caf Runs as a Double-Edged Sword during Infection Figure 6. Effect of overexpression of caf operon or individual caf genes on Salmonella antibiotic sensitivity. Strains P1-pF1, -pHF, -pSA, -pSF1, -pSM, and -pY were analyzed for their sensitivity to (A) erythromycin and (B) polymyxin B via determination of their MICs. The MIC differences were determined and statistically evaluated using the Tukey Kramer multiple comparisons test with * P,0.05, ** P,0.01, and *** P,0.001. Depicted are the mean 6 SEM of three independent experiments. doi:10.1371/journal.pone.0036283.g006 The overexpression of genes caf1A, caf1, or caf1M impacts Y. pestis Since overexpression of caf operon or its individual genes can attenuate heterologous bacterium Salmonella, we questioned whether their overexpression also exhibits unfavorable effects on the homologous bacterium Y. pestis. Thus, plasmid pF1, pHF, pSA, pSF1, pSM, and pY, carrying a chloramphenicol resistance marker, were introduced to the avirulent strain Y. pestis KIM6+ [21], which was generated by curing the virulent plasmid pCD1 from KIM-10 [22]. Strains KIM6+/pF1, pHF, pSA, pSF1, pSM, and pY were subjected to antimicrobial and temperature sensitivity assays. The results showed that KIM6+/pSA is susceptible to erythromycin (Figure 7A), PMB (Figure 7B), hydrogen peroxide (Figure 7C), bile (Figure 7D), and is unable to vigorously grow at 37uC (Figure 7E) relative to control pY. Strain KIM6+/pSF1 is sensitive to PMB (Figure 7B), and pSM is labile to peroxide hydrogen (Figure 7C), bile (Figure 7D), and high temperature (Figure 7E) when compared to pY. These observations suggest that KIM6+/pSA is the most delicate among the three individual genes recombinant strains, and KIM6+/pHF is even more fragile than strain pSA. In fact, KIM6+/pHF exhibited the most sensitive phenotypes among all the tested strains and all the tested stress conditions (Figure 7A– E). Strain KIM6+/pF1 was slightly sensitive to both hydrogen peroxide (Figure 7C) and high temperature (Figure 7E). Interest- ingly, all 6 strains, KIM6+/pF1, pHF, pSA, pSF1, pSM, and pY, were able to survive at 27uC on Brain Heart Infusion (BHI) agar with bile (Figure 7D) or at 37uC without bile (Figure 7E), but none survived at 37uC on BHI agar with bile (Figure 7F). This suggests that the double pressures from bile and the high temperature are lethal to all the recombinant strains, as well as the control. This clearly indicates overexpression of individual genes of caf1A, caf1, caf1M, or the whole caf operon produces strikingly deleterious effects to the homologous bacterium Y. pestis. Subsequently, the infection abilities and survivability of these strains were determined using macrophage RAW264.7 cells. KIM6+/pSA and pSF1 lost partial capability of infecting macrophages, but pSM did not when compared to control pY (Figure 8A). As expected, KIM6+/pHF displayed the lowest infectivity among all the tested strains, while pF1 showed no differences from pY (Figure 8A). This suggests that overexpression of either caf1A or caf1, particularly the entire caf operon, dramatically reduces the bacterial infectivity. KIM6+/pSF1 was cleared from macrophage very rapidly after infection. By 3 and 9 hrs post-infection, only 10.0% and 3.1% of the initial population of KIM6+/pSF1 remained alive, respectively (Figure 8B). Strain KIM6+/pSA, pHF, and pF1 also showed impaired survivability when compared to pY, while pSM exhibited no differences from pY. These results explicitly suggest that the caf operon and its individual gene caf1A and caf1 possess severe deleterious effects to Y. pestis. Discussion Since Y. pestis is classified as a category A select agent by the CDC [23], manipulation of wt Y. pestis requires a Biosafety Level 3 (BSL-3) facility, which limits access to and work with this pathogen. Thus, in our study, we established a platform for analyzing Y. pestis virulence factors by means of the wt BSL-2 pathogen, S. Typhimurium. As the caf operon has previously been demonstrated to be functional in Salmonella [10], use of wt Salmonella strains to analyze Yersinia virulence factors is feasible. To investigate whether caf possesses deleterious aspects during infection, we amplified the expression of caf operon in order to maximally expose these effects. Hence, the caf native promoter Pcaf1M was replaced with a strong fusion promoter, which increased F1 protein yield by 18.8- to 35.2-fold (Figure 2C). Results showed that overexpression of the caf operon heavily attenuates Salmonella, which was evidenced by P1-pHF being unable to proliferate in macrophages (Figure 3A) or colonize mouse spleens (Figure 3C), a similar attenuation impact only seen when highly virulent genes, such as phoP and rfaC, are deleted from the Salmonella genome [24]. This clearly demonstrates that potently deleterious effects are indeed encoded by the caf operon, as amplification of the caf operon renders the bacterium markedly PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e36283