Filamentous actin is a substrate for protealysin,a metalloprotease of invasive Serratia proteamaculansOlga Tsaplina1, Tatiana Efremova1, Ilya Demidyuk2 and Sofia Khaitlina1

1 Institute of Cytology, RAS, St Petersburg, Russia

2 Institute of Molecular Genetics, RAS, Moscow, Russia

Keywords

aluminium fluoride; bacterial proteases;

cytoskeleton; ECP32 ⁄ grimelysin; F-actin

dynamics

Correspondence

S. Khaitlina, Institute of Cytology, Russian

Academy of Sciences, Tikhoretsky av. 4,

194064 St Petersburg, Russia

Fax: +7 812 297 03 41

Tel: +7 812 297 42 96

E-mail: skhspb@yahoo.com

(Received 2 August 2011, revised 3

November 2011, accepted 4 November

2011)

doi:10.1111/j.1742-4658.2011.08420.x

Homologous bacterial metalloproteases ECP32 ⁄ grimelysin from Serratia

grimesii and protealysin from Serratia proteamaculans are involved in the

invasion of the nonpathogenic bacteria in eukaryotic cells and are sug-

gested to translocate into the cytoplasm [Bozhokina ES et al. (2011) Cell

Biol Int 35, 111–118]. The proteases have been characterized as actin-

hydrolyzing enzymes with a narrow specificity toward intact cell proteins.

However, cleavage of filamentous actin (F-actin) (i.e. the main actin species

in the cell) and the properties of the cleaved F-actin have not been investi-

gated previously. In the present study, we revealed the presence of proteal-

ysin in the cytoplasm of 3T3-SV40 cells infected with S. proteamaculans or

recombinant Escherichia coli expressing the protealysin gene. We also show

for the first time that purified protealysin and the lysates of the recombi-

nant E. coli producing protealysin cleave 20–40% of F-actin. Cleavage lim-

ited predominantly to the bond Gly42-Val43 efficiently increases the

steady-state ATPase activity (dynamics) of F-actin. AlF�4 abolishes this

effect and promotes the nucleation of protealysin-cleaved Mg-globular-

actin even in the absence of 0.1 M KCl, most likely as a result of the stabi-

lization of lateral intermonomer contacts of actin subunits. The results

obtained in the present study suggest that F-actin can be a target for protealysin

upon its translocation into the host cell.

Structured digital abstractl Protealysin cleaves Actin by protease assay (View interaction)

Introduction

Actin is involved in the majority of cell processes,

ranging from chromatin remodeling and transcription

regulation to intracellular transport, cell organization

and various types of cell movements and muscle con-

traction. It is natural, therefore, that the actin cyto-

skeleton is a target for drugs and toxins, as well as for

viruses and pathogenic bacteria interacting with

eukaryotic cells [1–5]. The effectors modify cytoskele-

ton dynamics through the modulation of small GTPases

and actin-binding proteins, or by direct interaction

with actin. Many bacterial effectors promote the acti-

vation of small GTPases Rho, Rac and Cdc42, follo-

wed by the activation of signaling cascades resulting in

actin polymerization [6–8]. Thereby bacterial effectors

often mimic natural activators of small GTPases or

directly stimulate the host signaling pathways by a

functional mimicry of host GTPases [7,8]. Other

bacterial effectors stimulate the efficient uptake of

bacteria in host cells by the formation of complexes

with actin-binding proteins [9], or by mediating

Abbreviations

F-actin, filamentous actin; FITC, fluorescein isothiocyanate; G-actin, globular actin; Pi, inorganic phosphate.

264 FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS

phosphorylation ⁄dephosphorylation of an actin-bind-

ing protein [10–13].

The other way in which many toxins and bacterial

factors modify the cytoskeleton is the direct interaction

of the effectors with actin. Latrunculin from sponges

reversibly and specifically disrupts actin cytoskeleton

by altering the actin monomer interface and thus

inhibiting polymerization [3,14]. Marine macrolide tox-

ins disrupt the cytoskeleton because they target both

globular actin and actin filaments with a high affinity

and either sterically prevent polymerization or mimic

fragmentation of filamentous actin (F-actin) by sever-

ing proteins [15,16]. Clostridium botulinum C2 toxin

and ADP-ribosyltransferase SpvB from Salmonella ent-

erica cause ADP-ribosylation of actin at Arg177,

thereby inhibiting actin polymerization [17,18]. A

unique mechanism of actin filament depolymerization

is exhibited by Vibrio cholerae RTX toxin, which cova-

lently cross-links actin monomers, thus interfering with

the formation of functional filaments [19]. On the

other hand, the bacterial protein SipA from Salmo-

nella typhimurium inhibits depolymerization of actin fila-

ments mechanically stabilizing the filament by

tethering actin subunits in opposing strands [20,21].

Even these few examples demonstrate the variability of

the mechanisms developed by bacteria and other

organisms to subvert actin dynamics for facilitating

the corresponding invasion or protection processes.

