Staying alive: bacterial inhibition of apoptosis during infection.

Abstract

The ability of bacterial pathogens to inhibit apoptosis in eukaryotic cells during infection is an emerging theme in the study of bacterial pathogenesis. Prevention of apoptosis provides a survival advantage because it enables the bacteria to replicate inside host cells. Bacterial pathogens have evolved several ways to prevent apoptosis by protecting the mitochondria and preventing cytochrome c release, by activating cell survival pathways, or by preventing caspase activation. This review summarizes the most recent work on bacterial anti-apoptotic strategies and suggests new research that is necessary to advance the field.

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Staying alive: bacterial inhibition of
apoptosis during infection
Christina S. Faherty and Anthony T. Maurelli
Department of Microbiology and Immunology, F. Edward He ´bert School of Medicine, Uniformed Services University of the Health
Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA
The ability of bacterial pathogens to inhibit apoptosis in
eukaryotic cells during infection is an emerging theme in
the study of bacterial pathogenesis. Prevention of apop-
tosis provides a survival advantage because it enables
the bacteria to replicate inside host cells. Bacterial
pathogens have evolved several ways to prevent apop-
tosis by protecting the mitochondria and preventing
cytochrome c release, by activating cell survival path-
ways, or by preventing caspase activation. This review
summarizes the most recent work on bacterial anti-
apoptotic strategies and suggests new research that is
necessary to advance the field.
Bacterial pathogens and apoptosis
The ultimate goal of all pathogens is to establish a site, or a
replicative niche, in the host where the pathogen can
multiply. Bacterial pathogens possess unique traits that
provide a survival advantage upon infection of the host.
Some pathogens manipulate the immune response,
whereas others attempt to avoid recognition. Some bac-
teria induce apoptosis in host cells. The ability of certain
bacteria to prevent apoptosis has recently emerged as a
new theme in pathogenesis.
Apoptosis, or programmed cell death, is a natural
phenomenon that occurs regularly in multicellular organ-
isms [1,2]. Apoptosis is characterized by DNA fragmenta-
tion, chromatin condensation, cytoplasmic shrinkage, and
cell death without lysis or damage to neighboring cells.
Apoptosis, which is activated by intrinsic and extrinsic
pathways (Figure 1), differs from other forms of cell death
[3,4].
Bacteria, viruses [5] and parasites [6] can either induce
or prevent apoptosis to augment infection. Many bacterial
pathogens that cause apoptosis target immune cells such
as macrophages [7] and neutrophils [8] because these cells
would otherwise kill the pathogens [9,10]. Bacterial induc-
tion of apoptosis has been studied extensively, and several
comprehensive reviews on pro-apoptotic bacterial patho-
gens are available [10,11]. However, new research has
shown that many bacterial pathogens can in fact prevent
apoptosis during infection. This research grew out of the
observations that some organisms induce cell death in one
cell type but not so in others (see below). It is becoming
more evident that the ability to inhibit apoptosis during
infection provides the bacterial pathogen with a survival
tool in vivo. Given that there are now many examples of
apoptosis inhibition during bacterial infection (Table 1,
Figure 2), we review the various ways that bacterial
pathogens inhibit apoptosis. Bacterial pathogens can be
grouped into three classes based on the mechanisms
employed to inhibit apoptosis. Here, we describe these
three mechanisms and provide examples of bacterial
pathogens that employ each method. The diversity of
bacterialpathogensthatinhibitapoptosisandthemultiple
mechanisms they have evolved underscore the importance
of this trait to bacterial pathogenesis.
Protection of the mitochondria and prevention of
cytochrome c release
One mechanism utilized by Chlamydia and Neisseria to
prevent apoptosis is the use of a secreted product that
prevents cytochrome c release. This mechanism has been
studied extensively in Chlamydia [12–14]. Infected epi-
thelial cells were exposed to either staurosporine (STS) or
ultraviolet(UV)irradiationtodetermineifthebacteriacan
preventapoptosisinducedbythesestimuli.Initially,itwas
discovered that Chlamydia trachomatis inhibits the pro-
apoptotic host proteins Bax and Bak and prevents them
from permeabilizing the mitochondrial membrane. Cyto-
chrome c release and subsequent caspase activation are
inhibited in infected cells [12]. Further research indicated
that Chlamydia targets pro-apoptotic proteins with a BH3
domain (i.e. Bik, Puma and Bim) for degradation [15–17].
