bacterial pathogens commandeer rab gtpases to establish intracellular niches

Traffic 2012; 13: 1565–1588 © 2012 John Wiley & Sons A/S

doi:10.1111/tra.12000

Review

Bacterial Pathogens Commandeer Rab GTPasesto Establish Intracellular Niches

Mary-Pat Stein1,∗, Matthias P. Muller2

and Angela Wandinger-Ness3

1Department of Biology, California State University,Northridge, Northridge, CA, USA2Department of Physical Biochemistry, Max PlanckInstitute of Molecular Physiology, Dortmund, Germany3Department of Pathology, University of New MexicoHSC, Albuquerque, NM, 87131, USA*Corresponding author: Mary-Pat Stein,[email protected]

Intracellular bacterial pathogens deploy virulence factors

termed effectors to inhibit degradation by host cells and

to establish intracellular niches where growth and dif-

ferentiation take place. Here, we describe mechanisms

by which human bacterial pathogens (including Chlamy-

diae; Coxiella burnetii ; Helicobacter pylori; Legionella

pneumophila; Listeria monocytogenes; Mycobacteria;

Pseudomonas aeruginosa, Salmonella enterica) modu-

late endocytic and exocytic Rab GTPases in order to

thrive in host cells. Host cell Rab GTPases are critical for

intracellular transport following pathogen phagocytosis

or endocytosis. At the molecular level bacterial effectors

hijack Rab protein function to: evade degradation, direct

transport to particular intracellular locations and monop-

olize host vesicles carrying molecules that are needed for

a stable niche and/or bacterial growth and differentiation.

Bacterial effectors may serve as specific receptors for Rab

GTPases or as enzymes that post-translationally modify

Rab proteins or endosomal membrane lipids required for

Rab function. Emerging data indicate that bacterial effec-

tor expression is temporally and spatially regulated and

multiple virulence factors may act concertedly to usurp

Rab GTPase function, alter signaling and ensure niche

establishment and intracellular bacterial growth, making

this field an exciting area for further study.

Key words: bacterial secretion, cytoskeletal motors,

membrane trafficking, pathogen containing vacuole or

inclusion, phagosome, post-translational modification,

regulation, replication

Received 21 March 2012, revised and accepted for

publication 13 August 2012, uncorrected manuscript

published online 17 August 2012, published online 13

September 2012

Rab-GTPase-Regulated Traffickingto Lysosomes is a Normal Host DefenseMechanism

Rab GTPases are central to the organization, maintenanceand dynamics of the cellular endomembrane system

through their functions in regulating specific membranetransport pathways (Figure 1, Table 1) (1,2). In bacte-rial infection, Rab proteins play a pivotal role in hostimmunity, internalization by endocytosis or phagocytosisand directing the transport of phagocytosed pathogens tolysosomes for degradation (Table 1). The normal transportpathway to lysosomes utilizes numerous Rab proteinsto efficiently deliver pathogen-containing vacuoles (PCV)from an early phagocytic compartment to a Rab5-positiveearly-endosomal compartment. Pathogens destined fordegradation are then shuttled through a Rab7-positive late-endosome prior to reaching their final destination, the lyso-somal compartment. Phagosomal maturation along thispathway has been analyzed by examination of the tempo-ral recruitment of protein and lipid markers to phagosomescontaining heat-killed pathogens, latex beads or pathogensthat do not block transport to the lysosome. Targetedmanipulation of Rab GTPase function through mutant pro-tein overexpression or siRNA depletion performed in par-allel has defined a large number of participating GTPasesand some of their functions in phagosome maturation.

Latex bead-containing phagosomes have been exten-sively studied over the last decade and a half to identifyproteins and lipids recruited to model phagosomes dueto the high degree of purity with which they can bepurified from higher density cellular membranes (47,48).Proteomic studies on purified latex bead-containingphagosomes have documented the recruitment of over40 Rab GTPases in mouse macrophages followingvariable uptake times (10–120 min) and monitoringkinetics of maturation after internalization for 10–180 min(49–52) (Figure 1). Some of the key principles thathave emerged are that: (i) Rab GTPases associate withmaturing phagosomes in a dynamic manner and changeover time; (ii) heterogeneity among phagosomes makesit difficult to discern the molecular sequence of eventswith absolute precision; (iii) all-or-no changes in Rabcompositions are rare suggesting subtle changes in con-centration are biologically significant; (iv) post-translationalmodifications, including phosphorylation can impact Rabassociation with phagosomes and (v) Rab GTPasesassociated with maturing phagosomes derive to varyingdegrees from nearly all endomembranes in the cell (endo-somes/lysosomes > plasma membrane > endoplasmicreticulum (ER) > Golgi > mitochondria) (52–54).

Studies on the maturation of phagosomes containingheat-inactivated, mutant or non-pathogenic bacteria usingimmunofluorescence and western blot analyses show

www.traffic.dk 1565

Stein et al.

Nucleus

ER

Golgi

Rab2Rab1

Rab7

Rab9

Rab4

Rab22a

Rab5Rab21

Late endosomeMVB

Lysosome

Autophagosome

Early endosome

Melanosome

Recycling endosome

Earlyphagosome

14

14

3

6

Tight junction

SV

27Rab17 Rab7

Rab36

Rab24Rab33

Rab7Rab34

Rab78

11

811

10

Cilium formation

14

14SG810

35

35

Cytokinesis

Rab11a (apical)

34

37

39

39

39

23

23

Phagolysosome

Rab11b (basolateral)

11

Intermediatephagosome

17

27b

21

5

23

23

23

23

18

7 9

34

39

27

37

vATPasecathepsinD

32

20

Mitochondrion

Rab32

20

Lipid droplet

MAM

43

25(epithelia)

Rab15

14

8

8

11

11

13

5

11

13

20

22

Rab22b

22b

22a

22a

22b

5

7 9

22b

415

36

32

Rab38

38

32

32

38

43

7

7

9

34

39

10-20

30-45

60-90

120-180

PhagocytosisEndocytosis

Exocytosis

35

21

1Time, min

13

Figure 1: Rab GTPase regulated pathways. Over 60 Rab GTPase family members regulate membrane transport on the exocytic,endocytic, phagocytic and recycling pathways. Shown are the normal functions (arrows) and localizations (black circles) of Rab GTPasesthat are targeted by bacterial pathogens as detailed in the text and Table 3. Phagosome and autophagosome maturation depend on thesequential fusion with early endosomes, late endosomes and lysosomes. Additional components needed for phagosome maturationare likely recruited from interactions with the secretory pathway based on the involvement of Rab GTPases with primary functionsin exocytosis, organelle biogenesis, ER and Golgi dynamics, and mitochondrial function (Rab20, Rab32, Rab38, Rab43). Over 40 RabGTPases have been identified on phagosomes at various stages of maturation; depicted are 24 Rab GTPases whose kinetic acquisitionand functions have been characterized by analyses of latex bead and non-pathogenic bacterial phagosomes (see text for detail).Some Rab GTPases may be acquired in a biphasic manner (51) and others transit gradually with small changes in concentration andphosphorylation state triggering changes in activity (12,53,55). MAM, mitochondria associated membrane (thought to be of ER origin);SV, synaptic vesicle; SG, secretory granule.

significant agreement with studies on latex bead phago-somes (51,53,55). However, molecular events at earlytime points after internalization, as well as the functions ofmany of the Rab GTPases on phagosomes have remainedelusive. Recently, detailed proteomic analyses of phago-somal compartments transporting live Staphylococcusaureus to lysosomes firmly established the kinetics ofRab protein recruitment to a non-pathogenic phagosomeand the requirements for Rab proteins in acidificationand degradative enzyme recruitment (Figure 1) (12). All

Rab proteins identified on S. aureus phagosomes, withthe exceptions of Rab8, Rab11 and Rab27, were alsofound on latex bead phagosomes, indicating that non-pathogenic bacteria are suitable models for phagosomaldynamics (52). Rab5 and Rab22 localized to S. aureus-containing phagosomes as early as 10 min after infectionwith a transient recruitment of various other Rab proteinsobserved for up to 1 h following infection (Rab8, Rab8b,Rab11, Rab11b, Rab13, Rab14, Rab20, Rab22a, Rab32,Rab38 and Rab43) (12). Recruitment and accumulation

1566 Traffic 2012; 13: 1565–1588

Co-opting Rab Protein Function

Tab

le1:

Rab

GTP

ases

:nor

mal

func

tions

and

asta

rget

sof

bact

eria

lpat

hoge

ns

Rab

GTP

ase

GTP

ase

part

ners

Loca

lizat

ion

Maj

ortr

affic

king

rout

eN

orm

alfu

nctio

nR

ole

inba

cter

ial

path

ogen

esis

Ref

eren

ces

revi

ew

Rab

GTP

ase

revi

ews

(1,2

)

Rab

1a,b

Rab

2E

RE

xocy

tosi

sE

Rto

Gol

gitr

ansp

ort

Pos

t-tr

ansl

atio

nally

mod

ified

byL.

pneu

mop

hila

toes

tabl

ish

nich

ean

dga

innu

trie

nts;

recr

uite

dto

chla

myd

iali

nclu

sion

s

(3–9

)

Rab

3a,R

ab3b

,R

ab3c

,Rab

3dR

ab26

,Rab

27,

Rab

37S

ecre

tory

gran

ules

,sy

napt

icve

sicl

es

Exo

cyto

sis

Reg

ulat

edse

cret

ion

Invi

tro

targ

etof

P.ae

rugi

nosa

Exo

S(1

0,11

)

Rab

4aR

ab5a

,Rab

11a,

Rab

14E

arly

endo

som

esan

dre

cycl

ing

endo

som

es

End

ocyt

osis

and

recy

clin

gR

egul

ates

sort

ing

and

endo

cytic

recy

clin

gto

the

plas

ma

mem

bran

e;tr

affic

king

ofhu

man

P-g

lyco

prot

ein

resp

onsi

ble

for

mul

tidru

gre

sist

ance

oftu

mor

s;fu

nctio

nsw

ithR

ab14

thro

ugh

shar

edef

fect

orR

UFY

1/R

abip

4

Invi

tro

targ

etof

P.ae

rugi

nosa

Exo

S(1

0)

Rab

5a,b

,cR

ab4a

,Rab

11a,

Rab

15,R

ab21

Pla

sma

mem

bran

e,cl

athe

rinco

ated

vesi

cles

and

early

endo

som

es

End

ocyt

osis

and

recy

clin

gE

ndoc

ytos

is,e

arly

endo

som

efu

sion

,nu

clea

rsi

gnal

ing

thro

ugh

AP

PL

Ear

lyph

agoc

ytos

is;e

xclu

ded

from

L.m

onoc

ytog

enes

phag

osom

esan

dch

lam

ydia

linc

lusi

ons;

Rec

ruite

dto

S.en

teric

a,M

.tub

ercu

losi

san

dC

.bu

rnet

iiP

CV

(12

–18)

Rab

6R

ab11

Gol

giE

xocy

tosi

sG

olgi

tran

spor

tR

ecru

ited

toch

lam

ydia

linc

lusi

ons,

with

effe

ctor

Bic

D1

regu

late

sch

lam

ydia

lpro

tein

synt

hesi

san

dnu

trie

ntde

liver

y;re

crui

ted

byL.

pneu

mop

hila

LidA

(8,1

9,20

)