Previously, we reported that spontaneously isolated

nonpathogenic bacteria Serratia grimesii and Serratia

proteamaculans synthesize thermolysin-like metallopro-

teases ECP32 ⁄ grimelysin [22,23] or protealysin [24–26],

which are characterized by a high specificity toward

actin. The appearance of the proteases correlated with

the capability of the bacteria to invade eukaryotic cells

and remodel their cytoskeleton [26,27]. Moreover, non-

invasive E. coli transformed by the grimelysin or the

protealysin gene confer the invasive phenotype [28].

We have also shown that protealysin introduced into

the culture medium does not induce the internalization

of non-invasive E. coli [28], which led us to suggest

that the enzyme translocates into the eukaryotic cell

where it modifies cytoplasmic proteins.

Proteases ECP32 ⁄grimelysin and protealysin are

actin-hydrolyzing enzymes with a narrow specificity

toward intact cell proteins [22,26]. Limited proteolysis

of globular actin (G-actin) by protease ECP32 abol-

ishes or slows down polymerization [29,30] and

enhances the dynamics of the filaments formed by

the cleaved monomers [31], thus producing changes in the

actin cytoskeleton favorable for bacteria to enter

the host cell. However, under physiological conditions,

actin cannot exist as a free monomer but forms fila-

ments (i.e. F-actin) whose accessibility to proteolysis is

very low [32,33]. The properties of cleaved F-actin

have not been investigated previously. In the present

study, we took advantage of using antibodies against

protealysin, purified recombinant protealysin and

lysates of recombinant E. coli expressing the protealy-

sin gene, aiming to elucidate the localization of prote-

alysin in the infected cell culture and the effects of

protealysin on F-actin dynamics.

The results obtained demonstrate for the first time

that the infection of eukaryotic cells with protealysin-

producing bacteria is accompanied by the translocation

of protealysin into the host cells. Furthermore, we

show that up to 40% of F-actin is digested with prote-

alysin in vitro within a time approximately correspond-

ing to that of the invasion experiments. F-actin is

cleaved by protealysin mainly at Gly42-Val43 within

the DNase I-binding loop, which strongly enhances the

filament dynamics. AlF�4 stabilizes the filaments and

promotes the nucleation step of actin polymerization,

most likely enhancing the effect of magnesium ions on

the lateral intermonomer contacts along the actin fila-

ment. These properties of protealysin-cleaved actin did

not depend upon whether purified protealysin or the

lysates of E. coli expressing the protealysin gene were

used. Therefore, we conclude that protealysin appears

to be the only component of the bacterial lysates to

affect actin dynamics. Taken together, these results

suggest that, as protealysin translocates into the host

cell, both G-actin and F-actin are targets for the

enzyme.

Results

Localization of protealysin in the infected cells

In invasion experiments, cultured cells are incubated

with the fluorescein isothiocyanate (FITC)-conjugated

bacteria, fixed with formaldehyde, stained with rhoda-

mine–phalloidin, and examined under a confocal

microscope to visualize bacteria and cytoskeleton

structures [26,28]. We used a similar protocol to reveal

the localization of protealysin in the infected cells by

staining the enzyme with anti-protealysin serum. Fig-

ure 1 represents the results of a typical experiment in

which cultured Balb 3T3 SV40 cells were incubated

either with the wild-type protealysin-producing bacte-

ria S. proteamaculans 94 (Fig. 1A) or with recombi-

nant E. coli BL21 (DE3) (pProPlnHis6) expressing the

protealysin gene (Fig. 1B). Lysates of these bacteria

demonstrated efficient and specific proteolytic activity

toward G-actin (Fig. 1D). As a control, we used

recombinant E. coli BL21 (DE3) transformed with

O. Tsaplina et al. Cleavage of F-actin with protealysin

FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS 265

plasmid pET23b not carrying the protealysin gene

(Fig. 1C). Lysates of E. coli BL21 (DE3) (pET23b) did

not exhibit any actin-hydrolyzing activity (Fig. 1D). In

line with our previous results [26–28], within 2 h of

incubation, the actin-containing structures within the

cells were rearranged, indicating that the stress fibres

had been destroyed (Fig. S1) and � 10% of the cells

were invaded by either the wild-type or recombinant

bacteria. Anti-protealysin serum revealed the enzyme

in both the cells infected by S. proteamaculans

(Fig. 1A) and those infected by the recombinant E. coli

synthesizing protealysin (Fig. 1A,B). In addition, colo-

calization of antibodies with bacteria was sometimes

observed. By contrast, E. coli BL21 (DE3) (pET23b)

not carrying the protealysin gene did not enter 3T3

SV40 cells and, in these samples, no protealysin could

be detected (Fig. 1C). These data imply that invasion

of the protealysin-producing bacteria is accompanied

by translocation of the enzyme into eukaryotic cells.

