C4-Dicarboxylates as Growth Substrates and Signaling Molecules for Commensal and Pathogenic Enteric Bacteria in Mammalian Intestine

Abstract

The C₄-dicarboxylates (C4-DC) l-aspartate and l-malate have been identified as playing an important role in the colonization of mammalian intestine by enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, and succinate as a signaling molecule for host-enteric bacterium interaction. Thus, endogenous and exogenous fumarate respiration and related functions are required for efficient initial growth of the bacteria. l-Aspartate represents a major substrate for fumarate respiration in the intestine and a high-quality substrate for nitrogen assimilation. During nitrogen assimilation, DcuA catalyzes an l-aspartate/fumarate antiport and serves as a nitrogen shuttle for the net uptake of ammonium only, whereas DcuB acts as a redox shuttle that catalyzes the l-malate/succinate antiport during fumarate respiration. The C4-DC two-component system DcuS-DcuR is active in the intestine and responds to intestinal C4-DC levels. Moreover, in macrophages and in mice, succinate is a signal that promotes virulence and survival of S. Typhimurium and pathogenic E. coli. On the other hand, intestinal succinate is an important signaling molecule for the host and activates response and protective programs. Therefore, C4-DCs play a major role in supporting colonization of enteric bacteria and as signaling molecules for the adaptation of host physiology.

Keywords: C4-dicarboxylates; Escherichia coli; Salmonella; Salmonella Typhimurium; fumarate respiration; initial growth; intestine colonization; l-aspartate; nitrogen assimilation; succinate.

FIG 1

Exogenous and endogenous fumarate respiration (FR) by E. coli. For endogenous FR (fumarate produced during hexose fermentation) up to 15% of the PEP formed during hexose fermentation (37) is carboxylated to yield OAA, which is then converted by the reductive branch of the anaerobic citric acid cycle to succinate. For exogenous FR, l-aspartate, l-malate, fumarate, l-tartrate, or citrate are taken up by antiporters from the medium, and succinate is excreted in an electroneutral antiport. Enzymes and feeding reactions for fumarate formation are shown in blue and red, respectively, joint reactions in green. Details are described in the text and in reviews (3, 8, 31, 35). At concentrations > 0.1 mM fumarate, l-aspartate or l-malate, the transporters DcuA, DcuB, and DcuC are able to replace each other. DcuA, DcuB, and DcuC are present in S. Typhimurium as well (19), whereas citrate and tartrate are used only by E. coli (6, 46), but not by S. Typhimurium for FR (47, 48, 100). AspA, aspartase; CitT, citrate/succinate antiporter; CL, citrate lyase; DcuA, C4-DC antiporter DcuA; DcuB, C4-DC antiporter DcuB; DcuC, C4-DC transporter DcuC; FR, fumarate respiration; Frd, fumarate reductase FrdABCD; FumB, fumarase B; Hyb, hydrogenase; Nuo, NuoA-N; PtsG, glucose transporter of the phosphotransferase system; TtdAB, tartrate dehydratase; TtdT, l-tartrate/succinate antiporter; MK, menaquinone; MKH₂, menaquinol.

FIG 2

Scheme for the DcuA/AspA, DcuB/AspA, and DcuB/FumB metabolons of E. coli. Complex formation between AspA and FumB with the Dcu transporters is based on interaction studies (43), suggesting metabolon formation and metabolic channeling. The l-aspartate/fumarate antiport used during nitrogen assimilation by DcuA results in net uptake of ammonium (“nitrogen or ammonium shuttle”), the fumarate/succinate or l-malate/succinate antiport in the net uptake of 2 [H] (“H or redox shuttle”) for the sake of fumarate respiration. Figure modified from Schubert and Unden (43). AspA, aspartase; DcuA, C4-DC transporter; DcuB, C4-DC transporter; FrdABCD, fumarate reductase; GS-GOGAT, glutamine synthetase (GS)-glutamine 2-oxoglutarate aminotransferase (GOGAT) pathway; Fum, fumarate; FumB, fumarase B; L-Asp, l-aspartate; L-Mal, l-malate; MKH₂, menaquinol; Succ, succinate.

FIG 3

Ammonium assimilation from l-aspartate using DcuA-AspA for uptake and intracellular ammonium release, ammonium assimilation by GS-GOGAT, and the GlnB regulatory system. The scheme shows the uptake of l-aspartate by DcuA and ammonium release by the DcuA-AspA metabolon, the ammonium assimilation via the common GS-GOGAT pathway yielding L-Glu, and the regulation of AspA by the GlnB regulatory system and regulatory factors. N↓, nitrogen-limited conditions; N↑, nitrogen-saturated conditions; 2-OG, 2-oxoglutarate; UTP, uridine-triphosphate; PP_i, diphosphate; P_i, phosphate; UMP, uridine-monophosphate; GlnD, uridylyltransferase/uridylyl-removing enzyme; Fum, fumarate; PII, nitrogen regulator GlnB; DcuA, aerobic l-aspartate transporter; AspA, aspartate ammonium-lyase; GS, glutamine synthetase GlnA; GOGAT, glutamine 2-oxoglutarate aminotransferase GltBD.

FIG 4

Promoter regions of dctA (A) and dcuB (B) and binding sites for transcriptional regulators DcuR, cAMP-CRP, FNR, ArcA, NarL and Lrp. The binding sites have been determined experimentally (solid line) (101–103) or by the presence of consensus sites (broken lines). Transcriptional regulators exerting positive (green) or negative (red) regulation on the promoter are annotated. Numbering gives the position relative to the transcriptional start sites of the promoters. The location of the binding sites for NarL and Lrp at dcuB have not been identified.

FIG 5

C₄-dicarboxylates as substrates or products of metabolism, and as signaling molecules for host/microbiota interaction in the intestine. Hexose fermentation, fumarate respiration (FR) and microaerobic respiration run in parallel under the microaerobic conditions of the intestine. The intestinal C4-DCs (black) serve as stimuli of the DcuS regulated metabolism of enteric bacteria, and of chemotaxis by Tar. Succinate produced by the enteric bacteria (red) or other microbiota is used for signaling or for communication with host cell, and succinate of host cells (macrophages) stimulates virulence and pathogenicity of S. Typhimurium. See the text for details. CytBD, microaerobic Cyt bd oxygen reductase; Fo, formate; ROS, reactive oxygen species; other abbreviations as in Fig. 1 to 4.