Rab

7aR

ab5a

,Rab

9a,

Rab

34,A

rf6,

Rac

1;R

ab27

a,R

ab33

Late

endo

som

esan

dly

soso

mes

;st

age

Iand

IIm

elan

osom

es;

surf

acta

nten

docy

tosi

san

dsi

gnal

ing

Aut

opha

gyan

dde

grad

atio

n;ly

soso

me

and

lyso

som

e-re

late

dor

gane

llebi

ogen

esis

;re

gula

ted

secr

etio

n

Tran

spor

tfr

omea

rlyto

late

endo

som

esan

dla

teen

doso

me

toly

soso

me

fusi

on;b

idire

ctio

nal

tran

spor

tof

sign

alin

gen

doso

mes

,au

toph

agos

omes

,mul

tives

icul

arbo

dies

,and

mel

anos

omes

onm

icro

tubu

les

inas

soci

atio

nw

ithdy

nein

and

kine

sin

mot

orpr

otei

ns;

axon

viab

ility

;pho

spho

inos

itide

hom

eost

asis

;lip

idtr

ansp

ort;

activ

atio

nof

mTO

Rsi

gnal

ing;

lung

inna

tean

dad

aptiv

eim

mun

ity

Pha

goso

me

acid

ifica

tion

and

cath

epsi

nD

recr

uitm

ent;

phag

osom

em

atur

atio

nan

dfu

sion

with

lyso

som

alsy

stem

;dis

soci

ated

from

M.t

uber

culo

sis

phag

osom

es;

RIL

Pef

fect

orin

tera

ctio

nbl

ocke

dby

S.en

teric

a;fa

cilit

ates

H.p

ylor

iand

C.b

urne

tiini

che

form

atio

n

(12,

21–2

7)

Rab

8a,R

ab8b

Rab

10,R

ab11

aG

olgi

,bas

eof

cilia

,ce

ntro

som

e,de

ndrit

es

Cel

lpol

ariz

atio

nP

olar

ized

tran

spor

tfr

omG

olgi

toba

sola

tera

lpla

sma

mem

bran

ean

dci

liain

epith

elia

and

phot

orec

epto

rs,

pola

rized

neur

iteou

tgro

wth

and

post

-syn

aptic

recy

clin

g

Tran

sien

tre

crui

tmen

tto

inte

rmed

iate

S.au

reus

and

M.t

uber

culo

sis

cont

aini

ngph

agos

omes

;rec

ruite

dby

L.pn

eum

ophi

laLi

dA

(8,1

2,28

,29)

Traffic 2012; 13: 1565–1588 1567

Stein et al.

Tab

le1:

Con

tinue

d

Rab

GTP

ase

GTP

ase

part

ners

Loca

lizat

ion

Maj

ortr

affic

king

rout

eN

orm

alfu

nctio

nR

ole

inba

cter

ial

path

ogen

esis

Ref

eren

ces

revi

ew

Rab

9a;R

ab9b

Rab

7aLa

teen

doso

mes

End

ocyt

osis

and

recy

clin

gTr

ansp

ort

from

endo

som

eto

tran

s-G

olgi

netw

ork;

lipid

tran

spor

t;ly

soso

me

and

lyso

som

ere

late

dor

gane

llebi

ogen

esis

Rec

ruitm

ent

toin

term

edia

tean

dla

teS.

aure

usan

dM

.tub

ercu

losi

sph

agos

omes

;ant

agon

ized

byS.

ente

rica

SifA

and

excl

uded

from

chla

myd

iali

nclu

sion

s

(12,

16,3

0)

Rab

10R

ab8a

,Rab

11a

Bas

eof

prim

ary

cilia

,Gol

giC

iliog

enes

isan

dci

liary

traf

ficki

ng;i

mm

une

syna

pse

form

atio

n;cy

toki

nesi

s

Pla

sma

mem

bran

ere

cycl

ing;

func

tions

inco

ncer

tw

ithR

ab8

and

Rab

11a;

insu

lin-s

timul

ated

GLU

T4tr

ansl

ocat

ion;

phag

osom

em

atur

atio

n;W

eibe

l-Pal

ade

body

form

atio

nan

dse

cret

ion

ofvo

nW

illeb

rand

fact

or;r

egul

ated

surf

ace

expr

essi

onof

Toll-

like

rece

ptor

(TLR

)4

Ear

lyph

agos

omes

;on

early

M.b

ovis

phag

osom

esan

dso

me

chla

myd

ial

incl

usio

nsvi

aC

Pn0

585

(16,

29,3

1,32

)

Rab

11a,

Rab

11b

(neu

ron

spec

ific)

Arf

4,R

ab6,

Rab

8a,

Rab

10,C

dc42

Gol

gian

dre

cycl

ing

endo

som

es,e

arly

endo

som

es,

phag

osom

es

End

ocyt

osis

and

recy

clin

g;ce

llpo

lariz

atio

nan

dci

lioge

nesi

s;im

mun

esy

naps

efo

rmat

ion;

cyto

kine

sis

Traf

ficki

ngfr

omth

etr

ans-

Gol

gine

twor

kto

apic

alre

cycl

ing

endo

som

esan

dpl

asm

am

embr

ane;

dopa

min

etr

ansp

orte

ran

dbe

ta2-

adre

nerg

icre

cept

ortr

affic

king

;pol

ariz

edtr

affic

king

inep

ithel

ia;p

hago

cyto

sis

inm

acro

phag

es;f

unct

ions

inco

ncer

tw

ithR

ab8

and

Rab

10pr

imar

yci

lioge

nesi

s;ci

liary

traf

ficki

ng

Tem

pora

llyre

gula

ted

asso

ciat

ion

with

chla

myd

iali

nclu

sion

sm

edia

ted

byC

Pn0

585;

inhi

bitio

nof

Rab

11-d

epen

dent

recy

clin

gby

H.

pylo

riC

agA

inhi

bitio

nof

Rab

11-F

IPef

fect

orin

tera

ctio

ns;e

xclu

ded

byM

.tub

ercu

losi

sby

ES

AT-

6

(12,

16,2

0,32

,33)

Rab

13Ti

ght

junc

tions

,Gol

gi,

endo

som

esC

ellp

olar

izat

ion

Ass

ocia

ted

with

tight

junc

tions

and

func

tions

ontr

ans-

Gol

gi-e

ndos

ome

circ

uit

inpo

lariz

edce

lls

Tran

sien

tlyon

S.au

reus

phag

osom

es(1

2)

Rab

14R

ab4,

Rab

39E

arly

endo

som

e,G

olgi

End

ocyt

osis

and

recy

clin

gE

ndoc

ytic

recy

clin

gof

tran

sfer

rin;

MH

Ccl

ass

Icro

ss-p

rese

ntat

ion

inde

ndrit

icce

lls;T

GN

toap

ical

traf

ficki

ngin

epith

elia

;sur

fact

ant

secr

etio

nin

alve

olar

cells

;in

sulin

-dep

ende

ntG

LUT4

tran

sloc

atio

n;fu

nctio

nsco

oper

ativ

ely

with

Rab

4th

roug

hsh

ared

effe

ctor

RU

FY1/

Rab

ip4;

regu

latio

nof

embr

yoni

cde

velo

pmen

tth

roug

hin

tera

ctio

nw

ithK

if16B

and

tran

spor

tof

FGF

Tran

sien

tlyas

soci

ated

with

inte

rmed

iate

S.au

reus

phag

osom

es;p

artic

ipat

esin

phag

osom

ear

rest

ofM

.tu

berc

ulos

isph

agos

omes

;fou

ndon

chla

myd

iali

nclu

sion

san

dL.

pneu

mop

hila

PC

V;m

ayse

rve

inlip

idre

crui

tmen

t

(12,

26,3

4,35

)

Rab

20P

hago

som

es,

mito

chon

dria

,ER

endo

som

es

End

ocyt

osis

and

recy

clin

gV

acuo

lar

ATP

ase

traf

ficki

ngin

kidn

ey;

targ

etof

HIF

inhy

poxi

ain

duce

dap

opto

sis;

phag

osom

eac

idifi

catio

nan

dm

atur

atio

n;G

apju

nctio

nbi

ogen

esis

Pha

goso

me

acid

ifica

tion

and

cath

epsi

nD

recr

uitm

ent

thro

ugh

lyso

som

efu

sion

;exc

lude

dfr

omph

agos

omes

byE

SA

T-6

(12)

1568 Traffic 2012; 13: 1565–1588


Tab

le1:

Con

tinue

d

Rab

GTP

ase

GTP

ase

part

ners

Loca

lizat

ion

Maj

ortr

affic

king

rout

eN

orm

alfu

nctio

nR

ole

inba

cter

ial

path

ogen

esis

Ref

eren

ces

revi

ew

Rab

22a

Rab

5a,R

ab7a

Ear

lyen

doso

me,

plas

ma

mem

bran

eE

ndoc

ytos

isan

dre

cycl

ing

Tran

spor

tof

tran

sfer

rinfr

omso

rtin

gen

doso

mes

tore

cycl

ing

endo

som

es;p

atho

gen

phag

ocyt

osis

;enr

iche

din

glia

;sh

ares

effe

ctor

san

dG

EFs

with

Rab

5(R

abex

-5,E

EA

1)

Tran

sien

tlyas

soci

ated

with

inte

rmed

iate

S.au

reus

phag

osom

es;a

ccum

ulat

eson

M.

tube

rcul

osis

phag

osom

esan

dpa

rtic

ipat

esin

phag

osom

ear

rest

(12,

36–3

8)

Rab

22b

tran

s-G

olgi

End

ocyt

osis

and

recy

clin

gG

olgi

-pla

sma

mem

bran

ere

cycl

ing

Ear

lyph

agoc

ytos

is;C

athe

psin

Dre

crui

tmen

tto

phag

osom

es;

tran

sien

tlyas

soci

ated

with

early

M.

tube

rcul

osis

phag

osom

e

(12,

28)

Rab

23P

lasm

am

embr

ane

and

endo

som

esC

iliog

enes

isan

dci

liary

Traf

ficki

ng;i

mm

une

syna

pse

form

atio

n;cy

toki

nesi

s

Traf

ficki

ngof

soni

che

dgeh

ogsi

gnal

ing

com

pone

nts;

cent

ral

nerv

ous

syst

emde

velo

pmen

tan

dci

liary

traf

ficki

ng

Ear

lyph

agoc

ytos

is;a

ssoc

iate

dw

ithS.

aure

usph

agos

omes

thro

ugho

uttr

ansi

tto

lyso

som

esfo

rdeg

rada

tion;

tran

sien

tlyas

soci

ated

with

early

M.

tube

rcul

osis

phag

osom

es

(12,

29)

Rab

27a,

bR

ab7a

,Rab

17R

ab32

,Rab

38M

elan

osom

es,l

ysos

ome

rela

ted

orga

nelle

sLy

soso

me

and

lyso

som

e-re

late

dor

gane

llebi

ogen

esis

;re

gula

ted

secr

etio

n

Mel

anos

ome

biog

enes

isan

dtr

affic

king

;pro

stat

em

arke

rse

cret

ion

S.au

reus

late

endo

cytic

traf

ficki

ngan

dph

agos

ome

mat

urat

ion;

excl

uded

from

Myc

obac

teria

phag

osom

esby

ES

AT-

6

(12)

Rab

29(R

ab7L

1)G

olgi

and

vacu

oles

,ov

erex

pres

sed

upon

sucr

ose

indu

ced

cell

vacu

olat

ion

Exo

cyto

sis

Str

ess

regu

late

dex

pres

sion

,bac

teria

lto

xin

traf

ficki

ngE

xpor

toft

ypho

idto

xin

ince

llsin

fect

edw

ithS.

ente

rica

sero

var

Typh

i;cl

eave

dby

Gtg

Ea

type

IIIse

cret

ion

effe

ctor

expr

esse

din

broa

d-ho

stS.

ente

rica,

but

not

S.ty

phi

(39,

40)

Rab

32M

itoch

ondr

ia,a

utop

hagi

cve

sicl

esA

utop

hagy

and

degr

adat

ion

Pos

t-G

olgi

traf

ficki

ngof

mel

anog

enic

enzy

mes

;ER

stre

ssm

edia

ted

apop

tosi

s;m

itoch

ondr

iald

ynam

ics

Cat

heps

inD

recr

uitm

ent

toph

agos

omes

(12,

41)

Rab

34R

ab7a

,Rab

36G

olgi

and

endo

som

esE

ndoc

ytos

isan

dre

cycl

ing

End

osom

es,m

acro

pino

som

efo

rmat

ion,

phag

osom

em

atur

atio

n,ly

soso

me

mor

phog

enes

is,f

unct

ions

with

Rab

36an

dR

ab7

thro

ugh

shar

edef

fect

or(R

ILP

)

Cat

heps

inD

recr

uitm

ent

toph

agos

omes

(12)

Rab

35C

dc42

End

osom

esan

dpl

asm

am

embr

ane

End

ocyt

osis

and

recy

clin

g;ci

lioge

nesi

san

dci

liary

Traf

ficki

ng;

Imm

une

syna

pse

form

atio

n;cy

toki

nesi

s

Fast

endo

cytic

recy

clin

g;cy

toki

nesi

s;im

mun

esy

naps

efu

nctio

n;M

HC

clas

sIa

ndII

endo

cyto

sis

and

recy

clin

g;T

cell

rece

ptor

recy

clin

g;ph

osph

oino

sitid

ere

gula

tion;

neur

iteou

tgro

wth

thro

ugh

inte

rfac

esw

ithC

dc42

;act

inre

mod

elin

gth

roug

hfa

scin

effe

ctor

lead

ing

tofil

opod

iafo

rmat

ion

Pho

spho

chol

inat

edby

L.pn

eum

ophi

laA

nkX

and

reve

rsed

byLe

m3/

lpg0

696

(4,4

2,14

6)

Traffic 2012; 13: 1565–1588 1569

Stein et al.

