Salmonella enterica serovar typhimurium response involved in attenuation of pathogen intracellular proliferation

Salmonella enterica serovar Typhimurium proliferates within cultured epithe lial and macrophage, cells. Intracellular bacterial proliferation is, howev er, restricted within normal fibroblast cells. To characterize this phenome non in detail, we investigated the possibility that the pathogen itself mig ht contribute to attenuating the intracellular growth rate. S. enterica ser ovar Typhimurium mutants were selected in normal rat kidney fibroblasts dis playing an increased intracellular proliferation rate. These mutants harbor ed loss-of-function mutations in the virulence-related regulatory genes pho Q, rpoS, slyA, and spvR. Lack of a functional PhoP-PhoQ system caused the m ost dramatic change in the intracellular growth rate. phoP- and phoQ-null m utants exhibited an intracellular growth rate 20- to 30-fold higher than th at of the wild-type strain. This result showed that the PhoP-PhoQ system ex erts a master regulatory function for preventing bacterial overgrowth withi n fibroblasts. In addition, an overgrowing clone was isolated harboring a m utation in a previously unknown serovar Typhimurium open reading frame, nam ed igaA for intracellular growth attenuator. Mutations in other serovar Typ himurium virulence genes, such as ompR, dam, crp, cya, mviA, spiR (ssrA), s piA, and rpoE, did not result in pathogen intracellular overgrowth. Nonethe less, lack of either SpiA or the alternate sigma factor RpoE led to a subst antial decrease in intracellular bacterial viability. These results prove f or the first time that specific serovar Typhimurium virulence regulators ar e involved in a response designed to attenuate the intracellular growth rat e within a nonphagocytic host cell. This growth-attenuating response is acc ompanied by functions that ensure the viability of intracellular bacteria.