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Intracellular pathogens hijack molecular motors 25

retrograde movement towards the nucleus (Kristensson

2 has been involved in the movement of melanosomes et al., 1986). Disruption of microtubule function leads to a

and the endoplasmic reticulum to Golgi trafficking (Le Bot reduced transport of the HSV capsid to the nucleus and

et al., 1998; Tuma et al., 1998). the capsids remain scattered within the cytosol (Sodeik

Although less abundant other kinesin-related proteins et al., 1997; Mabit et al., 2002). Indeed, the HSV-1 capsid

play an important role in specific vesicle transports. associates with cytoplasmic dynein during this phase

Unc104/Kif1A, a member of the Kinesin-3 familly, is one

µm 1 (Sodeik et al., 1997; Dohner et al., 2002). Consistently

of the fastest described kinesin motor (about 2 s ) with this concept, disruption of dynein function resulted in

(Klopfenstein et al., 2002). It uses a lipid binding pleckstrin impaired transport of various viral particles to the perinu-

homology (PH) domain to bind and then to transport syn- clear area (Kristensson et al., 1986; Suomalainen et al.,

aptic vesicles precursors from the cell body to the nerve 1999; Dohner et al., 2002; McDonald et al., 2002). Con-

terminal (Hall and Hedgecock, 1991). Unc104/Kif1A is versely, inhibition of kinesin function resulted in a

monomeric in solution but may dimerize via a predicted decrease of transport of different viral particles to the

coiled-coil region in the presence of a sufficient level of periphery (Rietdorf et al., 2001; Jouvenet et al., 2004).

Pi(4,5)P2 as the result of its concentration in lipid rafts

β1 A huge step towards the comprehension of the mech-

(Klopfenstein et al., 2002). In mouse, Kif13A binds the anism of capsid transport along microtubules was over-

subunit of the clathrin adaptor AP-1. It has been reported come with the tracking of individual viral particles in living

to play a role in the post-Golgi transport of the mannose cells (Suomalainen et al., 1999). Precise studies of the

6-phosphate receptor (Nakagawa et al., 2000). mobility of individual capsids of an herpesvirus (pseudo-

One of the most poorly understood aspects of kinesin- rabies virus PRV) during both retrograde (Smith et al.,

based transport is how the motor activity is regulated. In 2004) and anterograde transports (Smith et al., 2001)

some specific cases, kinesin motors utilize rab GTPases have shown that PRV capsids were moving bidirectionaly

or their activity to link themselves to their cargo. with a bias towards the cell body during entry and a bias

Rabkinesin6, for example, interacts with constitutively towards the terminal ends of the axons during escape.

active rab6 in yeast two-hybrid and may be necessary for PRV transport is highly processive and consistent with the

the COP1-independent vesicular transport from the Golgi presence of several motors on the capsid. Such fast

apparatus and the endoplasmic reticulum (Echard et al., bidirectional transports have also been described for

1998). KIF16 is a recently identified member of the kine- adenovirus (Suomalainen et al., 1999) and vaccinia intra-

sin-3 family. It transports early endosomes to the cell cellular mature virus between the cytoplasmic viral factory

periphery and this process is regulated by the small and the site of envelopment in the juxtanuclear area

GTPase rab5. Indeed KIF16B has a C-terminal PhoX (Ward, 2005).

homology motif known to bind PI(3)P. It is recruited to Numerous molecular interactions between viral proteins

PI(3)P-containing early endosomes in response to rab5 and microtubule motors-associated host proteins have

and its effector, the phosphatidylinositol−3-OH kinase been described. Particularly, the dynein light chain protein

hVPS34 (Hoepfner et al., 2005). TcTex seems to be a target used by different virus. Indeed,

the HSV-1 capsid protein VP26 has been shown to inter-

Involvement of the microtubule cytoskeleton in act with dynein light chains RP3 and Tctex1. The involve-

viral infections ment of this viral protein has been demonstrated by

microinjecting virions containing or not VP26 protein in

Most viruses replicate in the nucleus or the perinuclear +

living cells. Whereas VP26 virions undergo a rapid migra-

area, at a distant point from their peripheral entry site. The tion towards the perinuclear area, no orientated migration

step in between thus requires their transport towards the –

was observed for VP26 virion (Douglas et al

., 2004). The

replication site. Because of the high viscosity of the cyto- poliovirus receptor CD55 interacts with TcTex (Mueller

sol, viruses cannot rely on passive diffusion to reach the et al

., 2002; Ohka et al

., 2004) suggesting that poliovi-

perinuclear area. This is particularly true for neurotropic ruses could use CD55 both for receptor-mediated endocy-

viruses, which can enter the host cytosol in a dendrite or tosis and retrograde transport. Interaction of viral proteins

an axon at a huge distance from the cell body. Thus with the dynein intermediate chain (Ye et al., 2000) and

viruses hijack the host cytoskeleton and the associated the dynein light chain polypeptide LC8 (Jacob et al., 2000;

molecular motors to be transported to their replication site Raux et al., 2000; Alonso et al., 2001) have also been

