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Cytoplasmic access by intracellularbacterial pathogens

Jennifer Fredlund and Jost EnningaUnite

‘Dynamique

des

interactions

ho

te–pathoge

ne’,

Institut

Pasteur,

25

rue

du

Dr.

Roux,

75724

Paris,

France

Entry into host cells is a strategy widely used by bacterial

pathogens, after which they either remain within mem-

brane-bound compartments or rupture the endocytic

vacuole to reach the cytoplasm. During recent years,

cytoplasmic

access

has

been

documented

for

an

increas-

ing number of pathogens. Here we review how classical

cytoplasmic bacterial pathogens rupture their endocytic

vacuoles

as

well

as

the

mechanisms

used

to

accomplish

this

task

by

bacterial

species

for

which

host

cytoplasmiclocalization has only recently been identified. We also

discuss the consequences for pathogenesis resulting

from this change in intracellular localization, with a

particular focus on the role of the host. What emerges

is that cytoplasmic access plays an important role in the

pathophysiology

of

an

increasing

number

of

intracellular

bacterial pathogens.

Intracellular localization of bacterial pathogens

Bacterial pathogens reside in two major niches during host

infection:

they

either

remain

extracellular

or

are

interna-

lized

via

active

or

passive

pathways

[1].

When

internalized,

they are surrounded by host cell membranes. Subse-

quently, pathogens can remain vacuole-bound throughthe

entire

course

of

infection

by

blocking

delivery

of

the

lysosome or by modulating the physiological conditions of

the

vacuolar

niche.

An

alternative

strategy

is

rupture

of

the

vacuole,

or

phagolysosome,

giving

the

bacterium

access

to the host cell cytoplasm. The decision to remain within an

endomembrane

compartment

or

to

escape

into

the

cyto-

plasm has important consequences for both the invader

and

the

infected

cell.

The

physiological

environment

of

each niche differs drastically with regard to nutritional

access,

differential

pathogen

recognition,

and

spread

to

neighboring

cells.

Consequently,

induced

innate

and

adap-

tive immune responses occur in different ways depending

on

the

bacterial

localization.Until

recently,

intracellular

pathogens

were

neatly

separated into two groups, either cytoplasmic or vacuolar.

This

rigid

definition

has

been

challenged

by

a

series

of

scientific

reports

(Figure 1) suggesting

that

access

to

the

host

cytoplasm

is

more

frequent

than

previously

anticipated.

Bacterial pathogens that reach the host cytoplasm: the

classics

For

a

number

of

bacteria,

themechanismsof

cytosolic

access

have been studied in detail.We discuss known bacterial and

host

factors

important

for

this process

and present

a

brief

overview

of

howthey function

during vacuolar rupture.

The

proteins involved that are discussed in this and in the

following

section

are highlighted in Table

1.

Shigella triggers its entry to rapidly escape from the

intracellular

vacuole

Host

cellular

entry

into

both

epithelial

cells

and

macro-

phages by Shigella species has been studied for decades,

mostly

using

Shigella

flexneri

[2,3].

S. flexneri

invades

cells

via a trigger mechanism that involves injecting approxi-

mately 25

effector

proteins

directly

into

the

host

cytoplasm

through the mxi-spa type III secretion system (T3SS).

Cytoplasmic localization of Shigella within different cell

types

was

first

observed

by

transmission

electron

micro-

scopy (Box 1, Figure 1 A) [2]. Furthermore,

Shigella induces

‘comet

tails’

composed

of

host

actin

via

the

bacterial

factor

IcsA,

and

these

have

been

commonly

used

as

markers

for

bacterial cytoplasmic localization.

Initially

it

was

proposed

that

bacterial

effectors

play

a

major

role

in cytoplasmic

access

and

vacuolar

rupture

by

Shigella

[4]. In

particular,

the

T3SS

translocator

proteins

IpaB

and

IpaC

have

been

shown

to

destabilize

eukaryotic

cell membranes using red blood cell lysis assays at very

high concentrations

of

the

bacterial

proteins

[5].

Recent in

vitro studies on IpaB have suggested that its assembly into

multiprotein

complexes

[6,7]

allows

an

influx

of

potassium

ions

through

endolysosomal

membranes,

suggesting

pore

formation

and

an

involvement

in

vacuolar

rupture

[6].Underlining

the

possibility

that

other

factors

may

be

involved

in

the

vacuolar

rupture

process,

the

T3SS

effector

IpaH7.8 has also been associated with cytoplasmic access;

however,

its

function

is

not

yet

established

[8].

Further-

more, a non-characterized region of the Shigella virulence

plasmid

allowed

uncoupling

of

bacterial

entry

and

vacuolar

rupture,

hinting

at

potential

involvement

of

additional

factors in the rupture process [9]. On vacuolar rupture,

a

pool

of

smaller

vesicles

formed

from

the

vacuolar

mem-

brane remnants suggests that the autophagy machinery is

recruited

to

the

site

of

ruptured

membranes

[10,11]. Taken

together,

current

data

suggest

that

the

rupture

process

represents a signaling platform with important roles for

Review

0966-842X/$ – see front matter

2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.01.003

Corresponding author: Enninga, J. ( [email protected]).

