Carbon metabolism influences Shigella flexneri pathogenesis

The gram negative bacterium Shigella flexneri is an etiological agent of bacillary dysentery, and causes destruction of the human intestinal epithelium. S. flexneri is primarily transmitted via the fecal-oral route to its primary infective site in the colon. The bacterium invades and replicates within colonic epithelial cells, ultimately ulcerating the mucosal epithelium. To successfully establish infection, S. flexneri must quickly adapt to different environments in the host, including adjusting metabolism in response to changes in available carbon sources. In this study, the importance of the glycolytic and gluconeogenic pathways in S. flexneri pathogenesis was examined. The metabolic regulators CsrA and Cra reciprocally regulate the glycolytic and gluconeogenic pathways. The post-transcriptional regulator Cra activates expression of genes involved in gluconeogenesis and represses glycolysis. Conversely, CsrA activates glycolysis and represses gluconeogenesis. The absence of Cra increased S. flexneri attachment and invasion of cultured epithelial cells. In contrast, the csrA mutant was significantly impaired in both adherence and invasion. Both the csrA and cra mutants formed small, turbid plaques, suggesting that both regulators are required for plaque formation. The opposing phenotypes of the csrA and cra mutants suggested a correlation between invasion and glycolysis. The role of glycolysis in S. flexneri pathogenesis was confirmed by directly examining the first committed step in the pathway. The glycolytic enzyme phosphofructokinase I (PfkI, encoded by pfkA) is repressed by Cra and activated by CsrA. Glycolysis was critical for S. flexneri pathogenesis, as a mutation in pfkA rendered the bacterium noninvasive. The invasion defect of the csrA and pfkA mutants was due to reduced expression and secretion of the Shigella invasion plasmid antigen (Ipa) effectors. Expression of the master virulence regulators virF and virB was significantly reduced in the pfkA mutant, and is the principle reason for decreased invasion. The data presented show that glycolysis is required for invasion, but that plaque formation requires both glycolysis and gluconeogenesis. Because expression of the master virulence regulators is repressed in the pfkA mutant, S. flexneri may use carbon as an environmental regulator of virulence gene expression.