PerspectiveHost-Pathogen Interactions

Bacterial-Modulated Signaling Pathways in Gut Homeostasis

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Science Signaling  27 May 2008:
Vol. 1, Issue 21, pp. pe24
DOI: 10.1126/stke.121pe24

Abstract

Symbiotic mutualism with gut microbes occurs in all metazoans, and it is well established that commensal bacteria influence multiple aspects of host gut physiology such as innate immunity and development. However, our understanding of these coevolved interactions between prokaryotes and eukaryotes remains unclear. One mechanism by which commensal bacteria modulate host intracellular signaling pathways in order to avoid excess inflammation has now been determined. In this process, bacterial-induced reactive oxygen species in gut epithelial cells act as key messengers that inhibit the cullin-1–dependent protein degradation machinery, which in turn results in the stabilization of a master negative regulator of inflammation, inhibitor of nuclear factor-κB (IκB). Furthermore, this bacterial-mediated system also appears to be involved in the stabilization of a key developmental regulator, β-catenin. These findings provide new insights into the molecular mechanisms by which commensal microbes shape host cellular physiology.

The gut epithelia of all metazoans have evolved to form mutually beneficial relationships with microorganisms. These stable interactions between microbes and the alimentary epithelia of animals began more than 1 to 2 billion years ago. The human intestine contains a microbial community of enormous number and diversity, consisting of ~1014 cells from over 500 species of prokaryotic organisms (1). More than 100 years ago, Elie Metchnikoff suggested that certain microflora have beneficial roles in the physiology of gut epithelia and in host fitness (2). Indeed, several studies have now shown that commensal microbes profoundly modulate the expression of many host genes involved in diverse functions, thereby actively shaping host physiology (38). However, numerous important questions still remain unanswered. For example, how and why does the host gut immune system tolerate the existence of commensal microbes? The mechanisms by which commensal microbes positively affect gut cell development are also not understood. Finally, the modulation by commensal microbes of diverse signaling pathways that are essential for host gut homeostasis has yet to be dissected at the molecular level. Our understanding of the interaction between the gut and commensal microbes is limited, primarily because of a lack of knowledge of the signaling pathway(s) that link commensal microbes to host physiology. Kumar et al. now suggest that commensal bacterial–induced reactive oxygen species (ROS) generated in gut epithelial cells act as key messengers that modulate the protein-degradation machinery of several essential signaling components, which in turn influences diverse physiological processes of the host cells (9).

Inflammation is usually initiated when host cells come in contact with microbes and is dependent on the activation of the evolutionarily conserved nuclear factor κB (NF-κB) signaling pathway (10). NF-κB functions as the master transcription factor that controls the expression of genes encoding various inflammatory mediators such as cytokines. In the absence of an immune-activating signal, NF-κB exists in the cytoplasm in a complex with inhibitor of NF-κB (IκB). Upon signaling activation, IκB is phosphorylated by IκB kinase (IKK), and the phosphorylated form of IκB is subsequently subjected to ubiquitin-dependent protein degradation. The freed NF-κB translocates to the nucleus, where it promotes the transcription of various target genes that initiate the host inflammatory response. Commensal microbes produce many molecules that contain pathogen-associated molecular patterns, such as lipopolysaccharide and peptidoglycan, which normally provoke NF-κB–dependent inflammation (11). However, it is well known that commensal microbes coexist with the gut epithelia without causing much, if any, inflammation. An increasing number of studies suggest that commensal microbes use multiple strategies to modulate NF-κB signaling (9, 1215). For example, bacterial-mediated inhibition of the NF-κB pathway in human intestinal cells is achieved by promoting nuclear-cytoplasmic redistribution of NF-κB through the peroxisome proliferator-activated receptor-γ pathway (12). In addition, in the gut epithelia of Drosophila, the expression of NF-κB target genes, such as those that encode antimicrobial peptides, is repressed by the homeobox gene Caudal, which enables preservation of both the structure of the normal microbial community and the health of the host (14, 15). Thus, modulation of NF-κB signaling appears to be a common theme in ensuring mutualism between gut microbes and the host. In support of this view, Kumar et al. have described a mechanism involving a specific block of the polyubiquitination of IκB that was mediated by bacterial-induced production of ROS by host cells, which resulted in the inhibition of the NF-κB pathway (9).

