Small-molecular virulence inhibitors show divergent and immunomodulatory effects in infection models of Salmonella enterica serovar Typhimurium

The virulence-associated Salmonella pathogenicity island 2 (SPI2) type III secretion system supports intracellular replication of Salmonella enterica serovar Typhimurium in macrophage-like RAW264.7 cells. In contrast, the salicylidene acylhydrazide INP0010 and the benzimidazole omeprazole prevent virulence factor-mediated replication of S. Typhimurium in these cells. Here we show that INP0010 enhances expression of inducible nitric oxide synthase (iNOS), nitric oxide (NO) production, the oxidative burst and tumour necrosis factor-alpha (TNFα) release in infected RAW264.7 cells. INP0010 also inhibited SPI2 activity in RAW264.7 cells. The ability of INP0010 to suppress bacterial intracellular replication correlated with NO production. The iNOS inhibitor N-monomethyl-l-arginine restored SPI2 activity and antagonised the bacteriostatic effect of INP0010. Omeprazole, which inhibited iNOS expression in RAW264.7 cells, likewise antagonised INP0010. In infected epithelioid MDCK cells that did not express NO upon infection, INP0010 enhanced bacterial intracellular replication. In Caenorhabditis elegans, INP0010 significantly attenuated the virulence of S. Typhimurium. In this infection model, the attenuating effect of INP0010 was further enhanced by omeprazole. These results demonstrate that chemically unrelated virulence inhibitors may act in an antagonistic or additive manner, that their effect depends on the infection model applied, and that the attenuating effects of INP0010 in part relate to its ability to promote the SPI2 antagonist NO.