Limited proteolysis of F-actin

Previously, we showed that protealysin cleaves globu-

lar actin between Gly42 and Val43 within the DNase

I-binding loop, generating two fragments of 36 and

5 kDa. At a high enzyme ⁄ actin ratio, further proteoly-

sis of the 36-kDa fragment yields a 33-kDa product as

a result of cleavage of the peptide bonds Thr66-Leu67

and Gly63-Ile64 in the nucleotide cleft [26]. In the

actin polymer (F-actin), the sites Gly42-Val43 and

Gly63-Ile64 are involved in the monomer–monomer

contacts [34,35], which can protect them from

A B C

D

a b

Fig. 1. Localization of protealysin during the invasion of protealysin-producing bacteria in eukaryotic cells. S. proteamaculans 94 grown for

45 h at 30 �C with aeration (A, a), E. coli BL21 (DE3) (pProPlnHis6) carrying the protealysin gene (B, b) and E. coli BL21 (DE3) (pET23b) not

carrying the protealysin gene (C) grown for 18 h at 37 �C with aeration were stained with FITC as described in the Materials and methods.

Balb 3T3-SV40 fibroblasts were incubated with bacteria for 2 h, fixed with formaldehyde and stained with polyclonal rabbit anti-protealysin

serum (blue) and rhodamine-phalloidin (red). (a, b) Higher magnification of the regions delimited by the white frames in (A) and (B), respec-

tively. Images of the optical sections were taken over the middle of the cells. Scale bar = 15 lm. (D), Limited proteolysis of G-actin with

lysates of S. proteamaculans 94 (a), E. coli BL21 (DE3) (pProPlnHis6) (b) and E. coli BL21 (DE3) (pET23b) (c), corresponding to the invasion

experiments shown in (A, a), (B, b) and (C), respectively. G-actin (1 mgÆmL)1) was incubated with an equal volume of the lysate for 2 h at

22 �C. Ac, nondigested actin.

Cleavage of F-actin with protealysin O. Tsaplina et al.

266 FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS

proteolysis by protealysin. Consistently with this, only

the 36-kDa fragment was produced upon incubation

of F-actin with protealysin (Fig. 2A), which indicates

the poor accessibility to the enzyme of the sites within

the nucleotide cleft. Compared to G-actin, the accessi-

bility of the bond Gly42-Val43 within the DNase

I-binding loop in F-actin was also diminished (Fig. 2A).

However, quantification of the cleavage results showed

that � 20% and up to 40% of F-actin was digested at

enzyme ⁄ actin mass ratios of 1 : 50 and 1 : 5, respec-

tively, within a time approximately corresponding to

that taken in the invasion experiments. In line with the

earlier data [36], F-actin containing Mg2+ as a tightly

bound cation exhibited an increased protection of the

DNase I-binding loop from the cleavage. Importantly,

the 36-kDa fragment was also the main digestion prod-

uct upon incubation of F-actin with the lysates of

recombinant bacteria expressing the protealysin gene,

although, in this case, a small amount of the 33-kDa

fragment was also formed (Fig. 2B). This additional

cleavage is either the result of some contaminating

proteolytic activity or a higher concentration of prote-

alysin in the lysates that is difficult to relate to the

concentration of the purified enzyme.

Because many proteases are known to cut off the

C-terminal segment of the actin molecule [37] that is

involved in maintaining the filament structure, we also

monitored the cleavage of F-actin with protealysin

by using actin modified at the penultimate Cys374

with N-iodoacetyl-N-(5-sulfo-l-naphthy1)ethylenediamine

(IAEDANS). Independent of whether F-actin was

incubated with the purified protealysin or bacterial

lysates, in the AEDANS-labelled actin, Cys374, to

which AEDANS was conjugated, was preserved

(Fig. 2B). The C-terminus was also preserved in the

smaller fragments of G-actin where it is fairly accessible

for proteolysis (Fig. 2C) thus indicating the absence of

contaminating proteolytic activity in bacterial lysates.

Because the crude bacterial lysates might still con-

tain factors interfering with Mg-actin polymerization,

the latter was tested in both lysate-treated and proteal-

ysin-treated actin samples. As shown in Fig. 3, the

actin samples cleaved to a similar extent were proven

to polymerize similarly, indicating that other compo-

nents of the bacterial lysates do not interfere with the

properties of actin.

Dynamics of the cleaved F-actin

Globular actin cleaved between Gly42 and Val43 is

almost nonpolymerizable if it contains Ca2+ as a

tightly bound cation [22,29,30]. The cleaved actin con-

Fig. 2. Limited proteolysis of F-actin with protealysin. (A) Ca-G-

actin and Ca- or Mg-F-actins (24 lM) polymerized with KCl or MgCl2

as described in the bottom rows were incubated with protealysin

at 1 : 50 and 1 : 5 mass ratios for 2 h at 22 �C. (B) AEDANS-

labelled Ca-G-actin or Mg-F-actin (as indicated in the top row) was

incubated with the lysate of recombinant E. coli BL21 (DE3)

(pProPlnHis6) expressing the protealysin gene at 1 : 3000 and

1 : 100 volume ratios (as indicated in the bottom row). The same

gel was viewed in UV light and stained with Coomassie brilliant

blue G-250. (C) AEDANS-labelled G-actin was incubated with prote-

alysin at the mass ratios indicated in the bottom row for 2 h at

22 �C. The same gel was viewed in UV light and stained with

Coomassie brilliant blue G-250. Ac, nondigested actin.