The chlamydial proteasome-like activity factor (CPAF), a
protease that is secreted by C. trachomatis, was identified
as the bacterial product required for the degradation of the
pro-apoptotic proteins [18]. In addition, Chlamydia could
also upregulate the inhibitor of apoptosis proteins (IAPs)
when tumor necrosis factor a (TNF-a) was used as an
inducer of the extrinsic pathway of apoptosis [19]. It is
unknown if both CPAF and upregulation of the IAPs are
required for the anti-apoptotic activity of Chlamydia, and
this should be addressed. Moreover, protein interaction
studies of CPAF and BH3 domain proteins might shed
light on how Chlamydia specifically targets these host
factors.
In addition to blocking apoptosis, Chlamydia-infected
cells continue to undergo DNA synthesis and mitosis up to
40 h post-infection, which aids in establishing a persistent
infection[20].Preventing apoptosiswouldhelpensurethat
the eukaryotic cell continues to divide in the presence of
apoptotic signals from the host (see Concluding Remarks,
below). Obligate intracellular pathogens like Chlamydia
rely on the survival of the host cell. Therefore, it is import-
ant not only to inhibit apoptosis, but also to ensure that the
cell maintains the division cycle given that cell cycle arrest
Review
Corresponding author: Maurelli, A.T. (amaurelli@usuhs.mil).
0966-842X/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.tim.2008.02.001 Available online 18 March 2008
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often leads to apoptosis. It would be interesting to
determine if Chlamydia still prevents apoptosis in non-
dividing cells. Identification of bacterial proteins required
for maintaining mitosis and evaluation of whether these
proteins are required for or complement apoptosis inhi-
bition should be performed to further our understanding of
apoptosis inhibition and persistent infection.
Neisseria also inhibits apoptosis by preventing cyto-
chrome c release [21,22]. Neisseria meningitidis uses its
outer membrane protein porin PorB to prevent apoptosis
by targeting it to the mitochondria, which results in inhi-
bition of cytochrome c release. When Neisseria-infected
HeLacellsareincubatedwithSTS,mitochondrialintegrity
remains, there is no cytochrome c release, and a normal
mitochondrial membrane potential is maintained [22]. In
addition, when purified PorB is incubated with host cells
for 24 h, PorB enters the cell and prevents STS-induced
apoptosis. Purified PorB localizes to the mitochondrial
membraneandPorBco-immunoprecipitateswiththemito-
chondrial protein voltage-dependent anionic channel
(VDAC), which is a component of the mitochondrial per-
meability transition pore (PT). The authors suggested that
PorB interacts directly with the mitochondrial PT or
indirectly with the PT by binding to VDAC, and that this
interaction stabilizes the mitochondria in the presence of
STS [21]. Since this publication, however, additional stu-
dies on the PT and the role of VDACs [23,24] suggest that
VDACs might not be important for mitochondrial mem-
brane permeabilization during cell death. Therefore, PorB
localizes to some other protein in the PT and how PorB
specifically interacts with the PT to prevent STS-induced
apoptosis remains to be determined.
The PorB homolog in Neisseria gonorrhoeae, porin IB,
also translocates to the mitochondria, but one group found
that it causes apoptosis in Jurkat T cells and HeLa cells
[25]. However, Massari et al. [21] speculated that differ-
ences in cell types or porin purification explain the dis-
crepancies between the results. In addition, serum
deprivation was used, which makes the cells more sensi-
tive to stress and apoptotic stimuli. Massari et al. repeated
the experiments with PorB and porin IB using both serum-
deprived and normal experimental conditions, and found
the same results as he described for PorB. They concluded
that N. gonorrhoeae has the same ability to stabilize the
mitochondria through porin IB as N. meningitidis does
with PorB [21,22].
