caf Runs as a Double-Edged Sword during Infection Figure 2. Effects of overexpression of F1 capsule on Salmonella phenotypes. (A) Overexpression of F1 capsule resultant in anti- agglutination phenotype of Salmonella bacilli. After centrifugation, P1- pF1 and -pY were pelleted to the tube bottom while -pHF was not. (B) Detection of F1 capsular expression by Western blotting. Strain P1-pF1 did not express detectable F1 capsular proteins until 12 hrs post- inoculation, but F1 capsular proteins were visible from -pHF as early as 4 hrs post-inoculation. Control P1-pY did not produce F1 capsule. (C) Quantification of F1 capsule. The F1 capsular protein yields of P1-pF1 and -pHF were determined and their differences were analyzed using the Tukey Kramer multiple comparisons test with ** P,0.01. Depicted are the mean 6 SEM of three independent experiments. doi:10.1371/journal.pone.0036283.g002 Strain P1-pHF is highly attenuated in macrophages and mice To determine whether Salmonella encapsulation results in elevated resistance to phagocytosis, a macrophage RAW264.7 cell infection assay was performed. RAW264.7 macrophages were infected with P1-pF1, -pHF, and -pY (Figure 3A). At 0 hr, significantly fewer Salmonella bacilli were recovered from macro- phages infected with P1-pHF or -pF1 than from those infected with -pY (Figure 3A), suggesting expression of the F1 capsule inhibits entry into macrophages, in agreement with previous findings [12]. Surprisingly, the intracellular P1-pHF could not survive within macrophages, while -pF1 proliferated in macro- phages, but exhibited significant defects compared to -pY at 8 and 24 hrs post-infection (Figure 3A). To test whether the in vivo virulence of the Salmonella was affected by F1 encapsulation, we performed a virulence assay in mice. BALB/c mice were observed for survival after they were infected with 1.06109 colony forming units (CFUs) of P1-pHF, -pF1, or -pY via oral route. All mice given P1-pHF survived (9/9), while none of the -pF1 or -pY-infected mice survived (0/10 and 0/ 10, respectively) (Figure 3B). These results further demonstrate that overexpression of caf operon attenuates Salmonella in vivo. To address the mechanism by which mice survived from the encapsulated wt Salmonella, we performed an experiment to analyze the capability of P1-pHF to colonize mouse tissues using -pF1 and -pY as controls. The BALB/c mice were given P1-pHF, - pF1, or -pY with dose and route identical to the above survival assay. At 4 days post-administration, spleens, Peyer’s patches (PP), and livers were analyzed for bacterial CFUs (Figure 3C). Strain P1-pHF was not able to colonize the spleen. Colonization of PP by P1-pHF was reduced by 2,492-fold and 5,417-fold when compared to the -pY and -pF1 infected mice, respectively, while Figure 3. Effect of overexpression of caf operon on wt Salmonella virulence. (A) Caf1-mediated Salmonella attenuation in vitro. Strains P1-pF1, -pHF, and -pY were used for infecting RAW264.7 cells at a ratio of 1:1. Bacteria recovered from macrophages were determined and statistically evaluated using the Tukey Kramer multiple comparisons test with *** P,0.001 for P1-pF1 or -pHF vs -pY; and "" P,0.01 and """ P,0.001 for -pHF vs -pF1. Depicted are the mean 6 SD of three independent experiments. (B) Caf1-attenuated Salmonella was nonlethal to mice. Survival fractions of P1-pHF-administered mice (n = 9) vs -pF1 (n = 10) or -pY (n = 10) dosed mice were obtained and statistically evaluated using Mantel-Haenszel test with ** P,0.01 for - pHF vs -pY and "" P,0.01 for -pHF vs -pF1. Depicted are results from two independent experiments. (C) Caf1-attenuated Salmonella was unable to colonize mice. At 4 days post oral administration with 1.06109 CFUs of P1-pF1, -pHF, and -pY, mouse splenic, PP, and liver bacterial CFUs were determined and statistically evaluated using the Tukey Kramer multiple comparisons test with * P,0.4 and ** P,0.2. ND, not detected. Depicted are the mean 6 SEM of two independent experiments. doi:10.1371/journal.pone.0036283.g003 colonization of liver by P1-pHF was reduced by 123,580-fold and 9,965-fold when compared to the -pY and -pF1 infected mice, respectively. This study clearly demonstrated that P1-pHF is highly disabled and unable to disseminate and replicate within murine tissues. The gene in the caf operon responsible for Salmonella attenuation is determined Among the three caf genes harbored in pHF (Figure 1), we sought to determine the one responsible for the observed Salmonella attenuation. To this end, the capsular gene caf1, chaperone-like gene caf1M, and usher gene caf1A were deleted in-frame from pHF, and the plasmids pSA, pSF1, and pSM were created for overexpression of Caf1A, F1, and Caf1M, respectively (Figure 4). Compared to P1-pY, the -pSA, -pSF1, and -pSM strains showed no differences in growth rates (Figure 4E and 4F), but they all lost the ability to proliferate within macrophages (Figure 5A, P,0.001 for these 3 strains at any time points). All mice given P1-pSA (10/ 10), -pSF1 (10/10), or -pSM (10/10) survived, unlike all -pY- infected mice (0/10), which succumbed to infection (Figure 5B, P,0.01 for all these 3 strains vs control). Furthermore, strain P1- PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e36283