

pH~4,5

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Consequncias da no acidificao
1. No aquisio de enzimas lisossomais 2. Mesmo que adquira algumas como Catepsina, estas no so activas Trfego intracelular bloqueado em fases precoces no matura para formar o fagolisossoma 3. No entra em contacto com vias de processamento/apresentao MHCII e MHCI ou muito reduzido 4. Micobactrias camufladas do Sist. Imunitrio 5. Micobactrias

sobrevivem intracelularmente

TRANSPORTE DAS VESCULAS E MATURAO VIA CITOSQUELETO DE ACTINA VIA MICROTBULOS

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FROM LATEX BEAD PHAGOSOMES TO MYCOBACTERIUM TUBERCULOSIS

And the role of late actin events???

Papel da actina nas fases tardias da fagocitose/movimento de vesculas

1)Actin Track model 2)Formao de cometas de actina

Quick Time Matrix/Fred0

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Actin Track model


endosome Myosin Va? Phagosome

Griffiths

M. tuberculosis containing phagosomes and actin in infected macrophages


Heat-killed LIVE

Actin: rhodamin-phalloidin (red)

nucleus: DAPI

M.tuberculosis- GFP (green)

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Actin nucleation

fusion

acidification

killing

Only 0-5% of M.tb phagosomes nucleate actin

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A HOLISTC VIEW OF CELL SIGNALING

Efeito de lpidos pro-inflamatrios


Ceramida Esfingosina Esfingosina 1 fosfato c. araquidnico

Aumenta o nmero de M.tb fagossomas que nucleiam actina at 25%

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EM fusion assay in J774 Cells

Only 8-10% of live Mtb phagosomes Dead Mtb phagosomes actively fuses with lysosomes fuses with lysosomes

Lipidos proinflamatrios ou IFNgama aumentam para 20-40% os fagossomas com Mtb vivo que fundem com lisossomas

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Anes et al, (2003) 5(9):793-802

TRANSPORTE DAS VESCULAS E MATURAO


VIA CITOSQUELETO DE ACTINA Tratamento com citocalasina A 50% das vesculas fundem

VIA MICROTBULOS
Labis, curtos: periferia da clula Longos: entre MTOC e periferia

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Rab4 Rab5 Rab7 Rab9 Rab11 Etc.

EE e E/reciclagem V clatrina e EE LE EE/LE LE/transgolgi transgolgi/apical e basolateral

Rab11 RE Rab4?

Rab5
Rab15 CV Rab4

Rab11? TGN Rab9 Rab6 EE

Rab7
LE

David Russell

Rab5-GFP Rab7-GFP M.avium-TIRTC

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Maximiliano Gutierrez Cell, 2004; 119:753-766

Schematic of inducible macroautophagy

An environmental stress related signal (example: low nutrients, lack of sunlight) leads to the induction of autophagy and the sequestration of cytosolic components into autophagosomes. These autophagosomes (double membraned lytic vesicles) then dock and fuse with the vacuolar membrane where the contents undergo breakdown. This pathway induces killing of intracellular TB (Gutierrez et al. Cell 2004)

Mycobacterum tuberculosis

Macrophage

Phagosome

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Macrophage Mycobacterium tuberculosis

Phagosome

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O fagossoma micobacteriano uma vescula imatura, no acdica bloqueada entre fases controladas por Rab5 e Rab7 Que factores exporta a micobactria que vo modular a resposta da membrana do fagossoma e alterar o trfego intracelular?

Como podemos contrariar a informao/sinalizao do M.tb na membrana do fagosoma e potenciar as capacidades bactericidas do macrfago?

APPLIED PHAGOCYTOSIS

Questions?

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