Figure 1. The apoptosis pathway. Apoptosis is activated by intrinsic and extrinsic pathways. In the intrinsic pathway, certain apoptotic stimuli alter the normal status of the
Bcl-2 family of proteins (Box 1), which leads to permeabilization of the mitochondrial membrane. Under normal circumstances, the pro-survival proteins of the Bcl-2 family
protect the mitochondrial membrane. Once cytochrome c (CytoC) is released into the cytosol, it binds to the apoptosome [1], a complex of proteins made up of the
apoptosis activating factor-1 (Apaf1) protein and the initiator caspase-9. A morphological change in the apoptosome results from cytochrome c binding, which causes
caspase-9 to become activated. Caspase-9 activates the effector caspase-3, leading to apoptosis [1]. Caspase-3 is known as the ‘executioner caspase’, because it activates or
cleaves various protein targets, which is detrimental to the cell and results in death [1]. Activation of caspase-9 and caspase-3 are normally inhibited by a family of proteins
known as the inhibitor of apoptosis proteins (IAPs), and XIAP (X-linked IAP) is the most potent IAP [1]. In the extrinsic pathway, ligands such as Fas ligand (FasL) or tumor
necrosis factor a (TNF-a) bind to death receptors on the membrane of the host cell. Trimerization of the death receptors follows, forming what is known as the death-
inducing signaling complex (DISC), which includes the Fas-associated death domain (FADD) adaptor protein, the Flice-like inhibitory protein (FLIP), and procaspase-8 [2].
Caspase-8 is activated, which in turn directly activates caspase-3 [1]. In addition, caspase-8 activates the pro-apoptotic protein Bid, which stimulates the intrinsic pathway to
enhance the apoptotic signal, because Bid activation eventually leads to cytochrome c release [1].
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Other groups have found that porin IB might function
differently to inhibit apoptosis. N. gonorrhoeae increases
theexpressionofthehostanti-apoptoticgenesbfl-1,cIAP-2
and cox-2 through nuclear factor kappa B (NF-kB) acti-
vation [26,27]. Bfl-1 interacts with pro-apoptotic proteins
such as Bid and Bax to inhibit cytochrome crelease, cIAP-2
prevents caspase-3 activation, and Cox-2 activates the
phosphatidylinositol 3-kinase/Akt pathway (PI3K/Akt)
[26]. This pathway results in Akt phosphorylation, which
in turn prevents pro-apoptotic proteins from permeabiliz-
ing the mitochondrial membrane [28]. NF-kB activation is
required for the induced expression of the pro-survival
genes, and an NF-kB activation inhibitor prevents
upregulation of the genes [27]. Finally, apoptosis is also
Table 1. Classification of bacteria that inhibit apoptosis
Pathogens grouped by class
Protection of mitochondria
Chlamydia sp.
Neisseria sp.
Activation of cellular pathways
Salmonella enterica
Anaplasma phagocytophilum
Ehrlichia chaffeensis
Rickettsia rickettsii
Wolbachia
Bartonella sp.
Helicobacter pylori
Porphyromonas gingivalis
Listeria monocytogenes
Interaction with caspases
Shigella flexneri
Legionella pneumophila
Further investigation required
Mycoplasma fermentans
Brucella suis
Escherichia coli K1
Coxiella burnetii
Cell typea
Proposed or demonstrated mechanism Refs
Hep2, HeLa
HeLa, UEC
Inhibits and degrades pro-apoptotic proteins
Prevents cytochrome c release
[15–20]
[21,22,25–27,29]
HeLa, IEC-6
Neutrophils
THP-1
HUVEC
Neutrophils
Mono Mac 6, HUVEC
MKN45
GEC
J774
Activates PI3/Akt pathway
Activates p38 MAPK, ERK, PI3/Akt, NF-kB pathways
Activates NF-kB and upregulates pro-survival genes
Prevents cytochrome c release
Prevents caspase-3 activation
Activates NF-kB pathway, induces cIAP-1, cIAP-2 expression
Induces cIAP-2 expression through NF- kB activation
Activates PI3/Akt pathway
Activates PI3/Akt and NF-kB pathways
[30]
[32–34]
[35]
[36,37]
[38,39]
[40–42]
[53]
[55]
[56]
HeLa, T84
U937
Inhibits caspase-3 activation despite cytochrome c release
Activates NF-kB pathway and upregulates pro-survival genes
[44]
[46–48]
U937
THP-1
THP-1, RAW 264.7
HeLa, THP-1
Inhibits TNFa-induced apoptosis
Upregulates pro-survival genes
Upregulates pro-survival genes
Prevents cytochrome c release
[57,58]
[59]
[60]
[61,62]
aHeLa, UEC, IEC-6, Hep2, T84 and GEC are epithelial cell lines. MKN45 is a gastric adenocarcinoma cell line. Mono Mac 6, THP-1 and U937 are monocytic cell lines. UVEC are
primary human umbilical vein endothelial cells. J774 and RAW 264.7 are macrophage cell lines.