Gut epithelial cells generate ROS (1618), and intestinal redox balancing through the dynamic generation and elimination of ROS is critical for gut immune homeostasis in Drosophila during continuous gut-microbe interactions (18, 19). ROS are unstable and diffusible molecules that often act as intracellular messengers to transiently modulate various signaling pathways (20, 21). Several studies have shown that endogenous H2O2 alters the signaling potential of cells through the oxidative inactivation of critical signaling proteins that harbor ROS-sensitive, low-pKa Cys residues (20, 21). One of the best-known examples of this phenomenon is the inhibition, in response to peptide growth factors, of protein tyrosine phosphatases that contain a low-pKa Cys residue at their active sites (2022). Furthermore, catalytic Cys residues in the E1 and E2 enzymes responsible for the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to target proteins are also subject to redox control (23). Because ubiquitination and SUMOylation play key roles in affecting the stability and/or activity of target signaling molecules (24), the spatial and temporal control of ROS production could act as an essential determinant of diverse cellular signaling pathways. Kumar et al. showed that mouse gut epithelia produced ROS within minutes of coming into contact with enteric commensal bacteria (9). It is important to note that bacterial-stimulated ROS production inhibited the neddylation of cullin-1 [the covalent attachment of the ubiquitin-like NEDD8 protein to a Lys residue of cullin-1 (25, 26)] in the mucosa of mice within 30 min of contact between the bacteria and epithelial cells (9). Cullins are essential scaffold molecules that together with other proteins form multisubunit E3 ubiquitin ligases, so-called Cullin-RING ligase complexes. Among the Cullin-RING ligase superfamily, Skp1-Cdc53/cullin-F box (SCF) ubiquitin ligase contains the cullin-1 isotype out of the seven known cullin members (24). Neddylation of cullin-1 is required for the correct assembly of enzymatically active SCF complexes (24), and the NEDD8-conjugating E2 enzyme, Ubc12, enables cullin-1 neddylation by transferring NEDD8 (attached through a thioester bond to the catalytic Cys111 residue of Ubc12) to the Lys site of cullin-1 (27). Kumar et al. elegantly showed that Cys111 of Ubc12 was sensitive to ROS and that oxidation of Cys111 by bacterial-induced ROS prevented formation of the NEDD8-Ubc12 thioester complex and caused the subsequent inactivation of the neddylation machinery, which in turn resulted in a shift of equilibrium toward the cullin-1 deneddylation state in the bacteria-laden gut epithelia (9). Given that the bacterial-modulated abundance of neddylated cullin-1 correlates directly with the activity of the E3-SCFβ-TrCP ligase complex (28) and that the NF-κB–dependent gut inflammatory response is largely dependent on E3-SCFβ-TrCP–controlled IκB stability (13), ROS-mediated control of the cullin-1 neddylation and deneddylation cycle provides a mechanistic link between commensal microbes and gut immune homeostasis (Fig. 1).

Fig. 1.