Tab

le1:

Con

tinue

d

Rab

GTP

ase

GTP

ase

part

ners

Loca

lizat

ion

Maj

ortr

affic

king

rout

eN

orm

alfu

nctio

nR

ole

inba

cter

ial

path

ogen

esis

Ref

eren

ces

revi

ew

Rab

37S

ecre

tory

gran

ules

(insu

lin,

mas

tce

lls,

mac

roph

ages

)

Reg

ulat

edse

cret

ion

Deg

ranu

latio

n;re

gula

tion

ofw

ntsi

gnal

ing

and

angi

ogen

esis

,in

activ

ated

bym

ethi

onin

eam

inop

eptid

ase-

2(M

etA

P-2

),TN

Falp

hase

cret

ion

S.au

reus

late

endo

cytic

traf

ficki

ng;

late

phag

osom

em

atur

atio

n;in

crea

sed

expr

essi

onin

duce

dby

H.

pylo

riin

fect

ion

(12,

43,4

4)

Rab

38R

ab7a

,Rab

27a

Rab

32Ty

rosi

nase

posi

tive

mel

anos

omes

;su

rfac

tant

cont

aini

ngve

sicl

es

Lyso

som

ean

dly

soso

me-

rela

ted

orga

nelle

biog

enes

is;

regu

late

dse

cret

ion

Tran

s-G

olgi

tom

elan

osom

etr

ansp

ort;

lung

surf

acta

ntse

cret

ion;

func

tions

with

Rab

32an

dR

ab7

inm

elan

osom

ebi

ogen

esis

Cat

heps

inD

recr

uitm

ent

toph

agos

omes

(12,

41)

Rab

39R

ab14

Gol

gian

dea

rlyen

doso

mes

,AP

1m

embr

ane

dom

ains

;ly

soso

mes

End

ocyt

osis

and

recy

clin

gC

aspa

se-d

epen

dent

-IL-1

beta

secr

etio

n;ho

mol

ogy

toR

ab14

;ph

agos

omal

acid

ifica

tion

Pha

goso

me

acid

ifica

tion

(12)

Rab

43E

ndos

omes

,Gol

gi,

phag

osom

esE

ndoc

ytos

isan

dG

olgi

recy

clin

g,au

toph

agy

and

degr

adat

ion

ER

-Gol

gitr

ansp

ort;

retr

ogra

detr

ansp

ort

onth

eex

ocyt

icpa

thw

ay;

asso

ciat

esw

ithdy

nein

/dyn

actin

;ca

thep

sin

Dtr

ansp

ort

toph

agos

omes

Cat

heps

inD

recr

uitm

ent

toph

agos

omes

;tra

nsie

ntly

asso

ciat

edw

ithin

term

edia

teM

.tub

ercu

losi

sph

agos

omes

(12,

45,4

6)

1570 Traffic 2012; 13: 1565–1588


of Rab7, Rab9, Rab34 and Rab39 after 30 min wassubsequently observed and Rab27 and Rab37 were onlyobserved on S. aureus phagosomes 1 h post-infection.Rab23 localized to S. aureus-containing phagosomes atall time points analyzed (12). Rab39 was found importantfor phagosome acidification while Rab22b, Rab32, Rab34and Rab38 were crucial for Cathepsin D recruitment.Rab7 and Rab20 were central to phagosomal maturationand lysosomal fusion. These data demonstrate that theconcerted actions of multiple Rab GTPases results inthe acquisition of acidic pH and degradative enzymesand fusion of S. aureus-containing phagosomes withlysosomes for pathogen clearance. Furthermore, theinvolvement of Rab GTPases with primary functions inexocytosis, organelle biogenesis, ER and Golgi dynamics,and mitochondrial function (Rab20, Rab32, Rab38, Rab43)suggests that some of the components needed forphagosome maturation are recruited from interactionswith the secretory pathway (51,53–55). One interestingplayer in this respect is Rab32, which modulatesER calcium handling and cargo shuttling betweenmitochondria associated membranes and the peripheralER, providing a source for newly synthesized lipids andcalcium (an important cofactor in regulated fusion) (56).Together, the kinetic data for Rab recruitment providesbenchmarks one can use to classify when, where andhow specific intracellular pathogens arrest phagosomalmaturation and modulate their niche for intracellularsurvival.

Intracellular Pathogens and the Cell Typesthey Invade

Intracellular pathogens often utilize phagocytic cellssuch as macrophages as hosts to gain intracellularaccess. Pathogens such as Legionella pneumophila andMycobacterium tuberculosis are internalized by alveolarmacrophage leading to establishment of an intracellularniche (Figure 2A). However, other pathogens utilizenon-phagocytic cells or more than one cell type astheir homes, gaining intracellular access by receptor-mediated endocytosis, lipid-raft mediated internalizationor by manipulating host cell actin dynamics throughhost GTPases such as Rho, Rac and Cdc42, to achieveinternalization (reviewed in 57–59). Salmonella entericaserovar Typhi, for example, may infect and replicate inintestinal epithelia or in some cases traverse the intestinalbarrier by transcytosis, whereupon dendritic cells andmacrophages can phagocytose bacteria and establish avacuolar replicative niche (60,61). Systemic disseminationof Salmonella typhi causes human typhoid fever and canresult in persistent infection in the bone marrow and gallbladder for life. S. enterica serovar Typhimurium is usedextensively as an experimental model as it commonlycauses self-resolving gastroenteritis in humans due toinfection and replication in epithelia and can be studiedin mice (62,63). Pathogens such as Helicobacter pylorispecifically invade mucosal epithelia (Figure 2B). No

matter what cell type serves as the host, for intracellularsurvival pathogens such as Chlamydiae, H. pylori,L. pneumophila, Mycobacteria, Pseudomonas aeruginosa,and S. enterica modulate the transport of their vacuolesto evade transport to and degradation in lysosomes,and to establish an environment allowing for growthand differentiation (Table 2). Two notable exceptionsare Listeria monocytogenes, which escapes from thephagosome/vacuole to the cytoplasm, and Coxiellaburnetii, which capitalizes on the acidic environmentin lysosomes. This review focuses on mechanismsby which all of these pathogens modulate host RabGTPase activities to establish an intracellular niche whereacquisition of nutrients, lipids and other necessary factorsprepare the pathogen for egress from host cells.

Pathogen Requirements for IntracellularSurvival and Growth

One mechanism that pathogens use to avoid destructionand promote growth and multiplication is to prohibittransport of the PCV down the endocytic pathway tolysosomes. The selective recruitment of Rab or Rabeffector proteins to the PCV and the direct modulation ofRab protein activity are mechanisms utilized by pathogenvirulence factors to regulate the transport of the PCVthrough the host cell (Table 3). In addition to evadingdegradation, pathogens actively direct their transportto intracellular sites where assembly of the appropriateenvironment for bacterial differentiation and growth mayoccur. Pathogens direct trafficking of the PCV to specificintracellular locales utilizing Rab-regulated host cytoskele-tal motor proteins. Pathogens also modulate signaling anddirect the recruitment of host vesicles laden with proteinsand lipids to modify the PCV and to provide nutrients forbacterial growth. Thus, the establishment of an appro-priate intracellular niche involves multiple steps wherehost-bacterial protein interactions modulate Rab activities.

Intracellular bacterial pathogens employ complex secre-tion systems (summarized in Table 2) for conveyingvirulence factors into the host cell cytoplasm wherethey contact Rab proteins and modulate Rab GTPasefunctions. Some of the intracellular pathogens discussedin this review include Gram-negative bacteria (Chlamy-diae, P. aeruginosa and S. enterica), which rely on type3 (III) secretion systems (T3SS) comprised of flagella-like machines for protein injection (65,66). Salmonellavirulence depends on two interdependent T3SS sys-tems, T3SS1 and T3SS2 effectors that are involved inpathogen vacuole biogenesis (62,78). Other intracellularGram-negative bacteria utilize a type 4 (IV) secretionmachinery (T4SS), which resembles bacterial conjuga-tion pili (H. pylori) or in the case of C. burnetii andL. pneumophila use a specialized type 4B (T4BSS) assem-bly of Dot and Icm proteins (68,70). Mycobacteria andL. monocytogenes are Gram-positive and utilize gen-eral secretory (Sec) and twin-arginine translocation (Tat)

Traffic 2012; 13: 1565–1588 1571

Stein et al.

Nucleus

ER

Golgi

Helicobacter pylori

Pseudomonasaeruginosa

TJ

AJ

PI3K

ZO-1, Jam-A

Listeria monocytogenes

PM blebs

Chlamydiatrachomatis,pneumoniae

MTOC

Chlamydial inclusion

vacuole

A Macrophage Host B Epithelial Host

Mycobacterium tuberculosis

Legionellapneumophila

Nucleus

ER

Golgi

Salmonella entericaM

icrotubuleMicrotubule

Coxiella burnetii

EE

EE

Cp-Rab1Rab10Rab11

Rab7/RILPRab37

Rab3Rab4Rab5

Ct-Rab4Rab6Rab11Rab14

Rab5

Rab5Rab7

Rab5Rab11Rab13Rab14Rab20Rab22Rab27

Rab24

Rab1Rab6Rab8Rab35 LL

LE

LE

Rab7/RILP/dyneinRab9

Rab7/SKIP

kinesin/Arl8b

AP

Figure 2: Intracellular bacterial pathogens create specialized niches in macrophage and epithelial hosts by modulating Rab