Figure 2. Mechanisms by which bacterial pathogens inhibit apoptosis at different points along the apoptotic pathway. Chlamydia secretes the chlamydial proteasome-like
activity factor (CPAF) to inhibit and degrade the pro-apoptotic proteins with one BH3 domain. These pro-apoptotic proteins inhibit the pro-survival Bcl-2 proteins upon
activation. The outer membrane protein porin PorB of Neisseria meningitidis prevents cytochrome c (CytoC) release. Salmonella secretes the effector SopB through a type
III secretion system, resulting in the activation of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway. This pathway prevents cytochrome c release. Anaplasma also
activates the PI3K/Akt pathway in addition to activating nuclear factor kappa B (NF-kB). NF-kB prevents the release of cytochrome c and activates the inhibitor of apoptosis
proteins (IAPs). Bartonella, Ehrlichia, and Rickettsia activate NF-kB as well. Shigella inhibits caspase-3 activation. Legionella directly activates caspase-3 to enhance
infection, but inhibits apoptosis through NF-kB. Red lines indicate inhibition in the pathway while green arrows indicate activation. The bacterial proteins that specifically
participate in apoptosis inhibition are shown, where known. The shapes of the bacteria, all shown in red, represent their morphology. P, phosphate.
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inhibited in N. gonorrhoeae-infected human fallopian tube
epithelial cells when tumor necrosis factor a (TNF-a) is
used to induce apoptosis [29], indicating that Neisseria can
also inhibit the extrinsic pathway, and therefore could
possess several mechanisms to inhibit apoptosis.
Future research will be needed to address the varying
results between the two species of Neisseria. Perhaps N.
meningitidis also requires NF-kB activation. It is also
possible that despite the close relatedness of the two
species, these organisms use quite different strategies
to inhibit apoptosis. Identification of the eukaryotic
proteins that bind to PorB and porin IB is required to
determine if these porins function differently to inhibit
apoptosis. Experiments in which the porins are switched
between the two species of Neisseria could beperformed. In
any event, Neisseria and Chlamydia are prototypes of
bacteria that inhibit apoptosis by preventing cytochrome
c release through the secretion of a bacterial product.
Activation of cell survival pathways
The second mechanism by which bacteria inhibit apoptosis
is through exploitation of the cell survival pathways natu-
rally present in the host. Salmonella, Anaplasma, Ehrli-
chia, Rickettsia, Wolbachia and Bartonella are examples of
bacteria with this ability. Salmonella enterica serovar
Typhimurium inhibits camptothecin-induced apoptosis
in HeLa and rat small intestine epithelial cells by activat-
ing the PI3K/Akt pathway [30]. This pathway prevents
cytochrome c release, which inhibits activation of the
caspase cascade. Akt phosphorylation and activation
during infection occurs due to secretion of a type III
secretion system (T3SS) effector, SopB. A DsopB mutant
is unable to prevent camptothecin-induced apoptosis, and
Akt is not activated in cells infected with this mutant [30].
Future studies on SopB should determine if this Salmo-
nella protein functions like eukaryotic proteins that acti-
vate the PI3K/Akt pathway, for example, Cox-2. These
studies will help to further the understanding of the role
of this bacterial protein in apoptosis inhibition, and could
enable identification of other bacterial proteins that func-
tion in a similar manner. Finally, it is interesting to note
that Salmonella induces cell death in macrophages [31],
which enables the bacteria to escape immune cells. There-
fore, Salmonella infection of a eukaryotic cell leads to
differentoutcomesdependingonwhichcelltypeisinfected.
Anaplasma phagocytophilum prevents apoptosis in
neutrophils [32–34]. No inducer of apoptosis is required
because neutrophils isolated from human donors spon-
taneously undergo apoptosis by 24 h in cell culture, and
A.phagocytophiluminfectionoftheseneutrophilsprevents
the spontaneous apoptosis normally observed [32]. Protein
and gene expression studies showed that the p38 mitogen-
activated protein kinase (MAPK) pathway is activated by
the bacteria during infection. p38 MAPK activation leads
to the inhibition of caspase-3 activation, but other sig-
naling pathways might also be involved [32]. Not only is
p38 MAPK upregulated, but the extracellular signal-
regulated kinase (ERK), PI3K/Akt and NF-kB pathways
are also upregulated [33]. A bacterial product has not been
identified for this upregulation of cell-survival pathways,
butactiveinfectionisrequiredbecauseheat-killedbacteria
do not prevent neutrophil apoptosis as efficiently as live
bacteria [33]. Identifying a bacterial protein required for
apoptosis inhibition could shed light on which of the above
pathways are essential for apoptosis inhibition. In
addition, A. phagocytophilum can inhibit the extrinsic
pathway of apoptosis by preventing caspase-8 cleavage
[34], and therefore ensures its survival by blocking both
pathways of apoptosis.
