Mice Naturally Resistant to Yersinia pestis pgm Strains Commonly Used in Pathogenicity Studies

Plague in humans is caused by the gram-negative bacterium Yersinia pestis (6). The disease is 50 to 100% fatal if untreated and potentially could be caused on an epidemic scale by a malicious act. Accordingly, there is a need to understand the pathogenesis of Y. pestis and to enhance resistance to plague in people. A major virulence property of the plague-causing bacteria is the production of a set of six proteins called Yops that act within host cells to negate and modulate signaling pathways that orchestrate crucial host innate defense mechanisms (1). We had been studying YopM and had recently found that YopM counteracts innate host defenses, specifically causing a global loss of natural killer (NK) cells that could play a crucial defense role early in infection (4). In pursuit of that finding, we have been evaluating virulence of YopM Y. pestis in several strains of mice with defects in potential innate defense mechanisms that might be directly targeted by YopM. We fortuitously found a substrain (129S2/P2) of mouse strain 129 that is resistant to systemic plague due to Yersinia pestis KIM5. The 129S2/P2 mouse strain was of interest because it lacked expression of grancalcin (Gca / ) (10), a protein that may modulate actin bundling in bone marrow-derived cells, such as polymorphonuclear neutrophils (PMNs) and macrophages. Accordingly, we tested whether YopM is necessary for virulence in Gca / mice greater than 5 weeks old infected intravenously with Y. pestis KIM5 or a YopM mutant as described previously (4). (The Y. pestis KIM5 strain is highly attenuated when given by a peripheral route of infection due to deletion of the chromosomal pgm locus but is essentially fully virulent when it is given by an intravenous route [1, 11].) On the basis of data from two such experiments, the 50% lethal doses (LD50s) (9) for both the parent Y. pestis KIM5 and the YopM mutant were between 5 10 and 5 10 CFU. This is in contrast to an LD50 of 50 CFU for Y. pestis KIM5 versus 5 10 CFU for the YopM mutant in C57BL/6 mice (4). The Gca knockout had been made in a genetic background (129S2/P2) that we had not previously used in our studies. Accordingly, we tested whether it was the Gca / mutation or this background that was responsible for the plague resistance. The closest available match to the background strain was 129S2/Sv.Hsd (Harlan, Indianapolis, IN). As was the case for the Gca / mice, the LD50 for Y. pestis KIM5 in these mice was high, 2 10 CFU, and for the YopM mutant bacteria, it was 6 10 CFU (on the basis of the results of one experiment with female mice). We tested a second 129 mouse strain that has received extensive genetic characterization, 129P3/J (Jackson Laboratory, Bar Harbor, ME), with single doses of 10 CFU of the parent Y. pestis KIM5, and all four infected female mice survived, although they showed signs of illness (hunched posture and matted fur) (two experiments). A single similar test was made using a different pgm Y. pestis strain, CO993015.S5 (obtained from CDC, Ft. Collins, CO; a derivative of Y. pestis CO92), with the same outcome of all four mice becoming sick but surviving an intravenous challenge of 10 CFU. In a repetition of the experiment with a slightly higher dose of Y. pestis pgm strain CO99-3015.S5, all four mice died. These results suggested that the plague resistance likely was conferred by the 129 mouse genetic background but that different 129 substrains differed in the robustness of their resistance. This finding was interesting because there is no previous report of plague-resistant mice other than IL-10 / mice (7), and we have not seen evidence or reported polymorphisms that would indicate that these 129 mice lack interleukin 10 (IL-10). Dynamics of the infection. We infected groups of three female 129S2/P2 (Gca / ) mice intravenously with 5 10 CFU of Y. pestis KIM5 or Y. pestis KIM5-3002 (YopM ) and analyzed the number of viable bacteria (as CFU) in liver and spleen, leukocyte populations in spleen by flow cytometry, and histopathology in liver, all as previously described (4), on day 4 postinfection (two experiments). The numbers of viable bacteria for both bacterial strains in both liver and spleen were 10to 100-fold lower than those typically seen for infection of the susceptible C57BL/6 mouse strain (4, 7), as though the net growth of the Y. pestis strains were being limited or the bacteria were even being cleared. A robust acute inflammatory response dominated by infiltrating * Corresponding author. Mailing address: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY 40536-0298. Phone: (859) 323-6538. Fax: (859) 2578994. E-mail: [email protected]. † Present address: Bluegrass Community & Technical College, Cooper Drive, Lexington, KY 40506. ‡ Present address: The Blood Center of Southeastern Wisconsin, Inc., 638 N. 18th Street, P.O. Box 2178, Milwaukee, WI 53201-2178. Published ahead of print on 5 September 2006.