GTPases. Pathogens alter Rab GTPase functions to escape degradation and obtain essential nutrients for growth and survival. A)Macrophage host. Legionella pneumophila enters alveolar macrophages by coiling phagocytosis and creates a replicative niche in closeapposition to the endoplasmic reticulum (ER) by modulating the activity of Rab1 and Rab35. Legionella evades fusion with lysosomes(L), although some exchange with endosomes may take place and pH is mildly acidic. S. enterica can infect enterocytes or traverse theintestinal epithelial barrier by transcytosis, and in the subluminal Peyer’s patches be phagocytosed by macrophages and dendritic cellsthat can promote systemic dissemination and infection. S. enterica coopts active Rab7-regulated, microtubule transport to establish areplicative niche in the peri-Golgi region and form tubules called Sifs that promote cell-to-cell spread. M. tuberculosis and C. burnetii bothpreferentially infect alveolar macrophages, although Mycobacteria allow only early endosome (EE) fusion and induce phagosome arrestby selective Rab GTPase recruitment to avoid fusion with late endosomes (LE) and lysosomes. Coxiella-containing phagosomes on theother hand fuse with late endosomes, lysosomes and autophagosomes (AP), therefore, C. burnetii are adapted to thrive in an acidicniche. B) Epithelial host. L. monocytogenes infects macrophages, intestinal epithelia and hepatocytes; gaining entry by specific binding toand internalization with E-cadherin or Met receptors and evading degradation by blocking Rab5 before release to the cytoplasm. H. pyloriinfect intestinal epithelia through the apical recruitment of tight junction proteins (ZO-1 and Jam-A) and after internalization establish areplicative niche via the vacuolating toxin VacA and Rab7-mediated fusion with endosomes. Chlamydia trachomatis (Ct) or pneumonia(Cp) infect epithelia from the apical surface and utilize Rab-regulated, microtubule transport to establish a specialized inclusion in theperi-Golgi region that depends on Rab-regulated fusion with early endosomes, late endosomes and Golgi-derived vesicles. P. aeruginosarecruits the phosphatidylinositol 3-kinase to the apical plasma membrane (PM) where it resides within plasma membrane blebs andblocks endocytosis by ribosylation of Rab5.

pathways for translocation of unfolded and folded proteinsinto the extracytoplasmic-cell wall space, respectively.Mycobacteria have additional secretory systems (SecA2and ESX) that serve in the secretion of select proteinslacking N-terminal signal sequences such as ESAT-6.Bacterial virulence factors that are delivered via special-ized secretion systems, have several discrete functionaldomains and act in concert with other virulence factors tomodulate host cell functions are collectively termed bac-terial effectors, and are distinguished from toxins, whichcan act extracellularly (78). Here, we provide mechanisticexamples of how intracellular pathogens manipulate Rab

proteins through bacterial effector protein interactions todirect their intracellular lifestyle.

Normal Rab GTPase Function

Rab GTPases govern vesicular trafficking through a cycleof activation (GTP-binding), inactivation (GTP-hydrolysis)and cytosolic recycling (1). Membrane-dependent activa-tion is controlled by guanine nucleotide exchange factors(GEF), while inactivation is regulated by GTPase acti-vating proteins (GAP) that accelerate the hydrolysis of

1572 Traffic 2012; 13: 1565–1588


Tab

le2:

Intr

acel

lula

rba

cter

ialp

atho

gen

caus

eddi

seas

esan

dni

che

requ

irem

ents

Mic

robe

Gra

mst

ain

Sec

retio

nsy

stem

Targ

etce

llty

peIn

trac

ellu

lar

nich

eR

equi

rem

ents

for

repl

icat

ion

Hum

andi

seas

e/pa

thol

ogy

Ref

eren

ces

Chl

amyd

ia(tr

acho

mat

isan

dpn

eum

onia

e)

Neg

ativ

eT3

SS

Cer

vica

land

lung

epith

elia

peri-

Gol

gias

soci

ated

incl

usio

nS

peci

aliz

edin

clus

ion;

inhi

bitio

nof

lyso

som

alfu

sion

;hos

tde

rived

lipid

s(s

phin

gom

yelin

,ch

oles

tero

l,gl

ycer

opho

spho

lipid

san

dne

utra

llip

ids)

Sex

ually

tran

smitt

eddi

seas

e;bl

indn

ess;

pneu

mon

ia

(64

–66)

C.b

urne

tiiN

egat

ive

Dot

/Icm

T4B

SS

(IVB

),sp

ecia

lized

type

IVse

cret

ion

syst

emde

pend

ent

onD

otan

dIc

mpr

otei

nspr

esen

ton

lyin

Cox

iella

and

Legi

onel

la.

Mac

roph

age

Par

asito

phor

ous

vacu

ole/

phag

osom

e,C

oxie

llare

plic

ativ

eva

cuol

e,au

toph

agic

path

way

Low

pHan

dox

ygen

;ch

oles

tero

lric

hm

embr

ane;

met

abol

ites

from

auto

phag

y;sp

acio

usca

vity

devo

idof

lyso

som

alen

zym

es;

conv

ersi

onto

larg

ece

ll

Tran

smis

sion

via

inha

latio

n;Q

-fev

erin

clud

ing

pneu

mon

ia,

hepa

titis

,car

diac

dise

ase

(15,

67,6

8)

H.p

ylor

iN

egat

ive

T4S

S(c

onta

ct-a

ndpi

li-de

pend

ent)

Gas

tric

and

inte

stin

alep

ithel

iaLa

teen

docy

ticva

cuol

esA

ccum

ulat

ion

ofos

mot

ical

lyac

tive

wea

kba

ses

tofo

rmla

rge

spac

ious

vacu

ole;

inhi

bitio

nof

lyso

som

efu

sion

Gas

tric

ulce

rsan

dca

ncer

(69,

70)

L.pn

eum

ophi

laN

egat

ive

Dot

/Icm

T4B

SS

(IVB

)spe

cial

ized

type

IVse

cret

ion

syst

emde

pend

ent

onD

otan

dIc

mpr

otei

nspr

esen

ton

lyin

Legi

onel

laan

dC

oxie

lla

Lung

epith

elia

ER

and

Gol

gias

soci

ated

vacu

ole

Legi

onel

la-c

onta

inin

gva

cuol

ew

ithre

mod

eled

phos

phoi

nosi

tides

and

recr

uitm

ent

ofbi

osyn

thet

icve

sicl

esan

dho

sttr

affic

king

mac

hine

ryth

roug

hre

vers

ible

aden

ylyl

a-tio

n/de

aden

ylyl

atio

n

Tran

smis

sion

via

inha

latio

n,an

dw

ater

cont

aini

ngin

fect

edam

oeba

e;Le

gion

naire

s’di

seas

e,pn

eum

onia

,GI

infe

ctio

nsan

ddi

arrh

ea

(68,

71,7

2)

L.m

onoc

ytog

enes

Pos

itive

Gen

eral

secr

etor

y(S

ec)p

athw

ayfo

rtr

ansl

ocat

ion

from

cyto

solt

oex

trac

ytop

lasm

ic-

cell

wal

lsp

ace

Inte

stin

al,c

ervi

cal,

corn

eala

ndlu

ngep

ithel

ia;

hepa

tocy

tes,

nerv

ous

tissu

em

acro

phag

e

Ear

lyph

agos

omes

and

cyto

sol

Inhi

bitio

nof

phag

osom

em

atur

atio

n;es

cape

tocy

toso

l;P

I(3,4

,5)P

3

requ

irem

entf

orin

fect

ion

Food

-bor

nelis

terio

sis;

men

ingo

ence

phal

its;

fata

lin

20–3

0%of

case

s

(65,

73)

Traffic 2012; 13: 1565–1588 1573

Stein et al.

Tab

le2:

Con

tinue

d

Mic

robe

Gra

mst

ain

Sec

retio

nsy

stem

Targ

etce

llty

peIn

trac

ellu

lar

nich

eR

equi

rem

ents

for

repl

icat

ion

Hum

andi

seas

e/pa

thol

ogy

Ref

eren

ces

M.t

uber

culo

sis

Aci

d-fa

st,

Gra

m-

posi

tive

(lack

oute

rce

llm

em-

bran

e)

Gen

eral

secr

etor

y(S

ec)a

ndtw

in-a

rgin

ine

tran

sloc

atio

n(T

at)

path

way

sfo

rtr

ansl

ocat

ion

ofun

fold

edan

dfo

lded

prot

eins

,re

spec

tivel

y,fr

omcy

toso

lto

extr

acyt

opla

smic

-ce

llw

alls

pace

;S

ecA

2an

dE

SX

expo

rtsy

stem

sfo

rse

cret

ion

ofse

lect

prot

eins

such

asE

SA

T-6

lack

ing

N-t

erm

inal

sign

alse

quen

ces

Mac

roph

age

Arr

este

dea

rlyph

agos

omes

PI(3

)Psy

nthe

sis

inhi

bite

dan

dba

cter

ial

phos

phat

ases

secr

eted

topr

even

tph

agos

omal

mat

urat

ion

atea

rlyst

age;

spec

ializ

edba

cter

iall

ipid

sal

low

cont

inuo

usfu

sion

ofM

ycob

acte

rium

cont

aini

ngva

cuol

esw

ithea

rlyen

doso

mes

Tube

rcul

osis

,GIt

ract

infe

ctio

nsca

usin

gdi

arrh

eaan

dm

alab

sorp

tion

(65,

74)

P.ae

rugi

nosa

Neg

ativ

eT3

SS

Lung

,ski

n,ur

inar

ytr

act

and

corn

eal

epith

elia

Pla

sma

mem

bran

ebl

ebs

Api

calp

lasm

am

embr

ane

rem

odel

ing

into

baso

late

ral-l

ike

mem

bran

evi

aP

I3-

kina

sere

crui

tmen

tan

dA

DP

-rib

osyl

atio

nto

inac

tivat

eR

abpr

otei

nsan

dpr

even

tin

tern

aliz

atio

n

Opp

ortu

nist

icpu

lmon

ary

and

urin

ary

trac

tin

fect

ions

inim

mun

eco

mpr

omis

edpa

tient

s,m

ayca

use

seps

is

(66,

75,7

6)

S.en

teric

aN

egat

ive

T3S

S(fl

agel

la-li

kein

ject

isom

e),

Salm

onel

laT3

SS

effe

ctor

sar

een

code

dby

two

path

ogen

icity

isla

nds,

T3S

S1

and

T3S

S2

that

func

tion

coor

dina

tely

inin

vasi

onan

din

trac

ellu

lar

surv

ival

Inte

stin

alep

ithel

ia,

mac

roph

ages

are

are

serv

oir

Late

endo

cytic

Salm

onel

laco

ntai

ning

vacu

ole

Act

ivat

ion

ofP

I(3)P

synt

hesi

sto

recr

uit

host

traf

ficki

ngm

achi

nery

;m

anip

ulat

ion

ofm

otor

prot

eins

and

cyto

skel

etal

traf

ficki

ng

Hum

anfo

od-b

orne

illne

ss(s

erov

arTy

phim

uriu

m,

sero

var

Ent

eriti

dis)

,hu

man

syst

emic

dise

ase

and

typh

oid

feve

r(s

erov

arTy

phi)

(62,

70,7

7,78

)

1574 Traffic 2012; 13: 1565–1588


Tab

le3:

Alte

ratio

nof

Rab

GTP

ase

func

tions

byba

cter

iale

ffec

tor

prot

eins

inni

che

form

atio

n

Mic

robe

Viru

lenc

epr

otei

n(e

ffec

tor)

Act

ivity

Hos

tce

llpa

rtne

rC

onse

quen

ceof

inte

ract

ion

Ref

eren

ces

Mod

ulat

ion

ofm

embr

ane

traf

ficki

ngth

roug

hse

lect

ive

Rab

recr

uitm

enta

ndm

embr

ane

fusi

onB

ruce

llaab

ortu

sR

icA

Dot

/Icm

Type

IVse

cret

edpr

otei

nbi

nds

Rab

2R

ab2

Spe

cific

recr

uitm

ent

ofR

ab2

toP

CV

(79)

C.p

neum

onia

eC

Pn0

585

Incl

usio

nm

embr

ane

prot

ein

(Inc)

;Rab

GTP

ase

bind

ing

via

two

over

lapp

ing

cyto

solic

Rab

bind

ing

dom

ains

with

hom

olog

yto

host

Rab

bind

ing

prot

eins

GM

130,

FIP

3,go

lgin

-84

Rab

1,R

ab10

,Rab

11G

TP-d

epen

dent

recr

uitm

ent

ofR

ab11

toba

cter

ial

incl

usio

nfo

rpe

ri-nu

clea

rtr

ansp

ort

early

inin

fect

ion;

subs

eque

ntin

tera

ctio

nw

ithR

ab1

and

Rab

10to

mai

ntai

nM

TOC

loca

lizat

ion

and

acce

ssto

mem

bran

elip

ids

(32)