Three other obligate intracellular pathogens inhibit
apoptosis by activating cell survival pathways. First, Ehr-
lichia chaffeensis inhibits apoptosis in the human mono-
cyte cell line THP1 through upregulation of NF-kB, as
determined using microarray analysis. In addition, other
pro-survival genes such as bcl-2 and other bcl-2-related
genes are also induced during infection (Box 1) [35].
Further analysis is needed to verify the microarray data
and to identify the E. chaffeensis proteins required for
apoptosis inhibition. Next, the obligate intracellular
pathogen Rickettsia rickettsii prevents apoptosis in endo-
thelial cells in an NF-kB-dependent manner, which results
in the upregulation of pro-survival proteins, the down-
regulation of pro-apoptotic proteins, and a lack of cyto-
chrome c release and caspase activation [36,37]. It has not
been determined exactly how infection leads to NF-kB
activation, or what bacterial products are required to
inhibit apoptosis. Future studies identifying the bacterial
protein required for NF-kB activation will not only be
important to understand the strategy used by R. rickettsii,
butwillenablecomparisonswithotherbacterialpathogens
that depend on NF-kB activation for the inhibition of
apoptosis.Finally,Wolbachiaisanendosymbiontoffilarial
nematode parasites, and inhibits apoptosis in these nema-
todes [38]. The presence of Wolbachia is important during
parasitic infections in humans. Interestingly, the Wolba-
chia surface protein inhibits apoptosis in human neutro-
phils. Only the lack of caspase-3 activation was reported
[39], and additional studies are clearly needed to deter-
mine how the surface protein is anti-apoptotic. The
Wolbachia surface protein could serve as a means to
identify the proteins required for apoptosis inhibition in
Box 1. The Bcl-2 family of proteins
The Bcl-2 family of proteins is defined by the presence of at least
one Bcl-2 homology (BH) domain. The pro-survival proteins (Bcl-2,
Bcl-XL, Mcl-1, Bfl-1/A1, Bcl-W and Boo/Diva) comprise the Bcl-2
subfamily [63,64]. Bcl-2, Bcl-XL and Bcl-W have a hydrophobic
domain on the carboxyl terminus that targets the proteins to the
cytoplasmic face of the outer mitochondrial membrane, the
endoplasmic reticulum and the nuclear envelope. Bcl-2 is an integral
membrane protein whereas Bcl-XL and Bcl-W become tightly
associated with these membranes only after a cytotoxic signal
[63]. The pro-apoptotic proteins are broken down into two
subfamilies: the Bax subfamily in which the proteins have multiple
BH domains (Bax, Bak, Bok and Bcl-XS) and the BH3-only subfamily,
which comprise proteins with a single BH3 domain (Bid, Bad, Bmf,
Bim, Bik, Puma, Hrk and Noxa) [63,64]. Both subfamilies of pro-
apoptotic proteins are required for apoptosis initiation. The BH3-
only proteins are direct antagonists and act by binding to and
inhibiting the pro-survival proteins in response to apoptotic signals.
Once the BH3-only proteins neutralize Bcl-2, Bcl-XLand Bcl-W, the
pro-apoptotic Bax and Bak proteins form heterodimers and homo-
dimers within the mitochondrial membrane, which results in the
release of cytochrome c and other pro-apoptotic factors [63,64].
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Anaplasma and Ehrlichia through homology studies
because these organisms are closely related.
It is intriguing to note that not all obligate intracellular
pathogens inhibit apoptosis in a similar manner. As men-
tioned above, Chlamydia utilizes a different strategy to
prevent apoptosis during infection. One key difference
might be because Chlamydia remains inside a vacuole
during infection whereas other obligate intracellular
pathogens, such as Rickettsia, replicate in the cytoplasm
of the host cell. Perhaps future studies will determine if
thisdifferencecorrelateswiththedifferencesseenbetween
apoptosis inhibition for these organisms.
