Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species.

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including beta-catenin and the NF-kappaB inhibitor IkappaB-alpha. Ubiquitination of these proteins as well as others is catalyzed by the SCF(betaTrCP) ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-kappaB and beta-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.

The bacterial fermentation product butyrate influences epithelial signaling via reactive oxygen species-mediated changes in cullin-1 neddylation.

The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-kappaB pathway by blockade of IkappaB-alpha ubiquitination, a process catalyzed by the E3-SCF(beta-TrCP) ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and resulted in suppressive effects on the NF-kappaB pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3- SCF(beta-TrCP) ubiquitin ligase, the NF-kappaB inhibitor IkappaB-alpha. Mechanistically, we show that physiological concentrations of butyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways.

CatM regulation of the benABCDE operon: functional divergence of two LysR-type paralogs in Acinetobacter baylyi ADP1.

Two LysR-type transcriptional regulators, BenM and CatM, control benzoate consumption by the soil bacterium Acinetobacter baylyi ADP1. These homologs play overlapping roles in the expression of multiple genes. This study focuses on the benABCDE operon, which initiates benzoate catabolism. At this locus, BenM and CatM each activate transcription in response to the catabolite cis,cis-muconate. BenM, but not CatM, additionally responds to benzoate as an effector. Regulation by CatM alone is insufficient for growth on benzoate as the sole carbon source. However, three point mutations independently increased CatM-activated benA transcription and enabled growth on benzoate without BenM. Two mutations generate variants with one amino acid change in the 303-residue CatM, CatM(V158M) and CatM(R156H). These substitutions affected regulation of benA differently than that of catB, another CatM-regulated gene involved in benzoate catabolism. In relation to CatM, CatM(V158M) increased cis,cis-muconate-dependent transcription of benA but decreased that of catB. CatM(R156H) increased effector-independent expression of catB compared to CatM. In contrast, cis,cis-muconate was required with CatM(R156H) to activate unusually high benA expression. Thus, induction by cis,cis-muconate depends on both the sequence of CatM and the promoter. A point mutation at position -40 of the benA promoter enhanced CatM-activated gene expression and altered regulation by CatM(R156H). BenM and CatM bound to the same locations on ben region DNA. The frequency with which spontaneous mutations allow CatM to substitute for BenM might predict that one regulator would be sufficient for controlling benzoate consumption. This prediction is discussed in light of current and previous studies of the BenM-CatM regulon.

Cutting edge: bacterial modulation of epithelial signaling via changes in neddylation of cullin-1.

The human enteric flora plays a significant role in intestinal health and disease. Certain enteric bacteria can inhibit the NF-kappaB pathway by blockade of IkappaB-alpha ubiquitination. IkappaB-alpha ubiquitination is catalyzed by the E3-SCF(betaTrCP) ubiquitin ligase, which is itself regulated via covalent modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Neddylation is a biochemical event associated with diverse cellular processes related to cell signaling, however, physiological regulation of cullin neddylation has not been described in mammalian systems. We report that interaction of nonpathogenic bacteria with epithelial cells resulted in a rapid loss of neddylated Cul-1 and consequent repression of the NF-kappaB pathway. This observation may explain the ability of intestinal bacterial communities to influence diverse eukaryotic processes in general and inflammatory tolerance of the mammalian intestinal epithelia specifically.

Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-kappa B pathway.

Secreted prokaryotic effector proteins have evolved to modulate the cellular functions of specific eukaryotic hosts. Generally, these proteins are considered virulence factors that facilitate parasitism. However, in certain plant and insect eukaryotic/prokaryotic relationships, effector proteins are involved in the establishment of commensal or symbiotic interactions. In this study, we report that the AvrA protein from Salmonella typhimurium, a common enteropathogen of humans, is an effector molecule that inhibits activation of the key proinflammatory NF-kappaB transcription factor and augments apoptosis in human epithelial cells. This activity is similar but mechanistically distinct from that described for YopJ, an AvrA homolog expressed by the bacterial pathogen Yersinia. We suggest that AvrA may limit virulence in vertebrates in a manner analogous to avirulence factors in plants, and as such, is the first bacterial effector from a mammalian pathogen that has been ascribed such a function.

Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella.

The gastroenteritis-causing pathogen Salmonella typhimurium induces profound transcriptional changes in intestinal epithelia resulting in the recruitment of neutrophils whose presence is the histopathologic hallmark of salmonellosis. Here we used cDNA microarray expression profiling to define the molecular determinants that mediate such changes in model intestinal epithelia. Enteropathogenic Salmonella induced a classical proinflammatory gene expression program similar to that activated by the canonical proinflammatory agonist TNF-alpha. Nonproinflammatory bacteria, both commensals (Escherichia coli) and systemic pathogens (S. typhi), did not activate this expression profile. While S. typhimurium strains lacking the SPI-1-encoded type III system were fully proinflammatory, strains lacking the genes for the flagellar structural component flagellin were nearly devoid of proinflammatory signaling. Lastly, the epithelial proinflammatory response could be largely recapitulated by basolateral addition of purified flagellin. Thus, S. typhimurium flagellin is the major molecular trigger by which this pathogen activates gut epithelial proinflammatory gene expression.

Lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis.

The anti-inflammatory eicosanoid lipoxin A(4) (LXA(4)), aspirin-triggered 15-epi-LXA(4), and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA(4) analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA(4) analog pretreatment attenuated induction of approximately 50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA(4) analog pretreatment is regulated by NF-kappaB, suggesting that LXA(4) analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA(4) analog reduced NF-kappaB-mediated transcriptional activation in a LXA(4) receptor-dependent manner and inhibited induced degradation of IkappaBalpha. LXA(4) analog did not affect earlier stimulus-induced signaling events that lead to IkappaBalpha degradation, such as S. typhimurium-induced epithelial Ca(2+) mobilization or TNF-alpha-induced phosphorylation of IkappaBalpha. To establish the in vivo relevance of these findings, we examined whether LXA(4) analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA(4) analog (15-epi-16-para-fluoro-phenoxy-LXA(4), 10 microg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA(4) analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-kappaB pathway can be therapeutic for diseases characterized by mucosal inflammation.