Anthrax Lethal Toxin Increases Superoxide Production in Murine Neutrophils via Differential Effects on MAPK Signaling Pathways

Abstract

The combination of lethal factor and its receptor-binding partner, protective Ag, is termed lethal toxin (LT) and has critical pathogenic activity during infection with Bacillus anthracis. We herein report that anthrax LT binds and enters murine neutrophils, leading to the cleavage of mitogen-activated protein kinase kinase/MEK/MAPKK 1–4 and 6, but not mitogen-activated protein kinase kinase 5 and 7. Anthrax LT treatment of neutrophils disrupts signaling to downstream MAPK targets in response to TLR stimulation. Following anthrax LT treatment, ERK family and p38 phosphorylation are nearly completely blocked, but signaling to JNK family members persists in vitro and ex vivo. In contrast to previous reports involving human neutrophils, anthrax LT treatment of murine neutrophils increases their production of superoxide in response to PMA or TLR stimulation in vitro or ex vivo. Although this enhanced superoxide production correlates with effects due to the LT-induced blockade of ERK signaling, it requires JNK signaling that remains largely intact despite the activity of anthrax LT. These findings reveal a previously unrecognized mechanism through which anthrax LT supports a critical proinflammatory response of murine neutrophils.

Anthrax Lethal Toxin Inhibits Translation of Hypoxia-inducible Factor 1α and Causes Decreased Tolerance to Hypoxic Stress

“…2C), we next confirmed the proximal targets of LT activity in HepG2 cells. Consistent with our previous study (36), LT treatment caused cleavage of MKK1, MKK2, MKK3, MKK4, MKK6, but not MKK7 (Fig. 5A).…”

“…Cell extracts were then separated on 4 -12% NuPAGE BisTris gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad). The membranes were probed with antibodies of interest using standard Western blotting techniques as described previously (36). Western blotting films were scanned, and the intensity of protein bands was quantified using FluorChem TM Q software (Protein Simple Inc., San Jose, CA).…”

“…It should be noted that phosphorylation of RSK at multiple sites is required for normal kinase function (38), and its phosphorylation at Thr-573 depends on both ERK1/2 and ERK5 activity (39). It is likely that the more modest effect of LT in reducing phosphorylation at the Thr-573 site is due to persistent ERK5 signaling, as its upstream signaling component, MKK5, is resistant to the proteolytic effect of LT (36).…”

Inhibition of Interleukin 1β (IL-1β) Expression by Anthrax Lethal Toxin (LeTx) Is Reversed by Histone Deacetylase 8 (HDAC8) Inhibition in Murine Macrophages

“…PA is a carrier protein that incorporates LF into the cytoplasm; LF is a metalloprotease that cleaves all MAPK kinases (MEK1-7), except MEK5 (11) and MEK7 (12). Inactivation of these MEKs results in almost complete inactivation of MAPKs, including the ERKs and p38 MAPK (p38), but partial or no effects on JNK/SAPKs in macrophages (13,14) and other cell types (12,15). Inhibition of ERKs and p38 by LeTx suppresses expression of various inflammatory cytokines in macrophages (16 -18).…”

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

“…Additionally, purified ET reduces the neutrophil's oxidative burst. Recombinant LT's role in the neutrophil oxidative burst has also been examined with two discordant studies showing it either increases or decreases the oxidative burst in human neutrophils [199,200]. Purified LT also reduces the amount of proinflammatory cytokines and chemokines in NB-4 cells, a neutrophil-like immortal cell line [201,202].…”