Subtilase Cytotoxin Enhances Escherichia coli Survival in Macrophages by Suppression of Nitric Oxide Production through the Inhibition of NF-κB Activation

Abstract

e Subtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenic Escherichia coli (STEC), cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress and caspase-dependent apoptosis. SubAB alters the innate immune response. SubAB pretreatment of macrophages inhibited lipopolysaccharide (LPS)-induced production of both monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor ␣ (TNF-␣). We investigated here the mechanism by which SubAB inhibits nitric oxide (NO) production by mouse macrophages. SubAB suppressed LPS-induced NO production through inhibition of inducible NO synthase (iNOS) mRNA and protein expression. Further, SubAB inhibited LPS-induced IB-␣ phosphorylation and nuclear localization of the nuclear factor-B (NF-B) p65/p50 heterodimer. Reporter gene and chromatin immunoprecipitation (ChIP) assays revealed that SubAB reduced LPS-induced NF-B p65/p50 heterodimer binding to an NF-B binding site on the iNOS promoter. In contrast to the native toxin, a catalytically inactivated SubAB mutant slightly enhanced LPS-induced iNOS expression and binding of NF-B subunits to the iNOS promoter. The SubAB effect on LPS-induced iNOS expression was significantly reduced in macrophages from NF-B1 (p50)-deficient mice, which lacked a DNA-binding subunit of the p65/p50 heterodimer, suggesting that p50 was involved in SubAB-mediated inhibition of iNOS expression. Treatment of macrophages with an NOS inhibitor or expression of SubAB by E. coli increased E. coli survival in macrophages, suggesting that NO generated by macrophages resulted in efficient killing of the bacteria and SubAB contributed to E. coli survival in macrophages. Thus, we hypothesize that SubAB might represent a novel bacterial strategy to circumvent host defense during STEC infection.

NF-κB activation via MyD88-dependent Toll-like receptor signaling is inhibited by trichothecene mycotoxin deoxynivalenol

“…MyD88-deficient mice are highly susceptible to infection with a broad range of pathogenic microorganisms (von Bernuth et al, 2008). In addition, NO contributes to protection against infection with bacteria including Mycobacterium tuberculosis, L. monocytogenes and certain strains of Shiga toxigenic E. coli (Boockvar et al, 1994;El Kasmi et al, 2008;Tsutsuki et al, 2012) and macrophage cells in response to LPS produce the mediator, which stimulates MyD88-dependent pathway via TLR4 (Jacobs and Ignarro, 2001;Akira et al, 2006). Taken together, in this report the results of MyD88 downregulation suggest that the trichothecene DON, which is frequently detected in cereals (Yazar and Omurtag, 2008), is able to trigger dysregulation of innate immune response mediated through the TLR-MyD88 signaling pathway, leading to impairment of the host systems responsible for protection against infectious diseases.…”

“…Nitric oxide (NO) produced by cells of the innate immunity system plays a role in protection against various pathogens (Boockvar et al, 1994;El Kasmi et al, 2008;Tsutsuki et al, 2012). Our previous study demonstrated that DON inhibited LPS-induced NO production by mouse macrophage cells and also reduced the LPSinduced reporter gene activity of interferon-β (IFN-β) which is essential for LPS-induced expression of inducible NO synthase (iNOS) gene (Sugiyama et al, 2010).…”

Recent Advances in Understanding Enteric Pathogenic Escherichia coli

“…The catalytic A subunit is a serine protease that cleaves BiP/GRP78, a regulator of the stress response in the ER, and ultimately leads to cell death. SubAB may also be involved in altering host immune responses, as it was shown to prevent antibody secretion from B cells (350) and to suppress NO in macrophages, leading to the hypothesis that it may promote the survival of STEC in macrophages (351).…”

DAP1, a Negative Regulator of Autophagy, Controls SubAB-Mediated Apoptosis and Autophagy

“…SubAB-dependent BiP cleavage is inhibited by brefeldin A (BFA), a Golgi complex-disrupting agent (15,16). SubAB-induced ER stress due to BiP cleavage causes activation of stress sensor proteins, followed by the induction of various cellular events leading to cell damage, e.g., transient inhibition of protein synthesis (17), G 0 /G 1 cell cycle arrest (15,17), caspase-dependent apoptosis via mitochondrial membrane damage (18), activation of Akt-NF-B signaling (19), downregulation of gap junction expression (20), activation of RNA-dependent protein kinase (PKR)-like ER kinase (PERK) followed by caspase-dependent apoptosis (12), and inhibition of lipopolysaccharide (LPS)-stimulated NO production through inhibition of NF-B nuclear translocation and inducible nitric oxide synthase (iNOS) expression (21).…”