## 3 Results  
Attenuated Virulence of \(\Delta\)fruR, \(\Delta\)ssrAB, and \(\Delta\)bfq Mutants in Mice Infection  
To induce substantial alterations in the physiological behavior of _Salmonella_, three global regulator genes encoding FruR, SsrAB, and Hfq were deleted individually in _Salmonella_ Typhimurium strain 1120 (ST1120), which was isolated from swine feces [19]. The virulence of regulatory mutants was evaluated by LD\({}_{50}\) estimation during BALB/c mouse infection. In comparison with WT _Salmonella_ (LD\({}_{50}\) = 10\({}^{3.1}\)), all three mutants exhibited a significantly attenuated virulence, with an increase in LD\({}_{50}\) of at least 146-fold in mice infected with the \(\Delta\)fruR mutant (Table 1). In murine salmonellosis, invasive _Salmonella_ penetrates the intestinal epithelial barrier and spreads to deeper tissues, such as the spleen and liver, causing systemic infection. Therefore, _Salmonella_-induced splenomegaly is a primary pathological alteration observed during the process of _Salmonella_ systemic infection [31; 32]. We compared the development of splenomegaly between WT and mutant _Salmonella_ strains by weighing the spleens harvested from infected mice. At day 7 after _Salmonella_ infection, the body weights of infected mice were comparable between the WT and mutant strains, despite the use of different doses (WT: 10\({}^{3}\) CFU/mouse vs. mutant: 10\({}^{4}\) CFU/mouse; Figure 1A). As expected, the spleens of mice infected with WT _Salmonella_ were enlarged, showing an approximately 4.7-fold increase in size. However, the three mutants (\(\Delta\)fruR, \(\Delta\)ssrAB, and \(\Delta\)bfq) scarcely caused splenomegaly, even at a 10-fold higher dose (Figure 1B). To examine bacterial growth in the spleen tissues, the infected organ was homogenized and plated onto selective agar, and _Salmonella_ colonies were counted (Figure 1C). The bacterial loads in the spleen were remarkably low in the mice infected with the \(\Delta\)fruR (5.9 \(\times\) 10\({}^{2}\) CFU/g of spleen) and \(\Delta\)bfq (not detected) mutants compared with those infected with the WT strain (2.2 \(\times\) 10\({}^{5}\) CFU/g of spleen). The \(\Delta\)ssrAB mutant strain exhibited a 10-fold reduction in the bacterial load of the spleens (2.2 \(\times\) 10\({}^{4}\) CFU/g of spleen). \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Strains**} & **Infection Dose (CFU/Mouse)** & **Number of Dead Mice/ Total Mice Number** & \multirow{2}{*}{**LD\({}_{50}\) (CFU/Dose)**} \\ \cline{1-1}  & \(10^{5}\) & 3/4 & \(10^{3.1}\) \\  & \(10^{4}\) & 3/4 & \\  & \(10^{3}\) & 0/4 & \\  & \(10^{2}\) & 0/4 & \\ \(\Delta\)fruR & \(10^{7}\) & 4/4 & \(10^{5.3}\) \\  & \(10^{6}\) & 4/4 & \\  
The \(\Delta\)fruR, \(\Delta\)ssrAB, and \(\Delta\)lifq Mutants Are Defective in Survival Inside Macrophages  
To gain insight into the mechanisms underlying the attenuated virulence of these three mutants in mice, the intracellular survival ability was compared using murine macrophage cells. The growth of the \(\Delta\)fruR, \(\Delta\)ssrAB, and \(\Delta\)lifq mutant strains was comparable to that of WT _Salmonella_ in LB medium broth in vitro (Figure S1). However, all three mutants were significantly attenuated regarding intracellular survival at 10 h post-infection (Figure 2A), which was likely attributable to decreased SPI-2 expression [20]. Moreover, multiple structural devices expressed at the outer membrane determine the fate of _Salmonella_ during host-pathogen interactions. The profiles of OMPs were compared between the WT and mutant strains (Figure 2B). Multiple proteins were differentially expressed in the absence of FruR, SsrAB, or Hfq. For example, the \(\Delta\)lifq mutant strain produced less flagellin proteins, including FljB (52.5 kDa) and FliC (51.6 kDa), whereas the \(\Delta\)ssrAB mutant strain reversed the flagellin type from FliC to FljB, which is probably attributable to flagellar-phase variation.