(Sodeik, 2000; Smith and Enquist, 2002). What is true for reported. Concerning anterograde movement, proteins

entry is also true for escape and viruses have also devel- from various viruses have been described to interact

oped strategies using the molecular motors to exit host directly with kinesin (Kim et al., 1998; Tang et al., 1999;

cells. Diefenbach et al., 2002; Ward and Moss, 2004; Koshizuka

More than 20 years ago it has been shown that the et al., 2005).

HSV-1 capsid requires the microtubule network for its

© 2005 The Authors Cellular Microbiology

, 8

, 23–32

Journal compilation © 2005 Blackwell Publishing Ltd,

26 T. Henry, J.-P. Gorvel and S. Méresse after the entry. It moves towards the nucleus in a micro-

Viruses have to be transported in opposite directions in tubule-dependent manner (Ewing et al., 1978). This

function of the phase of the infectious cycle. This imposes movement is striking for a bacteria residing in the host

a tight regulation of the microtubule-associated motors. cytosol and is very different of the well-known actin-based

The net transport of herpes viruses towards the nucleus motility developed by other cytosolic bacteria such as

during entry and towards the terminal end of the axon Listeria, Shigella and Rickettsia. Movement on microtu-

during exit probably reflects modulation of plus end molec- bules is used by another cytosolic bacterium Actinobacil-

ular motors while minus end motors do not seem to be lus actinomycetemcomitans. It escapes from the host cell

regulated (Smith et al., 2004). Conversely, upon entry of and spreads to adjacent cells via intercellular protrusions

adenovirus, PKA and p38/MAPK pathways are activated that are dependent on microtubule integrity (Meyer et al.,

and stimulate minus end-directed transport (Suomalainen 1999). As exit requires a plus end motor, kinesin could be

et al., 2001). Another step of well-described regulation involved in this phenomenon. Kinesin has also been

occurs when vaccinia switches from a transport on micro- involved in the establishment of the Chlamydia psittaci

tubules to an actin-based motility at the cell surface replicative niche. Indeed, intracellular delivery of antibod-

(Rietdorf et al., 2001). Release of kinesin occurs after ies against kinesin after host cell permeabilization has

activation of the host cell kinase Src by a viral protein B5R. been shown to inhibit C. psittaci replication. However, this

Src activation leads to phosphorylation of A36R, the viral may only result from the loss of the close apposition of

protein interacting with KLC and to localized release of mitochondria to the C. psittaci inclusions (Escalante-

the virus (Newsome et al., 2004). Ochoa et al., 1999). All in all, little is known about diver-

sion of molecular motor functions by intracellular bacteria

Involvement of the microtubule cytoskeleton in and Salmonella has given the most striking illustration of

bacterial infections such a phenomenon.

Much less is known about the use of microtubule-associ-

ated motors by intracellular bacteria. While most bacteria Focus on Salmonella

that direct their entry in non-phagocytic cells such as During systemic infection of the host, Salmonella typhimu-

Shigella or Salmonella modulate the host actin cytoskel- rium replicates inside macrophages mainly in the spleen

eton (Cossart and Sansonetti, 2004), microtubule-depen- and the liver. This capacity to replicate inside macroph-

dent entry into host cells has been described for ages is critical for its virulence (Leung and Finlay, 1991)

Campylobacter jejuni, Citrobacter freundii and Serratia and is based upon the establishment of a niche of re-

marcescens (Oelschlaeger et al., 1993; Hertle and plication inside the host cell. This niche is called the

Schwarz, 2004). Also, inhibition of dynein by a low spec- Salmonella-containing vacuole (SCV). It is a unique

ificity inhibitor decreases entry of C. jejuni in host cell compartment resulting from the modulation of host cell

suggesting the involvement of this molecular motor in the biology by bacterial effectors translocated in the host cy-

microtubule-dependent entry of Campylobacter in host tosol. The second type three secretion system (TTSS-2)

cells (Hu and Kopecko, 1999). encoded by the Salmonella pathogenicity island-2 (Shea