Keywords: intracellular bacteria; vacuolar rupture; membrane trafficking; host cell

death.

128 Trends in Microbiology, March 2014, Vol. 22, No. 3

http://dx.doi.org/10.1016/j.tim.2014.01.003

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both bacterial and host proteins, and with effectors of the

Shigella

T3SS

orchestrating

the

process.

Listeria: zippering into host cells and forming pores in

the vacuole

Invasion

by

the

human

pathogen

Listeria

monocytogenes

is

achieved

via

bacterial

surface

proteins

such

as

internalin

A

(InlA) and InlB that respectively bind to human E-cad-

herin

and

have

affinity

for

host

glycosaminoglycans,

the

receptor

for

the

human

globular

part

of

the

complement

component gC1q-1 (gC1q-R), and the Met receptor (the

main

receptor

for

InlB)

[12]. This

interaction

triggers

a

complex signaling cascade involving a battery of host

factors, including kinases, GTPases, and even clathrin,

leading to vacuolar formation that has been described in

detail

[12].

The secreted

protein

listeriolysin-O

(LLO)

is

the

key

bacterial factor leading to vacuolar rupture15 min after

Listeria

internalization

[12]. It

is

a

member

of

the

choles-

terol-dependent

cytolysin

family,

which

also

includes

streptolysin-O and perfringolysin-O. Even though a link

has

been

made

between

LLO

activity

and

vacuolar

pH

that

results

in

membrane

permeation,

it

is

still

not

clear

whether this effect is direct or requires additional factors

[13].

This

is

because

the

bacterial

phospholipases

PI-PLC

and

PC-PLC

are

also

necessary

to

achieve

efficient

cyto-

plasmic

access

[14]. One

explanation

for

this

could

be

that

initial membrane damage via LLO allows better cytoplas-

mic

access

for

the

mature

phospholipases.

Similar to Shigella infection, it is clear that Listeria

proteins

function

in

concert

with

host

factors

to

grant

the

bacterium

cytoplasmic

access.

For

example,

host

gamma-interferon-inducible

lysosomalthiol

reductase

(GILT)

che-

mically reduces LLO, thereby increasing LLO activity [15],

and

the

cystic

fibrosis

transmembrane

conductance

regu-

lator

(CFTCR)

increases

intravacuolar

chloride

concentra-

tions to potentiate LLO activity [16]. In addition,

subversion

of

the Listeria-containing vacuole

from

the

endosomal

pathway

requires

Ca2+ flux

through

the

LLO

pores [13]. These fluxes inhibit the normal vacuolar

maturation

that

would

eventually

result

in lysosomal

fusion and bacterial destruction. Finally, an inducible

increase in membrane resistance to pressure, termed reni-

tence, has been identified. This mechanism may limit

vacuolar

membrane

damage

and

appears

to

involve

the

host

heat

shock

protein

HSP70

[17]. Vacuolar

escape

within activated macrophages is reduced by the production

of reactive

oxygen

and

nitrogen

intermediates

(ROIs

and

RNIs)

[18]; however,

their

mode

of

action

in

this

process

needs to be further studied.

Rickettsia: concerted action of hemolysins and

phospholipases

Rickettsiales

represent

an

order

of

important

obligate

human

intracellular

pathogens

targeting

mainly

endothe-

lial

cells

and

macrophages

[19]. Owing

to

the

complex

handling requirements for this pathogen, mostly genomic

comparison

has

been

used

to

delineate

its

infection

Shigella WT

15 min

5 3 5 n m

4 5 0 n m

60 min

0,50 0

20

40

60

80

100

20

40

60

80

1,5

15 min

2,5

BS176 Afal

Key:

M90T Afal

Rao 450 nm/535 nm

N u m b e r o f c e l l s

N u m b e r o f c e l l s

3,5

4,5 0,5

1,5

2,5

Rao 450 nm/535 nm

3,5

4,5

(B)(A) (C)

(D)

60 minBS176 Afal

Key:

M90T Afal

TRENDS in Microbiology

Figure 1.

Measuring the cytoplasmic access by bacterial pathogens. (A) Transmissionelectronmicroscopy hasbeenextensively usedas evidenceof cytoplasmic access by

bacteria, for example forShigella (scale bar 500nm; reprintedwith permission from [3]). (B) The absence of markers for endosomal or lysosomal compartments hints at

cytoplasmic localization. Here, dendritic cells (blue nuclei) were infectedwithMycobacterium tuberculosis (green) for 4 h (upper panel) or 96h (lower panel). At early time

points, the bacteria colocalize with lysosomal associated protein 1 (LAMP1, red); later, they spread throughout the cytoplasm [38].

(C) Galectin-3 (green) flags damaged

vacuolar membrane structures after their rupture by Shigella (red) [83].

(D) A fluorescence resonance energy transfer (FRET)-based approach measures access to the

cytoplasm in HeLa cells infected with wild type (WT) Shigella .