C.t

rach

omat

isC

T119

(IncA

)si

mila

rto

CP

n018

6

Incl

usio

nm

embr

ane

prot

ein

(Inc)

;SN

AR

Em

imic

acts

inco

ntro

lling

mem

bran

efu

sion

VA

MP

3,V

AM

P7,

VA

MP

8(R

abG

TPas

esin

dire

ctly

aspa

rtof

SN

AR

Eco

mpl

ex)

Inte

ract

ion

with

host

Rab

/tet

herin

g/S

NA

RE

prot

ein

com

plex

eson

endo

som

esto

regu

late

deliv

ery

ofnu

trie

nts

for

incl

usio

ngr

owth

,pre

vent

recy

clin

gan

dly

soso

mal

deliv

ery

(80)

C.t

rach

omat

isC

T147

Incl

usio

nm

embr

ane

prot

ein

sim

ilar

toth

eR

ab5

effe

ctor

EE

A1;

cont

ains

Zn-fi

nger

for

mem

bran

ebi

ndin

gbu

tla

cks

Rab

5G

TPas

ebi

ndin

gdo

mai

n

unkn

own

Sug

gest

edto

func

tion

inen

doso

me

teth

erin

gbu

tpr

eclu

defu

sion

poss

ibly

bybl

ocki

ngR

ab5

(81)

C.t

rach

omat

isC

T229

Incl

usio

nm

embr

ane

prot

ein

(Inc)

;Rab

4bi

ndin

gvi

acy

toso

licca

rbox

y-te

rmin

aldo

mai

n

Rab

4G

TP-d

epen

dent

recr

uitm

ent

ofR

ab4

toin

clus

ion

mem

bran

eto

prom

ote

dyne

inde

pend

ent

tran

spor

ton

mic

rotu

bule

sto

peri-

Gol

gire

gion

and

inte

ract

ion

with

tran

sfer

rin-c

onta

inin

gen

doso

mes

(82)

C.t

rach

omat

isC

T813

Incl

usio

nm

embr

ane

prot

ein

(Inc)

;SN

AR

Em

imic

acts

inm

embr

ane

fusi

on

VA

MP

7(R

abG

TPas

esin

dire

ctly

aspa

rtof

SN

AR

Eco

mpl

ex)

Inte

ract

ion

may

inhi

bit

VA

MP

7m

edia

ted

fusi

onw

ithly

soso

mes

orpl

asm

am

embr

ane

(80)

Traffic 2012; 13: 1565–1588 1575

Stein et al.

Tab

le3:

Con

tinue

d

Mic

robe

Viru

lenc

epr

otei

n(e

ffec

tor)

Act

ivity

Hos

tce

llpa

rtne

rC

onse

quen

ceof

inte

ract

ion

Ref

eren

ces

C.t

rach

omat

is?

Rab

6,B

icD

1,R

ab11

,R

ab14

Form

atio

nof

repl

icat

ive

nich

e,S

phin

golip

idre

crui

tmen

tto

incl

usio

nvi

aR

ab14

,bio

synt

hetic

and

endo

cytic

carg

ovi

aR

ab6

and

Rab

11.

(19,

34)

C.b

urne

tii?

Rab

5,R

ab7,

Rab

24C

onve

rgen

cew

ithau

toph

agic

path

way

sugg

este

dto

bloc

kde

grad

atio

nin

lyso

som

esth

ough

som

ede

grad

atio

nre

sist

ant

varia

nts

reac

hly

soso

mes

(15,

83)

H.p

ylor

iC

agA

Lipi

d-ra

ftas

soci

ated

cyto

toxi

nS

rcS

rcsu

bstr

ate,

bloc

ksR

ab11

-FIP

asso

ciat

ion

tode

crea

sepa

thog

enre

cycl

ing

(33,

84)

L.pn

eum

ophi

liaD

rrA

/Sid

MG

EF

mim

icR

ab1

Act

ivat

esR

ab1

onP

CV

(6,8

5)M

.tub

ercu

losi

sE

SA

T-6

mem

bran

epo

refo

rmat

ion

Rab

5,R

ab11

,Rab

11b,

Rab

13,R

ab20

,Rab

27P

ore

form

atio

nm

aypr

omot

edi

ssoc

iatio

nof

indi

cate

dR

abpr

otei

nsfr

omm

ycob

acte

rialp

hago

som

es,

cont

ribut

ing

toph

agos

ome

arre

stan

dni

che

form

atio

n

(12)

M.t

uber

culo

sis

ES

X-1

secr

eted

fact

ors

spec

ific

rab

rece

ptor

s?R

ab14

,Rab

22a

Rab

14m

ayfu

nctio

nin

sphi

ngol

ipid

deliv

ery

asis

also

the

case

for

Chl

amyd

ia,R

ab22

apr

eclu

des

Rab

7ac

quis

ition

(35,

37)

S.en

teric

a(s

erov

ars

with

broa

dho

stsp

ecifi

city

,not

Typh

i)

Gtg

EP

rote

ase

Rab

29(R

ab7L

1)C

leav

esR

ab29

inse

rova

rsw

ithbr

oad

host

spec

ifici

tyle

adin

gto

incr

ease

dre

plic

atio

nin

mac

roph

ages

;ins

sero

var

Typh

ilac

king

Gtg

ER

ab29

isno

tcl

eave

dan

dm

edia

tes

typh

oid

toxi

nse

cret

ion

(40)

S.en

teric

a(s

erov

arTy

phim

uriu

m)

Sop

BP

hosp

hoin

ositi

deph

osph

atas

e,C

dc42

bind

ing

and

GD

Iact

ivity

,T3

SS

1ef

fect

or

Rab

5,(in

dire

ctly

Rab

8b,

Rab

13,R

ab23

and

Rab

35)

Red

uctio

nof

nega

tivel

ych

arge

dP

I(4,5

)P2

via

phos

phat

ase

enab

les

Rab

5/P

I3-k

inas

ehV

ps34

recr

uitm

ent

and

also

prev

ents

elec

tros

tatic

mem

bran

ein

tera

ctio

nof

othe

rR

abs

ther

eby

mod

ulat

esm

embr

ane

traf

ficki

ngre

gula

tors

and

inhi

bits

SC

V-ly

soso

me

fusi

on.

(86

–88)

S.en

teric

a(s

erov

arTy

phim

uriu

man

ddu

blin

)

Sop

EG

EF

mim

ic,T

3SS

1ef

fect

orR

ab5,

Cdc

42an

dR

ac1

Rab

5bi

ndin

gan

dre

crui

tmen

tto

SC

V,i

nvi

tro

prom

otes

fusi

onof

SC

Vw

ithea

rlyen

doso

mes

and

nucl

eotid

eex

chan

geon

Rab

5;R

ac1

and

Cdc

42G

EF

activ

ityim

port

ant

for

inva

sion

(18,

89)

1576 Traffic 2012; 13: 1565–1588


Tab

le3:

Con

tinue

d

Mic

robe

Viru

lenc

epr

otei

n(e

ffec

tor)

Act

ivity

Hos

tce

llpa

rtne

rC

onse

quen

ceof

inte

ract

ion

Ref

eren

ces

Sele

ctiv

elo

caliz

atio

nth

roug

hm

odul

atio

nof

Rab

regu

late

dcy

tosk

elet

altr

ansp

ort

C.t

rach

omat

is?

Src

,Rab

11an

dva

rious

FIP

effe

ctor

s?R

ecru

itmen

tof

p150

(glu

ed)d

ynac

tin/d

ynei

nco

mpl

exfo

rM

TOC

tran

spor

tof

chla

myd

ial

incl

usio

nin

conj

unct

ion

with

Rab

11?

Juxt

anuc

lear

posi

tioni

ngm

ayfa

cilit

ate

acce

ssto

nutr

ient

sfr

omG

olgi

and

recy

clin

gen

doso

mes

for

nich

efo

rmat

ion

(20,

90,9

1)

H.p

ylor

iV

acA

Vac

uole

form

atio

nR

ab7-

RIL

P,O

RP

1LV

acA

and

Rab

7-R

ILP

are

esse

ntia

lfor

bact

eria

lva

cuol

efo

rmat

ion

thou

ghdi

rect

inte

ract

ion

isno

tde

mon

stra

ted.

The

Rab

7ef

fect

orO

RP

1Lm

aypa

rtic

ipat

ein

vacu

ole

tran

spor

tth

roug

hits

chol

este

rols

ensi

tive

regu

latio

nof

dyne

in

(23,

24,9

2,93

)

M.b

ovis

?R

ab7-

RIL

PE

vasi

onof

lyso

som

alde

liver

yby

bloc

king

Rab

7an

d/or

RIL

Pre

crui

tmen

t.(2

5)

S.en

teric

a(s

erov

arTy

phim

uriu

m)

Pip

B2

T3S

S2

prot

ein

inte

ract

sw

ithki

nesi

nlig

htch

ain

ofth

eki

nesi

n-1

mot

oran

dco

oper

ates

with

SifA

Kin

esin

-1m

otor

(Rab

7vi

aS

ifA)

Rec

ruits

auto

inhi

bite

dki

nesi

n-1

toS

CV

for

prop

erS

CV

posi

tioni

ngth

roug

hco

ordi

nate

dki

nesi

nm

otor

activ

atio

nw

ithS

ifA/S

KIP

and

plus

-end

Sif

tubu

leex

tens

ion

(62,

94,9

5)

S.en

teric

a(s

erov

arTy

phim

uriu

m)

SifA

T3S

S2

prot

ein

with

N-t

erm

inal

SK

IPbi

ndin

gdo

mai

nan

dC

-ter

min

alR

hoG

EF

both

requ

ired

for

Sif

form

atio

n;pr

enyl

ated

CA

AX

mot

iffo

rm

embr

ane

bind

ing

Rho

A-G

DP

,Rab

7,S

ifAan

dki

nesi

nin

tera

ctin

gpr

otei

n(S

KIP

)/PLE

KH

M2,

Rab

9an

dR

ILP

anta

goni

st,

Arl8

b

Reg

ulat

edS

CV

mem

bran

etu

bula

tion

thro

ugh

mul

tiple

host

and

bact

eria

leff

ecto

rpr

otei

nin

tera

ctio

ns.R

ecru

itsan

dac

tivat

esho

stR

hoA

and

bact

eria

lSse

J.Th

eS

ifA-S

KIP

com

plex

activ

ates

the

host

kine

sin-

1m

otor

(rec

ruite

din

inac

tive

stat

eby

Pip

B2)

and

prom

otes

Sif

tubu

leex

tens

ion

tow

ard

cell

perip

hery

.SifA

-SK

IPbi

ndin

gan

tago

nize

sth

eno

rmal

host

SK

IP-R

ab9-

GTP

bind

ing

thro

ugh

aco

nser

ved

Wxx

xEdo

mai

nin

SifA

that

acts

asa

G-p

rote

inm

imic

and

isco

nser

ved

amon

gba

cter

ialp

rote

ins.

SifA

also

bind

sho

stR

ab7

and

bloc

ksho

stR

ILP

/dyn

ein/

dyna

ctin

asso

ciat

ion

and

ther

eby

cont

rols

tubu

ledy

nam

ics.

(22,

30,8

9,96

–105

)

Traffic 2012; 13: 1565–1588 1577

Stein et al.

