Inhibition of MAPK and NF-B Pathways Is Necessary for
Rapid Apoptosis in Macrophages Infected with Yersinia
1
Yue Zhang,* Adrian T. Ting,
‡
Kenneth B. Marcu,
†§
and James B. Bliska
2
*
Macrophages respond to infection with pathogenic Yersinia species by activating MAPK- and NF-B-signaling pathways. To
counteract this response, Yersiniae secrete a protease (Yersinia outer protein J (YopJ)) that is delivered into macrophages,
deactivates MAPK- and NF-B-signaling pathways, and induces apoptosis. NF-B promotes cell survival by up-regulating ex-
pression of several apoptosis inhibitor genes. Previous studies show that deactivation of the NF-B pathway by YopJ is important
for Yersinia-induced apoptosis. To determine whether deactivation of the NF-B pathway is sufficient for Yersinia-induced apo-
ptosis, two inhibitors of the NF-B pathway, IB superrepressor or A20, were expressed in macrophages. Macrophages ex-
pressing these proteins were infected with Yersinia pseudotuberculosis strains that secrete functionally active or inactive forms of
YopJ. Apoptosis levels were substantially higher (5- to 10-fold) when active YopJ was delivered into macrophages expressing IB
superrepressor or A20, suggesting that deactivation of the NF-B pathway is not sufficient for rapid Yersinia-induced apoptosis.
When macrophages expressing A20 were treated with specific inhibitors of MAPKs, similar levels of apoptosis (within 2-fold)
were observed when active or inactive YopJ were delivered during infection. These results suggest that MAPK and NF-B
pathways function together to up-regulate apoptosis inhibitor gene expression in macrophages in response to Yersinia infection and
that YopJ deactivates both pathways to promote rapid apoptosis. In addition, treating macrophages with a proteasome inhibitor
results in higher levels of infection-induced apoptosis than can be achieved by blocking NF-B function alone, suggesting that
proapoptotic proteins are stabilized when proteasome function is blocked in macrophages. The Journal of Immunology, 2005,
174: 7939 –7949.
Y
ersinia pestis, Yersinia pseudotuberculosis, and Yersinia
enterocolitica are the three Yersinia strains pathogenic to
humans. These bacteria share a 70-kb plasmid, which is
required for virulence. This plasmid encodes a type III secretion
system and a set of secreted effector proteins that antagonize
phagocytosis and cytokine production in macrophages (1). One of
the effector proteins, known as Yersinia outer protein (Yop)J
3
in Y.
pestis and Y. pseudotuberculosis and YopP in Y. enterocolitica, is
required by all three pathogenic Yersinia species to provoke apo-
ptosis of macrophages (Refs. 2 and 3; Y. Zhang and J. B. Bliska,
unpublished data). YopJ shares sequence similarity with a family
of avirulence proteins found in bacterial pathogens of plants or
animals (4). The YopJ-related avirulence proteins secreted by plant
pathogens are responsible for inducing the hypersensitive re-
sponse, an apoptosis-like process in plant cells. At the molecular
level, YopJ is required for Yersinia to deactivate multiple MAPK
pathways (5–7) and the transcription factor NF-B pathway (8, 9).
During infection of macrophages by Yersinia, TLR4 plays an
important role in activating MAPK and NF-B pathways and in
triggering the apoptotic response (10, 11). Macrophages use TLR4
to sense LPS, the major cell wall component of all Gram-negative
bacteria. Macrophages deficient in TLR4 function are more resis-
tant to Yersinia-induced apoptosis than their wild-type (WT) coun-
terparts (10, 11). TLR4 uses the TIR domain-containing adaptor
inducing IFN- adaptor to activate downstream apoptotic path-
ways (12, 13), whereas activation of NF-B and MAPK pathways
by TLR4 involves other adaptors (reviewed in Ref. 14). Under
resting conditions, IB sequesters NF-B in the cytoplasm. In
macrophages treated with LPS or infected with bacteria, IB is
phosphorylated by IB kinase (IKK) and subsequently is
polyubiquitinated and degraded by the proteasome. This frees
NF-B to translocate to the nucleus to promote transcription of
target genes, including IB (15). LPS stimulation or bacterial
infection of macrophages also leads to phosphorylation and acti-
vation of MAPKs, including ERK, JNK, and p38, by the respective
MAPK kinases (MKKs) (5, 6).
The mechanism by which YopJ deactivates signaling pathways
involving multiple MAPKs and NF-B is under intensive study. It
was found that YopJ interacts with multiple members of the MKK
family, as well as IKK (16). YopJ prevents activation of MKK
and IKK when expressed ectopically in mammalian cells (16).
Sequence analysis predicts structural similarity between YopJ and
a class of cysteine proteases that act on ubiquitin-like (Ubl) pro-
teins (17). Indeed, YopJ contains a conserved catalytic triad of His,
Glu, and Cys residues that are found in other members of this
protease family, such as the yeast Ubl-specific protease 1 and ad-
enovirus-2 protease. Mutations that disrupt the predicted catalytic
site of YopJ abolish its ability to inhibit signaling and to trigger
apoptosis (17). Orth et al. (17) have proposed that YopJ cleaves a
Departments of *Molecular Genetics and Microbiology, and Center for Infectious
Diseases, and
†
Biochemistry and Cell Biology, Institute for Cell and Developmental
Biology, State University of New York, Stony Brook, NY 11794;
‡
Immunobiology
Center, Mount Sinai School of Medicine, New York, NY 10029; and
§
CRBA Lab-
oratory, St. Orsola University Hospital, University of Bologna, Bologna, Italy
Received for publication December 9, 2004. Accepted for publication April 12, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work is supported National Institutes of Health Grants AI43389 and AI53759
(to J.B.B.) and GM066882 (to K.B.M.). K.B.M. is a Senior Scholar of the Institute of
Advanced Studies of the University of Bologna.
2
Address correspondence and reprint requests to Dr. James B. Bliska, Center for
Infectious Diseases, State University of New York, Stony Brook, NY 11794-5222.
E-mail address: jbliska@ms.cc.sunysb.edu
3
Abbreviations used in this paper: Yop, Yersinia outer protein; WT, wild type; IKK,
IB kinase ; MKK, MAPK kinase; IB SR, IB superrepressor; Km, kanamycin;
BMDM, bone marrow-derived macrophage; LDH, lactate dehydrogenase; Ct, thresh-
old; Tnfaip3, TNF--inducted protein 3; RIP, receptor-interacting protein; qRT-PCR,
qualitative RT-PCR; DUB, deubiquitination enzyme.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00