The cephalosporin-derived FRET probe is cleaved by b-lactamase on the surface of the invading bacteria,

resulting in a signal switch from green to blue (left panel). The fluorescent ratio (450 nm/535 nm) can be plotted against the number of infected cells to highlight vacuolar

rupture after 60min of infection. Shigella BS176 AfaI cannot enter host cells and M90T AfaI is the invasive strain [84].

Review Trends in Microbiology March

2014,

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No.

3

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general,

less

is

known

about

the

specific

escape

mechan-

isms

than

for

those

in

described

above.

Nevertheless,

the

medical relevance of cytoplasmic access makes this a fast-

moving

field

of

research.

Intracellular

trafficking

of

SalmonellaSimilar

to Shigella, Salmonella enterica

serovar

Typhi-

murium ( S. Typhimurium) can invade epithelial cells via

a trigger

mechanism,

although

other

modes

of

entry

can

also

be

employed.

Unlike Shigella, Salmonella

classically

resides

inside

a

modified

vacuole

called

the Salmonella-

containing

vacuole

(SCV)

[1].

To

survive

in the

SCV, Sal-

monella must modify its progression through the normal

host

endocytic

pathway

(Box 2). SifA

is

important

for

maintenance of the SCV, because sifA mutants escape

from the vacuole after a few hours but do not replicate

in

the

macrophage

cytosol

[27]. SifA

also

controls

position-

ing of the SCV within infected cells, mediated by its

interaction

with

the

mammalian

protein

SKIP,

which

binds

the

molecular

motor

kinesin

[28]. SifA

may

also

have

guanine nucleotide exchange factor (GEF) activity that is

important

for

membrane

tubule

formation

[29].

Is an intravacuolar lifestyle the only one that allows

Salmonella

replication?

It

has

long

been

understood

that

a

small

proportion

of

infecting Salmonella

escape

from

the

SCV, with some of these bacteria being coated in ubiqui-

tinated proteins

or

associated

with

galectin-8

and

subject

to autophagy [30,31]. Knodler et al. recently reported that

unlike

late-escaping

sifA

mutants,

a

proportion

of

S.Typhi-

murium

leave

the

SCV

early

during

infection

of

epithelial

cells and replicate very rapidly in the cytoplasm (20 min

per

generation)

[32]. Quantitative

microscopic

techniques

demonstrated that this subpopulation occurs in a similar

percentage

of

infected

HeLa

cells

for

both

the

wild

type

and

a

mutant

without

the

T3SS-2

secretion

system,

which

is

normally important for SCV maturation. In gentamycin

survival

assays,

which

kill

bacteria

not

taken

up inside

host cells, the wild type and sifA mutants show similar

numbers

of

surviving

bacteria

at

8

h

post

infection,

even

though sifA mutants are unable to replicate in SCVs [33].

Malik-Kale

et

al.

showed

that

T3SS-2

mutants,

which

should

not

express sifA,

hyper-replicate

at

similar

rates

to wild type bacteria, thereby masking the lack of SCV

replication

[34]. The

role

of

hyper-replicating

bacteria

during

infection

is

unclear,

but

the

phenotype

is

indepen-

dent of T3SS-2, suggesting that the decision to leave the

vacuole

is

upstream

of

SCV

maturation.

Mycobacterium tuberculosis membrane rupture

Many

bacterial

factors

important

for

shaping

the

intracel-

lular niche within host cells have been identified by study-

ing

M.

tuberculosis

virulence,

including

the

unique

cell

wall

and

several

proteins

[35,36]. The

traditional

paradigm

of

M. tuberculosis infection is that the bacteria take up resi-

dence

within

vacuoles

inside

professional

phagocytes

in

the

lungs,

blocking

fusion

with

lysosomes

and

delaying

Box 1. Locating intracellular microbes

Determination of whether a bacterium is endomembrane-bound or

free in the cytosol has been a major challenge, but novel techniques

are providing new insights into bacterial localization. Owing to their

high resolution, electron micrographs have traditionally provided

evidence about the intracellular localization of a number of bacterial

pathogens. This has contributed to our understanding that Shigella ,

Listeria , Rickettsia ,

and Francisella species access the

host cyto-

plasm upon invasion. More recently, fluorescent approaches inconjunction with differential permeabilization of host cells have

distinguished bacteria within endomembrane-bound compartments

from those in the cytoplasm [4]. However, some of these methods

can be difficult to interpret because they measure the absence of

markers of endomembrane structures around the internalized

bacteria [38]. For example, lysosomal-associated protein 1 (LAMP1)

has been used to infer Salmonella vacuolar localization, but the

absence of a signal may not necessarily mean cytoplasmic

localization, because LAMP1-negative but Salmonella -positive

membrane structures have recently been identified [87]. In addition,

fractionation procedures have separated cytoplasmic pathogens

from those

localized in other cellular compartments. To overcome

the problems with these methods, reporters have been developed

that monitor membrane damage or yield direct information on

bacterial access to the cytoplasm (see Figure 1C,D in main text)

[83,84]. First, fluorescent galectin-3 can be used to identify recentlygenerated membrane fragments. Alternatively, another approach

uses cells loaded with a FRET reporter that is cleaved by b-

lactamase and can therefore detect contact between bacteria

expressing this enzyme and the reporter-containing cytosol [84].