Schematic model of commensal bacterial-modulated signaling pathways in host cells. Cullin-1 neddylation by Ubc12 is required for the correct assembly of the enzymatically active SCF complex. Cullin-1–associated and neddylation-dissociated protein-1 (CAND1) binds to the deneddylated form of cullin-1 and sequesters it in an unassembled inactive state. Under inflammatory conditions, ubiquitin-dependent degradation of IκB is assured by an active SCF complex. After degradation of IκB, the nuclear-translocated NF-κB promotes the activation of various inflammatory genes. The SCF-dependent degradation pathway is also involved in maintaining the low abundance of β-catenin in the absence of signals that trigger stabilization of β-catenin. After contact with gut epithelial cells, commensal bacteria induce the production of ROS, which act as second messengers that inhibit the cullin-1–dependent protein degradation machinery. This process is mediated by oxidative inactivation of Ubc12, which contains a ROS-sensitive low-pKa Cys residue. This event prevents the formation of NEDD8-Ubc12 thioester complexes and subsequently inactivates the neddylation machinery. The consequent shift of equilibrium toward the cullin-1 deneddylation state leads to disassembly and inactivation of the SCF complex, which involves Skp1 and F-box–protein displacement and CAND1 binding. Finally, the reduced potential of protein degradation machinery promotes the stabilization of ΙκB and β-catenin, which enables the maintenance of gut homeostasis, including the reduction of gut inflammation and the promotion of gut development.

Bacterial-regulated stabilization of IκB through the oxidative inactivation of the cullin-1 neddylation machinery may explain how commensal microbes peacefully coexist within the gut epithelia without causing any detectable inflammation. In mammalian gut epithelia, dysregulation of the NF-κB signaling pathway is involved in the pathogenesis of chronic inflammatory bowel disease (29). The findings by Kumar et al. may provide an interesting explanation for the anti-inflammatory, probiotic effects of commensal bacteria on the many chronic inflammatory diseases frequently found in gut epithelia in contact with commensal bacteria. Furthermore, because the E3-SCFβ-TrCP complex controls the stability of a wide array of essential signaling players (24), redox-controlled and cullin-1–mediated signal modulation may not be limited to inflammatory signaling pathways. Indeed, nonpathogenic enteric bacteria attenuate the ubiquitination of β-catenin by inhibiting E3-SCFβ-TrCP activity (30). Given that β-catenin is a multifunctional transcriptional coactivator involved in diverse cellular processes including cell proliferation, bacterial-mediated and redox-dependent modulation of host cell signaling may expand beyond the NF-κB pathway to include β-catenin signaling (Fig. 1).

Several important questions remain unanswered. For example, the ROS-generating machinery stimulated by commensal bacteria in the gut has yet to be identified. The results of other studies suggest that the nicotinamide adenine dinucleotide phosphate (NADP)H oxidase (NOX) family, notably NOXs and dual oxidases (DUOXs), are primarily involved in the generation of ROS by various nonphagocytic cells, including those of the gut epithelia (1618, 31). Indeed, Duox is involved in microbe-induced ROS generation in vivo in Drosophila (18). Direct demonstration of the involvement of specific isoforms of Nox, Duox, or both in bacterial-induced intestinal ROS generation using a vertebrate knockout model is the next important step in this field. It is unlikely that only commensal microbes elicit ROS production in the gut epithelia. Also, certain commensal bacteria are likely to be more potent ROS-inducers than others (9). It is tempting to speculate that beneficial combinations of commensal microbes dynamically produce ROS at a tightly controlled adequate abundance that allows gut homeostasis, whereas harmful combinations of commensal microbes (due to genetic deficiency in the host or other environmental factors such as antibiotic treatments) or pathogens stimulate the production of ROS in pathophysiological quantities. However, the bacterial-derived agonists that initiate intestinal ROS generation are currently unknown. Elucidation of the molecular nature of such agonists will greatly enhance our understanding of bacterial-gut mutualism. Finally, because the complexity of bacterial-host mutualism can be analyzed meaningfully only in live animals, it is necessary to consider the use of simple genetic animal models to address specific questions. Studying bacterial-gut interactions using zebrafish and Drosophila model systems has already proven to be successful (14, 3234). Future progress in all of the aforementioned areas will allow us to better understand the mechanism by which bacterial-induced ROS and the subsequent modulation of gut signaling pathways impact the long-term physiology of the intestine and host fitness. Such an understanding may ultimately lead to the development of a novel and rational strategy for the treatment of chronic inflammatory diseases.

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