“In the course of a proteomic analysis designed to discover

2. “In the course of a proteomic analysis designed to discover spore coat proteins, we identified several previously described exosporium proteins.”

3. Rationale • Anthrax: infection by B. anthracis spores • Understanding of disease • Prevention of or response to deliberate release as a bioweapon

4. Exosporium background • Present in some Bacillus species • Significant variation in structure • Means of attachment to spore unknown • Functions little understood • Attachment to host cells • Resistance to oxidative burst • Reduces innate immune response • Mediates phagocytosis • Regulates stickiness • Affects germination • May contain enzymes

5. Exosporium proteins • 20 proteins and glycoproteins • Lipids, carbohydrates • Orthologs of B. subtilis coat proteins • CotE (attachment?) • CotO (assembly?) • CotY, ExsY • Unique B. anthracis proteins • BclA – major protein component • ExsFA – basal layer, BclA assembly and projections = BxpB • ExsFB – paralog of ExsFA • BclB – stability • ExsFA-BclA-ExsY complex

6. Hypothesis • No overall hypothesis • Objective: characterize the role of ExsFA in exosporium

7. Mutant construction • B. anthracis “Ames strain,” virulent • exsFA mutant is RG124 • B. anthracis “Sterne strain,” attenuated • exsFA mutant is Ames-JAB-5 TcR pMR6 exsFA 5′ flanking sequence exsFA 3′ flanking sequence KmR chromosome chromosome exsFA KmR

8. Results: Electron microscopy • Growth and sporulation unaffected • TEM: “nap” missing from spores of both strains • Same finding as Steichen et al. • Sylvestre et al. reported fewer projections Steichen et al. Sylvestre et al. DexsFA wt

9. Results: Atomic force microscopy (AFM) • Mechanical imaging of untreated spores DexsFA wt

10. Results: Atomic force microscopy (AFM) – Fig. 1 • Loss of ridges on mutant spore coat wt Sterne mutant

11. Results: Immunofluorescence microscopy (IFM) – Fig. 2 • BclA normally located around forespore by 7h 1 cell bright field + Hoechst dye: binds DNA, blue fluorescence forespore mother cell chromosome mouse anti-BclA mAb fluorescent goat anti-mouse Ab

12. Results: Immunofluorescence microscopy (IFM) – Fig. 2 free spores

13. Results: Immunofluorescence microscopy (IFM) – Fig. 2 • Some BclA in mother cell at 7h • BclA around forespore at 8h and in free spores but polar • Associated with “cap” portion of exosporium? free spores 7h 8h

14. Results: Germination – Fig. 3 • Syto-9 dye taken up by germinating spore during rehydration (early) • Increased green fluorescence = germination • Mutant shows reduced germination, especially in Ames strain

15. Results: Germination – Fig. 3 • Reduced germination by loss of OD in Sterne strain with RPMI-BHI medium

16. Results: Germination – Fig. 3 • Late events monitored by tetrazolium overlay • No defect in mutants sporulate colonies on plate, heat to 80 °C overlay agar with TTC

17. Results: Virulence – Fig. 4 • Infected guinea pigs by i.m. and inhalation routes • No difference in virulence between wild-type and mutant intramuscular inhalation

18. gfp Fusion construction PCR from chromosome PCR from pKL147 exsFA 3′ end gfp pRG25 chromosome exsFA chromosome exsFA gfp

19. Results: Localization of ExsFA and ExsFB – Fig. 5 WT exsFA-gfp fusion DNA stain vegetative DNA stain 3 hrs DNA stain 6 hrs GFP DNA stain spores GFP

20. Results: Localization of ExsFA and ExsFB – Fig. 5 exsFA-gfp fusion exsFA-gfp fusion iunH-gfp fusion 6 hrs GFP spores GFP more spores

21. Results: Localization of ExsFA and ExsFB – Fig. 5 • IFM with anti-GFP antibody

22. Results: Localization of ExsFA and ExsFB – Fig. 5 • IFM with anti-GFP antibody in cotE and bclA mutants

23. What is the importance of this paper? • ExsFA (perhaps C terminus) required for exosporium “nap” • ExsFA plays a role in germination (contrary to others’ results) • ExsFA is not involved in virulence • ExsFA appears to be localized to the basal layer of the exosporium • ExsFB and IunH appear to be localized to the interspace

24. What is the importance of this paper? • Nap is dispensable for virulence: targeting the exosporium is a bad idea • Interesting but challenging to identify function of nap • Unusual paralogs (3rd in B. cereus) – adaptive role? • First step toward separating interspace and exosporium proteins/assembly