These newmethods have confirmed the cytoplasmic localization of

many bacteria classically known to reach the cytoplasm, and have

also revealed many other, previously unrecognized, bacterial

species that gain access to the cytoplasm.

Box 2. Disruption of membrane trafficking

To survive inside a vacuole, either temporarily or long term, an

intracellular bacteriummust interferewith normal host trafficking to

avoid detection and degradation. In the case of Listeria ,

its vacuole

(LCV) is altered via translocation of the bacterial factor Lmo2459,

which inhibits activation of the small GTPase Rab5a from its GDP

state into the GTP state through ADP-ribosylation [12]. It has been

suggested that this modification of the endocytic pathway prevents

recruitment of NADPH oxidase to the LCV, an enzyme that wouldlikely cause the vacuole to become toxic to the invader. Other Rab

GTPases have also been linked to steps of altered trafficking of the

LCV during infection [88]. It is thought that Francisella follows a

similar pathway to Listeria , and vacuolar membrane markers

include the GTPases Rab5 and Rab7, LAMP1, and the vacuolar

ATPpump [23]. To avoid the harsh environment of late endosomes/

lysosomes, the pathogen blocks activation of NADPH oxidase,

although this depends on the specific bacterial strain. In Shigella ,

recent studies indicate that IpaB alters exocytic membrane traffick-

ing and is involved in the dispersal of the Golgi complex during

infection, which disrupts cell-surface receptor trafficking and could

also disrupt maturation or trafficking of highly damaging lysosomal

proteases [89].

For bacteria that reside for longer periods inside the vacuole,

more extensive alteration of trafficking has been documented. For

instance, Salmonella targets multiple RabGTPases throughout thecourse of infection and SCVs are dynamically labeled, at times

containing themembranemarkers Rab5, EEA1, Rab7, andRab9, and

the lysosomalmarkers LAMP1 and vATPase. Yet they exclude other

lysosomal proteins, specifically the

highly damaging

mannose

phosphate receptor (MPR)-associated hydrolases [90]. The SifA–

SKIP complex was recently implicated in the mislocalization of

MPRs to late endosomes in both macrophages and epithelial cells

by acting as a Rab9 sink [91]. By disrupting the Syn10 retrograde

pathway, which requires Rab9, MPRs are not recycled back to the

trans-Golgi network (TGN). This allows SCVs to estab lish a

replication niche through fusion with late endosomes yet without

suffering degradation, because these compartments do not contain

hydrolases. Legionella , which also spends extended time in a

modified vacuole, specifically disrupts autophagy membranes, a

phenomenon described in detail in Box 3.


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maturation

of

the

phagolysosome.

Nevertheless,

other

mycobacteria,

including

the

fish

pathogen

M.

marinum,

do

access

the

host

cytosol,

even

forming

actin

comet

tails

[37]. The intravacuolar paradigm suggested for M. tuber-

culosis

has

recently

begun

to

shift.

Van

der

Wel et al.

were

the

first

to

convincingly

challenge

this

view

[38], although

some evidence had been seen before [39,40]. They con-

ducted

a

thorough

study

of

M.

tuberculosis

subcellularlocalization in human-derived dendritic cells (DCs) using

transmission

electron

microscopy

(TEM)

(Figure 1B). Sur-

prisingly,

over

50%

of

infected

DCs

contained

cytoplasmic

bacteria after 96 h of infection, whereas non-pathogenic

vaccine

strains

remained

in the

vacuole.

Cytoplasmic

release of M. tuberculosis was dependent on ESX-1, a type

VII secretion

system

(T7SS).

These

findings

were

independently

confirmed

in

macro-

phages using a fluorescence resonance energy transfer

(FRET)

reporter

(Box 1) to

detect

bacterial

contact

with

the

cytosol

[41]. Interestingly,

when

the

genomic

RD1

region containing ESX-1, which is known to restore viru-

lence

to

vaccine

strains,

was

added

to

the

attenuated

BCG

strain, these bacteria also contacted the host cytosol.Furthermore,

after

elimination

of

the

secretion

locus

ESX-1 and, more specifically, the C terminus of ESAT-6,

a

T7SS

substrate,

bacteria

remained

membrane-bound

[38,41].

This

agrees

with

other

evidence

that M. marinum

ESAT-6 alone forms small pores in macrophage vacuolar

membranes

[42]

and

that

markers

linked

to

membrane

damage,

such

as

galectin-3

and

ubiquitin,

colocalize

with

M. tuberculosis phagosomes [43]. In addition, ESAT-6 is

important

for

recognition

of M. tuberculosis

by the

cyto-

plasmic

autophagic

machinery

(see

below).