Bartonellahenselaeactivates
increased expression of cIAP-1 and cIAP-2 and inhibition
of caspase-3 activation and apoptosis [40]. Mitochondrial
integrity and the presence or absence of cytochrome c
release have not been tested. The B. henselae outer mem-
brane adhesin A (BadA) was identified as the bacterial
product required for the inhibition of apoptosis [40]. How-
ever, B. henselae and Bartonella quintana also inhibit
apoptosis in vascular endothelial cells through the BepA
protein, which is secreted through the type IV secretion
system [41]. Although infection activates NF-kB, BepA
translocates to the plasma membrane and increases the
cyclic adenosine monophosphate (cAMP) levels in the cell,
which results in the anti-apoptosis activity. cAMP is
known to increase the expression of cIAP-2, activate
protein kinase A (PKA) to inhibit the pro-apoptotic Bad
protein, and activate the ERK and p38 MAPK pathways
[41]. It was not determined if cIAP-2, PKA, ERK or p38
MAPK were affected by the BepA-dependent increase in
cAMP levels. The differences in results between the two
studies might be attributed to differences in cell types
used, but future studies should determine if one or both
of the Bartonella proteins are required for protection. B.
henselae and B. quintana cause human infections in which
angioproliferative lesions can occur. Interestingly, Barto-
nella vinsonii, Bartonella elizabethae and Bartonella clar-
ridgeiae are not associated with disease and do not inhibit
apoptosis in endothelial cells in the presence of actinomy-
cin D, a chemical inducer of apoptosis [42]. This obser-
vation further illustrates how bacterial pathogens have
evolved to inhibit apoptosis to establish a replicative niche
inside the host. Bartonella, along with Salmonella, Ana-
plasma, Ehrlichia, Wolbachia and Rickettsia, have found
ways to inhibit apoptosis through the exploitation of the
host cell survival pathways that normally function to block
apoptosis. This strategy of apoptosis inhibition is an indir-
ect but effective approach to prevent apoptosis during
infection.
NF-kB,leadingto
Interaction with cellular caspases
Unlike the two previous strategies, Shigella flexneri uses a
noveltactictoinhibitapoptosis.S.flexneriinhibitsapoptosis
in epithelial cells but causes cell death in macrophages,
which is a dichotomous trait it shares with Salmonella [43].
However,S.flexneriisabletopreventapoptosisinepithelial
cells downstream of cytochrome c release [44]. Upon in-
fection of HeLa cells and T84 cells, S. flexneri inhibits
STS-induced apoptosis by preventing the activation of cas-
pase-3.Cytochromecreleaseandcaspase-9activationoccur
in the presence of STS in infected HeLa cells; however,
caspase-3 activation is prevented. When STS is not used,
cytochrome c release is not detected, indicating that the
bacteria do not damage the mitochondria upon normal in-
fection. A DmxiE mutant of S. flexneri is unable to prevent
STS-induced apoptosis in infected HeLa cells. MxiE is a
transcriptional activator that induces the expression of 17
Shigella genes, whose gene products are secreted into the
cytoplasm of the hostcell through the T3SS[44]. The MxiE-
regulated genes required to inhibit apoptosis remain to be
identified. S. flexneri might blockapoptosis bytargeting the
activatedformofcaspase-9ortheinactiveformofcaspase-3
to prevent caspase-3 activation. In addition, preliminary
analysis with TRAIL (TNF-a-related apoptosis-inducing
ligand), an inducer of the extrinsic pathway, has shown
that S. flexneri is able to prevent apoptosis induced via this
pathway (C. Faherty, unpublished). This suggests that S.
flexneri inhibits both pathways of apoptosis by inhibiting
caspase-3 activation. It has also been reported that the
Shigella effector IpgD, which is homologous to the Salmo-
nella effector SopB (see above), activates the PI3K/Akt
pathway like Salmonella [45]. Although the activation of
thispathwaymighthavepro-survivalaffectsuponinfection,
it is not the primary mechanism of apoptosis inhibition
duringShigellainfection.IpgDactivatesthePI3K/Aktpath-
wayduringthefirsthourofinfection,whereasSopBinduces
a more sustained activation of the pathway for four hours
[30,45].Finally,aDipgDmutantisstillabletopreventSTS-
induced apoptosis [39].