Fig. 3. Polymerization of the protealysin cleaved G-actins obtained

by treatment with purified protealysin or with the lysate of recombi-

nant E. coli BL21 (DE3) (pProPlnHis6) expressing the protealysin

gene. Ca-G-actin (24 lM) was cleaved with protealysin at a 1 : 50

mass ratio or with the lysate of recombinant E. coli expressing the

protealysin gene at a 1 : 300 volume ratio for 20 h at 4 �C. Cleaved

Ca-G-actin was transformed into Mg-G-actin as described in the

Materials and methods and polymerized with 0.1 M KCl (arrow) at

22 �C. The inset shows the SDS ⁄ PAGE pattern of G-actin cleaved

with protealysin of bacterial lysates (1) and purified protealysin (2).

Ac, nondigested actin.

O. Tsaplina et al. Cleavage of F-actin with protealysin

FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS 267

taining tightly bound Mg2+ was found to polymerize,

although both the rate and extent of the reaction are

lower than those for intact actin [30]. Moreover,

assembly of the cleaved Mg-actin monomers results in

the formation of highly dynamic filaments as a result

of an enhanced turnover rate of the polymer subunits

[31]. To determine whether the cleavage of subunits

within F-actin results in the same effects, we compared

the properties of the cleaved F-actin with those of

F-actin assembled from the protealysin-cleaved mono-

mers. Figure 4 shows that the cleavage of F-actin is

accompanied by an � 30% decrease in the intensity of

both light scattering and pyrenyl fluorescence, which

reflects the shift in the steady-state monomer ⁄polymer

ratio toward shorter filaments and ⁄or the monomer.

Indeed, cleavage of G-actin may contribute to the

accumulation of the 33-kDa products during F-actin

cleavage (Fig. 4, inset). In addition, during ultracentrif-

ugation, � 90% of protealysin-cleaved F-actin was

recovered in the supernatant compared to 6–7% of the

total protein in the supernatant of intact F-actin sedi-

mented under the same conditions, although, morpho-

logically, the protealysin cleaved filaments were similar

to those of intact F-actin (not shown). This is consis-

tent with the changes in the polymer stability produced

by cleavage [30].

F-actin dynamics (i.e. the exchange of the filament

subunits with the monomer pool) is associated with

ATP hydrolysis after incorporation of ATP-containing

monomers into the polymer. Therefore, F-actin

dynamics can be evaluated by measuring the rates of

ATP hydrolysis in F-actin solutions at steady-state. As

shown in Fig. 5A, steady-state ATPase activity of

F-actin assembled from protealysin-cleaved actin

monomers was � 4 mol PiÆ(mol actin))1Æh)1, which is

similar to the ATPase activity of F-actin assembled

from the ECP32-cleaved monomers [31]. The 30% effi-

cient cleavage of F-actin with protealysin resulted in

the enhancement of steady-state ATPase activity to

� 1.2 molÆPiÆ(mol actin))1Æh)1 (Fig. 4B) compared to

0.02–0.04 molÆPiÆmol actin)1Æh)1 in the case of non-

modified F-actin under similar conditions [31,38].

Fig. 4. Effects of proteolysis on F-actin polymerization status. At

time 0, the lysate of recombinant E. coli BL21 (DE3) (pProPlnHis6)

expressing the protealysin gene was added to nonlabelled or 10%

pyrenyl-labelled Mg-F-actins (12 lM) at a 1 : 100 volume ratio, and

changes in pyrenyl fluorescence or light scattering intensities were

monitored at 22 �C. Inset: SDS ⁄ PAGE patterns of F-actin samples

withdrawn during the light scattering measurement. Ac, nondigested

actin.

Fig. 5. Steady-state ATP hydrolysis in solutions of F-actins. (A)

Ca-G-actin (24 lM) was incubated with the lysate of recombinant

E. coli BL21 (DE3) (pProPlnHis6) expressing the protealysin gene at

a 1 : 300 volume ratio for 20 h at 4 �C. The cleaved Ca-G-actin was

transformed into Mg-G-actin as described in the Materials and

methods. Mg-G-actin was polymerized with 0.1 M KCl for 40 min

and incubated further at 22 �C in the absence (circles) or presence

(triangles) of 1 mM AlF�4 . At the appropriate time intervals, aliquots

of the solutions were withdrawn for the determination of Pi con-

centration. (B) Mg-F-actin (24 lM) was treated with the lysate of

recombinant E. coli BL21 (DE3) (pProPlnHis6) expressing the protea-

lysin gene at a 1 : 100 volume ratio for 90 min at 22 �C, and the

cleaved F-actin was incubated for an additional 30 min at the same

temperature. During the incubation, aliquots of the cleaved F-actin

solutions were withdrawn for the determination of Pi concentration.

The results of three independent experiments are shown.