Together,

these

results suggest not only that vacuolar rupture is an active,

bacterially

mediated

process

but

also

that

it

is

essential

for

successful

infection

by

M.

tuberculosis

and

other

Mycobac-terium

species.

Legionella membranes

Legionella pneumophila

secretes

more

than

240

proteins

into host cells via its type IV secretion system (T4SS), Dot/

Icm,

some

of

which

direct

maturation

of

the Legionella-

containing

vacuole

(LCV)

via

prevention

of

lysosomal

fusion and recruitment of endoplasmic reticulum (ER)

membranes,

ribosomes,

and

mitochondria

(Box 3) [44].

Maintenance of the LCV is Dot/Icm-dependent, because

non-virulent Ddot/icm mutants are found in the cytosol of

human macrophages soon after infection [45]. Escape from

the

LCV

at

later

time

points

(12–18

h

post

infection)

is

also

seen for

wild

type

strains,

and

although

it

appears

to

be

a

regulated and important part of the infection cycle, there is

debate

about

what

factors

mediate

vacuolar

escape

[46].

It was

recently

shown

that

the

effector

SdhA

is

respon-

sible for maintaining the integrity of LCV membranes in

mouse

and

human

macrophages

through

antagonistic

action

to

the

effector

PlaA,

similar

to

the

interaction

between Salmonella SifA and SseJ [47]. However, this

does

not

explain

how Ddot/icm

mutants

could

gain

access

to the

cytosol.

Legionella

does

form

LCV

pores

during

infection

of

its

natural

ameobal

hosts

[48]

and

a

full-length

copy of the Legionella protein IcmT is important for both

pore

formation

and

infection

spread

in

mammalian

cells

[49,50], although

it

is

not

required

for

escape

from

the

LCV

[46]. icmT mutants replicate intracellularly to similar

levels

as

wild

type

bacteria,

but

remain

trapped

inside

the

host

cells

until

later

time

points

[51]. These

mutant

studies, in combination with cell biological techniques,

support

a

cytosolic

stage

of Legionella

infection

as

impor-

tant

for

progression

of

infection,

although

its

regulationremains

to

be

understood.

Consequences of cytoplasmic access of intracellular

pathogens

Cytoplasmic localization of a bacterium causes many per-

turbations

in

the

host

cell

(Figure 2). This

is

because

of

the

display

and

detection

of

bacterial

surface

signatures

by

a

specific set of cytoplasmic host recognition molecules dif-

ferent

from

the

receptors

at

the

cell

surface.

These

host

factors include receptors that activate the immune system

or autophagy. However, detection by host cell surveillance

systems

and

subsequent

host

cell

death

or

gene

expression

changes can be advantageous for the invading bacterium,

and can

even

be

an

essential

part

of

the

infection

process.

Cell death as a result of cytoplasmic access

Cell

death

occurs

via

multiple

pathways

in

mammalian

cells. Apoptosis is initiated by various signals resulting in

activation

of

the

cysteine

proteases

caspase-2,

caspase-8,

caspase-9,

and

caspase-10,

which

in

turn

activate

effector

caspases, ultimately resulting in blebbing and cell death

without

membrane

rupture.

However,

pyroptosis

is

mediated by activation of caspase-1 and results in inflam-

matory

cell

death.

In

this

case,

the

cell

membrane

is

perforated,

resulting

in

blebbing,

spilling

of

cell

contents,

and activation of cytokines. Necrosis is a form of cell death

Box 3.

Legionella and autophagy

Legionella infection is intimately involved with the autophagy

pathway, considering that LCV maturation is largely similar to

autophagosome formation. Therefore, it has been used as a major

model to study autophagy and bacterial infection. Many autophagy

markers are found on the LCV and it is thought that Legionella

effectively, or ineffectively, depending on the host, stalls autophagy

to establish its niche [92]. Ubiquitinated proteins are found on the

membranes soon after initial infection and multiple Legionella effectors contain motifs characteristic of E3 ubiquitin ligases. The

Legionella effector

AnkB

increases both the accumulation of

ubiquitinated proteins on the LCV and the ability of some Legionella

strains to survive [93], possibly mediated by proteasomal degrada-

tion of ubiquitin-labeled host proteins, a process that may provide

Legionella with amino acids that are essential for its intracellular cell

growth. LubX increases survival in Drosophila through ubiquitina-

tion and subsequent degradation of other bacterial effectors [94].

However, in Dictyostelium, the autophagy pathway has no effect on

Legionella growth [95], highlighting the different roles of autophagy

during infection of various hosts. The RavZ effector has an anti-

autophagic effect through specific cleavage of Atg8–LC3 at a bond

that not only unconjugates the protein from membrane phospha-

tidyl ethanolamine (PE) but also prevents its reattachment. How-

ever, a DravZ mutant does not display a growth defect in

macrophages,

suggesting that more effectors play a role inautophagy avoidance [96]. The effector LegA9 has the opposite

effect, targeting the LCV to the autophagy pathway via the p62

adaptor protein. A legA9 mutant is only the second mutant found,

besides flagellin mutants, that can grow in murine macrophages,

suggesting that autophagic clearance is a very important host

mechanism in fighting Legionella infection [97].