Legionella pneumophila seems to exploit the activation
of caspase-3 during intracellular replication in macro-
phages while at the same time preventing apoptosis until
the latestages of infection[46].L. pneumophila utilizes the
Dot/Icm type IV secretion system to activate caspase-3,
which in turn enables evasion of the endosomal-lysosomal
pathway through an unidentified mechanism [46]. L. pneu-
mophila is then free to grow inside a replicative niche
derived from the rough endoplasmic reticulum [46]. The
caspase-3 activation is independent of the intrinsic or
extrinsic pathways of apoptosis. During late stages of in-
fection, L. pneumophila escapes into the cytoplasm of the
host cell, and then apoptosis facilitates bacterial escape
and egress [46]. Apoptosis inhibition during early infection
despite caspase-3 activation is achieved through the acti-
vation of NF-kB, which is also dependent on the Dot/Icm
system [47]. Furthermore, the bacterial effector SdhA,
which is secreted by the Dot/Icm system, is required for
apoptosis inhibition [48]. L. pneumophila provides a fas-
cinating example of a bacterial pathogen that exploits
caspase-3 activation for replication while at the same time
inhibiting cell death, until the bacteria no longer need the
cell.
Shigella and Legionella are the first examples of bac-
teria that affect caspase-3 activation. The parasite Toxo-
plasma gondii also inhibits apoptosis by targeting caspase-
3 [49], and it will be interesting to see if S. flexneri uses the
same strategy as T. gondii. The consequence of this form of
protection is that the host cells are damaged when certain
apoptoticstimuliarepresent,becausecytochromecrelease
indicates that the mitochondria are no longer intact. Shi-
gella is a facultative intracellular pathogen and, therefore,
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it might not require prolonged apoptosis inhibition for
survival in vivo. This situation is different from obligate
intracellular pathogens such as Chlamydia in that these
organisms require longer apoptosis inhibition for their
longer replication cycle inside the host cell. Nevertheless,
the ability of pathogens to inhibit apoptosis at the point of
caspase-3 activation seems to be ideal for protecting the
host cells from both pathways of apoptosis.
Concluding remarks and future directions
An emerging theme in bacterial pathogenesis is the ability
of bacteria to inhibit apoptosis in eukaryotic cells to estab-
lish a replicativeniche inside the host.Bacterial pathogens
employ at least three different mechanisms to inhibit
apoptosis.Mitochondrialpermeabilizationandsubsequent
cytochrome c release are important events in apoptosis,
and also serve as a means to differentiate the various
mechanisms used to inhibit apoptosis. Bacterial pathogens
can secrete proteins to protect the mitochondria, upregu-
late cell survival pathways to prevent cytochrome c
release, or ignore this step and focus attention on the
caspases to inhibit apoptosis. Related organisms such as
A.phagocytophilumandE.chaffeensisusesimilarmethods
to inhibit apoptosis, whereas other related organisms such
as N. meningitidis and N. gonorrhoeae utilize different
mechanisms to inhibit apoptosis. Clearly, the method
employed by each bacterial strain does not have to be
the same, and each method probably represents the most
efficient means of inhibiting apoptosis for that pathogen in
that particular cell type.
Natural selection has a key role in determining how
each pathogen inhibits apoptosis. As the host evolves to
eliminate a bacterial infection, the pathogen must itself
evolvetocounteractthischangetomaintainthereplicative
niche inside the host. It would be constructive if future
researchcouldidentifykeyfactorsthatdictatethechoiceof
strategy. Does it depend on which cell type the pathogen
infects? Does it depend on how the pathogen enters that
cell type? Does it depend on whether the bacteria replicate
inside a vacuole or in the cytoplasm? Do homologous
proteins in different organisms prevent apoptosis in the
same manner? Do all obligate intracellular pathogens
inhibit apoptosis? Answers to these questions will further
our understanding of apoptosis inhibition as a facet of
bacterial pathogenesis and could lead to novel thera-
peutics. For example, an inhibitor that targets CPAF in
Chlamydia could enable the host cell to undergo apoptosis
inresponsetoinfectionandmighthaltpersistentinfection.
This inhibitor could therefore complement antibiotic
therapy. As new pathogens are discovered, the ability to
inhibit apoptosis should be considered as a feature of
pathogenicity of the organism. More importantly, known
pathogens should also be re-examined, especially given
that apoptosis inhibition was only recently reported for
S. flexneri.
There are two recurrent themes in bacterial pathogens
inhibiting apoptosis. First, many pathogens rely on NF-kB
activation to inhibit apoptosis. The Gram-negative bac-
terial cell surface component lipopolysaccharide (LPS)
activates the NF-kB pathway during infection [50].
