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

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

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

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

Hypothesis  No overall hypothesis  Objective: characterize the role of ExsFA in exosporium

Mutant construction  B. anthracis “Ames strain,” virulent exsFA mutant is RG124  B. anthracis “Sterne strain,” attenuated exsFA mutant is Ames-JAB-5 exsFA chromosome Km R chromosome pMR6 Km R exsFA 5′ flanking sequence exsFA 3′ flanking sequence Tc R

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. wt  exsFA Sylvestre et al.

Results: Atomic force microscopy (AFM)  Mechanical imaging of untreated spores wt  exsFA

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

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

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

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? 7h 8h free spores

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

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

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

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

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

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

spores Results: Localization of ExsFA and ExsFB – Fig. 5 exsFA-gfp fusion 6 hrsGFP exsFA-gfp fusioniunH-gfp fusion more spores

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

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

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

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 (3 rd in B. cereus) – adaptive role?  First step toward separating interspace and exosporium proteins/assembly