Cleavage of F-actin with protealysin O. Tsaplina et al.

268 FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS

Stabilization of F-actin cleaved with protealysin

Because F-actin dynamics is tightly coupled with ATP

hydrolysis and the release of Pi, the binding of Pi

analogue aluminum fluoride (AlF�4 ) in the nucleotide-

binding cleft mimics the ADP-Pi or ATP state and

stabilizes the filaments [39]. We have previously shown

that AlF�4 increases the thermal stability of F-actin

assembled from the monomers cleaved between Gly42

and Val43 [40]. Therefore, it was not unexpected that,

in the presence of AlF�4 , the steady-state ATPase activ-

ity of F-actin assembled from the protealysin-cleaved

monomers dropped to values characteristic of nonc-

leaved protein (Fig. 5A). However, in addition, AlF�4strongly accelerated the polymerization of protealysin-

cleaved Mg-G-actin, apparently as a result of the sta-

bilization of nuclei formed at the initial step of actin

polymerization, as demonstrated by the disappearance

of the long lag phase in the polymerization time course

characteristic of the cleaved actin polymerization

[Fig. 6, curve (Mg, Al) versus curve (Mg)]. Figure 6

also shows that the binding of AlF�4 does not acceler-

ate polymerization of the cleaved Ca-G-actin [Fig. 6,

curve (Ca, Al)], indicating a joint effect of Mg ions

and fluoride in actin stabilization.

To determine whether binding of AlF�4 is accompa-

nied by protection of subdomain 2 against proteolysis,

as shown earlier for the binding to F-actin of BeFx

[41], we probed the accessibility of subdomain 2 to

proteolysis with protealysin. Indeed, AlF�4 inhibited

cleavage of the bond Gly42-Val43 in F-actin subunits

(Fig. 6, inset), indicating that the stabilization of actin

filament with AlF�4 is a result of a tightening of the in-

termonomer contacts involving the DNase I-binding

loop.

Discussion

Serratia species are facultative pathogens that primar-

ily cause nosocomial infections. Serratia marcescens is

the most pathogenic species of this genus [42] and,

until recently, it had been the only Serratia species

shown to possess invasiveness mediated by a secreted

pore-forming cytolysin ShlA [43]. In addition, a num-

ber of reports describe human diseases from infections

with other Serratia species, including S. grimesii and

S. proteamaculans [42]. However, the mechanisms of

these infections are poorly understood. We have

recently found that S. grimesii and S. proteamaculans

can penetrate into eukaryotic cells, and their invasive

activity correlates with a specific proteolytic activity of

the bacterial lysates toward actin [26,27]. Moreover,

non-invasive E. coli became invasive upon transforma-

tion with the grimelysin or protealysin genes, indicat-

ing that the enzyme can be actively involved in

invasion [28]. In the present study, we demonstrate for

the first time that protealysin does translocate into the

cytoplasm of eukaryotic cells. We also show that

F-actin, usually resistant to proteolysis, is limitedly

digested either with purified protealysin or with the

lysates of recombinant E. coli expressing the protealy-

sin gene, whereas the lysates of recombinant E. coli

not carrying the protealysin gene do not display any

actin-hydrolyzing activity. These data indicate that the

actin-hydrolyzing activity of the E. coli lysates is

indeed produced by protealysin, and the lysates do not

appear to contain any other components actively

affecting actin proteolysis and dynamics.

Despite the high actin-hydrolyzing activity of the

bacterial lysates, the invasive activity of both wild-type

Serratia and recombinant E. coli is relatively low, with

only � 11% and 16% of cells invaded by Serratia and

recombinant E. coli, respectively [44]. The low level of

invasive activity does not allow us to quantify the

amount of the enzyme within the host cells. However,

a correlation between actin-hydrolyzing and invasive

activities was clearly seen in the properties of the

recombinant bacteria synthesized protealysin, with a

Fig. 6. Stabilization of protealysin-cleaved actin by AlF�4 . 5% pyre-

nyl-labelled Ca-G-actin (24 lM) was incubated with the lysate of

recombinant E. coli BL21 (DE3) (pProPlnHis6) expressing the prote-

alysin gene at a 1 : 300 volume ratio for 20 h at 4 �C. The cleaved

Ca-G-actin was transformed into Mg-G-actin as described in the

Materials and methods. At time 0, Ca- and Mg-actins were either

complemented with 1 mM AlF�4 (Ca, Al and Mg, Al, respectively)

followed by the addition of 0.1 M KCl (arrow) or directly polymerized

with 0.1 M KCl (Ca, Mg). The inset shows the effect of AlF�4 on the

cleavage of Mg-F-actin with protealysin. Mg-F-actin (24 lM) was

incubated with the lysates of recombinant E. coli expressing the

protealysin gene at a 1 : 200 volume ratio for 1.5 h in the absence

or presence of 1 mM AlF�4 . Ac, nondigested actin; n ⁄ s, actin

cleaved in the absence of fluoride.