Because NF-kB activation has many pro-survival effects
on the host cell, as outlined here, activation of the NF-kB
pathway by LPS might be a simple explanation of how
most bacteria inhibit apoptosis. Binnicker et al. [27]
addressed this aspect in their research with N. gonor-
rhoeae. When purified lipooligosaccharide (LOS) was
applied to the surface of host cells, there was no increase
in the expression of the pro-survival gene bfl-1. In addition,
infection of host cells with an LOS mutant resulted in the
same induced expression of bfl-1 as that seen in wild-type
bacteria. The authors speculated that different bacterial
products could promote the formation of different Rel/NF-
kB complexes during activation, which would interact with
different transcription factors and regulatory proteins to
regulate the transcription of a completely distinct set of
genes [27]. In addition, live extracellular L. pneumophila
and formalin-killed bacteria do not trigger NF-kB acti-
vation, further suggesting that the anti-apoptotic pheno-
typeinducedbypathogensthroughNF-kBactivationis not
mediated by LPS [47]. Understanding how NF-kB acti-
vation affects the ability of a pathogen to prevent apoptosis
needs to be further elucidated.
The second recurrent theme is the fact that most of
these studies use immortalized cell lines and chemical
inducers of apoptosis, especially in the studies involving
epithelial cells. The chemical inducers enable researchers
to apply an apoptotic stimulus in culture conditions to
study apoptosis inhibition. Most immortalized cell lines
already have a pro-survival phenotype. Positive controls,
that is, uninfected cells treated with the stimulus, enable
researchers to demonstrate that the bacterial pathogen
doesinfactinhibitapoptosisduringinfection.Thesestrong
stimuli can overcome the pro-survival phenotype of the
immortalized cell line. However, many of these stimuli are
not physiologically relevant. Therefore, researchers should
attempt to use more physiologically relevant conditions,
particularly after initial observations are made. For
instance, the use of primary human cells from the relevant
site of infection should be performed to confirm obser-
vations of apoptosis inhibition. Moreover, mimicking apop-
totic stimuli that these cells would normally encounter in
vivo should also be considered. For example, TNF-a is
secreted in response to many infections [51], and it induces
the extrinsic pathway of apoptosis. In addition, transmi-
gration of polymorphonuclear (PMN) leukocytes induces
apoptosis in T84 intestinal epithelial cells [52]. PMN
transmigration occurs during Shigella and Neisseria infec-
tions. Therefore, mimicking physiologically relevant con-
ditions is not only possible, but important for researchers
to enable an appropriate understanding of apoptosis inhi-
bition during infection.
Another significant area of research will be to determine
if the ability to inhibit apoptosis is associated with the
carcinogenic potential of the bacterial pathogen. Helicobac-
ter pylori [53] and B. henselae inhibit apoptosis, and these
organisms cause gastric carcinoma and bacillary angioma-
tosis, respectively. Furthermore, Chlamydia seems to
stimulate mitosis in infected cells in addition to inhibiting
apoptosis. Do certain organisms have the potential to cause
cancer through apoptosis inhibition? Some viruses with the
ability to inhibit apoptosis do cause cancer. An example is
theEpstein–Barrvirus,whichpromotesanti-apoptosisinB
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Page 7

lymphocytes and can cause Burkitt’s lymphoma [54].
Drawing connections between apoptosis inhibition and
thedevelopmentofcancerwillbeimportantforunderstand-
ing the full potential of bacterial pathogens.
Clearly, apoptosis inhibition is an emerging theme in
bacterial pathogenesis. Prevention of apoptosis enables
bacteria to replicate and survive in an environment that
is otherwise detrimental to the pathogen. This review clas-
sifies the different mechanisms of apoptosis inhibition into
three main categories, but other methods might exist. We
are only starting to understand the mechanisms employed
by bacteria to inhibit apoptosis and the importance of such
survival strategies to the pathogens. A clear understanding
ofthemolecularbasisofapoptosisinhibitionisneeded.With
all the outstanding questions that remain, future research
couldrevealthatbacterialinhibitionofapoptosisisfarmore
important than we currently suspect.
Acknowledgements
We thank the members of the Maurelli laboratory for their input in
discussions regarding the inhibition of apoptosis by bacterial pathogens.
We thank the Uniformed Services University and the Henry M. Jackson
Foundation for the Val Hemming Fellowship awarded to C.S.F.
Additional funding is provided by the National Institutes of Allergy and
Infectious Diseases grant AI24656. The opinions or assertions contained
herein are the private ones of the authors and are not to be construed as
official or reflecting the views of the Department of Defense or the
Uniformed Services University of the Health Sciences.
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