A multi-omic analysis reveals the role of fumarate in regulating the virulence of enterohemorrhagic Escherichia coli

The enteric pathogen enterohemorrhagic Escherichia coli (EHEC) is responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Several molecular mechanisms have been described for the pathogenicity of EHEC; however, the role of bacterial metabolism in the virulence of EHEC during infection in vivo remains unclear. Here we show that aerobic metabolism plays an important role in the regulation of EHEC virulence in Caenorhabditis elegans. Our functional genomic analyses showed that disruption of the genes encoding the succinate dehydrogenase complex (Sdh) of EHEC, including the sdhA gene, attenuated its toxicity toward C. elegans animals. Sdh converts succinate to fumarate and links the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) simultaneously. Succinate accumulation and fumarate depletion in the EHEC sdhA mutant cells were also demonstrated to be concomitant by metabolomic analyses. Moreover, fumarate replenishment to the sdhA mutant significantly increased its virulence toward C. elegans. These results suggest that the TCA cycle, ETC, and alteration in metabolome all account for the attenuated toxicity of the sdhA mutant, and Sdh catabolite fumarate in particular plays a critical role in the regulation of EHEC virulence. In addition, we identified the tryptophanase (TnaA) as a downstream virulence determinant of SdhA using a label-free proteomic method. We demonstrated that expression of tnaA is regulated by fumarate in EHEC. Taken together, our multi-omic analyses demonstrate that sdhA is required for the virulence of EHEC, and aerobic metabolism plays important roles in the pathogenicity of EHEC infection in C. elegans. Moreover, our study highlights the potential targeting of SdhA, if druggable, as alternative preventive or therapeutic strategies by which to combat EHEC infection.

chloramphenicol. The plates were incubated at 37°C for antibiotic screening and to induce the loss of pKD46. In order to generate EDL933 multiple gene mutants or prevent the polar effects on upstream and downstream gene expression of target genes, it is necessary to remove the resistance cassette with pCP20. The Flp recombinase expression plasmid pCP20 is also a temperature-sensitive plasmid, and the expression of Flp recombinase is induced at 43°C (2)(3)(4). Flp recombinase recognizes the FRT sites and removes the FRT site-flanked antibiotic resistance gene, generating an in-frame deletion mutant. The selected colonies were sensitive to Ampicillin and Kanamycin or chloramphenicol for absence of pCP20 and the resistance gene.  Figure S1. Screening of the EDL933 transposome mutant library. C. elegans glp-4 (bn2) L1 stage larvae were cultured on the Enriched Nematode Growth (ENG) medium plates at the restrictive temperature (25°C) at Day 1. At the same day, the EDL933 transposome mutant library, stored in 96-well plates and in -80°C freezers, was replicated in LB broth containing 50 µg/mL Kanamycin (Kan) and put in a 37°C incubator for 16 to 18 hours. At Day 2, the entire library was triplicated in 96-well plates containing LB broth with 50 µg/mL Kan and cultured at 37 °C for another 16 to 18 hours. At Day 3, when C. elegans glp-4 (bn2) animals reached to L4 larvae/young adult stage, the worms were washed off from ENG plates by M9 buffer and collected. These worms were mixed with each transposon mutant clones in 96-well plated, which was centrifuged and resuspended in S medium. Each well contained approximately 20 worms. Then, the 96-well plates were placed at 25°C with shaking at 70 rpm. After 8 days, the O.D. 595 values of each well were measured. The O.D. 595 value was close to 0.5 when worms were cultured with E. coli strain OP50 (as negative control). In contrast, the O.D. 595 value was around 1.0 when the worms were fed with EHEC wildtype EDL933 (as positive control). The hits/candidates with a decreased pathogenic phenotype toward C. elegans were selected with the O.D. value that was significantly lower compared to the EHEC wild-type EDL933 positive controls (P<0.05).

