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

Abstract

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.

UvrY is required for the full virulence of Aeromonas dhakensis

“…The expression of the UvrY-regulated genes and gyrB (internal control) was measured using quantitative real time-PCR (qRT-PCR,) as described previously [26,33]. Approximately 3 ml of LB broth containing bacteria that had been cultivated overnight were collected for RNA extraction.…”

Pathogenicity of urinary tract infectionEscherichia coliinCaenorhabditis elegans

“…Here, we adopted a liquid pathogenicity assay in which C. elegans and E. coli were co-incubated, and the turbidity was compared before and after the incubation. Although the method has been applied for a mutant library derived from one parent strain [12], this is the first report to apply it to numerous clinical isolates. Since the clinically isolated E. coli associated with UTIs showed significantly higher pathogenicity than the commensal strains, the results of the simple method in this study reflected the pathogenicity in the patient (Fig 2).…”

“…The pathogenicity assay method has been previously described in detail [12]. In brief, C. elegans glp-4 ( bn2 ts), which is temperature sensitive for pregnancy, was used to fix C. elegans generation during the assay.…”

“…From a technical, economical, and an ethical viewpoint, however, the model is not suitable for use in large-scale studies. The Caenorhabditis elegans model is cheap, easy to handle, and has been applied in various bacterial pathogenicity studies such as with Pseudomonas aeruginosa [3], Enterococcus [4], Serratia marcescens [5], and E. coli [6-12] including UPEC [9, 10]. For the UPEC study, the results revealed that the pathogenicity in C. elegans was associated with the number of VFs, which were identified as virulence genes involved in the pathogenicity in mammal [9].…”

“…In this method, C. elegans incubated with the lawn of interested bacteria was harvested with liquid medium from the plate after incubation for 1 day, and the survivability of C. elegans in the liquid medium was observed in a time-dependent manner. To further simplify the assay, we recently developed another liquid pathogenicity assay to screen a transposon mutant library of E. coli o157 comprised of 17,802 mutants [12]. Pathogenicity was evaluated by measuring the OD 595nm value of a mixture of mutant E. coli and C. elegans in 96-well plates after 8 days of incubation.…”