O. Tsaplina et al. Cleavage of F-actin with protealysin

FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS 269

point Glu113 ⁄Ala mutation in the active site that pre-

vented removal of the propeptide from the protealysin

precursor [45]. The recombinant bacteria synthesizing

inactivated protealysin were not found to be invasive

unless a small amount of actin-hydrolyzing activity

could be detected in their lysates at the late post-loga-

rithmic growth stage [28]. As the time of infection

increased, the efficiency of the invasion augmented

concomitantly with the enhancement of actin cleavage.

Furthermore, changes in cell morphology and cytoskel-

eton rearrangements accompanying the invasion of

E. coli synthesizing wild-type protealysin were more

pronounced than those produced by the E. coli synthe-

sizing mutant enzyme [28]. These results suggest that

cleavage-produced modifications of the properties of

actin promote bacterial internalization.

Limited proteolysis of G-actin within the DNase I-

binding loop has been shown to impair polymerization

[22,29,46] and enhance the polymer dynamics [31], as

well as decrease the affinity of gelsolin [47], myosin

subfragment 1 [48] and tropomyosin [49] to actin. It

has been suggested that the properties of F-actin trea-

ted with protease would be similar to those of F-actin

assembled from the proteolytically modified mono-

mers. The data reported in the present study provide

experimental support for this suggestion, which is

important in view of the translocation of the proteo-

lytic enzymes into the eukaryotic cell where F-actin is

a major actin species. Most importantly, cleavage of

F-actin with protealysin is accompanied by an efficient

enhancement of the filament dynamics. On the other

hand, aluminium fluoride, known to mimic a stable

ADP-Pi or ATP conformation of actin filaments

[39,41], accelerates the polymerization of protealysin-

cleaved actin monomers whose nucleation is impaired

by cleavage. Together with the increased dynamics of

the cleaved F-actin, these effects provide mechanisms

that may facilitate the invasion of eukaryotic cells by

bacteria.

Actin dynamics is determined by the polarity of the

actin filament where the protein subunits are added

predominantly to the ‘barbed end’, and dissociate

mainly from the other end, termed the ‘pointed end’.

Cleavage of actin between Gly42 and Val43 resulted in

enhancement of the turnover rate of polymer subunits

by one order of magnitude, primarily as a result of a

substantial increase in the rate constants for the disso-

ciation of actin subunits from the filament ends. The

dissociation rate constants were found to be approxi-

mately ninefold and more than fivefold higher for the

cleaved actin at the barbed and pointed ends, respec-

tively [31]. These effects appear to be determined by

the direct involvement of the cleavage site in the

intermonomer contacts of the filament and changes in

the overall conformation of subdomain 2 [31]. In addi-

tion, the recently published 3D structure of the pointed

end of the actin filament may shed light on the acceler-

ated release of the cleaved ADP-actin subunits from

the pointed end of the actin filament [50]. In this

model, the terminal subunit is tilted relative to the heli-

cal symmetry of the whole actin filament in such a

way that the hydrophobic loop of this subunit and the

DNase I-binding loop of the adjacent subunit are rear-

ranged to form a tighter contact, which may be

responsible for the low dissociation rate constants at

the pointed end of the actin filament [50]. Impairment

of the DNase I-binding loop by proteolysis may pre-

vent the tightening of the contact and result in faster

dissociation of the cleaved subunits and accelerated

filament dynamics.

Another important side of actin dynamics is the sta-

bilization of filaments by actin-binding proteins or low

molecular weight compounds. Recently, we have

shown that aluminium fluoride greatly increased the

thermal stability of F-actin assembled from the cleaved

actin monomers [40]. In the present study, we show

that this effect is associated with the inhibition by

AlF�4 of the filament dynamics and is likely a result of

tightening of the intermonomer contacts involving the

DNase I-binding loop. This is evident from the strong

protection of the protealysin cleavage site between

Gly42 and Val43 in subunits of AlF�4 -F-actin and is

consistent with the stabilization of actin subdomain 2

by BeFx, as demonstrated by the 3D reconstruction of

the filament structure [51] and biochemical approaches

[41].

The penetration of bacteria into eukaryotic cells

involves the disassembly of actin structures at the site

of bacterial–cell contact followed by actin polymeriza-

tion in cell surface protrusions, which rise around the

bacterial body and allow its engulfment in a macropin-

ocytic-like process [11,52]. The results obtained in the

present study show that F-actin can be limitedly

cleaved with protealysin within the DNase I-binding

loop, and that the cleavage strongly enhances the

dynamics of actin filaments and promotes their depoly-

merization. These effects can lead to the disassembly

of the actin cytoskeleton at the site of bacterial–cell

contacts and provide actin monomers for filament

assembly in cell surface protrusions. During this latter

step, the reversibility of the effects is of primary impor-

tance: assembly of the cleaved actin monomers into fila-

ments may be promoted by the low molecular weight

effectors mimicking Pi (present study) and actin-bind-

ing proteins [47,53] that stimulate actin polymerization

and stabilize the filaments. Thus, the limited cleavage

Cleavage of F-actin with protealysin O. Tsaplina et al.

