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Phosphoenolpyruvate carboxykinase controls Edwardsiella tarda virulence via oxaloacetate

ABSTRACT: Understanding the metabolic basis of bacterial virulence is essential for developing anti-infective strategies. Phosphoenolpyruvate carboxykinase (PEPCK), encoded by pckA, is a key enzyme at the metabolic junction between the pyruvate cycle and the TCA cycle, but its role in virulence regulation remains largely unexplored. In this study, we generated a pckA deletion mutant, ΔpckA, in Edwardsiella tarda and investigated its impact on virulence, metabolism, and vaccine potential. The ΔpckA mutant exhibited increased antibiotic sensitivity, faster growth, reduced autoaggregation, and significantly attenuated virulence in a tilapia infection model. Quantitative proteomics revealed global downregulation of type III and type VI secretion systems (T3SS and T6SS) and upregulation of flagellar proteins. Mechanistically, accumulation of oxaloacetate (OAA) in the mutant suppressed T3SS/T6SS expression without affecting flagellar motility. Importantly, ΔpckA induced a robust immune response in zebrafish and conferred strong protection against lethal challenge, with relative percent survival values of 64% and 81.82% in zebrafish and tilapia, respectively. Taken together, our findings identify PEPCK as a critical metabolic regulator of E. tarda virulence and highlight ΔpckA as a promising live attenuated vaccine candidate.

ORGANISM(S): Edwardsiella Tarda Eib202

SUBMITTER: Bo Peng

PROVIDER: PXD078087 | iProX | Wed May 06 00:00:00 GMT+01:00 2026

REPOSITORIES: iProX

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Project description:Many Gram-negative bacteria utilize the ubiquitous and versatile type VI secretion systems (T6SS) for diverse purposes, such as outcompeting other microbes or to successfully colonize hosts. T6SS of common pathogens are classified as T6SSi, but an outlier is the T6SSii and it is present only in the genus Francisella. The highly virulent human pathogen Francisella tularensis harbors a variant of the T6SSii distinct from that of many other species of the genus, e.g., aquatic pathogens and tick endosymbionts. At the gene level, the most distinctive difference between the two variants is the presence in F. tularensis of a putative T6SS effector denoted PdpC. We here provide bioinformatic evidence that PdpC contains a conserved amino acid triad homologous to motifs present in toxins of the family Make Caterpillars Floppy. By site-directed mutagenesis, each of the three amino acids was substituted in cis in the live vaccine strain, LVS, of F. tularensis. Since the T6SS of F. tularensis is essential for the intracellular life cycle and virulence of the pathogen, the effects of the mutations were tested in mammalian cells and in a mouse model. It was observed that the resulting mutants demonstrated distinct phenotypes, evidenced as delayed, or no intramacrophage phagosomal escape, variable degrees of intramacrophage replication, and marked attenuation in mice. The corresponding mutants were also generated in the related species F. novicida, a commonly used model for F. tularensis, which conveniently allows studies of T6SS-mediated secretion. A mass spectrometry analysis of secreted proteins demonstrated that PdpC is one of several T6SS-dependent substrates, and that the lack of PdpC, or the presence of each of the amino acid-substituted mutant PdpC proteins, did not affect the secretion pattern of F. novicida. Collectively, the data demonstrate that the markedly attenuated phenotypes of the mutants were due to essential roles of the amino acid triad of PdpC.

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Phosphoenolpyruvate carboxykinase controls Edwardsiella tarda virulence via oxaloacetate

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