Anaerobic metabolism is dispensable for the full virulence of EHEC in C. elegans
During anaerobic metabolism, the TCA cycle is repressed and nitrate catalyzed by nitrate reductase (Nar) and fumarate catalyzed by fumarate reductase (Frd) can both act as the alternative terminal electron acceptors other than oxygen (5); or alcohol dehydrogenase, encoded by the adhE gene, can regenerate NAD + for glycolysis and control fermentation in E. coli (6). Moreover, the transcriptional regulator Fnr (fumarate/nitrate reduction regulator) is required for anaerobic respiration and controls the switch from aerobic to anaerobic respiration (7), and the ribonucleotide reductase class III, encoded by nrdD and nrdG, is essential for a strictly anaerobic environment in E. coli (8). To test whether anaerobic metabolism, including anaerobic respiration and fermentation, also plays roles in the pathogenesis of EHEC in C. elegans, five isogenic mutants with narHJI, frdA, adhE, fnr, and nrdDG deletion (EDL933:ΔnarHJI, EDL933:ΔfrdA, EDL933:ΔadhE, EDL933:Δfnr, and EDL933:ΔnrdDG) were generated and tested. We noted that these isogeneic mutants were as toxic as the parental wildtype EDL933 ( Figure S3). Given the potential redundancy of these genes in controlling anaerobic metabolism, a compound mutant was also generated. Our results showed that the isogeneic EDL933:ΔnarHJIΔfrdAΔadhEΔfnrΔnrdDG mutant strain was as toxic as the wild-type EDL933 ( Figure S3). Together, our current data suggested that anaerobic metabolism is dispensable for the full virulence of EHEC in C. elegans. Figure S3. Deletion of genes involved in anaerobic metabolism did not alter EHEC toxicity in C. elegans. 5 The survival of N2 worms fed with the wild-type EDL933 (EDL933) and the isogenic deletion strains of narHJI (EDL933:ΔnarHJI), frdA (EDL933:ΔfrdA), adhE (EDL933:ΔadhE), fnr (EDL933:Δfnr), and nrdDG (EDL933:ΔnrdDG) were examined. Deletion of narHJI (median N2 lifespan = 6.0 ± 0.1 days, P=0.205), frdA (median N2 lifespan = 6.7 ± 0.6 days, P=0.129), adhE (median N2 lifespan = 6.0 ± 0.1 days, P=0.413), fnr (median N2 lifespan = 6.0 ± 0.1 days, P=0.448), and nrdDG (median N2 lifespan = 6.5 ± 0.7 days, P=0.908) were as toxic as the parental wild-type EDL933 (median N2 lifespan = 6.2 ± 0.5 days). "ns" represents no statistically significant difference examined by the Log-rank test. 6 The effect of fumarate is specific to EHEC The survival curves of C. elegans animals did not change when fed on the succinate or fumarate treated OP50 ( Figure S4A). These results suggested that the effect of fumarate was on EDL933:ΔsdhA mutant directly. We also generated the isogeneic sdhA mutant strain of E. coli OP50 (OP50:ΔsdhA) to examine whether the effect of fumarate is specific to EHEC. Our results showed that the survival curves of C. elegans animals fed on the wild-type OP50 and the OP50:ΔsdhA mutant were similar ( Figure S4B). Moreover, the survival curves of C. elegans animals fed on succinate or fumarate treated OP50:ΔsdhA were similar to the untreated control, which suggested that the sdhA gene is specifically required for the pathogenesis of EHEC in C. elegans. Figure S4 strain (OP50, N2 median lifespan = 20.5 ± 0.7 days) toward C. elegans animals. Worms on succinate-treated OP50:ΔsdhA strain (OP50:ΔsdhA+Succinate, N2 median lifespan = 20.0 ± 0.1 days, P=0.842) and fumarate-treated OP50:ΔsdhA strain (OP50:ΔsdhA+Fumarate, N2 median lifespan =20.5 ± 0.7 days, P=0.878) all exhibited similar lifespan compared to the untreated control (OP50:ΔsdhA, N2 median lifespan = 20.0 ± 1.4 days). "ns" represents no statistically significant difference examined by the Log-rank test. 8 The three putative C4-dicarboxylates sensor-regulator systems are dispensable The dcuSR operon (also known as yjdHG) encodes a two-component sensorregulator system (DcuS-DcuR) which can sense fumarate and lead to activation of the fumarate-succinate antiporter DcuB expression in E. coli (9,10). If fumarate restores sdhA mutant toxicity/virulence through the DcuSR two-component system, deletion of dcuSR in the sdhA mutant background cannot restore its toxicity after supplement of fumarate. We therefore generated the sdhAdcuSR isogenic mutant and examined its toxicity to C. elegans under fumarate supplement. As shown in Figure S5A, the toxicity of sdhAdcuSR mutant to C. elegans was significantly attenuated compared with wildtype EHEC (P<0.0001) but was similar to the sdhA single mutant (P=0.151). Moreover, addition of 2.5 mM fumarate not only restored the toxicity of sdhA mutant but also the sdhAdcuSR mutant which suggested that the dcuSR two-component system is not involved in sensing fumarate to regulate the virulence of EHEC.
Another DctS-DctR two-component system, which encoded by dctS and dctR genes, is required for high-affinity C4-dicarboxylate transport in Rhodobacter capsulatus (9,11). We blasted the amino acid sequence of DctS and DctR to the EDL933 amino acid sequence and identified YhiF (Z4909, yhiF) as a close homolog of DctR, but could not identify any homolog of DctS. The DctB-DctD sensor-regulator controls the expression of the dctA gene encoding C4-dicarboxylate transporter DctA in Rhizobia (11). We also blasted the amino acid sequence of DctB and DctD to EDL933 protein sequence and identified HyfR (Z3751, hyfR) as having the closest homology to DctD. However, we could not identify any DctB homolog in EDL933. Therefore, we generated the isogenic mutant of dctR (yhiF) and dctD (hyfR) in the sdhA mutant background to examine whether fumarate regulates EDL933 virulence through SdhA via these twocomponent systems. As shown in Figure S5B, dctRsdhA double mutant is less toxic to C. elegans compared with wild-type EHEC (P < 0.0001) but is similar to the sdhA single mutant (P=0.96). Supplement of 2.5 mM fumarate to the dctRsdhA double mutant restored its toxicity to that of the sdhA single mutant (P=0.57), suggesting that the DctS-DctR two-component sensing pathway is not required for fumarate to regulate EHEC toxicity.
We also generated dctD isogenic mutant in the sdhA mutant background and examined its toxicity toward C. elegans when supplied with 2.5 mM fumarate. In the same manner as the dctRsdhA double mutant, addition of fumarate to the dctDsdhA double mutant rescued its toxicity to that of the sdhA single mutant (P=0.86) ( Figure  S5C).   Tables   Table S1. Nematode strains used in this study.

Strain
Relevant characteristics Source or reference N2 C. elegans wild-type strain (12)