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Pathogenic bacteria can repli-

cate either outside or inside

susceptible host cells. Outside

the cells, they must evade or inhibit

circulating host defense molecules

such as antibodies, complement, and

lectins, as well as professional phago-

cytes that function to kill the invading

pathogens (1, 2). But bacteria with an

intracellular life-style face challenges

too. They must get adequate nutrition

from their intracellular milieu and

spread to new host cells to sustain

themselves in this niche. However,

rupture of the host cell exposes bacte-

ria to potent immune effectors in the

extracellular milieu. To overcome this

problem, bacteria have evolved strate-

gies for direct cell-to-cell transmis-

sion, chiefly by exploiting the host

cell’s actin cytoskeleton (3). On page

1729 of this issue, Hagedorn et al. (4)

describe a new actin-based strategy to

promote spread of infection.

The best understood cell-to-cell

transmission of bacteria is by

Listeria monocytogenes, a food-

borne occasional human pathogen.

The bacterium polymerizes host

cell actin to generate motive force

that ultimately leads to a host cell

membrane protrusion containing

the microbe. The protrusions are

subsequently ingested by neighbor-

ing cells. In the newly infected cell, Listeria

are contained within a double-membrane

vacuole, which they escape by lysing the vac-

uolar membranes. The bacteria thus take up

residence in the cytosol of the new cell,

where they polymerize actin and repeat the

infectious cycle (5, 6).

Much less is known about how other cyto-

plasmic bacteria spread from cell to cell, a

problem addressed by Hagedorn et al. Using

the genetically tractable model system of

infection—Dictyostelium discoideum amoeba

as a host cell and Mycobacterium marinum as

the infectious microbe—the authors identi-

fied a bacteria-induced actin-containing

structure—the “ejectosome”—by which cyto-

solic M. marinum exit infected cells in a non-

lytic process (see the figure). Previous studies

have shown that M. marinum escape from

phagosomes and polymerize actin in the cyto-

plasm, leading to bacterial motility and cell-

to-cell spread (7, 8). Actin polymerization

and motility depend on the host molecule

Arp2/3, as is the case for Listeria. Activation

of Arp2/3-mediated actin polymerization by

M. marinum requires a member of the

Wiskott-Aldrich syndrome protein (WASP)

family, whereas Listeria bypasses this step for

host Arp2/3 complex activation (8). The spe-

cific molecules on the surface of M. marinum

required to activate actin polymerization and

motility are not known, but the lipid-rich

“waxy coat” of the bacterium is thought to

participate because a lipid-binding region of

N-WASP is necessary for bacterial recruit-

ment of the Arp2/3 complex (8).

Pathogenic mycobacteria have a

specialized secretion system required

for virulence, called ESX-1, or type

VII secretion (9). For both M. marinum

and M. tuberculosis, ESX-1 is required

for efficient cell-to-cell spread

(10–12), but its role in this process

has been poorly understood. ESX-1

causes direct host membrane damage

(10), which may explain both phago-

some escape (10) and lysis of the host

cell plasma membrane (13), leading

to spread of infection. However,

this lytic model is challenged by

Hagedorn et al., who suggest a funda-

mentally different ESX-1–dependent

exit strategy.

In Dictyostelium, M. marinum

induces formation of a barrel-shaped,

actin-containing structure surround-

ing a bacterium as it crosses the host

cell plasma membrane in an outward

journey. Importantly, although this

process requires the ESX-1 secretion

system, analysis with impermeable

probes shows that bacterial egress is

nonlytic—possibly because the host

plasma membrane provides a dy-

namic and tight seal around the bacte-

ria as they are exiting. The barrel-

shaped ejectosome is thus fundamen-

tally distinct from actin-mediated

bacterial motility, and its genesis

involves the host cell molecules

RacH, Myosin IB, and coronin, but not

Arp2/3. Although a few bacteria with short

actin tails were observed, bacteria in the

process of ejection were rarely associated

with actin tails. Thus, the source of the force

required to propel a bacterium through an

ejectosome remains unknown. In Dictyo-

stelium, ejection often occurred into the

extracellular milieu. However, in infected

metazoan hosts, if the ejection were to occur

near a susceptible host cell, which is likely,

this could facilitate spread of bacterial in-

fection. Indeed, ESX-1 attracts uninfected

macrophages to adhere to infected cells

(14), and thus could facilitate this process.

Does M. marinum coordinate both distinct

mechanisms of actin polymerization during

infection? The dependence of its cell-to-cell

transmission on WASP proteins implies that

Arp2/3-mediated actin polymerization has a

Bacteria can pass from one cell to another

through a cytoskeletal structure that prevents

host cell destruction.The Art of Making an ExitFredric Carlsson and Eric J. Brown

CELL BIOLOGY

Cell-to-cell

spread

Cell-to-cell

spread

WASP, Arp2/3

Actin Bacterium

Actin-based

motility

Ejectosome

Host cell

Nucleus

Actin barrel

(Myosin IB, coronin)

Moving out and about. M. marinum escapes from intracellular vac-

uoles in infected host cells and polymerizes actin into a tail that propels

it forward, leading to membrane protrusions containing the bacterium.

The protrusion is engulfed by adjoining cells. An independent mecha-

nism of cell-to-cell spread is the ejectosome, an actin barrel through

which a bacterium exits the host cell in a nonlytic process.