Right after entry, movement towards the microtubule- et al., 1996) plays a critical role in this process. In the last

organizing centre has been described for several intracel- 3 years an increasing amount of evidence has shown that

lular bacteria such as Chlamydia trachomatis (Clausen the microtubule network and the associated motors play

et al., 1997; Grieshaber et al., 2003), C. jejuni (Hu and a critical role in the maturation of the SCV consistent with

Kopecko, 1999), Orientia tsutsugamushi (Kim et al., an early study showing the requirement of an intact

2001), all dependent of the dynein motor. Such a move- microtubule network for the establishment of the S. typh-

ment has also been described for latex-bead phagosomes imurium replication niche (Garcia-del Portillo et al., 1993).

in a manner dependent on microtubule integrity and

molecular motors (Blocker et al., 1996; 1998). However,

two striking characteristics can be highlighted among The early SCV migrates to the perinuclear area and

intracellular bacteria using dynein to reach the perinuclear escapes the fusion with lysosomes

area. Whereas overexpression of p50 dynamitin inhibits Whereas phagosomes containing latex beads or non-

most cargo transport by uncoupling the dynein complex pathogenic bacteria rapidly fuse with lysosomes, the SCV

from its cargo vesicle, it has no effect on transport of the matures in a different way. The fusion of the latex-bead

Chlamydia inclusion. This observation suggests that bac- phagosome with the late endosomal/lysosomal compart-

terial proteins included in the membrane of the inclusion ment is dependent on the recruitment of active rab7 and

couple this cargo directly with dynein independently of the its association with RILP/dynein–dynactin complex (Har-

dynactin complex (Grieshaber et al., 2003). O. tsutsuga- rison et al., 2003). The mature SCV is a Lamp-1-positive

mushi lyses its vacuole and escapes in the cytosol shortly © 2005 The Authors

Cellular Microbiology

, 8

, 23–32

Journal compilation © 2005 Blackwell Publishing Ltd,

Intracellular pathogens hijack molecular motors 27

participate in the formation and/or the maturation of these

compartment but is devoid of lysosomal enzymes (Garcia- structures (Guy et al., 2000; Jiang et al., 2004; Birming-

del Portillo and Finlay, 1995). The maturation of the SCV ham et al., 2005; Knodler and Steele-Mortimer, 2005).

in a Lamp-1-positive compartment is dependent on rab7 Finally, the microtubule network is reorganized in Salmo-

(Meresse et al., 1999). Three different teams have looked nella-infected cells with the formation of bundles of micro-

recently at the involvement of the rab7/RILP/dynein cas- tubules around clustered SCVs (Guignot et al., 2004;

cade at different steps in the maturation of the SCV. While Kuhle et al., 2004). SseF and SseG are necessary for this

they all found that the rab7/RILP/dynein cascade was phenotype (Kuhle et al., 2004) while it was originally

essential for the bacterial replication, their results differ described as independent of TTSS-2 effectors (Guignot

particularly in the outcome of the SCV in cells overex- et al., 2004).

pressing RILP. At early time post-invasion, the SCV

migrates into a perinuclear area (Fig. 2A) in a RILP/

dynein-dependent manner (Harrison et al., 2004). Early Role of molecular motors in the SCV membrane dynamics

overexpression of RILP triggers the recruitment of dynein The importance of the TTSS-2 effectors in the regulation

on the SCV and increases the frequency of fusion with of the SCV membrane dynamics was first uncovered with

lysosomes leading to inhibition of replication (Marsman the identification of SifA (Stein et al., 1996). Both in mac-

et al., 2004). At later post-invasion times, Salmonella rep- rophages and epithelial cells, the late phase of Salmonella

lication is associated with the formation of tubular exten- infection is marked by a dramatic redistribution of lysoso-

sions of the SCV membrane named Sifs (Fig. 2B) (Garcia- mal membrane proteins towards the SCVs, as well as by

del Portillo et al., 1993), the formation of which is also a disappearance of the lysosomal vesicular pattern

dependent on rab7 (Brumell et al., 2001). Surprisingly (Fig. 2B). This process is dependent on SifA functions as

