## 4 Discussion
_Salmonella_ lacking Hfq exhibited a marked attenuation of virulence during mouse infection and cleared out from the spleen at 7 dpi, yielding decreases in intracellular survival and motility; however, its ability to stimulate an immune response was marginal, indicating that the _alftg_ mutant strain failed to protect mice against lethal _Salmonella_ infection. Conversely, the _AfruR_ and _sssrAB_ strains exhibited a reduced virulence, but exerted sufficient immunogenicity providing a protection efficacy of 100% (_\(\Delta\)fruR_) and 75% (_\(\Delta\)ssrAB_) survival against infection with virulent _Salmonella_. Considering the role of SsrAB as the master regulators of SPI-2 and its associated genes, the attenuated virulence of the _sssrAB_ mutant strain was likely attributable to a malfunctioning type 3 secretion system (T3 SS) encoded by SPI-2. Thetemporal and spatial translocation of virulence effectors via T3 SS during host infection is critical for _Salmonella_ to colonize and persist successfully inside the host animals [46]. FruR, which was initially known as the transcriptional repressor of the _fru_ operon, is a dual regulator with both activation and repression activities that is implicated in the regulation of a large number of genes associated with carbon metabolism [17]. Because of these multifaceted roles of FruR in carbon metabolism, it is also termed catabolite repressor/activator (Cra). In pathogenic Gammaproteobacteria, including _Salmonella enterica_, _Shigella flexneri_, and enterohemorrhagic _Escherichia coli_, the lack of FruR also impairs bacterial virulence during host infection [20, 47, 48]. Carbohydrates are not only incorporated in energy metabolism pathways, but also exploited as structural building blocks, such as d-glucosamine-6-phosphate for bacterial peptidoglycan, lipopolysaccharide, and biofilm production. Therefore, the ability to maintain carbon homeostasis in response to extracellular environments is important for bacterial growth and resistance. _Salmonella_ devoid of FruR might be incompetent in proliferation in vivo because of an imbalanced carbon flow; moreover, _Salmonella_ may also undergo structural alterations in peptidoglycan and LPS compositions. However, despite the plausible defects that occur in the absence of FruR, the _AfruR_ mutant strain, as a prophylactic vaccine, was able to stimulate IgG and IgM production more promptly and strongly than were the WT and _AssrAB_ mutant strains, and afforded the best protection capacity among the tested strains against lethal _Salmonella_ infection. The innate immune system, which harnesses macrophages and neutrophils, is the primary line of defense against _Salmonella_ infection in the gut; however, subsequently, the adaptive immune system plays a greater role in eradicating _Salmonella_ by providing effective protection to subsequent challenges with related _Salmonella_ groups [16]. In this context, the importance of CD4\({}^{+}\) Th-cell-mediated immunity has gained attention [10]. Naive CD4\({}^{+}\) T cells are activated into the two subsets of Th1 and Th2 cells depending on the repertoires of cytokines produced by these Th cells. Th1 cells secrete IL-2, IFN-\(\gamma\), and TNF-\(\alpha\) and stimulate cell-mediated immunity and inflammation, whereas Th2 cells secrete IL-4, IL-5, and IL-13 and promote humoral immunity via B-cell activation and differentiation. Given that invasive NTS exploits host cells (even macrophages) to evade various immune responses, both humoral and cell-mediated immune responses are required for proficient protection against fatal _Salmonella_ infection. The ratio of IgG2a/IgG1 is typically used as a surrogate marker to predict the type of immune responses dominated by Th1 or Th2 cells. The _AfruR_ and _AssrAB_ mutants showed lower IgG2a/IgG1 ratios than did WT _Salmonella_, despite the comparable IgG2a levels among them, indicating that these two mutant strains can successfully induce Th2-cell-mediated humoral immunity as well as Th1-cell-mediated cellular immunity.