Department of Microbial Pathogenesis, Genentech Inc.,South San Francisco, 1 DNA Way, CA 94080, USA. E-mail:carlsson.fredric@gene.com; brown.eric@gene.com

Published by AAAS

role in transmission (8), and Hagedorn et al.

implicate the ejectosome in this process

as well. The authors did not observe promi-

nent actin tails in infected Dictyostelium;

whether actin-based motility plays less of a

role in cell-to-cell spread in this host, or

whether the shorter actin tails imply a differ-

ent equilibrium between actin polymeriza-

tion and depolymerization in Dictyostelium,

is unclear. It could be that decreased motility

facilitates ejectosome formation or that the

prominent membrane protrusions induced

by rapidly motile bacteria, as previously

observed for M. marinum in macrophages

(7), make ejectosomes more difficult to

observe. Understanding whether these two

modes of cell-to-cell spread represent alter-

native or complementary virulence strate-

gies will shed light on the diverse biological

functions of ESX-1.

Intriguingly, Hagedorn et al. also provide

evidence that M. tuberculosis might use ejec-

tosomes as an exit strategy, both in

Dictyostelium and mammalian cells. M.

tuberculosis is a major threat to human

health globally, and there is an urgent need to

improve our basic understanding of its

pathogenesis. M. tuberculosis is unable to

form actin tails (15), implying that this fea-

ture of M. marinum virulence might not

apply to M. tuberculosis pathogenesis. Even

the ability of M. tuberculosis to escape

phagosomes in mammalian cells is ex-

tremely controversial. Characterizing the

molecular mechanisms of ejectosome-medi-

ated exit in mammalian cells, and addressing

the importance of ESX-1–mediated ejecto-

some escape to mycobacterial pathogenesis

in general, and M. tuberculosis in particular,

will be important challenges for the future.

References1. M. W. Hornef, M. J. Wick, M. Rhen, S. Normark, Nat.

Immunol. 3, 1033 (2002).

2. D. M. Underhill, A. Ozinsky, Annu. Rev. Immunol. 20,

825 (2002).

3. J. M. Stevens, E. E. Galyov, M. P. Stevens, Nat. Rev.

Microbiol. 4, 91 (2006).

4. M. Hagedorn, K. H. Rohde, D. G. Russell, T. Soldati,

Science 323, 1729 (2009).

5. E. Gouin, M. D. Welch, P. Cossart, Curr. Opin. Microbiol.

8, 35 (2005).

6. L. G. Tilney, D. A. Portnoy, J. Cell Biol. 109, 1597 (1989).

7. L. M. Stamm et al., J. Exp. Med. 198, 1361 (2003).

8. L. M. Stamm et al., Proc. Natl. Acad. Sci. U.S.A. 102,

14837 (2005).

9. A. M. Abdallah et al., Nat. Rev. Microbiol. 5, 883 (2007).

10. L. Y. Gao et al., Mol. Microbiol. 53, 1677 (2004).

11. K. M. Guinn et al., Mol. Microbiol. 51, 359 (2004).

12. N. van der Wel et al., Cell 129, 1287 (2007).

13. I. C. Koo et al., Cell Microbiol. 10, 1866 (2008).

14. H. E. Volkman et al., PLoS Biol. 2, e367 (2004).

15. L. M. Stamm, E. J. Brown, Microbes Infect. 6, 1418

(2004).

10.1126/science.1172254

www.sciencemag.org SCIENCE VOL 323 27 MARCH 2009 1679

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Enzyme-catalyzed reactions are typi-

cally stereospecific, affecting only

one of a given pair of mirror-image

isomers. This arises from a close fit between

substrate molecules and the binding sur-

faces of proteins. But some enzymes do not

conform to this expectation. For example, P-

glycoprotein (P-gp) catalyzes the movement

of dozens of distinct classes of com-

pounds—from peptides to steroids, includ-

ing pairs of stereoisomers—across cellular

membranes. How does it perform this feat?

On page 1718 of this issue, Aller et al. (1)

report crystal structures of a mammalian P-

gp, both with and without a stereo pair of

inhibitor molecules bound to it. The results

show how P-gp recognizes multiple sub-

strates via specific, but multiple and par-

tially overlapping, binding sites.

P-gp is the leading cause of multidrug

resistance in cancer, where a single genetic

change can lead to its overexpression, frustrat-

ing chemotherapy treatments. Since P-gp was

first identified in the 1970s (2), there has been

some understandable disbelief that a single

protein could interact directly with the full

range of molecules whose intracellular con-

centrations it controls.

P-gp is a membrane-bound adenosine

triphosphatase that is a member of a large fam-

ily of adenosine triphosphate (ATP)–dependent

transporter proteins, the ABC (ATP-binding

cassette) gene family. Members of this family

are present in all living cells, where they carry

out various essential roles by transporting mol-

ecules across cellular membranes (3). Indeed,

the three-dimensional structures presented by

Aller et al. strongly resemble those of related

multidrug resistance proteins from bacteria,

suggesting that the mechanisms coupling sub-

strate recognition with transport may be the

same throughout this protein family (4, 5).

Molecular genetic and biochemical studies

of P-gp in model systems—including in vitro

Crystal structures elucidate how some enzymes

can bind many different molecules, including

mirror-image isomers.Through a Mirror, DifferentlyJonathan A. Sheps

BIOCHEMISTRY

A B

P-glycoprotein

CYTOPLASM

Direct model Indirect models

Modifying enzyme

or group of enzymes

Modified drug molecule

Drug molecules

Different views of P-gp action. The direct transport model (A) posits that many different drug molecules arerecognized by different sites on P-gp before efflux. In contrast, indirect models (B) suggest either that drugmolecules are modified by enzymes in the cell, creating a common recognition feature for recognition andefflux by P-gp, or that the activity of P-gp alters the cell membrane so as to render it impermeable to the drugs.The P-gp crystal structures reported by Aller et al. provide support for the direct transport model (panel A).

Cancer Genetics and Developmental Biology, BC CancerResearch Centre, British Columbia Cancer Agency,Vancouver, BC V5Z 1L3 Canada. E-mail: jsheps@bccrc.ca

Published by AAAS