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that also results in inflammation but is caspase-indepen-

dent.

It

was

generally

thought

to

be

uncontrolled

by

the

affected

cell,

but

recent

evidence

suggests

this

may

not

be

the

case

[52].

Pyroptosis via caspase-1 is often induced by detection of

cytosolic

bacteria

and

can

be

initiated

when

pathogens

escape from a vacuole. In myeloid cells, Shigella triggers

cell

death

through

caspase-1

following

endolysosomal

membrane

damage

[6].

However,

Shigella

triggers

cell

death

in

non-myeloid

cells

via

two

mechanisms:

(i)

a

caspase-1 dependent pathway triggered by ruptured

vacuolar

membranes,

which

also

participate

in

autophagy

Pepdoglycan

Damaged

membranes

Necrosis-likeShigella

Non-canonical

Pyroptosis

Salmonella

Shigella

Legionella

Mycobacteria

Casp 11

Salmonella

Burkholderia

Legionella

AIM2

Francisella

Legionella

Salmonella

GAS

Listeria

Mycobacteria

ShigellaShigella

Salmonella

AutophagyCell death

(B)

(A)

Bacterial effectors

RNA

DNA

Flagellin

TRENDS in Microbiology

Figure 2.

The impact of cytoplasmic access by intracellular bacteria. (A) Host signaling pathways induced after vacuolar rupture are triggered by bacterial effector proteins,

lipid A, flagellin, peptidoglycan, bacterial nucleic acids, the vacuolarcontents or damaged vacuolarmembranes. (B) Black font: different typesof cell death,such asnecrosis

and pyroptosis, are a consequence of cytoplasmic accessby bacterial pathogens. Purple font: the autophagy pathway also plays a major role in pathogen recognition and

control. Left: some bacteria block autophagy. Middle: successful autophagy. Green ovals represent LC3, small circles represent ubiquitin, and squares represent NDP52.

Right: very small green circles represent septins, which form cages around Shigella and show interdependencewith autophagy. Abbreviations: Casp 11, caspase-11; GAS,

group A Streptococcus ;

AIM2, absent in melanoma 2.


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activation

[10]; and

(ii)

a

caspase-independent

pathway

involving

loss

of

mitochondrial

membrane

potential,

simi-

lar

to

oxidative

stress.

Interestingly,

the

bacterium

also

induces pro-survival signals through Nod1/NFkB/IKKb

that

counteract

this

necrotic

cell

death

until

relatively

late

in infection

[53]. M. tuberculosis

cytosolic

access

has

only

recently begun to be studied, but it appears that this

change

in

localization

results

in

macrophage

cell

death[41]. In particular, the ESX-1 and ESX-5 T7SSs are impor-

tant

for

initiation

of

cell

death

during

M.

tuberculosis

and

M. marinum

infection,

likely

through

release

of

lysosomal

proteases from the ruptured phagosome into the cytosol

[54]. In

fact,

inhibition

of

cathepsin

L

[55]

or

cathepsin

B

[56] reduces macrophage cell death in response to myco-

bacterial

infection,

although

these

results

are

dependent

on bacterial

load.

ESX-1

plays

a

role

in

activation

of

inflammation and cell death through nod-like receptor

family,

pyrin-domain-containing

3

(NLRP3)

[43,57], which

supports

the

observation

that

bacteria

are

present

in

the

host cytosol via phagosomal escape, because NLRP3 moni-

tors

the

cytoplasm

and

activates

the

inflammasome

in

response to cytoplasmic stimuli. Interestingly, deletionof

ESX-5

effectors

had

a

more

significant

impact

on

cell

death, and it is thought that these factors only gain access

to host

proteins

after

vacuolar

escape

[54].

Salmonella

also

causes

cell

death

as

a

consequence

of

cytoplasmic access in the subset of infected host cells

harboring

hyper-replicating

bacteria.

Transcriptional

fusions

of

GFP

to

various Salmonella

promoters

revealed

that many hyper-replicating cytosolic bacteria express

both

flagellar

proteins

and

T3SS-1

genes

and

initiate

caspase-1-mediated

pyroptosis

and

interleukin-18

(IL-18)

release [32]. This suggests a connection between cytoplas-

mic

access

and

the

massive

inflammation

that

accompa-

nies

Salmonella

infection. Legionella

activates

host

cell-death

pathways

as

well,

apparently via multiple mechanisms. Cytosolic flagellin is

primarily

responsible

for

activation

of

caspase-1;

this

requires

the

NLRC4/Ipaf

and

Naip5

inflammasome

com-

ponents, and limits growth in restrictive cells [58,59].

Interferon b

(IFNb) is

increased

after

infection

in

a

Dot/

Icm-dependent,

but

not

flagellin-dependent,

manner

and

this increase is important for Legionella restriction in the

lung

epithelium

[60]. In

a

DsdhA

mutant,

which

has

an

unstable LCV, flagellin is not required for induction of host

immune response and cell death, nor for a large IFNb

response, which instead appears to be mediated by contact

of bacterial

DNA

with

the

cytosol

and

subsequent

activa-

tion

of

inflammation

via

the

AIM2

inflammasome

[61].

