Figure 7. Effect of overexpression of caf operon or individual caf genes on Yersinia antimicrobial and temperature suscepti- bilities. Six strains of KIM6+/pF1, pHF, pSA, pSF1, pSM, and pY were analyzed for their sensitivities to (A) erythromycin, (B) PMB, (C) hydrogen peroxide, (D) bile salt, (E) temperature of 37uC, and (F) bile salt combined with temperature of 37uC. (A, B) The erythromycin and PMB MICs of the 6 strains were respectively determined. (C) The 6 strains were incubated with 2.5 mM H2O2 for 1 hr and the survival percentages were determined in comparison with the non-treated controls, respectively. (D) The 6 strains were dropped onto BHI agar containing 1% bile and were incubated at 27uC for 48 hrs for CFU enumeration in comparison with those grown on BHI agar without bile, respectively. (E) The 6 strains were dropped onto BHI agar and were incubated at 37uC for CFU enumeration in comparison with those grown on BHI agar at 27uC, respectively. (F) The 6 strains were dropped onto BHI agar containing 1% bile and were incubated at 37uC for CFU enumeration in comparison with those grown on BHI agar without bile at 27uC, respectively. All experiments (A to F) were statistically analyzed for significant differences among these 6 strains using the Tukey Kramer multiple comparisons test with * P,0.05, ** P,0.01, and *** P,0.001. Depicted (A to F) are the mean 6 SEM of three independent experiments. doi:10.1371/journal.pone.0036283.g007 attenuated in vitro and in vivo. To circumvent the deleterious effect of caf, Y. pestis cleverly uses a weak and inducible promoter, Pcaf1M, to precisely govern caf operon expression. Through Pcaf1M, the vulnerability associated with the caf operon is adequately camouflaged allowing the P1-pF1 to still possess similar virulence to -pY in vivo. As there are three structural genes encoded in the caf operon, we queried which one was responsible for the caf operon-mediated Salmonella attenuation. Our data showed that overexpression of any caf Runs as a Double-Edged Sword during Infection Figure 8. Macrophage infection and survival assays of the recombinant Y. pestis KIM6+ strains. (A). infectivity assay. Strains KIM6+/pF1, pHF, pSA, pSF1, pSM, and pY were used for infecting macrophage RAW264.7 cells. At 1 hr post-infection, the intracellular bacterial CFUs were compared among these 6 strains. The Tukey Kramer multiple comparisons test was used for significant difference calculation with * P,0.05, ** P,0.01, and *** P,0.001. Depicted are the mean 6 SEM of three independent experiments. (B) Survival assay. Bacteria survived within the macrophages at 3 and 9 hrs post-infection were compared with the 0 hr CFU counts, and the percentages were calculated. Differences in survival rates among the 6 strains were calculated using the Tukey Kramer multiple comparisons test. Values depict the mean 6 SEM of three independent experiments with * P,0.05, ** P,0.01, and *** P,0.001 for the caf operon or caf gene expression strains vs control KIM6+/pY. doi:10.1371/journal.pone.0036283.g008 genes of the caf operon, including chaperone-like protein encoding gene caf1M, outer membrane protein encoding gene caf1A, and the capsule encoding gene caf1, was able to undermine the virulence of Salmonella (Figure 5). We then investigated the mechanisms of why overexpression of these individual genes, as well as the caf operon, could attenuate Salmonella. Seemingly, the impairments induced by overexpression of caf or its components in vitro and in vivo were associated with the elevated cell membrane permeability (Figure 6). When compared with P1-pY, the erythromycin MICs were significantly decreased for all strains other than P1-pF1, suggesting that -pF1 carefully regulates its vulnerability. The erythromycin MIC for strain P1-pSA expressing caf1A alone was identical to - pHF, suggesting these two strains share similar membrane permeability. The reason P1-pSA and -pHF possess similar membrane permeability is possibly due to the usher Caf1A alone being able to self-assemble into functional channels in the outer membrane without the assistance of the chaperone Caf1M. The over-installed Caf1A channels in the outer membrane facilitated the free entrance of the large molecule erythromycin and resulted in a decreased MIC. The Caf1A impairment to the membrane may dominate the overall detrimental effects associated with the caf operon shadowing the impairment of the Caf1M and F1 proteins, to the cell membrane. Thus, P1-pSA and -pHF display indifferent erythromycin sensitivities. Others have previously recognized that elevated curli expression increases the erythromy- cin sensitivity [18]. Thus, by combining previous observations with our results, we may conclude that overexpression of the cell surface appendages will proportionally enhance the channel expression level, and the elevated channels in the cell outer membrane facilitate entry of erythromycin into the cell, resulting in heightened bacterial susceptibility. Introduction of membrane permeabilizer antibiotic PMB into the channeled cell membrane PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e36283