Figure 4. Schematic maps of plasmid pSMA, pSA, pSM, and pSF1 and growth rates of Salmonella harboring these plasmids. (A) pSMA was generated by deletion of caf1 gene from pHF. (B) pSA was constructed by deletion of caf1M gene from pSMA. (C) pSM was created by deletion of caf1A gene from pSMA. (D) pSF1 was produced by cloning caf1 gene from pF1 to pHF to replace the caf1Mcaf1Acaf1 genes. The caf1(-), caf1M(-), and caf1A(-), respectively, indicate that the inner DNA sequences of gene caf1, caf1M, and caf1A were in-frame deleted. (E, F) Comparison of growth rates of P1-pSA, -pSM, -pSF1, and -pY. The bacterial growth rates were determined by measuring the OD600 every half hour (E) or determining bacterial CFU every hour (F), and the statistical differences of the growth rates of these strains were calculated using the Tukey Kramer multiple comparisons test. No significant differences of growth rates were discerned among these four strains. doi:10.1371/journal.pone.0036283.g004 pSA only slightly colonized mouse spleen, PP, and liver, whereas - pSF1 and -pSM were undetectable in spleen, PP, and liver (Figure 5C). These results suggest overexpression of any caf gene can attenuate the bacterial host. The mechanism of caf1A-, caf1-, caf1M-, and caf operon- mediated wt Salmonella attenuation is analyzed We hypothesized that the observed Salmonella attenuation due to the overexpression of genes caf1A, caf1, caf1M, or the caf operon might result from augmented membrane permeability. To test this hypothesis, strains P1-pF1, -pHF, -pSA, -pSF1, and -pSM were analyzed for sensitivity to erythromycin and polymyxin B (PMB), with -pY as a control. Erythromycin does not efficiently cross the outer membrane unless there is a breach of outer membrane integrity [18], while PMB exhibits elevated detrimental effects on the compromised bacterial outer membrane [19]. Results showed that the erythromycin minimum inhibitory concentration (MIC) of caf Runs as a Double-Edged Sword during Infection Figure 5. Effect of overexpression of caf genes on wt Salmonella virulence. (A) Overexpression of any caf genes was able to inactivate wt Salmonella in vitro. Strains P1-pSA, -pSF1, -pSM, and -pY were used for infecting RAW264.7 cells. Bacteria recovered from macrophages were determined and statistically evaluated using the Tukey Kramer multiple comparisons test with *** P,0.001 for P1-pSA, -pSF1, or -pSM vs -pY. Depicted are the mean 6 SD of three independent experiments. (B) Overexpression of any caf genes resultant in Salmonella nonlethal to mice. Mice orally dosed with 1.06109 CFUs of P1-pSA, -pSF1, and -pSM all survived, while those dosed with -pY all succumbed to infection. Survival fraction obtained from P1-pSA, -pSF1, or -pSM-dosed mice was compared with -pY-dosed mice and significance was respectively determined: ** P,0.05, "" P,0.01, and {{ P,0.01. Each group had total of 10 mice. Depicted are the mean of two independent experiments. (C) Overexpression of any caf genes resultant in Salmonella unable to colonize mice in vivo. At 4 days post oral administration with 1.06109 CFUs of P1-pSA, -pSF1, -pSM, or -pY, mouse splenic, PP, and liver bacterial CFUs were determined and statistically evaluated using the Tukey Kramer multiple comparisons test with * P,0.4, ** P,0.2, and *** P,0.1. ND, not detected. Depicted are the mean 6 SEM of two independent experiments. doi:10.1371/journal.pone.0036283.g005 strain P1-pF1 was not different from control -pY (Figure 6A). However, the MICs of strains P1-pHF, -pSA, -pSF1, and -pSM were all significantly lower than -pY. This result correlated with the observation that P1-pF1 maintains robust virulence similarly to that of -pY (Figure 3), while -pHF, -pSA, -pSF1, and -pSM are attenuated when compared with -pY (Figure 5). Due to the increased susceptibility to erythromycin, we speculated that P1-pHF, -pSA, -pSF1, and -pSM might also exhibit enhanced sensitivity to other antibiotics that target the cell membrane since, the antibiotic which permeabilizes the mem- brane may assist in further disrupting the integrity of the membrane. PMB is an antibiotic that is able to permeabilize the bacterial outer membrane and induce bactericidal activity [20]. The result showed that the PMB MICs of strains P1-pF1 and - pSF1 exhibited no differences in comparison with control -pY, while -pHF, -pSA, and -pSM displayed significantly lower MICs compared to -pY. Therefore, this experiment provides further evidence that the Salmonella attenuation may result from the disruption of the membrane. PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e36283