This is consistent with bacterial degradation on LCV exit,

and the

growth

of

this

mutant

is

severely

limited

within

macrophages.

However,

the

timing

of

cytosolic

contact

and

the number of bacteria entering the cytosol may be impor-

tant

for

infection

progression

and

the

ability

to

overwhelm

host

defense

mechanisms,

induce

inflammatory

responses,

and achieve cellular escape. In support of recent question-

ing

of

LCV

integrity, icmT mutants,

which

cannot

form

pores

in the

membranes

of

mammalian

cells,

do

cause

apoptosis

via

caspase-3

during

early

infection

[51], but

little necrotic morphology is seen during late infection.

In

addition,

they

do

not

cause

red

blood

cell

lysis

or

disease

in

mice.

Together,

these

data

suggest

that

cytosolic

access

is required

for

triggering

events

later

in infection.

Dealing with autophagy

Another

cytoplasmic

cell

process

is

autophagy,

a

major

catabolic

pathway

activated

in

response

to

nutrient

deple-

tion, misfolded proteins, and other signals. Autophagy

allows

for

recycling

of

cellular

contents

to

free

up

requiredmolecules or to remove harmful protein intermediates or

aggregates.

The

autophagic

machinery

recognizes

ubiqui-

tinated

proteins

and

surrounds

them

with

a

double

mem-

brane that later fuses with lysosomes, thereby digesting

the

contents.

In

general,

the

proteins

p62

and

NDP52

are

considered adaptors that bind to both ubiquitin and LC3,

with

LC3

then

directing

the

autophagic

complex

[62].

Interesting

work

using Shigella

links

autophagy

to

the

actin-dependent accumulation of host septins around cyto-

solic

bacteria

[63]. siRNA

knockdown

of

certain

septins

or

of

autophagy

genes

resulted

in

loss

of

both

septin

caging

and autophagy of Shigella, indicating an interdependence

between

the

two

systems

for

bacterial

clearance.

However,

this was not true for other bacteria examined, including Listeria,

which

is

also

autophagocytosed

but

independently

of actin and septins. Similar to Listeria, group A Strepto-

coccus

(GAS)

utilizes

a

pore-forming

toxin,

SLO,

that

is

important

for

escape

from

its

endosomally

derived

uptake

vacuoles. On access to the cytosol, these bacteria also

induce

autophagy,

and

are

quickly

marked

with

LC3

and limited

for

growth

during

early

infection

in

epithelial

cells [64].

Francisella

also

interferes

with

the

autophagy

pathway

during

its

cytoplasmic

stage

and

can

persist

within

the

cytoplasm for more than 10 h without detection [24,65].

The

polysaccharidic

O-antigen

plays

a

main

role

in autop-

hagy

evasion

via

the

Atg5

pathway;

however,

the

surfacemolecules

that

are

recognized

need

to

be

identified

[66].

Interestingly, in mouse primary macrophages, a percen-

tage of

Francisella

can

‘retranslocate’

in

a

membrane-

bound compartment

displaying

lysosomal

and

autophago-

somal features [65].

Autophagic

responses

to

pathogens

are

often

to

cytosolic

bacteria,

although

they

can

also

occur

in

response

to

damaged vacuolar membranes [10,30,31]. For instance,

ubiquitin

coats

cytosolic S.

Typhimurium

in

both

epithelial

cells and macrophages. In macrophages, this leads to

recruitment of proteasomes and subsequent bacterial

degradation [67]. In HeLa cells, the pathway has been

recently

characterized.

First,

host

sugar

molecules

exposed

through

damaged

SCV

membranes

are

labeled

with

galec-

tin-8 [31] and cytosolic Salmonella are also coated with

linear

ubiquitin

chains

[68]. Then

galectin-8

recruits

the

autophagy

adapter

NDP52

[31]

and

ubiquitin-coated

cyto-

solic bacteria recruit the additional autophagy adapters

p62, NDP52,

TBK1,

and

optineurin

[69]. Furthermore,

NDP52

recognizes

LC3C

[70]

and

TBK1

phosphorylates

optineurin, which leads to increased binding of the autop-

hagy

receptor

LC3

and

subsequent

autophagy

initiation

[69].

However,

Salmonella

can

also

counteract

autophagy

through

deubiquitination

by

the

effector

SseL

[71].

Autophagic targeting of M. tuberculosis has a similar

profile

to

that

of

Salmonella

and

requires

the

bacterial


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ESX-1

system.

Approximately

30%

of

intracellular M.

tuberculosis

immunostain

positive

for

autophagy

markers

4 h

post-infection

[72]

and

this

co-localization

is

dependent

primarily on ubiquitination by parkin [73]. This recruit-

ment

is

dependent

on

ESAT-6,

a

bacterial

effector

that

forms

pores

and

is

part

of

ESX-1,

and

expression

of L.

monocytogenes LLO restores some LC3 localization in an

D

esat6 strain

[72]. Taken

together,

these

data

suggest

thatautophagy is induced by mixing of vacuolar contents with

the

cytoplasm

due

to

vacuole

damage.

