[25] further increased cell permeability and resulted in decreased MICs, as evidenced by the decreased PMB MIC of strain P1-pSA and -pHF. The Salmonella attenuation caused by overexpression of genes caf1M and caf1 may be because these proteins are arrested within the periplasm. This may result in (1) other periplasmic proteins displaying abnormal function and (2) physical injuries to the membranes when they accumulate. Both of these possibilities can be applied to Caf1M since Caf1M is naturally located in periplasm due to its signal peptide guided transportation [26]. Thus, overexpression of caf1M in P1-pSM led to a decreased erythromycin and PMB MICs. The above two possibilities may also apply to F1 protein. Normally, the newly synthesized F1 capsular protein monomers are transported from cytoplasm to periplasm directed by its signal peptide and further traverse outer membranes to form polymers with the assistance of Caf1A and Caf1M [27]. In the absence of Caf1A and Caf1M, F1 proteins were not able to cross the outer membrane. Hence, F1 proteins trapped within the periplasm would display detrimental effects to the membrane, as in the case of P1-pSF1. Notably, P1-pSF1 showed no elevated sensitivity to PMB (Figure 6). One possible explanation for this observation, which will require further study, is that accumulated F1 proteins in the periplasm served as a protective layer against PMB disruption of the inner membrane. Nevertheless, since F1 proteins did not accumulate in the bacterial periplasm in the presence of Caf1A and Caf1M, F1-mediated Salmonella impairment might occur only sporadically. Since all three proteins overexpressed from the caf operon could impair Salmonella bacilli, we then questioned if proteins from any source could cause detrimental effects when accumulated within the periplasm. We observed no overt detrimental impact on Salmonella when chaperone gene cfaA or usher gene cfaC from the enterotoxigenic Escherichia coli (ETEC) cfa/I operon was overex- pressed in the periplasm [17]. Thus, impairment of Salmonella caused by overexpression of caf1M or caf1A seemed likely to be inherent in the caf operon. This deduction is virtually endorsed by the impacts of these individual genes on the Y. pestis strain KIM6+ (Figure 7). Overexpression of caf1A alone resulted in considerably high susceptibility of KIM6+ to both erythromycin and PMB. In addition to erythromycin and PMB, KIM6+/pSA also exhibited increased sensitivity to hydrogen peroxide, bile, and a high temperature of 37uC. This directly led to the lowered infectivity and survival of the KIM6+/pSA (Figure 8). Overexpression of caf1M alone in KIM6+ did not alter bacterial sensitivity to erythromycin and PMB, but resulted in the elevated sensitivity to bile, hydrogen peroxide, and high temperature. However, the macrophage infectivity and survival assays of KIM6+/pSM did not reveal any defects relative to control pY (Figure 8). Of interest is the strain KIM6+/pSF1 only showed slight susceptibility to PMB, but showed no sensitivity to erythromycin, hydrogen peroxide, bile, and high temperature. Nevertheless, KIM6+/ pSF1 displayed low infectivity similar to that of pSA, and its survival rate within macrophages was the lowest among all the recombinant Yersinia strains tested. This suggested the F1 antigen possessed the most potent detrimental effects in vitro among the three caf genes. As discussed above, the F1-mediated deleterious effects may not be fully implemented to Yersinia in the presence of the Caf1M and Caf1A, since it may be secreted out of the cells with the assistance of these two proteins [12,28]. By using KIM6+ to analyze the caf genes’ impact to the bacteria, we saw that these individual caf genes displayed a synergistically deleterious effect when co-expressed. This was demonstrated by the observation that KIM6+/pHF showed the most sensitive phenotypes to the hydrogen peroxide, bile, and high temperature among all the tested recombinant KIM6+ strains (Figure 7). Furthermore, caf Runs as a Double-Edged Sword during Infection KIM6+/pHF exhibited the lowest infecting capability compared with the other KIM6+ strains (Figure 8). This reduced infectivity may be associated with the overexpressed F1 capsular anti- phagocytic function [12], or may be associated with the following: (1) KIM6+/pHF may not be able to swim as vigorously as the other KIM6+ strains due to the viscous capsule on the cell surface. Previous study on Pseudomonas aeruginosa showed swimming motility is critical for the bacteria to establish a successful macrophage infection [29]. Thus, KIM6+/pHF may not effectively contact the macrophage to infect. (2) KIM6+/pHF may be neutralized by the macrophages after infection, particularly at the very early time points post-infection, i.e., during the infection incubation period. These two speculations may require a motile behavior assay and a dynamically microscopic observation for further investigation. In any case, this study implies that the vulnerability of caf operon found through Salmonella vector is applicable to the Yersinia. In summary, results achieved from this study do indicate that caf associates with serious vulnerabilities. In addition to the previously found immunogenic property of F1 capsular proteins which may potentially render Yersinia to an unfavorable condition during infection, this study suggests that the Caf1 apparatus may also be an Achilles’ heel when overexpressed. Yersinia benefits from caf operon expression via anti-phagocytic role of F1 capsule; however, it runs the risk of exposing the deleterious effects rooted in the Caf1 apparatus. Thus, the Caf1 apparatus may serve as a double- edged sword for Yersinia. Yersinia cautiously manages a balance between the advantages and disadvantages conferred by caf through regulating its expression with an elaborate promoter Pcaf1M, which determines when and where it should switch on and most importantly to what level it should transcribe, in order to take the advantage of F1 capsule while preventing the deleterious effects from being exposed. Materials and Methods Bacterial strains, media, and molecular manipulations The bacterial strains, plasmids, and their relevant characteristics are provided in Table 1. The primers used for polymerase chain reaction (PCR) are listed in Table 2. Diaminopimelic acid (DAP) (50 mg/ml) was used for E. coli H681 or S. Typhimurium P1 culture unless a plasmid containing asd was introduced. The asd+ plasmids pHF, pSA, pSF1, and pSM were constructed by means of H681 and transformed to P1 to generate Salmonella recombinant strains. Lysogeny broth (LB; 10 g of tryptone, 10 g of NaCl, and 5 g of yeast extract per liter) was used for recombinant E. coli and Salmonella growth at 37uC without DAP supplementation. Bacteria were cultured in LB and stored at 280uC in LB plus 20% glycerol. For growth rate comparison among the recombinant Salmonella strains, cells were inoculated from 280uC stock to LB agar for overnight incubation at 37uC. Salmonella bacilli were collected from plates, and the cell optical density at 600 nm (OD600) was adjusted to ,0.1. Bacterial inoculants were subjected to culture for 4 hrs at 37uC in BioScreen C (Lab Systems) with agitation at 150 rpm, and OD600 was measured every half hour. Data were downloaded and utilized for statistical comparison among these strains. Meanwhile, the bacterial CFUs were determined at one-hour intervals via colony enumeration after they were made serial dilutions on LB agar plates and incubation overnight at 37uC. To determine whether overexpression of caf operon or caf individual genes of caf1A, caf1, and caf1M impact the homologous bacterium Y. pestis, plasmids pF1, pHF, pSA, pSF1, pSM, and pY were transferred to KIM6+ after they were modified with a chloramphenicol resistance marker. Strain Y. pestis KIM6+ was a gift kindly provided by Robert D. Perry (University of Kentucky, PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e36283