Tab

le3:

Con

tinue

d

Mic

robe

Viru

lenc

epr

otei

n(e

ffec

tor)

Act

ivity

Hos

tce

llpa

rtne

rC

onse

quen

ceof

inte

ract

ion

Ref

eren

ces

S.en

teric

a(s

erov

arTy

phim

uriu

m)

Sop

D2

T3S

S2

prot

ein

requ

ired

for

intr

acel

lula

rgr

owth

,re

gula

tes

Sif

dyna

mic

s

?C

onne

ctio

nsto

SifA

and

Pip

B2,

may

mod

ulat

eR

ab7

and

mot

ors?

(106

)

S.en

teric

a(s

erov

arTy

phim

uriu

m)

Sse

FT3

SS

2pr

otei

nbi

nds

dyne

inin

com

plex

with

Sse

G

Rab

7/R

ILP

via

dyne

inor

kine

sin

inte

ract

ion?

Bin

dsS

seG

peri-

nucl

ear

loca

lizat

ion

ofS

CV

,dyn

ein

recr

uitm

ent

and

mic

rotu

bule

bund

ling

(62,

107)

S.en

teric

a(s

erov

arTy

phim

uriu

m)

Sse

GT3

SS

2pr

otei

nre

quire

dfo

rin

trac

ellu

lar

grow

th;

form

sco

mpl

exw

ithS

seF

tobi

nddy

nein

Rab

7/R

ILP

via

dyne

inor

kine

sin

inte

ract

ion?

Bin

dsS

seF;

requ

ired

for

dyne

inre

crui

tmen

tor

kine

sin

inhi

bitio

nan

dm

icro

tubu

lebu

ndlin

g

(62,

107)

S.en

teric

a(s

erov

arTy

phim

uriu

m)

Sse

JT3

SS

2pr

otei

nre

quire

dfo

rin

trac

ellu

lar

grow

th;

deac

ylas

e,ph

osph

olip

ase

and

acyl

tran

sfer

ase

activ

ities

alte

rS

CV

lipid

mem

bran

e;in

tera

cts

with

SifA

and

Pip

B2

inre

gula

ting

SC

Vm

embr

ane

tabu

latio

n

Rho

A-G

TP(R

ab7

via

SifA

and

Pip

B2?

)D

eacy

lase

activ

itym

edia

tes

recr

uitm

ent

ofR

hoA

and

faci

litat

esm

embr

ane

tubu

latio

n(S

ifs)m

aym

odul

ate

GTP

ase

func

tion.

(62,

100,

108)

Mod

ulat

ion

ofR

abfu

nctio

nth

roug

hpo

st-tr

ansl

atio

nalm

odifi

catio

nC

.pne

umon

iae

CP

n003

4;C

Pn0

367;

CP

n036

9;C

Pn0

370;

CP

n052

4

Unc

hara

cter

ized

prot

eins

cont

aini

ngM

acro

dom

ain

whi

chm

ayse

rve

inbi

ndin

gA

DP

-rib

osyl

ated

prot

eins

;Mac

rodo

mai

nm

aybe

regu

late

dth

roug

hm

ono-

AD

P-r

ibos

ylat

ion

Unk

now

n,A

DP

-rib

osyl

ated

GTP

ases

?P

aral

lels

toL.

mon

ocyt

ogen

esA

DP

-rib

osyl

atin

gen

zym

esp

ecifi

cfo

rR

ab5?

(13,

109,

110)

1578 Traffic 2012; 13: 1565–1588


Tab

le3:

Con

tinue

d

Mic

robe

Viru

lenc

epr

otei

n(e

ffec

tor)

Act

ivity

Hos

tce

llpa

rtne

rC

onse

quen

ceof

inte

ract

ion

Ref

eren

ces

C.t

rach

omat

isC

T058

Unc

hara

cter

ized

prot

ein

cont

aini

ngM

acro

dom

ain

whi

chm

ayse

rve

inbi

ndin

gA

DP

-rib

osyl

ated

prot

eins

;M

acro

dom

ain

may

bein

activ

ated

thro

ugh

mon

o-A

DP

-rib

osyl

atio

n

Unk

now

n,A

DP

-rib

osyl

ated

GTP

ases

?

Par

alle

lsto

L.m

onoc

ytog

enes

cont

ains

anA

DP

-rib

osyl

atin

gen

zym

esp

ecifi

cfo

rR

ab5?

(13,

109,

110)

L.pn

eum

ophi

laA

nkX

Pho

spho

chol

ine

tran

sfer

ase,

Type

IVD

ot/Ic

mpr

otei

nR

ab1,

Rab

35P

hosp

hoch

olin

atio

nof

Rab

1an

dR

ab35

,inh

ibiti

onof

GD

Ibin

ding

(4,4

2)

L.pn

eum

ophi

liaD

rrA

/Sid

MB

ifunc

tiona

lpro

tein

with

aden

ylyl

tran

sfer

ase

dom

ain

Rab

1A

ctiv

ates

Rab

1on

PC

V(6

,85)

L.pn

eum

ophi

laLe

pBG

AP

activ

ity,T

ype

IVD

ot/Ic

mpr

otei

nR

ab1

Inac

tivat

ion

ofR

ab1

(3,4

2)

L.pn

eum

ophi

laLe

m3/

lpg0

696

Dep

hosp

hoch

olin

atio

n,Ty

peIV

Dot

/Icm

prot

ein

Rab

1,R

ab35

Rev

erse

sph

osph

ocho

linat

ion

(4,4

2,14

6)

L.pn

eum

ophi

laLi

dAS

uper

effe

ctor

ofR

abpr

otei

ns,

Type

IVD

ot/Ic

mpr

otei

nR

ab1,

Rab

6,R

ab8

Bin

dsm

ultip

leR

abpr

otei

nsw

ithve

ryhi

ghaf

finiti

es(8

)

L.pn

eum

ophi

laS

idD

Dea

deny

lyla

tion

activ

ity,T

ype

IVD

ot/Ic

mpr

otei

nR

ab1

Rev

erse

sad

enyl

ylat

ion

(7,9

)

L.m

onoc

ytog

enes

GA

PD

Hfr

omlis

teria

Lmo2

459

AD

P-r

ibos

ylat

ion

Rab

5A

DP

-rib

osyl

atio

nof

Rab

5bl

ocks

Rab

5aex

chan

gefa

ctor

Vps

9an

dG

DIt

here

fore

bloc

ksph

agos

ome

endo

som

efu

sion

(13)

P.ae

rugi

nosa

Exo

ST3

SS

cyto

toxi

nw

ithhi

ghho

mol

ogy

toE

xoT;

N-t

erm

inal

GA

Pdo

mai

nth

atin

activ

ates

Rho

GTP

ases

;C-t

erm

inal

AD

P-r

ibos

yltr

ansf

eras

edo

mai

nth

atm

odifi

esho

stpr

otei

nsof

actin

cyto

skel

eton

,Ras

,Ral

and

Rab

GTP

ases

Rab

5,R

ab3,

Rab

4A

DP

-rib

osyl

atio

nof

Rab

5an

dot

her

Rab

prot

eins

cont

rolli

ngep

ithel

ialj

unct

ions

?R

ab3

and

Rab

4in

vitr

osu

bstr

ates

ofE

xoS

;Co-

imm

unop

reci

pita

tes

with

Rab

5,R

ab6

and

Rab

9;re

quire

dfo

rpl

asm

am

embr

ane

nich

efo

rmat

ion

inep

ithel

ia

(10,

75,7

6)

Traffic 2012; 13: 1565–1588 1579

Stein et al.

GTP to GDP. Activation in the case of Rab5, Rab7 andRab9 may be aided by a separate guanine nucleotidedissociation inhibitor (GDI) release factor (GDF) to promoteselective membrane recruitment and GTP-binding. ActiveGTP-bound Rab proteins are then able to interact with acarefully orchestrated sequence of downstream effectorsthat remodel membrane lipids or bind motor proteinsand in turn recruit additional factors to facilitate vesiculartranslocation on the cytoskeleton, targeting and fusion(reviewed in 2, 111). Following Rab-GTP hydrolysis, acommon GDI extracts GDP-bound Rab proteins andshields the membrane anchoring prenyl groups duringcytosolic recycling. The hierarchical cooperation betweenRab GTPases is coordinated through integrated cascadesthat depend on the spatial and temporal recruitment ofGEF and GAP proteins that act on sequential Rab GTPasesin the pathway and thus ensure seamless transitions(reviewed in 2). Bacterial pathogens have devised intricatestrategies for altering various aspects of the Rab-activationand functional cycle.

Selective Rab Recruitment to EvadeLysosomal Transport

Direct binding of bacterial effectors to host Rab

GTPases co-opts function

Chlamydia trachomatis and Chlamydia pneumoniae aresignificant human pathogens causing blindness, sexu-ally transmitted disease and pneumonia (Table 2) (64).Chlamydia species enter host mucosal epithelial cells ormacrophages and establish residence in a compartmenttermed the ‘inclusion’ that is required for Chlamydiaegrowth and differentiation (Figure 2). Formation of chlamy-dial inclusions and avoidance of transport to lysosomesboth require protein synthesis by Chlamydiae, suggest-ing that bacterial effector proteins facilitate remodelingof the chlamydial inclusion (112). Numerous Rab proteins(Rab1, Rab4, Rab6, Rab10 and Rab11) localize to chlamy-dial inclusions with some displaying species specificity(16). Direct binding of C. trachomatis inclusion membraneprotein (Inc), CT229 specifically to Rab4 was shown byyeast 2-hybrid and immunofluorescence studies (82). Asecond Inc protein, Cpn0585, sequentially binds to Rab1,Rab10 and Rab11 and thereby modulates transport (32).Structural studies suggest C. trachomatis CT147 mayact as a mimic of the Rab5 effector early endosomalantigen (EEA1) (81). CT147 likely can tether endosomestogether but precludes endosome fusion because it lacksthe structural equivalent of a Rab5-binding domain presentin EEA1, thus blocking normal protein recruitment andendosome fusion. As illustrated by these examples, directbinding of bacterial inclusion proteins to host Rab proteinsand downstream effectors is emerging as an importantmechanism for remodeling of bacterial inclusion mem-branes through regulated vesicle recruitment and fusionand is a fruitful area for further investigation (32,80–82)(Table 3).

Similarly, L. pneumophila, the causative agent of a poten-tially lethal pneumonia called Legionnaires’ disease thatafflicts primarily the elderly and the immunocompromised,translocates multiple proteins into host cells using itsDot/Icm type IV secretion apparatus. Currently, morethan 250 secreted proteins are known (113,114). Sev-eral of these proteins interact with Rab1 (Table 3), whichusually regulates vesicular trafficking between the ERand the Golgi apparatus. One such Rab1 interacting pro-tein, DrrA (defect in Rab1 recruitment protein A, alsocalled SidM) was originally described as a bifunctionalprotein containing GDI-displacement and GEF activitiesfor Rab1 (6,115). Further research showed that theobserved GDI-displacement activity was actually a resultof the GEF activity of DrrA and that no active displace-ment occurs (Figure 3A) (85). Another protein secretedby L. pneumophila, the protein LepB, acts as a Rab1GAP (3). Besides Rab1, the small GTPases Arf1, Rab7,Rab8 and Rab14 have been shown to be localized atthe Legionella-containing vacuole (LCV) during infection,although mechanisms of recruitment and functions ofthese proteins at the LCV require further study (26,116).Notably, L. pneumophila also secretes a protein calledLidA that is considered a ‘supereffector’ of Rab proteinsbased on its low picomolar affinity and extended pro-tein interaction interface (8). LidA binds Rab8a and Rab6,which are important in late exocytic events from theGolgi (8). LidA binds multiple Rab proteins in the GDP-and GTP-bound states with very high affinities and maythereby provide spatiotemporal regulation during infection.Thus, L. pneumophila produces a whole set of proteinsfor the subversion of Rab1-function and potentially otherGTPases during infection in order to support intravacuolargrowth.