Further

character-

ization

demonstrated

that

cytosolic

bacterial

DNA

is

a

key

factor for both autophagy induction [72] and cytokine

production

[74]. Interestingly,

Irf3 / mice,

which

have

a

limited immune response, did not die as a result of myco-

bacterial

infection,

suggesting

that

cytokine

production

and inflammation,

a

result

of

cytosolic

access,

are

critical

to the bacterium for successful infection [74]. However,

Atg5 / mice,

which

are

impaired

for

autophagy,

were

extremely

sensitive

to

infection

[72], in

agreement

with

earlier studies in macrophages demonstrating the impor-

tance

of

autophagy

in

the

control

of

mycobacteria.

This

highlights the careful balance involved in clearing infec-tion,

because

both

cytokine

production

and

autophagy

are

activated by cytosolic bacterial DNA, but one process sup-

ports,

while

the

other

restricts,

bacterial

growth.

Autophagy

is

also

initiated

by

recognition

of

peptido-

glycan by the intracellular surveillance proteins Nod1 and

Nod2.

These

sensors

subsequently

recruit

ATG16L

to

bac-

terial

entry

sites

to

allow

nucleation

during

autophagy.

Recognition of peptidoglycan also mediates autophagy

initiation

during L. monocytogenes

infection

of Drosophila,

but

through

the

pattern

recognition

receptor

protein

PGRP-LE rather than Nod [75]. One group recently found

that an

amino

acid

stress

response

was

activated

in

response

to

membrane

damage

during

infection

by

eitherthe

cytosolic

bacterium Shigella

or

SCV-bound Salmonella

[76]. In the case of Salmonella, membrane damage was

SPI-1-dependent

and

the

bacterium

later

relieved

autop-

hagy

activation

through

increased

amino

acid

uptake.

For

Shigella, membrane damage was a result of vacuolar

escape,

with

the

bacterial

effector

IcsB

shielding

it

from

degradation

[77]. It

is

clear

that

autophagy

is

an

important

mechanism of bacterial clearance for the host. Because it is

also

a

consequence

of

cytoplasmic

access,

bacteria

that

trigger autophagy must also have a strategy to combat

or subvert it to effect successful infection. These strategies

are numerous and varied and will be exciting new areas of

research

in

pathogenesis.

Non-canonical inflammasome activation

Caspase-11

is

an

understudied,

non-canonical

inflamma-

some

with

an

important

role

in

limiting

bacterial

infection

[78]. Salmonella can induce cell death via caspase-11 [79]

and cytosolic sifA Salmonella

mutants

cause

increased

pyroptosis

independent

of

the

inflammasome

adaptors

Nlrc4 and ASC, but dependent on caspase-11 [80]. It

was

recently

shown

that

Gram-negative

lipid

A activates

caspase-11,

but

the

compartment

in

which

this

activation

occurs

remains

unclear,

although

it

has

been

suggested

that it is in the cytosol [81]. In support of this, caspase-11 /

mice

are

sensitive

to

the

cytosolic

pathogen

Burkholderia

[80]. In

addition,

caspase-11

limits

growth

during Legio-

nella infection

in a

flagellin/Dot/Icm-dependent

manner

[82]. These

authors

reported

a

very

different

mechanism

of action, suggesting that caspase-11 promotes depho-

sphorylation

of

cofilin,

which

leads

to

actin

depolymeriza-

tion

around

the

LCV

and

fusion

of the

LCV

with

lysosomes.

Concluding

remarksThrough the use of new approaches, it has become appar-

ent

that

there

are

no

longer

two

distinct

groups

of

bacteria:

those

that

access

the

cytoplasm

and

those

that

do

not.

Emerging evidence indicates that a much larger group of

pathogens

escapes

from

the

vacuole.

One

consequence

of

this localization change is that it can resolve previously

unexplained

data

about

the

induction

of

immune

responses,

for

example

via

major

histocompatibility

com-

plex class I (MHC-I). It also leads to many new questions,

including

the

exact

escape

mechanisms

of the

newly

iden-

tified

pathogens

and

whether

rupture

itself,

bacterial

con-

tact with the cytosol, or both is the true cause of observed

host

responses.

Note

added

in

proof

While this review was in proof stage, an article by Knodler

et

al.

quantifying

and

further

characterizing

hyper-repli-

cation

of Salmonella

was

published

that

may

be

of

interest

to readers [98].

Acknowledgements

We apologize for not being able to cite all recent work in this fast moving

field because of space limitations.We would like to thank the members of

J.E.’s team forhelpful discussionandBeatrice deCougny for thedesign of

Figure 2. This work was funded by a grant from the European Research

Council to J.E. (ERC StG No. 261166) and by a Pasteur Foundation

fellowship to J.F.

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