RILP was not detected on Sifs despite the presence of it is not observed in cells infected with a corresponding

rab7 suggesting a local uncoupling of RILP from rab7 –

mutant (Fig. 2C). In addition, the sifA

(Harrison et al., 2004). This may explain why a later over- mutant progres-

expression of RILP has been found not to affect/inhibit sively loses its vacuole during the course of the infection

bacterial growth (Harrison et al., 2004; Marsman et al., resulting in its release into the host cytosol (Beuzon et al.,

2004). 2000). In macrophages, it thus gets exposed to a cytosolic

killing activity that is not active in HeLa cells allowing

replication of a sifA mutant in this latter cell line (Beuzon

Positioning of the late SCV –

et al., 2002). The disruption of the sifA vacuole is depen-

While dynein is clearly involved in the positioning in the dent on the activities of both plus and minus end-directed

perinuclear area at early time points, the localization of molecular motors. This assumption is supported by the

the SCV is also highly dependent on TTSS-2-secreted correlation between the recruitment of kinesin and the

effectors. Particularly, SseG targets the SCV in the peri- loss of the vacuole (Fig. 2E) and by the stabilization of the

nuclear area and is required for the intimate association sifA vacuole upon inhibition of either kinesin or dynein

between the SCV and the Golgi membranes (Salcedo and activities (Guignot et al., 2004; Boucrot et al., 2005). While

Holden, 2003). A possible involvement of SseG in the the molecular link between SifA and kinesin is mediated

control of molecular motors has not been addressed in by SKIP, a regulator of kinesin recruitment (Boucrot et al.,

this study. A mutant for another TTSS-2 effector SifA has 2005), SifA has also been suggested to form a complex

recently been shown to loose the perinuclear localization. with rab7, and thereby to regulate the local uncoupling of

The movement of the sifA RILP/dynein from rab7 (Harrison et al., 2004). Consistent

SCV towards the cell periphery with the hypothesis of SifA regulating the two opposite

is associated with a strong recruitment of kinesin on the molecular motors associated with the SCV, overexpres-

SCV indicating that the SifA protein is inhibiting kinesin sion of dominant negative forms of rab7 or RILP destabi-

recruitment (Figs 1 and 2D). The molecular interaction lizes the Salmonella vacuolar membrane (Guignot et al.,

sustaining this phenotype has been deciphered leading to 2004). Finally, while in control cells, each bacterium of a

the identification of a novel host protein named SKIP (for microcolony is clearly present in an individual Lamp-1-

SifA and Kinesin Interacting Protein) that inhibits kinesin positive vacuole, inhibition of dynein leads to the formation

recruitment on the SCV (Boucrot et al., 2005). Interest- of enlarged vacuoles containing several bacteria (Guignot

ingly, the SifA protein is also involved in the formation of et al., 2004). Thus SifA emerges as a major player of the

Sifs (Stein et al., 1996). These filamentous structures are modulation of molecular motors. However, it is likely that

formed along microtubules and depolymerization of the other TTSS-2 effectors are also involved in this process.

microtubule network disrupts Sifs (Garcia-del Portillo –

Indeed, the recruitment of kinesin on the sifA

et al., 1993). Even if the relevance of the Sifs in the Sal- vacuole is

monella pathogenesis is still unknown several other effec- dependent on the integrity of the TTSS-2 indicating that

tors including SopD2, PipB2, SpvB, SseJ, SseF and SseG other secreted effectors are responsible for the enrich-

© 2005 The Authors

Journal compilation © 2005 Blackwell Publishing Ltd, Cellular Microbiology, 8, 23–32

28 T. Henry, J.-P. Gorvel and S. Méresse

A 09.00 11.00 13.00 15.00 17.00

B C

% SCV positive for kinesin/ lamp1

D E

wt

sifA-

75

50 sifA-

25 wt

0 time (h)

2 4 6 8 10 © 2005 The Authors

Journal compilation © 2005 Blackwell Publishing Ltd, Cellular Microbiology, 8, 23–32


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Corso di laurea: Corso di laurea magistrale in biologia applicata alla ricerca biomedica
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A.A.: 2008-2009

I contenuti di questa pagina costituiscono rielaborazioni personali del Publisher Atreyu di informazioni apprese con la frequenza delle lezioni di Microbiologia Cellulare e Vaccinologia e studio autonomo di eventuali libri di riferimento in preparazione dell'esame finale o della tesi. Non devono intendersi come materiale ufficiale dell'università La Sapienza - Uniroma1 o del prof Bernardini Maria Lina.

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