Salmonella enterica are Gram-negative bacteria that aremost often associated with food-borne illnesses resultingin diarrhea, fever and abdominal cramps. S. enterica areinternalized into gastric epithelial cells or macrophages ina membrane-bound compartment termed the Salmonella-containing vacuole (SCV). S. enterica species utilizeT3SS secretion systems to deliver bacterial effectorproteins into host cells. The recruitment of Rab5 tothe SCV is associated with the generation of PI(3)P bya multifunctional T3SS1 protein, SopB. A phosphatasedomain in SopB reduces PI(4,5)P2 levels on the SCV(87,117). In addition, SopB has a Cdc42-binding domainthat acts as a Cdc42 guanine nucleotide dissociationinhibitor (GDI) (88). Thus, the activities of SopB directlyregulate actin polymerization and indirectly result in therecruitment of selective Rab5 effector proteins to the SCV,including hVPS34 phosphatidyl inositol 3-kinase. SopBmutant bacteria increasingly recruit and retain Rab8b,Rab13, Rab23 or Rab35 in contrast to wild-type S. entericasuggesting that modulation of the phosphoinositidesPI(4,5)P2 and PI(3)P on the SCV is also important toprohibit recruitment of specific Rab proteins and directthe maturation of the SCV (86).

1580 Traffic 2012; 13: 1565–1588


Rab

Rab

effector

ATP

DrrA (ATase)

LidA?

GDP

GDI

GDP

GDI

vesicle

GAPs (e.g. LepB),Mical-3

cytoplasm

DrrA (GEF)

SidD

GTPAMP

GTP

GTP

Legionella containing vacuole / other compartment

Rab

PC

CDP-choline

Lem3 (lpg0696)

LidA?

GDP

GDI

GDP

GDP

AnkX

phosphocholination adenylylation

connecdenn 1

Figure 3: Schematic of bacterial effector proteins secreted by Legionella pneumophila to subvert Rab function. The figureillustrates the modification of Rab proteins by (left) phosphocholine mediated by AnkX and (right) adenosine monophosphate mediatedby DrrA. Phosphocholination strongly inhibits GEF catalysis by connecdenn 1 while adenylylation strongly impairs binding of the humaneffector protein Mical-3 and inactivation by GAPs (e.g. LepB). In contrast, the ‘supereffector’ LidA can bind Rab1 also in the modifiedstates and might act as a tethering factor. Interaction with GDI can only occur after removal of the modifications, indicating a possiblerole of the modifications in recruitment and entrapment of Rab proteins at the surface of endogenous membranes. All proteins encodedby Legionella pneumophila for subversion of Rab function are indicated in red letters.

These examples reveal interactions of bacterial secretedeffector proteins with specific host cell Rab proteins onvacuolar membranes resulting in the modulation of PCVtransport early after internalization. The direct binding ofChlamydiae Inc proteins to Rab proteins, the enzymaticmodulation of Rab GTPases and accessory factors byLegionella, and Salmonella modulation of membranephosphoinositides illustrate the selective recruitment andactivation of Rabs and their effectors, which play animportant role in early alterations necessary for PCVmaturation.

Bacterial effector proteins post-translationally modify

Rab protein structure

In addition to the direct binding and recruitment of Rabproteins to the PCV membrane, pathogens have acquiredthe ability to control the activity of Rab proteins bydirectly altering Rab structure through post-translationalmodifications. L. monocytogenes, often a food-bornepathogen, infects macrophages and resides in the hostphagosome for only a brief time prior to escapinginto the host cell cytosol (13). Inhibition of Rab5aGEF activity was demonstrated to result in Listeriaintracellular survival (118) and this inhibition is dependenton Listeria glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) protein (p40, Lmo2459). Lmo2459 binds andrecruits Rab5 and ADP-ribosylates Rab5a, inhibitingexchange of GDP for GTP by inhibiting the interactionof Rab5a with Vps9 (13). Similarly, P. aeruginosa ExoSis a multidomain protein with an ADP-ribosyl transferasedomain that modulates multiple Rab GTPases (Table 3)(75). Thus, ADP-ribosylation may be a common bacterialstrategy for modulating trafficking and Rab GTPases thatrequires further study (e.g. in Chlamydiae, Table 3).

Reversible adenylylation (also called AMPylation) isanother post-translational modification and is used byL. pneumophila to modulate Rab GTPases. The Legionellaeffector DrrA, besides having GEF activity, possessesan N-terminal adenylyltransferase activity toward Rab1bTyr77. Adenylylation of Rab1 prolongs the GTP-boundactive state by preventing inactivation of Rab1 byGAPs including L. pneumophila LepB protein (Figure 3B)(5,7,9,119). The binding of the human effector proteinMical-3 is also impaired by Rab1b adenylylation, whereasthe Legionella effector protein LidA still binds Rab1-AMPwith high affinity (5,8). AnkX, another protein secreted byL. pneumophila catalyzes the covalent attachment of aphosphocholine moiety to the adenylylation-site-adjacentSer76 in Rab1b and Rab35. Although phosphocholination

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had only moderate effects on Rab1b binding to effectorproteins Mical-3 and LidA and interactions with GAPsand GEFs, phosphocholination of Rab35 strongly impairedconnecdenn 1 GEF activity (4,42). Interestingly, bothadenylylation and phosphocholination drastically reducethe affinity for GDI (120). This finding indicates that L.pneumophila encode two enzymatic activities that causethe recruitment and entrapment of Rab proteins on thesurface of intracellular membranes by inhibiting bindingand extraction of the Rab proteins from membranes byGDI. While DrrA is localized to the LCV, the localization ofAnkX during infection is not clear yet, although cell cultureexperiments indicate a function in vesicular traffickingto or from the Golgi apparatus (121). Adenylylation andphosphocholination of Rab1 can be reversed by theLegionella proteins SidD and Lem3 (lpg0696), respectively(7,9,42,122,146). Thus, L. pneumophila subverts Rabfunction in a temporally and spatially controlled manner bysecreting both Rab-interacting and -modifying proteins.

The emerging data identify pathogen catalyzed post-translational modifications as a pivotal mechanismwhereby pathogens modulate and usurp Rab GTPases.Taking a hint from studies on L. pneumophila, it isinteresting to consider that M. tuberculosis encodes over60 adenylylating enzymes that are considered key drugtargets (123). While half of the enzyme activities arethought to function in fatty acid modification, analyses ofRab GTPase adenylylation upon M. tuberculosis infectionhave not yet been undertaken.

Pathogens Usurp Rab GTPases to DirectTrafficking to Specific Intracellular Locales

Salmonella enterica and Chlamydiae actively manipulatecytoskeletal, motor-driven transport of their PCV tolocalize to specific regions of infected cells, whileMycobacteria actively block motor recruitment. Pathogen-directed transport is important in both evading degradationand obtaining nutrients for survival (124,125). Microtubule-dependent transport of endocytic compartments to theperi-nuclear Golgi and MTOC regions is dependenton Rab GTPase-effector complexes that in turn binddynein/dynactin to facilitate transport (126). In contrast,transport to the cell periphery depends on Rab GTPase-mediated regulation of kinesin and myosin motors(27,126,127).

Salmonella enterica utilizes dynein- and kinesin-dependenttransport for its peri-nuclear localization close to the MTOCearly in infection and to remodel its niche through mem-brane tubulation late in infection (reviewed in 62, 95).The early SNX3-dependent recruitment of Rab7 and Rab7-interacting lysosomal protein (RILP) to the SCV enablesdynein/dynactin motor binding and peri-nuclear transport(22,128). Later in infection, the activity of the T3SS2 pro-tein SifA in conjunction with multiple bacterial (PipB2,SseF, SseG, SseJ and SopD2) and host proteins facilitates

the extension of long tubules termed Salmonella-induced-filaments (Sifs) that are important for bacterial growthand ultimately cell-to-cell spread (99,104,105,107). SifAwhen present on the SCV binds Rab7 and interfereswith RILP/dynein/dynactin interactions, while promotingkinesin-dependent Sif membrane tubule extension onmicrotubules toward the cell periphery. SifA interac-tion with host protein SifA-and-kinesin-interacting-protein(SKIP/PLEKHM2) antagonizes SKIP-Rab9-GTP binding,which normally directs recycling between late endosomesand the trans-Golgi (30). The SifA-SKIP complex insteadactivates kinesin-1 in concert with the bacterial proteinPipB2 that functions upstream to recruit autoinhibitedkinesin to the SCV (30,95,97). Multiple kinesin motors(Kif5B, Kif11 and Kif24), as well as interactions with addi-tional host proteins [Arf family GTPase Arl8b and secretoryvesicle membrane proteins, (SCAMP) 2 and 3] are crucialfor both late endosomal dynamics and tubulation duringS. enterica infection (101,103,129,130). There is a closecooperation between Arf and Rab family GTPases in theintegration of membrane remodeling, e.g. in the caseof Rab7 and Arl8b through the shared HOPS effector(102). Therefore, further elucidation of how S. entericamodulates its niche by co-opting GTPase function is ofsignificant interest.

The early localization of the chlamydial inclusions to a peri-Golgi location near the MTOC is also regulated by dynein-dependent transport and requires active Src kinases(91,125). Recently, Chlamydiae inclusion membraneproteins (Inc) have been identified to bind both activeSrc family kinases and centrosome components (91,109).We speculate that recruitment of Src kinase togetherwith Rab11 serves to regulate the cytoskeletal motility ofchlamydial inclusions (20). Src kinase is normally activatedon Rab11 and RhoB containing endosomes where itfacilitates actin nucleation, and offers the potential forswitching between microtubule and actin based motilityof inclusions. Src could promote transport through SH2domain interactions as has been demonstrated forlysosome clustering (131) or through phosphorylationdependent motor recruitment as demonstrated for motilityof enveloped viruses (132,133). Alternatively, Src mayserve to increase the pool of activated Rab11, as itnormally does for Rab5 and Rab7, but which are noton inclusions (16,134). Active Rab11 would be expectedto have increased interaction with effectors such asthe Rab11 family interacting proteins (FIP2, FIP3 andFIP5), which in turn interact with dynein and kinesin-II to control transport on microtubules (127,135) andmyosin motors (MyoVb) to control actin based motility(136). Thus, the roles of Rab11 and Src in the cytoskeletaltransport of chlamydial inclusions are fruitful areas forfurther investigation and are expected to elucidatemechanisms for maintenance and maturation of thechlamydial intracellular niche.

Inhibition of Rab7-RILP interactions and reducedRab7 recruitment are suggested to prohibit the

1582 Traffic 2012; 13: 1565–1588


dynein-dependent transport of PCV to peri-nuclear lyso-somes and contribute to phagosomal maturation arrestas exemplified in mycobacterial infections (12,25). Gram-positive Mycobacteria cause severe pulmonary and intesti-nal infections in humans that are highly contagious andoften lethal. M. tuberculosis infects alveolar macrophages.The related Mycobacterium bovis causes pulmonary infec-tions in cattle, but may be spread to humans throughaerosols and non-pasteurized milk and is a commoncause of human tuberculosis in developing countries. Non-tuberculosis causing Mycobacteria include 20 speciesthat cause human and animal disease, among themsubspecies of Mycobacterium avium are implicated inchronic human lung diseases, chronic intestinal Crohn’sdisease, and a primary cause of morbidity and mortal-ity in immune compromised patients (137). All of thesepathogenic Mycobacteria species reside and multiply inmacrophages where they inhibit phagosome-lysosomefusion for intracellular survival. The cause of the phago-somal arrest is attributed to altered Rab7 function onMycobacteria-containing phagosomes (12,17,138,139). InM. tuberculosis infections, reduced phagosomal Rab7 lev-els are suggested to account for reduced RILP-mediatedtransport, while in M. bovis infections RILP recruitmentwas blocked due to a prevalence of inactive Rab7 onMycobacteria-phagosomes. Rab22a on the M. tubercu-losis phagosome contributed to the inhibition of Rab7recruitment to Mycobacteria-phagosomes, although theeffect might be indirect (37). Rab22a normally recruits oneof several Rab5 GEFs (Rabex-5) to early endosomes andis implicated in increasing early endosome fusion (38).In this regard it is interesting that M. avium depends onearly endosome fusion for an adequate supply of ironand inhibition of phagosome maturation (14). However, ifand how iron and recruitment of Rab22a by Mycobacteriamight impact Rab5 to Rab7 conversion remains unclear.In Mycobacteria-infected cells, the expression of bacte-rial lipids with similarity to glycosylphosphatidylinositolsantagonizes the recruitment of Rab5 and Rab7 effectorsthat synthesize (hVPS34) and recognize (EEA1) PI(3)P,which augment the block in phagosome maturation (140).The cumulative data suggest that mycobacterial proteinsand phagosomal membrane lipids may reduce Rab5-Rab7conversion, specifically block recruitment of RILP to Rab7(25), or mycobacterial proteins may act as Rab7-GAPsto inactivate Rab7 (25) and inhibit interaction with RILP.Further work remains to elucidate Mycobacteria species-specific mechanisms that depend on their niches and theroles of other Rab GTPases associated with Mycobacteria-phagosomes.