270 FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS

of F-actin with bacterial enzymes allows actin to pre-

serve its functional properties and produce changes in

the actin cytoskeleton that could be used by bacteria

to enter the host cell. This specific activity of the

enzyme toward actin, taken together with its narrow

substrate specificity toward intact cell proteins [54,55],

suggests that, upon translocation of protealysin into

the host cell, actin proteolysis is involved in the inter-

nalization of bacteria.

Materials and methods

Reagents

Tris, ATP (disodium salt), sodium azide, AlCl3, NaF, Triton

X-100, reagents for electrophoresis, FITC, rhodamine–phal-

loidin, gelatin and Mounting medium were purchased from

Sigma Chemical Co. (St Louis, MO, USA). Peptone and

yeast extract were obtained from Difco (Franklin Lakes, NJ,

USA). IAEDANS and N-(1-pyrenyl)iodoacetamide were

from Molecular Probes Inc. (Carlsbad, CA, USA).

Bacterial strains, cell culture and growth

conditions

S. proteamaculans 94 [24] were grown in LB medium at

30 �C, with aeration. The recombinant E. coli BL21 (DE3)

(pProPlnHis6) expressing the protealysin gene [45] and con-

trol recombinant E. coli BL21 (DE3) (pET23b) not carrying

the protealysin gene [45] were grown in LB medium at

37 �C, with aeration times specified as appropriate.

The cell line Balb 3T3-SV40 (SV40 transformed fibro-

blasts 3T3) was obtained from the Russian Cell Culture

Collection (Institute of Cytology, St Petersburg, Russia).

Cells were grown on glass cover slips in culture dishes in

the antibiotic-free DMEM (Biolot, Moscow, Russia) con-

taining 10% fetal bovine serum (Sigma) in a incubator at

37 �C supplemented with 5% CO2 for the time required to

form a monolayer (� 48 h).

Invasion assay

Bacteria were grown as described above until the actinase

activity of their extracts could be detected [26,28]. Thirty

minutes before the experiment, FITC (1 mgÆmL)1 of the

bacterial suspension) was added to the bacterial culture to

visualize bacteria. Bacteria were pelleted at 9600 g for

10 min; the pellets were washed with DMEM and added to

the host cells in a fresh portion of DMEM. The host cells

and bacteria were co-cultivated at 37 �C in 5% CO2 for the

times indicated as appropriate.

Rabbit polyclonal antibodies against protealysin precur-

sor were produced by Biotest Systems Ltd (Moscow,

Russia) in accordance with a standard protocol. For

immunization, recombinant protealysin precursor carrying

mutation Glu113 to Ala (inhibiting the precursor matura-

tion) and C-terminal His6-tag was purified as described by

Gromova et al. [45]. To visualize protealysin, 3T3-SV40

cells infected with S. proteamaculans 94, E. coli BL21

(DE3) (pProPlnHis6) expressing the protealysin gene or

control recombinant E. coli BL21 (DE3) (pET23b) that did

not carry the protealysin gene were washed with NaCl ⁄Pi,

fixed with 3.7% formaldehyde for 10 min and incubated

with 0.1% Triton X-100 for 5 min. After washing three

times with NaCl ⁄Pi, the samples were incubated in 1%

BSA for 30 min, and stained with polyclonal rabbit anti-

protealysin antibodies dissolved in 1% BSA, for 18 h at

4 �C. The samples were then washed with 0.05% Tween in

NaCl ⁄Pi three times and incubated for 1 h at 4 �C with

Alexa647-conjugated anti-rabbit IgG secondary serum

(Jackson ImmunoResearch Inc., Bar Harbor, ME, USA).

To visualize actin cytoskeleton, the antibody-stained sam-

ples were washed with 0.05% Tween in NaCl ⁄Pi three times

and with once with NaCl ⁄Pi and incubated with rhoda-

mine–phalloidin for 15 min at 37 �C in the dark. After

washing with NaCl ⁄Pi, the samples were mounted in the

mounting medium and analyzed with a confocal laser scan-

ning microscope.

Protein preparations

Rabbit skeletal muscle actin was isolated from acetone

dried muscle powder according to a standard procedure

[56]. G-actin was stored in buffer G (0.2 mM ATP, 0.1 mM

CaCl2, 5 mM Tris–HCl, pH 7.5, 0.02% NaN3) on ice for

1 week or as 0.2-mL aliquots (0.5–1.0 mgÆmL)1) frozen at

)20 �C for a single use. ATP-Ca-G-actin was transformed

into ATP-Mg-G-actin by 3–5 min of incubation with

0.2 mM EGTA ⁄ 0.1 mM MgCl2 at room temperature. Actin

labelled with N-(1-pyrenyl)iodoacetamide at Cys374 was

prepared as described previously [57]. Pyrenyl-labelled actin

was lyophilized in the presence of 2 mM sucrose and stored

at )70 �C. Before use, the lyophilized pyrenyl-labelled actin

was dissolved in buffer G and dialyzed against the same

buffer overnight. Actin labelled with IAEDANS at Cys374

was prepared as described previously [58]. To stabilize actin

with AlF�4 , G- or F-actin (24 lM) was incubated with 1 mM

ATP and 5 mM NaF for 10 min, which followed by the

addition of 1 mM AlCl3 [40]. The concentration of G-actin

was determined spectrophotometrically using an absorption

coefficient of 0.63 mLÆmg)1Æcm)1 at 290 nm [40] or by the

microbiuret method [59].

