caf Runs as a Double-Edged Sword during Infection Table 1. Bacterial strains, plasmids, and their characteristics. Bacterial strains E. coli H681 S. Typhimurium H71 S. Typhimurium P1 Y. pestis KIM6+ Plasmids pJGX15C-asd pHC pY pF1 pHF pSMA pSA pSM pSF1 Phenotypes and characteristics asd2. derived from parent strain X6212. Wild-type strain of S. Typhimurium. Dasd::kanR H71. pgm+, pst+, lcr2, fra+. derived from parent strain KIM-10. asd+, cfa/I+. asd+, cfa/I+. The PtetA in pJGX15C-asd was replaced by fusion promoter PM. asd+. cfa/I operon was removed from pJGX15C-asd. asd+, operon caf+. operon caf regulated by Pcaf1M. asd+, operon caf+. operon caf regulated by PM. asd+, gene caf12. caf1Mcaf1A regulated by PM. asd+, gene caf12, caf1M2. caf1A regulated by PM. asd+, gene caf12, caf1A2. caf1M regulated by PM. asd+, gene caf1+, caf1M2, caf1A2. gene caf1 regulated by PM. Sources or references [10] [42] [16] [43] [44] [16] This study [10] This study This study This study This study This study doi:10.1371/journal.pone.0036283.t001 that other vulnerabilities associated with caf operon that may not allow its expression beyond a definite threshold. To investigate the potential vulnerabilities, we overexpressed caf in wt Salmonella enterica serovar Typhimurium to determine whether overexpression of caf would display any impact on the bacterial host. Results show that overexpression of either the caf operon or any genes within this operon are able to dramatically debilitate Salmonella virulence. Results Construction of recombinant S. Typhimurium H71 strains that overexpress the F1 capsule To allow stable maintenance and expression of the caf operon, the asd-mediated balanced-lethal system was utilized in this study as previously described [15]. Plasmid pF1, pHF and pY containing asd genes were utilized (Table 1 and Figure 1). In plasmid pF1, caf operon is regulated by its native promoter upstream of caf1M (Pcaf1M) and its own regulator caf1R [10]. Plasmid pHF was constructed by replacing the Pcaf1M in pF1 with a strong constitutive fusion promoter PtetA,PpagC,PphoP (PM) [16]. Plasmid pY is an empty vector that was used as a control. The asd mutation strain Dasd::kanR S. Typhimurium H71 (P1) [16] was transformed with pF1, pHF, and pY, respectively, to obtain the recombinant strains P1-pF1, -pHF and -pY. Control strain P1-pY recovered full virulence to wt strain H71 in mice [17]. These three strains were grown in LB media at 37uC with agitation at 150 rpm for determination of growth rates. During lag and stationary growth phases, no differences in growth were observed among P1-pF1, -pHF, and -pY. However, P1-pF1 and - pHF proliferated slightly more slowly than -pY, and -pHF proliferated slightly more slowly than -pF1 in logarithmic (log) phase (Figure 1D and E), suggesting that encapsulation causes negligibly delayed multiplication. Overnight cell cultures of P1- pF1 and -pY formed pellets after centrifugation, while -pHF did not (Figure 2A), implying -pHF produces enormous F1 capsular proteins that prevent cells from agglutination. Western blot analysis confirmed that the fusion promoter enhanced F1 protein expression (Figure 2B) by ,35.2-fold and 18.8-fold in P1-pHF samples compared to -pF1 at 8 and 12 hrs post-inoculation, respectively (Figure 2C). Figure 1. Schematic maps of plasmid pF1, pHF, and pY and growth rates of Salmonella strains harboring these plasmids. (A) In pF1, the caf operon is regulated by its native promoter Pcaf1M. (B) In pHF, the caf operon is regulated by a fusion promoter pM. (C) Plasmid pY is an empty vector. (D, E) Comparison of growth rates of P1-pF1, pHF, and pY. The bacterial growth rates were determined by measuring the OD600 every half hour (D) or determining bacterial CFU every hour (E), and the statistical differences were analyzed using the Tukey Kramer multiple comparisons test with * P,0.05, ** P,0.01, and *** P,0.001 for P1-pF1 vs -pY; { P,0.05, {{ P,0.01, and {{{ P,0.001 for -pHF vs -pY; and " P,0.05, "" P,0.01, and """ P,0.001 for -pHF vs -pF1. Depicted are mean 6 SEM of three independent experiments. doi:10.1371/journal.pone.0036283.g001 PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e36283