Creation of an Intracellular Replicative Niche

Active remodeling of the PCV compartment throughthe modulation of Rab-dependent membrane traffickingfacilitates the formation of a replicative niche wherebacteria differentiate into their infective forms, acquirenutrients necessary for growth and actively replicate

in preparation for dissemination upon release fromhost cells. The late stage recruitment of Rab andRab effector proteins to PCV has been described andthrough expression of dominant-negative or constitutivelyactive Rab mutants or RNAi knockdown experiments,the necessity of Rab proteins derived from diverseendomembranes has been demonstrated to be crucial forpathogen growth. Here a few well-documented examples,where recruitment of host molecules is required forcreation of a replicative niche, are presented.

Productive formation of infectious Chlamydia (trachomatisand pneumoniae) requires a replicative niche wherebacterial differentiation and maturation take place anddepends on Rab6 and Rab11, GTPases involved in Golgitransport and endosomal recycling (20). Cells depleted ofRab6 and Rab11 by RNAi failed to allow C. trachomatismaturation, although the additional fragmentation ofthe Golgi, through loss of p115, rescued Chlamydiaematuration (20). Thus, in the absence of the redirectedtransport of newly synthesized host proteins and lipids byRab6 and Rab11 to chlamydial inclusions, the completedisruption of the exocytic pathway and an intracellularaccumulation of vesicular biosynthetic cargoes were ableto rescue the maturation of the chlamydial inclusion.Rab14, which functions in endosomal recycling, alsoplays a role in chlamydial inclusion formation by providingendogenously synthesized sphingolipids to the growinginclusion body (34). A role for Rab14 in preventinglysosomal transport, as shown for Mycobacteria, has notyet been demonstrated for Chlamydia. Recruitment ofboth Rab6 and its effector Bicaudal D1 (BicD1) requiredchlamydial protein synthesis and BicD1 recruitment wasindependent of Rab6, suggesting that BicD1 may facilitatethe recruitment of Rab6 to the inclusion in a mannerdistinct from normal Rab6-GTP mediated binding of BicD1to the Golgi (19). The data demonstrate that recruitmentof at least three Rab proteins to the inclusion membranefacilitates the growth and differentiation of Chlamydiaeby directing the transport and fusion of nutrient-richendosomal and Golgi derived vesicles to the inclusion.

Several Rab proteins also modulate Mycobacteria-phagosome maturation and the generation of a stableniche for bacterial growth. Active mechanisms forreducing recruitment of the endosomal Rab7 GTPase areimportant for evading lysosomal transport as discussedabove. Rab14 also plays a role in Mycobacteria-phagosome arrest (35), conceivably by modulatingsphingolipid transport as occurs in chlamydial inclusionformation (34). Mycobacteria express multiple lipidhydrolases (phospholipases and ceramidases) that canefficiently catabolize sphingolipids uniquely present inthe lung to fatty acids as an energy source (141).Ceramide generated from sphingolipid degradation mayalso impact host cell survival signaling (142). Thus,Rab14 may have a multi-functional role in precludingphagolysosomal fusion, enabling signaling, and providingnutrients and a favorable host cell environment for

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bacterial growth in the replicative niche. Mycobacteria-phagosomes exhibit enhanced early endosome fusionand altered recycling, which may coordinately ensure asupply of endocytosed nutrients important for bacterialsurvival. Recent studies show reduced Rab10 associationwith Mycobacteria-phagosomes, which together withenhanced early endosome fusion promoted by bacteriallipids is speculated to be important to the replicative niche(31,140). The observed mycobacterial exclusion of Rab10may be akin to Rab10 knockdown, which slows transferrinand glycosylphosphatidylinositol (GPI) anchored proteinremoval and recycling from phagosomes (31), therebyslowing recycling and ensuring access to transferrin andiron for Mycobacteria (14). GPI-anchored proteins maydeliver key nutrients such as folate or provide precursorsfor the biosynthesis of bacterial lipids [phosphatidylinositolmannoside (PIM), and its derivatives lipomannan (LM)and lipoarabinomannan (LAM)] that promote phagosomalarrest. The known function of Rab22a in modulatingcommunication between the biosynthetic and earlyendocytic pathways, may also make its recruitment tothe phagosome pivotal to bacterial growth though detailsremain to be established (36,37). These data suggest thatRab proteins on both the endocytic and Golgi pathwaysplay a role in Mycobacteria-phagosome maturation andgeneration of the replicative niche.

Helicobacter pylori is a Gram-negative bacterium thatcolonizes epithelial cells of the stomach and intestineresulting in massive inflammatory responses that causegastric ulcers and frequently, gastric cancer. Althoughpredominantly extracellular, H. pylori invade gastricepithelial cells and intracellular H. pylori persist in hostcells for long periods of time in large intracellular vacuolesformed with the aid of a bacterially encoded vacuolatingtoxin, VacA (143). Rab7 localization on VacA inducedvacuoles promotes homotypic fusion with Rab7-positivelate endosomes and recruitment of RILP, promoting peri-nuclear localization and ensuring the continuous deliveryof vesicular membranes required for VacA-dependentvacuolization (23,24,93). Selected late endosomal andlysosomal proteins such as Rab7, vacuolar ATPase,LAMP1 and Lgp110 localize to the H. pylori-vacuole,while Rab9, CI-M6PR and lysosomal enzymes such ascathepsin D are absent (23,93,144). Other Rab proteinshave also been implicated in the pathogenesis of H.pylori. Proteomic analyses demonstrated an increase ofRab37 expression in H. pylori-infected cells comparedto uninfected controls (43). Expression of H. pyloriCagA, a Type IV secretion system effector and targetof Src phosphorylation was observed to decreasethe association of Rab11-FIP with detergent-resistantmembranes and host membranes and may decreasepathogen recycling (33).

Coxiella burnetii is a Gram-negative obligate intracellularparasite that is the causative agent of Q-fever. Infectedindividuals most often are infected by inhalation ofaerosolized bacteria and display high fevers, headaches

and general malaise that may progress to pneumonia.Q-fever has a very low mortality rate (1–2%) foracute illness, but ∼65% of individuals who developchronic Q-fever succumb to the disease (67). Uponentry into host cells, C. burnetii initially resides in atight parasitophorous vacuole that over hours to daysmatures into a compartment resembling a lysosome.The parasitophorous vacuole initially acquires markersof early and late endosomes including Rab5 andRab7 (15,21). Data based on siRNA treatment andoverexpression of dominant active and inactive GTPasesindicate that both Rab5 and Rab7 play roles in thematuration of the C. burnetii parasitophorous vacuoleto a replicative parasitophorous vacuole. Between 6and 12 h after infection, the parasitophorous vacuoleacquires markers of autophagy including LC3 and Rab24(15,145), suggesting that the host autophagic pathway isnecessary to create the Coxiella parasitophorous vacuole.Defects in autophagy delay the maturation of Coxiellaparasitophorous vacuoles, however, further study will benecessary to understand the precise role of Rab24 andautophagy in early Coxiella infection. At 2 days post-infection, the parasitophorous vacuole is greatly enlarged(now referred to as a spacious parasitophorous vacuole)and has acquired lipid raft proteins (Flotillin1 and Flotillin2)as well as numerous lysosomal markers including 5′-nucleotidase, LAMP1 and LAMP2, and an acidic pHof ∼5 (83). Rab7, Rab24 and LC3 remain associatedat late time points (145). C. burnetii is currently theonly identified intracellular bacteria requiring a low-pHlysosome-like intracellular niche for growth, differentiationand maturation.

Pseudomonas aeruginosa is an important pathogen incystic fibrosis patients that creates an intracellular nicheat the cell surface on respiratory epithelia throughphosphatidylinositol 3-kinase recruitment and plasmamembrane remodeling (75,76). The bacterial ExoS proteinis pivotal in niche formation (75). ExoS is T3SScytotoxin with an N-terminal GAP domain that inactivatesRho GTPases and a C-terminal ADP-ribosyl transferasethat modifies Rab5 and thereby prevents bacterialinternalization while inducing plasma membrane blebbingto create a replicative niche. Although Rab3 and Rab4 arein vitro substrates of ExoS, their roles in niche formationare untested (10).

Summary

Rab GTPases, considered the master regulators ofmembrane trafficking, are important targets of bacterialpathogens. Soon after internalization, bacterial pathogensusurp one or more Rab GTPases to evade degradationand modulate intracellular localization. Later in infection,bacteria modulate Rab GTPase functions to obtainrequisite nutrients and create an environment that isconducive to intracellular bacterial survival and growth.Intracellular localization is important for evading host

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degradation and immune detection, as well as forpositioning the replicative niche in a subcellular locationthat facilitates bacterial access to host proteins, lipidsand inorganic molecules that are essential for bacterialgrowth and persistence. In most cases, bacterial effectorproteins, introduced by specialized secretion systems,modulate host Rab protein functions by acting as specificreceptors or by blocking normal Rab protein or Rabeffector function. Functional modulation of Rab GTPasesoccurs through changes in membrane lipid composition,Rab effector mimicry, post-translational modification ofRab GTPases, kinase signaling and alteration of Rabactivation cycles. The Rab GTPases required for pathogeninternalization, transport and growth are rapidly beingcataloged. Yet as highlighted here, many gaps remainin our knowledge of the precise mechanisms wherebyspecific Rab GTPases contribute to intracellular bacterialsurvival and growth. Although Rab GTPases and Rab-regulated pathways are important targets of disease, theyare as yet underexplored therapeutic targets. Thus, furtherstudy of Rab GTPases in bacterial infection is expectedto reveal insights into normal function as well as providenew ‘druggable’ targets.

Acknowledgments

MPS was supported by NIH SCORE 5SC2GM086312, MPM wassupported by IMPRS-CB (Dortmund, Germany) and AWN by NSFMCB0956027, NIDDK R01DK050141 and NINDS R21NS066435. Wegratefully acknowledge the support of Dr. Roger Goody (DirectorDepartment of Physical Biochemistry, Max Planck Institute of MolecularPhysiology, Dortmund, Germany). We apologize for omissions in thecitation of original work due to page and citation limitations.

We gratefully acknowledge Dr. Olivia Steele-Mortimer (NIAID, RockyMountain Laboratories) for the image of S. enterica induced Sifs and Dr.Robert A. Heinzen (Pathogenesis Section, NIAID) for the pseudocoloredscanning electron micrograph of C. burnetii contained in a lysosome-like parasitophorous vacuole. Images of L. pneumophila phagocytosis,structure of Rab1-AMP and cell graphic were prepared by the authors.

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1588 Traffic 2012; 13: 1565–1588

bacterial pathogens commandeer rab gtpases to establish intracellular niches

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