D. ,.

Figure 9 (Continued) (C) Defective binding and entry of macrophages by WI-I knockout yeasts is reversed following reexpression of WI-I. Unopsonized yeasts were incubated with macrophages for 6 hr; results are the mean ± SEM of three separate experiments. (D) Binding of yeasts to lung tissue ex vivo. Lungs of normal mice were removed and cryopreserved and 6-....m sections were applied to glass slides. The number of unopsonized yeasts attached to lung tissue sections was quantified. The extent of binding is defined as the number ofyeasts attached per 0.0 I mm2 of lung tissue, which was determined by inspecting a high-power field at 600 X magnification and counting 30 fields per strain in each experiment. (From Ref. 60.)

8. Indispensable Role of WI-l in Virulence It was hypothesized that WI-I knockout yeast unable to bind lung tissues and enter macrophages during infection would be less virulent. This hypothesis was tested in a murine model of lethal pulmonary blastomycosis [62J. All mice infected with the wild-type strain ATCC 26199 at a dose of IQ2 yeasts died from an overwhelming pulmonary and disseminated infection several weeks after inoculation, whereas all mice infected with the same dose of isogenic, WI-I knockout strain 55 lived and appeared healthy during observation over 72 days (Fig. lOA). Mice infected with either loJ or let yeasts of knockout strain 55, which are respectively 10 and 100 times the lethal dose of wild-type yeast, also survived and appeared healthy during the 72-day observation period. On postmortem examination, the lungs of mice infected with the knockout were macroscopically nonnal, but contained a small number of well-fonned granulomas with sequestered organisms (Fig. 108). The lungs of mice infected with the wild-type strain were filled with organisms and widespread inflammation. To confinn that the loss of WI-I is directly responsible for reduced virulence of strain 55 in vivo, it had to be shown that restored expression

Days post infection

Figure 10 Targeted gene replacement of WI-I reduces the pathogenicity of B. dermatitidis. (A) Survival following infection with wild-type strain 26199 and WI-I knockout strain 55. Male BALB/c mice (n = 15 mice/group) were infected intranasally with yeast cells of each strain, in varied doses. Survival was monitored for 72 days after infection. The two groups differ significantly (P <It: .(01) at each infectious dose tested. The experiment shown is representative of three independent experiments. The phenotype of knockout yeasts was stable; no revertants were identified among yeasts grown from mice infected with strain 55. (C) Survival after infection with wild-type strain 26199, WI-I knockout strain 55, and WI-I reconstituted strain 4155. Survival experiments were done as in (A), using a dose of 1<>4 yeasts to establish infection. The wild-type and WI-I reconstituted strains were significantly different from the WI-I knockout strain (P <It: .001 for each comparison). (From Ref. 60.)

Figure 10 (Continued) (B) Gross and microscopic pathology of mice infected with ATCC 26199 wildtype yeasts or WI-I knockout yeasts. Mice were analyzed 3 weeks postinfection. Lungs were stained with hematoxylin and eosin (H&E) to assess inflammation, and with Gomori Methenamine Silver (GMS) to visualize yeasts. The arrow denotes an isolated granuloma surrounded by normal lung tissue in a mouse infected with the WI-I knockout strain 55.