Protealysin was purified from E. coli BL21 (DE3)

(pProPlnHis6) cell lysate by sequential metal chelate affinity

chromatography on Ni2+-NTA-agarose (Qiagen, Valencia,

CA, USA) and gel filtration on a Superdex 75 HR 10 ⁄ 30column (Amersham Biosciences, Piscataway, NJ, USA) as

described previously [45]. Protealysin was stored in 25 mM

Tris–HCl buffer (pH 8.0) at 4 �C.

O. Tsaplina et al. Cleavage of F-actin with protealysin

FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS 271

Limited proteolysis assay

To estimate the susceptibility of F-actin to protealysin,

actin was polymerized under different conditions known to

affect the polymer structure [36]. G-actin (0.5 mgÆmL)1) in

buffer G containing Ca2+ as a tightly-bound cation (Ca-G-

actin) was polymerized with 0.1 M KCl for 2 h at room

temperature. G-actin containing Mg2+ as a tightly bound

cation (Mg-G-actin) was polymerized with 0.1 M KCl or

2 mM MgCl2 for 2 h at room temperature. F-actin was

incubated with protealysin (0.3 mgÆmL)1 in 25 mM Tris–

HCl, pH 8.0) at various enzyme ⁄protein mass ratios at

22 �C. At different time points, the digestion was stopped

by the addition of an equal volume of the electrophoresis

sample buffer containing 4% SDS, 125 mM Tris–HCl (pH

6.8) followed by 3 min of boiling. The digestion products

were analyzed by SDS ⁄PAGE [60]. The actinase activity

was determined by the appearance of specific actin frag-

ments. The small (5 kDa) product of the cleavage was not

seen on the gels.

To determine the ability of the protealysin-containing

bacterial extracts to cleave actin, bacteria were grown as

described above (usually for 18–24 h) and harvested by cen-

trifugation at 9600 g for 10 min. The pellets were resus-

pended in buffer G, and the bacteria were lysed by five

cycles of freezing and thawing. The lysates (bacterial

extracts) were clarified by centrifugation at 9600 g for 10

min. F-actin in the corresponding buffer (0.5 mgÆmL)1) was

mixed with an equal volume of the clarified lysate and incu-

bated for 2 h at room temperature or for 18 h at 4–6 �C.The reaction was stopped by the addition of the electropho-

resis sample buffer followed by 3 min of boiling. The diges-

tion products were analyzed by SDS ⁄PAGE as described

above.

ATP hydrolysis measurements

Protealysin-cleaved F-actin or Mg-G-actin, polymerized for

40 min to steady-state, were incubated at 22 �C. In aliqu-

ots of the F-actin solutions withdrawn after various time

intervals, the ATPase reaction was quenched by the addi-

tion of an equal volume of 0.6 M ice-cold perchloric acid,

precipitated protein was removed by centrifugation, and

released Pi was determined by the Malachite green method

[61].

Fluorescence microscopy

Cell samples were examined under a confocal scanning

microscope (Leica TCS SL; Leica Microsystems, Wetzlar,

Germany) using the argon ion (488 nm; green fluorescence)

and helium ⁄ neon (532 nm; red fluorescence; 633 nm, blue

fluorescence) laser system to visualize the FITC-stained

bacteria, rhodamine–phalloidin-stained cytoskeleton and

protealysin, respectively.

Fluorescence and light-scattering measurements

All fluorescence and light-scattering measurements were

performed in a Fluorat-02-Panorama spectrofluorimeter

(Lumex, St Petersburg, Russia). The fluorescence intensity of

pyrenyl-labelled actin was monitored at 407 nm after excita-

tion at 365 nm [35]. The intensity of light scattered at 90� wasmeasured at 350 nm. To visualize AEDANS-labelled fluores-

cence peptides, gels were photographed in UV light before

staining.

Acknowledgements

We thank Dr Ilya Nevzorov for discussions and his

valuable help in the preparation of the manuscript, as

well as the anonymous reviewers for their helpful

comments. The work was supported by the Russian

Foundation for Basic Research grants 11-04-00393 and

09-04-00734 and by the Program for Molecular and

Cell Biology of the Russian Academy of Sciences.

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Supporting information

The following supplementary material is available:

Fig. S1. Cytoskeleton rearrangements accompanying

the invasion of 3T3-SV40 cells with the protealysin-

producing bacteria.

This supplementary material can be found in the

online version of this article.

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Cleavage of F-actin with protealysin O. Tsaplina et al.

274 FEBS Journal 279 (2012) 264–274 ª 2011 The Authors Journal compilation ª 2011 FEBS